Chitinase-like proteins de-N-glycosylating CD36 modify cholesterol metabolism in atherosclerotic macrophages

Domain: neuroscience
Published: 2026-04-08
Editorial outcome: major_revision

Abstract
Polymorphisms of mouse chitinase-like protein 3 (Chil3), a member of the mammalian chitinase-like protein (CLP) family, have been demonstrated to be associated with inflammatory diseases by regulating lipid metabolism. However, the specific immunomodulatory impacts of CLPs, mainly mouse CHIL3 and its human functional homologue chitinase-3-like 2 (CHI3L2), on macrophage cholesterol metabolism and atherosclerosis have remained unclear. Here, we find CLPs (CHIL3 and CHI3L2) accelerate atherogenesis in a macrophage-dependent manner. Mechanistically, we identify an autocrine mechanism through which CLPs regulate cholesterol metabolism in macrophages. Macrophage-secreted CLPs exacerbate lipid uptake by binding to CD36. CLPs exhibit glycosidase activity, targeting and hydrolyzing N-glycosylated glycans on CD36, predominantly at sites N220 and N321, thereby enhancing lipid uptake. Increased lipid influx activates mTOR in macrophages, driving their transition to a pro-inflammatory phenotype while simultaneously suppressing peroxisome proliferator-activated receptor gamma (PPARγ) expression and thus impairing ABCG1-mediated cholesterol efflux. Single-cell sequencing reveals that CLPs increase atherosclerotic foamy macrophages, favoring vascular smooth muscle cells (VSMC) transformation into foam and osteoblast-like cells. Additionally, neutralizing antibodies targeting CHI3L2 prevent and treat atherosclerosis. These findings highlight the potential of CLPs as targets for disease diagnosis and therapy.

Introduction
Identification of key genes regulating metabolic diseases offers valuable insights into disease pathogenesis. In our previous study, we positionally cloned Chil3, encoding chitinase-like protein 3 (CHIL3/Ym1), as a susceptibility gene for inflammatory diseases through its modulation of macrophage lipid metabolism and alternative activation1. CHIL3 belongs to a family of chitinase-like proteins (CLPs), which lack chitinase activity due to mutations in putative enzyme sites2. Mammals possess several identified CLPs, including chitinase-3-like 1 (CHI3L1), CHI3L2, CHIL3, CHIL4, chitinase domain containing 1 (CHID1) and oviductal glycoprotein 1 (OVGP1)3. Notably, CHI3L2 is exclusive to humans and absent in rodents, whereas CHIL3 and CHIL4 are uniquely found in rodents4. Macrophages represent a significant source of both mouse and human CLPs. CHIL3 and CHI3L2 have emerged as markers of alternatively activated macrophages (AAMs)1,5, whereas CHI3L1 is prominently induced in classically activated macrophages (CAMs)6. CLPs, which contain a conserved glycosyl hydrolase family 18 (GH18) domain, participate in biological processes that contribute to chronic inflammatory and metabolic diseases through shared or molecule-specific immunometabolic functions3.

Atherosclerosis is a chronic, lipid-driven vascular inflammatory disease and a pivotal pathological precursor to cardiovascular diseases (CVD), involving the pathophysiological activation of macrophages, vascular smooth muscle cells (VSMCs) and endothelial cells (ECs)7,8. Dysregulation of cholesterol homeostasis in macrophages, mediated by the interplay of oxidized low-density lipoprotein (ox-LDL) uptake through scavenger receptors and cholesterol efflux, leads to macrophage dysfunction9,10. Furthermore, VSMCs are the main cellular component of advanced atherosclerotic aortas and are highly plastic cells that can undergo various phenotypic transformations, such as those into mesenchymal stem cells (MSCs), macrophage-like and osteochondrocyte-like cells11. These phenotypic transitions, which can be initiated by lipid uptake, enable VSMCs to contribute significantly to plaque development through enhanced lipid accumulation and calcification11. Identifying the molecular mechanisms controlling foam cell formation and retention within plaques holds promise for discovering potential clinical diagnostic markers and therapeutic targets for atherosclerosis. However, CLPs lacking chitinase activity have been poorly studied in atherosclerosis. Clinical studies have demonstrated a positive correlation between elevated serum CHI3L1/CHI3L2 levels and coronary artery disease development, plasma triglyceride (TG), and total cholesterol (TC) levels12-16. Only a few experimental studies have reported that CHI3L1 is associated with macrophage apoptosis, EC inflammation and activation of VSMCs, thereby exacerbating atherosclerosis17,18. Overall, there remains an absence of experimental evidence linking CLPs to immunometabolic regulation in chronic inflammatory diseases such as atherosclerosis. Our study aims to explore the involvement of CHIL3 and its human functional homologue CLP in the modulation of atherosclerosis.

Herein, we reveal that CLPs, specifically human-specific CHI3L2 and mouse-specific CHIL3, are markedly induced in atherosclerotic macrophages, and in particular alter the phenotypic transition of macrophages and VSMCs, skewing them towards a disease-promoting phenotype. Interventions targeting these CLPs can influence atherogenesis. Mechanistically, CLPs exhibit glycosidase activity, leading to scavenger receptor cluster of differentiation 36 (CD36) deglycosylation, thereby regulating lipid uptake. Additionally, CLPs play a crucial role in modulating cholesterol efflux and the immune-inflammatory state of macrophages. Moreover, the significant efficacy of neutralizing antibodies against CHI3L2 in treating atherosclerosis underscores the potential of CLPs as promising targets for precise diagnostic and therapeutic strategies. Together, these findings highlight the profound impact and intricate mechanisms of CLPs in atherosclerosis.

Results
Human CHI3L2 and mouse CHIL3 homologously exhibit parallel expression patterns in macrophage polarization and atherosclerosis

Our previous studies linked CHIL3 to lipid metabolism, suggesting a potential role in metabolic disorders such as atherosclerosis1. Here, we used atherosclerosis models to investigate the role of CHIL3, a mouse-specific CLP with no known human ortholog, and its human functional homologue, in lipid metabolism-related diseases. Integrated RNA sequencing (RNA-seq) database (GSE237219 and GSE20741420) showed significantly elevated expression levels of Chil1, Chil3 and Chid1 in the aortas of apolipoprotein E null (ApoE-/-) mice aged 32-40 weeks compared to wild-type (WT) mice (Supplementary Fig. 1a). The RNA-seq database (GSE28829)21 revealed that human CHI3L1 and CHI3L2 levels were significantly upregulated in advanced atherosclerotic plaque (AAP) compared with early atherosclerotic plaque (EAP) among human CLPs (Supplementary Fig. 1b). Based on the commonality of CLP expression in the human and mouse datasets, we prioritized Chil1/CHI3L1, Chil3, and CHI3L2 for further investigation. In the single cell RNA sequencing (scRNA-seq) dataset of the ApoE-/- and low density lipoprotein receptor null (Ldlr-/-) mouse models of atherosclerosis (GSE155513)11, Chil3 but not Chil1 expression was upregulated during atherosclerosis progression, particularly in atherosclerotic macrophages (Fig. 1a-f). Additionally, in the scRNA-seq dataset (GSE159677)22, CHI3L2 and CHI3L1 were abundant in the atherosclerotic core (AC) compared to proximally adjacent (PA) regions of human samples, and their expression in macrophages increased with disease progression (Fig. 1g-i). Based on these sequencing data, Chil3 and CHI3L2 demonstrated disease-associated induced expression.

Meanwhile, we also conducted a comparative analysis of CLPs between humans and mice from genetic and immunophysiological characteristics. The GH18 family includes chitinases and CLPs, with homologues loci between mice and humans identified, highlighting synteny between the mouse 1F4 and human 1q32 loci, and between mouse 3F3 and human 1p13 loci (Supplementary Fig. 1c)4,23. Notable orthologs in this family include CHIT1/Chit1, CHIA/Chia, CHI3LI1/Chil1, and OVGP1/Ovgp1 (Supplementary Fig. 1c). Although human CHI3L2 and mouse Chil3 are not sequence homologues, their closely matching chromosomal locations indicate that the GH18 family is widely distributed among mammals and retains conserved structural features, despite variations in expression across species. To further identify which human CLP share functions similar to CHIL3, we performed macrophage polarization experiments, leveraging the fact that CHIL3 is a typical marker of AAMs. Polarization of mouse peritoneal macrophages (PMφ) into CAMs using lipopolysaccharide (LPS) and interferon-γ (IFN-γ) or AAMs using interleukin (IL)-4 and IL-13 was conducted. CHIL3 levels were increased in AAMs but not in CAMs, indicating CHIL3 serves as a signature marker for AAMs (Supplementary Fig. 1d, e). Particularly, CHI3L2 rather than CHI3L1 was highly expressed in THP-1-derived AAMs, suggesting a consistent effect of CHI3L2 on macrophages akin to CHIL3 (Supplementary Fig. 1f, g). It was clear that CHIL3 and CHI3L2 showed consistent expression pattern during macrophage polarization.

Given the parallel expression pattern of human CHI3L2 and mouse CHIL3 in macrophage polarization and atherosclerosis, our prior focus was directed solely on these two among CLP family. Therefore, "CLPs" in this study specifically refer to mouse CHIL3 and human CHI3L2. The expression of CHIL3 and CHI3L2 was further confirmed in mouse models or human patients. We found that ApoE-/- mice on a Western diet (WD) for 3 months exhibited markedly higher circulating levels of CHIL3 protein compared to those on a normal diet (ND) (Fig. 1j). Additionally, CHI3L2 protein levels were markedly elevated in human atherosclerotic plasma (Fig. 1k), and immunofluorescence of human carotid endarterectomy specimens showed higher CHI3L2 in severe (atheromatous plaque stage, APS) versus fibrous plaque stage (FPSI) (Fig. 1l). Furthermore, CHI3L2 levels were similarly increased in CHI3L2 transgenic mice (ApoE-/-.Chil3∆.hCHI3L2tg) under WD conditions (Supplementary Fig. 1h), supporting its role in plaque formation.

Nature polymorphism of Chil3 and human functional homologue CHI3L2 influence atherogenesis in mice

Then the role of CLPs (CHIL3 and CHI3L2) on atherosclerosis was investigated. We used a Chil3 congenic deficient mouse strain (ApoE-/-.Chil3∆) which has a deletion of CHIL3 protein due to the nature polymorphism of Chil3 gene1, and also created a humanized CHI3L2 transgenic mouse strain (ApoE-/-.Chil3∆.hCHI3L2tg) crossed with ApoE-/-.Chil3∆ to exclude endogenous Chil3 interference. All male and female littermates were then subjected to a WD diet for 3 months. Metabolic parameters including serum TC, TG and high-density lipoprotein cholesterol (HDL-C) were comparable, while the levels of low-density lipoprotein cholesterol (LDL-C) and ox-LDL were significantly reduced in male and female ApoE-/-.Chil3∆ mice compared to ApoE-/- and ApoE-/-.Chil3∆.hCHI3L2tg mice (Fig. 2a, b). En-face Oil red O (ORO) staining of aortas and hematoxylin and eosin (H&E) and ORO staining of aortic roots revealed that the atherosclerotic lesion area of both male and female ApoE-/-.Chil3∆ mice was significantly less than that of ApoE-/- and ApoE-/-.Chil3∆.hCHI3L2tg mice (Fig. 2c-e). Compared with the male or female ApoE-/- and ApoE-/-.Chil3∆.hCHI3L2tg mice, in ApoE-/-.Chil3∆ mice, morphological analyses of the cross-sectional lesions showed an increase in collagen content by using Sirius red and contractile smooth muscle cell marker alpha smooth muscle actin (α-SMA) immunofluorescence staining, and a decrease in macrophages by using the macrophage marker CD68 immunofluorescence staining, respectively (Fig. 2d-g). Consistently, both male and female ApoE-/-.Chil3∆ mice exhibited reduced VCAM-1 (vascular cell adhesion molecule-1) expression in aortic root lesions compared to ApoE-/- and ApoE-/-.Chil3∆.hCHI3L2tg mice, while ICAM-1 (intercellular adhesion molecule-1) expression showed no significant difference (Fig. 2f, g), indicating that CLPs may preferentially regulate VCAM-1-associated leukocyte recruitment pathways, without broadly affecting ICAM-1-mediated endothelial activation.

In addition to genetic manipulation, we also administered recombinant human CHI3L2 (rhCHI3L2) protein supplementation to mice and observed its effects on the atherosclerosis model. We first placed 2-month-old ApoE-/-.Chil3∆ mice on a WD diet for 1 month and then injected the rhCHI3L2 or control protein intraperitoneally every three days for 2 months, while continuing the WD diet (Supplementary Fig. 2a). ELISA analysis demonstrated that exogenous rhCHI3L2 was successfully delivered and could be detected in the mouse circulation (Supplementary Fig. 2b). Our results showed that treatment with rhCHI3L2 significantly increased the lesion area in both the aorta and aortic root, while collagen content in the aortic root was significantly reduced (Supplementary Fig. 2c, d). Collectively, these findings highlight CHI3L2 can accelerate atherosclerosis progression.

CLPs alter the cell-specific phenotypic transition of macrophages and VSMCs

To delineate the role of CLPs in atherosclerotic cell fate, we performed scRNA-seq on aortic tissues from WD-fed littermate mice (ApoE-/-, ApoE-/-.Chil3∆ and ApoE-/-.Chil3∆.hCHI3L2tg). The analysis identified a diverse array of cell types, including T cells, ECs, macrophages, fibroblasts and VSMCs (Fig. 3a), with cell type annotation based on established markers (Supplementary Fig. 3a). Localization analysis of CLPs showed predominant localization of Chil3 in macrophages of ApoE-/- mice, contrasting with its absence in ApoE-/-.Chil3∆ and ApoE-/-.Chil3∆.hCHI3L2tg mice (Fig. 3a, b). CHI3L2 also exhibited predominant expression in macrophages of ApoE-/-.Chil3∆.hCHI3L2tg mice, distinguishing them from the other groups (Fig. 3a, b). Differences in cell type composition were observed in these three groups, with macrophages aggregating less in the aorta of CLP-deficient mice, consistent with our previous immunofluorescence results for CD68-positive macrophages (Fig. 3c, 2f, g).

We further classified macrophage subpopulations and performed pseudotime trajectory analysis, revealing seven distinct states based on gene expression dynamics (Supplementary Fig. 3b, c). The trajectory originated from Ly6C2+ monocyte-like, MHC-II, and resident macrophages, progressed through foamy and inflammatory macrophages, and later transitioned into VSMC-like and fibrochondrocyte (FC)-like macrophages (Supplementary Fig. 3c). Differential gene expression supported this trajectory: foam cell-related genes Lpl and Trem2 were upregulated in foamy macrophages, whereas Myh11 increased in later states (Supplementary Fig. 3d). Harmony integration of macrophages across the three experimental groups showed seven distinct cellular subpopulations aligned with the pseudotime trajectory (Fig. 3d). Notably, Chil3 was specifically enriched in Ly6C2+ monocyte-like macrophages in ApoE-/- mice, whereas CHI3L2 was mainly expressed in foamy, VSMC-like and FC-like macrophages in ApoE-/-.Chil3∆.hCHI3L2tg mice (Fig. 3e). Functionally, Chil3 increased inflammatory macrophages and reduced resident macrophages, while CHI3L2 expanded foam cell/FC-like clusters at the expense of resident and MHC-II macrophages (Fig. 3f). These findings suggest that CLPs not only influence macrophage phenotypic transition but also enhance macrophage infiltration during atherosclerosis progression.

The pseudotime trajectory analysis of VSMCs subpopulations derived from annotation of known markers revealed that the trajectory originated from contractile VSMCs, progressed through an intermediate cell state (ICS), and bifurcated into macrophage-like and FC-like VSMCs (Supplementary Fig. 3e, g). Genes associated with pseudotime progression were identified along these developmental trajectories. For instance, terminal VSMCs showed regulation of macrophage (Lgals3), osteogenic (Ibsp), and chondrogenic (Acan) genes (Supplementary Fig. 3f). Integration analysis demonstrated effective integration of VSMCs across the three experimental groups (Supplementary Fig. 3h). ICS cells exhibited elevated stem cell markers (Ly6a, Cd34) and reduced canonical SMC markers, indicating a transitional, multi-lineage phenotype with loss of the typical SMC phenotype (Supplementary Fig. 3g, i). Along the trajectory, increasing expression of FC-related (Col2a1, Ibsp, Acan) and macrophage-related (Lgals3, Tyrobp, Fcer1g) genes indicated the differentiation potential of ICS toward FC- or macrophage-like VSMCs (Supplementary Fig. 3g, i). Moreover, the presence of CLPs notably expanded the macrophage-like VSMCs and FC-like VSMCs, indicating that CLPs promote VSMC foam cell formation and calcification (Fig. 3g, h). To further validate the CLP-associated interaction between macrophages and VSMCs, we applied BMDM supernatants from ApoE-/-, ApoE-/-.Chil3∆ and ApoE-/-.Chil3∆.hCHI3L2tg mice on VSMCs to assess foam cell formation and calcification (Fig. 3i). The results were consistent with the scRNA-seq data, indicating that macrophage supernatants containing CLPs significantly promote lipid deposition and calcium deposition in VSMCs (Fig. 3j, k).

We subsequently examined fibroblast subpopulations and observed their differentiation into myofibroblasts (Supplementary Fig. 3j). Fibroblasts exhibited high expression of Ly6a and Bmp4, indicative of differentiation from MSCs or adipocytes (Supplementary Fig. 3k, i). Minor differences were detected between fibroblast subtypes across the three groups (Supplementary Fig. 3m, n). Overall, these findings suggest that CLPs can influence the phenotypic transition of atherosclerosis-associated cells, particularly macrophages and VSMCs.

CLPs accelerate atherogenesis in a macrophage-dependent manner

scRNA-seq highlights Chil3 and CHI3L2 expression predominantly in macrophages. This finding was further corroborated by co-localization experiments, which demonstrated the overlap of CHIL3 or CHI3L2 with CD68-positive macrophages in mouse atherosclerotic aortas (Fig. 4a and Supplementary Fig. 4a). Immunofluorescence staining of human atherosclerotic plaques demonstrated that CHI3L2 was highly expressed in plaque macrophages of APS compared to FPS (Fig. 4b and Supplementary Fig. 4b). Further investigation using ox-LDL-stimulated macrophages to induce foam cell formation showed markedly increased CHIL3 expression in foam cells derived from PMφ, bone marrow-derived macrophages (BMDMs), as well as high CHI3L2 expression in foam cells derived from PMφ, BMDMs in transgenic mice, and the human THP-1 cell line (Supplementary Fig. 4c, d). Additionally, analysis of the publicly available human RNA-seq dataset (GSE125126)24 revealed a significant upregulation of CHI3L2 expression in atherosclerotic macrophages (Supplementary Fig. 4e). These findings are consistent with public scRNA-seq data, which also demonstrate elevated expression of CHIL3 and CHI3L2 in macrophages within atherosclerotic lesions (Fig. 1c, f, i).

Then a bone marrow transplantation (BMT) model was used to assess the role of myeloid CLPs in atherosclerosis. Bone marrow (BM) cells from ApoE-/-, ApoE-/-.Chil3∆ and ApoE-/-.Chil3∆.hCHI3L2tg mice, all littermates expressing the CD45.1 alloantigen, were transplanted into lethally irradiated ApoE-/- recipients expressing the CD45.2 alloantigen (Fig. 4c), and the successful hematopoietic reconstitution was confirmed by flow cytometry (Supplementary Fig. 4f). After 12 weeks of WD feeding, H&E and ORO staining revealed reduced aortic lesion areas in mice receiving Chil3-deficient BM compared to those receiving CLP-expressing BM (Fig. 4d, e). Additionally, Chil3 deficiency also increased collagen deposition, with CHI3L2 overexpression reversing these effects (Fig. 4e). Given the macrophage-specific expression of Chil3, our animal model effectively demonstrates its impact on atherosclerosis. In contrast, CHI3L2 is broadly expressed; therefore, we generated a humanized CHI3L2 conditional knock-in mouse strain crossed with LysM-Cre mice in order to achieve macrophage-specific CHI3L2 expression (denoted as M-CHI3L2ki/wt). An additional model was established by injecting an adeno-associated virus-8 carrying a mutant PCSK9 gene (AAV8-PCSK9) into M-CHI3L2ki/wt and littermate control mice (M-CHI3L2wt/wt), followed by 3 months of WD feeding (Fig. 4f). Compared to controls, M-CHI3L2ki/wt mice developed more extensive lesions throughout the entire aortas and aortic roots, along with increased plaque burden and greater plaque instability (Fig. 4g, h). These findings further support a macrophage-centered role for CLPs in regulating lipid metabolism and atherosclerosis progression.

CLPs cause disequilibrium in cholesterol metabolism of macrophages though facilitating ox-LDL uptake and lipid deposition

The role of CLPs in macrophage cholesterol metabolism was then examined. A significant decrease in lipid deposition within PMφ and BMDMs with CLP deficiency, but a significant increase in CLP-enriched macrophages, was revealed by ORO and BODIPY493/503 staining, suggesting an enhancement of foam cell formation by CLPs (Supplementary Fig. 5a-c and Fig. 5a-c). Moreover, administration of recombinant CHIL3 protein (rCHIL3) or rhCHI3L2 in macrophages significantly intensified lipid deposition (Fig. 5d, e).

Using flow cytometry and a multiphoton microscope employing Dil-labeled ox-LDL (Dil-ox-LDL), we provided additional evidence supporting the involvement of CHIL3 and CHI3L2 in lipid uptake. Chil3 deficiency markedly impaired ox-LDL uptake in PMφ and BMDMs, whereas CHI3L2 overexpression heightened ox-LDL uptake compared to controls (Supplementary Fig. 5d, e and Fig. 5f, g). Additionally, lipid uptake in PMφ and BMDMs exhibited a gradient increase with escalating concentrations of rCHIL3 or rhCHI3L2, consistent with findings observed in THP-1-derived macrophages treated with rhCHI3L2 (Supplementary Fig. 5f and Fig. 5h, i). These collective observations suggest CLPs promote ox-LDL uptake and foam cell formation, disrupting macrophage cholesterol homeostasis.

CLPs interact with CD36 and promote CD36 endocytosis to enhance lipid uptake

Given their secretory nature, CLPs may affect macrophage cholesterol metabolism via interactions with membrane proteins. Utilizing immunoprecipitation (IP) and mass spectrometry (MS) analyses, we identified the top five candidate CHIL3-interacting cell-membrane proteins in PMφ, including CD36, a pivotal scavenger receptor managing ox-LDL internalization in macrophages (Fig. 6a). Co-IP with His-tagged rCHIL3/CHI3L2 in BMDMs confirmed interaction with CD36, but not with other scavenger receptors such as oxidized low-density lipoprotein receptor 1 (OLR1) and macrophage scavenger receptor 1 (MSR1) (Fig. 6b). Further validation through Co-IP with antibodies against CD36 confirmed the efficient interaction of these proteins (Fig. 6c). Establishment of a 293T cell line overexpressing His-tagged CHIL3/hCHI3L2 and Flag-tagged CD36 enabled detection of the association between His-CHIL3/hCHI3L2 and Flag-CD36 in these cells (Supplementary Fig. 6a and Fig. 6d). Moreover, immunofluorescence staining directly showed the interaction of CHIL3/CHI3L2 with CD36 in BMDMs or THP-1-derived macrophages (Supplementary Fig. 6b). Notably, interaction studies excluded the binding of CHIL3 or CHI3L2 with ox-LDL (Supplementary Fig. 6c, d).

Given the demonstrated interaction between CLPs and CD36, we hypothesized that CLPs facilitate CD36 internalization, and thus, lipid uptake. Notably, while total CD36 protein expression remained comparable in macrophages from ApoE-/-, ApoE-/-.Chil3∆ and ApoE-/-.Chil3∆.hCHI3L2tg mice (Supplementary Fig. 6e), surface CD36 was decreased upon ox-LDL treatment in ApoE-/- and ApoE-/-.Chil3∆.hCHI3L2tg macrophages, a phenomenon not observed in ApoE-/-.Chil3∆ macrophages, indicating that CLPs promote CD36 internalization (Fig. 6e, f). Furthermore, exogenous rCHIL3 or rhCHI3L2 treatment promoted CD36 internalization in a concentration-dependent manner (Fig. 6g, h). Immunofluorescence staining unveiled ox-LDL-induced trafficking of CD36 to early endosomes, with observed co-localization with early endosome antigen 1 (EEA1) and Ras-related protein Rab11 (a recycling endosome marker) exclusively in ApoE-/- macrophages following ox-LDL treatment, contrasting with the absence of such co-localization in ApoE-/-.Chil3∆ macrophages (Supplementary Fig. 6f).

Then we conducted rescue experiments based on the hypothesis that exogenous soluble CD36 protein would compete with cell surface CD36 for binding to CLPs and subsequently reduce the lipid uptake induced by CLPs. We utilized recombinant extracellular domain (ECD) of mouse or human CD36 proteins (Gly30-Lys439, rmCD36ECD or rhCD36ECD), to explore the potential competitive binding of exogenous CD36 to CLPs. Notably, administration of rCHIL3 protein significantly augmented cellular lipid uptake; however, pre-incubation of rCHIL3 protein with rmCD36ECD led to a marked reduction in cellular lipid uptake compared to the sole administration of rCHIL3 protein (Fig. 6i). Similarly, lentivirus-mediated knockdown of CD36 in BMDMs yielded comparable effects to those observed with rmCD36ECD treatment (Fig. 6j), with knockdown efficiency validated via Western blotting (Supplementary Fig. 6g). Furthermore, administration of rhCD36ECD resulted in competitive binding to CHI3L2, consequently attenuating lipid uptake in BMDMs relative to treatment with rhCHI3L2 protein alone (Fig. 6k). Collectively, these findings underscore the critical role of CLPs interaction with CD36 in mediating lipid uptake and cholesterol metabolism.

CLPs hydrolyze N-glycan of CD36 to promote lipid uptake

To delineate the mCD36 ECD responsible for the interaction with CHIL3, we generated several Flag-tagged mCD36 constructs, including full-length wild-type construct and truncated constructs with ECD partial deletion (denotated as Flag-mCD36, Flag-mCD36∆1, Flag-mCD36∆2, or Flag-mCD36∆3; see Fig. 7a). Each of these constructs was transfected into 293T cells along with His-tagged CHIL3, and subsequent Co-IP analyses were performed. Interestingly, no matter which segment of CD36ECD was truncated, the interaction between these truncated CD36ECD constructs and CHIL3 always remained similar to that of the full-length protein, suggesting that CHIL3 binding might be independent on the protein structure of CD36 (Fig. 7a). Considering the lectin-like properties of the CLP family, we hypothesized its potential binding to extracellular oligosaccharide moieties of CD36. Glycosylation serves as a crucial modifier of membrane proteins, and CD36 is a highly N-glycosylated integral membrane protein (Fig. 7a)25. To further confirm the dependence of CD36 N-glycosylation on CHIL3 interactions, the binding of rCLPs to PNGase F (an N-glycan endoglycosidase) treated CD36 protein was scrutinized, with untreated CD36 protein serving as a control. A remarkable gel shift of CD36 protein in SDS-PAGE indicated a change of glycosylation upon PNGase F treatment, accompanied by diminished interaction between CD36 and rCLP protein (Fig. 7b). Considering the extensively conserved nature of CLPs and the previously documented weak β-N-acetylglucosaminidase activity of CHIL3, alongside the interaction of CHI3L2 with the N-acetylglucosamine (GlcNAc) units26,27, the proteoglycan content was assessed using wheat germ agglutinin (WGA), a GlcNAc-specific lectin, revealing a significant reduction in rmCD36ECD/rhCD36ECD upon rCHIL3/rhCHI3L2 treatment (Fig. 7c). This diminution in CD36 glycan content caused by CLPs markedly enhanced the binding of CD36ECD to ox-LDL (Fig. 7d).

Label-free quantitation of intact glycopeptides and modified glycans by MS was employed to assess CD36 N-glycosylation levels with or without rCLP treatment. Glycans and three N-glycosylation sites (N220, N320, and N321) were identified in mouse CD36, and two sites (N220 and N321) were identified in human CD36 (Fig. 7e). Interestingly, treatment with rCHIL3 decreased N-glycosylation levels on N220 and N321 in rmCD36ECD, while treatment with rhCHI3L2 reduced N-glycosylation levels on N220 in rhCD36ECD (Fig. 7e). Meanwhile, most of the glycoforms at these sites were reduced using rCLP treatment compared to the control, confirming the ability of CLPs to diminish CD36 N-glycosylation (Fig. 7f).

We next established CD36 constructs with mutations at glycosylation sites and transfected them into the 293T cell line. Both the single mutation at N220 of mouse CD36 (CD36N220Q) and the double mutations at N220 and N321 (CD36N220Q-N321Q) did not affect the membrane translocation of CD36 (Fig. 7g). However, these mutations led to an increase in ox-LDL uptake compared to WT construct (Fig. 7h, i). Meanwhile, rCLP protein treatment could only increase lipid uptake in WT construct, and not in mutant constructs without glycosylation on N220 and N321, suggesting that CLPs target glycosylation sites to regulate CD36 function (Fig. 7h, i). In conclusion, these results demonstrate that CLPs with glycosidase hydrolytic activity bind and partially hydrolyze the N-glycosylated glycans of CD36, thereby promoting lipid uptake.

CLPs inhibit PPARγ-mediated cholesterol efflux through activating mTOR signaling pathway

In addition to the mechanism by which CLP targets CD36 to induce lipid intake, the secondary effects triggered by lipid overload in macrophages were further investigated based on our sequencing data. Gene set enrichment analysis (GSEA) of differentially expressed genes in macrophages from scRNA-seq revealed that CLP deficiency downregulated inflammation-related pathways, including the NOD-like receptor, chemokine, and cytokine-cytokine receptor interaction signaling pathways, while upregulating metabolism-related pathways such as oxidative phosphorylation, PPAR signaling, and glutathione metabolism (Supplementary Fig. 7a). This suggests that CLPs may be involved in regulating macrophage inflammatory responses, energy metabolism, and antioxidant capacity. In addition, the lysosome pathway was also markedly enhanced, reflecting increased cellular degradation and recycling capacity in CLP-deficient macrophages, which may indirectly support metabolic reprogramming and energy supply (Supplementary Fig. 7a). Further analysis showed that the core component of the NOD-like receptor family, the pyrin domain-containing 3 (Nlrp3) inflammasome, was significantly downregulated in macrophages from ApoE-/-.Chil3∆ mice, accompanied by markedly reduced expression of pro-inflammatory cytokines, including Il1b, Il6 and Tnf (Supplementary Fig. 7b), suggesting that the inflammasome may serve as a critical mediator through which CLPs regulate inflammatory cytokine expression in macrophages.

We subsequently performed bulk RNA-seq analysis of ox-LDL stimulated BMDMs isolated from ApoE-/- and ApoE-/-.Chil3∆ mice, revealing significant alterations in the expression of 1081 genes, with 463 downregulated and 618 upregulated genes in Chil3-deficient macrophages (Supplementary Fig. 7c). KEGG pathway enrichment analysis revealed the cytokine-cytokine receptor interaction pathway was the most significantly altered (Supplementary Fig. 7d). Notably, enrichment analysis of cholesterol metabolism genes suggested that CHIL3 may negatively regulate cholesterol transport and efflux. In Chil3-deficient macrophages, genes involved in cholesterol transport and hydrolysis, including Npc1/2 (Niemann-Pick C1/2), Stard3 (Star-related lipid transfer domain protein 3), Lipa (lysosomal acid lipase), and Nceh1 (neutral cholesterol ester hydrolase 1), as well as the efflux gene Abcg1 (ATP-binding cassette subfamily G member 1) were significantly upregulated, whereas cholesterol synthesis and uptake genes showed little change, with only Mylip (myosin regulatory light chain interacting protein, an E3 ubiquitin ligase promoting LDLR degradation) also being upregulated (Supplementary Fig. 8a). Upregulation of Soat1 (sterol O-acyltransferase 1/ACAT1) reflects coordinated cholesterol storage to maintain cellular homeostasis (Supplementary Fig. 8a). RT-qPCR further confirmed that Chil3 deficiency significantly upregulated Abcg1 and Mylip, and Western blotting showed that CLP deficiency significantly increased ABCG1 expression but not ABCA1 expression, suggesting a liver X receptor α (LXRα)-independent efflux pathway (Fig. 8a, Supplementary Fig. 8b-d). Previous study has shown that ABCG1 could, in certain contexts, occur independently of LXR activation28. This supports a role for CLPs in regulating ABCG1-mediated, LXRα-independent cholesterol efflux.

It has been reported that ABCG1 not only promotes cholesterol efflux but also contributes to lipid reprogramming and functional regulation29. Guided by this, we performed untargeted lipidomic profiling of macrophages and the results revealed that the loss of CLPs led to a marked reduction in lipid species associated with lipid storage, particularly cholesteryl esters (CE), accompanied by decreased levels of total cholesterol (Chol). These changes were consistent with the reduced foam cell propensity observed in our model (Fig. 8b, c). In contrast, membrane associated lipids, including phospholipids (phosphatidylethanolamine (PRE), phosphatidylglycerol (PG), phosphatidylserine (PS), phosphatidylinositol (PI)), lysophospholipids (lysophosphatidylcholine (LPC), lysophosphatidylethanolamine (LPE)), and sphingolipids (ceramides (Cer)), were increased (Fig. 8b, c). This indicates that the loss of CLPs leads to membrane-centric lipid remodeling, altering the composition and biophysical properties of the bilayer membrane. Correspondingly, cholesterol efflux to HDL (ApoB-depleted serum) was markedly enhanced in Chil3-deficient macrophages, further emphasizing the ABCG1-mediated cholesterol efflux (Fig. 8d).

Our previous research has shown that CHIL3 regulates PPARγ to coordinate macrophage polarization1, and PPARγ is involved in the cholesterol efflux30. Using Western blotting, we observed that the absence of CLPs led to elevated levels of PPARγ and ABCG1 in macrophages with ox-LDL stimulation (Fig. 8e). In addition, T0070907, an inhibitor of PPARγ, reduced ABCG1 expression in ApoE-/-.Chil3∆ macrophages but not ApoE-/- or ApoE-/-.Chil3∆.hCHI3L2tg macrophages (Fig. 8e). Macrophages respond to complex environmental cues by polarizing into distinct phenotypes. The nuclear receptor PPARγ drives robust phenotypic changes in macrophages following repeated stimulation with IL-4. In this context, the functional role of PPARγ is independent of ligand binding, and its upregulation under AAMs conditions is a common and conserved phenomenon31-34. We found that CLP deficiency significantly reduced the expression of inflammatory cytokines such as Il1b, Il6 and Tnf in foam cells, indicating an increased proportion of anti-inflammatory macrophages (Fig. 8f). In fact, as a negative regulator of AAMs, Chil3 deficiency enhances the AAM phenotype in mice1. Thus, we propose that enhanced PPARγ expression in CLP-deficient macrophages stems from increased anti-inflammatory macrophages rather than ligand-dependent mechanisms.

Lysosome-derived nutrients, including carbohydrates, lipids, and amino acids, are known to activate mammalian target of rapamycin (mTOR)35-37. In macrophage foam cells, lipids internalized via CD36 dissociate from the receptor in the acidic endosomal environment; CD36 is recycled to the plasma membrane, whereas ox-LDL is delivered to lysosomes for degradation38. The resulting free lipids, particularly cholesterol, are sensed within lysosomes and act as major inducers of mTOR activation. Our data provide functional validation for this lysosomal core model. Compared to ApoE-/-.Chil3∆ BMDMs, excessive ox-LDL strongly induced mTOR activation in ApoE-/- and ApoE-/-.Chil3∆.hCHI3L2tg BMDMs (Supplementary Fig. 8e). This mTOR activation was completely eliminated when lysosomal acidification and function were specifically inhibited using Baflomycin A1 (Baf-A1), underscoring the essential role of functional lysosomes in ox-LDL-driven mTOR signaling (Supplementary Fig. 8e). Recent studies have reported the critical role of mTOR nutrient sensing in regulating macrophage polarization, indicating that the mTOR pathway significantly promotes CAMs activation39-41. We found that the presence of mTOR inhibitor rapamycin significantly reversed the CLP-induced upregulation of inflammatory cytokines such as Il1b, Il6 and Tnf in foam cells, indicating that mTOR inhibits AAMs activation and may affect PPARγ (Fig. 8f). In fact, administration of rapamycin led to an upregulation in PPARγ and ABCG1 levels in ApoE-/- and ApoE-/-.Chil3∆.hCHI3L2tg macrophages (Fig. 8g). Furthermore, functional experiments demonstrated that treatment with rapamycin and PPARγ inhibitors reversed the effects of CLPs on macrophage lipid deposition (Supplementary Fig. 8f, g). These findings elucidate that CLPs reduce the proportion of anti-inflammatory macrophages by promoting mTOR signaling, thereby inhibiting PPARγ-mediated cholesterol efflux.

Neutralization of CHI3L2 protects against atherosclerosis and promotes atheroma resolution

To evaluate the impact of eliminating CHI3L2 expression on atherosclerosis, we tested the effects of a CHI3L2-neutralizing antibody both in vitro and in vivo. First, we assessed the in vitro efficacy of the antibody and found that rhCHI3L2 administration to THP-1-derived macrophages significantly increased cellular lipid deposition and lipid uptake (Fig. 9a, b). In contrast, co-administration of rhCHI3L2 and the CHI3L2-neutralizing antibody significantly reduced both lipid deposition and uptake compared to isotype control IgG antibodies (Fig. 9a, b). In vivo, injection of the CHI3L2-neutralizing antibody via tail vein into ApoE-/-.Chil3∆.hCHI3L2tg mice on a WD (Fig. 9c) significantly reduced overall plaque burden in the aorta, as assessed by en-face ORO staining, and decreased lesion area and plaque area in the aortic root, as shown by H&E and ORO staining (Fig. 9d, e). However, there was no effect on the collagen content of the aortic root, as determined by Sirius red staining, compared to isotype control IgG antibody injections (Fig. 9d, e). Given the macrophage cholesterol efflux can promote atherosclerosis regression, we also evaluated the therapeutic effect of CHI3L2 neutralizing antibody on atherosclerosis regression. ApoE-/-.Chil3∆.hCHI3L2tg mice were subjected to 3 months of WD followed by 1 month of ND feeding combined with CHI3L2 neutralizing antibody or IgG control treatment (Fig. 9f). Histological staining revealed that the presence of CHI3L2 antibody significantly reduced plaque size compared to IgG, although it had no significant effect on plaque stability (Fig. 9g, h). These results suggest the potential clinical therapeutic role of CHI3L2-neutralizing antibodies in atherosclerotic disease.

Methods
Materials
The reagents and antibodies used in this study are listed in Supplementary Table 4 (Antibodies and manufacturers) and Supplementary Table 5 (Reagents and manufacturers). All chemicals were of analytical grade and used without further purification unless otherwise specified.

Human plasma and aortic tissue samples
Human plasma and aortic tissue samples were collected with approval from the Ethics Committee of Xi’an Jiaotong University (No. LLSPJ-2023-304), and all participants provided written informed consent. Identifying information was removed to ensure privacy. Experiments complied with the Declaration of Helsinki. Plasma samples were obtained from healthy individuals (n=34, all healthy individuals underwent checkups that excluded cardiovascular diseases, autoimmune disorders, acute and chronic infections, malignant tumors, and liver or kidney insufficiency) and specimens with atherosclerosis (n=38), which were diagnosed by ultrasound. Human carotid endarterectomy specimens, including atheromatous plaque stage (APS, n=19) and fibrous plaque stage (FPS, n=19), were evaluated blindly by experienced pathologists according to the atherosclerosis staging system, were collected from the Department of Pathology, The First Affiliated Hospital of Xi'an Jiaotong University. Persons with diabetes or hypertension were excluded. The human sample collection was not restricted to a single sex, enhancing the generalizability of the study. Baseline characteristics are summarized in Supplementary Table 1 and Supplementary Table 2.

Mouse strains
Chil3-deficient congenic mice with B10.RIII background (C57Bl/10 H-2r (BR)) were generated in our previous positionally cloning studies1,57,58, and backcrossed with C57BL/10 H-2q.ApoE-/- mice (denoted as BQ.ApoE-/-) to replace the background to C57BL/10 H-2q (BQ), with ApoE deficiency to accelerate spontaneous atherosclerosis (denoted as BQ.ApoE-/-.Chil3∆, or ApoE-/-.Chil3∆). Human CHI3L2 transgenic mice were created by using a PiggyBac-on-BAC transgenic strategy (Cyagen, China). Human CHI3L2 coding sequence with its regulatory sequence was introduced into the genome of C57BL/6N mice. The transgenic mice were then backcrossed with BQ.ApoE-/-.Chil3∆ mice to generate BQ.ApoE-/-.Chil3∆.hCHI3L2tg (ApoE-/-.Chil3∆.hCHI3L2tg) mice, for the study of human CHI3L2 gene in mouse with a Chil3 deficient context. C57BL/6-Ly5.1-Pep3b (B6.CD45.1) mice, gifted by Prof. Baojun Zhang from Xi'an Jiaotong University, were crossed with BQ.ApoE-/-.Chil3∆.hCHI3L2tg mice to generate BQ.ApoE-/-.CD45.1 mice, BQ.ApoE-/-.Chil3∆.CD45.1 mice and BQ.ApoE-/-.Chil3∆.hCHI3L2tg.CD45.1 littermate mice. To achieve myeloid-specific overexpression of CHI3L2, we utilized a Cre-loxP-based conditional knock-in strategy (C57BL/6N (B6) genetic background). Briefly, mice harboring a loxP-flanked STOP (LSL) cassette upstream of the human CHI3L2 gene (Cyagen, China) were crossed with myeloid Lyz2 (also known as LysM)-Cre mice. For breeding, heterozygous floxed CHI3L2 knock-in mice (CHI3L2ki/wt) were paired with LysM-Cre mice to obtain offspring carrying both the knock-in allele and the Cre transgene (M-CHI3L2ki/wt). Littermates carrying the wild-type CHI3L2 allele together with LysM-Cre (M-CHI3L2wt/wt) served as controls.

In the initial phenotypic experiments, both male and female mice were used, but no significant sex differences were observed (Fig. 2). For subsequent experiments, only male mice were chosen to reduce variability and simplify result interpretation. We also ensured that the number of mice in each group was balanced. All these mouse strains were maintained in specific-pathogen free conditions in the Experimental Animal Center in Xi’an Jiaotong University. All mice were housed in a temperature-controlled environment at 22 °C under 12 h light/dark cycles. All animal experiments were performed according to the guidelines of the Institutional Animal Use and approved by the Institutional Animal Ethics Committee of Xi’an Jiaotong University (No.2023-1244).

Animal diet feeding and atherosclerotic lesions analysis
All male and female mice were fed a high-fat Western diet (WD; 41% kcal from fat, 0.15% kcal from cholesterol; Medicience, MD12015, China) starting at 8 weeks of age and maintained on the diet for 3 months. Our study examined male and female animals, and similar findings are reported for both sexes. To assess the effect of myeloid-specific CHI3L2 overexpression on atherosclerosis, each 8-week-old male mouse (M-CHI3L2ki/wt and M-CHI3L2wt/wt) was intravenously injected with 5×10¹¹ viral genomes of rAAV8-mPCSK9 (WZ Biosciences, China) and continued on WD for 3 months. CO2 was used to euthanize mice. After sacrifice, whole aortas were isolated and stained with Oil Red O (ORO), and en-face lesion area was measured by morphometry using Image J software (National Institutes of Health Software). Heart tissues containing aortic roots were carefully dissected and cryosectioned frozen sectioning. Serial cross-sections of 10 μm were cut through the aorta beginning at the origin of the aortic valve leaflets and stained with hematoxylin and eosin (H&E), ORO, Sirius Red staining and immunostained for α-smooth muscle actin (α-SMA), CD68, intercellular adhesion molecule 1 (ICAM-1), or vascular cell adhesion molecule 1 (VCAM-1) to quantify lesions in the aortic sinus. Image-Pro Plus 6.0 software (Media Cybernetics) was used for quantification.

Quantification of lipid levels
Serum TC, TG, LDL-C and HDL-C were measured using an automatic biochemical analyzer (Beckman Coulter AU5431) with corresponding enzymatic colorimetric assay kits. Briefly, TC was determined by hydrolysis of CE with cholesterol esterase, followed by oxidation of free cholesterol by cholesterol oxidase to generate hydrogen peroxide, which reacts with a chromogenic substrate to produce a color proportional to TC concentration. TG was measured via a glycerol phosphate oxidase-peroxidase method, in which TG are hydrolyzed to glycerol and fatty acids, and glycerol is oxidized to produce hydrogen peroxide for colorimetric detection. LDLR-C and HDL-C were quantified using homogeneous enzymatic colorimetric assays, in which specific surfactants and masking agents selectively solubilize non-target lipoproteins, allowing enzymatic oxidation of LDL-C or HDL-C. The resulting color intensity is proportional to LDL-C or HDL-C concentration.

Bone marrow transplantation (BMT)
Recipient CD45.2 male ApoE-/- mice at 8 weeks of age were irradiated with 2 doses of 4.25 Gy X-rays, separated by a 4-hour interval (Elekta Synergy, Sweden). BM cells were harvested from femurs and tibias of euthanized donor CD45.1 ApoE-/-, ApoE-/-.Chil3∆ or littermate ApoE-/-.Chil3∆.hCHI3L2tg mice under sterile conditions. Briefly, bones were flushed with Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum (FBS) and passed through a 40 µm filter. BM cells (5 × 106) were injected via the tail vein into each recipient ApoE-/- mouse 6 h post radiation exposure to replenish the hematopoietic system. Successful BMT was confirmed after 3 weeks by detecting CD45.1 from transplanted mice using flow cytometry. BMT mice could then undergo 12 weeks of WD.

Administration of rhCHI3L2 protein in vivo
The recombinant human CHI3L2 (rhCHI3L2) protein, labeled with hIgG1-Fc, was produced by Tsingke (Shanghai, China, endotoxin levels below 5 EU/mg). For these experiments, 2-month-old ApoE-/-.Chil3∆ male mice were initially fed a WD for 1 month. Subsequently, the mice received intraperitoneal injections of either hFc-rhCHI3L2 protein (0.25 µg/g) or hFc-IgG1 controls (0.25 µg/g) every 3 days during the final 2 months of WD feeding.

Evaluation of CHI3L2 antibody efficacy in vitro and in vivo
In in vitro studies, THP-1-derived macrophages were stimulated with rhCHI3L2 in the presence or absence of a CHI3L2-neutralizing antibody (Sino Biological, Beijing, China; endotoxin <1 EU/mg). Cellular lipid deposition and uptake were assessed by flow cytometry. For in vivo experiments, 2-month-old ApoE-/-.Chil3∆.hCHI3L2tg male mice were fed a WD for 1 month, then received monthly tail vein injections of CHI3L2-neutralizing antibody (5 μg/g) or IgG control (5 μg/g) for 2 months. To evaluate plaque regression, 2-month-old ApoE-/-.Chil3∆.hCHI3L2tg male mice were fed a WD for 3 months, then given a single tail vein injection of CHI3L2 neutralizing antibody or IgG, followed by 1 month on a ND.

Aortic cell isolation and Flow cytometry
Single-cell suspensions were prepared from the mouse aorta (whole aortic arch and thoracic aorta) using enzymatic digestion59. Briefly, aortas were cut into small pieces and incubated in 1 mL enzyme cocktail (450 U/mL collagenase I, 125 U/mL collagenase XI, 60 U/mL hyaluronidase I-S, 60 U/mL DNase-I; all in DMEM for 1 h at 37 °C. Digested tissues were filtered through a 40 µm strainer, and cells were centrifuged at 1200 × g, 4 °C for 10 min after erythrocyte lysis with ACK buffer. Cell pellets were used for flow cytometry. Anticoagulated blood was treated with ACK lysis and directly analyzed. BMT efficiency was confirmed by staining with PerCP anti-mouse CD45.1 and BV785 anti-mouse CD45.2 antibodies. Samples were acquired on a NovoCyte 3005 flow cytometer (Agilent) and analyzed using NovoSampler Pro software (Agilent).

scRNA-seq analysis
Aortic tissues from WD-fed male littermates were collected, and six aortas were pooled for scRNA-seq analysis at LC Bio Technology Co., Ltd (Hangzhou, China). Single-cell libraries were prepared using 10X Genomics protocols and sequenced on an Illumina NovaSeq 6000. Reads were aligned to the mm10 reference genome with Cell Ranger (v6.1.1), including the human CHI3L2 gene. Cells were filtered based on mitochondrial gene content (<5%) and gene counts (200-2,500) using Seurat 4.3.260. The top 2,000 variable genes (‘vst’ method) were used to compute principal components (PCs), and 33 significant PCs were selected via the ElbowPlot method. Batch effects were corrected with Harmony v1.0. Clustering and UMAP visualization were performed using Seurat functions, and cluster-specific marker genes were identified with FindAllMarkers. Pseudotime trajectory analysis was conducted with Monocle 3. Public scRNA-seq datasets (GSE155513, GSE159677) were analyzed similarly, using tSNE for visualization.

Immunofluorescence staining
In brief, after EDTA antigen retrieval, sections were permeabilized with 0.1% Triton X-100 for 15 min and blocked with 5% BSA for 30 min at room temperature (paraffin sections were routinely dewaxed and hydrated). Sections were incubated overnight at 4 °C with primary antibodies in a humidified chamber. After three PBS washes, sections were incubated with Alexa Fluor 488 (AF488)- or Rhodamine-conjugated secondary antibodies for 2 h at room temperature. For directly labeled antibodies, AF488 anti-mouse CD68 was added after blocking. For co-staining, APC-CD36 or AF488 CD68 antibodies were added simultaneously with secondary antibodies. Nuclei were stained with DAPI. Images were captured on a multiphoton microscope (SP8 DIVE, Leica) and acquired/analyzed with Leica LAS X 3.5.7. Quantification was performed using Image-Pro Plus 6.0.

Macrophage acquisition and cultivation
Mouse peritoneal macrophages (PMφ) were obtained from 6-to-8-week-old mice following intraperitoneal injection of 2 mL 3% thioglycolate broth. Three days later, mice were euthanized, and peritoneal cells were harvested by lavage with 5 mL ice-cold PBS/EDTA, centrifuged (1,200 × g, 10 min, 4 °C), lysed with ACK buffer, washed, and cultured in DMEM with 10% FBS and 1% penicillin-streptomycin (PS). After 6 h, non-adherent cells were washed off with pre-warmed PBS, and fresh medium was added for experiments.

Bone marrow-derived macrophages (BMDMs) were generated by flushing femurs and tibias with sterile PBS, filtering through a 40 µm strainer, centrifuging, and lysing with ACK buffer. Cells were seeded at 2 ×106 cells/mL in DMEM with 10% FBS and macrophage colony-stimulating factor (M-CSF, 10 ng/mL) for 7 days.

THP-1 monocytes (human monocytic leukemia cell line) were cultured in RPMI 1640 medium containing 10% FBS and 1% PS, then differentiated into macrophages using 10 ng/mL phorbol 12-myristate 13-acetate (PMA) for 48 h before harvesting for experiments.

Foam cell formation, lipid uptake and cholesterol efflux
Macrophages were cultured in DMEM with 10% FBS and 1% PS, and treated with or without ox-LDL (30 μg/mL) for 48 h. Cells were fixed in 4% paraformaldehyde (PFA) for 10 min at room temperature and washed with PBS. For ORO staining, cells were immersed in ORO solution for 20 min, rinsed with PBS, counterstained with hematoxylin, washed again with PBS, and visualized by light microscopy. Foam cells were identified by the presence of ORO-positive lipid droplets and quantified as a percentage of total cells. For neutral lipid staining, cells were incubated with BODIPY493/503 for 30 min at 37 °C and analyzed by flow cytometry or multiphoton microscopy. Foam cell formation was quantified based on the mean fluorescence intensity (MFI) of BODIPY staining. For lipid uptake assays, cells were incubated with DiI-ox-LDL (30 μg/mL) for 2 h at 37 °C, followed by analysis using flow cytometry or multiphoton microscopy. Where indicated, cells were pretreated with recombinant proteins or signaling pathway inhibitors for 4 h prior to the addition of ox-LDL or DiI-ox-LDL.

The cholesterol efflux assay performed according to the manufacturer's instructions. Briefly, macrophages were seeded in 96-well plates (1×105 cells per well) and labeled with fluorescent cholesterol at 37 °C for 1 h, followed by equilibration for 16 h. Cells were then incubated with ApoB-depleted serum (HDL-containing) for an additional 4 h. Fluorescence intensities in the culture medium and cell lysates were measured using an Infinite M200 microplate reader (Ex/Em = 485/523 nm, Tecan), and cholesterol efflux was calculated based on the ratio of fluorescence in the medium to total fluorescence.

VSMC acquisition, cultivation and stimulation
Primary vascular smooth muscle cells (VSMCs) were isolated from 6-8-week-old mice. Briefly, the entire aorta was excised, cleaned of connective tissue, and rinsed in cold PBS. After removal of the endothelial layer by gentle luminal scraping, aortas were minced and digested in DMEM containing collagenase II (1 mg/mL), elastase (0.5 mg/mL), and DNase-I (60 U/mL) at 37 °C for 1 h. The cell suspension was filtered, centrifuged, and cultured in DMEM supplemented with 10% FBS and 1% PS. Cells were cultured at 37 °C with 5% CO2, and medium was replaced every 2-3 days. VSMCs between passages 3 and 6 were used for experiments.

To assess macrophage-VSMC crosstalk, BMDMs were treated with ox-LDL (50 μg/mL) for 48 h, and the conditioned media were applied to VSMSCs for an additional 48 h. Lipid accumulation in VSMCs was evaluated by BODIPY staining and flow cytometry. For calcification assays, BMDMs were cultured for 10 days in the presence of ox-LDL (50 μg/mL) and calcification medium (CaP medium, 10 mM β-glycerophosphate and 1.5 mM CaCl2)61. The resulting conditioned media were used to stimulate VSMCs for 14 days, and calcification was assessed by Alizarin Red staining. For Alizarin red staining, VSMCs in 6-well plates were fixed in paraformaldehyde for 15 min at room temperature, incubated with 2% Alizarin red for 10 min and washed with double-distilled H2O. Positively stained cells appeared to have a reddish/purple color.

Macrophage polarization and signaling pathway studies
For macrophage polarization, lipopolysaccharide (LPS, 100 ng/mL) and interferon-γ (IFN-γ, 10 ng/mL) were treated to macrophage to polarize the CAMs, while IL-4 (20 ng/mL) and IL-13 (50 ng/mL) were treated to macrophage to polarize the AAMs.

To study the cell signaling activation, macrophages were treated with or without PPARγ inhibitor T0070907 (1 μM), lysosomal function inhibitor Bafilomycin A1 (Baf-A1, 100 nM) or mTOR inhibitor rapamycin (100 nM) in the presence of ox-LDL for indicated times.

RNA extraction, quantitative real-time PCR and RNA-seq
Total RNA was extracted from aortic tissues or macrophages by TRIzol reagent and reversely transcribed into cDNA using RevertAid First strand cDNA Synthesis Kit according to the manufacturer’s instructions. RT-qPCR was performed using FastStart Universal SYBR Green Master. All primer sequences used for RT-qPCR are listed in Supplementary Table 3. Relative mRNA expression was normalized to β-actin and calculated by the 2-ΔΔCt method.

For RNA-seq, ApoE-/- or ApoE-/-.Chil3∆ BMDMs treated with ox-LDL were dissolved in TRIzol and the RNA libraries were sequenced on the illumina Novaseq 6000 platform by LC Bio Technology CO., Ltd (Hangzhou). Bioinformatic analysis was performed using the OmicStudio tools at https://www.omicstudio.cn/tool.

Protein extraction and Western blotting
Proteins were extracted by ice-cold radioimmunoprecipitation assay (RIPA) buffer containing proteinase inhibitor and/or phosphatase inhibitors, and quantified using a BCA kit. Equal amounts of protein were separated by 7.5% or 10% SDS-PAGE and transferred onto PVDF membranes. Membranes were blocked with 10% nonfat milk in TBST for 2 h at room temperature and incubated with primary antibodies overnight at 4 °C, followed by HRP-conjugated secondary antibodies. After washing with TBST three times for 10 min each, the blots were visualized and imaged using ECL Plus Kit and Genegenome XRQ NPC Chemiluminescent Imaging Analysis System (Syngene). The proteins were normalized with β-actin or β-Tubulin and respective total protein content. Densitometric analysis of the gels was done using Image J software. Antibody information is provided in the Supplementary Information, and uncropped blots are available as Source Data or Supplementary Information.

Plasmid construction and transfection
Recombinant lentiviruses targeting mouse CD36 (Lv-sh mCD36) or control were generated by GenePharma (Shanghai, China). The shRNA target sequence is listed in Supplementary Table 3. BMDMs were infected at 70-80% confluency and analyzed 72 h post-transduction. Cells were then treated as indicated for subsequent experiments. Plasmids were transfected into 293T cells using Lipofectamine 8000, and cells were harvested 48 h post-transfection for further analyses. Truncated and mutant Flag-mCD36 constructs were generated by Tsingke Biotech (Beijing, China) and sequence-verified.

Enzyme-linked immunosorbent assay (ELISA) assay
CHIL3 and CHI3L2 levels were quantified using commercial ELISA kits according to the manufacturers’ instructions. CHI3L2 values were log-transformed to normalize data distribution. Mouse serum ox-LDL levels were measured using an ELISA kit. Absorbance was read at 405 nm using an Infinite M200 plate reader. In some experiments, chemiluminescent enzyme immunoassay was used.

For in vitro binding assays of CLPs to ox-LDL, ox-LDL (5 μg/mL) was encapsulated in a 96-well plate at 4 °C overnight. Then 5 μg/mL of His-rCHIL3 (positive control His & hFc-rmCD36) or His-rhCHI3L2 protein (positive control His & hFc-rmCD36) was added and incubated for 3 h at room temperature. Then HRP-conjugated mouse anti-His antibody and HRP substrate were sequentially added to the wells and incubated for 1 h at room temperature.

To test the effect of CLPs on the binding of CD36ECD to ox-LDL, His & hFc-rmCD36 with/without His-rCHIL3, hFc-rhCD36 with/without His-rhCHI3L2, PNGase F-treated rmCD36 or rhCD36 were firstly incubated at 37 °C for 24 h. ox- LDL (5 μg/mL) was encapsulated on a 96-well plate at 4 °C overnight. Then approximately 5 μg/mL of CLP-treated or non-treated rCD36 was added and incubated at room temperature for 3 h after blocking using 1% BSA. PNGase F-treated rCD36 was used as a positive control. Peroxidase AffiniPure goat anti-human IgG, Fcγ fragment specific antibody and HRP substrate were then added to the wells and incubated for 1 h.

Immunoprecipitation and mass spectrometry (IP-MS)
Cell lysates were prepared by using ice-cold cell lysis buffer for Western and IP supplemented with proteinase inhibitor. To identify endogenous CLP-interacting proteins in macrophages, cell lysates were incubated with His-tagged CHIL3 for 1 h at room temperature. Control or specific antibodies (anti-IgG/anti-His) were added to the lysates and incubated for 12 h at 4°C with constant rotation. Then, 30 µL of pre-washed Pierce Protein A/G beads were added and the mixture was incubated for an additional 4 h at 4°C. The antigen-antibody-bead complexes were washed five times with PBS and sent for mass spectrometry (MS) analysis, analyzed by Shanghai Bioprofile Technology (China). Briefly, the samples (1 biological replicate for IgG and His groups) were enzymatically digested, and the resulting peptides were desalted using C18 StageTip and vacuum-dried. The peptides were separated using an Easy nLC 1200 system with solvent A (0.1% formic acid in water) and solvent B (80% acetonitrile, 0.1% formic acid in water) for gradient elution. The separated peptides were analyzed using a Q-Exactive mass spectrometer in DDA mode, with a precursor ion scan range of 350-1800 m/z, a resolution of 60,000 for MS1, and a resolution of 15,000 for MS2. Data were analyzed using MaxQuant 2.0.1.0, and the uniprot-Reference proteome-Mus musculus (Mouse) [10090]-55286-20230104.fasta database was used for peptide identification. Filtering criteria included PSM FDR ≤0.01 and protein FDR ≤0.01. The IP-MS identification results are provided in the Source Data, with a focus on membrane proteins.

For the co-IP experiments used in Western blotting, proteins were extracted from 293T cells transfected with the relevant plasmids. Alternatively, recombinant CD36 proteins were deglycosylated and incubated with recombinant CHIL3 or CHI3L2 at 37°C for 1 h prior to IP. After the immune complex formation, immunocomplexes were eluted with 2× loading buffer by boiling at 95°C for 5 min, separated by SDS-PAGE, and analyzed by immunoblotting with appropriate antibodies.

N-glycosylation removal assay
Non-denaturing deglycosylation of rCD36 was performed. Briefly, rCD36 proteins were mixed with GlycoBuffer 2 (10×) and PNGase F, and the mixture was incubated for 24 h at 37°C. The deglycosylated samples were then used for subsequent experiments.

Determination of CD36 surface glycan content
rCD36 was incubated/not incubated with equal amounts of rCHIL3 or rhCHI3L2 for 24 h at 37°C, with PNGase F-treated rCD36 as a positive control. Proteoglycan content was assessed using wheat germ agglutinin (WGA). The glycan content was detected by Western blotting using the biotin-affinity protein system. Incubation with 5 μg/mL bio-WGA was performed for 2 h, followed by 1 h of incubation with Streptavidin HRP.

Glycomics and analysis
Label-free quantitation of intact glycopeptides and modified glycans by MS was performed by incubating rCD36 with/without rCHIL3 or rhCHI3L2 at 37°C for 24 h. Glycan mass spectrometry was done and quantitatively analyzed by Shanghai Bioprofile Technology (China). The quantitative analysis results of glycosylation levels at each site after correcting for protein concentration were shown by column diagram, and the quantitative results of glycoforms at each site after correcting for protein concentration were shown by annular heat map. The glycoforms information is presented in the Source Data.

Non-targeted lipidomic analysis
Non-targeted lipidomic analysis was performed by LC Bio Technology Co., Ltd. (Hangzhou, China). Briefly, cell samples were extracted using a modified methyl tert-butyl ether method. Lipids were separated on an ACQUITY UPLC CSH C18 column using a gradient of mobile phase A (acetonitrile/water, containing ammonium formate and formic acid) and mobile phase B (isopropanol/acetonitrile, containing ammonium formate and formic acid). The separated lipids were analyzed by high-resolution mass spectrometry in both positive and negative electrospray ionization modes, with full-scan and data-dependent acquisition. Raw data were processed using XCMS and metaX for peak detection, alignment, and retention time correction, and metabolites were annotated by matching to KEGG and HMDB databases. During data processing, for each genotype, lipid abundances in ox-LDL treated samples were normalized to their corresponding untreated control groups to obtain relative lipid change values.

Quantification and statistical analysis
All data are shown as mean ± standard deviation (SD). Normality was assessed using the Shapiro-Wilk test, while the equality of variances was determined using the F-test. The statistical significance of the difference between means was assessed using the two-tailed Student’s t-test (for normally distributed data) or Mann Whitney U test (for not normally distributed data). One-way ANOVA with appropriate post-hoc correction was utilized for comparing the effect of treatment within a group. Two-way ANOVA with appropriate post-hoc correction was used when two conditions were considered between the groups. Adjusted P values were used for the post-hoc tests. Statistical analysis was performed using GraphPad Prism, version 9.0. Specific statistical information is provided in the Source Data.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "Summary:\nIn the submission entitled \"Chitinase-like proteins de-N-glycosylating CD36 rewire cholesterol metabolism in atherosclerotic\nmacrophages \" by Wang et al., the authors describe the role of two mammalian chitinase-like proteins (i.e., mouse Chil3 and\nhuman CHIL3L2) in atherosclerosis. Specifically, the authors identify an autocrine mechanism by which macrophage\nchitinase-like proteins increase cholesterol uptake through CD36. This activity drives mTOR signaling to inhibit cholesterol\nefflux. The authors also use single cell RNA-seq to show foamy cells derived from macrophage and vascular smooth muscle\ncells. Lastly, antibody targeting of CHI3L2 neutralized atherosclerotic progression.\nOverall, this substantial submission describes novel findings that provide molecular insights into the mechanisms underlying\natherogenesis and identifies a potential therapeutic target. The results generally support the conclusions, and the\nexperiments appear to be rigorous and appropriate, with several notable exceptions detailed below. Other suggestions and\ncomments are also included that may help improve the submission:\nMajor:\n1. Did the authors only utilize male mice from the Chil3 congenic mouse strain as this was the only gender affected by the\ndeficiency? If not, then why were female mice not studied (i.e., other than in the isolated macrophage experiments)?\n2. The CD68 staining in Figure 1h appears non-specific, as the green fluorescence shows strong background signal, making\nit difficult to assess true CD68 expression. Since CD68 is a macrophage marker expressed in atherosclerotic lesions, it is\nimportant to clearly demonstrate specific staining. Additionally, co-staining CD68 with α-SMA could better illustrate the\nstability of the plaques within the lesion instead of IHC staining of α-SMA. In Figure 1i, the lesion boundaries are not clearly\ndefined—adding an outline would improve clarity. It is also unclear whether the selected image accurately represents\nreduced lesion size in Chil3-deficient mice. How is the expression of ICAM1 and VCAM-1 levels in the lesions? The above\ncomment is applicable to the other figures (2i,j…)\n3. The authors used a collection time of 5 months after Western diet feeding. Did earlier time points fail to show the changes\nobserved at 5 months? If so, what does this say about the role of CLPs in atherosclerosis initiation? At the same time, the\ntreatment regimen described in Figure 9c started at 3 months, which only accounts for the prevention of plaque progression.\nWhile interesting, the most clinically important model would assess plaque regression following dietary alteration (e.g., to a\nND).\n4. Single-cell RNA sequencing (scRNA-seq) data indicate stronger interactions between macrophages and vascular smooth\nmuscle cells (VSMCs) in wild-type and CHI3L2 transgenic mice, but not in Chil3-/- mice. These findings might be benefit by\nco-culturing primary macrophages and VSMCs isolated from all three groups.\n5. Quantification of western blotting results is needed throughout the manuscript (eg. Fig6b-d, …).\n6. In addition to CD36 and Abcg1, the protein expression levels of other lipid uptake receptors (e.g., SR-B1, LDLR) and\ncholesterol efflux transporters (e.g., ABCA1) should be examined, as their expression may be influenced by post-\ntranslational regulation. Assessing only mRNA levels by qRT-PCR is insufficient.\nMinor:\n1.While the manuscript is largely well-written, there are several instances of the use of unusual terminology. For instance,\nthe term \"atherosclerogenesis\" is unusual. The commonly used term for this process is atherogenesis. In addition, the use of\nthe term \"fate mapping\" seems incorrect. The authors appear to mean \"fate\" as fate mapping are methods that allow the\nidentification of cells throughout differentiation or transition. The pluralization of expression to describe changes in gene\noutput is also non-standard (i.e., \"... the expression of X and Y were ...\" is preferable)\n2.ORO and histological staining data in Figure 1f, g, and i should be enlarged. There also appears to be a discrepancy in the\nscale bar length in panels h and i. The above comment is applicable to the other figures, which should also be checked for\nscale bar accuracy in length and use the same position in each panel and a consistent line thickness.\n3. The human plasma and tissue cohorts (n=34 healthy, n=38 atherosclerotic) are modest. Excluding patients with\ndiabetes/hypertension limits clinical relevance, as these conditions frequently coexist with atherosclerosis. The authors\nshould address whether findings generalize to broader populations.", "reviewer_2": "In this study, Wang et al. reported the impact of Chitinase-like protein 3 (Chil3) and its human functional homolog chitinase-\n3-like 2 (CHI3L2) on macrophage lipid metabolism and atherosclerosis. Using numerous elegant experimental approaches\nas well as in in vitro and in vivo models, the authors proposed a new autocrine mechanism in which secreted CLP regulates\ncholesterol uptake in macrophages through binding to CD36. Importantly, Wang et al. provided important new therapeutic\ninsights with the use of antibodies targeting CHI3L2 which attenuate atherosclerosis. This group previously demonstrated\nthat Chil3 polymorphisms were associated with inflammatory diseases by regulating lipid metabolism. This study is in the\ncontinuation of their previous works by positioning Chil3 in the development of atherosclerosis. The overall study is very\ninteresting but several points must be clarified by the authors.\nMajor points.\n1. Previous numerous studies characterized several macrophage populations in atherosclerotic plaques by single-cell RNA-\nSequencing (PMID: 38639096, PMID: 29545366, PMID: 31591603, PMID: 32981416….). Was Chil3 expression associated\nwith a specific subtype cluster of macrophages in atherosclerotic plaques (Trem2+ macrophages (PMID: 38646596)) ? Was\nthis latter altered upon Chil3 deficiency ? Analysis of Chil3 expression in CAM (classically activated macrophages) and\nAAM (alternatively activated macrophages) is indeed very limited (Extended Data Fig. 2e-h).\n2. Because lipid uptake in macrophages has a profound impact on intracellular lipid homeostasis beyond foam cell\nformation, it is important to further describe the consequences of the alteration of Chil3 expression in the lipidome of\nmacrophages, especially in membrane lipid composition. Then, lipidome of WT and Chil3  macrophages must be explored\nin order to decipher the role of Chil3 in macrophages. Such an analysis is even more essential since Chil3 deficiency allows\nan increased expression of Abcg1, a transporter recently identified as playing a key role in lipid rewiring and functions of\nmacrophages (beyond cholesterol efflux) (PMID: 39661702).\n3. Through the uptake of oxidized lipids and free fatty acids, CD36 plays major roles in several pathways controlling the\nmetabolic phenotype of macrophage (fatty acid oxidation and storage, ROS production, energy metabolism….). The authors\nmust provide information about the impact of Chil3 deficiency on metabolic pathways in macrophages (RNA-sequencing\nand/or seahorse analyses for example).\n4. The authors did not explain and discuss the role of Chil3 on inflammation, especially on the inflammatory phenotype of\nmacrophages. Because CD36, the main target of Chil3, is involved in NLRP3 activation (PMID: 23812099), the reduced IL-\n1β expression observed in aorta of Chil3  mice (Fig1e) could result from an impaired activation of the NLRP3 pathway.\nAlthough the role of Chil3 in foam cell formation is very likely involved in the attenuated atherosclerosis development, the\nauthors cannot exclude the role of inflammation in their mechanism since antiiflammatory targeting IL-1β led to a reduction of\natherosclerotic cardiovascular diseases (PMID: 28845751). The authors must explore and/or discuss this aspect.\nOthers major points.\n5. In mice with a total Chil3 deficiency, what tissue drives the circulating Chil3 / CHI3L2 concentrations in mice (Fig. 1a, \nExtended data Fig. 2i……) ? Same question in humans (Fig. 2c)?\n6. Extended Data Fig. 1(a). What was the diet of ApoE-/- and WT mice ?\n7. Fig. 1c. The title of the Y-axis must be clarified. What class of lipid was\nquantified ? Cholesterol ? How was quantified the\nlipid profile? How do the authors explain the reduced serum LDL (likely LDL-C) in BQ.ApoE-/-.Chil3-/- mice in comparison to\nBQ.ApoE-/- mice ? Same comments in Fig. 2e).\n8. Same question with oxLDL (Fig. 1d).\n9. Fig. 2k. The efficiency of the treatment with rhCHI3L2 must be validated by quantification of CHI3L2 in the circulation of\nmice following intraperitoneal injection.\n10. Figure 3. Single-cell RNA-Sequencing data are interesting but it would be\nuseful to know the biological pathways that\nare altered in macrophages in the three mouse models (WT, Chi  and Chil3 .CHI3L2tg). A same comment can be done in\nBMDM and PM cells (Figure 5). As a whole, impact of Chil3 deletion as well as expression of CHI3L2 in Chil3-deficient cells\non the metabolic phenotype of macrophages is lacking. In this context, it is very intriguing that no cholesterol metabolism (or\nlipid metabolism) pathways were found altered by RNA-sequencing in oxLDL stimulated Chil3  versus WT BMDM\n(Extended Data Fig. 7b). These findings weaken a significant role of Chil3 in macrophage lipid uptake as proposed by the\nauthors.\n11. Figure 6. Although not in the top 5 candidate, were P2Y6R and TREM2 (or some TLRs) identified in IP and MS analyses\nsince these receptors promote lipid uptake and atherosclerosis (PMID: 38036416, PMID: 38646596) ?\n12. As a follow up of my previous comment, may Chil3 promote hydrolysis of N-glycosylated glycans of others membrane\nproteins without physically interacting with them (and then not to be detected by co-IP) ?\n13. Figure 8a. How do the authors explain that the impaired cholesterol uptake in Chil3  BMDM was not accompanied by an\nincreased expression of Ldlr and Hmgcoar genes; the expression of these genes being downregulated by cholesterol.\n14. Figure 8d. Although the method used to quantify cholesterol efflux is highly questionable, the results are quite intriguing.\nIndeed, a 45% cholesterol efflux during a 4h incubation with HDL is extremely high (about 10% is expected) and suggests\nan alteration of cell viability. The authors must describe the method used (what type of HDL used ? what is the % of\ncholesterol efflux without HDL ? ……..).\n15. Extended Data Fig. 7d and Figure 8. The authors proposed that the increased Abcg1 expression resulted from an\nelevation of Ppar  expression. If it is the case, Abca1 expression would be also increased since induction of Ppar  stimulates\nAbca1 expression (PMID: 11135616)\n16. Figures 8e-g. It is difficult to conclude on these experiments in the absence of the WT BMDM. More importantly, the\nauthors proposed that the reduced cholesterol uptake (in the presence of oxLDL) in Chil3  BMDM increases Abcg1\nexpression through alteration of mTOR and induction of Ppar  expression. Such a mechanism is not coherent with the fact\nthat oxysterols are potent activators of Abcg1 expression through the LXR pathway (PMID: 16024918). Indeed, Abcg1\nexpression is increased when macrophages are incubated with oxLDL (source of oxysterols) as confirmed in Fig. 8e-f. Then,\nit would be expected to observe a reduced Abcg1 expression in Chil3  BMDM as a consequence of the impaired oxLDL\nuptake. How do the authors reconcile these potential conflicting observations ?", "reviewer_3": "This study focuses on the role of chitinase-like proteins in atherosclerosis.\nMajor:\n1) General: how is LDL-cholesterol measured? This usually does not accumulate at high levels in the plasma of ApoE-/- \nmice. The authors need to be far more thorough here, especially since they state that they find differences.\n2) The authors several times refer to WT mice. It is well known that WT mice do not develop atherosclerosis, so it does not\nmake sense that atherosclerosis would be studied in this particular model. The authors need to clarify this for each and\nevery study. Fig 2k-m. Were these wild-type mice? Or Ldlr-/- mice? This needs to be clarified in the text. Line 207. Again, I\nam confused as to the mouse models here. WT mice do not develop atherosclerosis. The authors need to really clarify this\nbetter. Fig 4c-g. What was the genotype of the recipients of the mice for bone marrow transplantation? Please clarify. It is\nimpossible that these were just CD45.2 mice without any mutation since these mice do not develop atherosclerosis.\n3) The number of mice for any analysis in atherosclerosis experiments needs to be at least n=10. In very many figures, these\nnumbers are lower; real conclusions can therefore not be drawn.\n4) The authors need to provide more definitive evidence, preferably in vivo, that the upregulation of CD36 is what accounts for their phenotype. Was CD36 staining increased in atherosclerotic plaques?\n5) Extended Data Fig 1a. These data on Chil3 being upregulated are not that convincing. I understand the use of public\ndatasets, which in general makes things easier, but it seems to me that the n would need to be increased to really conclude\nthat Chil1 and Chid1 are not affected. These experiments are not hard to do and would need to be performed in order for the\nauthors to really draw conclusions here.\n6) Fig 2c. How has plasma from ‘control’ and ‘atherosclerosis’ been defined? This would require a far more extensive\ndescription. Also here the x-axis crosses the y-axis at 3.0, with the intention to magnify a small, though significant, increase.\nWhat is the extent of the increase shown in Fig 2c? Why were the data log-transformed?\n7) Fig 2f. Again, the x-axis is not crossing 0. Why making so much of the small difference in oxLDL? What is the extent of the\nincrease? And what is the extent of the increase in LDL in Fig 2e?\n8) Fig 2i-j. Too few datapoints have been included. The data in Fig 2g-h suggest more mice were included in these studies;\nthe same number would need to be included in Fig 2i-j.\n9) Fig 1a. This is a very strange way to display the data with the x-axis crossing the y-axis at -200. This would need to be\nchanged to 0. If there are values lower than 0 they would need to be adjusted to 0.\n10) Extended Fig 2d. What do the authors mean with a proliferative cell?\n11) Extended Fig 2i. This is a minimal difference. The x-axis should not cross at 4.0, but at 0.0; otherwise the authors make\ntoo much of a maybe not even 5% difference, which I do not view as meaningful.\n12) Extended Fig 2f and 2h. The number of replicates should at least be increased to n=4. Preferably n=6. N=3 is\ninsufficient. Data on THP-1 cells would need to be complemented by data on PBMCs differentiated in what the authors refer\nto as AAMs or CAMs; THP-1 cells are already very activated since they are differentiated into macrophages employing PMA.\n13) Fig 4a. More examples on co-localization would need to be shown. The examples in Fig 4b on co-localization are too\nunclear to draw conclusions.\n14) Fig 4b. What is meant by APS and FPS? Again, there is an unclarity here. This makes it hard to read and properly\nreview this manuscript.\n15) Fig 4h. The chlodronate experiment is usually performed as a way to study monocyte infiltration into atherosclerotic\nplaques. The transferred monocytes are labeled with beads, so that homing of monocytes to plaques can be studied. Such\nan experiment is actually never performed with bone marrow derived macrophages. If performed in this way, macrophages\nneed to be labeled so their fate can be studied better. It is unclear what the authors have done in Fig 4h-k. It seems rather to\nme that they have performed a bone marrow transplantation without performing the irradiation first. If the authors claim that\nthe phenotype is macrophage-driven, they will need to create a macrophage-specific knockout. That is only the proper way\nthat is widely accepted in the field.\n16) Extended Figure 4f. The flow cytometry plots are insufficient in showing that the transfer with macrophages actually led to\naccumulation of considerable amount of macrophages in the aorta. The method needs to be established first.\n17) Extended Fig 5a-c. These calculated interactions actually do not tell us that much. What is exactly happening here and\nwhat is the biological nature of these interactions.\n18) Extended Fig 5d. If the authors really want to draw a conclusion on macrophages, they need to perform laser capture\nmicroscopy on atherosclerotic plaques, or single cell RNASeq, but they cannot do this by performing whole RNA extraction\non the aorta.\n19) Extended Fig 5e. When were these macrophages isolated? It is impossible to directly isolate BMDMs; then these cells\nwould be bone marrow monocytes. This would need to be clarified. How was the experiment performed?\n20) Extended Fig 5f-j. Same questions – how were these experiments performed? This needs to be stated in the figure\nlegends or better described in the main text. If the authors claim more oxLDL uptake, ideally they perform mass-spec on their\nmacrophages to show that free cholesterol and cholesteryl esters are increased. This is the only definitive measure. These\nmfi measurements tell us very little, and again, in extended Fig 5j, differences are minor for BMDMs. I do not understand the\nsignificant p-values. The authors are making too much of these data.\n21) Fig 6i-k. Were these intracellular stainings? Ideally the authors would complement these data with mass spec\nmeasurements of free cholesterol and cholesteryl esters.\n22) Text line 474. The authors imply that Mylip would be involved in the cholesterol efflux pathway while it in fact induces the\ndegradation of the Ldl receptor.\n23) Fig 8. It is of interest that mainly Abcg1 is affected in Chil-3 deficiency. How do the authors explain that this particular\ntransporter is affected and not Abca1? What would the mechanism be underlying this finding?\n24) Related to this, Abcg1 deficiency does not affect atherosclerotic lesion area or decreases it. This does not align with the\nfindings of the authors. Please explain.\n25) How would lipid deposition activate mTOR?\n26) Line 557 – the authors are discussing ‘inflammatory foamy macrophages’. But the foamy macrophages are generally\nconsidered to be anti-inflammatory.\nMinor:\n1) Keywords: atherosclerogenesis (also in lines 101 and 110). This is not a usual word. It is atherosclerosis or\natherogenesis.\n2) Introduction: line 82 – it probably needs to state: various transformations, such as those into…\n3) Also in lines 83-84 it is implied that the switch to other phenotypes accounts for SMCs taking up lipid. This is simply not\ntrue. Lipid uptake by SMCs accounts fort heir differentiation into macrophages for example. Please adjust the text\naccordingly."}, "author_response": "Answer: We thank you for raising this important point. Our initial focus on male\nmic in studies of chitinase-like proteins (CLPs, mainly Chil3 and CHI3L2) was\nbased on methodological and conceptual considerations, not an assumption of\nsex-specific effects. Additional experiments showed that Chil3-deficient mice\nare resistant to atherosclerosis in both sexes. While female mice generally\nexhibit a more protective response due to hormones (e.g., estrogen)1, we also\nevaluated female mice in a 3-month Western diet (WD) model across three\ngenetically modified strains (ApoE-/-, ApoE-/-.Chil3∆ and ApoE-/-.Chil3∆.CHI3L2tg\nmice) and observed effects comparable to males, despite smaller plaque areas\n(Fig. 1 in this letter). These findings suggest that CLPs exert no major sex-\nspecific effect on atherosclerosis. The new results have been included in the\nrevised manuscript as Fig. 2.\nOur core hypothesis posits that Chil3 deficiency exerts its anti-atherosclerotic\neffects through intrinsic macrophage mechanisms. To test this, we conducted\nin vitro studies using macrophages from male and female mice. For early\nanalyses such as lipid accumulation, primary macrophages were isolated\nseparately from male and female mice within the same batch, and the effect of\nChil3 on lipid deposition was assessed. Consistent results were observed\nacross both sexes (Fig. 2 in this letter, also in the revised manuscript as\nExtended Data Fig. 5a), strongly indicating that the core cellular function\nof Chil3 is not sex-dependent. Therefore, the mechanisms described are\nlikely applicable to both male and female mice. Subsequent experiments used\nthe same batch of cells and aimed for balanced male-to-female ratios across\ngenotypes, with the sex of each macrophage source indicated in the figure\nlegend.\nFig. 1. CLPs promote the formation of atherosclerosis.\nFig. 2. Chil3 promotes lipid deposition in macrophages.\n2. The CD68 staining in Fig. 1h appears non-specific, as the green fluorescence\nshows strong background signal, making it difficult to assess true CD68\nexpression. Since CD68 is a macrophage marker expressed in atherosclerotic\nlesions, it is important to clearly demonstrate specific staining. Additionally, co-\nstaining CD68 with α-SMA could better illustrate the stability of the plaques\nwithin the lesion instead of IHC staining of α-SMA. In Fig. 1i, the lesion\nboundaries are not clearly defined—adding an outline would improve clarity. It\nis also unclear whether the selected image accurately represents reduced\nlesion size in Chil3-deficient mice. How is the expression of ICAM1 and VCAM-\n1 levels in the lesions? The above comment is applicable to the other Figures\n(2i, j…)\nAnswer: Thanks. We have performed additional experiments and image\nanalyses to address each point raised, as detailed below.\nPoint 1: CD68/α-SMA co-staining\nResponse: We conducted new double immunofluorescence staining for CD68\nand α-SMA on aortic root sections from ApoE-/-, ApoE-/-.Chil3∆ and ApoE-\n/-.Chil3∆.hCHI3L2tg littermate mice (Fig. 1f-g in this letter). The co-staining\nclearly shows the distribution of macrophage (CD68+) and α-SMA, supporting\nour conclusion that CLP deficiency promotes a more stable plaque phenotype\nwith reduced macrophage content.\nPoint 2: Lesion boundaries and representation of lesion size reduction\nResponse: Lesion boundaries in representative images were delineated with\nwhite or black dashed lines for clarity (For representative images, please refer\nto Fig. 1, 3 in this letter). For further details, please refer to the revised\nmanuscript, which includes all animal experimental results. Additionally, we\nhave examined the selected representative images to ensure they accurately\nreflect the severity of lesions in each genetically modified mouse.\nPoint 3: ICAM-1 and VCAM-1 expression\nResponse: Additional immunofluorescence staining showed a significant\nincrease in VCAM-1, but not ICAM-1, in lesions of ApoE-/- and ApoE-\n/-.Chil3∆.hCHI3L2tg mice compared to ApoE-/-.Chil3∆ mice (Fig. 1f-g in this\nletter). These findings indicate that CLPs preferentially regulate VCAM-1-\nassociated leukocyte recruitment pathways, without broadly affecting ICAM-1-\nmediated endothelial activation.\n3. The authors used a collection time of 5 months after Western diet feeding.\nDid earlier time points fail to show the changes observed at 5 months?\nIf so,\nwhat does this say about the role of CLPs in atherosclerosis initiation? At the\nsame time, the treatment regimen described in Fig. 9c started at 3 months,\nwhich only accounts for the prevention of plaque progression. While interesting,\nthe most clinically important model would assess plaque regression following\ndietary alteration (e.g., to a ND).\nAnswer: We apologize for the confusion caused by our original schematic\ndiagram. The atherosclerosis model consistently used a 3-month induction\nperiod: 2-month-old mice were fed a WD for 3 months, reaching 5 months of\nage at the endpoint. We have recognized that the schematic diagram is\nmisleading and somewhat redundant in presenting the genotype-phenotype\nrelationship. Therefore, we have removed this diagram while ensuring that the\n that the\nmodeling period remains 3 months. However, the schematic diagram for the\nneutralizing antibody efficacy experiment has been corrected (Fig. 3c in this\nletter).\nIn this study, all models used a 3-month WD, a standard early- to mid-stage\natherosclerosis model in many classic studies2,3, facilitating data comparison.\nWithin 3 months, lipid deposition, inflammatory responses, macrophage\nactivation, and cholesterol metabolism changes are evident, providing a\nconvenient window to assess interventions. WD beyond 3 months leads to\nmore mature plaques with calcification, modeling late-stage lesions. Thus, we\nfocused here on CLP effects in early- to mid-stage atherosclerosis, while our\nrecent work addresses late-stage disease, including VSMC calcification.\nFor evaluating the CHI3L2 neutralizing antibody, treatment was initiated in 2-\nmonth-old mice after 1 month of WD feeding to induce CHI3L2 expression,\nbased on our previous findings that circulating CLPs are elevated in\natherosclerotic serum and in ox-LDL-stimulated macrophage-derived foam\ncells. Mice continued with WD for additional 2 months, with the antibody\nadministered via tail vein injection at the start of the second and third months.\nThis model was primarily designed to assess the preventive effect of the\nantibody (Fig. 3c-e in this letter).\nFollowing your suggestion, we also tested the therapeutic potential of the\nCHI3L2 antibody to promote plaque regression, which is clinically relevant.\nApoE-/-.Chil3∆.hCHI3L2tg mice were fed WD for 3 months to establish plaques,\nthen randomized to ND for 1 month with either isotype IgG or CHI3L2\nneutralizing antibody (Fig. 3f). CHI3L2 antibody treatment markedly enhanced\nplaque regression and reduced lesion volume compared to diet switching alone\n(Fig. 3g-h in this letter).\nThese findings support our central hypothesis and enhance the clinical\ntranslational value of our work. The results indicate that targeting CHI3L2\nnot only prevents disease progression but, in combination with dietary\nintervention, can actively reverse pre-existing lesions. The new results\nhave been included in the revised manuscript as Fig. 9.\nFig. 3. Targeting CHI3L2 could block the progression of atherosclerosis.\n4. Single-cell RNA sequencing (scRNA-seq) data indicate stronger interactions\nbetween macrophages and vascular smooth muscle cells (VSMCs) in wild-type\nand CHI3L2 transgenic mice, but not in Chil3-/- mice. These findings might be\nbenefit by co-culturing primary macrophages and VSMCs isolated from all three\ngroups.\nAnswer: Thanks. We performed co-culture experiments. Primary bone\nmarrow-derived macrophages (BMDMs) were isolated from ApoE-/-, ApoE-\n/-.Chil3∆ and ApoE-/-.Chil3∆.hCHI3L2tg mice, while primary VSMCs were\nobtained from the aorta of ApoE-/-.Chil3∆ mice.\nFoam cell formation: BMDMs with different genotypes were treated with 50\nμg/mL ox-LDL for 48 hours. The conditioned media were collected and applied\nto VSMCs for 48 hours (Fig. 4a in this letter). BODIPY flow cytometry showed\nthat conditioned media from BMDMs of ApoE-/- and ApoE-/-.Chil3∆.hCHI3L2tg\nmice significantly increased lipid deposition in VSMCs compared with media\nfrom ApoE-/-.Chil3∆ BMDMs (Fig. 4b in this letter).\nCalcification: BMDMs were cultured with ox-LDL and calcification medium for\n10 days. Conditioned media applied to VSMCs for 14 days enhanced\ncalcification (Fig. 4a in this letter). Alizarin red staining demonstrated that,\nrelative to conditioned media from ApoE-/-.Chil3∆ BMDMs, media from ApoE-/-\nand ApoE-/-.Chil3∆.hCHI3L2tg BMDMs significantly enhanced VSMC\ncalcification (Fig. 4c in this letter).\nThe new results have been included in the revised manuscript as Fig. 3i-k.\nCertainly, in addition to the autocrine regulatory effects of CLPs on\nmacrophages, their regulatory influences on other cell types, including VSMCs,\nare also interesting and significant. While this study focuses on macrophages,\nthe mechanisms by which CLPs regulate VSMCs will be explored in future work.\nFig. 4. CLPs promote the phenotypic transformation of VSMCs.\n5. Quantification of western blotting results is needed throughout the\nmanuscript (eg. Fig6b-d, …).\nAnswer: Thank you for your careful attention. We wish to clarify that Fig. 6b-d,\nFig. 7a-b, and Extended Data Fig. 6a in our manuscript present results from co-\nimmunoprecipitation (co-IP) experiments, not standard Western blotting\nquantifying protein expression levels. Co-IP primarily demonstrates qualitative\nprotein-protein interactions: that is, whether one protein physically binds to\nanother. The data are typically presented as a pair of blots from the same\nexperiment: one showing the immunoprecipitated complex and another\nshowing the input controls. The presence or absence of a band, rather than its\nintensity, indicates binding. Band intensity can be influenced by IP efficiency\nand does not linearly reflect binding affinity.\nKey co-IP experiments were independently replicated three times, with all\nreplicates consistently confirming the interaction pattern; representative blots\nare shown in the manuscript. Furthermore, we confirm that all other Western\nblotting images intended to show expression or activity levels have been\nsubjected to quantitative analysis. Detailed results are provided in Fig. 7, 8 and\nExtended Data Fig. 1, 6, 8 of the revised manuscript.\n6. In addition to CD36 and Abcg1, the protein expression levels of other lipid\nuptake receptors (e.g., SR-B1, LDLR) and cholesterol efflux transporters (e.g.,\nABCA1) should be examined, as their expression may be influenced by post-\ntranslational regulation. Assessing only mRNA levels by qRT-PCR is insufficient.\nAnswer: Thanks. We have now quantified the protein expression levels of key\nlipid uptake receptors (LDLR, MSR1, OLR1 and SR-B1) and core cholesterol\nefflux transporters (ABCG1, ABCA1) in macrophages under experimental\nconditions via Western blotting (Fig. 5 in this letter). Key findings indicate that,\nexcept for ABCG1, CLPs did not significantly alter the levels of other\nreceptors, consistent with the mRNA trends we initially reported. The new\nresults have been included in the revised manuscript as Fig. 8a.\nFig. 5. The effect of CLPs on receptors involved in cholesterol metabolism.\nMinor:\n1. While the manuscript is largely well-written, there are several instances of\nthe use of unusual terminology. For instance, the term \"atherosclerogenesis\" is\nunusual. The commonly used term for this process is atherogenesis. In addition,\nthe use of the term \"fate mapping\" seems incorrect. The authors appear to\nmean \"fate\" as fate mapping are methods that allow the identification of cells\nthroughout differentiation or transition. The pluralization of expression to\ndescribe changes in gene output is also non-standard (i.e., \"... the expression\nof X and Y were ...\" is preferable)\nAnswer: Thanks. We have revised the manuscript accordingly to adhere to the\ncommon use.\n \"Atherosclerogenesis\" has been replaced with the standard and widely\naccepted term \"atherogenesis\" throughout the manuscript.\n “Fate mapping” has been replaced with precise descriptions; we now\ndescribe our data as revealing the “phenotypic transition” of cells (Lines\n219-279 in the revised manuscript).\n The sentence structure has been corrected to the standard form, \"...the\nexpression of CHI3L2 and CHI3L1 in macrophages was... (as shown in\nlines 137-138 in the revised manuscript.)\" (treating \"expression\" as a\nsingular entity), and we have carefully checked the entire text.\n2.ORO and histological staining data in Fig. 1f, g, and i should be enlarged. There also appears to be a discrepancy in the\nscale bar length in panels h and i. The above comment is applicable to the other Figures, which should also be checked for\nscale bar accuracy in length and use the same position in each panel and a consistent line thickness.\nAnswer: We apologize for the oversight in using representative image\npresentation. We have now enlarged key histological images (Fig. 2c-f, 4, 9,\nand Extended Data Fig. 2 in the revised manuscript, Fig. 1, 3 in this letter) to\nbetter highlight cellular details. All scale bars have been carefully reviewed and\ncorrected to ensure accurate reporting. These updates have been incorporated\ninto the revised manuscript, and we sincerely appreciate your constructive\nfeedback.\n3. The human plasma and tissue cohorts (n=34 healthy, n=38 atherosclerotic)\nare modest. Excluding patients with diabetes/hypertension limits clinical\nrelevance, as these conditions frequently coexist with atherosclerosis. The\nauthors should address whether findings generalize to broader populations.\nAnswer: Thanks. We fully agree that the cohort size in this study is limited.\nHowever, this research was designed as a mechanism-driven, proof-of-concept\ninvestigation rather than a large-scale epidemiological study. Our primary goal\nwas to minimize confounding variables during the initial discovery phase to\nobtain a clear, interpretable signal regarding the specific role of CHI3L2 in\natherosclerosis. Using a tightly controlled, focused cohort is a well-established\napproach in early biomarker and mechanistic research, establishing\nfoundational insights before extending to larger, more heterogeneous\npopulations.\nGiven that diabetes and hypertension frequently coexist with atherosclerosis,\ntheir exclusion was a deliberate methodological choice, allowing us to examine\nthe direct contribution of CHI3L2 without interference from strong confounding\nsignals associated with major metabolic and stress pathways. This controlled\ndesign strengthens the internal validity of our study and increases confidence\nthat the observed associations are attributable to atherosclerosis itself rather\nthan secondary effects of hyperglycemia or hypertension.\nIn response to your concern, we have explicitly acknowledged this limitation in\nthe revised discussion section of the manuscript (Lines 688-695). Notably,\ngrowing evidence suggests that the biological pathways examined in our study\nmay indeed be relevant to broader patient groups. For instance, CHI3L1 (YKL-\n40) is established as a key mediator of inflammation and vascular pathology in\natherosclerosis, metabolic syndrome and diabetes4. This precedent offers a\nstrong biological rationale for hypothesizing that CHI3L2 may likewise play an\nimportant role in populations with comorbidities. We are actively planning\nfollow-up translational clinical studies to test this hypothesis.\nReviewer #2 (Remarks to the Author):\nIn this study, Wang et al. reported the impact of Chitinase-like protein 3 (Chil3)\nand its human functional homolog chitinase-3-like 2 (CHI3L2) on macrophage\nlipid metabolism and atherosclerosis. Using numerous elegant experimental\napproaches as well as in in vitro and in vivo models, the authors proposed a\nnew autocrine mechanism in which secreted CLP regulates cholesterol uptake\nin macrophages through binding to CD36. Importantly, Wang et al. provided\nimportant new therapeutic insights with the use of antibodies targeting CHI3L2\nwhich attenuate atherosclerosis. This group previously demonstrated that Chil3\npolymorphisms were associated with inflammatory diseases by regulating lipid\nmetabolism. This study is in the continuation of their previous works by\npositioning Chil3 in the development of atherosclerosis. The overall study is\nvery interesting but several points must be clarified by the authors.\nMajor points.\n1. Previous numerous studies characterized several macrophage populations\nin atherosclerotic plaques by single-cell RNA-Sequencing (PMID: 38639096,\nPMID: 29545366, PMID: 31591603, PMID: 32981416….). Was Chil3\nexpression associated with a specific subtype cluster of macrophages in\natherosclerotic plaques (Trem2+ macrophages (PMID: 38646596))? Was\nthis latter altered upon Chil3 deficiency? Analysis of Chil3 expression in CAM\n(classically activated macrophages) and AAM (alternatively activated\nmacrophages) is indeed very limited (Extended Data Fig. 2e-h).\nAnswer: We thank you for the insightful comments. In our scRNA-seq dataset,\nwe identified seven macrophage subpopulations in atherosclerotic aortas:\nLy6C2+ monocyte-like, resident, MHC-II, inflammatory, foamy, VSMC-like, and\nfibrochondrocyte (FC)-like macrophages. Chil3 was specifically enriched in\nLy6C2+ monocyte-like macrophages in ApoE-/- mice, whereas CHI3L2 was\nmainly expressed in foam cells, VSMC-like macrophages, and FC-like\nmacrophages in ApoE-/-.Chil3∆.hCHI3L2tg mice. Functionally, Chil3 increased\ninflammatory macrophages and reduced resident macrophages, while CHI3L2\nexpanded foam cell/FC-like clusters at the expense of resident and MHC-II\nmacrophages (Fig. 6 in this letter). These differences are consistent with the\nfact that Chil3 and CHI3L2 are not direct orthologs and exhibit subtype-specific\npatterns and consequences. The new results have been included in the revised\nmanuscript as Fig. 3d-f and Extended Data Fig. 3b.\nFig. 6. scRNA-seq analysis of aortic macrophage phenotyping across\nthree genotypes.\nThe Chil3+ macrophage population we identified co-expressed canonical Ly6C+\nmonocyte markers (Ly6c2, Ccr2, Ccl2) and inflammatory genes (Il1b, Ifitm\nfamily), corresponding to a pro-inflammatory monocyte-like state similar to the\n“monocyte-macrophage transitional state” described by Yang et al., who also\nreported high Chil3 expression in this cluster5. In our model, Chil3 deficiency\ndid not markedly change this monocyte-like cluster but reduced inflammatory\nmacrophages and increased resident macrophages, consistent with prior\nobservations that Ly6Chi monocytes dominate healthy arteries, whereas\nLy6Clow monocytes predominate in atherosclerosis6.\nThese data suggest that Chil3’s effects on inflammatory and resident\nmacrophages, rather than monocyte-like cells, drive disease progression.\nAlthough foam cell clusters were not significantly altered, foam cell formation\ndecreased, indicating that Chil3 regulates macrophage functional programs\nrather than cluster identity.\nChil3’s role is context-dependent. In acute injury, Chil3 Ly6C monocytes are\nimmunoregulatory and pro-repair7, while in chronic inflammation such as\n⁺ ⁺\natherosclerosis, Chil3 is expressed in pro-inflammatory Ly6C monocyte-\nderived macrophages, and its deficiency attenuates plaque development. This\n⁺\nreflects the influence of the tissue microenvironment: acute injury fosters a\nresolution-oriented milieu that permits Chil3+ monocytes to adopt reparative\nprograms, whereas the lipid-rich, oxidative, necrotic environment of\natherosclerotic plaques redirects these cells toward pro-inflammatory fates.\nWe also mapped CHI3L2 expression in atherosclerotic plaques. scRNA-seq\ndata show that macrophages are highly plastic8,9. Prior studies identified lipid-\npoor inflammatory and Trem2 foam macrophages, with Trem2 cells linked to\nadvanced lesions10,11. Consistent with this, we identified a Trem2+ foam cell\n⁺ ⁺\npopulation (also called foamy macrophages in our revised Fig.3). CHI3L2 was\npredominantly expressed within these late-stage Trem2+ foam cells, and its\nexpression greatly expanded this population in ApoE-/-.Chil3∆.hCHI3L2tg mice.\nWe have already covered this topic in the Discussion section (Lines 593-606).\nMacrophage polarization experiments (Extended Data Fig.1g-j in the revised\nmanuscript) were performed not to replicate scRNA-seq clusters but to identify\nthe human functional homolog of mouse-specific Chil3. Using chromosomal\nsynteny and functional polarization assays, we found that CHI3L2, not CHI3L1,\naligns with Chil3: Chil3 and CHI3L2 are established markers of alternatively\nactivated macrophages (AAMs)12,13, whereas CHI3L1 is mainly associated with\nclassically activated macrophages (CAMs)14. These data support focusing on\nthe Chil3/CHI3L2 functional axis.\n2. Because lipid uptake in macrophages has a profound impact on intracellular\nlipid homeostasis beyond foam cell formation, it is important to further describe\nthe consequences of the alteration of Chil3 expression in the lipidome of\nmacrophages, especially in membrane lipid composition. Then, lipidome of WT\nand Chil3∆ macrophages must be explored in order to decipher the role of Chil3\nin macrophages. Such an analysis is even more essential since Chil3 deficiency\nallows an increased expression of Abcg1, a transporter recently identified as\nplaying a key role in lipid rewiring and functions of macrophages (beyond\ncholesterol efflux) (PMID: 39661702).\nAnswer: We sincerely appreciate your insightful suggestion. In response, we\nperformed non-targeted lipidomics on macrophages from ApoE-/-, ApoE-\n/-.Chil3∆ and ApoE-/-.Chil3∆.hCHI3L2tg mice. Neutral lipids including cholesterol,\ncholesteryl esters (CE), and saturated fatty acids (USFA) were significantly\nreduced, consistent with the decreased lipid droplet formation and foam cell\ntendency observed in our model (Fig. 7a in this letter). In contrast, membrane-\nassociated lipid species were markedly increased in ApoE-/-.Chil3∆\nmacrophages, including several classes of phospholipids\n(Phosphatidylethanolamine (PE), Phosphatidylglycerol (PG),\nPhosphatidylserine (PS), Phosphatidylinositol (PI)), lysophospholipids\n(Lysophosphatidylcholine (LPC), Lysophosphatidylethanolamine (LPE)), and\nsphingolipids (Ceramides (Cer)) (Fig. 7b in this letter). These findings indicate\nthat loss of CLPs drives a membrane-focused rewiring of lipid metabolism,\nresulting in increased membrane lipid enrichment and altered biophysical\nproperties. These results have been added to the revised manuscript (Fig. 8b,\nc).\nFig. 7. The effects of CLPs on macrophage lipids.\nDahik et al. reported impaired PE metabolism and reduced phosphatidylcholine\n(PC) in ABCG1-deficient BMDMs15, consistent with our model in which CLP\nexpression suppresses ABCG1 and lowers these phospholipids. They also\nshowed that ABCG1 loss redistributes saturated fatty acids (SFA) from\nmembranes to lipid droplets, increasing USFA, decreasing membrane rigidity,\nand reducing pro-inflammatory lipids15. Interestingly, in contrast, we observed\nthat CLPs (with low ABCG1) increase SFA, likely reflecting the context-\ndependent role of ABCG116. In atherosclerosis, ABCG1 primarily facilitates\ncholesterol transport, and its inhibition is pro-inflammatory17, whereas in\nmetabolic disease, it regulates fatty acid metabolism, where inhibition can be\nanti-inflammatory15.\nOur data indicate that CLPs broadly regulate membrane lipid distribution. While\nour primary aim was to dissect how CLPs modulate cholesterol pathways via\nthe mTOR-PPARγ-ABCG1 axis, the lipidomic changes suggest CLPs may also\ninfluence PPARγ indirectly through membrane composition. We propose that\nloss of CLPs increases membrane phospholipids and sphingolipids, triggering\ncompensatory ABCG1 upregulation and reducing SFA and cholesterol,\nestablishing a distinct lipid-rewiring state that may further shape macrophage\ninflammatory function. We now highlight in the revised Discussion (Lines 658–\n666) that future studies should examine fatty acid saturation ratios, membrane\nfluidity, lipid droplet dynamics, and ABCG1 inhibition consequences.\n3. Through the uptake of oxidized lipids and free fatty acids, CD36 plays major\nroles in several pathways controlling the metabolic phenotype of macrophage\n(fatty acid oxidation and storage, ROS production, energy metabolism….). The\nauthors must provide information about the impact of Chil3 deficiency on\nmetabolic pathways in macrophages (RNA-sequencing and/or seahorse\nanalyses for example).\nAnswer: Thank you for your suggestion. Gene set enrichment analysis (GSEA)\nof differentially expressed genes in macrophages from scRNA-seq, revealed\nthat CLPs deficiency downregulated inflammation-related pathways, including\nthe NOD-like receptor, chemokine, and cytokine-cytokine receptor interaction\nsignaling pathway, while upregulating metabolism-related pathways such as\noxidative phosphorylation, PPAR signaling, and glutathione metabolism (Fig. 8\nin this letter). This suggests that CLPs may be involved in regulating\nmacrophage inflammatory responses, energy metabolism, and antioxidant\ncapacity. In addition, the lysosome pathway was also markedly enhanced,\nreflecting increased cellular degradation and recycling capacity in CLP-deficient\nmacrophages, which may indirectly support metabolic reprogramming and\nenergy supply (Fig. 8 in this letter). The supplementary results are presented\nin Extended data Fig. 7a in the revised manuscript.\nFig. 8. GSEA analysis.\nResearch on the mechanisms linking CLPs to energy metabolism remains in\nits early stages. This study primarily focused on identifying a novel mode of\naction for CLPs: a protein exhibiting glycosidase-like hydrolytic activity that\nregulates the N-glycosylation modification of CD36, along with its subsequent\nrole in the PPARγ-ABCG1 signaling pathway. Our current experiments\ndemonstrate CLPs' role in regulating obesity and non-alcoholic fatty liver\ndisease (NAFLD). Models of obesity and NAFLD effectively illustrate how CLPs\ninfluence energy metabolism biological processes. Direct metabolic flux and\nenzyme activity assays (e.g., via Seahorse analysis) will further elucidate these\nmechanisms, which we plan to validate in subsequent work. However, the\nprimary focus of this paper remains the novel CLP receptor and its effects on\nthe receptor.\n4. The authors did not explain and discuss the role of Chil3 on inflammation,\nespecially on the inflammatory phenotype of macrophages. Because CD36,\nthe main target of Chil3, is involved in NLRP3 activation (PMID: 23812099),\nthe reduced IL-1β expression observed in aorta of Chil3∆ mice (Fig1e) could\nresult from an impaired activation of the NLRP3 pathway. Although the role of\nChil3 in foam cell formation is very likely involved in the attenuated\natherosclerosis development, the authors cannot exclude the role of\ninflammation in their mechanism since anti-inflammatory targeting IL-1β led to\na reduction of atherosclerotic cardiovascular diseases (PMID: 28845751). The\nauthors must explore and/or discuss this aspect.\nAnswer: Thanks. CD36-mediated lipid uptake promotes intracellular\ncholesterol crystallization and activates the NLRP3 inflammasome, enhancing\nIL-1β maturation and secretion18. As shown in Fig.8 of this letter, the NOD-like\nsignaling pathway was significantly upregulated in the presence of CLPs.\nFurther analysis showed that the core component of the NOD-like receptor\nfamily, the pyrin domain-containing 3 (Nlrp3) inflammasome, was significantly\ndownregulated in macrophages from ApoE-/-.Chil3∆ mice, accompanied by\nmarkedly reduced expression of pro-inflammatory cytokines, including Il1b, Il6\nand Tnf (Fig. 9a in this letter). These results suggest that the inflammasome\nmay serve as a critical mediator through which CLPs regulate inflammatory\ncytokine expression in macrophages. However, the expression of Il6 and Tnf\ndoes not entirely depend on NLRP3 inflammasome activation19. RNA\nsequencing also revealed a significant enrichment of inflammatory signaling\npathways (Fig. 9b in this letter). These results are presented in the Externed\nData Fig. 7b-d of the revised manuscript.\nFig. 9. The effects of CLPs on macrophage inflammation.\nIntegrating these data, we propose a “polarization-metabolic axis” model: Chil3\ndeficiency reduces CD36-mediated lipid uptake, decreases lysosomal lipid load,\nand inhibits mTOR signaling. This shifts macrophages toward an anti-\ninflammatory state, increasing PPARγ expression and enhancing ABCG1-\nmediated cholesterol efflux (detailed explanation provided in responses to\nQuestions 15 and 16). The overall attenuation of pro-inflammatory\ntranscriptional programs (Fig. 9 in this letter) explains the coordinated\ndownregulation of Il1b, Il6 and Tnf. Previous studies show that CAMs provides\nan “initiation signal” for inflammasome activation, whereas anti-inflammatory or\npartially AAM-biased programs limit it20,21. Thus, reduced Nlrp3 expression in\nChil3-deficient macrophages reflects an overall shift toward an anti-\ninflammatory state, and we highlight the potential role of inflammasome\nsignaling in the Discussion section (Lines 682-687).\nOthers major points.\n5. In mice with a total Chil3 deficiency, what tissue drives the circulating Chil3 /\nCHI3L2 concentrations in mice (Fig. 1a, Extended data Fig. 2i……)? Same\nquestion in humans (Fig. 2c)?\nAnswer: Under physiological conditions, Chil3 is predominantly expressed in\nmacrophages of the lung, bone marrow, and spleen22, whereas CHI3L2 is\npredominantly expressed in articular chondrocytes as well as lung and heart\nmacrophages23. Although their basal expression is low, both genes are\nmarkedly upregulated in disease settings. In atherosclerosis models, we\npropose that locally recruited macrophages are the primary source driving the\nincrease in circulating Chil3 (CHI3L2). Numerous studies have shown that\nactivated macrophages are the major source of CLPs under inflammatory\nconditions14. Our scRNA-seq data reveal that Chil3 expression predominantly\narises from monocyte-derived macrophage subsets, whereas CHI3L2\nexpression is largely confined to late-stage macrophages (Fig. 6 in this letter).\nThese macrophages may undergo apoptosis during atherosclerosis\nprogression, potentially releasing their CLP content into the circulation.\nAccordingly, we infer that circulating CHI3L2 in humans is also primarily derived\nfrom inflammatory macrophages involved in atherosclerotic lesions. While\nplaque macrophages represent the main source, other tissues such as Kupffer\ncells in the liver and macrophages in adipose tissue may also contribute\nmodestly in a systemic inflammatory context, as we observed that CLPs also\naffect obesity and NAFLD.\n6. Extended Data Fig. 1(a). What was the diet of ApoE-/- and WT mice ?\nAnswer: All mice were maintained on a normal diet (ND) for 32 weeks. To\nexamine CLP family expression, ApoE-/- mice on ND or WD would normally be\nselected. We reviewed numerous GEO datasets of mouse atherosclerosis\nmodels (ApoE-/- on ND or WD), but large-scale detection of CLP family genes\n(Chil1, Chil3, Chil4, Chid1 and Ovgp1) was largely undetectable, likely due to\nthey are secreted proteins and mRNA levels are relatively low. In the GSE2372\ndataset24, all screened CLP family members were detectable; this dataset\nincludes ApoE-/-and WT mice on ND at 32 weeks. Given that ApoE-/- mice on\nND can spontaneously develop atherosclerosis by this age, we used them as\nthe model group, with WT mice as controls. Notably, GSE2372 had a small\nsample size (n=3), so we integrated it with another public dataset,\nGSE20741425, which includes ApoE-/- and WT mice on ND for 40 weeks and is\ncomparable to GSE2372 (Fig. 10 in this letter, Extended Data Fig. 1a, b in the\nrevised manuscript).\nFig. 10. Expression of CLPs in mice and humans in the GEO dataset.\n7. Fig. 1c. The title of the Y-axis must be clarified. What class of lipid was\nquantified ? Cholesterol ? How was quantified the\nlipid profile? How do the authors explain the reduced serum LDL (likely LDL-C) in BQ.ApoE-/-.Chil3-/- mice in comparison to\nBQ.ApoE-/- mice ? Same comments in Fig. 2e).\nAnswer: Sorry. We have modified the Y-axis label to define it as lipid\n(cholesterol & TG) profiles in serum (mmol/L). HDL and LDL should also be\nreplaced with HDL-C and LDL-C, because the test measures cholesterol in\nthese components (Fig. 11 in this letter). As stated in the manuscript, plasma\nlipid levels were measured using a direct homogeneous assay on a Beckman\nAU5431 automatic biochemistry analyzer, which employs specific surfactants\nand enzymes to quantify cholesterol primarily within the HDL/LDL fraction. We\nhave changed all instances of HDL or LDL in the revised manuscript to HDL-C\nor LDL-C (Fig. 2a, b).\nFig. 11. lipid (cholesterol & TG) profiles in serum (mmol/L). TC, total\ncholesterol; TG, triglyceride; LDL-C, low-density lipoprotein cholesterol;\nHDL-C, high-density lipoprotein cholesterol.\nInflammation could be a key driver of hepatic VLDL (a precursor to LDL in\ncirculation), and decreased inflammation may reduce VLDL/LDL production26,27.\nWe have found that Chil3 deficiency significantly attenuates both local and\nsystemic inflammation (Fig. 8, 9 in this letter), which may explain the reduced\nLDL-C levels in mice. In addition, downregulation of systemic inflammation may\nalso improve hepatic lipid metabolism, enabling the liver to more effectively\nclear circulating LDL. Our recent study on CLPs in NFLAD showed that CLPs\nsignificantly reduced hepatic lipid deposition, suggesting that CLP deficiency\ncontributes to improved hepatic lipid metabolism. However, more observations\nare still required in future in this study to ascertain the conclusive roles and\nmechanisms.\n8. Same question with oxLDL (Fig. 1d).\nAnswer: We propose several potential mechanisms: (1) Reduced oxidative\nenvironment for ox-LDL generation. In atherosclerosis, ox-LDL generation is\ndriven by inflammation-induced oxidative stress. Chil3 deficiency markedly\nattenuates systemic and lesion-associated inflammation (Fig. 8, 9 in this\nletter), lowering oxidative conditions necessary for LDL modification and\nreducing ox-LDL production. (2) Reduced hepatic VLDL/LDL secretion leading\nto a smaller LDL substrate pool. As noted above, Chil3 deficiency may impair\nthe liver’s capacity to secrete VLDL, decreasing circulating LDL levels and\nconsequently limiting the substrate available for oxidative modification. (3)\nPotential enhancement of antibody-mediated clearance of ox-LDL. Natural\nantibodies in the circulation can bind ox-LDL to form immune complexes, which\nare then efficiently cleared by macrophages in the liver and spleen28. Chil3\ndeficiency may indirectly improve the efficiency of this immunological clearance\npathway. (4) More efficient non-pathological clearance of ox-LDL. ox-LDL may\nalso be removed through more effective, non-inflammatory scavenger\npathways. For example, functional macrophages or hepatic Kupffer cells may\nclear ox-LDL more efficiently through alternative receptors without becoming\nlipid-overloaded, as Chil3 deficiency improves hepatic immunometabolic\nhomeostasis. Notably, several of these mechanisms are supported by our\nindependent research demonstrating that CLP deficiency improves hepatic lipid\nhomeostasis, but more work is still needed to understand these mechanisms.\n9. Fig. 2k. The efficiency of the treatment with rhCHI3L2 must be validated by\nquantification of CHI3L2 in the circulation of mice following intraperitoneal\ninjection.\nAnswer: Thanks. We assessed circulating CHI3L2 levels following\nintraperitoneal injection of an hIgG1-Fc-tagged rhCHI3L2, which was designed\nto extend its plasma half-life in vivo. Two-month-old ApoE-/-.Chil3∆ mice were\nfed a WD for 1 month before initiating protein administration. During the\nsubsequent 2 months of WD feeding, mice received intraperitoneal injections\nevery three days of either hFc-rhCHI3L2 (0.25 µg/g) or an hFc-IgG1 control\n(0.25 µg/g). Serum collected on days 2 and 3 post-injection was analyzed using\na commercial ELISA kit, and we found that serum CHI3L2 levels on day 2 post-\ninjection reached 35.64% of initial treatment containing 0.50 µg/mL CHI3L2\nprotein. By day 3, the level decreased to 12.85%. No detectable CHI3L2 signal\nwas observed in the control group on day 2 (Fig. 12 in this letter). This\nconfirms that our intraperitoneal delivery protocol successfully introduces the\nrecombinant protein into the systemic circulation, supporting the biological\nrelevance of the observed phenotypic effects. We have referenced the newly\nadded supplementary data in the Results section (Extended Data Fig. 2b).\nFig. 12. Validation of recombinant protein injection into the body.\n10. Fig. 3. Single-cell RNA-Sequencing data are interesting but it would be\nuseful to know the biological pathways that are altered in macrophages in the\nthree mouse models (WT, Chil3∆ and Chil3∆.CHI3L2tg). A same comment can\nbe done in BMDM and PM cells (Fig. 5). As a whole, impact of Chil3 deletion\nas well as expression of CHI3L2 in Chil3-deficient cells on the metabolic\nphenotype of macrophages is lacking. In this context, it is very intriguing that\nno cholesterol metabolism (or lipid metabolism) pathways were found altered\nby RNA-sequencing in ox-LDL stimulated Chil3∆ versus WT BMDM\n(Extended Data Fig. 7b). These findings weaken a significant role of Chil3 in\nmacrophage lipid uptake as proposed by the authors.\nAnswer: The scRNA-seq results are shown in Fig.8 of this letter. We\nreanalyzed the RNA-seq data, focusing on gene sets related to cholesterol\nmetabolism, and observed upregulation of key genes regulating intracellular\ncholesterol processing in ox-LDL-stimulated ApoE-/-.Chil3Δ BMDMs: including\nNpc1, Npc2 (lysosomal cholesterol efflux), Stard3 (lysosomal-endoplasmic\nreticulum cholesterol transport), Lipa (lysosomal cholesterol esterification),\nNceh1 (cytoplasmic cholesterol esterification), Soat1 (Acat1) (cholesterol re-\nesterification), Abcg1 (cholesterol efflux). In contrast, changes in cholesterol\nuptake and biosynthesis genes were minimal (Fig. 13 in this letter). This\npattern suggests that Chil3 deficiency promotes rapid lysosomal cholesterol\nefflux, efficient cholesterol esterification, and enhanced delivery of cholesterol\nefflux mechanisms such as ABCG1. These results are presented in Extended\nData Fig. 8 of the revised manuscript.\nFig. 13. RNA-seq data.\n11. Fig. 6. Although not in the top 5 candidate, were P2Y6R and TREM2 (or\nsome TLRs) identified in IP and MS analyses since these receptors promote\nlipid uptake and atherosclerosis (PMID: 38036416, PMID: 38646596)?\nAnswer: We appreciate your insightful question. As highlighted in the\nsuggested references, P2RY6 and TREM2 are recognized promoters of lipid\nuptake and atherosclerosis3,11. In our IP-MS analysis aimed at identifying high-\nconfidence Chil3-interacting proteins, neither receptor appeared in statistically\nsignificant matches, indicating that under our experimental conditions, they do\nnot form stable or direct complexes with Chil3 detectable by this method. While\nexploring additional potential receptors is scientifically valuable, the primary\ngoal of this study was to move from large-scale screening to mechanistic\nvalidation. CD36 emerged as the top candidate in our IP-MS screen, guiding us\nto focus subsequent functional experiments on this high-probability target. This\napproach allowed us to rigorously demonstrate that CLPs (Chil3 and CHI3L2)\ndirectly bind CD36, remodel its N-glycans, and thereby enhance CD36-\nmediated lipid uptake, a pathway critical for in vivo atherosclerosis development.\nCD36 thus serves as a clear example to elucidate the novel mechanism of\nCLPs in atherosclerosis.\nWe have not excluded the involvement of other receptors. As stated, “Although\nwe did not observe any interaction between CLPs and the scavenger receptors\nOLR1 and MSR1, the possibility of their interactions cannot be ruled out” (Fig.\n6b in the revised manuscript, Discussion section, Lines 610-611). Current data\ndo not support direct interactions between Chil3 and P2RY6 or TREM2, but\nindirect effects or context-dependent interactions remain possible. Investigating\nadditional CLP-associated receptors represents an important direction for\nfuture research.\n12. As a follow up of my previous comment, may Chil3 promote hydrolysis of\nN-glycosylated glycans of others membrane proteins without physically\ninteracting with them (and then not to be detected by co-IP)?\nAnswer: The possibility you raise is plausible. Lectin-glycan interactions are\ninherently transient, so Chil3 could theoretically act on other glycoproteins after\nmodifying CD36. However, Chil3’s three-dimensional structure restricts binding\nto specific glycan conformations; if other membrane proteins do not present\ncompatible glycans, Chil3 will not engage or act on them29.\nOur functional data indicate that CD36 is the primary target of Chil3 in\natherosclerosis models, likely due to its highest affinity for CD36’s specific\nglycan structure. If Chil3 broadly hydrolyzed N-glycans across many membrane\nproteins, we would expect widespread effects on multiple receptors. Instead,\nwe observe a highly specific phenotype: impaired CD36-mediated lipid uptake\nwithout significant changes in other scavenger receptor functions. This supports\na specific regulatory role via the Chil3-CD36 axis rather than broad, non-\nspecific glycan remodeling.\nWhile broader substrate activity cannot be entirely excluded, we are planning\nin vitro glycan array and cryo-electron microscopy analyses to systematically\ndefine its complete enzymatic substrate spectrum30. Within this study,\ncombined genetic, biochemical, and functional evidence confirms the Chil3-\nCD36 axis as a key pathway in macrophage lipid metabolism and\natherosclerosis.\n13. Fig. 8a. How do the authors explain that the impaired cholesterol uptake in\nChil3∆ BMDM was not accompanied by an increased expression of Ldlr and\nHmgcr genes; the expression of these genes being downregulated by\ncholesterol.\nAnswer: In classical cholesterol homeostasis, reduced intracellular cholesterol\nupregulates LDLR and HMGCR via SREBP2 activation31. This represents a\ncrucial feedback regulatory mechanism for maintaining cellular cholesterol\nhomeostasis. However, in ApoE-/- and ApoE-/-.Chil3∆ BMDMs with or without ox-\nLDL stimulation, we found that Ldlr/Hmgcr were downregulated compared to\ntheir respective controls. Yet, no differences in Ldlr/Hmgcr expression were\nobserved among the two genotypes after ox-LDL treatment (Fig. 14 in this\nletter). We propose that ox-LDL-stimulated BMDMs represent a foam cell\nmodel, where high ox-LDL exposure causes substantial intracellular cholesterol\naccumulation, creating a “cholesterol overload” state. In this context, SREBP2\nactivity is suppressed by intracellular cholesterol, as reflected by\ndownregulated Ldlr and Hmgcr in all ox-LDL-treated BMDMs (Fig. 14 in this\nletter). This confirms that the classical cholesterol feedback pathway is\ninhibited under foam cell conditions.\nFig. 14. The expression of cholesterol-related molecules.\nThus, the key question is not why Chil3 deficiency fails to upregulate Ldlr/Hmgcr,\nbut how it still reshapes macrophage cholesterol metabolism under cholesterol\noverload. Our data show that Chil3-deficient macrophages reduce net\ncholesterol accumulation by two mechanisms: limiting CD36-mediated uptake\nand enhancing the PPARγ-ABCG1 efflux axis. This positions Chil3 as a critical\ncholesterol “fine-tuner,” regulating a pathway that can override the classical\nSREBP2 feedback. Even when SREBP2 is suppressed, Chil3 specifically\nbalances cholesterol import (CD36) and export (ABCG1).\nAdditionally, upregulation of the E3 ligase Mylip in Chil3-deficient macrophages\npromotes LDLR degradation32, explaining why LDLR levels remain unchanged\ndespite reduced uptake (Fig. 13 in this letter).\n14. Fig. 8d. Although the method used to quantify cholesterol efflux is highly\nquestionable, the results are quite intriguing. Indeed, a 45% cholesterol efflux\nduring a 4h incubation with HDL is extremely high (about 10% is expected) and\nsuggests an alteration of cell viability. The authors must describe the method\nused (what type of HDL used? what is the % of\ncholesterol efflux without\nHDL ? ……..).\nAnswer: The cholesterol efflux assay was performed following the\nmanufacturer's instructions (ab196985; Abcam). Macrophages were seeded in\n96-well plates (1×10⁵ cells/well), loaded with fluorescent-labeled cholesterol at\n37°C for 1 h, incubated for 16 h, and then treated with serum-free medium\ncontaining HDL (ApoB-depleted serum) for 4 h. Fluorescence of supernatant\nand cell lysates were measured (Ex/Em = 485/523 nm, Infinite M200, Tecan).\nCulture medium and cell lysate cholesterol efflux were calculated based on the\nfluorescence intensity of the medium and that of cell lysates, in accordance with\nthe manufacturer’s instructions (Methods section, Lines 910-918).\nWe fully understand your concerns regarding the relatively high cholesterol\nefflux rate (45%) observed in our experiments, which indeed exceeds the\napproximately 10% typically reported using conventional radiolabeling methods.\nHowever, we wish to clarify that this result is consistent with the data range\nreported in multiple published studies using the same fluorescent assay kit we\nemployed33-35. Under similar conditions (HDL acceptor, 4 h incubation),\nreported efflux rates range 30-50%. This is not an anomaly but a characteristic\nof this methodology. HDL-mediated cholesterol efflux capacity (CEC) can\nprevent cardiovascular disease. However, the lack of standardization in CEC\nassays hinders their application in large cohorts and comparability across\nstudies. Cell-based CEC assays are influenced by numerous factors:\ncholesterol donor cells, cholesterol tracers, and HDL samples36,37.\nTwo main CEC assay methods exist: BODIPY-cholesterol consistently shows\nhigher efflux than [³H]-cholesterol: 11.8 ± 0.6%/h vs. 2.7 ± 0.3%/h with 2%\napoB-depleted serum, reflecting its higher sensitivity and suitability for high-\nthroughput screening to link cholesterol efflux with atherosclerosis38.\n15. Extended Data Fig. 7d and Figure 8. The authors proposed that the increased\nAbcg1 expression resulted from an elevation of Ppar expression. If it is the case,\nAbca1 expression would be also increased since induction of Ppar stimulates\nAbca1 expression (PMID: 11135616)\nAnswer: In exploring how CLPs (Chil3 and CHI3L2) regulate macrophage lipid\nhomeostasis, we observed that CLP deficiency reduces lipid uptake while\npromoting lipid efflux. RT-qPCR and Western blotting of macrophages under\nox-LDL stimulation showed that among efflux receptors, only ABCG1 was\nsignificantly upregulated, while ABCA1 remained unchanged in CLP-deficient\ncells (Fig. 14, 15a in this letter), indicating CLPs primarily rely on the ABCG1\nmediated cholesterol efflux pathway. Cholesterol uptake and synthesis-related\nmolecules showed no significant differences across groups (Fig. 14, 15a in this\nletter).\nWe previously found that Chil3 deficiency upregulates PPARγ in pneumonia\nand psoriasis mouse models12,39. Here, under ox-LDL, CLP deficiency also\nincreased PPARγ expression, and PPARγ inhibitors reversed ABCG1\nupregulation and lipid reduction, confirming its central role (Fig. 16 in this\nletter).The physiological ligand of PPARγ, ox-LDL, activates ligand-dependent\ntranscription40. However, when CLPs are absent, lipid intake decreases and the\nincreased PPARγ does not have sufficient ligands to activate it, suggesting a\nligand-independent signaling. PPARγ expression levels can also be modulated\nby microenvironmental cues such as proinflammatory cytokines, leading to\nalterations in PPARγ-mediated events through a ligand-independent pathway12.\nOur study reveals the existence of the ligand-independent PPARγ\ntranscriptional activation mechanism involving mTOR, a key regulator of lipid\nmetabolism and lipogenesis41.\nAlthough classical PPARγ signaling involves the LXRα-ABCA1 axis42, studies\nshow that it can also regulate cholesterol efflux via ABCA1-independent\npathways43. Although ABCG1 and ABCA1 are both LXRα target genes, their\nsensitivities to different signals, lipid environments, and transcription factor\ncombinations are distinct. One study demonstrated that PPARγ agonists\nsuppress foam cell formation without altering LXRα or ABCA1 expression43,\nproviding a plausible mechanism for the effects on LXR-independent and LXR-\ndependent macrophage foam cell formation44.\nRT-qPCR showed a trend toward increased Abca1 expression in ApoE-/- or\nApoE-/-.Chil3∆ BMDMs compared to their respective controls (without ox-LDL).\nHowever, in Western blotting, ABCA1 protein expression in each genotype\nshowed no change after ox-LDL treatment compared to the untreated group\n(Fig. 14, 15a in this letter), suggesting in the ox-LDL-stimulated foam cell\nmodel, ABCA1 protein may undergo complex post-translational regulation,\nsuch as ubiquitination or lysosomal degradation. Furthermore, LXRα\nexpression showed no significant changes, indicating that the classical LXRα-\nABCA1 axis was not effectively activated (Fig. 15b in this letter).\nHeterogeneity in LXRα and ABCA1 expression may result from foam cells\naccumulating high ox-LDL, which can suppress LXRα expression or promoter\nactivity and limit ABCA1 protein changes45. Alternatively, foam cell-like\nmacrophages may increase phagocytosis, removing LXRα agonists45.\nFig. 15. The expression of cholesterol-related molecules.\nIn summary, our model does not overturn classical theory but complements it\nby revealing a refined transcriptional program in CLP-deficient macrophages.\nHere, enhanced PPARγ signaling specifically upregulates ABCG1 via an LXR-\nindependent pathway. This section has been updated in the Results and\nDiscussion (Fig. 8a and Extended Data Fig. 8a–d, line 651-658 in the revised\nmanuscript).\n16. Figures 8e-g. It is difficult to conclude on these experiments in the absence\nof the WT BMDM. More importantly, the authors proposed that the reduced\ncholesterol uptake (in the presence of ox-LDL) in Chil3∆ BMDM increases\nAbcg1 expression through alteration of mTOR and induction of Ppar expression.\nSuch a mechanism is not coherent with the fact that oxysterols are potent\nactivators of Abcg1 expression through the LXR pathway (PMID: 16024918).\nIndeed, Abcg1 expression is increased when macrophages are incubated with\nox-LDL (source of oxysterols) as confirmed in Fig. 8e-f. Then, it would be\nexpected to observe a reduced Abcg1 expression in Chil3∆ BMDM as a\nconsequence of the impaired ox-LDL uptake. How do the authors reconcile\nthese potential conflicting observations?\nAnswer: We appreciate your insightful comment. In the original manuscript,\nsignaling pathway studies were conducted in two main groups: Chil3-deficient\n(ApoE-/-.Chil3∆) macrophages and their controls (ApoE-/-); CHI3L2 transgenic\n(ApoE-/-.Chil3∆.hCHI3L2tg) macrophages and their controls (ApoE-/-.Chil3∆),\nfollowing strict littermate principles. During revision, we combined BMDMs from\nall three littermate strains for Western blotting analysis, providing a more\nintuitive presentation (Fig. 16 in this letter).\nTo elucidate CLPs’ direct mechanisms, we performed IP-MS, identifying CD36\nas a receptor target. Functional experiments confirmed that CLPs specifically\nhydrolyze CD36 N-glycans, enhancing CD36-mediated lipid uptake. This\nrepresents the most significant innovation in our work: proposing a novel\nbiological function for CLPs: glycosidase-like activity.\nUp-taken lipids enter endosomes, dissociate from CD36 in the acidic\nenvironment, and are delivered to lysosomes for degradation46. These free\nlipids (particularly cholesterol) are subsequently sensed in the lysosomal lumen\nvia well-established mechanisms involving the lysosomal cholesterol transport\ncomplex (NPC1/NPC2) and the v-ATPase-Ragulator-Rag GTPase axis47-49.\nNutrients generated by lysosomal degradation (such as carbohydrates, lipids,\nand amino acids) have been demonstrated to activate mTOR50-52. In this study,\nwe propose that exogenous lipids (especially ox-LDL) are the primary drivers\nof mTOR activation in macrophages. Our data shows that compared to ApoE-\n/-.Chil3∆ BMDMs, excessive ox-LDL significantly induced mTOR activation in\nApoE-/- and ApoE-/-.Chil3∆.hCHI3L2tg BMDMs. This mTOR activation was\ncompletely eliminated when lysosomal acidification and function are specifically\ninhibited using Bafilomycin A1 (Baf-A1) (Fig. 16a in this letter). This critical\nfinding underscores the absolute necessity of functional lysosomes in\ntransmitting ox-LDL-driven mTOR signaling.\nUnder ox-LDL stimulation, CLPs deficiency significantly increases PPARγ-\nmediated lipid efflux (Fig. 16b in this letter), reflecting a non-ligand-dependent\nPPARγ pathway. The relatively minor ox-LDL activation of PPARγ caused by\nCLPs deficiency would be insufficient to follow a ligand-dependent PPARγ\npathway. PPARγ drives macrophage polarization independently of ligands, and\nthe IL-4/STAT6 axis promotes PPARγ expression and AAM-associated gene\ntranscription53-55. This indicates that PPARγ upregulation under AAMs\nconditions is a common and conserved phenomenon53,54. These findings\ncollectively explain why CLPs deficiency induces a non-ligand-dependent\nPPARγ pathway in macrophages, consistent with AAMs. In our atherosclerosis\nresearch, we found that the presence of CLPs significantly increases the\nexpression of inflammatory cytokines such as Il1b, Il6 and Tnf in macrophage-\nlike foam cells, indicating an increased proportion of pro-inflammatory\nmacrophages (Fig. 16c in this letter).\nmTORC1 nutrient sensing plays a key role in macrophage polarization,\npromoting CAM activation56. Within CAMs, hypoxia inducible factor 1 alpha\n(HIF-1α), which can be activated by mTORC1, is essential for the transcription\nof proinflammatory cytokine genes57. Concurrently, high-protein diet-induced\natherosclerosis relies on macrophage mTORC158, and lysosomal cholesterol\nloading independently activates mTORC159,60. As a negative regulator of AAMs,\nChil3 deficiency enhances the AAM phenotype12. Consistently, mTOR inhibitor\nrapamycin reverses CLP-induced pro-inflammatory macrophage increases\n(Fig. 16c in this letter) and upregulates PPARγ and ABCG1 in ApoE-/- and\nApoE-/-.Chil3∆.hCHI3L2tg macrophages (Fig. 16d in this letter).\nThe new results have been included in the revised manuscript as Fig. 8 and\nDiscussion (Lines 667-681).\nFig. 16. CLPs inhibit PPARγ-mediated cholesterol efflux through\nactivating mTOR signaling pathway.\nTherefore, allow us to reiterate our proposed mechanism. CLPs target and\nhydrolyze CD36 N-glycans, enhancing CD36-mediated ox-LDL uptake. At this\npoint, ox-LDL entering the lysosomes activates mTOR, reducing anti-\ninflammatory macrophages and PPARγ expression via a ligand-insensitive\nmechanism. This triggers a non-LXR-dependent decrease in ABCG1-mediated\ncholesterol efflux. The combined effects of increased uptake and reduced efflux\ndrive macrophage lipid overload, promoting foam cell formation.\nReviewer #3 (Remarks to the Author):\nThis study focuses on the role of chitinase-like proteins in atherosclerosis.\nMajor:\n1) General: how is LDL-cholesterol measured? This usually does not\naccumulate at high levels in the plasma of ApoE-/-mice. The authors need to be\nfar more thorough here, especially since they state that they find differences.\nAnswer: We thank you for this insightful comment. Plasma LDL-C was\nmeasured using a direct homogeneous assay on a Beckman AU5431 analyzer,\nwhich quantifies cholesterol primarily in the LDL fraction. We fully acknowledge\nyour accurate point that the ApoE-/- mouse model is characterized primarily by\nthe accumulation of cholesterol-rich VLDL and remnant lipoproteins, and that\nclassic LDL is not the dominant fraction in the plasma of these mice under\nstandard conditions61. However, it is important to note that the \"LDL-C\" value\nobtained from such direct homogeneous assays in dyslipidemia models like the\nApoE-/- mouse often captures not only true LDL but also a portion of the\ncholesterol present in small, dense, atherogenic remnant lipoproteins that fall\nwithin the measurable size/density range of the assay. Therefore, in the context\nof our study, the reported \"LDL-C\" is best interpreted as a practical and widely\naccepted index of the total atherogenic lipoprotein burden that is responsive to\nexperimental interventions, rather than solely the concentration of classic LDL\nparticles.\nIn response, we revised the Methods section(Lines 768-771) to clarify that “LDL\ndetection in ApoE-/ mice may include cholesterol from both classic LDL as well\nas small, dense, atherogenic remnant lipoproteins, and thus serves as a\nfunctional index of total atherogenic lipoprotein burden.” The significant\ndifference in LDL-C we observed strongly suggests that CLPs effectively\nmodulated this broader atherogenic lipoprotein pool (Fig. 17a in this letter).\nThis is a biologically relevant and meaningful finding. Our interpretation is\nconsistent with numerous studies in the field that report and discuss changes\nin \"LDL-C\" measured by direct assays in ApoE-/- mice. For example: Xin-Yu\nYang et al. reported a significant decrease in plasma LDL-C in ApoE-/- mice\nafter hydroxyurea therapy, using a comparable direct method62.\nAlthough our method does not distinguish classic LDL-C from dense remnants,\nwe further investigated lipoprotein atherogenicity by measuring plasma ox-LDL\nusing a highly specific and sensitive commercial ELISA kit (Novus Biologicals,\nNBP3-18035). This method directly quantifies the oxidized lipoproteins, which\nare the key drivers of foam cell formation and atherosclerosis progression. Our\ndata revealed that CLPs specifically promote the oxidation of atherogenic\nlipoproteins (which reside within the broader \"LDL-C\" measurable pool). This\noxidation process is a central mechanism in atherosclerosis progression (Fig.\n17b in this letter).\nFig. 17. Lipid level in mouse sera.\n2) The authors several times refer to WT mice. It is well known that WT mice\ndo not develop atherosclerosis, so it does not make sense that atherosclerosis\nwould be studied in this particular model. The authors need to clarify this for\neach and every study. Fig 2k-m. Were these wild-type mice? Or Ldlr-/- mice?\nThis needs to be clarified in the text. Line 207. Again, I am confused as to the\nmouse models here. WT mice do not develop atherosclerosis. The authors\nneed to really clarify this better. Fig 4c-g. What was the genotype of the\nrecipients of the mice for bone marrow transplantation? Please clarify. It is\nimpossible that these were just CD45.2 mice without any mutation since these\nmice do not develop atherosclerosis.\nAnswer: Thank you for your comments. We apologize for the confusion\nregarding “WT” mice. In our study, all mice used for atherosclerosis\nexperiments were on an ApoE-/- background. Our intention in using the term\n\"WT\" was solely to denote that these mice carry wild-type allele of Chil3 or\nCHI3L2 gene. We fully agree that the use of \"WT\" in this context was imprecise\nand could be misinterpreted as referring to the overall mouse model. To resolve\nthis unambiguously, we have now revised the entire manuscript accordingly.\nThe term \"WT, Chil3∆ and Chil3∆.CHI3L2tg\" has been replaced with the precise\ngenotype designation: \"ApoE-/-, ApoE-/-.Chil3∆ and ApoE-/-.Chil3∆.CHI3L2tg\".\nPoint-by-point clarification:\n Fig. 2k-m and Line 207: Fig. 2k-m in the previous manuscript showed\nresults from recombinant hCHI3L2. We administered the recombinant\nprotein via tail vein injection in ApoE-/-.Chil3∆ mice. Line 207: Replace\nWT with ApoE-/-. The main text and Figure legends (Extended Data Fig.\n2) have been updated to reflect this change.\n Fig. 4c-g (Bone Marrow Transplantation): The recipient mice for the\nbone marrow transplantation study were ApoE-/- mice. The text has been\nrevised to explicitly state: \"ApoE-/- recipient mice...\" to eliminate any\ndoubt (Fig. 4c in the revised manuscript).\n3) The number of mice for any analysis in atherosclerosis experiments needs\nto be at least n=10. In very many Figures, these numbers are lower; real\nconclusions can therefore not be drawn.\nAnswer: Our animal experiments used inbred mice, with all experimental mice\nhaving the same genetic background, and strictly followed the principle of\nlittermate controls to minimize variability within groups. We fully agree that the\nnecessary number of animals per group in experiments is required to ensure\naccuracy of results, but it is also necessary to balance the 3R principles.\nWe have now expanded sample sizes to n ≥ 10 per group for all key\natherosclerosis analyses. The updated results, now included in Fig. 18-20, 33\nbelow in this letter, robustly confirm our original conclusions. With this increased\nsample size, the difference in lesion size between mice lacking CLPs and those\nexpressing CLPs remains highly statistically significant. All relevant figures,\nstatistical analyses, and the Methods section have been updated accordingly\nin the revised manuscript. The new results have been included in the revised\nmanuscript as Fig. 1, 4, 9 and Extended Data Fig. 2.\nFig. 18. CLPs promote the formation of atherosclerosis.\nFig. 19. CHI3L2 recombinant proteins promote the formation of\natherosclerosis.\nFig. 20. Neutralizing CHI3L2 could block the progression of\natherosclerosis.\n4) The authors need to provide more definitive evidence, preferably in vivo, that\nthe upregulation of CD36 is what accounts for their phenotype. Was CD36\nstaining increased in atherosclerotic plaques?\nAnswer: Our data indicate that the observed phenotype is driven not by\nincreased CD36 expression but by CLP-mediated post-translational\nmodification enhancing CD36-dependent lipid internalization. As macrophage-\nsecreted proteins, CLPs likely act in an autocrine manner.\nUsing IP-MS, we identified and validated a direct interaction between CLPs and\nCD36. Total CD36 protein levels remained unchanged, indicating increased ox-\nLDL uptake is not due to elevated biosynthesis (Fig. 21a in this letter).\nPrevious studies have demonstrated that CD36 levels on the cell membrane\ndecrease during lipid endocytosis2. To investigate this, we used flow cytometry\nto assess CD36 levels on the cell surface of primary macrophages from mice\nof different genotypes (with or without ox-LDL). A reduction in CD36 levels on\nthe cell membrane would indicate increased internalization of CD36, and\nconsequently, more intracellular lipid uptake2. Our results show that CLPs\ninduce a decrease in CD36 levels on the membrane surface, suggesting they\nenhance the endocytosis of CD36 (Fig. 21b, c in this letter).\nImmunofluorescence co-staining with the early endosome marker EEA1\nconfirmed increased CD36-EEA1 colocalization in Chil3-expressing cells,\nproviding direct evidence that CLPs promote CD36 trafficking into early\nendosomes (Fig. 21d in this letter).\nFig. 21. CLPs affect CD36 endocytosis.\nOf particular significance is our discovery of a glycosyltransferase-like function\nfor CLPs. Building on the lectin-like properties of the CLP family, we established\nthat their binding to CD36 is N-glycan-dependent. Label-free intact\nglycopeptide mass spectrometry revealed that recombinant CLPs (rCLPs)\nsignificantly reduce N-glycosylation at CD36 residues N220 and N321 (Fig. 22a\nin this letter). To determine the functional relevance of these sites, we\ngenerated CD36 expression vectors carrying glycosylation-site mutations and\ntransfected them into 293T cells. Compared with the wild-type construct, both\nthe single-point mutant CD36N220Q and the double mutant CD36N220Q-N321Q\nexhibited enhanced ox-LDL uptake (Fig. 22b, c in this letter). Moreover, rCLPs\ntreatment enhanced lipid uptake only in wild-type CD36-expressing cells, with\nno effect in mutants lacking the N220 and N321 glycosylation sites (Fig. 22b, c\nin this letter). These findings delineate a finely-tuned regulatory mechanism\nthat will be the central subject of our ongoing and future investigations.\nAccordingly, a primary and immediate objective is the generation of mouse\nmodels carrying targeted mutations at the critical glycosylation sites (N220 and\nN321) of CD36, which we anticipate will provide definitive in vivo validation of\nthe pathway uncovered here.\nFig. 22. CLPs affect the glycosylation of CD36.\n5) Extended Data Fig 1a. These data on Chil3 being upregulated are not that\nconvincing. I understand the use of public datasets, which in general makes\nthings easier, but it seems to me that the n would need to be increased to really\nconclude that Chil1 and Chid1 are not affected. These experiments are not hard\nto do and would need to be performed in order for the authors to really draw\nconclusions here.\nAnswer: We agree that drawing conclusive findings requires adequate sample\nsizes. In the revised manuscript, we have strengthened our initial findings by\nintegrating two independent public datasets, which together provided a\ncombined sample size of n=7 (Extended Data Fig. 1a, b in the revised\nmanuscript). This integrated analysis revealed significant upregulation of Chil1,\nChil3, and Chid1 in CLP-enriched atherosclerotic aortas of mice (Fig. 23a in\nthis letter). Cross-referencing human atherosclerosis databases further\nconfirmed notable upregulation of CHI3L1 and CHI3L2 in human coronary\natherosclerotic plaques (Fig. 23b in this letter). These consistent findings\nacross species guided our focus toward these differentially expressed CLPs.\nFig. 23. CLPs levels in public RNA-seq dataset.\nIn determining our research focus, we considered several factors regarding\nChid1/CHID1:\n Its expression shows notable inter-species heterogeneity between mice\nand humans\n The upregulation observed in murine atherosclerosis was relatively\nmodest compared to other CLPs (only 1.29-fold, whereas Chil1 and\nChil3 are upregulated by 8.78-fold and 4.94-fold, respectively)\n Existing literature coverage of Chid1 in CLP family studies remains\nlimited\n Chil1 (CHI3L1), Chil3, and CHI3L2 are the most widely distributed and\nbest characterized members of the CLP family, whereas research on\nChid1/CHID1 remains extremely limited\n Our primary objective is to prioritize these common CLPs, subsequently\napplying insights analogously to all altered CLP molecules.\nOur study was initially motivated by mouse Chil3, which previous research has\nshown to influence alternative macrophage activation through regulation of lipid\nmetabolism12. This led us to hypothesize that Chil3 might play a role in lipid\nmetabolism disorders such as atherosclerosis. This prediction was\nsubsequently confirmed by our expanded research findings.\nAs Chil3 is mouse-specific, identifying its functional human homolog was\nessential. Analysis of human atherosclerosis transcriptomic data revealed\nsignificant overexpression of both CHI3L1 and CHI3L2 in atherosclerotic\nplaques (Fig. 23b in this letter). Subsequent chromosomal synteny analysis\nand macrophage polarization experiments demonstrated that CHI3L2, rather\nthan CHI3L1, exhibits a functional pattern in macrophage polarization that\nclosely mirrors that of mouse Chil3. This functional distinction is well-supported\nin the literature: both Chil3 and CHI3L2 are established markers of alternatively\nactivated macrophages (AAMs)12,13, while CHI3L1 is predominantly associated\nwith classically activated macrophages (CAMs)14. Therefore, we focused\nspecifically on the functional axis comprising mouse Chil3 and human CHI3L2.\nIndeed, the roles of CLPs in atherosclerosis remain poorly explored. Only a\nhandful of studies have suggested that Chil1 exacerbates atherosclerosis by\ninfluencing macrophage apoptosis, endothelial inflammation, and vascular\nsmooth muscle cell activation63,64. Thus, our identification\nof Chil3 and CHI3L2 represents the first evidence for the involvement of CLP\nfamily members beyond Chil1 in this disease. While we do not rule out a\ncontributory role for CHI3L1, the present work is centered on the human\nhomolog most functionally analogous to Chil3, as determined by genomic and\nfunctional criteria. Future studies will prioritize elucidating the mechanisms of\nother CLPs, such as CHID1, in atherogenesis.\nWe have revised the Discussion section to more accurately articulate\nconclusions regarding Chil1 and Chid1, and have added a discussion of the\nlimitations of the current analysis (Lines 580-592). Future studies will\nsystematically explore the role of other cholesterol lipoprotein binding proteins,\nincluding CHID1, in atherosclerosis development.\n6) Fig 2c. How has plasma from ‘control’ and ‘atherosclerosis’ been defined?\nThis would require a far more extensive description. Also here the x-axis\ncrosses the y-axis at 3.0, with the intention to magnify a small, though\nsignificant, increase. What is the extent of the increase shown in Fig 2c? Why\nwere the data log-transformed?\nAnswer: We appreciate your comments regarding the definition of the plasma\ncohort. In the Methods section (Lines 708-720), we have explicitly stated that\nthe control group was recruited from healthy individuals (n=34; all healthy\nsubjects were excluded from cardiovascular disease, autoimmune diseases,\nacute and chronic infections, malignant tumors, and liver or kidney\ninsufficiency), while the atherosclerosis group comprised patients diagnosed by\nultrasound (n=38). We have supplemented the demographic and clinical\ncharacteristics of both groups (e.g., age, gender, lipid levels) in the Extended\nData Table1 to ensure data transparency (Table 1 in this letter).\nYou correctly noted the log-transformation of the y-axis values. This was a\nnecessary step for the statistical analysis. Specifically, serum CHI3L2 levels\nwere quantified using a highly sensitive chemiluminescence assay, which yields\nraw data in relative light unit (RLU). The distribution of these raw RLU values\nwas strongly right-skewed and did not meet the assumption of normality\nrequired for parametric testing. Log-transformation was therefore applied to\nnormalize the data distribution, enabling the use of a generalized linear model\nadjusted for age, which was a key confounding variable between our control\nand atherosclerosis groups (Table 1 in this letter).\nTable 1. Baseline characteristics of the study participants (plasma\nsamples).\nUnadjusted model Adjusted model*\nHealthy AS Healthy AS\nCharacteristics P-value P-value\n(N=34) (N=38) (N=34) (N=38)\nlgCHI3L2** 3.77 ± 0.22 3.93 ± 0.30 0.012 3.79 ± 0.27 3.92 ± 0.27 0.045\nAge, years 55.29 ± 12.33 64.58 ± 9.63 <0.001 − − −\nMale 25 (73.53) 26 (68.42) 0.634 − − −\nTC, mmol/L 3.90 ± 0.61 3.58 ± 0.94 0.088 − − −\nTG, mmol/L 1.10 ± 0.66 1.18 ± 0.60 0.581 − − −\nHDL-C, mmol/L 1.44 ± 1.07 1.06 ± 0.22 0.035 − − −\nLDL-C, mmol/L 2.36 ± 0.59 2.08 ± 0.91 0.129 − − −\nValues are Mean ± SD or No. (%). AS, atherosclerosis; TC, total cholesterol;\nTG, triglyceride; LDL-C, low-density lipoprotein cholesterol; HDL-C, high-\ndensity lipoprotein cholesterol.\n*Generalized linear model with adjustment for age was constructed to compare\nCHI3L2 level between the groups. **CHI3L2 levels were converted to\nlogarithmic parameters.\nWe acknowledge that the log-transformed difference appears visually small,\nand have revised the figure to better represent the actual, biologically relevant\ndifference (displaying raw RLU values) (Fig. 24 in this letter, Fig. 1h in the\nrevised manuscript). While the difference on the log-scale is modest, it\ncorresponds to a substantial and physiologically significant increase in the raw\nRLU values. Of course, Table 1 still presents log-transformed values, primarily\nto eliminate age-related variations.\nFig. 24. CLPs levels in human plasma.\n7) Fig 2f. Again, the x-axis is not crossing 0. Why making so much of the small\ndifference in ox-LDL? What is the extent of the increase? And what is the extent\nof the increase in LDL in Fig 2e?\nAnswer: We sincerely thank this critical and insightful comment. Upon careful\nre-examination of our data analysis process, we identified a significant error in\nthe construction of the ELISA standard curve. Specifically, the initial use of an\ninappropriate quadratic fitting model led to inaccurate quantification of ox-LDL\nconcentrations, with reported values exceeding the valid range of the standard\ncurve (Fig. 25a-c in this letter). We deeply regret this oversight and sincerely\napologize for the resulting inaccuracies.\nTo thoroughly address this issue, we have reprocessed all raw absorbance data\nand reconstructed the standard curve using a scientifically sound four-\nparameter logistic (4-PL) curve fitting model (Fig. 25d in this letter). All ox-LDL\nconcentrations have been recalculated based on this corrected standard curve,\nensuring the values now fall within the validated measurement range (Fig. 25e\nin this letter).\nFig. 25. Processing of ox-LDL raw data.\nWe have generated a revised version of the Figure with the corrected data (Fig.\n26b in this letter). While the overall trend in concentration changes remains\nconsistent with our initial observations, all numerical values have now been\nproperly calibrated to fall within the established linear range of the standard\ncurve. Furthermore, we have adjusted the x-axis to originate at zero in the new\nFigure to prevent any potential visual misinterpretation.\nFig. 26. Lipid levels in mouse serum.\nBased on the revised data, we can now provide a precise quantification of the\nobserved increases. In male mice (Fig 26b in this letter), serum ox-LDL levels\nin mice expressing Chil3 and CHI3L2 were significantly elevated compared to\nthose lacking CLPs (1.44 ± 1.07 × 10³ ng/mL), reaching concentrations of 3.35\n± 1.66 × 10³ ng/mL and 3.03 ± 1.78 × 10³ ng/mL, respectively. This corresponds\nto a 2.33-fold increase in the Chil3 group and a 2.10-fold increase in the CHI3L2\ngroup. A similar trend was observed in female mice. Relative to the CLP-lacking\ncontrol group (1.10 ± 0.78 × 10³ ng/mL), serum ox-LDL concentrations in\nfemales expressing Chil3 and CHI3L2 showed increases of 64% (1.80 ± 0.76\n× 10³ ng/mL) and 97% (2.17 ± 0.71 × 10³ ng/mL), respectively.\nThe raw LDL-C concentration data are as follows: In male mice, serum LDL-C\nlevels increased by 82% in the Chil3 group (13.89 ± 4.67 mmol/L) and by 51%\nin the CHI3L2 group (11.53 ± 2.92 mmol/L), relative to controls (7.62 ± 3.27\nmmol/L). Similarly, in female mice, serum LDL-C concentrations were elevated\nby 61% in the Chil3 group (10.82 ± 3.39 mmol/L) and by 72% in the CHI3L2\ngroup (11.59 ± 5.72 mmol/L), compared to the control group (6.72 ± 2.82\nmmol/L) (Fig 26a, 27 in this letter).\nFig. 27. Raw data of LDL-C.\nTherefore, we fully concur with your assessment that discussing minor\ndifferences based on the initial and erroneous data was not appropriate.\nHowever, with the implementation of corrected values, the observed increase\nin ox-LDL demonstrates both statistical significance and biological importance.\nThese robust findings provide a solid foundation for the experimental\nconclusions drawn in our subsequent sections. These results are shown in\nFig.2a, b of the revised manuscript.\n8) Fig 2i-j. Too few datapoints have been included. The data in Fig 2g-h suggest\nmore mice were included in these studies; the same number would need to be\nincluded in Fig 2i-j.\nAnswer: We have now included all available animal samples in the study, which\nwere utilized for data generation in Fig. 2f, g in the revised manuscript. The\nupdated Figure is provided as Fig. 18 in this response letter for your reference.\n9) Fig 1a. This is a very strange way to display the data with the x-axis crossing\nthe y-axis at -200. This would need to be changed to 0. If there are values lower\nthan 0 they would need to be adjusted to 0.\nAnswer: We sincerely apologize for the oversight in our original data\npresentation. In fact, our actual dataset contains no negative values (raw data\nprovided in Fig. 28a in this letter). The appearance of a negative range on the\nY-axis was an artifact caused by the default settings of GraphPad Prism during\nviolin plot generation, which we neglected to adjust manually.\nFig. 28. Raw data of Chil3 level.\nTo rectify this issue, we have replaced the original violin plot with a bar chart\nand properly set the Y-axis origin to zero. This presentation format offers a more\naccurate and intuitive visualization, as all measured values are unequivocally\npositive. Furthermore, following your suggestion, we have systematically\nrefitted and reprocessed all ELISA data throughout the manuscript using a 4-\nPL model (raw fitting data shown in Fig. 28b in this letter). The revised Figure\nis presented below (Fig. 29 in this letter) and has been updated accordingly\nin the main text (Fig. 1a in the manuscript).\nFig. 29. The Chil3 level in mouse serum.\n10) Extended Fig 2d. What do the authors mean with a proliferative cell?\nAnswer: In our manuscript, the term \"proliferative cell\" refers to a\ntranscriptional state of cells that are actively progressing through the cell cycle,\nrather than a distinct cellular lineage. This definition is robustly supported by\nour scRNA-seq data. The cluster we annotated as \"proliferative\" is\ncharacterized by a strong and coordinated expression of genes that are\nhallmark indicators of active cell division. The gene list provided by you is\nexemplary of this signature, including:\n Core mitotic regulators: Mki67 (a universal marker for proliferation),\nTop2a, Nusap1, Aspm, Ube2c, and Pttg1.\n Histone genes: Hist1h1b/c/d/e, Hist1h4c (synthesized during DNA\nreplication in S phase).\n Chromatin assembly and structural proteins: Hmgb1/2, Hmgn2, H2afv, Tuba1b, Tubb (critical for chromosome segregation and cytokinesis in M\nphase).\nThe enrichment of this gene set is a well-established signature for cells in the\nG2 and M phases of the cell cycle. We observed this proliferative cluster in\nspatial proximity to T cell-rich regions in human atherosclerotic plaques, which\nmay suggest localized cell division activity within the plaque microenvironment.\n11) Extended Fig 2i. This is a minimal difference. The x-axis should not cross\nat 4.0, but at 0.0; otherwise the authors make too much of a maybe not even\n5% difference, which I do not view as meaningful.\nAnswer: We apologize for any confusion caused by the scaling of the y-axis.\nAs detailed in our response to Comment #6, the raw chemiluminescence data\n(RLU) for CHI3L2 were highly right-skewed. Log-transformation was applied to\nnormalize the distribution prior to conducting the age-adjusted generalized\nlinear model, which is a standard and recommended practice for analyzing such\nnon-normally distributed data. While the difference seems small on the log-\nscale, it corresponds to a substantial and statistically significant increase in the\nraw, untransformed data. The use of a non-zero y-axis intercept was solely\nintended to visualize this statistically significant log-transformed difference\nmore clearly, not to overstate its biological importance.\nUpon careful consideration, we recognized a key distinction between this\ndataset and the human plasma data addressed in Question #6. The human\nplasma analysis required log-transformation and age adjustment due to\nsignificant age differences within the cohort. In contrast, the data in Extended\nFig. 2i represent CHI3L2 expression levels in transgenic mice, which is a\nrelatively homogeneous \"small-sample event.\" For this dataset, a direct\npresentation of the raw data is more appropriate and transparent.\nWe thank you for this comment. We have re-analyzed the data for Extended\nFig. 2i (Extended Data Fig. 1k in the revised manuscript) and agree that the\nlog-transformation was unnecessary in this specific context (Fig. 30 in this\nletter).\nFig. 30. The CHI3L2 level in mouse serum.\n12) Extended Fig 2f and 2h. The number of replicates should at least be\nincreased to n=4. Preferably n=6. N=3 is insufficient. Data on THP-1 cells would\nneed to be complemented by data on PBMCs differentiated in what the authors\nrefer to as AAMs or CAMs; THP-1 cells are already very activated since they are\ndifferentiated into macrophages employing PMA.\nAnswer: We agree with you that increasing the number of biological replicates\nstrengthens statistical confidence. In direct response to this point, we have\nrepeated the key experiments and increased the sample size from n=3 to n=4-\n6 (Fig. 31 in this letter, Extended Data Fig. g-j in the revised manuscript). It\nshould be particularly noted that in the THP-1 cell line experiment (Fig. 31c in\nthis letter), an average value was obtained after each technical repeat well.\nThe current data was repeated three times, and the average values of the six\ntimes were analyzed in combination with the results of the previous three times.\nFig. 31. The CLP level in macrophages.\nWe appreciate your valid point about the activated nature of PMA-differentiated\nTHP-1 macrophages. In this specific part of our study, the experimental goal\nwas not to fully model primary human macrophage biology, but rather to\nperform a controlled, initial functional screening to identify the human CLPs that\nmost closely mirrors the polarization behavior of mouse Chil3. While PMA\ninduces a degree of activation, these cells are widely recognized as resembling\nM0 macrophages, with low expression of canonical M1 or M2 markers\ncompared with fully polarized CAMs/M1 or AAM/M2 cells65-67, making them a\nstandard model for studying macrophage polarization. Consistent with this, our\ndata show that CHI3L2, but not CHI3L1, induces marker expression patterns\nthat faithfully recapitulate the polarization behavior observed for mouse Chil3,\nconfirming the validity of this screening system12-14. In addition, PBMCs also\nneed to differentiate their monocytes into macrophages in vitro using PMA, and\nthen further polarize them into different phenotypes. Of course, we agree with\nyou and have also noted the potential impact of PMA stimulation, but we believe\nit does not affect our conclusion in this study.\n13) Fig 4a. More examples on co-localization would need to be shown. The\nexamples in Fig 4b on co-localization are too unclear to draw conclusions.\nAnswer: Thanks. Specifically, we have now collected a substantial number of\nadditional images confirming the co-localization of CLPs with macrophages in\nmouse atherosclerotic plaques (n=4 biological replicates) (Fig. 32a, b in this\nletter, Fig. 4a and Extended Data Fig. 4a in the revised manuscript). For human\natherosclerotic tissue samples, we have provided enlarged views of the\nimmunofluorescence staining for clearer visualization. White arrows have been\nadded to explicitly indicate the co-localization of CHI3L2 with CD68-positive\nmacrophages (Fig. 32c in this letter, Fig. 4b and Extended Data Fig. 4b in the\nrevised manuscript).\nFig. 32. Colocalization of CLPs and macrophages.\n14) Fig 4b. What is meant by APS and FPS? Again, there is an unclarity here.\nThis makes it hard to read and properly review this manuscript.\nAnswer: The abbreviations APS (atheromatous plaque stage) and FPS\n(fibrous plaque stage) were defined when initially introduced in the manuscript\n(Lines 170-171) and have been maintained consistently thereafter.\n15) Fig 4h. The chlodronate experiment is usually performed as a way to study\nmonocyte infiltration into atherosclerotic plaques. The transferred monocytes\nare labeled with beads, so that homing of monocytes to plaques can be studied.\nSuch an experiment is actually never performed with bone marrow derived\nmacrophages. If performed in this way, macrophages need to be labeled so\ntheir fate can be studied better. It is unclear what the authors have done in Fig\n4h-k. It seems rather to me that they have performed a bone marrow\ntransplantation without performing the irradiation first. If the authors claim that\nthe phenotype is macrophage-driven, they will need to create a macrophage-\nspecific knockout. That is only the proper way that is widely accepted in the\nfield.\nAnswer: In this manuscript, we employed the macrophage adoptive\ntransferring experiment primarily to investigate the role of macrophage-derived\nCLPs in atherosclerosis. Specifically, 8-week-old male recipient ApoE-/-.Chil3∆\nCD45.1 mice were fed a WD for 13 weeks. At the start of weeks 12 and 13, the\nrecipient mice were intraperitoneally injected with clodronate liposomes to\ndeplete macrophages. Macrophage-scavenging agents, such as clodronate\nliposomes (Liposoma BV), are a class of compounds specifically designed to\ndeplete macrophages. Clodronate liposomes are well-established, convenient,\nand cost-effective tools for macrophage depletion across various tissues,\nincluding the liver, spleen, lungs, and blood. After two days of clodronate\nliposome treatment, macrophage depletion is nearly complete. Following this,\nBMDMs from donor CD45.2 (ApoE-/-, ApoE-/-.Chil3∆ and ApoE-\n/-.Chil3∆.hCHI3L2tg) mice were transferred into the recipient mice via tail vein\ninjection to assess the impact of macrophage CLPs on atherosclerosis. The\nsuccessful accumulation of transferred CD45.2 macrophages in the aorta was\ndetected by flow cytometry shown in Extended data Fig. 4f in the revised\nmanuscript. This experimental technique has been validated in various disease\nmodels, including atherosclerosis, non-alcoholic fatty liver disease and\ninflammatory lung diseases12,68,69.\nWe fully agree with your proposal to establish a macrophage-specific\nknockout model. Given the specific expression of Chil3 in macrophages, we\nbelieve our animal model strategy effectively demonstrates the impact of\nmacrophage-derived Chil3 on atherosclerosis. In contrast, CHI3L2 is widely\nexpressed; therefore, to specifically investigate the role of myeloid-derived\nCHI3L2 in atherosclerosis progression, we additionally generated mice with\nmacrophage specific knock in CHI3L2 (M-CHI3L2ki/wt). We established another\nmouse model by injecting an adeno-associated virus-8 carrying a mutant\nPCSK9 gene (AAV8-PCSK9) into M-CHI3L2ki/wt and control mice\n(M-CHI3L2wt/wt). These AAV8-PCSK9 mice were fed a WD for three months,\nafter which plaque burden was assessed (Fig. 33a in this letter). Compared\nto M-CHI3L2wt/wt controls, M-CHI3L2ki/wt mice exhibited more extensive lesions\nthroughout the entire aorta and aortic root, along with increased plaque burden\nand greater plaque instability (Fig. 33b, c in this letter). These new results are\nincluded in the Fig. 4f-h in the revised manuscript.\nFig. 33. Effects of macrophage-specific knock-in CHI3L2 on\natherosclerosis.\n16) Extended Fig. 4f. The flow cytometry plots are insufficient in showing that\nthe transfer with macrophages actually led to accumulation of considerable\namount of macrophages in the aorta. The method needs to be established first.\nAnswer: In our study, CD45.1 recipient mice were intravenously injected with\nCD45.2 BMDMs following macrophage depletion. The presence of CD45.2+\ncells in both circulation and aortic tissue, as detected by flow cytometry,\nprovides direct evidence for the successful recruitment and accumulation of\ntransferred macrophages. The control group (without CD45.2+ BMDM injection)\nshowed no detectable CD45.2+ cells, confirming the specificity of our detection.\nTherefore, the presence of CD45.2+ cells in recipient mice unequivocally\ndemonstrates the successful accumulation of transferred macrophages in the\naorta. This methodology has been previously validated and widely applied in\natherosclerosis research, as referenced in our manuscript68.This result showed\nin Extended data Fig. 4h of the revised manuscript. In addition, as mentioned\nin question 15, we have incorporated the CLP macrophage-specific knock-in\nexperiment to further substantiate the significant role of macrophages in CLP-\nregulated atherosclerosis.\n17) Extended Fig 5a-c. These calculated interactions actually do not tell us that\nmuch. What is exactly happening here and what is the biological nature of these\ninteractions.\nAnswer: Thanks for pointing it out, we basically agree. Our primary aim was to\ninvestigate how CLPs influence macrophages and to identify their key cellular\ntargets within the atherosclerotic aortic environment. Because CLPs are\npredominantly expressed and secreted by macrophages, we explored the\npotential for auto/paracrine signaling. The major novel finding of this study is\nthat CLPs target the macrophage LDL receptor CD36, exerting glycosidase\nactivity to modify CD36 N-linked glycans, regulate CD36-mediated lipid\nendocytosis, and secondarily influence the mTOR-PPARγ-ABCG1 lipid efflux\npathway. We agree that while the CellChat interaction data are informative, they\ndo not yet provide the mechanistic depth expected at this stage. Accordingly,\nwe have removed the corresponding interaction diagrams and signaling\nstrength calculations from the manuscript to better align with the article’s scope\nand focus.\n18) Extended Fig 5d. If the authors really want to draw a conclusion on\nmacrophages, they need to perform laser capture microscopy on\natherosclerotic plaques, or single cell RNASeq, but they cannot do this by\nperforming whole RNA extraction on the aorta.\nAnswer: We thank you for this comment and wish to clarify that the data in\nExtended Data Fig. 5d (Fig. 8f in the revised manuscript) were obtained from\nprimary macrophages treated with ox-LDL in vitro, not from bulk RNA\nsequencing of aortic tissue. Therefore, the conclusions drawn from this figure\nspecifically reflect the inflammatory response of macrophages under controlled\nconditions. We have revised the figure legend and main text to clearly indicate\nthe experimental source to prevent any potential misunderstanding.\nFurthermore, our scRNA-seq analysis of aortic macrophages from\natherosclerotic mice confirmed that CLP expression enhances the transcription\nof key inflammatory factors, including Il1b, Il6, and Tnf (Fig. 34 in this letter).\nFig. 34. The inflammatory factors levels.\n19) Extended Fig 5e. When were these macrophages isolated? It is impossible\nto directly isolate BMDMs; then these cells would be bone marrow monocytes.\nThis would need to be clarified. How was the experiment performed?\nAnswer: The two primary murine macrophage types used in this study, namely\nperitoneal macrophages (PMφ) and BMDMs, are well-established models\nwidely employed in atherosclerosis-related in vitro research3,70,71. Both cell\ntypes were isolated from 6- to 8-week-old genetically modified mice maintained\non a standard chow diet, rather than a Western diet. To evaluate the role of\nCLPs in macrophage lipid accumulation, we stimulated these primary\nmacrophages with ox-LDL to induce foam cell formation in vitro.\nPMφ were obtained following intraperitoneal injection of 2 mL 3% thioglycolate\nbroth. Three days later, mice were euthanized, and peritoneal cells were\nharvested via lavage with 5 mL ice-cold PBS/EDTA and centrifuged at\n1,200 rpm for 10 min at 4 °C. Cells were then lysed with ACK buffer, centrifuged\nagain, and cultured in DMEM supplemented with 10% FBS and 1% PS. After\n6 h, non-adherent cells were removed, and fresh DMEM was added for\nsubsequent experiments.\nBMDMs were generated by flushing the bone marrow cavities of femurs and\ntibias under aseptic conditions, filtering the cell suspension through a 40 μm\nstrainer, centrifuging at 1,200 rpm for 10 min, lysing with ACK buffer, and\nwashing with PBS. Cells were then seeded at 2 ×106 cells/mL in DMEM\ncontaining 10% FBS and macrophage colony-stimulating factors (M-CSF, 10\nng/mL) for 7 days. As detailed in the Methods section (Lines 866-882), BMDMs\nwere generated by differentiating bone marrow cells in vitro for 7 days in\nmedium containing M-CSF. The resulting adherent cells are widely recognized\nand referred to as BMDMs in the literature3,70,71.\n20) Extended Fig 5f-j. Same questions-how were these experiments performed?\nThis needs to be stated in the Figure legends or better described in the main\ntext. If the authors claim more ox-LDL uptake, ideally, they perform mass-spec\non their macrophages to show that free cholesterol and cholesteryl esters are\nincreased. This is the only definitive measure. These MFI measurements tell us\nvery little, and again, in extended Fig 5j, differences are minor for BMDMs. I do\nnot understand the significant p-values. The authors are making too much of\nthese data.\nAnswer: Macrophages were pretreated with varying concentrations of\nrecombinant Chil3 protein for 4 h at 37 °C, followed by incubation with Dil-ox-\nLDL (30 μg/mL) for 2 h. Cells were then detached with 0.25% trypsin-EDTA,\npelleted by centrifugation at 1000 × g for 5 min, washed three times with ice-\ncold PBS, and resuspended for flow cytometry analysis (full details in Methods,\nLines 904-909).\nWhile MS is the gold standard for quantifying intracellular cholesterol, the Dil-\nox-LDL uptake assay measures a distinct but equally important process:\nreceptor-mediated lipoprotein internalization. The mean fluorescence intensity\n(MFI) reflects the total ox-LDL actively internalized and localized inside the cell,\nproviding a direct functional readout of scavenger receptor activity72,73. An\nincrease in Dil-MFI indicates enhanced CD36-dependent endocytosis, the\nupstream event driving lipid accumulation detected by mass spectrometry.\nFollowing your suggestion, we also quantified cellular lipids by MS (Fig. 35 in\nthis letter). These results confirm that CLPs significantly elevate intracellular\nfree cholesterol and cholesteryl esters in macrophages, consistent with\nincreased lipid uptake. These data are included in the ABCG1-related\nmechanism section (Fig. 8c in the revised manuscript), which addresses the\nmetabolic consequences of lipid loading, whereas the current section focuses\non the initial effects of CLPs on lipid uptake.\nFig. 35. Intracellular lipid levels using MS.\nFinally, we clarify the interpretation of our data. Although the MFI differences in\nBMDMs were more modest than those in PMφ, this variation may reflect factors\nsuch as the batch of recombinant Chil3 protein, macrophage lineage, or\nvariations in the Dil-ox-LDL preparation used. Nevertheless, the statistical\nsignificance in BMDMs arises from the low variability and high consistency\nwithin each group, where even small shifts in mean values can yield robust p-\nvalues, reflecting reproducible biological effects.\nImportantly, the enhancing effect of recombinant Chil3 on ox-LDL uptake was\nconsistently observed across two independent primary macrophage types.\nWhile the response was strong and highly significant in PMφ, the same\ndirectional trend, milder but statistically significant, was reproducibly detected\nin BMDMs. This consistency supports the conclusion that Chil3 promotes lipid\nuptake, evidenced by both a pronounced effect in peritoneal macrophages and\na reproducible, statistically supported effect in BMDMs.\n21) Fig 6i-k. Were these intracellular staining? Ideally the authors would\ncomplement these data with mass spec measurements of free cholesterol and\ncholesteryl esters.\nAnswer: We thank your comment, which allows us to clarify the principles of\nthe Dil-ox-LDL flow cytometry assay. This method does not represent a\nconventional intracellular staining protocol, but rather provides a quantitative\nassessment of receptor-mediated internalization. Dil, a lipophilic fluorescent\ndye, is covalently incorporated into the lipid core of ox-LDL. When incubated\nwith macrophages at 37°C, Dil-ox-LDL binds scavenger receptors such as\nCD36 and is subsequently internalized via receptor-mediated endocytosis. The\nfluorescence detected by flow cytometry originates from internalized particles\nwithin endosomal/lysosomal compartments, as membrane-bound ox-LDL is\nremoved by stringent washing. Thus, MFI directly reflects scavenger receptor\nactivity by indicating the extent of ox-LDL uptake3.\nRegarding CD36, literature shows that it contains a large, glycosylated\nextracellular domain that harbors the ligand-binding site74,75. CD36 also exists\nin a soluble form corresponding to its extracellular domain (ECD), generated\nthrough proteolytic cleavage76. In our rescue experiments, we used a\nrecombinant CD36 protein encoded by the mouse or human ECD sequence\n(Gly30–Lys439) with an N-terminal signal peptide and a C-terminal His tag.\nTherefore, rCD36Gly30-Lys439 and rCD36ECD refer to the same protein.\nOur findings suggest that Chil3 and CHI3L2 bind to CD36 and promote its\nendocytosis. To further test this, we performed a competition assay using\nexogenous recombinant CD36 (rCD36ECD). Chil3 or CHI3L2 were pre-\nincubated with species-matched rCD36ECD, allowing the formation of CLP–\nCD36 complexes and reducing the number of free CLPs. This mixture was then\napplied to macrophages, followed by Dil-ox-LDL uptake analysis. Notably, the\ncomplex solution markedly reduced lipid uptake compared with recombinant\nCLPs alone, supporting our conclusion that Chil3 and CHI3L2 compete for\nCD36 binding and modulate lipid uptake. Details are provided in the revised\nmanuscript (Lines 385-401).\nWe also agree that MS quantification of intracellular free cholesterol and\ncholesteryl esters would be an excellent way to evaluate downstream lipid\naccumulation and foam cell formation. These approaches address distinct but\ncomplementary questions: our current data examine the “input” step (uptake),\nwhereas your suggestion examines the “storage” step (accumulation). While\nour results clearly show that CLPs enhance lipid uptake, assessing their impact\non subsequent lipid storage is an important direction for future work. We\nappreciate this valuable suggestion.\n22) Text line 474. The authors imply that Mylip would be involved in the\ncholesterol efflux pathway while it in fact induces the degradation of the Ldl\nreceptor.\nAnswer: We thank you for the insightful comment and for pointing out our\nmisinterpretation of MYLIP function. MYLIP (IDOL) primarily regulates\ncholesterol uptake by promoting LDL receptor ubiquitination and degradation,\nrather than directly affecting ABCA1/ABCG1-mediated cholesterol efflux. We\napologize for this oversight and have accordingly reclassified Mylip under\ncholesterol influx (Fig. 36 in this letter, Extended Data Fig. 8a in the revised\nmanuscript).\nFig. 36. Heatmap of cholesterol-related genes.\n23) Fig 8. It is of interest that mainly Abcg1 is affected in Chil3 deficiency. How\ndo the authors explain that this particular transporter is affected and not Abca1?\nWhat would the mechanism be underlying this finding?\nAnswer: When examining cholesterol-metabolism-related proteins in BMDMs\nof different genotypes (ApoE-/-, ApoE-/-.Chil3∆ and ApoE-/-.Chil3∆.hCHI3L2tg)\nunder ox-LDL stimulation, we found that among the major cholesterol efflux\nreceptors, only ABCG1 was markedly upregulated in CLP-deficient cells,\nwhereas ABCA1 remained unchanged (Fig. 37a in this letter). This pattern\nsuggests that CLPs primarily influence cholesterol handling through the\nABCG1-mediated efflux pathway.\nWe further observed that CLP deficiency significantly increased PPARγ\nexpression in BMDMs under ox-LDL stimulation. While the classical view\nattributes PPARγ-mediated cholesterol efflux mainly to the LXRα-ABCA1 axis42,\ngrowing evidence indicates that PPARγ also suppresses foam-cell formation\nthrough ABCA1-independent mechanisms43. Although both ABCG1 and\nABCA1 are LXRα target genes, their transcriptional sensitivities and responses\nto lipid environments differ substantially. Notably, prior studies have shown that\nPPARγ agonists can reduce foam cell formation without altering LXRα or\nABCA1 expression43. These findings offer a mechanistic explanation for both\nLXR-independent and LXR-dependent regulation of macrophage foam cell\nformation44.\nIn our RT-qPCR analysis, we observed a modest increase in ABCA1 mRNA\nlevels in ApoE-/- or ApoE-/-.Chil3∆ BMDMs compared with their respective\nuntreated controls. However, Western blotting showed no corresponding\nchange in ABCA1 protein expression after ox-LDL stimulation across all\ngenotypes (Fig. 37a in this letter, Extended data Fig. 8b in the revised\nmanuscript). This strongly suggests that in the ox-LDL-stimulated foam cell\nmodel, ABCA1 protein may undergo complex post-translational regulation.\nFor instance, its ubiquitination degradation pathway could be activated, or it might\nbe degraded within lysosomes, leading to accelerated protein turnover and\npreventing net protein accumulation.\nMoreover, LXRα expression remained unchanged across all genetically\nmodified BMDMs (Fig. 37b in this letter), indicating that the classical LXRα–\nABCA1 transcriptional axis was not effectively activated. Several mechanisms\nmay account for this heterogeneity in LXRα and ABCA1 regulation. First, foam\ncells may accumulate high levels of ox-LDL, whose cytotoxic effects can\nsuppress LXRα expression or promoter activity, thereby limiting Abca1 protein\ninduction45. Alternatively, foam cell-like macrophages may become more\nphagocytic, consistent with known dendritic cell characteristics. This could lead\nto phagocytosis of LXRα agonists (ligands)45. These results have been included\nin the revised manuscript as Fig. 8a, Extended Data Fig. 8d and Discussion\nsection (Lines 646-658).\nFig. 37. The expression of cholesterol-related molecules.\n24) Related to this, Abcg1 deficiency does not affect atherosclerotic lesion area\nor decreases it. This does not align with the findings of the authors. Please\nexplain.\nAnswer: We acknowledge that recent studies have reported seemingly\nconflicting roles of macrophage ABCG1 in murine atherosclerosis77,78. However,\nthese investigations assessed ABCG1 deletion as an isolated variable. In\ncontrast, our model reflects a more integrated pathophysiological context:\nABCG1 downregulation is not an initiating event in atherogenesis but a\ndownstream consequence of a potent CLP-induced signaling cascade.\nIn our system, CLPs enhance CD36-dependent lipid uptake, driving substantial\nox-LDL internalization and subsequent mTOR activation. This axis promotes a\npro-inflammatory macrophage state, suppresses PPARγ, and ultimately\nreduces ABCG1 expression. Thus, decreased ABCG1 is one element of a\nbroader pro-atherogenic program.\nIn conventional Abcg1-/- models, lifelong transporter deficiency triggers\nsystemic and cellular compensation, including increased ABCA1 and ApoA1\nexpression, which can mitigate lipid accumulation and lesion progression under\ncertain dietary conditions77. The resulting phenotype therefore reflects both the\nprimary mutation and long-term adaptive remodeling.\nBy contrast, in our setting, ABCG1 downregulation occurs in macrophages\nsimultaneously subjected to overwhelming CD36-driven lipid influx. Under\nthese conditions, residual or compensatory efflux pathways are rapidly\nsaturated, and the loss of ABCG1 synergizes with excessive lipid uptake to\nexacerbate lipid retention, inflammation, and plaque progression.\n25) How would lipid deposition activate mTOR?\nAnswer: Nutrients derived from lysosomal degradation such as sugars, lipids,\nand amino acids are well-established activators of mTOR, particularly the\nmTORC1 complex50-52. In our study, we propose that exogenous lipids,\nespecially ox-LDL, serve as the primary trigger for mTOR activation in\nmacrophages. After internalization, ox-LDL is delivered to lysosomes, where\ncholesterol esters are hydrolyzed by lysosomal acid lipase (LAL), releasing free\ncholesterol and fatty acids46. These liberated lipids, particularly cholesterol, are\nsensed within the lysosomal lumen through the established NPC1/NPC2\ntransport system and the v-ATPase-Ragulator-Rag GTPase axis47-49. Lipid\nsensing induces conformational changes in Rag GTPases that recruit mTORC1\nto the lysosomal membrane, enabling full activation in the presence of\nadditional growth signals such as Akt pathway activity79.\nOur data functionally validate this lysosome-dependent nutrient-sensing model.\nAs shown in the newly added figure (Fig. 38 in this letter), ox-LDL-induced\nmTOR activation was completely abolished by bafilomycin A1 (Baf-A1), which\nblocks lysosomal acidification and function. Any mTOR activation arising from\nnon-lysosomal inputs, such as signaling through surface receptors or upstream\nkinases, would remain intact under Baf-A1 treatment. Thus, our results strongly\nsupport the conclusion that ox-LDL activates mTOR in macrophages through\nthe canonical lysosomal nutrient-sensing machinery. The result has been\nincluded in the revised manuscript as Extended Data Fig. 8e and are discussed\nin the Discussion section (Lines 675-687).\nFig. 38. The effect of Baf-A1 on mTOR.\n26) Line 557 – the authors are discussing ‘inflammatory foamy macrophages’.\nBut the foamy macrophages are generally considered to be anti-inflammatory.\nAnswer: Sorry. We have re-analyzed our scRNA-seq data (Fig. 39 in this\nletter). We identified seven macrophage subpopulations in atherosclerotic\naortas: Ly6C2+ monocyte-like, resident, MHC-II, inflammatory, foamy, VSMC-\nlike, and fibrochondrocyte (FC)-like macrophages. Chil3 was specifically\nenriched in Ly6C2+ monocyte-like macrophages in ApoE-/- mice, whereas\nCHI3L2 was mainly expressed in foam cells, VSMC-like macrophages, and FC-\nlike macrophages in ApoE-/-.Chil3∆.hCHI3L2tg mice. Functionally, Chil3\nincreased inflammatory macrophages and reduced resident macrophages,\nwhile CHI3L2 expanded foam cell/FC-like clusters at the expense of resident\nand MHC-II macrophages (Fig. 39 in this letter). These differences are\nconsistent with the fact that Chil3 and CHI3L2 are not direct orthologs and\nexhibit subtype-specific patterns and consequences. The new results have\nbeen included in the revised manuscript as Fig. 3d-f and Extended Data Fig.\n3b.\nFig. 39. The scRNA-seq data.\nMinor:\n1) Keywords: atherosclerogenesis (also in lines 101 and 110). This is not a\nusual word. It is atherosclerosis or atherogenesis.\nAnswer: Thanks. We have corrected this throughout the manuscript. The term\n“atherosclerogenesis” has been replaced with “atherogenesis” in all relevant\nsections (including lines 101 and 110) to accurately describe plaque formation.\n2) Introduction: line 82 – it probably needs to state: various transformations,\nsuch as those into…\nAnswer: Thanks. We have changed it to “such as those into”.\n3) Also in lines 83-84 it is implied that the switch to other phenotypes accounts\nfor SMCs taking up lipid. This is simply not true. Lipid uptake by SMCs accounts\nfor their differentiation into macrophages for example. Please adjust the text\naccordingly.\nAnswer: Thanks. We have changed it to “These phenotypic transitions, which\ncan be initiated by lipid uptake, enable VSMCs to contribute significantly to\nplaque development through enhanced lipid accumulation and calcification”."}, {"round": 2, "reviewer_comments": {"reviewer_1": "I have carefully reviewed the revised manuscript and the authors’ point-by-point responses. The revisions have adequately\naddressed all previously raised concerns. I have no further comments.", "reviewer_2": "The authors have made a huge work in order to address all the Reviewer’s comments. As a whole, the numerous new data\nand explanation strengthen the main message of this manuscript. Few last points must however be clarified.\n1. As suggested by the Reviewer, the authors performed a lipidomic analysis that clearly helps to decipher the underlying\nmechanisms through which Chil3 modulates lipid metabolism in macrophages. However, non-targeted lipidomic profiling\nmust be detailed in the Methods section. In addition it is not clear how were expressed the lipidomic data in Fig. 8b-c.\nMol of lipids ? % of total lipids ?\n2. Importantly, the following sentence in line 499 must be modified : “The results revealed the loss of CLPs reduced neutral\nlipids (Cholesterol (Chol), cholesteryl esters (CE), saturated fatty acids (SFA)) which was ……..”. Only cholesterol esters\n(CE) are neutral lipids whereas cholesterol (total cholesterol I guess) is a combination of CE and free cholesterol which is a\nhydrophilic lipid (not a neutral lipid). Moreover, SFA are carried out by both neutral lipids such as triglycerides and CE and\namphiphilic lipids such as phospholipids and sphingolipids (not neutral lipids).\n3. Figure 2a. How do the authors explain that LDL-C was reduced but not total cholesterol in Chil3 deficiency mice upon\nwestern diet. In addition, such a reduction of total cholesterol was not compensated by an increased HDL-C.\n4. Extended Figure 2b. This new Figure is not confusing. The Y-axis is labelled as “Change relative to positive control (%)”.\nWhat is the positive control here ? Moreover, why CHI3L2 protein was not quantified before the initial injection ?\n5. Methods section, line 913. This sentence must be corrected. It is written that cells were “treated with serum-free medium\ncontaining HDL (ApoB-depleted serum)”. Is it “serum-free medium” which is depleted in all lipoproteins including HDL or\n“ApoB-depleted serum” which is only depleted in ApoB-containing lipoproteins but not HDL?\nI guess that it is the second\nproposition.\n6. The authors propose that the unexpected downregulation of Ldlr and Hmgcr in Chil3 deficient mice while intracellular\ncholesterol content was reduced could be explained by the increased expression of Mylip (Extended Data Fig. 8a). Was this\nincreased Mylip expression also observed in peritoneal macrophages in Extended Data Fig. 8b ?", "reviewer_3": "This manuscript has improved, but still several points remain.\nMajor:\n1) My first point referred to how LDL-c was measured. The authors are not providing any clarity here. They basically state\nthat they used a Beckman AU5431 analyzer. This analyzer is not used exclusively for cholesterol measurements so\nbasically the authors have not provided a clear response to my comment. Which assay did the authors carry out exactly?\nAlso, the authors cannot simply acknowledge my point that they have measured other apoB containing lipoproteins than\nLDL-C and still call this LDL-C. That is inaccurate. So, please provide an accurate response to my question as to how LDL-\nC is measured and which other lipoproteins are measured concomitantly.\n2) I do not agree with the response given to my question 12. PMA definitely activates monocytes/macrophages. PBMCs\nrequire M-CSF to be differentiated in macrophages. The authors can simply acknowledge the caveat of the THP-1\nmacrophages here.\n3) My problem with the response to point 16 (extended Fig 4f) is that macrophages usually do not circulate in blood.\nMonocytes circulate in blood and differentiate into macrophages upon recruitment into tissues. I also do not agree with the\nauthors that the injection of BMDMs into blood is an established way to study the role of macrophages in atherosclerosis.\nSince the authors have now included the myeloid specific knockout, the manuscript would be improved if the experiment on\nthe in vivo transfer of BMDMs would be omitted.\n4) In response to point 21, the issue is that DiI is in the phospholipid component of oxLDL while the authors claim effects on\ncholesterol (the core of the oxLDL) particle. Again, these data would be strengthened by intracellular cholesterol\nmeasurements.\n5) The response to my question 23 is just merely speculation. The discovery of the role of ABCG1 in cholesterol efflux was\nafter reference 43 was published. It would also suffice to state that the authors cannot really explain this discrepancy. It is\nalso simply not the case that ‘Although both ABCG1 and ABCA1 are LXRα target genes, their transcriptional sensitivities\nand responses to lipid environments differ substantially.’ This statement should not be included in the manuscript. The\nauthors are also making too much of a difference between data on ABCA1 at the mRNA level and assessed via Western\nblot. It is perfectly possible that a slight increase in ABCA1 mRNA does not translate into differences in ABCA1 at the\nprotein level. Please omit any statements addressing this discrepancy from the manuscript. Also the new text in the discussion in\nlines 651-674 is highly speculative and needs to be shortened considerably so that all speculations are removed here and\nonly the data that have been obtained are discussed briefly.\n6) Response to point 24. Again here, this response is not really satisfactory since there is no experimental evidence to\nsupport it. The authors can also simply state that they cannot really explain this and recognize the caveat."}, "author_response": "Response: Thanks for your previous great suggestions. It helps us a lot to\nimprove our work.\nResponse: We thank you for your time and the constructive\nsuggestions. Your input has greatly helped us strengthen our work.\nResponse: We appreciate your insightful comments that strengthened our\nwork."}, {"round": 3, "reviewer_comments": {"reviewer_2": "The authors have clarified the remaining points. I have no further comments.", "reviewer_3": "The authors have addressed my comments."}, "author_response": "Response: Thanks for your previous great suggestions. It helps us a lot to\nimprove our work.\nResponse: We appreciate your insightful comments that strengthened our\nwork."}]

Decision letter