Identification of apolipoprotein B-reactive CDR3 motifs allows tracking of atherosclerosis-related memory CD4+T cells in multiple donors.

Introduction: Atherosclerosis is a major pathological condition that underlies many cardiovascular diseases (CVDs). Its etiology involves breach of tolerance to self, leading to clonal expansion of autoreactive apolipoprotein B (APOB)-reactive CD4+T cells that correlates with clinical CVD. The T-cell receptor (TCR) sequences that mediate activation of APOB-specific CD4+T cells are unknown. Methods: In a previous study, we had profiled the hypervariable complementarity determining region 3 (CDR3) of CD4+T cells that respond to six immunodominant APOB epitopes in most donors. Here, we comprehensively analyze this dataset of 149,065 APOB-reactive and 199,211 non-reactive control CDR3s from six human leukocyte antigen-typed donors. Results: We identified 672 highly expanded (frequency threshold > 1.39E-03) clones that were significantly enriched in the APOB-reactive group as compared to the controls (log10 odds ratio ≥1, Fisher's test p < 0.01). Analysis of 114,755 naïve, 91,001 central memory (TCM) and 29,839 effector memory (TEM) CDR3 sequences from the same donors revealed that APOB+ clones can be traced to the complex repertoire of unenriched blood T cells. The fraction of APOB+ clones that overlapped with memory CDR3s ranged from 2.2% to 46% (average 16.4%). This was significantly higher than their overlap with the naïve pool, which ranged from 0.7% to 2% (average 1.36%). CDR3 motif analysis with the machine learning-based in-silico tool, GLIPHs (grouping of lymphocyte interactions by paratope hotspots), identified 532 APOB+ motifs. Analysis of naïve and memory CDR3 sequences with GLIPH revealed that ~40% (209 of 532) of these APOB+ motifs were enriched in the memory pool. Network analysis with Cytoscape revealed extensive sharing of the memory-affiliated APOB+ motifs across multiple donors. We identified six motifs that were present in TCM and TEM CDR3 sequences from >80% of the donors and were highly enriched in the APOB-reactive TCR repertoire. Discussion: The identified APOB-reactive expanded CD4+T cell clones and conserved motifs can be used to annotate and track human atherosclerosis-related autoreactive CD4+T cells and measure their clonal expansion.

Mild Hypoxia Accelerates Cerebral Cavernous Malformation Disease Through CX3CR1-CX3CL1 Signaling.

BACKGROUND: Heterogeneity in the severity of cerebral cavernous malformations (CCMs) disease, including brain bleedings and thrombosis that cause neurological disabilities in patients, suggests that environmental, genetic, or biological factors act as disease modifiers. Still, the underlying mechanisms are not entirely understood. Here, we report that mild hypoxia accelerates CCM disease by promoting angiogenesis, neuroinflammation, and vascular thrombosis in the brains of CCM mouse models. METHODS: We used genetic studies, RNA sequencing, spatial transcriptome, micro-computed tomography, fluorescence-activated cell sorting, multiplex immunofluorescence, coculture studies, and imaging techniques to reveal that sustained mild hypoxia via the CX3CR1-CX3CL1 (CX3C motif chemokine receptor 1/chemokine [CX3C motif] ligand 1) signaling pathway influences cell-specific neuroinflammatory interactions, contributing to heterogeneity in CCM severity. RESULTS: Histological and expression profiles of CCM neurovascular lesions (Slco1c1-iCreERT2;Pdcd10fl/fl; Pdcd10BECKO) in male and female mice found that sustained mild hypoxia (12% O2, 7 days) accelerates CCM disease. Our findings indicate that a small reduction in oxygen levels can significantly increase angiogenesis, neuroinflammation, and thrombosis in CCM disease by enhancing the interactions between endothelium, astrocytes, and immune cells. Our study indicates that the interactions between CX3CR1 and CX3CL1 are crucial in the maturation of CCM lesions and propensity to CCM immunothrombosis. In particular, this pathway regulates the recruitment and activation of microglia and other immune cells in CCM lesions, which leads to lesion growth and thrombosis. We found that human CX3CR1 variants are linked to lower lesion burden in familial CCMs, proving it is a genetic modifier in human disease and a potential marker for aggressiveness. Moreover, monoclonal blocking antibody against CX3CL1 or reducing 1 copy of the Cx3cr1 gene significantly reduces hypoxia-induced CCM immunothrombosis. CONCLUSIONS: Our study reveals that interactions between CX3CR1 and CX3CL1 can modify CCM neuropathology when lesions are accelerated by environmental hypoxia. Moreover, a hypoxic environment or hypoxia signaling caused by CCM disease influences the balance between neuroinflammation and neuroprotection mediated by CX3CR1-CX3CL1 signaling. These results establish CX3CR1 as a genetic marker for patient stratification and a potential predictor of CCM aggressiveness.

Breaking tolerance: the autoimmune aspect of atherosclerosis.

Atherosclerotic cardiovascular disease (ASCVD) is a chronic inflammatory disease of the arterial walls and is characterized by the accumulation of lipoproteins that are insufficiently cleared by phagocytes. Following the initiation of atherosclerosis, the pathological progression is accelerated by engagement of the adaptive immune system. Atherosclerosis triggers the breakdown of tolerance to self-components. This loss of tolerance is reflected in defective expression of immune checkpoint molecules, dysfunctional antigen presentation, and aberrations in T cell populations - most notably in regulatory T (Treg) cells - and in the production of autoantibodies. The breakdown of tolerance to self-proteins that is observed in ASCVD may be linked to the conversion of Treg cells to 'exTreg' cells because many Treg cells in ASCVD express T cell receptors that are specific for self-epitopes. Alternatively, or in addition, breakdown of tolerance may trigger the activation of naive T cells, resulting in the clonal expansion of T cell populations with pro-inflammatory and cytotoxic effector phenotypes. In this Perspective, we review the evidence that atherosclerosis is associated with a breakdown of tolerance to self-antigens, discuss possible immunological mechanisms and identify knowledge gaps to map out future research. Rational approaches aimed at re-establishing immune tolerance may become game changers in treating ASCVD and in preventing its downstream sequelae, which include heart attacks and strokes.

Dysregulated cellular metabolism in atherosclerosis: mediators and therapeutic opportunities.

Accumulating evidence over the past decades has revealed an intricate relationship between dysregulation of cellular metabolism and the progression of atherosclerotic cardiovascular disease. However, an integrated understanding of dysregulated cellular metabolism in atherosclerotic cardiovascular disease and its potential value as a therapeutic target is missing. In this Review, we (1) summarize recent advances concerning the role of metabolic dysregulation during atherosclerosis progression in lesional cells, including endothelial cells, vascular smooth muscle cells, macrophages and T cells; (2) explore the complexity of metabolic cross-talk between these lesional cells; (3) highlight emerging technologies that promise to illuminate unknown aspects of metabolism in atherosclerosis; and (4) suggest strategies for targeting these underexplored metabolic alterations to mitigate atherosclerosis progression and stabilize rupture-prone atheromas with a potential new generation of cardiovascular therapeutics.

Single Cell High Dimensional Analysis of Human Peripheral Blood Mononuclear Cells Reveals Unique Intermediate Monocyte Subsets Associated with Sex Differences in Coronary Artery Disease.

Monocytes are associated with human cardiovascular disease progression. Monocytes are segregated into three major subsets: classical (cMo), intermediate (iMo), and nonclassical (nMo). Recent studies have identified heterogeneity within each of these main monocyte classes, yet the extent to which these subsets contribute to heart disease progression is not known. Peripheral blood mononuclear cells (PBMC) were obtained from 61 human subjects within the Coronary Assessment of Virginia (CAVA) Cohort. Coronary atherosclerosis severity was quantified using the Gensini Score (GS). We employed high-dimensional single-cell transcriptome and protein methods to define how human monocytes differ in subjects with low to severe coronary artery disease. We analyzed 487 immune-related genes and 49 surface proteins at the single-cell level using Antibody-Seq (Ab-Seq). We identified six subsets of myeloid cells (cMo, iMo, nMo, plasmacytoid DC, classical DC, and DC3) at the single-cell level based on surface proteins, and we associated these subsets with coronary artery disease (CAD) incidence based on Gensini score (GS) in each subject. Only frequencies of iMo were associated with high CAD (GS > 32), adj.p = 0.024. Spearman correlation analysis with GS from each subject revealed a positive correlation with iMo frequencies (r = 0.314, p = 0.014) and further showed a robust sex-dependent positive correlation in female subjects (r = 0.663, p = 0.004). cMo frequencies did not correlate with CAD severity. Key gene pathways differed in iMo among low and high CAD subjects and between males and females. Further single-cell analysis of iMo revealed three iMo subsets in human PBMC, distinguished by the expression of HLA-DR, CXCR3, and CD206. We found that the frequency of immunoregulatory iMo_HLA-DR+CXCR3+CD206+ was associated with CAD severity (adj.p = 0.006). The immunoregulatory iMo subset positively correlated with GS in both females (r = 0.660, p = 0.004) and males (r = 0.315, p = 0.037). Cell interaction analyses identified strong interactions of iMo with CD4+ effector/memory T cells and Tregs from the same subjects. This study shows the importance of iMo in CAD progression and suggests that iMo may have important functional roles in modulating CAD risk, particularly among females.

Single cell transcriptomics reveals recent CD8T cell receptor signaling in patients with coronary artery disease.

Coronary artery disease (CAD) is a major cause of death worldwide. The role of CD8+ T cells in CAD is unknown. Recent studies suggest a breakdown of tolerance in atherosclerosis, resulting in active T cell receptor (TCR) engagement with self-antigens. We hypothesized that TCR engagement would leave characteristic gene expression signatures. In a single cell RNA-sequencing analysis of CD8+ T cells from 30 patients with CAD and 30 controls we found significant enrichment of TCR signaling pathways in CAD+ subjects, suggesting recent TCR engagement. We also found significant enrichment of cytotoxic and exhaustion pathways in CAD cases compared to controls. Highly significant upregulation of TCR signaling in CAD indicates that CD8 T cells reactive to atherosclerosis antigens are prominent in the blood of CAD cases compared to controls.

Pairing of single-cell RNA analysis and T cell antigen receptor profiling indicates breakdown of T cell tolerance checkpoints in atherosclerosis.

Atherosclerotic plaques form in the inner layer of arteries triggering heart attacks and strokes. Although T cells have been detected in atherosclerosis, tolerance dysfunction as a disease driver remains unexplored. Here we examine tolerance checkpoints in atherosclerotic plaques, artery tertiary lymphoid organs and lymph nodes in mice burdened by advanced atherosclerosis, via single-cell RNA sequencing paired with T cell antigen receptor sequencing. Complex patterns of deteriorating peripheral T cell tolerance were observed being most pronounced in plaques followed by artery tertiary lymphoid organs, lymph nodes and blood. Affected checkpoints included clonal expansion of CD4+, CD8+ and regulatory T cells; aberrant tolerance-regulating transcripts of clonally expanded T cells; T cell exhaustion; Treg-TH17 T cell conversion; and dysfunctional antigen presentation. Moreover, single-cell RNA-sequencing profiles of human plaques revealed that the CD8+ T cell tolerance dysfunction observed in mouse plaques was shared in human coronary and carotid artery plaques. Thus, our data support the concept of atherosclerosis as a bona fide T cell autoimmune disease targeting the arterial wall.

Identification of human exTreg cells as CD16+CD56+ cytotoxic CD4+ T cells.

In atherosclerosis, some regulatory T (Treg) cells become exTreg cells. We crossed inducible Treg and exTreg cell lineage-tracker mice (FoxP3eGFP-Cre-ERT2ROSA26CAG-fl-stop-fl-tdTomato) to atherosclerosis-prone Apoe-/- mice, sorted Treg cells and exTreg cells and determined their transcriptomes by bulk RNA sequencing (RNA-seq). Genes that were differentially expressed between mouse Treg cells and exTreg cells and filtered for their presence in a human single-cell RNA-sequencing (scRNA-seq) panel identified exTreg cell signature genes as CST7, NKG7, GZMA, PRF1, TBX21 and CCL4. Projecting these genes onto the human scRNA-seq with CITE-seq data identified human exTreg cells as CD3+CD4+CD16+CD56+, which was validated by flow cytometry. Bulk RNA-seq of sorted human exTreg cells identified them as inflammatory and cytotoxic CD4+T cells that were significantly distinct from both natural killer and Treg cells. DNA sequencing for T cell receptor-ß showed clonal expansion of Treg cell CDR3 sequences in exTreg cells. Cytotoxicity was functionally demonstrated in cell killing and CD107a degranulation assays, which identifies human exTreg cells as cytotoxic CD4+T cells.

Single-cell profiling reveals inflammatory polarization of human carotid versus femoral plaque leukocytes.

Femoral atherosclerotic plaques are less inflammatory than carotid plaques histologically, but limited cell-level data exist regarding comparative immune landscapes and polarization at these sites. We investigated intraplaque leukocyte phenotypes and transcriptional polarization in 49 patients undergoing femoral (n = 23) or carotid (n = 26) endarterectomy using single-cell RNA-Seq (scRNA-Seq; n = 13), flow cytometry (n = 24), and IHC (n = 12). Comparative scRNA-Seq of CD45+-selected leukocytes from femoral (n = 9; 35,265 cells) and carotid (n = 4; 30,655 cells) plaque revealed distinct transcriptional profiles. Inflammatory foam cell-like macrophages and monocytes comprised higher proportions of myeloid cells in carotid plaques, whereas noninflammatory foam cell-like macrophages and LYVE1-overexpressing macrophages comprised higher proportions of myeloid cells in femoral plaque (P < 0.001 for all). A significant comparative excess of CCR2+ macrophages in carotid versus plaque was observed by flow cytometry in a separate validation cohort. B cells were more prevalent and exhibited a comparatively antiinflammatory profile in femoral plaque, whereas cytotoxic CD8+ T cells were more prevalent in carotid plaque. In conclusion, human femoral plaques exhibit distinct macrophage phenotypic and transcriptional profiles as well as diminished CD8+ T cell populations compared with human carotid plaques.

Cell-autonomous regulation of complement C3 by factor H limits macrophage efferocytosis and exacerbates atherosclerosis.

Complement factor H (CFH) negatively regulates consumption of complement component 3 (C3), thereby restricting complement activation. Genetic variants in CFH predispose to chronic inflammatory disease. Here, we examined the impact of CFH on atherosclerosis development. In a mouse model of atherosclerosis, CFH deficiency limited plaque necrosis in a C3-dependent manner. Deletion of CFH in monocyte-derived inflammatory macrophages propagated uncontrolled cell-autonomous C3 consumption without downstream C5 activation and heightened efferocytotic capacity. Among leukocytes, Cfh expression was restricted to monocytes and macrophages, increased during inflammation, and coincided with the accumulation of intracellular C3. Macrophage-derived CFH was sufficient to dampen resolution of inflammation, and hematopoietic deletion of CFH in atherosclerosis-prone mice promoted lesional efferocytosis and reduced plaque size. Furthermore, we identified monocyte-derived inflammatory macrophages expressing C3 and CFH in human atherosclerotic plaques. Our findings reveal a regulatory axis wherein CFH controls intracellular C3 levels of macrophages in a cell-autonomous manner, evidencing the importance of on-site complement regulation in the pathogenesis of inflammatory diseases.

V4 C3 MXene Immune Profiling and Modulation of T Cell-Dendritic Cell Function and Interaction.

Although vanadium-based metallodrugs are recently explored for their effective anti-inflammatory activity, they frequently cause undesired side effects. Among 2D nanomaterials, transition metal carbides (MXenes) have received substantial attention for their promise as biomedical platforms. It is hypothesized that vanadium immune properties can be extended to MXene compounds. Therefore, vanadium carbide MXene (V4 C3 ) is synthetized, evaluating its biocompatibility and intrinsic immunomodulatory effects. By combining multiple experimental approaches in vitro and ex vivo on human primary immune cells, MXene effects on hemolysis, apoptosis, necrosis, activation, and cytokine production are investigated. Furthermore, V4 C3 ability is demonstrated to inhibit T cell-dendritic cell interactions, evaluating the modulation of CD40-CD40 ligand interaction, two key costimulatory molecules for immune activation. The material biocompatibility at the single-cell level on 17 human immune cell subpopulations by single-cell mass cytometry is confirmed. Finally, the molecular mechanism underlying V4 C3 immune modulation is explored, demonstrating a MXene-mediated downregulation of antigen presentation-associated genes in primary human immune cells. The findings set the basis for further V4 C3 investigation and application as a negative modulator of the immune response in inflammatory and autoimmune diseases.

HIV infection and cardiovascular disease have both shared and distinct monocyte gene expression features: Women's Interagency HIV study.

Persistent inflammation contributes to the development of cardiovascular disease (CVD) as an HIV-associated comorbidity. Innate immune cells such as monocytes are major drivers of inflammation in men and women with HIV. The study objectives are to examine the contribution of circulating non-classical monocytes (NCM, CD14dimCD16+) and intermediate monocytes (IM, CD14+CD16+) to the host response to long-term HIV infection and HIV-associated CVD. Women with and without chronic HIV infection (H) were studied. Subclinical CVD (C) was detected as plaques imaged by B-mode carotid artery ultrasound. The study included H-C-, H+C-, H-C+, and H+C+ participants (23 of each, matched on race/ethnicity, age and smoking status), selected from among enrollees in the Women's Interagency HIV Study. We assessed transcriptomic features associated with HIV or CVD alone or comorbid HIV/CVD comparing to healthy (H-C-) participants in IM and NCM isolated from peripheral blood mononuclear cells. IM gene expression was little affected by HIV alone or CVD alone. In IM, coexisting HIV and CVD produced a measurable gene transcription signature, which was abolished by lipid-lowering treatment. In NCM, versus non-HIV controls, women with HIV had altered gene expression, irrespective of whether or not they had comorbid CVD. The largest set of differentially expressed genes was found in NCM among women with both HIV and CVD. Genes upregulated in association with HIV included several potential targets of drug therapies, including LAG3 (CD223). In conclusion, circulating monocytes from patients with well controlled HIV infection demonstrate an extensive gene expression signature which may be consistent with the ability of these cells to serve as potential viral reservoirs. Gene transcriptional changes in HIV patients were further magnified in the presence of subclinical CVD.

Integrated single-cell analysis-based classification of vascular mononuclear phagocytes in mouse and human atherosclerosis.

AIMS: Accumulation of mononuclear phagocytes [monocytes, macrophages, and dendritic cells (DCs)] in the vessel wall is a hallmark of atherosclerosis. Using integrated single-cell analysis of mouse and human atherosclerosis, we here aimed to refine the nomenclature of mononuclear phagocytes in atherosclerotic vessels and to compare their transcriptomic profiles in mouse and human disease. METHODS AND RESULTS: We integrated 12 single-cell RNA-sequencing (scRNA-seq) datasets of immune cells isolated from healthy or atherosclerotic mouse aortas, and data from 11 patients (n = 4 coronary vessels, n = 7 carotid endarterectomy specimens) from two studies. Integration of mouse data identified subpopulations with discrete transcriptomic signatures within previously described populations of aortic resident (Lyve1), inflammatory (Il1b), as well as foamy (Trem2hi) macrophages. We identified unique transcriptomic features distinguishing aortic intimal resident macrophages from atherosclerosis-associated Trem2hi macrophages. Also, populations of Xcr1+ Type 1 classical DCs (cDC1), Cd209a+ cDC2, and mature DCs (Ccr7, Fscn1) with a 'mreg-DC' signature were detected. In humans, we uncovered macrophage and DC populations with gene expression patterns similar to those observed in mice. In particular, core transcripts of the foamy/Trem2hi signature (TREM2, SPP1, GPNMB, CD9) mapped to a specific population of macrophages in human lesions. Comparison of mouse and human data and direct cross-species data integration suggested transcriptionally similar macrophage and DC populations in mice and humans. CONCLUSIONS: We refined the nomenclature of mononuclear phagocytes in mouse atherosclerotic vessels, and show conserved transcriptomic features of macrophages and DCs in atherosclerosis in mice and humans, emphasizing the relevance of mouse models to study mononuclear phagocytes in atherosclerosis.

Kindlin2 enables EphB/ephrinB bi-directional signaling to support vascular development.

Direct contact between cells expressing either ephrin ligands or Eph receptor tyrosine kinase produces diverse developmental responses. Transmembrane ephrinB ligands play active roles in transducing bi-directional signals downstream of EphB/ephrinB interaction. However, it has not been well understood how ephrinB relays transcellular signals to neighboring cells and what intracellular effectors are involved. Here, we report that kindlin2 can mediate bi-directional ephrinB signaling through binding to a highly conserved NIYY motif in the ephrinB2 cytoplasmic tail. We show this interaction is important for EphB/ephrinB-mediated integrin activation in mammalian cells and for blood vessel morphogenesis during zebrafish development. A mixed two-cell population study revealed that kindlin2 (in ephrinB2-expressing cells) modulates transcellular EphB4 activation by promoting ephrinB2 clustering. This mechanism is also operative for EphB2/ephrinB1, suggesting that kindlin2-mediated regulation is conserved for EphB/ephrinB signaling pathways. Together, these findings show that kindlin2 enables EphB4/ephrinB2 bi-directional signal transmission.

Single-cell profiling of CD11c+ B cells in atherosclerosis.

Circulating CD11c+ B cells, a novel subset of activated B cells, have been linked to autoimmunity and shown to expand with age. Atherosclerosis is an age-associated disease that involves innate and adaptive immune responses to modified self-antigens. Yet, the expression of CD11c on specific B-cell subtypes and its link to atherosclerosis are poorly understood. In this study, we characterized the frequency of CD11c+ B cells in tissues in mice with aging. We observed an age-associated increase in CD11c+ B cells in the spleen and bone marrow of ApoE-/- mice, and this was associated with an increase in aortic plaque. In addition, we also utilized single-cell multi-omics profiling of 60 human subjects undergoing advanced imaging for coronary artery disease (CAD) to subtype CD11c+ B cells and determine their frequency in subjects with high and low severity of CAD. Using unsupervised clustering, we identified four distinct clusters of CD11c+ B cells, which include CD27 and IgD double negative 2 (DN2), age-associated (ABC), CD11c+ unswitched memory (USWM), and activated Naïve (aNav) B cells. We observed an increase in the frequency of both ABC B cells and DN2 B cells in patients with high CAD severity. Pathway analysis further demonstrated augmentation of autophagy, IFNg signaling, and TLR signaling in DN2 cells in high-severity CAD patients. On the other hand, an increase in the negative regulator of BCR signaling through CD72 was found in ABC cells in low-severity CAD patients. Through investigating scRNAseq of atheroma, these DN2 cells were also found to infiltrate human coronary atheroma.

Titration of 124 antibodies using CITE-Seq on human PBMCs.

Single-cell RNA-sequencing (scRNA-Seq) is widely used to characterize immune cell populations. However, mRNA levels correlate poorly with expression of surface proteins, which are well established to define immune cell types. CITE-Seq (cellular indexing of transcriptomes and epitopes by sequencing) utilizes oligonucleotide-tagged antibodies to simultaneously analyze surface phenotypes and transcriptomes. Considering the high costs of adding surface phenotyping to scRNA-Seq, we aimed to determine which of 188 tested CITE-Seq antibodies can detect their antigens on human peripheral blood mononuclear cells (PBMCs), a commonly interrogated cell population in immunology, and find the optimal concentration for staining. The recommended concentration was optimal for 76 antibodies, whereas staining quality of 7 antibodies improved when the concentration was doubled. 33 and 8 antibodies still worked well when the concentration was reduced to 1/5 or 1/25, respectively. 64 antigens were not detected at any antibody concentration. Optimizing the antibody panel by removing antibodies not able to detect their target antigens and adjusting concentrations of the remaining antibodies will improve the analysis and may reduce costs. In conclusion, our data are a resource for building an informative and cost-effective panel of CITE-Seq antibodies and use them at their optimal concentrations in future CITE-seq experiments on human PBMCs.

Deleting interleukin-10 from myeloid cells exacerbates atherosclerosis in Apoe-/- mice.

Atherosclerosis is initiated by subendothelial retention of lipoproteins and cholesterol, which triggers a non-resolving inflammatory process that over time leads to plaque progression in the artery wall. Myeloid cells and in particular macrophages are the primary drivers of the inflammatory response and plaque formation. Several immune cells including macrophages, T cells and B cells secrete the anti-inflammatory cytokine IL-10, known to be essential for the atherosclerosis protection. The cellular source of IL-10 in natural atherosclerosis progression is unknown. This study aimed to determine the main IL10-producing cell type in atherosclerosis. To do so, we crossed VertX mice, in which IRES-green fluorescent protein (eGFP) was placed downstream of exon 5 of the Il10 gene, with atherosclerosis-prone Apoe-/- mice. We found that myeloid cells express high levels of IL-10 in VertX Apoe-/- mice in both chow and western-diet fed mice. By single cell RNA sequencing and flow cytometry analysis, we identified resident and inflammatory macrophages in atherosclerotic plaques as the main IL-10 producers. To address whether IL-10 secreted by myeloid cells is essential for the protection, we utilized LyzMCre+Il10fl/fl mice crossed into the Apoe-/- background and confirmed that macrophages were unable to secrete IL-10. Chow and western diet-fed LyzMCre+Il10fl/fl Apoe-/- mice developed significantly larger atherosclerotic plaques as measured by en face morphometry than LyzMCre-Il10 fl/flApoe-/-. Flow cytometry and cytokine measurements suggest that the depletion of IL-10 in myeloid cells increases Th17 cells with elevated CCL2, and TNFα in blood plasma. We conclude that macrophage-derived IL-10 is critical for limiting atherosclerosis in mice.