Tissue-resident macrophage survival depends on mitochondrial function regulated by SerpinB2 in chronic inflammation

Domain: neuroscience
Published: 2026-02-12
Editorial outcome: major_revision

Abstract
How cellular metabolism facilitates tissue-resident macrophage maintenance remains elusive. Here we show that visceral adipose tissue (VAT)-resident macrophages, unlike monocyte-derived macrophages, are enriched with mitochondrial-specific antioxidant enzymes restraining inflammation and promoting VAT homeostasis and insulin sensitivity. Additionally, VAT resident macrophages express high levels of plasminogen activator inhibitor type 2, encoded by SerpinB2, which is involved in the blood coagulation cascade. SerpinB2 promotes adipose resident macrophage survival by regulating mitochondrial oxidative phosphorylation and preventing the release of pro-apoptotic cytochrome c from the mitochondria into the cytoplasm via antioxidant glutathione production. Chronic inflammation, such as obesity, diminishes SerpinB2 expression in VAT macrophages in patients and mice, leading to the decline of this macrophage subset. Mechanistically, interferon-γ elevation in diabetes induces Ikaros, a transcriptional suppressor, which binds to the SerpinB2 promoter and decreases SerpinB2 expression. Congruently, selective depletion of the IFN-γ receptor in myeloid cells or supplementation of macrophage-specific SerpinB2 deficient mice with N-acetylcysteine, a glutathione precursor, restores VAT resident macrophage survival, decreases adipocyte size, and improves glucose tolerance and insulin sensitivity. Our data thus reveal an unexpected function of SerpinB2 in the regulation of mitochondrial function and survival of tissue-resident macrophages.

Introduction
Tissue-resident macrophages are important in health and disease, including tissue homeostasis, organ regeneration, removal of cellular debris, injury, and infection. A recent study showed that peritoneal resident macrophages migrate to the heart after myocardial infarction to mitigate cardiac fibrosis. Cardiac resident macrophages support mitochondrial homeostasis and electrical conduction. A novel CX3C chemokine receptor-positive resident synovial macrophages have been reported to provide a protective barrier against inflammatory reactions. Similarly, microglia, which are resident macrophages in the brain, execute important functions such as synapse plasticity and elimination, neuronal activity, and tissue repair. Moreover, resident dermal macrophages promote the regeneration of local nerves after mechanical injury and are renewed by local proliferation. Adipose macrophages are indispensable in tissue homeostasis, metabolic inflammation, and insulin resistance. Triggering receptors expressed on myeloid cells 2+ lipid-associated adipose macrophages have recently been demonstrated to elicit a protective response in obesity.

Results
VAT monocyte-derived and resident macrophages have different transcriptome profiles

Using various mouse strains, such as CX3CR1+/GFP and CX3CR1creER/+ ROSAtdTomato mice, and techniques like parabiosis and genetic fate mapping, we observed that CX3CR1+ CCR2+ and CX3CR1− CCR2− VAT macrophages are monocyte-derived and tissue-resident, respectively. We performed a lineage tracing experiment in CX3CR1-CreER-TdTomato mice to probe the conversion of CX3CR1+ CCR2+ macrophages into CX3CR1− CCR2− macrophages (Supplementary Fig. 1B). Four weeks after tamoxifen injection, most tdTomato+ macrophages were CX3CR1YFP+. Only about 12% of them were CX3CR1YFP−. These data indicate that CX3CR1+ macrophages convert to CX3CR1− macrophages, albeit at low levels. RNA sequencing revealed that these two subsets of macrophages express discrete transcriptome profiles (Fig. 1D and Supplementary Fig. 1C) (GSE 118226). CX3CR1+ CCR2+ monocyte-derived macrophages expressed genes encoding inflammatory molecules such as matrix metalloproteinases, tumor necrosis factor superfamily 14, and IFNB1 (Supplementary Fig. 1C) and other pro-inflammatory genes typically expressed by monocyte-derived macrophages (Fig. 1E). Further, CX3CR1− CCR2− VAT resident macrophages expressed genes involved in collagen synthesis (Supplementary Fig. 1C) and other genes typically expressed by tissue-resident macrophage (Fig. 1E). Analysis of publicly available bulk RNA sequencing data (GSE 112396) revealed that anti-inflammatory CD206+ and pro-inflammatory CD206− VAT macrophages exhibited a similar gene signature (Fig. 1F). Consistent with their pro-inflammatory gene expression, CX3CR1+ CCR2+ and CD206− macrophages expressed high levels of the genes, which positively correlate with insulin resistance (Fig. 1G, H and Supplementary Fig. 1D). In contrast, CX3CR1− CCR2− and CD206+ macrophages expressed similar transcriptomic profiles including higher levels of insulin sensitivity and anti-inflammatory genes (Fig. 1G–J and Supplementary Fig. 1D, E). Altogether, these data demonstrate that VAT CX3CR1+ CCR2+ macrophages are pro-inflammatory while CX3CR1− CCR2− macrophages are anti-inflammatory and express high levels of the genes, which promote systemic insulin sensitivity.

Chronic inflammation leads to apoptosis of VAT resident macrophages

The balance between monocyte-derived infiltrating and tissue-resident macrophages in metabolic tissues is a key contributing factor in the maintenance of metabolic health. We first aimed to assess the changes in the macrophage subsets in VAT in chronic inflammatory diseases such as obesity. We observed an increase in CCR2+ monocyte-derived macrophages and a decrease in CCR2− VAT resident macrophages in omental adipose tissue of obese patients (Fig. 1K, L and Supplementary Fig. 1F). The changes in these macrophage subsets followed a similar pattern in obese mouse VAT (Fig. 1M and Supplementary Fig. 1G–I). We also calculated the frequencies and numbers of CX3CR1− CCR2+ macrophages in lean and obese mice (Supplementary Fig. 1J). Although the frequencies of these macrophages decreased, their numbers were not significantly changed in obese mice. Furthermore, we also assessed CD9-expressing lipid-associated macrophages and TIM4+ VAT resident macrophages at one and two months after HFD initiation. The numbers of both subsets of macrophages increased significantly at one month after HFD (Supplementary Fig. 1K). However, although CD9-expressing lipid-associated macrophages remained significantly increased at two months after HFD, the abundance of TIM4+ VAT resident macrophages diminished at this time. We further investigated the dynamics of VAT resident macrophages at different time points after HFD feeding in CX3CR1creER/+ ROSAtdTomato mice using intravital microscopy (Fig. 1N). The frequency of monocyte-derived macrophages (tdTomato+) increased whereas the proportions of adipose resident macrophages (tdTomato−) decreased at 2 and 4 months after HFD initiation (Fig. 1O and Supplementary videos 1 and 2).

Further, to understand if apoptosis contributed to the decline in CX3CR1− CCR2− tissue-resident macrophage numbers in obese mice, we measured annexin V, a late apoptosis marker, and caspase 3, an early apoptosis marker, in this macrophage population. Obese mice had augmented proportions of annexin V+ (Fig. 1P and Supplementary Fig. 1L) and caspase 3+ (Fig. 1Q, R and Supplementary Fig. 1M, N) resident macrophages in VAT compared to lean mice, indicating a higher rate of apoptosis in this macrophage subset in obesity. In contrast, the frequency of caspase 3+ CCR2+ macrophages did not significantly change in obese mice (Fig. 1Q). We next determined whether the change in the frequency of the VAT macrophage subsets after HFD feeding is permanent. As expected, HFD feeding significantly increased monocyte-derived macrophages in VAT while the resident macrophage population was decreased (Supplementary Fig. 1S and O). Interestingly, the frequencies of the macrophage subsets were restored two months after HFD withdrawal (Fig. 1S and Supplementary 1R). To ascertain whether the emergence of newly generated VAT resident macrophages after HFD withdrawal is due to their local proliferation or higher differentiation of infiltrated monocytes, we carried out the following experiments. We performed parabiosis between two congenically different mice and quantified the frequency of VAT resident macrophages derived from the parabiont partner three months after HFD initiation, and two and four months after withdrawal of the diet (Supplementary Fig. 1P). Parabiosis allows us to discern if a cell population is derived from circulating hematopoietic cells. HFD withdrawal did not significantly change parabiont-derived VAT resident macrophage frequencies, ruling out the possibility of differentiation of recruited monocytes into this macrophage subset after the diet withdrawal. VAT resident macrophages after HFD withdrawal exhibited augmented Ki-67 staining (Fig. 1T and Supplementary Fig. 1Q), indicating their higher proliferation after HFD withdrawal.

Loss of VAT resident macrophages enhances insulin resistance in diet-induced obesity

As CX3CR1− CCR2− tissue-resident macrophages are enriched for insulin sensitivity genes, we were interested in deciphering the functions of these macrophages in diet-induced insulin resistance. To this end, we generated LysMcre/+ Chr2fl/fl mice, which express the channelrhodopsin-2 (ChR2) protein in macrophages after exposure to blue light (450–490 nm) (Fig. 2A). Photostimulation of this protein leads to prolonged action potential firing activity, resulting in cellular apoptosis. Extended blue light exposure (BLE) resulted in the reduction of VAT-resident macrophages but not monocyte-derived macrophages possibly due to a high turnover and increased apoptosis of the latter cell population compared to the white light exposure (WLE) (Fig. 2B). Indeed, blue light exposure in obese LysMcre/+ ChR2fl/fl mice significantly enhanced Annexin V expression, a marker of early apoptosis, in CX3CR1+ CCR2+ VAT macrophages (Supplementary Fig. 2A). We next examine if blue light exposure also affects TIM4+ adipose resident macrophages and CD9+ lipid-associated macrophages (LAM), which are crucial for adipose biology. We observed that TIM4+ macrophage abundance was lower in VAT of LysMcre/+ ChR2fl/fl mice after blue light exposure as expected (Supplementary Fig. 2B). Moreover, the number of CD9+ LAMs, which are recruited in obesity and play protective roles, diminished significantly upon blue light exposure. Additionally, we have examined if blue light exposure in VAT of the optogenetic mice alters the expression of Annexin V, an early marker of apoptosis, in macrophages residing in distant organs. This treatment did not significantly change Annexin V expression in microglia, Kupffer cells, and splenic macrophages (Supplementary Fig. 2C). Consistently, we observed no significant difference in total hepatic macrophage and Kupffer cell numbers (Supplementary Fig. 2D). VAT-resident macrophage depletion resulted in glucose intolerance and insulin resistance (Fig. 2C and Supplementary Fig. 2E). Consistently, these mice had higher levels of triglycerides, free fatty acids, and free glycerol (Fig. 2C). Furthermore, pAkt and glut4 expression, which is associated with insulin sensitivity, was impaired after blue light exposure (Fig. 2D and Supplementary Fig. 2F, G). The levels of Slc2a4, Irs1, 2 and 3, Sfrp5, Adpn, and Ces1e, which promote glucose clearance, were significantly decreased after VAT resident macrophage depletion with blue light illumination (Fig. 2E). Transcript analysis further showed that blue light-exposed VAT samples exhibited higher levels of proinflammatory cytokines associated with obesity-mediated insulin resistance (Fig. 2F). Collectively, these data suggest that VAT resident macrophages improve insulin sensitivity.

CX3CR1− CCR2− VAT resident macrophages express SerpinB2 at high levels

We noticed that CX3CR1− CCR2− macrophages expressed high levels of Pdgfc, Gata1, and Gata2 that promote tissue-resident macrophage survival by inhibiting the activation of caspases and subsequent apoptosis (Fig. 3A). Among all cell survival genes, SerpinB2 was highly expressed in VAT resident macrophages (Fig. 3A and Supplementary Fig. 3A). Various organs, such as the liver and muscle, important in glucose homeostasis did not express detectable levels of SerpinB2 (Fig. 3B). Among all cells in adipose tissue, which we tested, macrophages expressed very high levels of SerpinB2 (Fig. 3C). Macrophages differentiated from THP-1 human monocytes using phorbol-12-myristate-13-acetate also exhibited high SerpinB2 expression; however, SerpinB2 was not detectable in the cell culture conditioned medium (Fig. 3D) although copious amounts of pro-inflammatory cytokines were present in the culture, suggesting that SerpinB2 is poorly secreted. To validate the RNA sequencing data, we measured SerpinB2 expression in the macrophage subsets in gonadal adipose tissue of lean mice. Adipose tissue-resident macrophages expressed significantly higher levels of SerpinB2 mRNA and protein (Fig. 3E–G) than monocyte-derived macrophages. To discern if SerpinB2 is also expressed at high levels in human adipose tissue-resident macrophages, we collected human omental adipose tissue. Human CCR2− macrophages also expressed higher levels of SerpinB2 mRNA and protein than CCR2+ macrophages (Fig. 3H–J).

IFN-γ-mediated SerpinB2 downregulation leads to VAT resident macrophage apoptosis in chronic inflammation

We next sought to delineate the mechanisms behind enhanced apoptosis of VAT resident macrophages in chronic inflammation precipitated by obesity. To determine if SerpinB2 levels in VAT resident macrophages alter in obesity, we first quantified SerpinB2 in CCR2− macrophages in omental adipose tissue of healthy and diabetic/obese patients and observed a significant downregulation of SerpinB2 in these macrophages of diabetic/obese patients (Fig. 3K–M and Supplementary Table 1). Furthermore, using bivariate analysis, we observed a significant negative correlation between the frequency of SerpinB2-expressing macrophages in VAT and the BMI of the patients (Fig. 3N). Congruent with the reduction in SerpinB2 expression in VAT CCR2− macrophages in obese humans, gonadal adipose tissue of obese mice harbored fewer SerpinB2-expressing macrophages (Fig. 3O, P). Additionally, adipose tissue-resident macrophages in obese patients and mice downregulated the expression of SerpinB2 (Fig. 3Q, R). It has been reported that obesity can increase endogenous palmitic acid levels. This excess palmitic acid can promote inflammation. Because exaggerated inflammation can reduce SerpinB2 expression, we sought to test whether palmitic acid can reduce the expression of this gene. Indeed, palmitate treatment in bone marrow-derived macrophages of mice suppressed SerpinB2 expression (Fig. 3S).

Adipose tissue secretes high levels of pro-inflammatory cytokines such as IFN-γ and TNF in obesity. THP1 macrophages treated with IFN-γ, but not TNF, expressed lower levels of SerpinB2 whereas inhibition of IFN-γ with Bay-117082 increased SerpinB2 expression (Fig. 4A, B, and Supplementary Fig. 3B). Consistently, fibroblasts and macrophages produced high levels of IFN-γ in obese mice (Fig. 4C). We hypothesized that IFN-γ binds to its receptor on VAT macrophages to decrease SerpinB2 expression. Consistently, obese mice with myeloid-specific deficiency in the IFN-γ receptor (Ifngr1) had elevated SerpinB2 expression in VAT resident macrophages compared to wild type control mice (Fig. 4D and Supplementary Fig. 3C). In line with this finding, this macrophage subset in myeloid-specific Ifngr1-deficient mice was more abundant and less apoptotic (Fig. 4D, E and Supplementary Fig. 3C–E) while the total number of adipose macrophages did not change in these mice compared to the control mice (Supplementary Fig. 4A). Additionally, LysMcre/+ Ifngr1fl/fl mice had reduced fasting blood glucose levels, improved glucose tolerance, and reduced levels of triglycerides, free glycerol, and free fatty acids in the serum; however, their body weights were unchanged compared to LysM+/+ Ifngr1fl/fl mice (Fig. 4F and Supplementary Fig. 3F). LysMcre/+ Ifngr1fl/fl mice also had higher pAkt, glut4, adiponectin, and Ppary expression compared to the control group, indicating improved insulin signaling (Fig. 4G, H and Supplementary Fig. 3G). In line with these findings, the expression of insulin sensitivity-promoting genes like Slc2a4, Irs1, Irs3, Sfrp5, Adipoq, Ces1d, Abca4, Abcg1, and Abcg4 were increased in VAT of these mice (Fig. 4I). In addition, Cd36, Msr1, Olr1, Ccl3, Ccl5, Cxcl1, Cxcl2, Il1b, Il6, and Tnf transcripts, which are associated with increased risk of obesity and insulin resistance, were markedly diminished in VAT of LysMcre/+ Ifngr1fl/fl mice (Fig. 4I, J). Our in-silico analysis showed that the SerpinB2 promoter has binding motifs for Ikaros, a transcriptional suppressor (Fig. 4K and Supplementary Fig. 3H). A chromatin immunoprecipitation assay confirmed the binding of Ikaros to the SerpinB2 promoter (Fig. 4L), and IFN-γ treatment significantly increased Ikaros expression in BMDM (Fig. 4M). Treatment with lenalidomide, a known inhibitor of Ikaros, significantly blocked IFN-γ-mediated SerpinB2 downregulation (Fig. 4N). Congruently, monocyte-derived macrophages, which express SerpinB2 at lower levels, were enriched for the genes encoding the receptors for IFN-γ (Fig. 4O). Altogether, these data indicate that IFN-γ-induced Ikaros decreases SerpinB2 expression in VAT resident macrophages. To determine whether SerpinB2 is indeed involved in the survival of macrophages, we analyzed apoptosis of BMDM, THP-1 macrophages, and VAT resident macrophages. We observed elevated caspase 3 levels in BMDM lacking SerpinB2 (Fig. 4P) and decreased proportions of apoptotic THP-1 macrophages after SerpinB2 overexpression (Fig. 4Q). Furthermore, obese SerpinB2−/− mice harbored an augmented frequency of apoptotic VAT resident macrophages (Fig. 4R). In contrast, the numbers of Kupffer cells in obese SerpinB2+/+ and SerpinB2−/− mice were similar (Supplementary Fig. 4B). In aggregate, these data suggest that SerpinB2-deficient macrophages are apoptotic.

Increased mitochondrial ROS in SerpinB2−/− macrophages induces apoptosis

Next, we investigated the mechanisms of enhanced apoptosis in SerpinB2−/− macrophages. To understand how SerpinB2 mediates macrophage survival, we measured cytochrome c levels in cytosol and mitochondria. The release of cytochrome c from the mitochondria into the cytosol has been reported to be a major mechanism of cellular apoptosis. We observed increased concentrations of cytosolic cytochrome c while the levels of mitochondrial cytochrome c decreased in SerpinB2−/− BMDM compared to SerpinB2+/+ BMDM (Fig. 5A, B). These findings suggest that loss of SerpinB2 results in cytochrome c release from the mitochondria into the cytosol. Cytochrome c release into the cytosol depends on mitochondrial oxidative stress. To understand the mechanisms of cytochrome c translocation from mitochondria to the cytoplasm in the absence of SerpinB2, we quantified the genes encoding antioxidant enzymes in the VAT macrophage subsets. We ascertained that CX3CR1− CCR2− VAT macrophages of lean mice were enriched with anti-oxidant enzymes such as superoxide dismutase (Sod3), glutathione-S-transferases (Gstm, Gsta, Gstp, and Gstt), mitochondrial membrane-associated Mgst1, metallothioneins (Mt1 and Mt2), and mitochondrial specific antioxidant enzymes like glutathione peroxidase (Gpx7), malic enzyme 1 (Me1), and isocitrate dehydrogenase 2 (Idh2) (Fig. 5C, D). Similarly, CX3CR1− CCR2− VAT macrophages had elevated expression of the GSH encoding genes (Fig. 5E). Additionally, the expression of the GSH-encoding genes in CX3CR1− CCR2− VAT macrophages was decreased in obesity (Fig. 5F). Moreover, GSH gene expression in CX3CR1− CCR2− VAT macrophages and glucose clearance were correlated (Fig. 5G). Taken together, these results demonstrate that CX3CR1− CCR2− VAT macrophages are enriched in GSH in lean mice. Among the genes involved in GSH precursor import, only Slc7A13, which mediates the transport of glutamic acid, one of the precursors for GSH synthesis, was upregulated in CX3CR1− CCR2− VAT macrophages (Fig. 5H). Notably, SerpinB2 deficiency significantly curtailed the levels of these antioxidant genes in VAT macrophages (Fig. 5I). Consistently, SerpinB2−/− BMDM isolated from lean or obese mice had higher levels of mitochondrial ROS than SerpinB2+/+ BMDM (Fig. 5J, K and Supplementary Fig. 4C, D). Mitotracker green MFI was significantly decreased in SerpinB2−/− BMDM treated with palmitate, indicating lower mitochondrial volume in these cells compared to SerpinB2+/+ BMDM (Supplementary Fig. 4E). To examine the function of mitochondrial ROS in cytochrome c release into the cytosol from the mitochondria, we treated BMDM derived from SerpinB2+/+ and SerpinB2−/− mice with mitotempol, a mitochondrial ROS scavenger. This treatment significantly decreased cytosolic cytochrome c levels while the concentrations of mitochondrial cytochrome c were increased in SerpinB2−/− BMDM (Fig. 5L) indicating suppressed cytochrome c release from the mitochondria. Concomitantly, the treatment caused a non-significant decrease in the level of annexin V, an early apoptosis marker, in SerpinB2−/− BMDM (Fig. 5M). Nevertheless, annexin V mean fluorescent intensities in SerpinB2+/+ BMDM were unchanged after the treatment (Fig. 5M). To understand if SerpinB2 deficiency alters the inflammasome, which can trigger pyroptotic cell death, we measured Nlrp3, which is involved in inflammasome signaling. The expression of Nlrp3 in SerpinB2+/+ and SerpinB2−/− adipose resident macrophages was similar (Supplementary Fig. 4F), suggesting that this pathway does not contribute to increased apoptosis of this macrophage subset in absence of SerpinB2. Altogether, these data imply that the lack of SerpinB2 in macrophages elevated mitochondrial ROS, which triggered cytochrome c release into the cytosol.

SerpinB2 deficiency aggravates inflammation

Adipose tissue inflammation mediated by macrophages contributes to insulin resistance. OXPHOS analysis using SeaHorse XF bioanalyzer in BMDM obtained from lean and obese C57BL/6 mice showed that BMDM of obese mice have elevated oxygen consumption rates (Fig. 6A and Supplementary Fig. 5A). Interestingly, OCR and inflammatory cytokines are strongly correlated (Fig. 6A). From these observations, we hypothesized that enhanced OXPHOS activity in macrophages exaggerates inflammation in obesity. Consistently, obese LysMcre/+ COX10fl/fl mice that lack the mitochondrial complex IV activity exhibited decreased inflammation, and improved glucose clearance, insulin sensitivity, and insulin levels compared to obese LysM+/+ COX10fl/fl mice although the body weights were similar between the groups (Fig. 6B and Supplementary Fig. 5B, C). We observed depressed basal and maximal respiration and spare capacity while extracellular acidification rates were unchanged in Cox10-deficient BMDM compared to WT BMDM (Supplementary Fig. 5D, E). Similarly, SerpinB2 deficiency increased OCR (Supplementary Figs. 6C and 5F) and inflammation (Supplementary Figs. 5G and H) in BMDM while SerpinB2 overexpression in THP-1 macrophages depressed inflammation (Fig. 6D). In line with these findings, we observed that SerpinB2−/− adipose macrophages expressed higher levels of inflammatory genes, such as Il1b, Tnf, Ccl2, Ccl3, iNOS, etc., and lower levels of pro-resolving genes, such as Il10 and Arg1, than SerpinB2+/+ adipose macrophages (Supplementary Fig. 5I, J). In aggregate, these data support the premise that SerpinB2 is essential for suppressing macrophage-mediated inflammation.

SerpinB2 deficiency in macrophages exacerbates glucose intolerance in diet-induced obesity

Our data demonstrate that VAT resident macrophages are crucial to the maintenance of insulin sensitivity (Fig. 1). Because SerpinB2 expression is critical for the survival of this macrophage subset, and there is an inverse correlation between macrophage-specific SerpinB2 expression in omental adipose tissue and BMI in patients, we hypothesize that the loss of SerpinB2 expression in macrophages results in glucose intolerance. A glucose tolerance test revealed that SerpinB2−/− mice had delayed glucose clearance and higher fasting insulin levels compared to SerpinB2+/+ mice whereas the body weights were unaltered (Fig. 6E and Supplementary Fig. 6A). To specifically test the contributions of myeloid-specific SerpinB2, we generated the mice that lack SerpinB2 in myeloid cells (Supplementary Fig. 6B). Glucose and insulin tolerance tests revealed that these mice had delayed glucose clearance and aggravated insulin resistance, respectively, compared to age-matched control mice while the body weights of the mice in both groups were similar (Fig. 6F and Supplementary Fig. 6C, D). SerpinB2 deficiency in myeloid cells also led to increased levels of fasting serum insulin, triglycerides, free fatty acids, and free glycerol suggesting increased whole-body insulin resistance (Fig. 6F). These findings were further confirmed by a hyperinsulinemic-euglycemic clamp study. During this experiment, the glucose infusion rate to match plasma glucose levels was significantly less in LysMcre/+ SerpinB2fl/fl mice (Fig. 6G), demonstrating whole body insulin resistance in these mice. Additionally, plasma insulin and free fatty acid levels were higher in LysMcre/+ SerpinB2fl/fl mice (Fig. 6G) without noticeable differences in the body weights, steady-state whole-body glucose uptake measurements, and fasting rates of endogenous (hepatic) glucose production (Supplementary Fig. 6E). However, hepatic glucose production during steady-state was markedly greater in LysMcre/+ SerpinB2fl/fl mice during the clamp, indicating that the failure to suppress hepatic glucose production accounted for the differences in whole-body insulin sensitivity between the groups (Fig. 6G). In line with this, we observed decreased p-Akt in VAT and muscle (Fig. 6H and Supplementary Fig. 6F), diminished Glut4, Pparγ, and adiponectin in VAT, and reduced Glut4 in muscle (Fig. 6I and Supplementary Fig. 6F). Furthermore, insulin sensitizing Slc2a4, Irs1, Irs2, Sfrp5, Adipoq, and Abcg4 transcripts were repressed in VAT of LysMcre/+ SerpinB2fl/fl mice (Fig. 6J). Furthermore, the lack of SerpinB2 in myeloid cells elevated pro-inflammatory gene expression in VAT (Fig. 6J). Similarly, sorted macrophages from VAT from these mice had suppressed expression of anti-inflammatory genes and heightened pro-inflammatory genes (Fig. 6K). SerpinB2 deficiency augmented apoptosis of VAT resident but not monocyte-derived macrophages (Supplementary Fig. 6G). Altogether, these data corroborate the significance of macrophage SerpinB2 expression in maintaining systemic insulin sensitivity.

IL-4 reverses exaggerated diet-induced insulin resistance in the absence of myeloid SerpinB2

To investigate the molecular mechanisms of insulin sensitivity exerted by VAT resident macrophages, we assessed the expression of the cytokines promoting insulin sensitivity from the RNA sequencing analysis data of sorted CX3CR1+ CCR2+ and CX3CR1− CCR2− VAT macrophages. We observed that VAT-resident macrophages expressed higher levels of Il4, Il33, and Il34 (Fig. 6L). To discern whether increased insulin resistance in LysMcre/+ SerpinB2fl/fl mice is due to lack of IL-4, we supplemented these mice with IL-4 (Supplementary Fig. 6C). IL-4 supplementation accelerated glucose clearance, encouraged insulin sensitivity, and decreased fasting glucose concentrations (Fig. 6F and Supplementary Fig. 6D) while no noticeable improvements in insulin, triglycerides, and free glycerol levels were observed (Fig. 6F). Consistent with these findings, IL-4 replenishment boosted p-Akt, Glut4, and Pparγ expression in VAT, and pAkt and glut4 levels in muscle (Fig. 6H, I and Supplementary Fig. 6H). As expected, the expression of insulin sensitivity-promoting genes was enhanced while the transcripts of the proteins stimulating inflammation and insulin resistance were drastically reduced in VAT (Fig. 6J). Altogether, these findings reveal that IL-4 can reverse aggravated insulin resistance in the absence of myeloid SerpinB2.

Loss of adipose resident macrophages provokes VAT expansion in diet-induced obesity

Because VAT expansion in obesity is associated with insulin resistance, we deciphered the role of adipose resident macrophages in VAT growth. Towards this end, we depleted VAT resident macrophages in LysMcre/+/Chr2fl/fl mice with blue light exposure. Blue light-exposed LysMcre/+/Chr2fl/fl mice had heavier VAT, bigger adipocytes, and higher expression of adipose expansion genes, reflecting the adipocyte enlargement in the absence of VAT resident macrophages (Fig. 7A). In agreement with these data, myeloid-specific SerpinB2 deficiency, which we found to deplete the adipose resident macrophage pool, heightened VAT weight and adipocyte diameter whereas IL-4 replenishment in LysMcre/+ SerpinB2fl/fl mice restored these parameters (Fig. 7B). Consistent with the finding that Ifngr1 deficiency prevents adipose resident macrophage apoptosis in obesity and improves insulin sensitivity, mice lacking this receptor in myeloid cells had lower VAT weights and smaller adipocytes (Supplementary Fig. 7A). Interestingly, adipose resident macrophages expressed low levels of Il1b, which stimulates adipocyte growth, and high levels of Tgfb2, which inhibits adipocyte growth, compared to monocyte-derived macrophages (Supplementary Fig. 7B). As cytoskeleton remodeling is a prerequisite step during the morphological transition of fibroblast-like preadipocytes into lipid-filled spherical mature adipocytes, we measured cytoskeleton proteins like beta-tubulin, beta-actin, and vimentin. Beta-tubulin and beta-actin expression was significantly suppressed while vimentin levels were elevated in mice lacking VAT resident macrophages and mice deficient in myeloid SerpinB2 (Fig. 7C and Supplementary Fig. 7C). The absence of myeloid Ifngr1 and IL-4 replenishment in LysMcre/+ SerpinB2fl/fl mice inflated the expression of beta-tubulin and beta-actin and diminished vimentin levels (Supplementary Fig. 7D, E). Supporting these observations, adipocytes residing near CCR2− VAT resident macrophages were smaller (Fig. 7D).

Glutathione (GSH) negatively regulates adipocyte hypertrophy and expansion of VAT in diet-induced obesity

Given that CX3CR1− CCR2− VAT macrophages express high levels of GSH, SerpinB2 deficiency lowers these antioxidants in VAT macrophages, and depletion of VAT resident macrophages increased adipocyte size, we hypothesized that the anti-inflammatory and suppressed adiposity properties are attributable to GSH. N-acetylcysteine (NAC), an aminothiol and synthetic precursor of intracellular cysteine and GSH, dose-dependently abrogated the differentiation of adipocytes (Fig. 7E and Supplementary Fig. 7F). Concordant with the inhibitory effect on adipocyte development, NAC treatment promoted the expression of insulin sensitivity-promoting genes and blunted pro-inflammatory and adipose expansion genes in VAT depots (Fig. 7F). These results imply the significant role played by GSH in confining the expansion of adipocytes in obesity. To ascertain the protective role of GSH, obese LysMcre/+ SerpinB2fl/fl mice were supplemented with 1% of NAC for 8 weeks in drinking water. NAC supplementation significantly curtailed fasting glucose levels, and improved glucose tolerance and insulin sensitivity compared to vehicle treatment (Fig. 7G and Supplementary Fig. 7G). Interestingly, NAC supplementation significantly reduced the body weight (Supplementary Fig. 7G). Moreover, insulin, triglycerides, free fatty acids, and free glycerol levels were reduced in the serum of NAC-treated obese LysMcre/+ SerpinB2fl/fl mice (Fig. 7G). Further, Akt activation was enhanced in muscle and VAT of NAC-supplemented LysMcre/+ SerpinB2fl/fl mice (Fig. 7H and Supplementary Fig. 7H). Consistently, Glut4 and adiponectin protein levels (Fig. 7H and Supplementary Fig. 7H) and insulin sensitivity-promoting gene expression (Fig. 7I) raised while the transcripts of inflammatory, adipose expansion, and insulin resistance-promoting genes were suppressed (Fig. 7I) in VAT of NAC-treated LysMcre/+ SerpinB2fl/fl mice. Furthermore, NAC supplementation escalated the number of tissue-resident macrophages in VAT (Fig. 7J and Supplementary Fig. 7I), reduced macrophage apoptosis (Supplementary Fig. 7J), and limited VAT weight (Supplementary Fig. 7K) and adipocyte size (Supplementary Fig. 7L). These results imply the significant role played by GSH in confining the expansion of adipocytes in obesity. Moreover, adipocytes cultured in the presence of conditioned media from LysMcre/+ SerpinB2fl/fl BMDM expanded more compared to LysM+/+ SerpinB2fl/fl BMDM (Supplementary Fig. 7M, N). However, adipocyte expansion was significantly attenuated with conditioned medium of NAC-supplemented LysMcre/+ SerpinB2fl/fl BMDM. In cluster, these findings endorse that the pro-inflammatory and glucose intolerance phenotype in the absence of myeloid SerpinB2 could be attributed to the lack of GSH.

Methods
Mouse strains, cellular fate mapping, and treatments

The control and experimental mice were co-housed in a barrier facility at the University of Pittsburgh. Both male and female mice (16–50 weeks old) wild type and transgenic mice have been used for the experiments. To investigate the origin of adipose tissue macrophages, female ROSA-tdTomato mice were mated with male Cxcr1creER mice. We injected tamoxifen (50 µl of 20 mg/ml solution, Sigma, T5648) dissolved in corn oil (Sigma, C8267) in the offspring to induce permanent tdTomato expression in Cxcr1+ macrophages. Cxcr1GFP/GFP, LysMcre/cre, Ifngrfl/fl (Jackson Lab, #025394), and SerpinB2−/− (Jackson Lab, #007234) mice were purchased. SerpinB2Tm1c mice (KOMP project ID CSD66707) were bred with Flp recombinase-expressing mice (Jackson Lab, #009086) to generate conditional-ready Tm1a mice. Tm1a mice were crossed with wild-type female mice purchased from the Jackson Lab at least for five generations. After this, Tm1a mice were bred with LysMcre/cre mice to generate LysMcre/+/LysM+/+ SerpinB2fl/fl mice. Eight to twelve weeks old male and female mice were fed with either a chow or HFD (Research Diets Inc, #D12492) for four months. Ten weeks old LysMcre/+ SerpinB2fl/fl mice were fed with an HFD for two months followed by either an i.p infusion of 2 doses/week of 1 ng IL-4 (R&D, #404-ML-050/CF) dissolved in 100 microliters of PBS or supplementation of 1% NAC (Research Products, #A10040) dissolved in water for 8 weeks as previously. The mice were randomized among various groups, and most analyses were blindfolded. The mice were euthanized using carbon dioxide. The sources and identifiers of all reagents and resources used in this study have been included in Supplementary Table 2.

Study approval

All animal experimental protocols were approved by the Institutional Animal Care and Use Committee of the University of Pittsburgh. Omental adipose tissue from patients with diabetes and normal individuals was collected via a warm autopsy program directed by Dr. Mauricio Rojas. His team obtained consents from decedents’ next-of-kin or legal representatives. These patients were classified as either lean (<25 BMI) or obese (>30 BMI) based on their BMI. Collection of the tissue samples from deceased patients was approved by the University of Pittsburgh Committee for Oversight of Research and Clinical Training Involving Decedents (CORID#724).

Parabiosis

CD45.1 (Jackson Lab, #002014) and CD45.2 (Jackson Lab, #000664) mice were surgically joined. Following anesthesia and disinfection using betadine scrub and 70% isopropanol, an incision was made from the ear to the base of the tail on the adjacent sides of the mice. The subcutaneous fascia was loosened, and the skin was sutured using a 5–0 silk suture. The olecranon and knee joints were attached to avoid stress on the suture lines. Non-absorbable sutures were removed on day 7 after the surgery, and chimerism was ensured on day 21 after parabiosis by flow cytometric analyses of peripheral blood leukocytes.

Intravital microscopy

tdTomato expression was induced in CX3CR1-expressing cells by injecting 100 µl of 20 mg/ml of tamoxifen (Sigma) dissolved in corn oil (Sigma) once a day for five consecutive days into CX3CR1CreER/+ ROSAtdTomato/+ mice. By using betadine and 70% ethanol, the abdominal skin was disinfected, and an incision was made on the linea alba. Gonadal adipose tissue was retracted and placed in sterile 0.9% saline. Gonadal adipose tissue was imaged for 30–60 minutes per mouse by using a two-photon microscope that was equipped with a heated mouse holder to maintain a body temperature of 37°C. To maintain contact between the tissue and the microscope objective, Methocel 2% gel was used during imaging. The total imaging duration was 30–60 minutes per mouse. Data analysis was performed using ImageJ.

Gamma irradiation and bone marrow transplantation

Non-anesthetized mice were placed in a gamma irradiator and exposed to 10 Gy irradiation. Then the mice were returned to the housing facility and injected with one million bone marrow cells/mouse i.v under anesthesia.

Organ harvesting, flow cytometry, and cell sorting

Mice were euthanized and perfused thoroughly with 30 mL of ice-cold PBS through the left ventricle after incising the right atrium to remove circulating blood from solid organs such as adipose tissue. Gonadal and mesenteric adipose tissues were harvested and digested in PBS containing 1 mg/ml of collagenase IV (Worthington, #LS004209) and 20 µM HEPES buffer at 37°C on thermoblock by rotating at 750 rpm for 30 minutes. After red blood cell lysis for three minutes using RBC lysis buffer (BioLegend, #420302), blood cells were washed and resuspended in cold FACS buffer (PBS containing 0.5% BSA). Cell staining and analysis were performed as described previously. All antibodies used in this study were purchased from eBioscience, BioLegend, and BD Biosciences. Anti-mouse and human CCR2 antibodies were used in 1:30 dilutions, and all other antibodies were used in 1:600 dilutions in FACS buffer before flow cytometry acquisition. We used these following antibodies: anti-CD11b (BD Biosciences, M1/70 # 557657), CD11c (BioLegend, N418 # 117338, BD Biosciences, HL3 # 553800), Ly6G (BD Biosciences, 1A8 # 563979), CD115 (eBioscience, AFS98 # 46-1152-82), Ly-6C (BioLegend, HK 1.4 # 128006), CD19 (BD Biosciences, 1D3 # 563148), MHC class II (BioLegend, M5/114.15.2 # 107620), CD64 (BD Biosciences, X54-5/7.1 # 558455), F4/80 (BioLegend, BM8 # 123114), CD45.1 (BioLegend, A20 # 110730), CD45.2 (BioLegend, 104 # 109820, BD Biosciences, 104 # 560693), CCR2 (R&D Systems, # FAB5538A) and streptavidin (BD Biosciences, #563260, 563261). Neutrophils and monocytes were gated as CD11b+ Ly6G+ and CD11b+ CD115+, respectively. CD45+ CD11b+ CD64+ F4/80+ cells in adipose tissue were considered as macrophages. Human omental leukocytes were extracted using the same digestion method mentioned above and stained with antibodies against CD45 (BD Biosciences, HI30 # 564585), CCR2 (BioLegend, K036C2 #357206), CD24 (BD Biosciences, ML5 # 561647), CD14 (BD Biosciences, #555399), CD16 (BD Biosciences, 3G8 # 560195), CD11c (BD Biosciences, B-ly6 # 563404), CD206 (BD Biosciences, 19.2 # 564063), and HLA-DR (BD Biosciences, G46-6 # 565127). CD45+ CD11c+ CD206+ cells in adipose tissue obtained from patients were deemed as macrophages. Fortessa Flow Cytometers at the Unified Flow Cytometry Core at the University of Pittsburgh were used to perform data acquisition. Data were analyzed using FlowJo software (TreeStar). To assess the apoptosis of adipose tissue macrophages, the cells were first stained with antibodies mentioned above. FITC Annexin V apoptosis detection I (BD Biosciences, #556547) and PE-active Caspase 3 apoptosis (BD Biosciences, #550914) kits were used according to the manufacturer’s instructions.

Whole-mount imaging of VAT using confocal microscopy

Following collection of VAT from patients and mice, the samples were fixed with 4% formalin for 2 hours and stored in 30% sucrose solution containing 0.05% sodium azide overnight before storing at −80°C. Small pieces of formalin-fixed VAT were cut, permeabilized with 0.1% triton X-100 for 4 hours and blocked overnight in PBS containing 2% BSA in 96 well cell culture plates. Tissues were incubated for 48 hours with the primary antibodies against proteins like F4/80 (Invitrogen, #MAI-91124), cleaved caspase 3 (Abcam, #ab13847), CD11b (Abcam, #ab133357), CX3CR1 (Abcam, #ab8021), CCR2 (Bio-Rad, #AAM72), Ki67, CD68 (ThermoFisher Scientifics, # 14-0688-82), and SerpinB2 (Invitrogen, #PA5-27857) followed by washing with PBS and incubation for 24 hours at 4°C with Alexa fluor 488, 594 and 647-conjugated secondary antibodies. Tissue sections were stained and mounted with Vectashield DAPI (Vector Laboratories, #H-1200). Nikon confocal laser microscopy was used to obtain images of the stained tissue sections. We used ImageJ and NIS-Elements to discern the frequency and number of cell populations and calculate the mean fluorescence intensities of proteins. For measuring adipocyte size, small pieces of VAT were stained with 1 µg/ml BODIPY (ThermoFisher Scientifics, #D3922) for 15–30 minutes followed by PBS washing in 96-well plates and mounting with Vectashield DAPI. About 10–15 images were randomly selected for every sample, 100–200 adipocytes were selected randomly from every image, and adipocyte size was measured using ImageJ.

Glucose and insulin tolerance tests

Two-month-old mice that were fed with a 60% calorie-rich HFD for 120 days were fasted for 6 hours before glucose and insulin tolerance tests, Blood was collected from these mice by tail nicking to measure glucose concentrations. Fasting blood glucose concentrations were measured and considered as a 0-minute time point. Sterile glucose solution (1 g/kg body weight dissolved in PBS to make a 10% solution) was injected intraperitoneally, and glucose concentrations were measured at various time points. For the insulin tolerance test, mice were injected with insulin (0.25 U/kg body weight, Fisher Scientifics, #12-585-014) and blood glucose levels were measured at 15, 30, 60, 90, and 120 minutes after injection.

Assays to measure serum concentrations of insulin

Mice were anesthetized using 2% isoflurane mixed with oxygen. Blood was collected via retro-orbital bleeding using non-heparin-coated capillary tubes and allowed to clot. Samples were centrifuged (3000 rpm for 10 minutes at 4°C) to separate serum, which was stored at −80°C until further analyses. An ELISA-based assay was performed to measure serum insulin concentrations (Mercordia Diagnostics, #10-1247-01).

Estimation of serum triglycerides, free fatty acids, and free glycerol concentrations

Following serum separation, triglycerides, free glycerol, and free fatty acids were quantified using the following reagents or kits: triglycerides liquid reagent (Pointe Scientific, #T7532120), EnzyChrom free glycerol assay kit TM (Bioassay Systems, #EGLY-200), and EnzyChrom free fatty acid assay kit TM (Bioassay Systems, #EFFA-100).

Cell sorting and quantitative RT-PCR

We obtained single-cell suspensions of adipose tissue as described above. The cells were stained for macrophage markers. VAT monocyte-derived and resident macrophages were sorted using a FACSAria into RNAse-free tubes containing 100 µl of RNA extraction buffer. mRNA was extracted using a commercially available kit (Applied Biosystems, #12204-01) and cDNA was made by a reverse transcription assay using high-capacity cDNA kit (ThermoFisher Scientific, #4368813). Genes encoding cytokines were quantified with a Powerup TM SYBR TM Green master mix (Applied Biosystems™, #A25778)-based quantitative RT-PCR assay (Applied Biosystems). Ct values were normalized to the housekeeping gene Gapdh and 18sRNA, and gene expression was ascertained. Excel and Prism were used to generate heatmaps based on the gene expression values. The primer sequences have been provided in Supplementary Table 3.

Cell sorting and RNA-seq analysis

We obtained single-cell suspensions of adipose tissue as described above. The cells were stained for macrophage markers. VAT monocyte-derived and resident macrophages were sorted using a FACSAria into RNAse-free tubes containing 100 µl of RNA extraction buffer. mRNA was extracted using a commercially available PicoPure TM RNA isolation kit (Applied Biosystems, #12204-01). mRNA sequencing was performed by the Health Science Sequencing Core at UPMC Children’s Hospital of Pittsburgh and FASTQ files have been deposited to GSE 118226. Using CLC Genomics Workbench 21, RNA seq data analysis was performed, and a QC report was generated to check the quality of the data. The adaptor sequences (CTGTCTTATA) were trimmed from the 3’ end of the reads. The trimmed reads were then mapped against the Mus musculus CLC reference genome version 86. Gene expression was normalized as reads per kilobase of transcript, per million mapped reads (RPKM), and the genes that met a cut-off of FDR P < 0.05 and 2 log2 fold change in CX3CR1+ CCR2+ compared to CX3CR1− CCR2− adipose macrophages were calculated using the software. A Volcano plot to depict differential gene expression was made, and a heatmap showing the genes having at least 2-fold differences between the macrophage subsets and less than FDR P value of 0.05 was generated using CLC Genomics Workbench. Next, we used Ingenuity Pathway Analysis to find canonical and disease pathways differentially expressed between the two subsets of macrophages.

Gene expression analysis using quantitative RT-PCR

Following collection, organs were stored overnight in RNA later (Sigma, #R0901) and transferred to −80°C after removing RNA later. mRNA was extracted using an RNEasy kit (Qiagen, #74104), and cDNA was made by a reverse transcription assay. The genes encoding cytokines were quantified with a SYBR Green-based quantitative RT-PCR assay (Applied Biosystems). Ct values were normalized to the housekeeping gene Gapdh and 18sRNA, and gene expression was ascertained. Excel and Prism were used to generate heatmaps based on the gene expression values. The primer sequences have been provided in Supplementary Table 3.

Apoptosis induction in VAT resident macrophages

Single-cell suspensions of VAT were obtained using collagenase digestion as written above. The cells were cultured in 1 ml DMEM containing 20 µM camptothecin (Sigma, #C9911) for 2 hours to induce apoptosis. The cells were stained with antibodies against leukocyte markers. The apoptosis kits were used to quantify apoptotic macrophages by flow cytometry. For annexin V staining (BD Biosciences, #556547), the cells were suspended in 100 µl of 1x binding buffer and stained with 5 µl annexin V Alexa Fluor 488 and 1 µl of 100 µg/ml propidium iodide (BioLegend, #421301). The cells were incubated at room temperature for 15 minutes, and 400 µl of 1x binding buffer was added and analyzed by flow cytometry immediately. For caspase 3 staining (BD Biosciences, #550914), the cells were incubated in 500 µl of BD cytofix/cytoperm buffer for 20 minutes on ice and were centrifuged at 370 RPM for 7 minutes at 4°C followed by washing in 500 µl of the perm/wash buffer. The cells were then incubated in 100 µl of perm/buffer containing 5 µl of caspase 3 antibody for 30 minutes at room temperature. The cells were washed in 1 ml of perm/wash buffer and diluted in 400 µl of perm/wash buffer before flow cytometry analysis.

Insulin stimulation in the vena cava

Two months old mice LysM+/+ SerpinB2fl/fl and LysMcre/+ SerpinB2fl/fl, LysM+/+ Ifngr1fl/fl and LysMcre/+ Ifngr1fl/fl mice were fed on 60% calorie-rich high-fat diet for 16 weeks. These mice were fasted overnight for 16 hours and anesthetized with isoflurane and injected with or without insulin (0.25 U/kg body weight, Fisher Scientifics, #12-585-014). Fifteen minutes after insulin injection, liver, muscle, and visceral adipose tissue were harvested. These tissues were processed for immunoblot to check the activation status of Akt using antibodies that detect total and phosphorylated Akt.

BMDM culture and treatments

Femurs and tibias were collected from SerpinB2+/+, SerpinB2−/−, LysM+/+ SerpinB2fl/fl, and LysMcre/+ SerpinB2fl/fl mice after left ventricular perfusion with PBS to collect bone marrow cells, which were RBC lysed and plated in cell culture dishes. After six hours, floating cells were transferred into new dishes and these cells were used for BMDM culture. Adherent cells were cultured in DMEM containing 5.5 mM glucose and supplemented with 20% L929 conditioned medium for 6–8 days. Similarly, BMDM were cultured in 5.5 mM glucose containing DMEM along with 20% L929 conditioned medium. BMDM obtained from the mice mentioned above were grown in 60 mm dishes for 2 days, and the levels of IL-1β (ThermoFisher Scientifics, #88-7013-88) and IL-6 (ThermoFisher Scientifics #88-7064-88) were measured using ELISA. Additionally, cells were collected, RNA was made using an RNEasy kit (Qiagen, #74104), and gene expression was quantified. Similarly, BMDM from bone marrow cells of C57BL/6 mice were cultured and treated with 250 milliunits of IFN-γ (Invitrogen, #PHC4031) or various chemicals such as palmitate (Acros Organics, #416700050), cycloheximide (Alfa Aesar, #J6690103), and 250 µM mitotempol (Fisher Scientific, #NC1229394) for 0-24 hours in either 6 well plates or chamber slides. In a separate experiment, BMDM were treated with or without Ikaros inhibitor lenalidomide (5 µM) (MedChemExpress, #HYA0003) for 2 hours followed by stimulation with 250 milliunits of IFN-γ for 24 hours.

THP-1 cell culture: THP-1 monocytes were seeded at a density of 2×105/ml and differentiated into macrophages in the presence of 100 nM Phorbol-12-myristate-13-acetate (PMA) (Calbiochem, # 524400) for 2 days in RPMI low glucose media in 6-well plates or 4-well chambered slides as described previously. Following differentiation, macrophages were cultured without any treatment for 2 more days by changing medium for every 24 hours. Then the cells were treated with or without NF-kB inhibitor Bay-117082 (MedChemExpress, #HY-13453) for 2 hours followed by stimulation with 250 milliunits of IFN-γ for 24 hours. The cells were either fixed in 4% paraformaldehyde for immunofluorescence or used for gene expression analysis.

3T3L1 mouse fibroblasts culture and treatment

3T3-L1 fibroblasts were cultured in ZenBio Inc 3T3-L1 preadipocyte Medium (#PM1L1) until they reached about 50% confluence in 6 well plates, followed by IL4 (10 ng/ml) or NAC treatment and differentiation with ZenBio Inc adipocytes differentiation medium (#DM2L1). After two days, differentiated cells were maintained in ZenBio Inc adipocytes maintenance medium (#OMAM) for 10 days with periodic changes of the culture medium, IL4, and NAC every two days as previously. To delineate tissue-resident macrophages’ effects on adipocyte development, two days after differentiation 3T3-L1 fibroblasts derived adipocytes were cultured in the presence of conditioned media of BMDM derived from LysM+/+ SerpinB2fl/fl, LysMcre/+ SerpinB2fl/fl mice, and NAC-treated LysMcre/+ SerpinB2fl/fl mice. The BMDM was cultured in the conditioned medium along with the maintenance medium (1:1 ratio) for 6–8 days. BODIPY staining and confocal microscopy were performed to assess adipocyte size.

Analysis of mitochondrial volume, membrane potential, ROS, and cytochrome c in VAT macrophages and BMDM using flow cytometry and confocal microscopy

Single-cell suspensions from VAT were obtained using the method discussed above. Cells were incubated with the antibodies against Ly-6C, F4/80, and CD11b. Following staining with the antibodies, the cells were treated with 200 nM mitotracker green (Invitrogen, #M7514, Excitation/emission: 490/516, in FITC channel) to detect all mitochondria, 200 nM mitotracker deep Red (Invitrogen, #M22426, Excitation/emission: 644/665, in APC channel) to detect mitochondrial membrane potential, and 5 µM mitosox red (PE) (Invitrogen, #M36008, Excitation/emission: 644/665 in APC channel) to detect mitochondrial reactive oxygen species. For confocal microscopy, BMDM were treated with or without 250 µM palmitate for 24 hours and incubated with 200 nM mitotracker green, 200 nM mitotracker deep Red (Excitation/emission: 644/665, in the Cy3 channel), and 5 µM of mitosox red (PE), and counterstained with 300 nM DAPI (Invitrogen, #D1306) for 30 minutes in PBS. To quantify nuclear vs. cytoplasmic cytochrome c, we gently dropped 100 µl of cytochrome c antibody (Novus Biologicals, #SC69-08) solution into the wells containing BMDM and incubated overnight at 4°C. After washing with PBS containing 0.5% BSA (PBB) five times, the cells were incubated for 60 minutes with goat anti-mouse secondary antibody conjugated with Cy3. Images were taken using a Nikon 216.3 confocal microscope.

Hyperinsulinemic euglycemic clamps studies

These were performed according to guidelines established by the Mouse Metabolic Phenotyping Center Consortium. Briefly, an indwelling jugular vein catheter was surgically implanted five days before the clamp. Mice were fasted overnight prior to the study and received an infusion of [3-3H]-glucose (HPLC purified; Perkin-Elmer Life Sciences) at a rate of 0.05 µCi/min for 120 min followed by blood sampling via tail vein massage to determine basal (fasted) rates of glucose turnover. Next, mice received a primed/continuous infusion of insulin and [3-3H]-glucose (Novolin; Novo Nordisk; 10.5 mU/kg/min and 0.36 µCi/min prime for 4 min; 4.5 mU/kg/min and 0.1 µCi/min continuous) and a variable infusion of 20% dextrose to maintain euglycemia. Blood glucose was sampled at 10 min intervals for the 120 min infusion and plasma samples were collected every 10 min during the last 40 min of the clamp for determination of glucose enrichment for turnover calculations and plasma insulin levels.

Immunoblot

Following euthanization and perfusion with PBS through the left ventricle of mice, the liver and quadriceps muscles were harvested and snap frozen in liquid N2. Tissues and BMDM were homogenized in RIPA lysis buffer (Alfa Aesar, #J63324) containing phosphatase and protease (Pierce, #88666) inhibitors. Homogenates were subjected to SDS-PAGE, and blots were probed with primary antibodies against adiponectin (CST, #2789), beta-tubulin (CST, #2128), beta-actin (CST, #4970), vimentin (Sigma, #SAB4200716), glut4 (Fisher Scientifics, #MA183191), SerpinB2 (Invitrogen, #PA5-27857), Akt (CST, #4691), p-Akt ser473 (CST, #4060), and GAPDH (CST, #2118). For measuring cytochrome c levels in the cytoplasm and mitochondria, cells were subjected to cytosol/mitochondria fractionation using a cytosol/mitochondria extraction kit (Biovision, #K256-25) and SDS-PAGE was incubated with a cytochrome c antibody (Novus Biologicals, #SC69-08) for overnight at 4°C. Subsequently, we incubated the blots with secondary IgG-HRP antibody, and images were captured by molecular imaging system as described previously.

Cellular mitochondrial bioenergetics of BMDM

Bioenergetic analysis of oxidative phosphorylation was performed using Seahorse XF96 Bioanalyzer. Cells were seeded in collagen type I–coated Seahorse plate and treated when confluent. Following treatment, cells were washed with warm media, and the growth medium was replaced with a bicarbonate-free medium. After attaining equilibration for 60 minutes in a 37°C incubator without CO2, cells were sequentially treated with 20 µM oligomycin, 20 µM FCCP at 45 minutes, and 20 µM rotenone at 62 minutes in the presence or absence of etomoxir, a carnitine palmitoyltransferase 1b (CPT1b) inhibitor. Baseline respiration, respiratory ATP production, and maximal respiration rates were measured and normalized to protein content by performing protein analysis.

VAT resident macrophages depletion in LysMcre/+ ChR2fl/fl mice

We generated LysMcre/+ ChR2fl/fl mice that express the channelrhodopsin-2 (ChR2) protein in macrophages and induced apoptosis in VAT resident macrophages by exposing epididymal adipose tissue to the blue light (440 nm) for 20 minutes as earlier. For control, epididymal adipose tissue was exposed to the white light for 20 minutes. To investigate the function of VAT resident macrophages in systemic glucose clearance and insulin sensitivity preservation in obesity, blue light-exposed (BLE) and white light-exposed (WLE) mice were fed on a high-fat diet for 15-60 days.

Calculations of insulin sensitivity, insulin resistance, and GSH gene expression

We combined the expression of insulin sensitivity and anti-inflammatory genes obtained by RNA sequencing and qPCR in the VAT macrophage subsets and considered as 100%. Based on this combined gene expression, we calculated the expression of individual genes as percentages in Fig. 1J. Similarly, for the GSH-encoding genes, we represented the combined the expression of these genes obtained from the RNA sequencing and qPCR experiments as 100%. Based on this average expression, we calculated the expression of individual genes and then averaged them to represent as a single value in Fig. 5E.

PCA Plots

The PCA plot for GTT (AUC) and GSH gene expression in CX3CR1− CCR2− VAT macrophages was made using the calculated percentage expression of the GSH-encoding genes (Gstm1, Gstm2, Gstm3, Gstm4, Gstp1, Gstp4, Mgst1, Gstt1, Gstt2, and Gstt3) for each sample. In the same way, the PCA plots for maximal OCR and secreted cytokines from BMDM were made from the OXPHOS data of Seahorse analysis and ELISA data by calculating the percentage of cytokines released from BMDM of lean and SerpinB2+/+ mice with respect to obese and SerpinB2−/− mice.

Chromatin immunoprecipitation

We performed ChIP as previously. Briefly, SerpinB2 (ThermoFisher Scientific, #PA5-27857) and IgG antibodies were used for chromatin precipitation. Genomic DNA was extracted from immunoprecipitated and non-immunoprecipitated (INPUT) samples, and qPCR was performed to quantify the promoter binding regions.

Statistics

The data were represented as mean ± SEM. The Mann Whitney test (two-tailed) was used to determine the significance between two groups. One-way ANOVA with Bonferoni’s post hoc correction test was performed to determine differences among data obtained from more than two groups. Results were considered statistically significant when P < 0.05.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "The manuscript reads well and contains an impressive amount of data. It seems that the conclusions of the manuscript are\npartly supported by the results. I do have several questions and suggestions that may help to further improve the current\nmanuscript.\n1. I do wonder about the novelty of the first part of the manuscript related to resident versus infiltration macrophages in\nadipose tissue during the development of obesity. Most of these results part of the first figure of the manuscript are more or\nless established. I would suggest to shorten this part of the manuscript and focus on the role of serpinB2 in promoting the\nsurvival of tissue resident macrophages.\n2. I am somewhat confused about the source/role of IFNy. It seems this originates from myeloid cells part of the adipose\ntissue, yet what cells would produce it? Newly recruited macrophages? Or could there also be a role for adipocytes or other\nnon-immune resident cells? And what signals would drive upregulation of IFNy production in adipose tissue?\n3. Why would the role of SerpinB2 be so specific for resident macrophages? Why would it not be expressed by non-resident\nmacrophages? It also seems a well-orchestrated event, so why would this mechanism exist since it seems to negatively\nimpact on adipose tissue health?\n4. How do these VAT resident macrophages restrain adipocyte growth? This is not clear from the data presented in the\ncurrent manuscript.\n5. The authors should be careful in concluding on the role of resident vs. infiltrating macrophages and their role in systemic\ninsulin sensitivity/resistance. This is all based on associations presented in figure 1.\n6. In figure 2, it would be important to indicate BW development of the animals after the depletion of macrophages. How\ndoes this procedure impact on macrophages in tissues other than the adipose tissue ?\n7. I do not really understand the added value of figure 3S. Yes, palmitate decreases SerpinB2 expression, yet do the authors\nnow suggest that fatty acids are the drivers of decreased expression in adipose tissue resident macrophages?\n8. Could the authors provide more clinical characteristics of the individuals with obesity that were included in the\nmanuscript?\n9. In figure 4H and 4I different metabolic and inflammatory genes are presented. Why are data presented in different layouts?\nAnd what is the added value of measuring these metabolic genes?\nIt would be helpful to learn more about the BW of the animals in the main figure and not in the supplemental figure 3.\n10. In figure 6 related to the metabolic profiles of the macrophages, it would be very informative to also present the ECAR\ndata of these experiments. I would also suggest to show the complete OCR graphs in the main figure, so not only the\nmaximal OCR. I am also somewhat confused about the data presented in figure 6, since all of these results are generated in\nex vivo/in vitro experiments. This would imply that the absence of SerpinB2 would also promote apoptosis/changes in\nmitochondrial respiration in other tissue macrophages in vivo. Have the authors examined other tissues of their in vivo\nstudies?\nIn general, the manuscript contains an impressive amount of data. For certain figures, the authors might consider to lower\ndata density helping readability of the manuscript. I would also suggest to remove the last part of the manuscript that is\nfocused on adipocyte size. This seems of little added value in conveying the message that resident VAT adipose tissue\nmacrophages control insulin sensitivity.\nThe results section is very extensive, yet the discussion is short and more or less a summary of the results. I think it also\nreflects the different results that are presented in the current manuscript. The third paragraph shortly deals with how\nmacrophages control adipocyte growth, while the next paragraph discuss the role of mitochondria in cytokine production by\nmacrophages without a clear connection between both paragraphs.", "reviewer_2": "The current study proposed to understand the impact of SerpinB2 on tissue-resident macrophage survival in obese adipose\ntissue. A set of transgenic mouse models were employed to determine the importance of SerpinB2 in CX3CR1+CCR2+ or\nCX3CR1+CCR2- macrophages. The authors first examined the two subsets of macrophages in visceral adipose tissue\n(VAT) transcriptome profiles and subsequently focused on SerpinB2-regulated macrophage survival and their impact on\nsystemic metabolic regulation. The study also zoomed in on SerpinB2-regulated oxidative stress mediated by mitochondria\nreleasing cytochrome C as the molecular mechanism. The overall study is interesting and likely to provide new aspects of\nhow SerpinB2 regulates resident macrophage function in obese visceral adipose tissue. Although some aspects of the topic\nwere supported by detailed characterization, major concerns should be addressed to support the primary argument as\nfollows:\n1. One key aspect of the study is to argue that SerpinB2 will selectively regulate residential vs monocyte-derived\nmacrophage functions in obese VAT. The study focused on these two macrophage subsets marked with CX3CR1 and\nCCR2 and suggested that CX3CR1+CCR2+ are monocyte derived macrophages while CX3CR1-CCR2- are residential\nmacrophages. However, this key point as shown in Fig 1 was rather not convincing. For example, the expression pattern of\nCCR2 in CX3CR1-GFP positive vs negative portions are comparative evidenced by MFI. As shown in Fig S1F-- a\nsignificant portion of the CX3CR1-CCR2+ population was not calculated in both lean and obese samples. These cells\nappear to have similar CCR2 MFI as those in CX3CR1-GFP+ populations.\n2. The comparison between CX3CR1+CCR2+/ CX3CR1-CCR2- to CD206+/CD206+ cells further argue the indicated\npopulations could be more suitable for inflammatory vs anti-inflammatory states than residential or monocyte-derived\nmacrophages. Furthermore, a good body of evidence suggest that monocyte-derived macrophage can be either\ninflammatory or anti-inflammatory.\n3. Authors suggested that a “decrease” in CCR2- population is likely due to increased apoptosis. However, almost all results\non this point were presented as proportion differences, which could be merely due to a significant increase in the CCR2+\npopulation. Meanwhile, authors only reported some minor increase in the proportion of caspase 3+ cells in the CCR2-\npopulation, but no results were provided for CCR2+ cells. Without considering obesity-induced stress in this context, data\npresented only on the CCR2 population are not convincing. Similarly, no results on the CCR2+ population were examined.\nIn Fig 2B, no differences were observed in CCR2+ cells, and the authors proposed that it was likely due to high turnovers.\nWith a significantly increased infiltration rate, it could also accompanied by a high rate of apoptosis.\n4. SerpinB2 is expressed in monocytes and macrophages and can be further induced under inflammatory conditions, as\nreported previously. Results in this study also reported that THP1 differentiated mac in vitro can express high levels of\nSerpinB2. Why was its expression in CCR2+ mac barely detectable (Fig 3)?\n5. SerpinB2-deficient macrophages are apoptotic, which could have various causal factors, including a direct impact on the\ninflammasome. The rationale for only examining cytochrome C levels was not clear.", "reviewer_3": "The manuscript by Vasamsetti et al explores the dynamics of adipose tissue macrophages (ATMs) during obesity and\nhighlights the role of SerpinB2 in obesity related macrophage dysfunction. Using a mouse model of diet induced obesity\nthey provide evidence that during obesity “resident” ATMs undergo apoptosis. Using an inducible model of macrophage cell\ndeath they suggest that loss of resident macrophages worsening obesity and insulin resistance. One of the genes enriched in\n“resident” ATMs was SerpinB2. The expression of SerpinB2 was suppressed by IFNg in culture and KO of the IFNgR in\nmyeloid cells increased in the number of SerpinB2 positive macrophages and improved measures of insulin resistance in\nvivo. KO of SerpinB2 also led to increased ATM OXPHOS, adipose inflammation and worse insulin resistance, which could\nbe rescued by IL-4. The authors provide evidence that Serpin is associated with improved mitochondrial function and less\nROS generation. Overall, the study tackles an important area of research. In addition, the authors should be commended for\ntheir combined use of several genetic models and inhibitors to address the role of SerpinB2 in ATMs. However, there are\nseveral points that should be addressed to improve the impact of the study.\nMajor Comments:\n1) The authors needs be careful with the terminology of resident vs recruited adipose macrophages purely based on\nexpression of CX3CR1 and/or CCR2. Although CX3CR1 and CCR2 are expressed on incoming recruited cells they can be\ndownregulated rapidly in the tissue. Therefore, cells that are CX3CR1 and CCR2 negative can still be monocyte-derived,\nrecruited cells. The “true” ATM resident population is very small in number (~ 10,000 cells per fat pad) whereas upon high fat\nfeeding the number of ATMs increases 10-fold and typically consists of CD9, TREM2 expressing ATMs (known as lipid\nassociated macrophages (LAMS; PMID31257031), which are monocyte derived. To address the conversion rate of Cx3Cr1\npos to Cx3Cr1 neg macrophages in adipose tissue during obesity, the authors should use their Cx3Cr1-YFP, Cx3CR1-\nCreER-TdT system for fate mapping. Using these mice, the authors could determine what percentage of the “red” cells\n(MdMs) are YFP-positive (i.e. actively expressing Cx3Cr1) at day 1 and day 7 post labeling with tamoxifen. In addition, it\nwould be useful to assess for CD9 expression via flow cytometry to identify “lipid associated macrophages” and TIM4 to\nassess for true resident macrophages. The authors also need to include the absolute number of cells per gram of tissue or\nper fat pad in addition to just percentages. This information is critical for interpreting the study and understanding adipose\ntissue macrophage dynamics.\n2) In Figure 2, the authors employ an innovative approach to delete macrophages from the adipose tissue of obese mice by\nleveraging a Cre-activatable expression of a photosensitive channel that triggers cells death upon exposure to blue light.\nHowever, the experimental design indicates that mice were fed a HFD for 1 month prior to exposure to light. During the\nmonth of feed the ATM compartment expands substantially via recruitment and proliferation of monocyte derived cells. As\ndiscussed in point #1, the majority of the macrophages in the adipose tissue at this time are likely CD9-positive LAMs (which\nare predominantly CCR2 low). As LysM expresses well in all macrophages but only poorly in monocytes (< 50%) it is\npossible that the failure of light to deplete CCR2 cells is both related to rapid turnover and poor expression in\nmonocytes/early macrophages. Either way the use of CCR2 IF to assess the specificity the deletion approach for resident vs.\nrecruited cells is flawed. Flow cytometry using other markers such as CD9 and TIM4 should be employed. My suspicion is\nthat this approach leads to the loss of LAMs, which are recruited macrophages that play a protective role in obese adipose\ntissue and liver (PMID 31257031; PMID36521495) . Without this information it is hard to harmonize this study with the larger\nliterature around ATMs and obesity.\n3) In general, the figures are difficult to follow even after reading the legend. As an example, in Figure 1 the A panel is hard\nto interpret as the Cx3CR1 pos dot plot contains 80% Cx3Cr1 negative cells. Panels B and C which included the tamoxifen\nlabeling system and parabiosis require going back to the main text to figure out what these plots represent. Adding\nschematics and/or better details in the legend is important. In addition, removing some of the panels from Figure 1 would\nimprove the impact of the data. For example, the value of Panel D and Panel F are not clear. This data could be in\nsupplemental. Also, for panels M and N the protocol for when tamoxifen was given relative to the start of HFD is unclear.\nHow many does were given? Tamoxifen can also influence metabolic phenotypes so this needs to be accounted for.\n4) What happened to total ATM content in the myeloid-IFNGR1 KO mice? Were total numbers of macrophages impacted or\njust the macrophage phenotype? Using flow cytometry to assess SerpinB2 expression in WT vs. KO ATMs would enhance\nstrength the semiquantitative IF data in Fig. 4 C (the provide flow data for SerpinB2 later in the manuscript showing\nfeasibility).\n5) The NAC experiments are somewhat hard to interpret as this is a systemic antioxidant. What happened to macrophage\nnumber and cell death with NAC? NAC is also poorly tolerated as a oral solution due to its smell. Could some of the\nobserved effects be related to changes in weight or diet intake?\n6) The authors use a myeloid COX10 KO mouse to provide evidence that enhanced OXPHOS may be detrimental to ATMs\nand lead to increased ROS and death. However, this data is only shown in supplemental and is missing the seahorse data\nto show the impact of COX10 KO on BMDM metabolism.\nMinor:\n1) In Figure 1 N why did the authors use autofluorescence? Macrophage specific staining with either CD68 or F4/80 would\nbe better here.\n2) As mentioned in major comments panel 1M should include both percentage and total number of macrophages as this will\nbe hugely different between STD vs. HFD fed animals.\n3) The authors should include the flow gating scheme with representative images for ATMs in the supplemental data.\n4) In figure 5 K they authors show Mitosox staining, which reveals subtle differences between WT and SerpinB2 KO\nmacrophages. They then report a ratio of Mitosox to Mitotracker. Was this based on flow determined MFI values? Does this\nmean that mitochondrial number was also different between genotypes to drive the ratio up?\n5) The protocol for IL-4 reconstitution is missing from the methods. Did they use IL-4/IL-4R complexes or just naked\ncytokine?\n6) In panel 5M the displayed MFI quantification is not congruent with the adjacent flow plot. The numbers for MFI must x\n1000. This should be indicated."}, "author_response": "We appreciate the overall enthusiasm of this reviewer for our study. We have addressed each\ncomment of this reviewer with additional data and explanation as discussed below.\n1. I do wonder about the novelty of the first part of the manuscript related to resident versus\ninfiltration macrophages in adipose tissue during the development of obesity. Most of these\nresults part of the first figure of the manuscript are more or less established. I would suggest\nto shorten this part of the manuscript and focus on the role of serpinB2 in promoting the\nsurvival of tissue resident macrophages.\nAs this reviewer recommended, we have now shortened the part of the manuscript containing the\nfunctions of resident and monocyte-derived macrophages to focus on the role of SerpinB2 in\npromoting the survival of tissue resident macrophages (lines 139-164).\nResponse Fig. 1: Obesity increases IFN-𝛄-expressing fibroblasts and macrophages in\nadipose tissue. VAT from lean and obese mice were stained for the markers of fibroblasts,\nmacrophages, and adipocytes along with IFN-𝛄. The frequencies of IFN-𝛄-expressing cells\nwere assessed by confocal microscopy. n=5/group. **p<0.01.\n2. I am somewhat confused about the source/role of IFNy. It seems this originates from\nmyeloid cells part of the adipose tissue, yet what cells would produce it? Newly recruited\nmacrophages? Or could there also be a role for adipocytes or other non-immune resident\ncells? And what signals would drive upregulation of IFNy production in adipose tissue?\nTo find the source of IFN-y, we stained VAT of lean and obese mice with the markers for\nadipocytes, fibroblasts, and macrophages. We observed that obesity significantly increased the\nfrequencies of IFN-𝛄-expressing fibroblasts and macrophages (Response Fig. 1 and Fig. 4C and\nlines 282-283 in the manuscript).\nSeveral mechanisms have been proposed for the production of IFN-y by obese adipose tissue. In\nobesity, adipocytes secrete CXCL12, which recruits natural killer cells and stimulates the secretion\nof IFN-y by these cells1. Besides natural killer cells, inflammatory macrophages and T helper cells\ncan infiltrate adipose tissue and secrete IFN-y in obesity2, 3. Additionally, the activation of HIF-1⍺\n1\nin response to the hypoxic environment in adipose tissue in obesity can stimulate the transcription\nof various genes including Ifng4. We have now included these mechanisms of IFN-y production in\nobesity in Discussion (lines 511-516 in the manuscript).\n3. Why would the role of SerpinB2 be so specific for\nresident macrophages? Why would it not be expressed\nby non-resident macrophages? It also seems a well-\norchestrated event, so why would this mechanism exist\nsince it seems to negatively impact on adipose tissue\nhealth?\nOur data provided in the manuscript demonstrate that IFN-y\nsuppresses SerpinB2 expression in macrophages (Fig. 4).\nBased this observation and the data showing that monocyte- Response Fig. 2: Monocyte-\nderived non-resident macrophages express low levels of derived macrophages express\nSerpinB2, we hypothesized that this macrophage population high levels of the Ifng\nhas high levels of the IFN-y receptors. Indeed, this receptors. Ifngr1 and Ifngr2\npopulation has high expression of Ifngr1 and Infgr2 were quantified by RNA seq in\ncompared to VAT resident macrophages (Response Fig. 2 monocyte-derived and resident\nand Fig. 4O, and lines 304-305 in the manuscript).\nn=3/group, *p<0.05.\nThe question of this reviewer regarding the significance of\nSerpinB2 expression by the macrophage subsets is\ninteresting. We have observed that IFN-𝛄, which is released\nin high quantity in infection, decreases SerpinB2 levels in\nVAT resident macrophages, resulting in their apoptosis. Loss\nof VAT resident macrophages can lead to infiltration of\nmonocyte-derived macrophages, which have better infection\nfighting ability and lower SerpinB2 expression, thereby are\nresistant to IFN-y-mediated apoptosis. Future studies will be\nResponse Fig. 3: VAT\nrequired to examine the significance of SerpinB2 expression\nmonocyte-derived and\nby the macrophage subsets in infection clearance. We have\nresident macrophages express\nnow included this point in Discussion (lines 516-522 in the\ndifferential levels of the\nmanuscript).\ncytokines regulating adipose\ngrowth. RNA sequencing was\n4. How do these VAT resident macrophages restrain\nperformed in monocyte-derived\nadipocyte growth? This is not clear from the data\nand resident macrophages sorted\npresented in the current manuscript.\nfrom VAT. The RPKM values\nWe appreciate this insightful comment of this reviewer.\nfor Il1b and Tgfb2 are plotted.\nAdipocyte expansion is regulated by several biological\nn=3/ group.\nprocesses including adipocyte apoptosis and necrosis,\nadipogenesis, lipolysis, and lipogenesis. Inflammatory cytokines produced by macrophages and\nmonocytes can regulate these processes. A recent study showed that PDDGcc produced by\nmacrophages can control adipocyte energy storage, which aids in the growth of adipose tissue in\nobesity5. We observed that adipose monocyte-derived and tissue-resident macrophages expressed\ndifferential amounts of cytokines affecting adipose growth. For example, adipose resident\nmacrophages express low levels of Il1b, which stimulates adipocyte growth6, and high levels of\nFig. 3 and Fig. S7B and lines 444-446 in the manuscript). However, how these inflammatory cues\nregulate adipocyte growth by affecting adipocyte apoptosis and necrosis, adipogenesis, lipolysis,\nor lipogenesis is beyond the scope of this study. We have now acknowledged this limitation of our\nstudy in Discussion (lines 558-566 in the manuscript).\n5. The authors should be\ncareful in concluding on\nthe role of resident vs.\ninfiltrating macrophages\nand their role in systemic\ninsulin\nsensitivity/resistance. This\nis all based on associations\npresented in figure 1.\nThis point of the reviewer is\nwell taken. We have\nspecifically depleted VAT Response Fig. 4: Blue light exposure to VAT does not affect\napoptosis of distant macrophages. A) Annexin V mean\nresident macrophages in an\noptogenetic LysMcre/+ fluorescence intensities were calculated by flow cytometry in\nChr2fl/fl mice and found that microglia, Kupffer cells, and splenic macrophages in LysMcre/+\nthe depletion of this Chr2fl/fl mice after white and blue light exposure to VAT. B)\npopulation led to systemic\nhepatic macrophages and Kupffer cells were enumerated by\nglucose intolerance and confocal microscopy in these mice. n=3-5/group.\ninsulin resistance (Fig. 2).\nAdditionally, we have acknowle dged the correlative nature of the data presented in Figure 1 in\nDiscussion (lines 569-572 in the manuscript).\n6. In figure 2, it would be important to indicate BW development of the animals after the\ndepletion of macrophages. How does this procedure impact on macrophages in tissues other\nthan the adipose tissue?\nWe now provide the body weight data in these mice (Figure S2E). Additionally, we have examined\nif blue light exposure in VAT of the optogenetic mice alters the expression of Annexin V, an early\nmarker of apoptosis, in macrophages residing in distant organs. This treatment did not significantly\nchange Annexin V expression in microglia, Kupffer cells, and splenic macrophages (Response Fig.\n4A and Fig. S2C and lines 226-229 in the manuscript). Consistently, we observed no significant\ndifference in total hepatic macrophage and Kupffer cell numbers (Response Fig. 4B and Fig. S2D\nand lines 229-230 in the manuscript).\n7. I do not really understand the added value of figure 3S. Yes, palmitate decreases SerpinB2\nexpression, yet do the authors now suggest that fatty acids are the drivers of decreased\nexpression in adipose tissue resident macrophages?\nWe have used palmitate as a stimulus for macrophages. It has been reported that obesity can\nincrease endogenous palmitic acid levels8. This excess palmitic acid can promote inflammation9.\nBecause exaggerated inflammation can reduce SerpinB2 expression, we sought to test whether\n3\npalmitic acid can reduce the expression of this gene. We have now clarified this rationale in the\ntext (lines 272-275 in the manuscript).\n8. Could the authors provide more clinical characteristics of the individuals with obesity that\nwere included in the manuscript?\nSince these patients were deidentified\ndeceased donors, we have limited\nPatient Age Sex BMI Diabetic\ndemographic and clinical information\nfor these patients. However, we were\nHAC-2 49 F 29.737 No\nable to retrieve the information on age,\nsex, BMI, and diabetes status in these\nHAC-4 51 M 27.31 No\npatients (Response Table 1 and Table\nS1 and lines 267 in the manuscript).\nHAC-6 91 M 28.1 Yes\nHAC-10 22 F 20.906 No\n9. In figure 4H and 4I different\nmetabolic and inflammatory genes HAC-12 25 F 25.712 Yes\nare presented. Why are data HAC-13 59 F 33.299 Yes\npresented in different layouts? And\nHAC-15 56 F 28.125 No\nwhat is the added value of\nHAC-17 19 M 22.531 No\nmeasuring these metabolic genes? It\nHAC-18 65 M 29.037 No\nwould be helpful to learn more\nabout the BW of the animals in the HAC-19 84 F 22.319 No\nmain figure and not in the HAC-20 73 M 34.6 Yes\nsupplemental figure 3.\nHAC-22 42 F 45.54 Yes\nWe appreciate this comment of the\nHAC-26 59 M 28 No\nreviewer. The metabolic genes shown\nHAC-28 19 F 20.5 No\nin new Fig. 4I are insulin-sensitivity\nHAC-30 83 M 22.4 No\npromoting genes, which were\nincreased in the absence of Ifngr1 in HAC-31 85 F 20.05 No\nmyeloid cells. New Fig. 4J shows the HAC-32 65 F 41.025 Yes\ninflammatory genes, which were\nHAC-33 76 M 25.6 Yes\nsuppressed in the absence of myeloid\nHAC-38 57 M 39 No\nIfngr1. As this reviewer suggested, we\nResponse Table 1: The demographic information of\nhave now moved the body weight data\npatients. Mesenteric adipose tissues from deceased\nto the main figure (Fig. 4F).\ndonors were collected. The demographic information\nof these patients is provided in this table.\n10. In figure 6 related to the\nmetabolic profiles of the\nmacrophages, it would be very informative to also present the ECAR data of these\nexperiments. I would also suggest to show the complete OCR graphs in the main figure, so\nnot only the maximal OCR. I am also somewhat confused about the data presented in figure\n6, since all these results are generated in ex vivo/in vitro experiments. This would imply that\nthe absence of SerpinB2 would also promote apoptosis/changes in mitochondrial respiration\nin other tissue macrophages in vivo. Have the authors examined other tissues of their in vivo\nstudies?\nwe were not successful in measuring these parameters from these sorted macrophages because\nthey are metabolically inactive just after the sort probably due to the stress of cell sorting. We have\nnow provided the OCR data in the main figure and ECAR traces in the supplement (Response Fig.\n5 and Fig. 6C and S5F and line 372 in the manuscript). Additionally, our new data demonstrated\nthat Kupffer cell numbers in the liver do not change in SerpinB2-/- obese mice (Response Fig. 6\nand Fig. S4B and lines 312-313 in the manuscript). The mechanisms of the absence of numerical\nchanges in Kupffer cells of these mice should be addressed by future studies (Discussion, lines\n575-577 in the manuscript).\n11. In general, the\nmanuscript contains an\nimpressive amount of\ndata. For certain figures,\nthe authors might\nconsider to lower data\ndensity helping\nreadability of the\nmanuscript. I would also\nsuggest to remove the last Response Fig. 5: SerpinB2 deficiency suppresses oxidative\npart of the manuscript phosphorylation and augments extracellular acidification\nthat is focused on rates. BMDM were derived from SerpinB2+/+ and SerpinB2-/-\nadipocyte size. This seems mice. OCR and ECAR were analyzed by the SeaHorse assay.\nof little added value in n=5/ group.\nconveying the message\nthat resident VAT adipose tissue\nmacrophages control insulin sensitivity.\nAs this reviewer recommended, we have\nnow extensively worked on the\nmanuscript and figures to make them less\ndense. However, since the editor wanted\nus to keep the adipocyte data, we have\nmoved the data involving adipocyte\ncharacteristics after NAC treatment to the\nsupplement (Fig. S7K-M) to focus on the\nmessage that resident VAT adipose tissue\nmacrophages control insulin sensitivity.\nResponse Fig. 6: SerpinB2 deficiency does not\n12. The results section is very extensive,\nalter Kupffer cell numbers. Kupffer cells in\nyet the discussion is short and more or\nSerpinB2+/+ and SerpinB2-/- mice were evaluated\nless a summary of the results. I think it\nby flow cytometry. n=4-7/ group.\nalso reflects the different results that\nare presented in the current manuscript. The third paragraph shortly deals with how\nmacrophages control adipocyte growth, while the next paragraph discusses the role of\nmitochondria in cytokine production by macrophages without a clear connection between\nboth paragraphs.\nextensively to clearly connect different parts of this section.\nReviewer #2 (Remarks to the Author):\nThe current study proposed to understand the impact of SerpinB2 on tissue-resident\nmacrophage survival in obese adipose tissue. A set of transgenic mouse models were\nemployed to determine the importance of SerpinB2 in CX3CR1+CCR2+ or CX3CR1-CCR2-\nmacrophages. The authors first examined the two subsets of macrophages in visceral adipose\ntissue (VAT) transcriptome profiles and subsequently focused on SerpinB2-regulated\nmacrophage survival and their impact on systemic metabolic regulation. The study also\nzoomed in on SerpinB2-regulated oxidative stress mediated by mitochondria releasing\ncytochrome C as the molecular mechanism. The overall study is interesting and likely to\nprovide new aspects of how SerpinB2 regulates resident macrophage function in obese\nvisceral adipose tissue. Although some aspects of the topic were supported by detailed\ncharacterization, major concerns should be addressed to support the primary argument as\nfollows:\nWe are glad to know that this reviewer found this\nstudy interesting. We have addressed the crucial\nconcerns of this reviewer with new experimental\ndata.\n1. One key aspect of the study is to argue that\nSerpinB2 will selectively regulate residential vs Response Fig. 7: The abundance of\nmonocyte-derived macrophage functions in CX 3 CR1- CCR2+ macrophages do not\nobese VAT. The study focused on these two change in obesity. The frequencies and\nmacrophage subsets marked with CX3CR1 numbers of CX 3 CR1- CCR2+ macrophages\nand CCR2 and suggested that were assessed by flow cytometry. n=4/\nCX3CR1+CCR2+ are monocyte derived group, *<p<0.05.\nmacrophages while CX3CR1-CCR2- are\nresidential macrophages. However, this key po int as shown in Fig 1 was rather not\nconvincing. For example, the expression pattern of CCR2 in CX3CR1-GFP positive vs\nnegative portions are comparative evidenced by MFI. As shown in Fig S1F-- a\nsignificant portion of the CX3CR1-CCR2+ population was not calculated in both lean and obese\nsamples. These cells appear to have similar CCR2 MFI as those in CX3CR1-GFP+\npopulations.\nThe comment of this reviewer regarding CCR2 expression in CX CR1+ cells is well taken. To\n3\ndistinguish the macrophage subsets in mouse VAT based on their CCR2 and CX CR1expression,\n3\nwe have now shown the gates for the following populations in Fig. S1H: CX CR1+ CCR2+,\n3 3\nCX CR1- CCR2+ macrophages in lean and obese mice. Although the frequencies of these\n3\nmacrophages decreased, their numbers were not significantly changed in obese mice (Response\n3\nand subsided in obesity, respectively (Response Fig. 8A and Fig. S1I and line 173 in the\nmanuscript).\n2. The comparison between CX3CR1+CCR2+/ CX3CR1-CCR2- to CD206+/CD206- cells\nfurther argue the indicated populations could be more suitable for inflammatory vs anti-\ninflammatory states than residential or monocyte-derived macrophages. Furthermore, a\ngood body of evidence suggest that monocyte-derived macrophage can be either\ninflammatory or anti-inflammatory.\nWe agree with this\ninterpretation that CX3CR1+\nCCR2+ and CX3CR1- CCR2-\nmacrophages are\ninflammatory and anti-\ninflammatory, respectively.\nWe have now also included\nthis information in Results\n(lines 161-163 in the\nmanuscript). Additionally,\nusing a mouse model of\nparabiosis, we demonstrate\nthat CX CR1- CCR2-\n3\nmacrophages are adipose\nresidents whereas CX CR1+\nhematopoietic cell-derived\n(Fig. 1C and line 141 in the\nmanuscript). Furthermore,\nwe previously used an array\nof techniques, including cell\nlineage tracing, to ascertain\nthat CX CR1- CCR2-\n3\nmacrophages are adipose\nresidents whereas CX CR1+\n3\nmonocyte-derived10. macrophages increases whereas CX CR1- CCR2-\n3\nmacrophage numbers decrease in obesity. A) The numbers\n3. Authors suggested that a of CX CR1+ CCR2+ and CX CR1- CCR2- VAT macrophages\n3 3\n“decrease” in CCR2- in obese mice (four months of HFD) and B-C) CD9+ and TIM4+\npopulation is likely due to VAT macrophages at one and two months after HFD feeding\nincreased apoptosis. were assessed by flow cytometry. n=4-10/ group, *<p<0.05,\nHowever, almost all results ***p<0.001, ****p<0.0001.\non this point were\npresented as proportion differences, which could be merely due to a significant increase in\nthe CCR2+ population. Meanwhile, authors only reported some minor increase in the\n7\nproportion of caspase 3+ cells in the CCR2- population, but no results were provided for\nCCR2+ cells. Without considering obesity-induced stress in this context, data presented only\non the CCR2 population are not convincing. Similarly, no results on the CCR2+ population\nwere examined. In Fig 2B, no differences were observed in CCR2+ cells, and the authors\nproposed that it was likely due to high turnovers. With a significantly increased infiltration\nrate, it could also accompanied by a high rate of apoptosis.\nAs this reviewer suggested, we have now included the absolute numbers of various macrophage\nsubsets in VAT of lean and obese mice (Response Fig. 8A and Fig. S1I and line 173 in the\nmanuscript). We have also shown the dynamics of the numbers of other adipose macrophage\nsubsets reported in the literature, including CD9+ lipid-associated macrophages and TIM4+\nresident macrophages (Response Fig. 8B & 8C and Fig. S1K and lines 175-180 in the manuscript).\nThe number of both subsets of macrophages increased significantly at one month after HFD.\nHowever, although CD9-expressing lipid-associated macrophages remained significantly\nincreased at two months after HFD, the abundance of TIM4+ VAT resident macrophages\ndiminished at this time. Our data indicate that, although there is an initial expansion of the TIM4+\nresident macrophage subset after HFD initiation, this macrophage population shrinks at latter\nstages.\nAdditionally, we have shown that the\nfrequency of caspase 3+ CCR2+\nmacrophages did not significantly\nchange in obese mice by flow\ncytometry (Response Fig. 9A and Fig.\n1Q and lines 191-192 in the\nmanuscript).\nMoreover, as this reviewer predicted,\nblue light exposure in obese LysMcre/+\nChR2fl/fl mice significantly enhanced Response Fig. 9: CXCR1+ CCR2+ macrophage\nearly apoptosis, in CX CR1+ CCR2+ initiation. The frequencies of apoptotic macrophages\nflow cytometry (Response Fig. 9B and light exposure in LysM+/+ ChR2fl/fl and LysMcre/+ ChR2fl/fl\nFig. S2A and lines 219-221 in the mice were calculated by flow cytometry. n=4/ group.\nmanuscript). We have now included\nthis information in the manuscript (lines 2 17-218).\n4. SerpinB2 is expressed in monocytes and macrophages and can be further induced under\ninflammatory conditions, as reported previously. Results in this study also reported that\nTHP1 differentiated mac in vitro can express high levels of SerpinB2. Why was its expression\nin CCR2+ mac barely detectable (Fig 3)?\nAlthough we observed high expression of SerpinB2 by VAT resident macrophages, CCR2+\nmacrophages also expressed SerpinB2, albeit at low levels. We found that this subset of\nmacrophages expresses high levels of the IFN-𝛄 receptors (Response Fig. 2 and Fig. 4O and lines\n304-305 in the manuscript). Since we have shown that IFN-𝛄 reduces SerpinB2 expression in\nmacrophages (Fig. 4N), low SerpinB2 expression by CCR2+ macrophages may be due to their high\nexpression of the IFN-𝛄 receptors. Additionally, the discrepancies of SerpinB2 expression by\naltered phenotypes of cultured macrophages. In line with this hypothesis, previous studies have\nshown that macrophages cultured in vitro exhibit significant changes in their epigenome11, 12, 13.\n5. SerpinB2-deficient macrophages are apoptotic, which could\nhave various causal factors, including a direct impact on the\ninflammasome. The rationale for only examining cytochrome C\nlevels was not clear.\nWe focused on cytochrome C because we observed SerpinB2\nmodulates mitochondrial function, which can regulate cytochrome C\nrelease from mitochondria. Additionally, we observed no change in\nthe expression of Nlrp3, which is involved in inflammasome\nsignaling, in SerpinB2-/- adipose resident macrophages compared to Response Fig. 10:\nSerpinB2 deficiency\nthe control (Response Fig. 10 and Fig. S4F and lines 352-356 in the\ndoes not significantly\nmanuscript). We have now discussed this in Results and\nalter Nlrp3\nacknowledged the limitations of not studying inflammasomes in\nexpression in adipose\nSerpinB2-deficient macrophage apoptosis in Discussion (lines 498-\nresident\n500 in the manuscript).\nmacrophages. The\nexpression of Nlrp3,\nwhich is involved in\nthe inflammasome\nReviewer #3 (Remarks to the Author):\npathway, was\nmeasured by qPCR.\nThe manuscript by Vasamsetti et al explores the dynamics of\nn=12=16/ group.\nadipose tissue macrophages (ATMs) during obesity and\nhighlights the role of SerpinB2 in obesity related macrophage\ndysfunction. Using a mouse model of diet induced obesity they provid e evidence that during\nobesity “resident” ATMs undergo apoptosis. Using an inducible mo del of macrophage cell\ndeath they suggest that loss of resident macrophages worsenin g obesity and insulin\nresistance. One of the genes enriched in “resident” ATMs was SerpinB2. The expression of\nSerpinB2 was suppressed by IFNg in culture and KO of the IFNgR in myeloid cells increased\nin the number of SerpinB2 positive macrophages and improved measures of insulin\nresistance in vivo. KO of SerpinB2 also led to increased ATM OXPHOS, adipose\ninflammation and worse insulin resistance, which could be rescued by IL-4. The authors\nprovide evidence that Serpin is associated with improved mitochondrial function and less\nROS generation. Overall, the study tackles an important area of research. In addition, the\nauthors should be commended for their combined use of several genetic models and\ninhibitors to address the role of SerpinB2 in ATMs. However, there are several points that\nshould be addressed to improve the impact of the study.\nWe appreciate the positive evaluation of our study by this reviewer. We have extensively revised\nthe manuscript with additional data and explanation from the literature to address the major and\nminor concerns mentioned below.\nMajor Comments:\n9\n1) The authors needs be careful with the terminology of resident vs recruited adipose\nmacrophages purely based on expression of CX3CR1 and/or CCR2. Although CX3CR1 and\nCCR2 are expressed on\nincoming recruited cells they\ncan be downregulated rapidly\nin the tissue. Therefore, cells\nthat are CX3CR1 and CCR2\nnegative can still be\nmonocyte-derived, recruited\ncells. The “true” ATM Response Fig. 11: Most monocyte-derived macrophages\nresident population is very do not downregulate CX CR1 in adipose tissue.\n3\nsmall in number (~ 10,000 CX3CR1CreER/+ ROSAtdTomato/+ mice were injected with\ncells per fat pad) whereas tamoxifen, and the flow cytometry analysis was performed\nupon high fat feeding the four weeks later. n=3/ group.\nnumber of ATMs increases 10-\nfold and typically consists of CD 9, TREM2 expressing ATMs (known as lipid associated\nmacrophages (LAMS; PMID31257031), which are monocyte derived. To address the\nconversion rate of Cx3Cr1 pos to Cx3Cr1 neg macrophages in adipose tissue during obesity,\nthe authors should use their Cx3Cr1-YFP, Cx3CR1-CreER-TdT system for fate mapping.\nUsing these mice, the authors could determine what percentage of the “red” cells (MdMs)\nare YFP-positive (i.e. actively expressing Cx3Cr1) at day 1 and day 7 post labeling with\ntamoxifen. In addition, it would be useful to assess for CD9 expression via flow cytometry to\nidentify “lipid associated macrophages” and TIM4 to assess for true resident macrophages.\nThe authors also need to include the absolute number of cells per gram of tissue or\nper fat pad in addition to just percentages. This information is critical for interpreting the study and\nunderstanding adipose tissue macrophage dynamics.\nWe appreciate the comment of this reviewer. We performed a parabiosis (Fig. 1C) experiment to\nexamine the contribution of monocytes to the differentiation of these two subsets of adipose\nmacrophages. Additionally, as this reviewer suggested, we conducted a lineage tracing experiment\nin CX CR1-CreER-TdTomato mice (Response Fig. 11 and Fig. S1B and lines 143-147 in the\n3\nmanuscript). These mice were injected with tamoxifen. Four weeks after tamoxifen injection, most\ntdTomato+ macrophages were CX CR1YFP+. Only about 12% of them were CX CR1YFP-.\n3 3\nThese\ndata indicate that CX CR1+ macrophages convert to CX CR1- macrophages, albeit at low levels.\n3 3\nAs this reviewer suggested, we have now shown the absolute numbers of the VAT macrophage\nsubsets per mg of tissue (Response Fig. 8A and Fig. S1I and lines 171-173 in the manuscript). The\nnumber of CX CR1+ CCR2+ (monocyte-derived) macrophages increased while the abundance of\ntwo months after HFD initiation. The number of both subsets of macrophages increased\nsignificantly at one month after HFD (Response Fig. 8B & 8C and Fig. S1K and lines 175-180 in\nthe manuscript). However, although CD9-expressing lipid-associated macrophages remained\nsignificantly increased at two months after HFD, the abundance of TIM4+ VAT resident\n10\nmacrophages diminished at this time. Our data indicate that although there is an initial expansion\nof the TIM4+ resident macrophage subset after HFD initiation, this macrophage population shrinks\nat latter stages possibly due to their high apoptosis. The flow cytometry plots (Response Fig. 8B)\nhave not been included in the manuscript due to lack of space.\n2) In Figure 2, the authors employ an innovative approach to delete macrophages from the\nadipose tissue of obese mice by leveraging a Cre-activatable expression of a photosensitive\nchannel that triggers cells death upon exposure to blue light. However, the experimental\ndesign indicates that mice were fed a HFD for 1 month prior to exposure to light. During the\nmonth of feed the ATM compartment expands substantially via recruitment and\nproliferation of monocyte derived cells. As discussed in point #1, the majority of the\nmacrophages in the adipose tissue at this time are likely CD9-positive LAMs (which\nare predominantly CCR2 low). As LysM expresses well in all macrophages but only poorly in\nmonocytes (< 50%) it is\npossible that the failure of light to deplete CCR2 cells is both related to rapid turnover and poor expression in\nmonocytes/early macrophages. Either way the use\nof CCR2 IF to assess the specificity the deletion approach for resident vs. recruited cells is\nflawed. Flow cytometry using other markers such as CD9 and TIM4 should be employed.\nMy suspicion is\nthat this approach leads to the loss of LAMs, which are recruited macrophages that play a protective role in obese adipose\ntissue and liver (PMID 31257031; PMID36521495). Without this information it is hard to harmonize this study with the larger\nliterature around ATMs and\nobesity.\nTo address this crucial concern,\nwe have performed confocal\nmicroscopy on VAT samples\nfrom these mice. Our new data\ndemonstrate that TIM4+\nmacrophage abundance was\nlower in VAT of LysMcre/+\nChR2fl/fl mice after blue light\nexposure as expected\n(Response Fig. 12 and Fig.\nS2B and lines 221-226 in the\nmanuscript). Moreover, as this\nreviewer suspected, the Response Fig. 12: Blue light exposure to VAT of LysMcre/+\nnumber of CD9+ LAMs ChR2fl/fl mice depletes adipose TIM4+ resident and CD9+\ndiminished significantly upon lipid-associated macrophages. VAT of LysMcre/+ ChR2fl/fl\nblue light exposure. We have mice was exposed to either white light (WLE) or blue light\nnow included this information (BLE). The numbers of TIM4+ and CD9+ macrophages were\nin Results14, 15 (Lines 221-226 enumerated by confocal microscopy. n=5/ group. **p<0.01.\nin the manuscript).\n3) In general, the figures are diffi cult to follow even after reading the legend. As an example,\nin Figure 1 the A panel is hard to interpret as the Cx3CR1 pos dot plot contains 80% Cx3Cr1\nnegative cells. Panels B and C wh ich included the tamoxifen labeling system and parabiosis\nrequire going back to the mai n text to figure out what these plots represent. Adding\n11\nschematics and/or better details in the legend is important. In addition, removing some of\nthe panels from Figure 1 would improve the impact of the data. For example, the value of\nPanel D and Panel F are not clear. This data could be in supplemental. Also, for panels M\nand N the protocol for when tamoxifen was given relative to the start of HFD is unclear. How\nmany does were given? Tamoxifen can also influence metabolic phenotypes so this needs to\nbe accounted for.\nWe apologize for this confusion. Fig. 1A shows CX CR1-GFP+ macrophages in CX CR1GFP/+ mice\n3 3\nthat express GFP under the CX CR1 promoter. Panels B and C show the data from the cell lineage\n3\ntracing and parabiosis experiments, respectively. As this reviewer suggested, we have now\nincluded experimental details in the figure legends. We have included the tamoxifen dosing regime\nin the figure legend. Finally, to address Comment #1 of Reviewer 1 on Figure 1, we have now\nshortened the part of the manuscript containing the functions of resident and monocyte-derived\nmacrophages to focus on the role of SerpinB2 in promoting the survival of tissue resident\nmacrophages (lines 140-164 in the manuscript).\n4) What happened to total\nATM content in the myeloid-\nIFNGR1 KO mice? Were\ntotal numbers of\nmacrophages impacted or\njust the macrophage\nphenotype? Using flow Response Fig. 13: The total number of adipose\ncytometry to assess SerpinB2 macrophages does not significantly change in obese mice\nexpression in WT vs. KO lacking myeloid Ifngr1. LysM+/+ Ifngr1fl/fl and LysMCre/+\nATMs would enhance Ifngr1fl/fl mice were fed with an HFD, and macrophages were\nstrength the semiquantitative analyzed by flow cytometry. n=4-5/ group.\nIF data in Fig. 4 C (the\nprovide flow data for SerpinB2 la ter in the manuscript showing feasibility).\nOur data indicate that the total nu mber of adipose macrophages did not alter in obese LysM+/+\nIfngr1fl/fl and LysMcre/+ Ifngr1fl/fl m ice (Response Fig. 13 and Fig. S4A and lines 288-289 in the\nmanuscript).\nBecause we were given a short period to revise this manuscript, we were unable to re-breed LysM+/+\n,\nIfngr1fl/fl and LysMcre/+ Ifngr1fl/fl mice to perform the flow cytometry experiment assessing\nSerpinB2 expression as suggested by the reviewer. However, we have shown a blinded analysis of\nthe IF data, which demonstrates that SerpinB2 expression was significantly upregulated in the\nobese mice lacking myeloid Ifngr1 (Figure 4D).\n5) The NAC experiments are somewhat hard to interpret as this is a systemic antioxidant.\nWhat happened to macrophage number and cell death with NAC? NAC is also poorly\ntolerated as a oral solution due to its smell. Could some of the observed effects be related to\nchanges in weight or diet intake?\nAs this reviewer requested, we have provided total and apoptotic macrophage numbers in mice\nafter NAC treatment. We observed that, although NAC treatment did not alter the total number of\n12\nadipose macrophages, it reduced the abundance of macrophages expressing caspase 3 (Response\nFig. 14A and Fig. S7J and lines 479-480 in the manuscript).\nNAC treatment reduced body weights in LysMcre/+ SerpinB2fl/fl mice (Response Fig. 14B and Fig.\nS7G and lines 471-\n472 in the\nmanuscript). Some of\nthe beneficial effects\nof NAC could be\nattributed to the\ndecreased body\nweights. We have\nnow acknowledged\nthis in Discussion\n(lines 584-585 in the\nmanuscript).\n6) The authors use a\nmyeloid COX10 KO\nmouse to provide\nResponse Fig. 14: NAC treatment reduces macrophage apoptosis\nevidence that\nand body weight in obese LysMcre/+ SerpinB2fl/fl mice. LysM+/+\nenhanced OXPHOS\nSerpinB2fl/fl and LysMCre/+ SerpinB2fl/fl mice were fed with an HFD and\nmay be detrimental\nsupplemented with water or NAC. A) Total and apoptotic VAT\nto ATMs and lead to\nmacrophages were determined by confocal microscopy. B) The body\nincreased ROS and\nweights are provided. n=4-10/ group. **p<0.01.\ndeath. However,\nthis data is only shown in supplemental and is missing the seahorse data to show the impact\nof COX10 KO on BMDM metabolism.\nResponse Fig. 15: COX10 deficiency suppresses oxidative phosphorylation in BMDM.\nBMDM was collected from LysM+/+ COX10fl/fl and LysMCre/+ COX10fl/fl mice and a seahorse\nassay was performed to measure oxygen consumption rate (A) and extra-cellular acidification\nrate (B) in these cells. n=4/ group. *p<0.05.\nWe have now performed the SeaHorse assay in BMDM isolated from WT and myeloid-specific\nCox10 KO mice. This experiment uncovered depressed basal and maximal respiration and spare\ncapacity while extracellular acidification rates were unchanged in Cox10-deficient BMDM\n13\ncompared to WT BMDM (Response Fig. 15 and Fig. S5D &S 5E and lines 370-372 in the\nmanuscript).\nMinor:\n1) In Figure 1 N why did the authors use autofluorescence? Macrophage specific staining\nwith either CD68 or F4/80 would be better here.\nFigure 1N represents the intravital microscopy data of the dynamics of adipose monocyte-derived\nand resident macrophages at different timepoints after HFD initiation. We were unable to stain the\nmacrophages in vivo with an antibody. However, we have performed flow cytometry in these mice\nto show that CCR2+ macrophages (F4/80+) express tdTomato (Response Fig. 11 and Fig. S1B and\nlines 143-147 in the manuscript).\n2) As mentioned in major comments panel 1M should include both percentage and total\nnumber of macrophages as this will be hugely different between STD vs. HFD fed animals.\nWe have now included the total number of both macrophage subsets (Response Fig. 8A and Fig.\nS1I and lines 171-173 in the manuscript).\n3) The authors should include the flow gating scheme with representative images for ATMs\nin the supplemental data.\nResponse Fig. 16: The gating strategy for adipose macrophages is provided. Single cell\nsuspensions from adipose tissue were stained with various markers of leukocytes, and the cells\nwere analyzed by flow cytometry.\nThe gating strategy for flow cytometry has been\nincluded in the supplemental data (Response Fig. 16\nand Fig. 1G).\n4) In figure 5 K they authors show Mitosox\nstaining, which reveals subtle differences between\nWT and SerpinB2 KO macrophages. They then\nreport a ratio of Mitosox to Mitotracker. Was this\nbased on flow determined MFI values? Does this\nResponse Fig. 17: SerpinB2\nmean that mitochondrial number was also\ndeficiency decreases mitochondrial\ndifferent between genotypes to drive the ratio up?\nvolume in macrophages. BMDM\nYes, these are flow cytometry data. We provided the\nisolated from SerpinB2+/+ and\nratios of mitosox to mitotracker to assess\nSerpinB2-/- mice were stained with\nmitochondrial ROS content per unit of mitochondria.\nmitotracker green and analyzed by\nAlthough we cannot determine mitochondrial\nflow cytometry. n=5-6/group.\nnumber, we can assess mitochondrial volume using\n14\nthe mitotracker green dye by flow cytometry. We now show that mitotracker green MFI was\nsignificantly decreased in SerpinB2-/- BMDM treated with palmitate (Response Fig. 17 and Fig.\nS4E and lines 342-344 in the manuscript).\n5) The protocol for IL-4 reconstitution is missing from the methods. Did they use IL-4/IL-4R\ncomplexes or just naked cytokine?\nHere is the protocol: Ten weeks old LysMcre/+SerpinB2 fl/fl mice were fed with an HFD for two\nmonths followed by i.p infusion of 1 ng IL-4 (R&D, #404-ML-050/CF, 2 doses/ week) dissolved\nin 100 microliters of PBS (lines 600-603 in the manuscript).\n6) In panel 5M the displayed MFI quantification is not congruent with the adjacent flow plot.\nThe numbers for MFI must x 1000. This should be indicated.\nWe apologize for this oversight. We have now corrected the Y axis label of the graph.\nReferences:\n1. Kim, H.H. et al. xCT-mediated glutamate excretion in white adipocytes stimulates\ninterferon-gamma production by natural killer cells in obesity. Cell Rep 42, 112636\n(2023).\n2. Chawla, A., Nguyen, K.D. & Goh, Y.P. Macrophage-mediated inflammation in\nmetabolic disease. Nat Rev Immunol 11, 738-749 (2011).\n3. Yao, J. et al. Macrophage IRX3 promotes diet-induced obesity and metabolic\ninflammation. Nat Immunol 22, 1268-1279 (2021).\n4. Lawler, H.M. et al. Adipose Tissue Hypoxia, Inflammation, and Fibrosis in Obese\nInsulin-Sensitive and Obese Insulin-Resistant Subjects. J Clin Endocrinol Metab 101,\n1422-1428 (2016).\n5. Cox, N. et al. Diet-regulated production of PDGFcc by macrophages controls energy\nstorage. Science 373 (2021).\n6. Hofwimmer, K. et al. IL-1beta promotes adipogenesis by directly targeting adipocyte\nprecursors. Nat Commun 15, 7957 (2024).\n7. Choy, L. & Derynck, R. Transforming growth factor-beta inhibits adipocyte\ndi^erentiation by Smad3 interacting with CCAAT/enhancer-binding protein (C/EBP)\nand repressing C/EBP transactivation function. J Biol Chem 278, 9609-9619 (2003).\n8. Annevelink, C.E., Sapp, P.A., Petersen, K.S., Shearer, G.C. & Kris-Etherton, P.M. Diet-\nderived and diet-related endogenously produced palmitic acid: E^ects on metabolic\nregulation and cardiovascular disease risk. J Clin Lipidol 17, 577-586 (2023).\n15\n9. Qiu, T. et al. Obesity-induced elevated palmitic acid promotes inflammation and\nglucose metabolism disorders through GPRs/NF-kappaB/KLF7 pathway. Nutr\nDiabetes 12, 23 (2022).\n10. Vasamsetti, S.B. et al. Apoptosis of hematopoietic progenitor-derived adipose tissue-\nresident macrophages contributes to insulin resistance after myocardial infarction.\nSci Transl Med 12 (2020).\n11. Amit, I., Winter, D.R. & Jung, S. The role of the local environment and epigenetics in\nshaping macrophage identity and their e^ect on tissue homeostasis. Nat Immunol\n17, 18-25 (2016).\n12. Lavin, Y. et al. Tissue-resident macrophage enhancer landscapes are shaped by the\nlocal microenvironment. Cell 159, 1312-1326 (2014).\n13. Yona, S. et al. Fate mapping reveals origins and dynamics of monocytes and tissue\nmacrophages under homeostasis. Immunity 38, 79-91 (2013).\n14. Jaitin, D.A. et al. Lipid-Associated Macrophages Control Metabolic Homeostasis in a\nTrem2-Dependent Manner. Cell 178, 686-698 e614 (2019).\n15. Wang, X. et al. Prolonged hypernutrition impairs TREM2-dependent e^erocytosis to\nlicense chronic liver inflammation and NASH development. Immunity 56, 58-77 e11\n(2023).\n16"}, {"round": 2, "reviewer_comments": {"reviewer_1": "The authors have adequately responded to many of the suggestions. However, I do still have\nseveral remaining issues.\nThe manuscript still remains (too) information dense making it hard to read and digest all data\nthat is presented. This also seems to go hand in hand with the fact that the manuscript basically\npresents two stories as one. The part on adipocyte size regulation seems a somewhat different\nstory and is also not mentioned in the abstract. I would advise to reconsider this part again.\nI also feel that the inclusion of the data obtained from human VAT samples is somewhat difficult\nto interpret. These samples were obtained from deceased people introducing many potential\nconfounders. Also, age and BMI is hugely variable and no information on cause of death is\npresented.\nNote, there is also no need to present BMI with 3 decimals. Also, in de M&M section the study\nparticipants are described as people with and without diabetes, while in the results section the\nparticipants are presented as lean vs. obese. Some of the study participants do have diabetes, yet\nbased on their BMI would be classified as overweight. Also, how is diabetes being classified and\nis this type 1 or type 2 diabetes?\nRelated to the animal study with the LysM +/+ Ifngr1fl/fl and LysMcre-/+ Ifngr1 fl/fl mice, some\nresults are difficult to interpret. Although total BW is similar, VAT adipose tissue is smaller.\nWhat about total adipose tissue mass? Anything known about activity, food intake of the animals\n? I guess multiple factors are at play here beyond the local effects of the macrophages in\nVAT.", "reviewer_2": "The authors have provided additional results and discussion, addressing each point raised in the\nfirst review, particularly the attributes used to define \"residential\" versus \"infiltration\"\nmacrophages. I have no further questions for the current version.", "reviewer_3": "In the revised manuscript the authors have largely addressed my concerns. The added significant\nnew data and reorganized the mansucript, which significantly improves the impact of the study. I\nhave no further comments."}, "author_response": "We appreciate the insightful comments from this reviewer. Addressing the concerns has helped\nus significantly improve the manuscript.\nThe manuscript still remains (too) information dense making it hard to read and digest all data\nthat is presented. This also seems to go hand in hand with the fact that the manuscript basically\npresents two stories as one. The part on adipocyte size regulation seems a somewhat different\nstory and is also not mentioned in the abstract. I would advise to reconsider this part again.\nWe have kept the adipocyte component in the manuscript in accordance with the previous\nsuggestion of the editor. However, the reviewer has accurately pointed out that the abstract\nlacked the information on adipocyte size. We have now included this information in the abstract.\nI also feel that the inclusion of the data obtained from human VAT samples is somewhat difficult\nto interpret. These samples were obtained from deceased people introducing many potential\nconfounders. Also, age and BMI is hugely variable and no information on cause of death is\npresented.\nWe acknowledge these limitations of the patient samples and include this text in Discussion:\n“Another limitation of this study is that we used samples from deceased patients, which\nmay introduce many potential confounders. These patients had highly variable BMI, and\nthe information regarding their causes of death was not available”\nNote, there is also no need to present BMI with 3 decimals. Also, in de M&M section the study\nparticipants are described as people with and without diabetes, while in the results section the\nparticipants are presented as lean vs. obese. Some of the study participants do have diabetes, yet\nbased on their BMI would be classified as overweight. Also, how is diabetes being classified and\nis this type 1 or type 2 diabetes?\nWe have now provided the BMI with 2 decimals. These patients had type 2 diabetes. We have\ncategorized these patients in either lean or obese based on their BMI. This information is now in\nMethods.\nRelated to the animal study with the LysM +/+ Ifngr1fl/fl and LysMcre-/+ Ifngr1 fl/fl mice, some\nresults are difficult to interpret. Although total BW is similar, VAT adipose tissue is smaller.\nWhat about total adipose tissue mass? Anything known about activity, food intake of the animals\n? I guess multiple factors are at play here beyond the local effects of the macrophages in\nVAT.\nThese are all excellent points that reviewers have noted. In response to this comment, we have\nacknowledged the effects of these variables in Discussion: “Although our data indicate that\nmyeloid-specific deficiency of Infgr1 resulted in improved insulin sensitivity by protecting\nadipose-resident macrophages from apoptosis, we cannot rule out the contributions of reduced\nfood intake, increased activity, and reduced fat mass to the beneficial effects.”.\nWe thank the reviewer for his constructive critiques, which helped us improve the manuscript\nsignificantly.\nWe appreciate the positive feedback of this reviewer."}]

Decision letter