TRIM13 in situ engineering boosts anti-inflammatory capacity of CAR-Ms for liver fibrosis therapy

Domain: chemistry
Published: 2026-02-27
Editorial outcome: major_revision

Abstract
Chimeric antigen receptor macrophages (CAR-Ms) therapy has shown great promise in liver fibrosis, however limited anti-inflammatory capacity of CAR-Ms in the fibrotic foci compromises their anti-fibrotic potency. We here report tripartite motif containing 13 (TRIM13) engineered CAR-Ms for effectively manipulating the anti-inflammatory phenotype of CAR-Ms, augmenting their anti-fibrosis efficacy. Specifically, our efferocytosis-sparked lipid nanoparticles (ESLNPs) efficiently engineered fibrosis-associated macrophages to anti-inflammatory CAR-Ms by co-delivering mRNA encoding TRIM13 and anti-fibroblast activation protein (FAP) CAR respectively. Our data demonstrated these reprogrammed CAR-Ms exhibited a sustained anti-inflammatory phenotype via blocking the mitochondrial DNA (mtDNA)-STING pathway through the overexpression of TRIM13, and showed notable FAP-targeted phagocytosis. Treatment with ESLNPs in male mice with liver fibrosis obviously ameliorated fibrosis through synergizing anti-fibrotic and inflammation-resolution activities, ultimately prompting substantial hepatic function restoration. In sum, our findings established that remodeling and sustaining the anti-inflammatory phenotype of CAR-Ms markedly elevated their therapeutic efficacy in liver fibrosis, benefiting CAR-Ms therapy with broad application in other fibrotic diseases.

Introduction
Liver fibrosis is an aberrant wound-healing response to chronic liver injury, afflicting millions of people worldwide, which ultimately progresses to cirrhosis and lead to lethal clinical complications. Activated hepatic stellate cells (aHSCs) are the primary effector cells of liver fibrogenesis, driving the excessive production of extracellular matrix (ECM) in response to chronic liver damage1,2. Chimeric antigen receptor (CAR) directs immunocytes to specifically track and eradicate target cells, which has shown notable efficacy in anti-tumor and anti-fibrosis therapy. However, elimination of aHSCs and other activated fibroblasts based on CAR cell therapies has only demonstrated modest efficacy at best, as CAR immunocytes fail to resolve chronic local inflammation that drives the continuous activation of HSCs. Furthermore, existing anti-fibrotic CAR cell therapies face a severe caveat about off-target side effects due to systemic circulation of CAR cells and the non-specificity of target antigens3–5. Specifically eliminating pathological aHSCs synergistic with reversing inflammatory milieu is a fruitful novel therapy against liver fibrosis, yet effective strategies are unexplored.

Macrophages are considered as a central hub for maintaining liver homeostasis and act as a “double-edged sword” in liver fibrosis progression. Upon liver injury, Kupffer cells release numerous inflammatory chemokines to recruit the Ly6C high expression (Ly6Chi) bone marrow-derived macrophages (BMDMs) to the injured area6,7, where recruited BMDMs trigger the activation of quiescent hepatic stellate cells (HSCs) via secreting transforming growth factor-β (TGF-β), platelet-derived growth factor (PDGF) and other profibrotic mediators, further exacerbating fibrosis. However, during fibrosis regression, the inflammatory Ly6Chi macrophages switch to anti-inflammatory Ly6C low expression (Ly6Clo) macrophages that attenuate the secretion of inflammatory cytokines to halt fibrosis progression and increase matrix metalloproteinases (MMPs) to degrade the deposited ECM8–11. Given the pervasive infiltration of macrophages in fibrotic liver and their phenotypic plasticity, we hypothesized that in situ transforming fibrosis-benefiting macrophages to anti-inflammatory aHSCs-specific CAR-macrophages (CAR-Ms) would achieve specific elimination of aHSCs and reverse inflammatory fibrosis niche.

cGAS-STING signaling axis detects pathogenic DNA and triggers innate immune responses, and the activation of this pathway drives the polarization of macrophages toward a profibrotic phenotype in the setting of tissue damage12. In fibrotic liver tissue, STING is largely upregulated and activated by massive mitochondrial DNA (mtDNA) released from injured hepatocytes, thereby driving the release of inflammatory cytokines and the activation of HSCs13–15. The homeostasis of STING is tuned by TRIM13 (an E3 ubiquitin ligase) that catalyzes Lys6-linked polyubiquitination and subsequent degradation of STING, and upregulating TRIM13 pushed mtDNA-induced profibrotic macrophages to turn into the anti-inflammatory phenotype accompanied by reduced production of proinflammatory and profibrotic mediators16. Hence, we proposed that downregulating STING in macrophages by TRIM13 overexpression could ameliorate the local inflammatory milieu and present an adjuvant therapeutic strategy for CAR-Ms therapy of liver fibrosis.

Fibroblast activation protein (FAP), a type II transmembrane glycoprotein, is a classical marker of activated fibroblasts and exhibits specific expression in aHSCs throughout the progression of liver fibrosis17,18. However, FAP-positive fibroblasts/aHSCs are also the key effector cells of a normal wound-healing process. The prolonged persistence and extralesional expression of anti-FAP CAR poses a risk of tissue injury or other unintended consequences. To address this substantial problem, we leveraged the power of messenger RNA (mRNA) to develop a transient anti-fibrotic CAR-Ms locoregional engineering strategy. Externalized phosphatidylserine (PS) on the outer leaflet of apoptotic cell membrane is arguably the most emblematic “eat-me” signal for macrophages during efferocytosis and has demonstrated high potency in enhancing targeted uptake of nanomedicines by macrophages19,20. However, prior research indicated that nanoformulations containing PS showed a natural spleen enrichment property due to the negative potential generated from ionization of phosphate and carboxyl groups21,22. Here, we constructed an efferocytosis-sparked lipid nanoparticle (ESLNP) with reactive oxygen species (ROS)-responsive PS exposure property, for locoregionally in situ generation of anti-inflammatory CAR-Ms in fibrotic areas. ESLNP-engineered anti-fibrotic CAR-Ms redirected phagocytic function toward FAP-positive aHSCs and sustained an anti-inflammatory phenotype, which ultimately thwarted fibrosis progression and promoted liver reestablishment. Arguably, our work presents a potential therapeutic approach for liver fibrosis and other inflammation-based fibrosis diseases, which warrants further assessment in clinical trials.

Results
Design and characterization of efferocytosis-sparked LNP
To achieve efficient delivery of mRNA drugs for exerting therapeutic activity, we first designed a class of novel ionizable lipid, termed as PnT14, which was created by reacting amine head and alkyl tails with Tris(hydroxymethyl) aminomethane (TRIS) by simple ammonolysis and esterification reactions (Fig. 1A and Supplementary Fig. S1). Their chemical structures were confirmed with 1H and 13C nuclear magnetic resonance (NMR) (Supplementary Fig. S2). We then synthesized PST (a helper lipid) by conjugating thiazolidine-2-one to PS, a key “eat me” signal displayed on the surface of apoptotic cells (Fig. 1B and Supplementary Figs. S3, 4). This strategic modification endowed PS with the capability of responsive exposure in ROS-enriched fibrotic regions, thus assuring targeted delivery of mRNA drugs to fibrosis-associated macrophages and avoiding spleen enrichment caused by negative charges23. To maximize LNP potency, a series of LNPs were prepared by mixing the ethanol phase containing PnT14, PST, cholesterol, and DMG-PEG with the aqueous phase containing enhanced green fluorescent protein (EGFP) mRNA via a microfluidic device following an orthogonal experimental method (Fig. 1C and Supplementary Table S1)24. The mRNA delivery efficiency of these LNPs in macrophages was evaluated via cytometry analysis, with the optimal formulation at a molar ratio of P3T14:cholesterol:DMG-PEG:PST = 25:35:5:40 (Fig. 1D).

We further characterized the size, morphology, surface charges and mRNA delivery efficiency of the optimal LNPs in PBS and ROS-simulated environment with 0.1 mM H2O2. The H2O2-treated LNPs exhibited an increased average size of 207.5 nm, compared to 163.9 nm for the PBS group, with both maintaining a polydispersity index (PDI) below 0.3 and displaying a uniform spherical morphology under transmission electron microscopy (TEM) (Fig. 1E, F). The mRNA encapsulation efficiency in the H2O2 group was 88.19 ± 1.62%, which was not statistically different from that in the PBS group (91.69 ± 3.10%) (Fig. 1G). In contrast, the zeta potential of LNPs dropped to −4.93 ± 1.45 mV in the H2O2 group compared with −1.81 ± 0.30 mV in the PBS group, while no similar trend was observed in LNPs with DOPE as a helper lipid (Fig. 1H). Further results substantiated the excellent stability in particle size, zeta potential and encapsulation efficiency of optimal LNPs (Supplementary Fig. S5). Subsequently, we quantified the PS exposure rate of ESLNPs via fluorescein isothiocyanate (FITC) conjugated-Annexin V that is specifically bound to PS. As depicted in Fig. 1I and Supplementary Fig. S6A, a remarkably increased FITC-positive proportion (60.33 ± 4.85%) was detected in the H2O2 group compared with merely 6.28 ± 1.23% in the PBS group, confirming the ROS-responsive deshielding and PS presentation of ESLNP. To explain the influence of PS exposure on mRNA delivery efficiency, we detected EGFP expression in macrophages incubated with ESLNPs in PBS or H2O2. The EGFP-positive cells percentage was 71.37 ± 2.06% in the H2O2 group, and for PBS group, the ratio was 64.80 ± 0.92% (Fig. 1J and Supplementary Fig. S6B). These data provided direct evidence that our ESLNPs specifically exposed PS under ROS stimulation, thus facilitating endocytosis by macrophages and in turn leading to increased transfection efficiency in the ROS environment.

We next utilized confocal laser scanning microscopy (CLSM) to visualize the cellular uptake and intracellular trafficking of mRNA in BMDMs and RAW 264.7 cells (Supplementary Fig. S7). As shown in Fig. 1K and Supplementary Fig. S8A, abundant carboxyfluorescein (FAM)-labeled mRNA signal was detected in the ESLNPs groups and substantial enhancement in mRNA signal intensity was achieved with the addition of H2O2, indicating the pro-internalization function of the exposed PS motif. With increased incubation time, the reduced colocalization of mRNA and lysosomes, along with wider mRNA distribution in the cytoplasm indicated the successful escape of ESLNPs from lysosomes (Fig. 1L and Supplementary Fig. S8B). Additionally, no discernible cytotoxic effect was observed on macrophages incubated with escalating doses of ESLNPs (Supplementary Fig. S9). Hence, ESLNPs demonstrated strong potential for broad application in mRNA therapeutics, offering excellent delivery efficiency and safety.

In vitro generation of anti-inflammatory aHSCs-specific CAR-Ms
We designed the sequences of TRIM13 mRNA and anti-FAP CAR mRNA that comprised the CD8α leader, hinge and transmembrane domains, the CD3ζ endodomain, the anti-FAP scFv (single-chain fragment variable) domain and a Myc-tag (Fig. 2A, Supplementary Fig. S10 and Supplementary Table S2)25. The CAR and TRIM13 expression in RAW 264.7 cells and BMDMs were assessed 12 h post-incubation with distinct formulations. As depicted in Fig. 2B and Supplementary Fig. S11 approximately 38.27 ± 1.19% of RAW 264.7 and 31.10 ± 2.51% of BMDMs were engineered with anti-FAP CAR in the CAR mRNA-laden ESLNP (C@ESLNP) group, which was further confirmed by the colocalization of Myc-labeled CAR and cell membrane in confocal images of RAW 264.7 cells treated with C@ESLNP (Fig. 2C). Western blot analysis demonstrated that TRIM13 protein was markedly upregulated in the T@ESLNP group (ESLNPs encapsulating TRIM13 mRNA), while STING protein levels and the phosphorylation of TBK1 and IRF3 were substantially reduced. In contrast, no discernible changes were observed following treatment with free TRIM13 mRNA or s@ESLNPs (ESLNP encapsulating scramble mRNA) (Fig. 2D). Moreover, TBK1 phosphorylation is reported to promote NF-κB activation, leading us to infer that TRIM13 might likewise suppress NF-κB pathway26,27. We then utilized flow cytometry to assess the expression level of IκBɑ, an essential regulator of NF-κB signaling, and found that the upregulation of TRIM13 partially rescued the downregulated IκBɑ level resulted by mtDNA, further supporting TRIM13’s inhibitory role in NF-κB pathway activation (Supplementary Fig. S12). These data provided direct evidence for the successful construction of anti-inflammatory CAR-Ms mediated by mRNA-laden LNPs.

Subsequently, we evaluated the anti-fibrotic activity of the engineered macrophages with anti-FAP CAR and TRIM13 in vitro. Beads anchored with FAP were first employed for phagocytic performance determination, and macrophages with CAR modification demonstrated enhanced phagocytosis toward FAP beads (Supplementary Fig. S13). Subsequently, we extracted the primary HSCs and established FAP-positive aHSC cell model by incubating mouse HSCs with TGF-β (10 ng/mL) for the following phagocytosis evaluation. The disparity in FAP expression between aHSCs and quiescent HSCs was verified by confocal images (Supplementary Fig. S14)17. Following a 12 h co culture of differently treated macrophages with quiescent HSCs or aHSCs, the percentages of macrophages that engulfed aHSCs in C@ESLNP and CT@ESLNP groups showed a notable elevation compared with the control group, indicating the phagocytic capacity of CAR-Ms (Fig. 2E, F). The targeting phagocytic property of CAR-Ms was also confirmed by the markedly increased fluorescence signal detected in CAR-Ms that was coincubated with aHSCs, whereas negligible fluorescence was observed in all experimental groups coculturing macrophages with quiescent HSCs (Fig. 2G). Meanwhile, we coincubated macrophages with apoptotic cells to investigate whether the PS exposure of ESLNP would affect macrophages’ normal ability to cleanup apoptotic cells. Confocal imaging revealed that ESLNP had no discernable effect on macrophage phagocytosis of apoptotic cells (Supplementary Fig. S15). Accordingly, CAR modification in macrophages initiated and redirected phagocytotic activity against FAP+ aHSCs, with exhibited excellent efficacy and safety, providing support for further application of this approach for macrophage programming to eliminate fibrotic pathological cells in situ.

We next assess the phenotypic switch of macrophages after reprogramming with CAR and TRIM13. We detected Ly6C expression on BMDMs by flow cytometry and identified a change in the proportion of Ly6Chi and Ly6Clo macrophages following various treatments (Fig. 2H, I). Specifically, the percentage of Ly6Chi macrophages dropped sharply to 45.97 ± 4.13% in the T@ESLNP group compared to 81.40 ± 5.00% in the PBS group, corresponding with the Ly6Clo macrophages proportion increasing remarkably to 54.03 ± 4.13%, 3-fold higher than that in the PBS group. A similar trend was also observed in macrophages with synergistic treatment of CT@ESLNP. The culture medium was then collected for ELISA analysis. As shown in Fig. 2J, the levels of inflammatory factors, including TNFα, IL-1β, IFN-γ, and IL-6, revealed a noteworthy reduction after T@ESLNP and combination treatment. The secretion of TGF-β, a crucial cytokine promoting the activation of HSCs, was considerably downregulated in the T@ESLNP and combination treatment groups. We subsequently established a macrophage-fibroblast co-culture model to assess the regulatory impact of TRIM13 on macrophage inflammatory responses in the fibrotic milieu. The results showed that co-culturing led to a significant increase in the proportion of pro-inflammatory macrophages, whereas co-expression of TRIM13 downregulated this proportion, further demonstrating that TRIM13 restrains the fibrotic microenvironment-driven polarization of macrophages toward pro-inflammatory phenotype (Supplementary Fig. S16). These findings sufficiently certified that TRIM13 overexpression drove the anti-inflammatory phenotype transformation of macrophages for reprogramming the fibrosis microenvironment. Together, the ESLNP-mediated combination of TRIM13 overexpression and CAR engineering in macrophages synergistically facilitated the precise elimination of aHSCs and enhanced anti-inflammatory function, thereby arousing a more robust anti-fibrotic effect.

In vivo macrophage reprogramming mediated by systemically injected ESLNP
In fibrotic livers, injured hepatocytes release ROS and ROS-responsive drug delivery has been reported as a feasible approach to target fibrotic liver28,29. As illustrated in Fig. 3A, ESLNPs expose phosphatidylserine (PS) upon passage through ROS-enriched fibrotic regions, enabling selective binding to PS receptors on macrophages and facilitating the in situ engineering of fibrosis-associated macrophages. To evaluate the specificity and efficiency of ESLNPs for in vivo delivery, a hepatic fibrosis model was established through intraperitoneal administration of CCl4. Subsequently, three lipid nanoparticles (LNPs) were formulated with DOPE, PST, and PS as helper lipids, respectively. The systemic safety of these LNPs was assessed via a hemolytic assay. All three LNP formulations demonstrated excellent biocompatibility, showing no hemolytic risk even at concentrations far exceeding the therapeutic dose, thereby confirming the feasibility of LNP-based investigations in vivo (Supplementary Fig. S17). These LNPs encapsulating luciferase (Luci) mRNA were intravenously administrated to both CCl4-induced fibrosis mice and olive oil-treated mice that were a non-fibrotic mouse model30. Mice from different treatment groups were imaged 24 h post-dosing using the in vivo imaging system (IVIS) spectrum. As expected, both the fibrotic mice and the control mice exhibited pronounced bioluminescent signal intensity (BLI) in the liver following the injection of ESLNP and DOPE-LNP. In contrast, administration of PS-LNP resulted in a marked enrichment in the spleen, which was consistent with the prior investigations (Fig. 3B–D)22. Moreover, compared with the control mice, BLI intensity was elevated in the fibrotic mice treated with ESLNP, yet no discernable difference between two mice models following DOPE-LNP injection. Next, we administered DOPE-LNPs and ESLNPs encapsulating Myc-tagged CAR mRNA to evaluate their cellular tropisms. Flow cytometry analysis revealed that ESLNP predominantly transduced liver macrophages, accounting for 81.66 ± 2.28% of Myc-positive cells, and that the proportion presented remarkable elevations in the fibrotic mice treated with ESLNP compared to both the ESLNP-treated control mice and DOPE-LNP treatment groups (Fig. 3E, F). These data highlighted that ESLNP with the property of exposing the original structure of PS in the ROS-abundant fibrotic milieu, exhibited excellent macrophage-targeted reprogramming capacity and biocompatibility in vivo, supporting the potential for translating this platform into fibrosis treatment.

Furthermore, we explored the expression kinetics of mRNA drug via detecting BLI at multiple time points post a single administration of ESLNP. As depicted in Fig. 3G, H, the BLI disappeared on day 5, which indicated a longstanding protein expression after ESLNP treatment and provided instruction for the design of therapeutics schedules and pharmacodynamic assessment experiments.

Therapeutic efficacy of ESLNP in liver fibrosis mice
The therapeutic efficacy of ESLNP encapsulating different mRNA drugs was comprehensively investigated in a CCl4-induced liver fibrosis mouse model. As shown in Fig. 4A, after four weeks of CCl4 administration, the mice were randomly divided into four groups and treated with various therapeutic modalities. The group without CCl4 administration was treated with PBS as the sham control. We first utilized immunofluorescence to test ESLNP-enabled in vivo expression of therapeutic proteins and their direct effect. Following administration of C@ESLNPs, the colocalization of Myc and F4/80 in the immunofluorescence images indicated the successful expression of Myc-labeled CAR in macrophages (Fig. 4B). The immunofluorescence images also revealed the colocalization of anti-FAP CAR and FAP, indicating the phagocytosis of aHSCs by engineered CAR-Ms, which was further confirmed by the notable reduction of FAP signals after C@ESLNP treatments (Fig. 4C and Supplementary Fig. S18). As to T@ESLNPs, the immunofluorescence imaging further substantiated a reduction in the colocalization of STING and F4/80, indicating a diminished expression of STING in macrophages (Fig. 4D, E). These results collectively confirmed the therapeutic potential of ESLNPs in targeting and reprogramming macrophages for antifibrotic treatment.

To assess the anti-fibrotic efficacy, the mice were sacrificed two weeks post-treatment, and liver tissue and blood samples were collected for subsequent analyses. Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) are well-established markers of hepatic injury, and the observed decline in their serum levels signifies restoration of liver function (Fig. 4F). Subsequently, hematoxylin and eosin (H&E), Masson, Sirius Red, and α-smooth muscle actin (α-SMA) staining were applied to liver slices to further evaluate antifibrotic efficacy. H&E staining results revealed that hepatocytes in the combinatory treatment group were well-aligned and structurally intact. Furthermore, mice in the combinatory treatment group exhibited a significant decrease in collagen deposition and α-SMA expression. The average percentages of positive area in Masson, Sirius Red, and α-SMA staining decreased about 8.52-fold, 4.99-fold, and 6.05-fold respectively in the combinatory treatment group than that in the PBS group (Fig. 4G–J). Quantitative PCR (qPCR) was also performed to assess the mRNA levels of Col1a1, Col2a1 and TIMP-1 in liver tissues from each group (Supplementary Table S3). Notably, the CT@ESLNP treatment elicited a pronounced downregulation of these fibrosis-related genes (Fig. 4K). The reduction of hydroxyproline (HYP) in harvested liver tissue further confirmed the potent collagen-degrading efficacy of CT@ESLNP treatment (Fig. 4L) Scanning electron microscopy (SEM) images revealed an increased proportion of thick fibril bundles in the decellularized liver ECM following CCl4 treatment. However, after CT@ESLNP treatment, the ECM exhibited a relatively finer and more uniform structure, resembling the fibrillar architecture found in healthy liver ECM. (Fig. 4M). Accordingly, anti-inflammatory aHSCs-specific CAR-Ms demonstrated exceptional efficacy in eliminating pathogenic aHSCs, accelerating the degradation of deposited collagen, halting fibrosis progression and thereby promoting fibrosis resolution.

Subsequently, we sought to assess the effect of macrophage reprogramming on liver function restoration. In Ki67 immunostaining sections, enhanced proliferation was detected in the livers of all treated mice, and the combinatory treatment promoted the most robust cell proliferation and liver regeneration (Fig. 4N, O). Furthermore, all treatment groups reversed the body weight loss that appeared in the PBS group, demonstrating the normalization of liver function in treated mice (Supplementary Fig. S19). Moreover, we constructed a high-fat, methionine-choline-deficient (HFMCD)-induced model to comprehensively test the therapeutic efficacy of ESLNP, which mimicked metabolism-associated liver fibrosis characterized by hepatocellular injury, lipid accumulation, inflammation, and progressive fibrosis. After 2 weeks of treatment, liver tissues were harvested for histological examination and HYP quantification (Fig. 4P). CT@ESLNP treatment reduced both microvesicular and macrovesicular steatosis in H&E-stained sections, significantly decreased Masson staining, and lowered the positive areas of Sirius Red and α-SMA staining (Fig. 4Q–T). Meanwhile, hepatic HYP levels were markedly decreased in CT@ESLNP-treated mice (Fig. 4U). These data collectively indicated the powerful therapeutic efficacy of ESLNP-edited anti-inflammatory CAR-Ms, which could promote fibrosis resolution and liver regeneration.

We further evaluated the biosafety of diverse treatment strategies in vivo. No conspicuous histopathological abnormality was detected in major organ sections of mice subjected to various treatments (Supplementary Fig. S20). Furthermore, there was no significant difference in serum kinase levels between the PBS group and ESLNPs-treated groups (Supplementary Fig. S21). These findings underscore the biocompatibility of ESLNPs and the biosafety of anti-fibrosis therapies. Taken together, the in situ macrophage engineering strategy with CT@ESLNP exhibited a commendable safety profile and proved marked efficacy in reversing hepatic fibrosis while improving liver function.

Anti-fibrotic immunity elicited by ESLNP in vivo
We next investigated the immunomodulatory potency of ESLNP-reprogrammed macrophages in liver fibrosis mice and sought to elucidate the specific mechanism of anti-fibrotic immune responses. Mice were euthanized after different treatments, and the liver tissues and peripheral blood were collected for flow cytometry analysis, immunohistochemical staining and ELISA, respectively. As depicted in Fig. 5A–C, mice in three treatment groups showed a notable switch of macrophages from profibrotic phenotype to anti-fibrotic phenotype, as evidenced by a decreased proportion of Ly6Chi/CD86+ macrophages and an increased proportion of Ly6Clow/CD206+ macrophages compared with that of the PBS-treated mice, signifying that both TRIM13 overexpression and CAR-mediated phagocytosis against aHSCs could induce macrophage polarization. Furthermore, the CT@ESLNP group exhibited an even more pronounced shift to anti-inflammatory phenotype, underscoring that synergizing TRIM13 overexpression and CAR modification could further reverse the inflammatory milieu. Furthermore, reduced serum levels of inflammatory factors, including TNF-α, IFN-γ, IL-6, and IL-1β, were detected in the CT@ESLNP-treated mice compared to that of PBS-treated mice (Fig. 5D). Therefore, CT@ESLNP treatment could effectively induce macrophage polarization towards an anti-inflammatory phenotype in the fibrotic liver of mice and thereby mitigate hepatic inflammation.

The immune cell composition in the liver following different treatments was further investigated through flow cytometry and histopathological immunostaining (Fig. 5E and Supplementary Figs. S22–25). As shown in Fig. 5E, F and Supplementary Fig. S22A, both C@ESLNP and CT@ESLNP treatments resulted in a substantial increase of neutrophils, with flow cytometry results showing 1.80-fold and 1.77-fold higher than that in the PBS group, respectively. Immunofluorescence images revealed a significant increase of hepatic dendritic cells (DCs) in the C@ESLNP and CT@ESLNP treatment groups (Fig. 5E, G), and flow cytometry results further demonstrated that C@ESLNP and CT@ESLNP effectively induced DCs maturation (Supplementary Fig. S22B). Considering the established role of macrophages as antigen-presenting cells, we next assessed the ability of CAR-Ms to recruit T cells. Elevation in the T cell infiltration was observed in mice treated with C@ESLNP and CT@ESLNP, with CD3+ cells increasing to 37.13 ± 1.56% and 33.50 ± 1.59% in the C@ESLNP and CT@ESLNP groups (Supplementary Fig. S22C), and the quantitative analysis of immunofluorescence images also revealed a significant increase in CD4+ and CD8+ T-cell infiltration (Fig. 5E, H, I). These results collectively suggested that the phagocytosis of aHSCs by CAR-Ms triggered the adaptive immune responses, promoted immunocytes infiltration and subsequent activation in the fibrosis foci for eliciting a robust anti-fibrotic immunity. Overall, the combinatory therapeutic approach, integrating anti-inflammatory phenotype-modulating strategy with CAR-Ms therapy, effectively reversed the inflammatory fibrotic niche and activated an adaptive anti-fibrotic cascade, terminating fibrosis progression and accelerating fibrosis resolution, ultimately promoting hepatic restoration.

ScRNAseq analyses of CT@ESLNP therapy
To further elucidate the anti-fibrotic mechanism of CT@ESLNP, we performed single-cell sequencing of liver tissues from mice treated with PBS and CT@ESLNP (Fig. 6A). After quality control, 21,580 cells were collected for further analysis. According to the classic markers of various cell types, 10 clusters, namely B cells, conventional dendritic cells (cDCs), endothelial cells, macrophages, monocytes, neutrophil cells, NK cells, plasmacytoid dendritic cells (pDCs), plasma cells and T cells, were identified (Fig. 6B and Supplementary Fig. S26A). Subsequently, we focused on the distribution of relevant cell subtypes in the myeloid cell population (Fig. 6C and Supplementary Fig. S26B). Among these clusters, the proportion of Ly6Chi cluster cells decreased in CT@ESLNP groups while the Ly6Clo cluster cells increased (Fig. 6D). We further scored a macrophage gene signature and found that CT@ESLNP treatment markedly decreased expression of genes associated with a pro-inflammatory phenotype, including Ccl3, Cxcl2 and Mrc1 (Supplementary Fig. S26C). Additionally, we evaluated alterations in macrophage phagocytic capacity induced by in situ generated anti-inflammatory CAR-Ms. Analyses of the phagocytosis-related genes, including Axl, Mertk, Fcgr1, and Cd5l, demonstrated that macrophages in the CT@ESLNP group exhibited substantially augmented phagocytosis relative to PBS-treated group. The reinforced phagocytic capacity of macrophages facilitated the clearance of aHSCs and apoptotic cells, thereby promoting pronounced mitigation of liver fibrosis progression (Fig. 6E).

In this study, we designed efferocytosis-sparked lipid nanoparticles (ESLNPs) by incorporating an ROS-responsive shielding group that concealed the negative charge of PS to avoid splenic sequestration yet exposed PS in the ROS-enriched fibrotic tissues, thereby enabling selective delivery of dual mRNAs encoding anti-FAP CAR and TRIM13 into fibrosis-associated macrophages and in situ generation of anti-inflammatory CAR-Ms. The engineered macrophages specifically eliminate aHSCs and resolve chronic hepatic inflammation, synergically arrests fibrosis progression and promotes liver recovery (Fig. 6F). These findings highlight the translational potential of this antifibrotic immunotherapy and underscore the need for its evaluation in other fibrotic diseases.

Methods
Materials
Fumaryl chloride, cholesterol, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DOPE) and 1,2-dimyristoyl-rac-glycero-3-methoxypolyethyleneglycol-2000 (DMG-PEG) were bought from Shanghai Macklin Biochemical Co., Ltd. 2,4,6-triisopropylbenzenesulfonyl chloride (TPSCL), 2H-3,4-dihydropyran, tert-butyldimethylsilyl chloride, Glyoxaline, 4-Methyl-1-piperazineacetic acid, 4-Methyl-1-piperazinepropanoic acid, 4-Methyl-1-piperazinebutanoic acid, Pyridin-1-ium 4-methylbenzenesulfonate (PPTS), tetradecanoic acid, N-Boc L-Ser, thiazolidine-2-one, 2-(2-Iminoimidazolidin-1-yl) acetic acid, (4-methylpiperazin-1-yl) acetic acid, 4-methyl-1-piperazinebutanoic acid, olive oil and carbon tetrachloride were purchased from Meryer, Bidepharm Co., Ltd and Mreda Technology Inc. Sodium (R)-2,3-bis(stearoyloxy) propyl hydrogenphosphate (DSPA) were purchased from AVT (Shanghai) Pharmaceutical Tech Co., Ltd. Recombinant murine granulocyte-macrophage colony-stimulating factor (GM-CSF) and TGF-β was purchased from PeproTech Inc and Solarbio. Cell Counting Kit-8 (CCK8), bicinchoninic acid (BCA) assay kit, and cell plasma membrane staining kit with DiD were provided by Beyotime. LysoTracker Red was provided by Dalian Meilun Biotech Co., Ltd. EGFP plasmids, anti-FAP CAR plasmids, TRIM13 plasmids were purchased from Shanghai GeneBiogist Co., Ltd. The Quant-iT Ribo-Green RNA Assay Kit was purchased from Thermo Fisher Scientific. The Hydroxyproline (HYP) content assay kit and Red Blood Cell Lysis Buffer were provided by Solarbio.

The antibodies used in this study were listed in Supplementary Table S4. Cell culture media and consumables were procured from GibcoBRL and CORNING. Cell-Tracker Red CMTPX and Cell-Tracker Green CMFPA were provided by Shanghai Maokang bio. The pHrodoTM Red was purchased from Thermo Fisher Scientific. Plasmid DNA was prepared using Plasmid Mini Kit Plus (Tsingke). In vitro transcription was performed with a T7 High Yield RNA Transcription Kit, and the resulting transcripts were capped using a Cap1 Capping System (both from Novoprotein). The ELISA kits were purchased from Wuhan Servicebio Technology Co., Ltd.

Cells
The RAW 264.7 cells were purchased from the National Collection of Authenticated Cell Cultures, Chinese Academy of Science (SCSP-5036). RAW 264.7 cells were cultured in DMEM (Gibco) containing 10% fetal bovine serum (HyClone), 1% penicillin/streptomycin (Gibco) and a 5% CO2 atmosphere at 37°C. Bone marrow-derived macrophages (BMDMs) were isolated from mice femurs and cultured in DMEM (Gibco) containing 10% fetal bovine serum (HyClone), 1% penicillin/streptomycin (Gibco) and 10 ng/ml GM-CSF (PeproTech) in a 5% CO2 atmosphere at 37°C. AML12 cells were purchased from National Collection of Authenticated Cell Cultures, Chinese Academy of Science (SCSP-550). AML12 cells were cultured in DMEM-F12 containing 10% fetal bovine serum, 0.5% ITS-G (100×), 40 ng/mL Dexamethasone, and 1% Penicillin/Streptomycin. Primary HSCs were isolated by differential centrifugation. Briefly, the mouse was sterilized and the liver was exposed, then perfused via the portal vein with 30 mL of calcium- and magnesium-free Hank’s solution followed by 1 mg/mL type IV collagenase (3 mL/min). The liver was excised, minced, and digested in a 37°C oscillating water bath for 30 min. The cell suspension was filtered through a 100-μm nylon mesh, washed, and centrifuged twice. HSCs were isolated by continuous 60% Percoll density gradient centrifugation (4°C, 800 × g, 20 min). HSCs were cultured in DMEM-F12 (Gibco) containing 10% fetal bovine serum (HyClone) and 1% penicillin/streptomycin (Gibco) in a 5% CO2 atmosphere at 37°C46.

Animals
Male C57BL/6J mice (6–8 weeks) were obtained from an accredited vendor (Ji’nan Pengyue Laboratory Animal Breeding Co., Ltd.). All animal procedures were reviewed and approved by the Institutional Laboratory Animal Ethical and Welfare Committee (Cheeloo College of Medicine, Shandong University) and conducted in accordance with institutional guidelines. For the CCl4-induced liver fibrosis model, mice were treated with intraperitoneal injection of a CCl4/olive oil (1/4, v/v) mixture at a dose of 2 μL/g twice a week for 6 weeks, with treatment commencing after the first 4 weeks of CCl4 administration. Male C57BL/6J mice (6–8 weeks) were fed a HFMCD diet (A06071301B, Research Diets) to establish the HFMCD model, while control mice received a normal diet (D11112201, Research Diets) under identical housing conditions. After model establishment, treatment was administered following the same protocol as used in the original CCl4-induced fibrosis model. Therapeutic outcomes were evaluated by histological analysis of liver sections and quantification of HYP content.

Synthesis and characterization of PnT14 and PST
For the synthesis of PnT14, P1T14 serves as an illustrative example. To a mixture of tert-butyldimethylsilyl chloride (44.6 mmol), imidazole (92.9 mmol), and TRIS (12.4 mmol), 5 mL of anhydrous N, N-dimethylformamide was added, and the reaction proceeded at room temperature for 12 h. The reaction mixture was then diluted with water and extracted with dichloromethane (CH2Cl2). The organic phase was separated, dried over anhydrous sodium sulfate (Na2SO4), and concentrated under reduced pressure. The resulting crude product was purified by silica gel column chromatography (ether/ethyl acetate), yielding the TRIS-TBS (94.21% yield).

For the synthesis of P1TBS, a mixture of 4-methyl-1-piperazineacetic acid (6.32 mmol) and dichloromethane (10 mL) was treated dropwise with 7.200 mL of triethylamine under stirring in an ice bath for 10 min. Subsequently, 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride (9.39 mmol) and 1-hydroxybenzotriazole (9.32 mmol) were added, and the reaction was carried out for 30 min. The TRIS-TBS (6.47 mmol) was dissolved in dichloromethane and added dropwise to the reaction mixture, which was then stirred for 24 h. The organic phase was diluted with saturated sodium bicarbonate (NaHCO3) and extracted with CH2Cl2. After drying with anhydrous Na2SO4, the solvent was removed under reduced pressure. The yellow oil was purified by silica gel column chromatography (dichloromethane/methanol), yielding the P1TBS (74.11% yield).

To a solution of 2.73 mmol of TBAF·3H2O dissolved in tetrahydrofuran (THF), the solution was added dropwise to a mixture of P1TBS (0.828 mmol) and 10 mL of THF under an ice bath. The reaction proceeded for 3 h and then removed the solvent under reduced pressure to obtain a light yellow oil. 9.39 mmol of 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride, 9.32 mmol of 1-hydroxybenzotriazole, and 3 mmol of tetradecanoic acid were added to this system, and the reaction was carried out for 24 h. The organic phase was diluted with saturated NaHCO3 and extracted with CH2Cl2. After drying with anhydrous Na2SO4, the solvent was removed under reduced pressure, and the product was purified by silica gel column chromatography (dichloromethane/methanol), yielding a yellow oily material P1T14 (24.22% yield).

For the synthesis of PST. N-Boc L-Ser (146 mmol), PPTS (146 mmol), and 2H-3,4-dihydropyran (146 mmol) were dissolved in 100 ml of CH2Cl2 and reacted at room temperature for 24 h. The mixture was removed under reduced pressure, and the residue was dissolved in ethyl acetate and washed with water. The organic phase was subsequently dried over anhydrous Na2SO4 and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography, yielding THP-Boc-Ser as a white solid in 31.42% yield.

TEA (1.110 mL) was added dropwise to THP-Boc-Ser (1.73 mmol) dissolved in CH2Cl2 at 0°C, followed by the addition of 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride (2.07 mmol), 1-hydroxybenzotriazole (2.07 mmol), and thiazolidine-2-one (1.73 mmol). The reaction was carried out for 24 h. The organic phase was diluted with saturated NaHCO3 and extracted with CH2Cl2. After drying with anhydrous Na2SO4, the solvent was removed under reduced pressure. The crude product was purified by silica gel column chromatography, yielding THP-Boc-Ser-T as a white solid in 40.58% yield.

THP-Boc-Ser-T (0.6 mmol) and PPTS (0.6 mmol) were dissolved in anhydrous ethanol and stirred at 55°C for 12 h. After evaporation under reduced pressure, the reaction mixture was diluted with saturated NaHCO3 and extracted with CH2Cl2. The organic phase was dried with anhydrous Na2SO4 and concentrated under reduced pressure, yielding Boc-Ser-T 92.15%.

DSPA-Na (0.131 mmol) and TPSCL (0.394 mmol) were placed in a dry vessel, and Boc-Ser-T (0.262 mmol) dissolved in 5 mL of anhydrous pyridine was added under N2 protection. The reaction proceeded overnight at 40°C. The solvent was removed under reduced pressure, and the residue was dissolved in CH2Cl2. The crude product was purified by silica gel column chromatography, yielding Boc-PST as a white solid in 35.88% yield.

Boc-PST (0.47 mmol) was dissolved in CH2Cl2, and 1 mL of trifluoroacetic acid was added dropwise to the system in an ice bath. The reaction was carried out for 2 h, and subsequently concentrated under reduced pressure. The residue was diluted with saturated NaHCO3 and extracted with CH2Cl2, the organic phase was dried over anhydrous Na2SO4 and concentrated to obtain a white solid product PST with a yield of 96.16%.

¹H and ¹³C NMR spectra for PnT14 and PST were acquired in CDCl₃ using Bruker DRX-400 (¹H) and Bruker AV-600 (¹³C) instruments.

mRNA construction
Anti-FAP CAR plasmids were linearized with MluI, purified, and used as templates for T7-driven in vitro transcription. The resulting RNA was subsequently Cap-1 capped and purified, and transcript integrity was assessed by agarose gel electrophoresis. TRIM13 mRNA was prepared identically to FAP-specific CAR mRNA.

Formulation, optimization and characterization of ESLNPs
LNPs containing EGFP mRNA were prepared based on different formulations shown in Supplementary Table S1 via a microfluidic device. RAW 264.7 cells were seeded in 48-well plates (1 × 105 cells/well) and cultured for 12 h, followed by incubation with LNPs for 16 h (300 ng mRNA/well). Flow cytometry (Gallios, Beckman, USA) was used to evaluate transfection efficiency. The formulations with the highest transfection efficiency were used for subsequent experiments. The ionizable lipid P3T14, PST, cholesterol, and DMG-PEG were dissolved in ethanol at a molar ratio of 25:30:30:1.5 and the mRNA was diluted in a citrate disodium hydrogen phosphate buffer (pH = 4). The LNPs were assembled by swiftly combining the ethanol phase and aqueous phase with a microfluidic device. Subsequently, it was dialyzed in a DEPC-PBS solution (MWCO 100 kDa) for 30 min to eliminate ethanol and free mRNA.

For the encapsulation efficiency of ESLNPs, the Quant-iT Ribo-Green RNA kit was mixed with either LNPs or LNPs demulsified with 2% Triton X-100. Upon excitation by a 480 nm laser, the fluorescence intensity at 520 nm was measured by a multimode plate reader (EnSight, PerkinElmer, Singapore), and the mRNA content was determined based on the standard curve. The mRNA content in LNP and LNP demulsified with 2% Triton X-100 is denoted as M1 and M2, respectively. The encapsulation efficiency was calculated with the formula: (M2−M1)/M2 × 100%. The morphology of ESLNPs was determined by transmission electron microscope (TEM). Particle size, polydispersity index (PDI), and zeta potential of ESLNP were measured by the Zetasizer Nano ZS90 (Malvern, UK). PtdSer presentation was validated by flow cytometry20. All the ROS treatment of LNPs was processed with 0.1 mM H2O2 for 12 h.

In vitro cytotoxicity assay
The cell toxicity of ESLNPs was detected by CCK-8 assay. The RAW 264.7 cells were seeded in 96-well plates (1 × 104 cells/well) and incubated with LNPs containing mRNA at different concentrations (62.5, 100, 250, 500, 1000, 1250 ng/mL). After 6 h of incubation, the treated medium was replaced with 100 μL of fresh medium, and cultured for additional 18 h. Subsequently, 10 μL of CCK-8 agent were added to each well, followed by incubation at 37°C for 2 h. The absorbance at 450 nm was then measured by a multimode plate reader (EnSight, PerkinElmer, Singapore).

Examination of cellular uptake
To visualize LNP-mediated mRNA cellular uptake, mRNA was labeled with carboxyfluorescein (FAM). RAW 264.7 cells (3.0 × 105 cells/well) and BMDMs (3.0 × 105 cells/well) were seeded on sterile glass bottom culture dishes and cultured overnight. PBS, FAM-labeled mRNA and LNPs were added to the dishes and incubated for 5 h. The cells were washed with PBS, and the cell membranes were stained with DiD. Then, extra DiD was removed by washing with PBS, and the nuclei were stained with DAPI. Images were acquired by High-Resolution Living Cell Imager (LSM900 with Airyscan 2, Zeiss, Germany).

Examination of endosome escape
Raw 264.7 cells (3.0 × 105 cells/well) were seeded on sterile glass bottom culture dishes and cultured overnight. The cells were treated with ESLNPs carrying FAM-labeled mRNA and subsequently incubated for 1 or 5 h. Cells were then washed with PBS and stained with LysoTracker Green (Invitrogen) to label acidic endo/lysosomal compartments, followed by DAPI staining for nuclear visualization. Images were acquired by High-Resolution Living Cell Imager (LSM900 with Airyscan 2, Zeiss, Germany).

mtDNA isolation and quantification
Mitochondrial DNA (mtDNA) was isolated using a mtDNA isolation Kit (Abcam) according to the manufacturer’s instructions. For immunofluorescence staining, isolated mtDNA was labeled with Cy5-dCTP following the manufacturer’s instructions (Amersham Cy5-dCTP, Cytiva).

Expression of anti-FAP CAR and the macrophage phagocytosis analysis
ESLNPs encapsulating anti-FAP CAR mRNA were prepared for in vitro transfection tests. RAW 264.7 cells and BMDMs cells were seeded into 6-well plates at 3.0 × 105 cells/well and transfected with LNPs (1 μg of mRNAs/well) for 12 h. Transfection efficiency was tested by flow cytometry, and the expression of Myc-labeled CAR was confirmed by CLSM.

HSCs (1.0 × 105 cells/well) were seeded on sterile glass bottom culture dishes, cultured overnight, and labeled with Cell-Tracker Red CMTPX dye (or pHrodoTM Red for CLSM). To activate quiescent HSCs, the cells were treated with 10 ng/mL TGF-β1 for 24 h. Subsequently, HSCs or aHSCs (1.0 × 105 cells/well) were added to BMDMs (1.0 × 105 cells/well) with different treatments as scheduled and incubated for 12 h. The cells were harvested and stained with anti-CD11b, and the percentage of BMDMs that phagocytosed HSCs and aHSCs was quantified by flow cytometry.

Phenotype changes of macrophages with different treatment
BMDMs were seeded in 6-well plates (3.0 × 105 cells/well) and cultured for 12 h. To mimic profibrotic phenotype macrophage in the fibrotic foci, RAW 264.7 cells and BMDMs were stimulated with mtDNA (100 ng/mL) for 12 h. The macrophages were then incubated with ESLNP encapsulating mRNAs as scheduled. After incubation, the medium was collected, and the concentration of TNF-α, IL-6 et al. was measured by ELISA (Servicebio, China) following the manufacturer’s instruction. The expression of Ly6C and CD86 was detected by flow cytometry (Gallios, Beckman, USA).

Apoptotic cell clearance capacity
AML12 cells were washed once with PBS and incubated with pre-warmed Cell-Tracker Red, protected from light, for 30 min in a 5% CO2 atmosphere at 37°C; cells were then washed twice with PBS and treated with 1968 µL complete DMEM containing 32 µL of 50 mM palmitic acid stock (final palmitate 0.8 mM) for 24 h.

Macrophages treated with or without ESLNP were labeled with Cell Tracker Green, and then co-incubated with Cell Tracker Red-labeled apoptotic AML12 cells at a 1:1 ratio for 4 h. The efferocytosis process was further visualized by CLSM.

The distribution of LNPs in vivo
To induce CCl4-related liver fibrosis, 6–8 weeks C57BL/6 mice were intraperitoneally (i.p.) injected with 2 μL/g CCl4 diluted in olive oil (1/4, v/v) twice a week. Olive oil injection served as the control group. Both healthy mice and fibrotic mice were injected with LNPs encapsulating Luci mRNA (0.3 mg/kg), and the bioluminescent signals were detected by the IVIS imaging system after luciferin injection (100 mg/kg).

To further determine CAR expression in the liver after systemic administration of C@ESLNPs (1 mg/kg), liver tissues were collected and the proportion of CAR+ cells was analyzed by flow cytometry. (Gallios, Beckman, USA). Macrophages were labeled with anti-F4/80-BV421, and CAR expression was detected with anti-Myc-FITC. CAR expression was further confirmed by immunofluorescence staining of liver tissue sections.

In vivo treatment of CCl4-induced fibrotic mice
Six to eight weeks C57BL/6 mice were intraperitoneally (i.p.) injected with 2 μL/g CCl4 diluted in olive oil (1/4, v/v) twice a week for 4 weeks. Then, the CCl4-treated mice were randomly divided into 4 groups and systemically injected with PBS, T@ESLNP, C@ESLNP and CT@ESLNP twice a week (1 mg/kg) for following two weeks with continued CCl4 stimulation. Body weight was monitored throughout the study. At the end of the treatment period, mice were euthanized and the liver tissues and blood samples were collected for subsequent analysis.

Liver tissue decellularization
To obtain decellularized whole-liver ECM from mice, we followed an in situ perfusion protocol. Briefly, mice subjected to various treatments were anesthetized, and the hepatic portal vein was cannulated with a polyethylene tube connected to a peristaltic pump. The flow rate was set at 3 ml/min, and the inferior vena cava was severed to allow for buffer perfusion. 1.9% EGTA solutions were perfused for 20 min to flush out blood. Then, sodium dodecyl sulfate (SDS) solutions at concentrations of 0.01%, 0.02%, and 0.05% were sequentially perfused over 12 h for each step. Deionized water was subsequently perfused for an additional 12 h to thoroughly rinse away any residual buffer. The decellularized tissues were collected and preserved in glutaraldehyde fixative (Biosharp, China) and further characterization was conducted by Shiyanjia, China.

HYP assays
The total collagen content of liver tissue was quantified by the HYP Content Assay kit (Solarbio, China) according to the manufacturer’s instructions.

In vivo flow cytometry
For the flow cytometry analysis, the tissues were collected and placed into a digestive enzyme cocktail, followed by shaking at 37°C for 1 h. Then the tissues were homogenized and the suspension was filtered through 70 μm nylon cell strainer, followed by centrifugation at 800 × g for 7 min. The pellet was resuspended in RBC lysis buffer and vortexed for 2 min. Afterward, PBS was added and repeated centrifugation step47. Finally, the sediments were resuspended into PBS and treated with FcR blocking reagent for 15 min (Miltenyi Biotec, Germany). Then, the cells suspension was incubated with desired antibodies for 1 h at room temperature. All samples were analyzed by flow cytometry (Gallios, Beckman, USA).

Immunofluorescence and immunohistochemistry
The cells were seeded on sterile glass bottom culture dishes and cultured for 12 h. After that, these cells were fixed in 4% paraformaldehyde and permeabilized with 0.2% Triton X-100. Non-specific binding was blocked with 2% bovine serum albumin (BSA), after which the cells were incubated with primary antibodies at 37°C for 1 h. Subsequently, fluorophore-labeled secondary antibodies were applied, and nuclei were counterstained with DAPI as needed.

For immunohistochemistry analysis, mice with various treatments were euthanized, and the liver tissues were harvested and fixed in 4% paraformaldehyde, embedded in paraffin, and sectioned for staining (conducted by Wuhan Servicebio Technology Co., Ltd). Subsequently, the stained sections were visualized and captured by the HD panoramic scan and image analysis system (VS120, Olympus, Japan).

Quantitative PCR
Total RNA from liver was extracted using RNA extraction solution (Wuhan Servicebio Technology Co., Ltd) following the manufacturer’s instructions. RNA samples (1 µg) were then reverse-transcribed into complementary DNA (cDNA) using SweScript All-in-One RT SuperMix for qPCR (Wuhan Servicebio Technology Co., Ltd). The data were normalized to housekeeping gene β-actin expression, and statistical analysis was calculated by the 2−ΔΔCt equation. The primers used in this study are shown in Supplementary Table S3.

Single-cell RNA sequencing
The samples were obtained from the liver from mice treated with PBS or CT@ESLNP. The harvested tissues were transferred to a Petri dish (kept on wet ice) prefilled with 1× PBS (RNase-free, Ca2+/Mg2+-free) and washed with 1× PBS to remove blood, grease, and other surface contaminants. Tissues were then diced into ~0.5 mm2 pieces and washed again with 1× PBS. The tissue pieces were incubated in dissociation solution (0.35% collagenase IV, 2 mg/mL papain, 120 U/mL DNase I) for 20 min at 37°C in a shaking water bath (100 rpm). Dissociation was terminated by adding PBS containing 10% fetal bovine serum. The samples were gently triturated 5-10 times with a pipette (carefully to avoid shear-induced cell death), filtered through a 70–30 µm cell sieve, and centrifuged at 300 × g for 5 min at 4°C The cell pellet was resuspended in 100 µL 1× PBS (0.04% BSA). To remove erythrocytes, 1 mL of 1× erythrocyte lysis solution (MACS 130-094-183, stock 10×) was added and incubated at room temperature or on wet ice for 2–10 min. After lysis, cells were centrifuged at 300 × g for 5 min at 4°C and the pellet collected. Dead Cell Removal MicroBeads (100 µL; MACS 130-090-101) were added, mixed, and incubated for 15 min at room temperature. Binding buffer was then added and the suspension applied to MS Columns (130-042-201) to remove reagents and dead cells. Cell pellets were collected by centrifugation at 300 × g for 5 min at 4°C, resuspended in 1× PBS (0.04% BSA), and centrifuged again at 300 × g for 5 min at 4°C (repeated twice). The resulting single-cell suspension was adjusted to 100 µL of 1× PBS (0.04% BSA). Cell viability was assessed by trypan blue staining and required to be >85%. Cell counts were obtained using a hemocytometer or a Countess II Automated Cell Counter, and the final cell concentration was 700–1200 cells/µL.

Single-cell suspensions were loaded onto a 10× Chromium chip following the manufacturer’s instructions for the 10× Genomics Chromium Single-Cell 3′ Kit (v3). cDNA amplification and library construction were performed according to standard protocols. Libraries were sequenced by LC-Bio Technology (Hangzhou, China) on an Illumina NovaSeq 6000 system (paired-end, 150 bp) to a minimum depth of 20,000 reads per cell. For bioinformatic analysis, raw Illumina base-call files were converted to FASTQ format using bcl2fastq (v5.0.1). scRNA-seq reads were aligned to the reference genome and cellular 3’-end transcripts were identified and counted using Cell Ranger (v7.0.0) (see 10× Genomics support). The Cell Ranger output expression matrix was imported into Seurat (v4.1.0) for quality filtering, scaling and clustering. Low-quality cells were removed using the following thresholds: >500 genes detected per cell and <25% mitochondrial gene content. Finally, cells were projected into two-dimensional space using t-SNE or UMAP.

Biosafety analysis
For the biosafety analysis, healthy mice were randomly divided into four groups and subjected to injections of PBS, C@ESLNP, T@ESLNP and CT@ESLNPs. The mice were euthanized, and the major organs (heart, liver, spleen, lungs, kidneys) were sectioned and stained with H&E. Whole blood was collected, and serum was isolated for the measuring of ALT, AST, CREA and BUN.

Statistical analysis
All statistical analyses of raw data were conducted by GraphPad Prism software. The statistical significance was analyzed by Student’s t test or ANOVA. Significant differences are indicated as ns P ≥ 0.05, *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "In the submitted manuscript, the authors have revealed that ESLNP-engineered anti-fibrotic CAR-Ms redirect phagocytosis to FAP-positive aHSC and maintain an anti-inflammatory phenotype, ultimately halting fibrosis progression and facilitating liver reconstruction, providing a new therapeutic target for liver fibrosis. However, there are some problems, which must be solved before it is considered for publication. If the following problems are well-addressed, this article believes that the basic contribution of this paper will be important for the cell-targeted therapy of liver fibrosis.\nSeveral major specific comments are listed below.\n1. The authors have constructed an efferocytosis-sparked lipid nanoparticle (ESLNP) with reactive oxygen species (ROS)-responsive PS exposure property, for locoregionally in situ generation of anti-inflammatory CAR-Ms in fibrotic areas. Does ESLNP block the ability of normal macrophages to clean up apoptotic cells?\n2. The authors should show FAP expression in a human cohort. It would be important to know if FAP expression is different in patients with and without fibrosis/cirrhosis and in the different etiologies.\n3. In the current paper, only one fibrosis model was used, and other liver fibrosis models such as BDL, DDC-diet and HFCF diet, should be used to test the therapeutic efficiency of ESLNP as well. This would have been very helpful to show efficacy in a clinically relevant model.\n4. Generation of CAR macrophages with purported advantages over CART cells should be established based on head-to-head comparison with the prior publication.\n5. Macrophages have high plasticity. Are there any cues from the microenvironment that promote this plasticity in vivo? Alternatively, this could be investigated either with in vivo co-culture with fibroblasts or from in vivo single cell isolation of the CAR-M.\n6. In this study, the therapeutic effects of ESLNP on mice with liver fibrosis were investigated by in vitro and in vivo experiments. However, the number of mice in each group of in vivo experiments is generally required to be n ≥ 6, and the number of mice in the in vivo experiments of this study did not meet the corresponding criteria.\n7. For the detection of changes in cell proportions as in Fig. 1D, Fig. 2I, and Fig. 3B the corresponding flow scatter plots should also be shown.\n8. The name of each experimental grouping in Figure 3D should be labeled above the WB data plot, and protein names should not overlap the WB data plot.\n9. Figure 3D detection of changes in protein level of STING, TRIM13 is not sufficient to explain the effect of T@ESLNP on the activation of the cGAS-STING pathway. On this basis, the changes in protein level of cGAS, p-sting, p-IRF3, and p-TBK should also be explored.\n10. For the therapeutic effect in fibrotic mice, changes in fibrosis-related genes such as collagen1, collagen2, and timp1 should also be detected using QPCR.\n11. In this study, the effects of ESLNP or CT@ESLNP on macrophage polarization and inflammatory factor release were detected by flow and ELISA. On this basis, liver tissues could be collected from fibrosis model mice and mice in each treatment group, and liver histiocytes could be isolated to further elucidate the molecular and cellular alterations induced by ESLNP or CT@ESLNP by single-cell RNA sequencing.", "reviewer_2": "This study proposes a novel TRIM13-engineered CAR-Ms therapy, utilizing lipid nanoparticles (ESLNPs) to achieve in situ engineering in fibrotic livers, demonstrating significant anti-inflammatory and anti-fibrotic effects. This approach is innovative in the treatment of liver fibrosis; however, further optimization of this work is required before it can be considered for acceptance.\nMajor:\n1. While the research primarily focuses on the regulation of macrophages, it is equally important to consider hepatocytes, which are the main functional cells of the liver, as well as hepatic stellate cells, which play a critical role in the pathogenesis of fibrosis. In Figure 4b, it seems that both DOPE and ES LNP have similar expression of mRNA, suggesting there might be a potential for hepatocytes uptake. The transfection efficiency and corresponding functional impacts in these cell types also deserve careful attention. Please show how much hepatocytes have been transfected and what’s the potential off-target effect?\n2. Engineering macrophages into Car is a very powerful strategy for treating liver fibrosis. If the macrophages in the spleen can also be powered with Car, can it enhance the therapeutic efficiency? Please comments on why macrophages in spleen have been avoided for expression of CAR.\nMinor\n3. The authors used H₂O₂ to simulate the ROS environment in fibrotic livers; however, the rationale for selecting the specific concentration of 0.1 mM H₂O₂ is not explicitly discussed in the manuscript. Please provide a detailed explanation to clarify the basis for this choice.\n4. The fluorescence images presented in the manuscript should be supported by quantitative data with n ≥ 3 to enhance the robustness and reliability of the findings.\n5. For IVIS, please display the actual bar values (specific numerical values) rather than using only \"high to low\" to represent signal intensity.\n6. In Figure 2J, the normal condition has >60% EGFP expression, and H2O2 only increased slightly EGFP expression, please explain why there’s still a high portion of cell taken up LNPs under normal condition, will that impact specificity?\n7. In Figure 5D, the differences between the various groups appear to be minimal, or the field of view in the images may be too small. Please adjust the presentation to provide a clearer and more distinct representation of the data.", "reviewer_3": "In the manuscript by Gao et al., the authors developed a new chimeric antigen receptor macrophages (CAR-Ms) therapy against liver fibrosis using efferocytosis-sparked lipid nanoparticles specifically activated under oxidative stress conditions. These nanoparticles are designed to expose phosphatidylserine to be incorporated by macrophages under oxidative stress conditions, and they confer CAR-Ms the ability to specifically engulf activated hepatic stellate cells (aHSCs) via anti-fibroblast activation protein (FAP). Overexpression of TRIM13 downregulated STING to sustain anti-inflammatory phenotype of CAR-Ms. Although this idea is potentially interesting, Gao et al. recently published a similar strategy using CAR-Ms expressing anti-FAP against cardiac fibrosis (Gao et al., Cell discovery, 2024 PMID:39134538). Moreover, Lee et al. also designed artificial liposomes to reduce the viability of aHSCs utilizing FAP to attenuate liver fibrosis (Lee et al., Nat Commun, 2022 Ref.17). Therefore, additional experiments will be necessary to establish this CAR-M system as a new and promising strategy to treat liver fibrosis.\nMAJOR POINTS\n1. This system uses TRIM13 overexpression to ubiquitylate and degrade intracellular STING as reported by Li et al. (Li et al., Sci Adv, 2022 Ref.16), thereby inactivating cGAS-STING pathway to maintain CAR-Ms in an anti-inflammatory state. However, TRIM13 was reported to ubiquitylate several other inflammatory proteins including TRAF6 (Huang and Baek, Mol Pharmacol, 2017 PMID: 28087809) and Nur77 (Huang et al., Sci Rep 2018 PMID: 30224829). Moreover, TRIM13 self-ubiquitylation was reported to be involved in p62-mediated ER-phagy (Ji et al., Mol Cell, 2019 PMID: 31375263). Therefore, TRIM13 overexpression can change the stability of various proteins other than STING and affect NF-κB signaling. The authors should discuss these possibilities, and if possible, check the amount of these proteins experimentally by Western blot, immunostaining. or FACS.\n2. For all the FACS data in the Figures (Fig1D, Fig2I, Fig3H, Fig4E, Fig6ABC, and FigS18), please describe both the antigens and fluorescent dyes in the axis as in FigS19-21.\n3. In Figure 3, the authors claim that FAP-positive activated HSCs are engulfed by macrophages treated with C@ESLNP or CT@ESLNP. However, it is difficult to distinguish whether the macrophages merely bind to the target cells or actually engulf them. To clarify this, the authors should label aHSCs or qHSCs with pHrodo Red and assess whether the fluorescence signal increases—indicating acidification in the lysosomes following phagocytosis. Alternatively, transmission electron microscopy should be performed to directly demonstrate the engulfment of aHSCs by macrophages.\n4. In the RESULTS section describing Fig4, the authors mainly used LNPs encapsulating Luciferase mRNA to detect bioluminescent signals. However, the FACS data in Fig4EF detected Myc signals using c@ESLNPs. Although this is described in the MATERIALS AND METHODS section, please also indicate this point in the RESULTS section to avoid confusion.\n5. The authors examined only one in vivo model of liver fibrosis (CCl4 injection). However, this in vivo model is highly inflammatory and artificial. Lee et al. used three models (CCl4, bile duct ligation, and CDAHFD) to show the effectiveness of their aHSC-targeted liposomes (Lee et al., Nat Commun, 2022 Ref.17). To further substantiate the authors’ conclusion and extend the future clinical application, the authors should test the efficacy using other experimental fibrosis models, such as CDAHFD-induced liver fibrosis. If the efferocytosis-sparked lipid nanoparticles established by the authors are also effective in lipid-rich steatohepatitis, this would increase the chance of future clinical applications for human diseases such as metabolic dysfunction-associated steatohepatitis (MASH).\n6. In the Cells part of the MATERIALS AND METHODS section, please describe the preparation method of primary HSCs in more detail. Also, the purity of primary HSCs should be checked by autofluorescence of vitamin A droplet or anti-Desmin antibody staining.\n7. Please indicate the source of mRNA sequence of anti-FAP CAR. As the nucleotide sequence in Table S2 is not found in the nucleotide database, the readers cannot be confident whether this protein binds to mouse FAP.\n8. In the cartoon of ESLNPs in Fig1F, Fig2D and Fig4A, PnT14 has two fatty acid tails. However, its structure suggests that PnT14 should have three fatty acid tails. Please confirm this point.\n9. The description of the statistical analysis is currently insufficient throughout the Figures. The authors should specify the statistical methods used for each figure; for example, two-tailed unpaired Student’s t-test (a) and one-way ANOVA with Tukey’s multiple comparison test for panel (b). Furthermore, since the data were obtained from multiple individual mice rather than from triplicate experiments using cell lines, the results should be expressed as mean ± standard error (SE), rather than standard deviation (SD).\n10. In Figure S17, the signal of the indicated antibody was too weak to allow proper evaluation. The authors should replace these immunohistochemistry results with more representative images.\nMINOR POINTS\n1. The authors refer to their own results, including specific figure numbers, in the Introduction. This practice does not align with the standard format of Nature Communications. The authors should relocate these descriptions to the Results section, where they are more appropriate.\n2. In the Author contributions section, the description of Maosen Han (MH) is missing.\n3. In the MATERIALS AND METHODS section, mixed usage of h/hours, min/minutes, and ×/* are found.", "reviewer_4": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts."}, "author_response": "Responsetoreviewers’comments:\nWe are grateful for the constructive feedback from the reviewers. According to\nthe reviewers’suggestions, we have added additional data to validate our conclusion.\nThe manuscript has been substantially revised. Below, please find our point-by-point\nresponses tothe reviewers’comments.\nReviewer#1:\nIn the submitted manuscript, the authors have revealed that ESLNP-engineered\nanti-fibrotic CAR-Ms redirect phagocytosis to FAP-positive aHSC and maintain\nan anti-inflammatory phenotype, ultimately halting fibrosis progression and\nfacilitating liver reconstruction, providing a new therapeutic target for liver\nfibrosis. However, there are some problems, which must be solved before it is\nconsidered for publication. If the following problems are well-addressed, this\narticle believes that the basic contribution of this paper will be important for the\ncell-targeted therapy of liver fibrosis. Several major specific comments are listed\nbelow.\nQ1: The authors have constructed an efferocytosis-sparked lipid nanoparticle\n(ESLNP) with reactive oxygen species (ROS)-responsive PS exposure property,\nfor locoregionally in situ generation of anti-inflammatory CAR-Ms in fibrotic\nareas. Does ESLNP block the ability of normal macrophages to clean up\napoptoticcells?\nA: We thank the reviewer for this detailed comment. In the revised manuscript, to\nassess macrophage-mediated clearance of apoptotic cells after ESLNP treatment, we\nfirst established an apoptotic cell model by coincubating with 0.8mM palmitic acid,1\nand then incubated them with ESLNP-coincubated or untreated macrophages for 4\nhours, after which the phagocytosis of apoptotic cells was recorded and quantified..\nConfocal imaging revealed that PS exposure of ESLNP had no discernable effect on\nmacrophage phagocytosis of apoptotic cells. The results have been added to the\nrevised manuscript as Fig. S15.\n“Meanwhile, we coincubated macrophages with apoptotic cells to investigate\nwhether the PSexposureofESLNPwould affect macrophages’normal ability to clean\nup apoptotic cells. Confocal imaging revealed that ESLNP had no discernable effect\nonmacrophagephagocytosis of apoptoticcells (Fig. S15)”\nFig. S15 (A) Representative CLSM images of macrophages co-incubated with\napoptotic cells. Apoptotic cells were labeled with CellTracker Red, and macrophages\nare labeled with CellTracker Green. Scale bar, 50 μm. (B) Quantification of the\ncolocalization area of apoptotic cells and macrophages (n=3). Data are presented as\nmean ± SD. Statistical significance among groups was assessed using Student’s t-test.\nSignificance levels were denoted as ns P≥ 0.05.\nQ2: The authors should show FAP expression in a human cohort. It would be\nimportant to know if FAP expression is different in patients with and without\nfibrosis/cirrhosis andinthedifferent etiologies.\nA: We sincerely appreciate the reviewer’s insightful comment. Indeed, evaluating\nFAP expression in a human cohort is of significant clinical relevance, particularly for\nvalidating the translational potential of FAP-targeted strategies. Numerous studies\nhave demonstrated that FAP is universally upregulated in patients with fibrosis and\ncirrhosis caused by different etiologies, such as HBV infection, HCV infection,\nprimary biliary cirrhosis and primary sclerosing cholangitis 2,3,4,5. FAPis a biomarker\noriginating from activated hepatic stellate cells, with trace expression in quiescent\nhepatic stellate cells.6 Furthermore, a recent study combining transcriptional analysis\nwith functional assays in clinical liver samples identified FAPα as a pathological\nbiomarker for liver fibrosis grading.7 Notably, FAPα showed a superior quantitative\nlinear correlation with fibrosis grade compared with conventional markers. These\nfindings demonstrated FAP as an ideal target for the diagnosis and treatment of liver\nfibrosis, highlighting thepotential ofFAP-targeted therapies forclinical application.\nQ3: In the current paper, only one fibrosis model was used, and other liver\nfibrosis models such as BDL, DDC-diet and HFCFdiet, should beused to test the\ntherapeutic efficiency of ESLNP as well. This would have been very helpful to\nshowefficacy inaclinically relevantmodel.\nA: We sincerely appreciate the reviewer’s constructive suggestion. To fully test the\ntherapeutic efficacy of ESLNP, we constructed a high-fat,\nmethionine-choline-deficient (HFMCD) model that mimicked metabolism-associated\nliver fibrosis,characterized by hepatocellular injury,lipid accumulation, inflammation,\nand progressive fibrosis. We believe this more clinically relevant model would\nprovide a more comprehensive evaluation of the therapeutic potential of our proposed\nstrategy. The associated experimental methods, results and discussion have been\nadded totheupdated manuscript.\n“Male C57BL/6J mice (6-8 weeks) were fed a HFMCD diet to establish the\nHFMCD model, while control mice received a normal diet under identical housing\nconditions. After model establishment, treatment was administered following the\nsame protocol as used in the original CCl -induced fibrosis model. Therapeutic\n4\noutcomes were evaluated by histological analysis of liver sections and quantification\nofHYPcontent..”\n“Moreover, we constructed a high-fat, methionine-choline-deficient\n(HFMCD)-induced model to comprehensively test the therapeutic efficacy of ESLNP,\nwhich mimicked metabolism-associated liver fibrosis characterized by hepatocellular\ninjury, lipid accumulation, inflammation, and progressive fibrosis. After 2 weeks of\ntreatment, liver tissues were harvested for histological examination and HYP\nquantification (Fig. 4P). CT@ESLNPs treatment reduced both microvesicular and\nmacrovesicular steatosis in H&E-stained sections, significantly decreased Masson\nstaining, and lowered thepositiveareas of SiriusRed and α-SMAstaining (Fig. 4Q-T).\nMeanwhile, hepatic HYP levels were markedly decreased in CT@ESLNPs-treated\nmice (Fig. 4U). These data collectively indicated the powerful therapeutic efficacy of\nESLNP-edited anti-inflammatory CAR-Ms, which could promote fibrosis resolution\nand liverregeneration.”\nFig.4 (P) Schematic illustration of the experimental design in HFMCD-induced liver\nfibrosis model. (Q) Representative images of H&E, Masson staining, SiriusRed\nstaining, and α-SMA staining in the liver tissues. Scale bar, 100μm. (R-T)\nQuantitative analysis of Masson staining (R), SiriusRed staining (S) and α-SMA\nstaining (T) in liver tissues at the end of the treatment (n=9). Data are presented as\nmean ± SE. Statistical significance among groups was assessed using one-way\nANOVA. Significance levels were denoted as ns P ≥ 0.05, * P < 0.05, ** P < 0.01,\n**** P < 0.0001. (U) HYP content in liver tissues of various treatment groups (n=6).\nData are presented as mean ± SE. Statistical significance among groups was assessed\nusing one-way ANOVA. Significance levels were denoted as* P < 0.05, **** P <\n0.0001.\nQ4: Generation of CAR macrophages with purported advantages over CART\ncells should be established based on head-to-head comparison with the prior\npublication.\nA: We thank the reviewer for this important comment. We fully acknowledge that a\ndirect head-to-head comparison between CAR-M and CAR-T would provide a more\nrigorous evaluation of the advantages of CAR-M in liver fibrosis treatment. Given the\nliver’s abundant macrophage reservoir, our primary objective was to develop and\ncharacterize the ESLNP-based approach for generating functional CAR-M and to\nevaluate theirtherapeutic efficacy in liver fibrosis models. Previous work has reported\nCAR-T-based antifibrotic approaches; instead of duplicating those studies,8, 9 we\naimed to develop a complementary strategy that leverages the distinct biological\nproperties of macrophages, including their innate homing to fibrotic tissues,\nphagocytic activity, and capacity to remodel the extracellular matrix. The therapeutic\nefficacy of CAR cell therapy is intricately linked to critical factors including target\nantigen selection, the methodology employed in cell preparation, and the expression\nlevel of CAR.Aformal head-to-head comparison between CAR-M and CAR-Twould\nrequire extensive additional optimization and standardization of CAR-T construction,\nmanufacturing and dosing regimens. However, these specific methods and details\nhave not been fully disclosed in the available CAR-T researches. We plan to pursue\nexperiments in future studies to comprehensively evaluate the therapeutic efficacy of\nCAR-M and CAR-T therapies in multiple liver fibrosis models, and we have added a\nstatement in thediscussion toclarify theexisting limitation and future direction.\nQ5: Macrophages have high plasticity. Are there any cues from the\nmicroenvironment that promote this plasticity in vivo? Alternatively, this could\nbe investigated either with in vivo co-culture with fibroblasts or from in vivo\nsinglecell isolationoftheCAR-M.\nA: We thank the reviewer for raising this important point. It is well-established that\nmacrophages demonstrate remarkable plasticity, and their phenotype can be\nprofoundly modulated by the surrounding microenvironment. In the fibrotic foci,\nactivated fibroblasts serve as a crucial source of pro-inflammatory mediators, such as\nIL-6 and CXCL10, which can either directly act on macrophages or cooperate with\nfibroblasts to establish a self-reinforcing circuit that perpetuates fibrogenesis.10,11. In\nthe revised manuscript, we performed co-culture experiments between macrophages\nand fibroblasts to investigate the effect of fibrotic environment on macrophages’\nplasticfunctionality,and the newly incorporated content isdetailed as follows.\n“We subsequently established a macrophage-fibroblast co-culture model to\nassess the regulatory impact of TRIM13 on macrophage inflammatory responses in\nthefibrotic milieu.The results showed that co-culturing led to a significant increase in\nthe proportion of pro-inflammatory macrophages, whereas co-expression of TRIM13\ndownregulated this proportion, further demonstrating that TRIM13 restrains the\nfibrotic microenvironment-driven polarization of macrophages toward\npro-inflammatory phenotype.”\nFig.S16 Representative flowcytometric plotsdepicting and quantitativeanalysis of\ntheCD86+macrophages withdifferent treatments (n=3). Dataare presented as mean ±\nSD.Statistical significance among groups was assessed usingone-wayANOVA.\nSignificance levels were denoted as ***P< 0.001,****P< 0.0001.\nQ6: In this study, the therapeutic effects of ESLNP on mice with liver fibrosis\nwere investigated by in vitro and in vivo experiments. However, the number of\nmice in each group of in vivo experiments is generally required to be n ≥ 6, and\nthe number of mice in the in vivo experiments of this study did not meet the\ncorresponding criteria.\nA: We sincerely appreciated the detailed and important comment. In response, we\nhave repeated the in vivo experiments and expanded the sample size to n = 6 mice per\ngroup. The updated data have been incorporated into the revised manuscript, with\ncorresponding changes clearly noted in the figure legends. We believe this revision\nwould increasethe reliabilityand reproducibility ofour findings.\nQ7:Forthe detection ofchanges in cell proportions as in Fig. 1D, Fig. 2I, and Fig.\n3B thecorresponding flowscatterplotsshould also beshown.\nA: We sincerely appreciate your careful review and detailed suggestion. Following\nthe reviewer’s suggestion, we have added the scatter plots to the revised\nsupplementary information, thereby enhancing the transparency and interpretability of\nour results. The data in the original Fig. 1 were mostly derived from preliminary\nmechanism‐verification experiments using other delivery vehicle in our earlier work.\nWe removed certain panels from the original Fig. 1 as these preliminary data\nduplicated information presented in Fig. 3 (Fig. 2 in the revised version).\nConsolidating them improves the manuscript’s narrative and the clarity of data\npresentation. This reorganization was done solely to improve presentation and does\nnot change the experimental findings. Thus, Fig. 1D has been deleted, and other flow\nscatter plotshave been added tothe supplementary information.\nFig. S6 (corresponding Fig. 3B in previous version) The ROS-responsive properties\ncharacterization and transfection efficiency detection of ESLNPs. (A) Representative\nflow cytometric plots depicting and quantitative analysis of PS-presenting of ESLNPs\ntreated with PBS or H O (n=3). (B) Representative flow cytometric plots depicting\n2 2\nand quantitative analysis of EGFP-positive cells transfected with PBS or H O -treated\n2 2\nESLNPs (n=3). Data are presented as mean ± SD. Statistical significance among\ngroups was assessed using one-way ANOVA. Significance levels were denoted as ns\nP≥ 0.05,**P <0.01,****P < 0.0001\nFig.S11 (corresponding Fig. 2I in previous version) Representative flow cytometric\nplots depicting CAR expression in RAW264.7 cells and BMDMs and quantitative\nanalysis (n=3). Data are presented as mean ± SD. Statistical significance among\ngroups was assessed using one-way ANOVA. Significance levels were denoted as ns\nP≥ 0.05,****P <0.0001.\nQ8: The name of each experimental grouping in Figure 3D should be labeled\nabovetheWB data plot, andproteinnames should notoverlap theWB data plot.\nA:Thanks for thereviewer’s detailed suggestion.Following thereviewer’ssuggestion,\nwe have revised Fig.3D by labeling each experimental group above the Western blot\ntoclearly exhibitexperimental grouping andWBbands.\nQ9: Figure 3D detection of changes in protein level of STING, TRIM13 is not\nsufficient to explain the effect of T@ESLNP on the activation of the\ncGAS-STING pathway. On this basis, the changes in protein level of cGAS,\np-sting,p-IRF3, and p-TBK shouldalso beexplored.\nA: We are grateful for the reviewer’s insightful comment. To systematically validate\nthe effect of T@ESLNP on the activation of the cGAS-STING pathway, we\nsupplemented the study with additional data on the protein level of cGAS, p-IRF3,\nand p-TBK.Thesupplemented dataand corresponding analysis are as follows.\n“Western blot analysis demonstrated that TRIM13 protein was markedly\nup-regulated in the T@ESLNP group (ESLNPs encapsulating TRIM13 mRNA),\nwhile STING protein levels and the phosphorylation of TBK1 and IRF3 were\nsubstantially reduced. In contrast, no discernible changes were observed following\ntreatment with free TRIM13 mRNA or s@ESLNPs (ESLNP encapsulating scramble\nmRNA)(Fig. 3D).”\nFig.3D Representative western blot analysis of TRIM13 and STING pathway in\nmtDNA-pretreated BMDMs from various treatment groups.\nQ10: For the therapeutic effect in fibrotic mice, changes in fibrosis-related genes\nsuchas collagen1, collagen2, andtimp1 should also bedetected using qPCR.\nA: We sincerely appreciate the reviewer's constructive suggestion. Collagen I,\nCollagen II, and TIMP1 are closely associated with fibrosis. We apologize for the\nomission of these proteins in the initial experimental design, and have now evaluated\ntheirexpression levels using qPCR.\n“Total RNA from liver was extracted using RNA extraction solution (Wuhan\nServicebio Technology Co., Ltd) following the manufacturer’s instructions. RNA\nsamples (1 µg) were then reverse-transcribed into complementary DNA (cDNA)\nusing SweScript All-in-One RT SuperMix for qPCR (Wuhan Servicebio Technology\nCo., Ltd). The data were normalized to housekeeping gene β-actin expression, and\nstatisticalanalysis was calculated bythe2−ΔΔCt equation.”\n“Quantitative PCR (qPCR) was also performed to assess the mRNA levels of\nCollagen I, Collagen II and TIMP-1 in liver tissues from each group. Notably, the\nCT@ESLNP treatment elicited a pronounced downregulation of these fibrosis-related\ngenes (Fig. 4K).”\nFig, 4K qPCRs for Col1a1, Col2a1 and TIMP-1 (n=6). Data are presented as mean ±\nSE. Statistical significance among groups was assessed using one-way ANOVA.\nSignificance levels were denoted as ns P ≥ 0.05, * P < 0.05, ** P < 0.01, **** P <\n0.0001\nQ11: In this study, the effects of ESLNP or CT@ESLNP on macrophage\npolarization and inflammatory factor release were detected by flow and ELISA.\nOn this basis, liver tissues could be collected from fibrosis model mice and mice\nin each treatment group, and liver histiocytes could be isolated to further\nelucidate the molecular and cellular alterations induced by ESLNP or\nCT@ESLNPby single-cell RNAsequencing.\nA: We thank the reviewer for the constructive suggestion. We fully agree that\nsingle-cell RNA sequencing would provide a powerful means to dissect the complex\ncellular and molecular landscapes induced by ESLNP or CT@ESLNP in the liver,\nparticularly in the context of macrophage plasticity and the multicellular interactions\nunderlying fibrosis resolution. In the revised manuscript, we employed single-cell\nRNA sequencing of liver tissues to further elucidate the molecular and cellular\nalterationsinduced byCT@ESLNP.The added content is as follows.\n“To further elucidate the anti-fibrotic mechanism of CT@ESLNP, we performed\nsingle-cell sequencing of liver tissues from mice treated with PBS and CT@ESLNP.\n(Fig. 6A). After quality control, 21850 cells were collected for further analysis.\nAccording to the classic markers of various cell types, 10 clusters, namely B cells,\nconventional dendritic cells (cDCs), endothelial cells, macrophages, monocytes,\nneutrophil cells, NK cells, plasmacytoid dendritic cells (pDCs), plasma cells and T\ncells, were identified (Fig. 6B and Fig. S26A). Subsequently, we focused on the\ndistribution of relevant cell subtypes in the myeloid cell population (Fig. 6C and Fig.\nS26B). Among these clusters, the proportion of Ly6Chi cluster cells decreased in\nCT@ESLNP groups while the Ly6Clo cluster cells increased (Fig. 6D). We further\nscored a macrophage gene signature and found that CT@ESLNP treatment markedly\ndecreased expression of genes associated with a pro-inflammatory phenotype,\nincluding Ccl3, Cxcl2 and Mrc1.(Fig. S26C).Additionally, we evaluated alterations in\nmacrophage phagocytic capacity induced by in situ generated anti-inflammatory\nCAR-Ms. Analyses of the phagocytosis-related genes, including Axl, Mertk, Fcgr1,\nand Cd5l, demonstrated that macrophages in the CT@ESLNP group exhibited\nsubstantially augmented phagocytosis relative to PBS-treated group. The reinforced\nphagocytic capacity of macrophages facilitated the clearance of aHSCs and apoptotic\ncells, thereby promoting pronounced mitigation of liver fibrosis progression. (Fig.\n6E).”\nFig.6 scRNA-seq reveals the profile and heterogeneity of macrophages. (A)\nSchematic diagram of the experimental procedure. (B) UMAP integrating single cells\nfrom PBS and CT@ESLNP treatment groups. Ten clusters indicated by colors are\nmarked with the presumed cell types. (C) Dot plots display the differentially genes of\nmyeloid cells. (D) Percentages of major subpopulations of myeloid cells. (E) Violin\nplot showing the expression of Axl, Mertk, Fcgr1 and CD5l in macrophage clusters\nfrom each group.\nFig. S26 Dot plots display the top marker genes in single-cell samples. (A) Dot\ncoloration and size denote the normalized mean expression level across cell\npopulations and the fraction of cells within each population expressing the gene,\nrespectively. (B) UMAP projection of the myeloid cells in each group. (C) Violin plot\nshowing the expression of Ccl3, Cxcl2 and Mrc1 in macrophage clusters from each\ngroup.\nReviewer#2:\nThis study proposes a novelTRIM13-engineered CAR-Ms therapy, utilizing lipid\nnanoparticles (ESLNPs) to achieve in situ engineering in fibrotic livers,\ndemonstrating significant anti-inflammatory and anti-fibrotic effects. This\napproach is innovative in the treatment of liver fibrosis; however, further\noptimization ofthiswork is requiredbeforeitcan beconsidered foracceptance.\nQ1: While the research primarily focuses on the regulation of macrophages, it is\nequally important to consider hepatocytes, which are the main functional cells of\nthe liver, as well as hepatic stellate cells, which play a critical role in the\npathogenesis offibrosis. In Figure4b,it seems thatboth DOPE andES LNPhave\nsimilar expression of mRNA, suggesting there might be a potential for\nhepatocytes uptake. The transfection efficiency and corresponding functional\nimpacts in these cell types also deserve careful attention. Please show how much\nhepatocytes havebeen transfected andwhat’s thepotential off-target effect?\nA: We sincerely appreciate the reviewer’s insightful comment. In this study, we\nconstructed a liver macrophage-targeting lipid nanoparticle (ESLNP) to deliver\nmRNA drugs for in situ engineering CAR-Ms, and flow cytometry analysis revealed\nthat approximately 80% of Myc-positive cells were macrophages, supporting the\nfeasibility of our targeting strategy (Fig. 3F). Although a fraction of ESLNPs was\ninevitably internalized by non-macrophages,likehepatocytes and hepatic stellate cells,\nthe CAR expression in these cells was negligible because its signaling domains\nfunctioned specifically as stimulatory signals in immunocytes. Moreover, given the\nextensive and high expression of FAP in activated hepatic stellate cells, the low\nexpression of CAR in the non-macrophages would not affect the recognition and\nphagocytosis of CAR-Ms toward FAP+ hepatic stellate cells. In contrast, the\nadditional expression of TRIM13 in non-macrophages would also exert\nanti-inflammatory effect via promoting STING ubiquitination and subsequent\ndegradation,thereby contributingtofibrosis resolution.\nQ2: Engineering macrophages into CAR is a very powerful strategy for treating\nliver fibrosis. If the macrophages in the spleen can also be powered with CAR,\ncan it enhance the therapeutic efficiency? Please comments on why macrophages\ninspleen havebeen avoidedforexpressionof CAR.\nA: We appreciate the reviewer's valuable comment. During fibrosis progression,\ntissue-resident macrophages are dynamically replenished by monocytes from\nperipheral blood andspleen,suggesting that CAR engineering of splenicmacrophages\ncould potentially contribute to the clearance of FAP+ hepatic stellate cells. However,\nFAP is not a highly specific target and expressed on a lot of normal cells.12, 13\nTherefore, circulating CAR-monocytes/ macrophages derived from the spleen could\nattack healthy FAP-expressing cells, posing significant on-target/ off-fibrosis toxicity\nrisks. Consequently, we developed a liver-restricted macrophage engineering platform\n(ESLNP)to minimizepotential damage to healthy tissues.\nQ3: The authors used H O to simulate the ROS environment in fibrotic livers;\n2 2\nhowever, the rationale for selecting the specific concentration of 0.1 mM H O is\n2 2\nnot explicitly discussed in the manuscript. Please provide a detailed explanation\ntoclarify thebasisforthis choice.\nA: We are truly grateful for the reviewer’s detailed comment. In the fibrotic liver\nenvironment, ROS levels are significantly elevated, and the concentration of 0.1 mM\nH₂O₂ has been used in other studies to induce the activation of HSCs.14,15 Moreover, a\nrelated study reported the use of 0.1 mM H O to investigate ROS-responsive drug\n2 2\ntherapy for liver fibrosis.16 These reports collectively support the use of 0.1 mM H₂O₂\nas an effective and reliable approach to mimic the ROS-enriched environment of\nfibrotic liver, providing a solid experimental basis for exploring fibrosis mechanisms\nand potential therapeuticstrategies..\nQ4: The fluorescence images presented in the manuscript should be supported\nby quantitative data with n ≥ 3 to enhance the robustness and reliability of the\nfindings.\nA: We sincerely appreciate the reviewer’s detailed suggestion. Following the\nreviewer’s suggestion, we performed quantitative analysis of the fluorescence signals\nin Fig.1K, 1L, S14 and S18, and have incorporated the data and corresponding\ndescriptions into therevised manuscript.\nFig. S8 (A) Quantitative analysis of mean fluorescence intensity (MFI) in RAW264.7\ncells and BMDMs co-incubated with free FAM-labeled mRNA or ESLNPs (n=3).\nData are presented as mean ± SD. Statistical significance among groups was assessed\nusing one-wayANOVA. Significance levels were denoted as ns P ≥ 0.05, * P < 0.05,\n*** P < 0.001, **** P < 0.0001 (B) Quantitative analysis of co-localization of\nFAM-labeled mRNA with lysosomes (n=3). Data are presented as mean ± SD.\nStatistical significance among groups was assessed using Student’s t test. Significance\nlevels were denoted as *P< 0.05.\nFig. S14 Representative CLSM images and quantitative analysis of FAPexpression in\nHSCs treated with 10ng/ml TGF-β for 0h, 24h and 48h. The FAP was labeled with\nanti-FAP (green). Scale bar, 20 μm. Data are presented as mean ± SD. Statistical\nsignificance among groups was assessed using one-way ANOVA. Significance levels\nwere denoted as ****P<0.0001.\nFig. S18 Immunofluorescence images and quantitative analysis illustrated the\nexpression of FAPin various treatment groups. Red, FAPlabeled with anti-FAP; blue,\ncell nuclei (n=9). Scale bar, 100 μm. Data are presented as mean ± SE. Statistical\nsignificance among groups was assessed using one-way ANOVA. Significance levels\nwere denoted as *P<0.05,**P<0.01,****P< 0.0001.\nQ5: For IVIS, please display the actual bar values (specific numerical values)\nratherthan usingonly \"highto low\"to representsignal intensity.\nA: We thank the reviewer for this comment and constructive suggestion. Following\nthe reviewer’s suggestion, we have added the actual bar values to the IVIS figures\naccordingly and updated thedata intherevised manuscript as follows.\nFig.3B IVIS images of mice 12h post-treatment of DOPE-LNP, ESLNP and PS-LNP\nencapsulating Luci mRNA.\nFig.3C Representative images of bioluminescence signal biodistribution in isolated\norgans.\nFig.3G IVIS images of mice treated with ESLNP encapsulating Luci-mRNA at\ndifferent time points. The Luci substrate was injected at 0h, 24h, 48h, 72h, 96h and\n120hintraperitoneally after ESLNPs treatment.\nQ6: In Figure 2J, the normal condition has >60% EGFP expression, and H O\n2 2\nonly increased slightly EGFP expression, please explain why there’s still a high\nportion of cell taken up LNPs under normal condition will that impact\nspecificity.\nA: We sincerely appreciate the reviewer’s insightful comment. Macrophages\ninherently possess the capacity to engulf foreign substances, enabling them to\nnon-specifically internalize nanodrugs, such as LNPs. Given the robustly non-specific\nphagocytosis by macrophages and the absence of other cells that were cocultured to\ncompetitively uptake LNPs, specific endocytosis mediated by exposed PS-receptor\ninteractions in the H O -mimicked ROS-enriched environment may not lead to a\n2 2\nnotable enhancement in the delivery efficiency. This may explain the only slightly\nincreased expression levels of encoded protein in targeting molecules-labelled LNPs\ncompared to undecorated LNPs, aphenomenon observed inpriorresearch.17\nUpon systemic administration, LNPs highly accumulate in spleen and the liver\ndue to the opsonization and subsequent sequestration by the mononuclear phagocyte\nsystem and hemorheological/blood vessel flow limitations. As to our ESLNPs, the\nexposed PS promotes the specific accumulation of LNPs in macrophages in the\nfibrotic liver, while the shielded PS reduced the enrichment of LNPs in spleen,\nthereby enhancing the in vivo editing specificity of LNPs, which was confirmed by\nthe reduced splenic accumulation and enhanced hepatic enrichment in the IVIS\nimages (Fig. 3B-D). Importantly, flow cytometry demonstrated that ESLNPs\nsubstantially increased the proportion of CAR-positive macrophages in fibrotic livers,\nindicating that targeting specificity was effectively maintained (Fig. 3Eand F).\nQ7: In Figure 5D, the differences between the various groups appear to be\nminimal, or the field of view in the images may be too small. Please adjust the\npresentationto provideaclearerandmoredistinctrepresentationof thedata.\nA: We sincerely appreciate the reviewer’s detailed suggestion. Following the\nreviewer’s suggestion, we have embarked on a thorough re-evaluation of the\nrepresentative fields presented in Fig. 5D (Fig. 4D in the revised version), and fully\nagree that the original images may not sufficiently highlight the differences between\ngroups due to the limited field of view. To address this, we have replaced the images\nwith larger and more representative fields that better capture the overall histological\ncharacteristics and more clearly reflect the distinctions among groups. Webelieve that\nthese modifications will significantly improve the quality of our manuscript and\nfacilitateabetter understanding ofour work byreaders.\nFig. 4D Immunofluorescence images of STING expression in various treatment\ngroups. Green, F4/80; red, STING; blue, cell nuclei. Scale bar,200μm.\nReviewer#3:\nIn the manuscript by Gao et al., the authors developed a new chimeric antigen\nreceptor macrophages (CAR-Ms) therapy against liver fibrosis using\nefferocytosis-sparked lipid nanoparticles specifically activated under oxidative\nstress conditions. These nanoparticles are designed to expose phosphatidylserine\nto be incorporated by macrophages under oxidative stress conditions, and they\nconfer CAR-Ms the ability to specifically engulf activated hepatic stellate cells\n(aHSCs) via anti-fibroblast activation protein (FAP). Overexpression of TRIM13\ndownregulated STING to sustain anti-inflammatory phenotype of CAR-Ms.\nAlthough this idea is potentially interesting, Gao et al. recently published a\nsimilarstrategy using CAR-Ms expressing anti-FAPagainstcardiac fibrosis (Gao\net al., Cell discovery, 2024 PMID:39134538). Moreover, Lee et al. also designed\nartificial liposomes to reduce the viability of aHSCs utilizing FAP to attenuate\nliver fibrosis (Lee et al., Nat Commun, 2022 Ref.17). Therefore, additional\nexperiments will be necessary to establish this CAR-M system as a new and\npromisingstrategy to treatliverfibrosis.\nQ1: This system uses TRIM13 overexpression to ubiquitylate and degrade\nintracellular STING as reported by Li et al. (Li et al., Sci Adv, 2022 Ref.16),\nthereby inactivating cGAS-STING pathway to maintain CAR-Ms in an\nanti-inflammatory state. However, TRIM13 was reported to ubiquitylate several\nother inflammatory proteins including TRAF6 (Huang and Baek, Mol\nPharmacol, 2017 PMID: 28087809) and Nur77 (Huang et al., Sci Rep 2018 PMID:\n30224829). Moreover, TRIM13 self-ubiquitylation was reported to be involved in\np62-mediated ER-phagy (Ji et al., Mol Cell, 2019 PMID: 31375263). Therefore,\nTRIM13 overexpression can change the stability of various proteins other than\nSTING and affect NF-κB signaling.The authors should discuss these possibilities,\nand if possible, check the amount of these proteins experimentally by Western\nblot, immunostaining.orFACS.\nA: We sincerely appreciate the reviewer’s insightful comment concerning the broader\nfunctional roles of TRIM13 that extend far beyond its regulation of STING. In the\nrevised manuscript, we initially conducted a systematic validation to ascertain the\nimpact of TRIM13 upregulation on the activation of the cGAS-STING pathway by\nquantifying theprotein level of cGAS, p-IRF3, and p-TBK viawestern blot.Moreover,\nwe utilized flow cytometry to assess the expression level of IκBɑ, an essential\nregulator of NF-κB signaling, to explore the regulatory effect of TRIM13 on other\ninflammatory signaling pathways in addition to the GAS - STING pathway. We\nbelieve that the further exploration of these molecular mechanisms would be valuable\nfor future studies to fully elucidate the pleiotropic roles ofTRIM13. The added results\nand analysis are as follows.\n“Western blot analysis demonstrated that TRIM13 protein was markedly\nupregulated in the T@ESLNPgroup (ESLNPs encapsulating TRIM13 mRNA),\nwhile STING protein levels and the phosphorylation of TBK1 and IRF3 were\nsubstantially reduced. In contrast, no discernible changes were observed following\ntreatment with free TRIM13 mRNA or s@ESLNPs (ESLNP encapsulating scramble\nmRNA) (Fig. 3D).”\nFig.3D Representative western blot analysis of TRIM13 and STING pathway in\nmtDNA-pretreated BMDMs from various treatment groups.\n“Moreover, TBK1 phosphorylation is reported to promote NF-κB activation,\nleading us to infer that TRIM13 might likewise suppress NF-κB pathway. We then\nutilized flow cytometry to assess the expression level of IκBɑ, an essential regulator\nof NF-κB signaling, and found that the upregulation of TRIM13 partially rescued the\ndownregulated IкBɑ level resulted by mtDNA, further supporting TRIM13’s\ninhibitoryrole inNF-κB pathway activation (Fig. S12).”\nFig.S12 Representative flow cytometric plot and quantitative analysis of IкBɑ\nexpression in macrophages with mtDNA co-incubation and further treated with PBS,\ns@ESLNP, T@ESLNP (n=3). Data are presented as mean ± SD. Statistical\nsignificance among groups was assessed using one-way ANOVA. Significance levels\nwere denoted as ns P ≥ 0.05,***P<0.001,****P< 0.0001.\nQ2: For all the FACS data in the Figures (Fig1D, Fig2I, Fig3H, Fig4E, Fig6ABC,\nand FigS18), please describe both the antigens and fluorescent dyes in the axis as\ninFigS19-21.\nA: We thank the reviewer for the detailed suggestion. In accordance with the\nreviewer’s advice, we have revised all relevant flow cytometry figures (Fig. 1D, Fig.\n2I, Fig. 3H, Fig. 4E, Fig. 6A–C, and Fig. S18) with the full annotation of both\nantigens and corresponding fluorescent dyes on the axes, consistent with the format\nused in Fig. S19–S21. This modification enhances the clarity and interpretability of\nthe flow cytometry data throughout the manuscript, and the modified and renumbered\nFACSplot are as follows.\nFig. 1I Flow cytometry analysis of ROS-responsive PS exposure of ESLNP with\nFITC-AnnexinV.\nFig. 2H Representative flow cytometric plots depicting the expression of Ly6C in\nBMDMs treated with PBS,T@ESLNP,C@ESLNPand CT@ESLNP.\nFig. 3E Representative flow cytometric plots depicting the percentage of macrophage\ninMyc-positive cells.\nFig. 5A-C Representative flow cytometric plots and quantitative analysis of\nmacrophages Ly6C (A), CD206 (B) and CD86 (C) expression inhepatic macrophages\nfrom micesubjected to various treatments (n=6).\nFig. S22 The changes in immune cell composition in livers after various treatments.\nRepresentative flowcytometry analysis and quantitative analysis of the percentages of\n(A) CD45+CD11b+Ly6G+ neutrophils, (B) CD45+CD11c+CD80+CD86+ dendritic cells\nand (C)CD45+CD3+Tcells in thelivertissues (n=6).\nQ3: In Figure 3, the authors claim that FAP-positive activated HSCs are\nengulfed by macrophages treated with C@ESLNPorCT@ESLNP. However, it is\ndifficult to distinguish whether the macrophages merely bind to the target cells\nor actually engulf them. To clarify this, the authors should label aHSCs or\nqHSCs with pHrodo Red and assess whether the fluorescence signal\nincreases—indicating acidification in the lysosomes following phagocytosis.\nAlternatively, transmission electron microscopy should be performed to directly\ndemonstrate theengulfment ofaHSCsbymacrophages.\nA: We sincerely appreciate this insightful suggestion. To further distinguish between\nthe binding and phagocytosis of HSCs, we repeated phagocytosis experiments using\npHrodo™ Red-labeled HSCs. As shown in the confocal images, the fluorescence\nsignal was substantially increased in CAR-Ms that was coincubated with aHSCs,\nindicating the successful engulfment. We believe this additional evidence strengthens\nour conclusion that engineered macrophages actively phagocytose FAP-positive\naHSCs.\n“The targeting phagocytic property of CAR-Ms was also confirmed by the markedly\nincreased fluorescence signal detected in CAR-Ms that was coincubated with aHSCs,\nwhereas negligible fluorescence was observed in all experimental groups coculturing\nmacrophages with quiescent HSCs(Fig. 2G).”\nFig.2G Representative CLSM images of HSCs or aHSCs phagocytosed by\nmacrophages orCAR-Ms.TheHSCs/aHSCs were stained with pHrodo Red. Scale bar,\n50μm.\nQ4: In the RESULTS section describing Fig4, the authors mainly used LNPs\nencapsulating Luciferase mRNA to detect bioluminescent signals. However, the\nFACS data in Fig4E-F detected Myc signals using c@ESLNPs. Although this is\ndescribed in the MATERIALSAND METHODS section, please also indicate this\npointinthe RESULTSsection toavoid confusion.\nA: We thank the reviewer for pointing out this important clarification. To avoid\npotential confusion, we have now revised the Results section corresponding to Fig.\n3E–F to explicitly state that, unlike the bioluminescence imaging experiments using\nluciferase mRNA, the flow cytometry analysis was performed using LNPs\nencapsulating Myc-tagged mRNA. This clarification, which was previously only\nmentioned in theMaterials and Methods section, has nowbeen included in theResults\nsection for improved clarity and consistency.\n“Next, we administered DOPE-LNPs and ESLNPs encapsulating Myc-tagged\nCAR mRNAto evaluate their cellular tropisms. Flow cytometry analysis revealed that\nESLNP predominantly transduced liver macrophages, accounting for 81.66 ± 3.94%\nof Myc-positive cells, and that the proportion presented remarkable elevations in the\nfibrotic mice treated with ESLNP compared to both the ESLNP-treated control mice\nand DOPE-LNPtreatment groups (Fig. 3Eand F).”\nQ5: The authors examined only one in vivo model of liver fibrosis (CCl4\ninjection). However, this in vivo model is highly inflammatory and artificial. Lee\net al. used three models (CCl4, bile duct ligation, and CDAHFD) to show the\neffectiveness of their aHSC-targeted liposomes (Lee et al., Nat Commun, 2022\nRef.17). To further substantiate the authors’ conclusion and extend the future\nclinical application, the authors should test the efficacy using other experimental\nfibrosis models, such as CDAHFD-induced liver fibrosis. If the\nefferocytosis-sparked lipid nanoparticles established by the authors are also\neffective in lipid-rich steatohepatitis, this would increase the chance of future\nclinical applications forhuman diseases such as metabolic dysfunction-associated\nsteatohepatitis (MASH).\nA: We sincerely appreciate the reviewer’s constructive suggestion, which was also\nhighlighted byReviewer 1.Tobetter capture clinically relevant disease characteristics,\nwe added the HFMCD model that mimicked the key features of metabolic-associated\nliver fibrosis. We believe this model provides a more physiologically relevant context\nfor evaluating the therapeutic potential of our strategy and thus strengthens the\ntranslational significance ofour findings.\n“Male C57BL/6J mice (6–8 weeks) were fed a HFMCD diet to establish the\nHFMCD model, while control mice received a normal diet under identical housing\nconditions. After model establishment, treatment was administered following the\nsame protocol as used in the original CCl -induced fibrosis model. Therapeutic\n4\noutcomes were evaluated by histological analysis of liver sections and quantification\nofHYPcontent.”\n“Moreover, we constructed a HFMCD-induced model to comprehensively test\nthe therapeutic efficacy of ESLNP, which mimicked metabolism-associated liver\nfibrosis characterized by hepatocellular injury, lipid accumulation, inflammation, and\nprogressive fibrosis. After 2 weeks of treatment, liver tissues were harvested for\nhistological examination and HYP quantification (Fig. 4P). CT@ESLNPs treatment\nreduced both microvesicular and macrovesicular steatosis in H&E-stained sections,\nsignificantly decreased Masson staining, and lowered the positive areas of Sirius Red\nand α-SMA staining (Fig. 4Q-T). Meanwhile, hepatic HYP levels were markedly\ndecreased in CT@ESLNPs-treated mice (Fig. 4U). These data collectively indicated\nthe powerful therapeutic efficacy of ESLNP-edited anti-inflammatory CAR-Ms,\nwhich could promote fibrosis resolution and liverregeneration.”\nFig.4 (P) Schematic illustration of the experimental design in HFMCD-induced liver\nfibrosis model. (Q) Representative images of H&E, Masson staining, SiriusRed\nstaining, and α-SMA staining in the liver tissues. Scale bar, 100μm. (R-T)\nQuantitative analysis of Masson staining (R), SiriusRed staining (S) and α-SMA\nstaining (T) in liver tissues at the end of the treatment (n=9). Data are presented as\nmean ± SE. Statistical significance among groups was assessed using one-way\nANOVA. Significance levels were denoted as ns P ≥ 0.05, * P < 0.05, ** P < 0.01,\n**** P < 0.0001. (U) HYP content in liver tissues of various treatment groups (n=6).\nData are presented as mean ± SE. Statistical significance among groups was assessed\nusing one-way ANOVA. Significance levels were denoted as* P < 0.05, **** P <\n0.0001.\nQ6: In the Cells part of the MATERIALS AND METHODS section, please\ndescribe the preparation method of primary HSCs in more detail. Also, the\npurity of primary HSCs should be checked by autofluorescence of vitamin A\ndropletoranti-Desmin antibody staining.\nA: We sincerely thank the reviewer for this detailed comment. According to the\nreviewer’s suggestion, we have revised the Materials and Methods section to provide\na more detailed description of the isolation procedure for primary hepatic stellate cells\n(HSCs), including perfusion, liver digestion, and density gradient centrifugation steps.\nIn addition, to address the reviewer’s concern regarding cell purity, we performed\nflow cytometry analysis to assess HSC identity using an anti-Desmin antibody. The\nresults confirmed that the isolated HSCs were of high purity,and these data have been\nincluded in therevised manuscript as Fig.S14.\n“Primary HSCs were isolated by differential centrifugation. Briefly, the mouse\nwas sterilized and the liver was exposed, then perfused via theportal vein with 30 mL\nof calcium- and magnesium-free Hank’s solution followed by 1 mg/mL type IV\ncollagenase (3 mL/min). The liver was excised, minced, and digested in a 37 ℃\noscillating water bath for 30 minutes. The cell suspension was filtered through a\n100-μm nylon mesh, washed, and centrifuged twice. Hepatic stellate cells (HSCs)\nwere isolated by continuous 60% Percoll density gradient centrifugation (4 ℃, 9600\nrpm, 20 min). HSCs were cultured in DMEM-F12 (Gibco) containing 10% fetal\nbovine serum (HyClone) and 1% penicillin/streptoMycin (Gibco) in a 5% CO\n2\natmosphere at 37℃”\n“Subsequently, we extracted the primary HSCs and established FAP-positive\naHSC cell model by incubating mouse HSCs with TGF-β (10 ng/mL) for the\nfollowing phagocytosis evaluation (Fig. S14).”\nFig. S14 (A) The purity of HSCs verified by flow cytometry (B) Representative\nCLSM images of FAP expression in HSCs treated with 10ng/ml TGF-β for 0h, 24h\nand 48h. The FAP was labeled with anti-FAP (green) (n=3). Scale bar, 20 μm. Data\nare presented as mean ± SD. Statistical significance among groups was assessed using\none-wayANOVA. Significance levels were denoted as, ****P<0.0001.\nQ7: Please indicate the source of mRNA sequence of anti-FAP CAR. As the\nnucleotide sequence in Table S2 is not found in the nucleotide database, the\nreaders cannotbeconfidentwhetherthisprotein bindsto mouseFAP.\nA: We thank the reviewer for the valuable comment. The amino acid sequence of the\nanti-FAP CAR used in our study is consistent with that of a previously reported\nantibody targeting murine FAP.18 The mRNA sequence was codon-optimized for\nexpression in mouse cells and synthesized accordingly, which might explain why the\nexact nucleotide sequence is not available in public databases. To enhance\ntransparency and avoid potential confusion, we have revised Table. S2 to display the\namino acid sequence of the CAR construct instead of the optimized nucleotide\nsequence. We have also included a reference to the original source of the protein\nsequence in the revised manuscript. We appreciate the reviewer’s suggestion, which\nhelped us clarify theorigin and specificity ofour CARdesign.\nTable. S2Amino acid sequence.\nName Sequence\nMASPLTRFLSLNLLLLGESIILGSGEAQVQLKESGGLVQPGGSLKL\nSCAASGFTFSSYGMSWVRQTADKRLELVATTNNNGGVTYYPDSV\nKGRFTISRDNAKNTLYLQMSSLQSEDTAMYYCARYGYYAMDYW\nGQGISVTVSSGGGGSGGGGSGGGGSDVLMTQTPLWLPVSLGDQA\nSISCRSSQSIVHSNGNTYLEWYLQKPGQSPKLLIYKVSNRFSGVPD\nAnti-FAP RFSGSGSGTDFTVKISRVEAEDLGVYYCFGGSHVPYTFGGGTKLEI\nCAR KEQKLISEEDLTTTKPVLRTPSPVHPTGTSQPQRPEDCRPRGSVKG\nTGLDFACDIYIWAPLAGICVALLLSLIITLIRAKFSRSAETAANLQDP\nNQLYNELNLGRREEYDVLEKKRARDPEMGGKQQRRRNPQEGVY\nNALQKDKMAEAYSEIGTKGERRRGKGHDGLYQGLSTATKDTYDA\nLHMQTLAPR\nMELLEEDLTCPICCSLFDDPRVLPCSHNFCKKCLEGLLEGNVRNSL\nWRPSPFKCPTCRKETSATGVNSLQVNYSLKGIVEKYNKIKISPKMP\nVCKGHLGQPLNIFCVTDMQLICGICATRGEHTKHVFSSIEDAYARE\nKNAFESLFQSFETWRRGDALSRLDTLETNKRKALQLLTKDSDKV\nKEFFEKLQHTLDQKKNEILSDFETMKLAVMQTYDPEINKINTILQE\nTRIM13\nQRMAFNIAEAFKDVSEPIIFLQQMQEFREKIKVIKETPLPHSNLPTS\nPLMKNFDTSQWGDIKLVDVDKLSLPQDTGVFTSKIPWYPYLLLM\nMVVLLGLLIFFGPTVFLEWSPLDELATWKDYLSSFNSYLTKSADFI\nEQSVFYWEQMTDGFFIFGERVKNVSLVALNNVAEFICKYKLL\nQ8: In the cartoon of ESLNPs in Fig1F, Fig2D and Fig4A, PnT14 has two fatty\nacid tails. However,its structuresuggests that PnT14 shouldhavethree fatty acid\ntails.Please confirmthispoint.\nA: We thank the reviewer for the detailed comment. We apologize for the\ninconsistency in the schematic representations shown in Fig. 1F (Fig. 6F in the\nrevised version), Fig. 2D (Fig. 1D in the revised version), and Fig. 4A(Fig. 3Ain the\nrevised version), where only two tails were illustrated for simplicity. We have now\nrevised the corresponding cartoons to accurately depict the three-tail structure of\nPnT14, consistent with its actual molecular configuration. These updated figures have\nbeen updated intherevised manuscript.\nFig. 6F Schematic illustration of in vivo generation of aHSCs-specific\nanti-inflammatory CAR-Ms mediated by ESLNPs for reversing liver fibrosis. In the\nROS-enriched fibrotic liver, the exposure of PS triggers LNPs internalization by\nmacrophages in an efferocytosis-like manner, which thus in situ engineering\nmacrophages with anti-FAP CAR and TRIM13 for reversing liver fibrosis based on\naHSCs-specific clearance and inflammatory resolution\nFig.1DESLNPsynthesis andtransfection efficacy evaluation in macrophages.\nFig. 3A Schematic diagram of ESLNP’s liver macrophages-targeting delivery.\nESLNPs circulating to the fibrosis areas exfoliate thiazolidine-2-one upon ROS\nstimulation, and then PS is exposed and induces macrophage internalization via\nbindingtophosphatidylserine receptor.\nQ9:The description of the statistical analysis is currently insufficient throughout\nthe Figures. The authors should specify the statistical methods used for each\nfigure;forexample, two-tailed unpaired Student’s t-test (a) andone-wayANOVA\nwith Tukey’s multiple comparison test for panel (b) Furthermore, since the data\nwere obtained from multiple individual mice rather than from triplicate\nexperiments using cell lines, the results should be expressed as mean ± standard\nerror(SE),ratherthanstandard deviation(SD).\nA: We sincerely appreciate the reviewer’s constructive suggestion. In the revised\nmanuscript, we have detailed the specific statistical methods in each figure legend to\nclarify the applied tests. Furthermore, we have reformatted data from animal\nexperimentsto reflect mean ± SE and corrected in therevised version.\nQ10: In Figure S17, the signal of the indicated antibody was too weak to allow\nproper evaluation. The authors should replace these immunohistochemistry\nresultswith morerepresentativeimages.\nA: We thank the reviewer for the thoughtful suggestion. We have deleted the\nimmunohistochemistry images with weak signal in the revised manuscript and\nemployed flow cytometry, a more sensitive detection technology, to quantify the\nexpression levels of M1 marker and M2 marker in macrophages following different\ntreatments, which provide a more robust and quantifiable evaluation of the relevant\ncellular changes.\nFig.5 B-C Representative flow cytometric plots and quantitative analysis of\nmacrophages CD206 (B) and CD86 (C) expression inhepatic macrophages from mice\nsubjected to various treatments (n=6). Data are presented as mean ± SE. Statistical\nsignificance among groups was assessed using one-way ANOVA. Significance levels\nwere denoted as *P<0.05,**P< 0.01,***P <0.001,****P< 0.0001.\nQ11:The authors refer to their own results, including specific figure numbers, in\nthe Introduction.This practice does notalign with the standardformat of Nature\nCommunications. The authors should relocate these descriptions to the Results\nsection, wherethey aremoreappropriate.\nA:We thank the reviewer for the detailed suggestion. In accordance with the journal’s\nguidelines, we have substantially revised the Introduction section to focus solely on\nbackground, rationale, and the broader context of the study. The data in the original\nFig. 1 were mostly derived from preliminary mechanism‐verification experiments\nusing other delivery vehicle in our earlier work. We removed certain panels from the\noriginal Fig. 1 as these preliminary data duplicated information presented in Fig. 3\n(Fig. 2 in the revised version). Consolidating them improves the manuscript’s\nnarrative and the clarity of data presentation. This reorganization was done solely to\nimprovepresentation and does notchange theexperimental findings.\nQ12: In theAuthorcontributions section, the description of Maosen Han (MH) is\nmissing.\nA: We appreciate the reviewer’s careful review and apologize for the oversight. We\nhave now supplemented the contribution of Maosen Han (MH) in the Author\nContributionssection ofthe revised manuscript.\n“M.H., X.D. and C.L. characterized theproperties ofthematerials.”\nQ13: In the MATERIALS AND METHODS section, mixed usage of h/hours,\nmin/minutes, and ×/* arefound.\nA: We thank the reviewer for the detailed comment. We have thoroughly\ndouble-checked and revised the Materials and Methods section to ensure consistent\nusageof unitsand symbols throughout themanuscript.\nReviewer#4:\nI co-reviewed this manuscript with one of the reviewers who provided the listed\nreports. This is part of the Nature Communications initiative to facilitate\ntraining in peer review and to provide appropriate recognition for Early Career\nResearchers whoco-reviewmanuscripts.\nA: We sincerely thank the reviewers for their invaluable time and expertise in\nevaluating our manuscript. The collaborative review process facilitated by Nature\nCommunications to support Early Career Researchers is greatly appreciated. All\ncomments have been systematically addressed in therevised manuscript, with detailed\npoint-by-point responses provided below and corresponding revisions highlighted in\nthemain text.Weremain available to providefurther clarification as required.\nReferences\n1. Wang, X. et al. Prolonged hypernutrition impairs TREM2-dependent efferocytosis to license\nchronicliverinflammationandNASHdevelopment.Immunity56,58-77.e11(2023).\n2. M T, L. et al. Fibroblast activation protein: a cell surface dipeptidyl peptidase and gelatinase\nexpressed by stellate cells at the tissue remodelling interface in human cirrhosis. Hepatology\n29(1999).\n3. Yang, A.T. et al. Fibroblast Activation Protein Activates Macrophages and Promotes\nParenchymal Liver Inflammation and Fibrosis. Cell Mol Gastroenterol Hepatol 15, 841-867\n(2023).\n4. Keane,F.M.etal.Quantitationoffibroblastactivationprotein(FAP)‐specificproteaseactivity\ninmouse,baboonandhumanfluidsandorgans.FEBSOpenBio4,43-54(2013).\n5. Levy MT, M.G., Marinos G, Gorrell MD. Intrahepatic expression of the hepatic stellate cell\nmarker fibroblast activation protein correlates with the degree of fibrosis in hepatitis C virus\ninfection.Liver22(2),93-101(2002).\n6. Levy, M.T., McCaughan, G.W., Marinos, G. & Gorrell, M.D. Intrahepatic expression of the\nhepatic stellate cell marker fibroblast activation protein correlates with the degree of fibrosis\ninhepatitisCvirusinfection.Liver22,93-101(2002).\n7. Gao, J. et al.A FAPalpha-activated MRI nanoprobe for precise grading diagnosis of clinical\nliverfibrosis.NatCommun15,8036(2024).\n8. Amor,C.etal.SenolyticCARTcellsreversesenescence-associatedpathologies.Nature583,\n127-132(2020).\n9. Zhang,Z.etal.Cardiolipin-mimiclipidnanoparticleswithoutantibodymodificationdelivered\nsenolyticinvivoCAR-Ttherapyforinflamm-aging.Cellreports.Medicine6,102209(2025).\n10. Efsen, E. et al.Agonist-specific regulation of monocyte chemoattractant protein-1 expression\nbycyclooxygenasemetabolitesinhepaticstellatecells.Hepatology33,713-721(2001).\n11. Kim, Y. et al.Anti-fibrotic activity and enhanced interleukin-6 production by hepatic stellate\ncells in response to imatinib mesylate. Liver international : official journal of the\nInternationalAssociationfortheStudyoftheLiver32,1008-1017(2012).\n12. Suh, M., Gil, J., Kang, Y.K., Choi, H. & Cheon, Gi J. Aged‐Related Fibroblast Activation\nProtein Expression in Skeletal Muscles Evaluated by PET Imaging. Journal of Cachexia,\nSarcopeniaandMuscle16(2025).\n13. Bae,S.etal.Fibroblastactivationproteinαidentifiesmesenchymalstromalcellsfromhuman\nbonemarrow.BritishJournalofHaematology142,827-830(2008).\n14. García-Trevijano, E.R. et al. Transforming growth factor beta1 induces the expression of\nalpha1(I)procollagenmRNAbyahydrogenperoxide-C/EBPbeta-dependentmechanisminrat\nhepaticstellatecells.Hepatology29,960-970(1999).\n15. Shao, M. et al. From liver fibrosis to hepatocarcinogenesis: Role of excessive liver H(2)O(2)\nandtargetingnanotherapeutics.Bioactivematerials23,187-205(2023).\n16. Hao, Y. et al. Reactive Oxygen Species-Responsive Polypeptide Drug Delivery System\nTargeted Activated Hepatic Stellate Cells to Ameliorate Liver Fibrosis. ACS Nano 16,\n20739-20757(2022).\n17. Wang, W. et al. In vivo FAP-CAR macrophages enhance chemotherapy and immunotherapy\nagainst pancreatic cancer by removing the fibrosis barrier. J Control Release 384, 113888\n(2025).\n18. Wang, L.-C.S. et al. Targeting FibroblastActivation Protein in Tumor Stroma with Chimeric\nAntigen Receptor T Cells Can Inhibit Tumor Growth and Augment Host Immunity without\nSevereToxicity.CancerImmunologyResearch2,154-166(2014)."}, {"round": 2, "reviewer_comments": {"reviewer_1": "The revised manuscript has already addressed all the concerns.", "reviewer_2": "The authors have addressed all my questions. I don't have further questions regarding the manuscript.", "reviewer_3": "In the revised manuscript by Gao et al., the authors have responded to reviewer comments and added new data—including HFMCD treatment and scRNA-seq—which substantially improve the manuscript. However, several additions lack essential methodological and data-availability details. The following issues should be addressed before the paper can be considered for acceptance.\nMajor Points:\n1. In Figure 4k, please provide the full nucleotide sequences of the primers for Col1a1, Col2a1, Timp1, and β-actin in a new Supplementary Table.\n2. List all antibodies used (flow cytometry/FACS, immunohistochemistry, and Western blot) in a new Supplementary Table, including suppliers and catalog numbers.\n3. In Figure 6, deposit the liver scRNA-seq dataset from PBS- and CT@ESLNP-treated mice to a public repository (e.g., GEO) and include the accession number in the manuscript.\n4. The revised manuscript (p. 20, line 4) states that 262 cells were collected, whereas the rebuttal letter (p. 11, line 1) reports 21,850 cells. Please reconcile this discrepancy. In general, 262 cells are insufficient for a robust scRNA-seq analysis.\n5. On p. 26, line 11, specify the manufacturers (and catalog numbers) of the HFMCD and normal control diets.\nMinor Point:\n• In the summary cartoon (Fig. 6F), replace “CCl₄-induced liver fibrosis” with “CCl₄- and HFMCD-induced liver fibrosis” or simply “liver fibrosis,” since the data show CAR-M also mitigates HFMCD-induced fibrosis.", "reviewer_4": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts."}, "author_response": "Response to reviewers’ comments:\nWe greatly appreciate the constructive feedback from the reviewers and editors,\nwhich helped us improve the quality of the manuscript. According to the reviewers’\nsuggestion, we have added additional data and further modified certain descriptions.\nBelow, please find our point-by-point responses to the reviewers’ comments.\nReviewer #1:\nThe revised manuscript has already addressed all the concerns.\nA: We sincerely thank the reviewer for all the thoughtful review and we are pleased\nto hear that the revised manuscript has successfully addressed all the concerns. We\ntruly appreciate the time and effort the reviewer has dedicated to evaluating our\nmanuscript, and we believe the revisions have strengthened the overall quality of our\nwork.\nReviewer #2:\nThe authors have addressed all my questions. I don't have further questions\nregarding the manuscript.\nA: We sincerely thank the reviewer for the positive feedback. We are pleased to hear\nthat all of your questions have been addressed, and we greatly appreciate the time and\neffort you have put into reviewing our manuscript. Your thoughtful evaluation has\nbeen invaluable in improving the quality of our work.\nReviewer #3:\nIn the revised manuscript by Gao et al., the authors have responded to reviewer\ncomments and added new data-including HFMCD treatment and\nscRNA-seq-which substantially improve the manuscript. However, several\nadditions lack essential methodological and data-availability details. The\nfollowing issues should be addressed before the paper can be considered for\nacceptance.\nQ1: In Figure 4k, please provide the full nucleotide sequences of the primers for\nCol1a1, Col2a1, Timp1, and β-actin in a new Supplementary Table.\nA: We thank the reviewer for this detailed comment. In the revised supplementary\ninformation, we have added the primer sequences for qPCR in Table. S3 to provide\nclearer and more comprehensive details regarding the experimental design.\nTable. S3 Primers used for qPCR\nName Sequence\nM-col1a1-F 5’-CTCCCAGAACATCACCTATCACT-3’\nM-col1a1-R 5’-GGAGGTCTTGGTGGTTTTGTATT-3’\nM-col2a1-F 5’-GGAAGAGCGGAGACTACTGGATT-3’\nM-col2a1-R 5’-GCTCTTGCTGCTCCACCAGTT-3’\nM-TIMP1-F 5’-GCAACTCGGACCTGGTCATAAG-3’\nM-TIMP1-R 5’-TCCCACAGCCTTGAATCCTTT-3’\nM-β-actin-F 5’-GTGACGTTGACATCCGTAAAGA-3’\nM-β-actin-R 5’-GTAACAGTCCGCCTAGAAGCAC-3’\nQ2: List all antibodies used (flow cytometry/FACS, immunohistochemistry, and\nWestern blot) in a new Supplementary Table, including suppliers and catalog\nnumbers.\nA: We thank the reviewer for this valuable suggestion. In response, we have added the\nTable. S4 that lists all antibodies used in this study, including the antibody names,\nsuppliers, and catalog numbers, to ensure full transparency and reproducibility of our\nexperimental methods.\nTable. S4 Antibodies usded in this study\nAntibody Supplier Catalog number\nPE/Dazzle™ 594 anti-mouse CD80 Biolegend Cat104738\nPE/Cy7 anti-mouse Ly-6G Biolegned Cat127617\nAPC/Cy7 anti-mouse CD45.2 Biolegned Cat109824\nAPC anti-mouse CD4 Biolegned Cat100412\nPercp anti-mouse Ly-6C Biolegned Cat128027\nPE/Cy7 anti-mouse CD86 Biolegend Cat105013\nBV241 anti-mouse F4/80 Biolegend Cat123132\nFITC anti-mouse CD8a Biolegend Cat100705\nAPC/Cy7 anti-mouse CD3 Biolegend Cat100222\nPE anti-mouse CD11c Biolegend Cat117307\nPercp/Cy5.5 anti-mouse CD45.2 Biolegend Cat109827\nFAP Polyclonal Antibody ThermoFisher PA5-99313\nMyc-tag FITC Abcam ab1394\nMyc-tag FITC Abcam ab1263\nAnti-STING Abcam ab288157\nAnti-IKB alpha Abcam ab32518\nTRIM13 antibody Santa Cruz sc-398129\nAnti- Phospho-TBK1/NAK Cell signaling 5483T\nAnti-Phospho-IRF-3 Cell signaling 4947T\nAnti-cGAS Cell signaling 31659T\nAnti-TBK1/NAK Cell signaling 3504T\nAnti-IRF-3 Cell signaling 4302T\nQ3: In Figure 6, deposit the liver scRNA-seq dataset from PBS- and\nCT@ESLNP-treated mice to a public repository (e.g., GEO) and include the\naccession number in the manuscript.\nA: We sincerely appreciate the reviewer’s suggestion. We have deposited the liver\nscRNA-seq dataset from PBS and CT@ESLNP-treated mice to the Genome Sequence\nArchive (GAS) database. The accession number has been included in the manuscript.\n“The scRNA-seq data have been deposited in the Genome Sequence Archive\n(Genomics, Proteomics & Bioinformatics 2025) in National Genomics Data Center\n(Nucleic Acids Res 2025), China National Center for Bioinformation / Beijing\nInstitute of Genomics, Chinese Academy of Sciences (GSA: CRA031089) that are\npublicly accessible at https://ngdc.cncb.ac.cn/gsa.”\nShared URL: https://ngdc.cncb.ac.cn/gsa/s/VRciPRQ8\nQ4: The revised manuscript (p. 20, line 4) states that 262 cells were collected,\nwhereas the rebuttal letter (p. 11, line 1) reports 21,850 cells. Please reconcile this\ndiscrepancy. In general, 262 cells are insufficient for a robust scRNA-seq\nanalysis.\nA: We thank the reviewer for pointing out this mistake. It was a typographical error in\nthe revised manuscript. The correct number of cells collected is 21,850, as shown in\nthe “Point-by-Point Response Letter”. We have made the necessary correction in the\nrevised manuscript to reflect the accurate number of cells and apologize for any\nconfusion this may have caused.\n“To further elucidate the anti-fibrotic mechanism of CT@ESLNP, we performed\nsingle-cell sequencing of liver tissues from mice treated with PBS and CT@ESLNP.\n(Fig. 6A). After quality control, 21580 cells were collected for further analysis.”\nQ5: On p. 26, line 11, specify the manufacturers (and catalog numbers) of the\nHFMCD and normal control diets.\nA: We sincerely appreciate the reviewer’s detailed suggestion. We have specified the\nmanufacturers and catalog numbers for the HFMCD and normal control diets as\nfollows.\n“Male C57BL/6J mice (6-8 weeks) were fed a HFMCD diet (A06071301B,\nResearch Diets) to establish the HFMCD model, while control mice received a normal\ndiet (D11112201, Research Diets) under identical housing conditions.”\nQ6: In the summary cartoon (Fig. 6F), replace “CCl -induced liver fibrosis” with\n4\n“CCl - and HFMCD-induced liver fibrosis” or simply “liver fibrosis”, since the\n4\ndata show CAR-M also mitigates HFMCD-induced fibrosis.\nA: We thank the reviewer for the detailed comment. We have updated the summary\ncartoon (Fig. 6F) to replace “CCl -induced liver fibrosis” with “liver fibrosis”. The\n4\nupdated figure has been updated in the revised manuscript.\nFig. 6F Schematic illustration of in vivo generation of aHSCs-specific\nanti-inflammatory CAR-Ms mediated by ESLNPs for reversing liver fibrosis. In the\nROS-enriched fibrotic liver, the exposure of PS triggers LNPs internalization by\nmacrophages in an efferocytosis-like manner, which thus in situ engineering\nmacrophages with anti-FAP CAR and TRIM13 for reversing liver fibrosis based on\naHSCs-specific clearance and inflammatory resolution\nReviewer #4:\nI co-reviewed this manuscript with one of the reviewers who provided the listed\nreports. This is part of the Nature Communications initiative to facilitate\ntraining in peer review and to provide appropriate recognition for Early Career\nResearchers who co-review manuscripts.\nA: We sincerely thank the reviewer for the involvement in the peer review process.\nWe appreciate the unique role you played in co-reviewing this manuscript as part of\nthe Nature Communications initiative. Your contribution, alongside the other\nreviewers, has been instrumental in improving the manuscript."}, {"round": 3, "reviewer_comments": {"reviewer_3": "The authors have carefully revised the manuscript in accordance with the reviewer's suggestions. I am satisfied with the revisions, and the manuscript is now suitable for publication in Nature Communications."}, "author_response": "A: We sincerely appreciate the reviewer’s positive assessment. We are gratified that\nthe revisions we submitted have satisfactorily addressed the reviewer’s concerns, and\nwe are grateful for the thorough and constructive critique. The reviewer’s insights\nhave substantially strengthened the manuscript."}]

Decision letter