Artery Tertiary Lymphoid Organs Control Aorta Immunity and Protect against Atherosclerosis via Vascular Smooth Muscle Cell Lymphotoxin ß Receptors.

Tertiary lymphoid organs (TLOs) emerge during nonresolving peripheral inflammation, but their impact on disease progression remains unknown. We have found in aged Apoe(-/-) mice that artery TLOs (ATLOs) controlled highly territorialized aorta T cell responses. ATLOs promoted T cell recruitment, primed CD4(+) T cells, generated CD4(+), CD8(+), T regulatory (Treg) effector and central memory cells, converted naive CD4(+) T cells into induced Treg cells, and presented antigen by an unusual set of dendritic cells and B cells. Meanwhile, vascular smooth muscle cell lymphotoxin ß receptors (VSMC-LTßRs) protected against atherosclerosis by maintaining structure, cellularity, and size of ATLOs though VSMC-LTßRs did not affect secondary lymphoid organs: Atherosclerosis was markedly exacerbated in Apoe(-/-)Ltbr(-/-) and to a similar extent in aged Apoe(-/-)Ltbr(fl/fl)Tagln-cre mice. These data support the conclusion that the immune system employs ATLOs to organize aorta T cell homeostasis during aging and that VSMC-LTßRs participate in atherosclerosis protection via ATLOs.

B-1b Cells Possess Unique bHLH-Driven P62-Dependent Self-Renewal and Atheroprotection.

BACKGROUND: B1a and B1b lymphocytes produce IgM that inactivates oxidation-specific epitopes (IgMOSE) on LDL (low-density lipoprotein) and protects against atherosclerosis. Loss of ID3 (inhibitor of differentiation 3) in B cells selectively promotes B1b but not B1a cell numbers, leading to higher IgMOSE production and reduction in atherosclerotic plaque formation. Yet, the mechanism underlying this regulation remains unexplored. METHODS: Bulk RNA sequencing was utilized to identify differentially expressed genes in B1a and B1b cells from Id3KO and Id3WT mice. CRISPR/Cas9 and lentiviral genome editing coupled with adoptive transfer were used to identify key Id3-dependent signaling pathways regulating B1b cell proliferation and the impact on atherosclerosis. Biospecimens from humans with advanced coronary artery disease imaging were analyzed to translate murine findings to human subjects with coronary artery disease. RESULTS: Through RNA sequencing, P62 was found to be enriched in Id3KO B1b cells. Further in vitro characterization reveals a novel role for P62 in mediating BAFF (B-cell activating factor)-induced B1b cell proliferation through interacting with TRAF6 (tumor necrosis factor receptor 6) and activating NF-κB (nuclear factor kappa B), leading to subsequent C-MYC (C-myelocytomatosis) upregulation. Promoter-reporter assays reveal that Id3 inhibits the E2A protein from activating the P62 promoter. Mice adoptively transferred with B1 cells overexpressing P62 exhibited an increase in B1b cell number and IgMOSE levels and were protected against atherosclerosis. Consistent with murine mechanistic findings, P62 expression in human B1 cells was significantly higher in subjects harboring a function-impairing single nucleotide polymorphism (SNP) at rs11574 position in the ID3 gene and directly correlated with plasma IgMOSE levels. CONCLUSIONS: This study unveils a novel role for P62 in driving BAFF-induced B1b cell proliferation and IgMOSE production to attenuate diet-induced atherosclerosis. Results identify a direct role for Id3 in antagonizing E2A from activating the p62 promoter. Moreover, analysis of putative human B1 cells also implicates these pathways in coronary artery disease subjects, suggesting P62 as a new immunomodulatory target for treating atherosclerosis.

Loss of Id3 (Inhibitor of Differentiation 3) Increases the Number of IgM-Producing B-1b Cells in Ischemic Skeletal Muscle Impairing Blood Flow Recovery During Hindlimb Ischemia.

OBJECTIVE: Neovascularization can maintain and even improve tissue perfusion in the setting of limb ischemia during peripheral artery disease. The molecular and cellular mechanisms mediating this process are incompletely understood. We investigate the potential role(s) for Id3 (inhibitor of differentiation 3) in regulating blood flow in a murine model of hindlimb ischemia (HLI). Approach and Results: HLI was modeled through femoral artery ligation and resection and blood flow recovery was quantified by laser Doppler perfusion imaging. Mice with global Id3 deletion had significantly impaired perfusion recovery at 14 and 21 days of HLI. Endothelial- or myeloid cell-specific deletion of Id3 revealed no effect on perfusion recovery while B-cell-specific knockout of Id3 (Id3BKO) revealed a significant attenuation of perfusion recovery. Flow cytometry revealed no differences in ischemia-induced T cells or myeloid cell numbers at 7 days of HLI, yet there was a significant increase in B-1b cells in Id3BKO. Consistent with these findings, ELISA (enzyme-linked immunoassay) demonstrated increases in skeletal muscle and plasma IgM. In vitro experiments demonstrated reduced proliferation and increased cell death when endothelial cells were treated with conditioned media from IgM-producing B-1b cells and tibialis anterior muscles in Id3BKO mice showed reduced density of total CD31+ and αSMA+CD31+ vessels. CONCLUSIONS: This study is the first to demonstrate a role for B-cell-specific Id3 in maintaining blood flow recovery during HLI. Results suggest a role for Id3 in promoting blood flow during HLI and limiting IgM-expressing B-1b cell expansion. These findings present new mechanisms to investigate in peripheral artery disease pathogenesis.

B Cell-Activating Factor Antagonism Attenuates the Growth of Experimental Abdominal Aortic Aneurysm.

B cell-activating factor (BAFF), part of a tumor necrosis factor family of cytokines, was recently identified as a regulator of atherosclerosis; however, its role in aortic aneurysm has not been determined. Here, the study examined the effect of selective BAFF antagonism using an anti-BAFF antibody (blocks binding of BAFF to receptors BAFF receptor 3, transmembrane activator and CAML interactor, and B-cell maturation antigen) and mBaffR-mFc (blocks binding of BAFF to BAFF receptor 3) on a murine model of abdominal aortic aneurysm (AAA). In a prevention strategy, the antagonists were injected before the induction of AAA, and in an intervention strategy, the antagonists were injected after the induction of AAA. Both strategies attenuated the formation of AAA. In the intervention group, BAFF antagonism depleted most of the mature B-cell subsets in spleen and circulation, leading to enhanced resolution of inflammation in AAA as indicated by decreased infiltration of B cells and proinflammatory macrophages and a reduced number of apoptotic cells. In AAA tissues, B cells and macrophages were found in close contact. In vitro, B cells, irrespective of treatment with BAFF, impaired the efferocytosis activity of macrophages, suggesting a direct innate role of B cells on macrophage function. Altogether, BAFF antagonism affects survival of the mature B cells, promotes resolution of inflammation in the aorta, and attenuates the growth of AAA in mice.

Diversification and CXCR4-Dependent Establishment of the Bone Marrow B-1a Cell Pool Governs Atheroprotective IgM Production Linked to Human Coronary Atherosclerosis.

RATIONALE: B-1 cell-derived natural IgM antibodies against oxidation-specific epitopes on low-density lipoprotein are anti-inflammatory and atheroprotective. Bone marrow (BM) B-1a cells contribute abundantly to IgM production, yet the unique repertoire of IgM antibodies generated by BM B-1a and the factors maintaining the BM B-1a population remain unexplored. CXCR4 (C-X-C motif chemokine receptor 4) has been implicated in human cardiovascular disease and B-cell homeostasis, yet the role of B-1 cell CXCR4 in regulating atheroprotective IgM levels and human cardiovascular disease is unknown. OBJECTIVE: To characterize the BM B-1a IgM repertoire and to determine whether CXCR4 regulates B-1 production of atheroprotective IgM in mice and humans. METHODS AND RESULTS: Single-cell sequencing demonstrated that BM B-1a cells from aged ApoE-/- mice with established atherosclerosis express a unique repertoire of IgM antibodies containing increased nontemplate-encoded nucleotide additions and a greater frequency of unique heavy chain complementarity determining region 3 sequences compared with peritoneal cavity B-1a cells. Some complementarity determining region 3 sequences were common to both compartments suggesting B-1a migration between compartments. Indeed, mature peritoneal cavity B-1a cells migrated to BM in a CXCR4-dependent manner. Furthermore, BM IgM production and plasma IgM levels were reduced in ApoE-/- mice with B-cell-specific knockout of CXCR4, and overexpression of CXCR4 on B-1a cells increased BM localization and plasma IgM against oxidation specific epitopes, including IgM specific for malondialdehyde-modified LDL (low-density lipoprotein). Finally, in a 50-subject human cohort, we find that CXCR4 expression on circulating human B-1 cells positively associates with plasma levels of IgM antibodies specific for malondialdehyde-modified LDL and inversely associates with human coronary artery plaque burden and necrosis. CONCLUSIONS: These data provide the first report of a unique BM B-1a cell IgM repertoire and identifies CXCR4 expression as a critical factor selectively governing BM B-1a localization and production of IgM against oxidation specific epitopes. That CXCR4 expression on human B-1 cells was greater in humans with low coronary artery plaque burden suggests a potential targeted approach for immune modulation to limit atherosclerosis.

B Lymphocytes and Adipose Tissue Inflammation.

The immune system plays an important role in obesity-induced adipose tissue inflammation and the resultant metabolic dysfunction, which can lead to hypertension, dyslipidemia, and insulin resistance and their downstream sequelae of type 2 diabetes mellitus and cardiovascular disease. While macrophages are the most abundant immune cell type in adipose tissue, other immune cells are also present, such as B cells, which play important roles in regulating adipose tissue inflammation. This brief review will overview B-cell subsets, describe their localization in various adipose depots and summarize our knowledge about the function of these B-cell subsets in regulating adipose tissue inflammation, obesity-induced metabolic dysfunction and atherosclerosis.

Novel Autoimmune IgM Antibody Attenuates Atherosclerosis in IgM Deficient Low-Fat Diet-Fed, but Not Western Diet-Fed Apoe-/- Mice.

OBJECTIVE: Oxidized phospholipids (OxPL), such as the oxidized derivatives of 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine, 1-palmitoyl-2-(5-oxovaleroyl)-sn-glycero-3-phosphorylcholine, and 1-palmitoyl-2-glutaroyl-sn-glycero-3-phosphorylcholine, have been shown to be the principal biologically active components of minimally oxidized LDL (low-density lipoprotein). The role of OxPL in cardiovascular diseases is well recognized, including activation of inflammation within vascular cells. Atherosclerotic Apoe-/- mice fed a high-fat diet develop antibodies to OxPL, and hybridoma B-cell lines producing natural anti-OxPL autoantibodies have been successfully generated and characterized. However, as yet, no studies have been reported demonstrating that treatment with OxPL neutralizing antibodies can be used to prevent or reverse advanced atherosclerosis. Approach and Results: Here, using a screening against 1-palmitoyl-2-(5-oxovaleroyl)-sn-glycero-3-phosphorylcholine/1-palmitoyl-2-glutaroyl-sn-glycero-3-phosphorylcholine, we generated a novel IgM autoantibody, 10C12, from the spleens of Apoe-/- mice fed a long-term Western diet, that demonstrated potent OxPL neutralizing activity in vitro and the ability to inhibit macrophage accumulation within arteries of Apoe-/- mice fed a Western diet for 4 weeks. Of interest, 10C12 failed to inhibit atherosclerosis progression in Apoe-/- mice treated between 18 and 26 weeks of Western diet feeding likely due at least in part to high levels of endogenous anti-OxPL antibodies. However, 10C12 treatment caused a 40% decrease in lipid accumulation within aortas of secreted IgM deficient, sIgM-/-Apoe-/-, mice fed a low-fat diet, when the antibody was administrated between 32-40 weeks of age. CONCLUSIONS: Taken together, these results provide direct evidence showing that treatment with a single autoimmune anti-OxPL IgM antibody during advanced disease stages can have an atheroprotective outcome.

B cells and atherosclerosis.

B cells have emerged as important immune cells in cardiovascular disease. Initial studies have suggested that B cells protect against atherosclerosis development. However, subsequent studies demonstrating aggravation of atherosclerosis by B-2 cells have shed light on the subset-dependent effects of B cells. Here, we review the literature that has led to our current understanding of B cell regulation of atherosclerosis, touching on the importance of subsets, local regulation, human translation, and therapeutic potential.

B-1b Cells Secrete Atheroprotective IgM and Attenuate Atherosclerosis.

RATIONALE: B cells contribute to atherosclerosis through subset-specific mechanisms. Whereas some controversy exists about the role of B-2 cells, B-1a cells are atheroprotective because of secretion of atheroprotective IgM antibodies independent of antigen. B-1b cells, a unique subset of B-1 cells that respond specifically to T-cell-independent antigens, have not been studied within the context of atherosclerosis. OBJECTIVE: To determine whether B-1b cells produce atheroprotective IgM antibodies and function to protect against diet-induced atherosclerosis. METHODS AND RESULTS: We demonstrate that B-1b cells are sufficient to produce IgM antibodies against oxidation-specific epitopes on low-density lipoprotein both in vitro and in vivo. In addition, we demonstrate that B-1b cells provide atheroprotection after adoptive transfer into B- and T-cell deficient (Rag1(-/-)Apoe(-/-)) hosts. We implicate inhibitor of differentiation 3 (Id3) in the regulation of B-1b cells as B-cell-specific Id3 knockout mice (Id3(BKO)Apoe(-/-)) have increased numbers of B-1b cells systemically, increased titers of oxidation-specific epitope-reactive IgM antibodies, and significantly reduced diet-induced atherosclerosis when compared with Id3(WT)Apoe(-/-) controls. Finally, we report that the presence of a homozygous single nucleotide polymorphism in ID3 in humans that attenuates Id3 function is associated with an increased percentage of circulating B-1 cells and anti-malondialdehyde-low-density lipoprotein IgM suggesting clinical relevance. CONCLUSIONS: These results provide novel evidence that B-1b cells produce atheroprotective oxidation-specific epitope-reactive IgM antibodies and protect against atherosclerosis in mice and suggest that similar mechanisms may occur in humans.

Artery Tertiary Lymphoid Organs Control Multilayered Territorialized Atherosclerosis B-Cell Responses in Aged ApoE-/- Mice.

OBJECTIVE: Explore aorta B-cell immunity in aged apolipoprotein E-deficient (ApoE(-/-)) mice. APPROACH AND RESULTS: Transcript maps, fluorescence-activated cell sorting, immunofluorescence analyses, cell transfers, and Ig-ELISPOT (enzyme-linked immunospot) assays showed multilayered atherosclerosis B-cell responses in artery tertiary lymphoid organs (ATLOs). Aging-associated aorta B-cell-related transcriptomes were identified, and transcript atlases revealed highly territorialized B-cell responses in ATLOs versus atherosclerotic lesions: ATLOs showed upregulation of bona fide B-cell genes, including Cd19, Ms4a1 (Cd20), Cd79a/b, and Ighm although intima plaques preferentially expressed molecules involved in non-B effector responses toward B-cell-derived mediators, that is, Fcgr3 (Cd16), Fcer1g (Cd23), and the C1q family. ATLOs promoted B-cell recruitment. ATLO B-2 B cells included naive, transitional, follicular, germinal center, switched IgG1(+), IgA(+), and IgE(+) memory cells, plasmablasts, and long-lived plasma cells. ATLOs recruited large numbers of B-1 cells whose subtypes were skewed toward interleukin-10(+) B-1b cells versus interleukin-10(-) B-1a cells. ATLO B-1 cells and plasma cells constitutively produced IgM and IgG and a fraction of plasma cells expressed interleukin-10. Moreover, ApoE(-/-) mice showed increased germinal center B cells in renal lymph nodes, IgM-producing plasma cells in the bone marrow, and higher IgM and anti-MDA-LDL (malondialdehyde-modified low-density lipoprotein) IgG serum titers. CONCLUSIONS: ATLOs orchestrate dichotomic, territorialized, and multilayered B-cell responses in the diseased aorta; germinal center reactions indicate generation of autoimmune B cells within the diseased arterial wall during aging.

Protective Role for B-1b B Cells and IgM in Obesity-Associated Inflammation, Glucose Intolerance, and Insulin Resistance.

OBJECTIVE: Little is known about the role(s) B cells play in obesity-induced metabolic dysfunction. This study used a mouse with B-cell-specific deletion of Id3 (Id3(Bcell KO)) to identify B-cell functions involved in the metabolic consequences of obesity. APPROACH AND RESULTS: Diet-induced obese Id3(Bcell KO) mice demonstrated attenuated inflammation and insulin resistance in visceral adipose tissue (VAT), and improved systemic glucose tolerance. VAT in Id3(Bcell KO) mice had increased B-1b B cells and elevated IgM natural antibodies to oxidation-specific epitopes. B-1b B cells reduced cytokine production in VAT M1 macrophages, and adoptively transferred B-1b B cells trafficked to VAT and produced natural antibodies for the duration of 13-week studies. B-1b B cells null for Id3 demonstrated increased proliferation, established larger populations in Rag1(-/-) VAT, and attenuated diet-induced glucose intolerance and VAT insulin resistance in Rag1(-/-) hosts. However, transfer of B-1b B cells unable to secrete IgM had no effect on glucose tolerance. In an obese human population, results provided the first evidence that B-1 cells are enriched in human VAT and IgM antibodies to oxidation-specific epitopes inversely correlated with inflammation and insulin resistance. CONCLUSIONS: NAb-producing B-1b B cells are increased in Id3(Bcell KO) mice and attenuate adipose tissue inflammation and glucose intolerance in diet-induced obese mice. Additional findings are the first to identify VAT as a reservoir for human B-1 cells and to link anti-inflammatory IgM antibodies with reduced inflammation and improved metabolic phenotype in obese humans.

B-Cell Depletion Promotes Aortic Infiltration of Immunosuppressive Cells and Is Protective of Experimental Aortic Aneurysm.

OBJECTIVE: B-cell depletion therapy is widely used for treatment of cancers and autoimmune diseases. B cells are abundant in abdominal aortic aneurysms (AAA); however, it is unknown whether B-cell depletion therapy affects AAA growth. Using experimental models of murine AAA, we aim to examine the effect of B-cell depletion on AAA formation. APPROACH AND RESULTS: Wild-type or apolipoprotein E-knockout mice were treated with mouse monoclonal anti-CD20 or control antibodies and subjected to an elastase perfusion or angiotensin II infusion model to induce AAA, respectively. Anti-CD20 antibody treatment significantly depleted B1 and B2 cells, and strikingly suppressed AAA growth in both models. B-cell depletion resulted in lower circulating IgM levels, but did not affect the levels of IgG or cytokine/chemokine levels. Although the total number of leukocyte remained unchanged in elastase-perfused aortas after anti-CD20 antibody treatment, the number of B-cell subtypes was significantly lower. Interestingly, plasmacytoid dendritic cells expressing the immunomodulatory enzyme indole 2,3-dioxygenase were detected in the aortas of B-cell-depleted mice. In accordance with an increase in indole 2,3-dioxygenase+ plasmacytoid dendritic cells, the number of regulatory T cells was higher, whereas the expression of proinflammatory genes was lower in aortas of B-cell-depleted mice. In a coculture model, the presence of B cells significantly lowered the number of indole 2,3-dioxygenase+ plasmacytoid dendritic cells without affecting total plasmacytoid dendritic cell number. CONCLUSIONS: The present results demonstrate that B-cell depletion protects mice from experimental AAA formation and promotes emergence of an immunosuppressive environment in aorta.

Artery tertiary lymphoid organs contribute to innate and adaptive immune responses in advanced mouse atherosclerosis.

Tertiary lymphoid organs emerge in tissues in response to nonresolving inflammation. Recent research characterized artery tertiary lymphoid organs in the aorta adventitia of aged apolipoprotein E-deficient mice. The atherosclerosis-associated lymphocyte aggregates are organized into distinct compartments, including separate T-cell areas harboring conventional, monocyte-derived, lymphoid, and plasmacytoid dendritic cells, as well as activated T-cell effectors and memory cells; B-cell follicles containing follicular dendritic cells in activated germinal centers; and peripheral niches of plasma cells. Artery tertiary lymphoid organs show marked neoangiogenesis, aberrant lymphangiogenesis, and extensive induction of high endothelial venules. Moreover, newly formed lymph node-like conduits connect the external lamina with high endothelial venules in T-cell areas and also extend into germinal centers. Mouse artery tertiary lymphoid organs recruit large numbers of naïve T cells and harbor lymphocyte subsets with opposing activities, including CD4(+) and CD8(+) effector and memory T cells, natural and induced CD4(+) regulatory T cells, and memory B cells at different stages of differentiation. These data suggest that artery tertiary lymphoid organs participate in primary immune responses and organize T- and B-cell autoimmune responses in advanced atherosclerosis. In this review, we discuss the novel concept that pro- and antiatherogenic immune responses toward unknown arterial wall-derived autoantigens may be organized by artery tertiary lymphoid organs and that disruption of the balance between pro- and antiatherogenic immune cell subsets may trigger clinically overt atherosclerosis.

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.

B-1b Cells Have Unique Functional Traits Compared to B-1a Cells at Homeostasis and in Aged Hyperlipidemic Mice With Atherosclerosis.

Immunoglobulin M (IgM) to oxidation specific epitopes (OSE) are inversely associated with atherosclerosis in mice and humans. The B-1b subtype of B-1 cells secrete IgM to OSE, and unlike B-1a cells, are capable of long-lasting IgM memory. What attributes make B-1b cells different than B-1a cells is unknown. Our objectives were to determine how B-1b cells produce more IgM compared to B-1a cells at homeostatic condition and to see the differences in the B-1a and B-1b cell distribution and IgM CDR-H3 sequences in mice with advanced atherosclerosis. Here, in-vivo studies demonstrated greater migration to spleen, splenic production of IgM and plasma IgM levels in ApoE-/-Rag1-/- mice intraperitoneally injected with equal numbers of B-1b compared to B-1a cells. Bulk RNA seq analysis and flow cytometry of B-1a and B-1b cells identified CCR6 as a chemokine receptor more highly expressed on B-1b cells compared to B-1a. Knockout of CCR6 resulted in reduced B-1b cell migration to the spleen. Moreover, B-1b cell numbers were significantly higher in spleen of aged atherosclerotic ApoE-/- mice compared to young ApoE-/- mice. Single cell sequencing results of IgHM in B-1a and B-1b cells from peritoneal cavity and spleen of atherosclerotic aged ApoE-/- mice revealed significantly more N additions at the V-D and D-J junctions, greater diversity in V region usage and CDR-H3 sequences in B-1b compared to B-1a cells. In summary, B-1b cells demonstrated enhanced CCR6-mediated splenic migration, IgM production, and IgM repertoire diversification compared to B-1a cells. These findings suggest that potential strategies to selectively augment B-1b cell numbers and splenic trafficking could lead to increased and more diverse IgM targeting OSE to limit atherosclerosis.

Chemokine Receptor-6 Promotes B-1 Cell Trafficking to Perivascular Adipose Tissue, Local IgM Production and Atheroprotection.

Chemokine receptor-6 (CCR6) mediates immune cell recruitment to inflammatory sites and has cell type-specific effects on diet-induced atherosclerosis in mice. Previously we showed that loss of CCR6 in B cells resulted in loss of B cell-mediated atheroprotection, although the B cell subtype mediating this effect was unknown. Perivascular adipose tissue (PVAT) harbors high numbers of B cells including atheroprotective IgM secreting B-1 cells. Production of IgM antibodies is a major mechanism whereby B-1 cells limit atherosclerosis development. Yet whether CCR6 regulates B-1 cell number and production of IgM in the PVAT is unknown. In this present study, flow cytometry experiments demonstrated that both B-1 and B-2 cells express CCR6, albeit at a higher frequency in B-2 cells in both humans and mice. Nevertheless, B-2 cell numbers in peritoneal cavity (PerC), spleen, bone marrow and PVAT were no different in ApoE-/-CCR6-/- compared to ApoE-/-CCR6+/+ mice. In contrast, the numbers of atheroprotective IgM secreting B-1 cells were significantly lower in the PVAT of ApoE-/-CCR6-/- compared to ApoE-/-CCR6+/+ mice. Surprisingly, adoptive transfer (AT) of CD43- splenic B cells into B cell-deficient µMT-/-ApoE-/- mice repopulated the PerC with B-1 and B-2 cells and reduced atherosclerosis when transferred into ApoE-/-CCR6+/+sIgM-/- mice only when those cells expressed both CCR6 and sIgM. CCR6 expression on circulating human B cells in subjects with a high level of atherosclerosis in their coronary arteries was lower only in the putative human B-1 cells. These results provide evidence that B-1 cell CCR6 expression enhances B-1 cell number and IgM secretion in PVAT to provide atheroprotection in mice and suggest potential human relevance to our murine findings.

In vivo liposomal delivery of PPARα/γ dual agonist tesaglitazar in a model of obesity enriches macrophage targeting and limits liver and kidney drug effects.

Macrophages are important regulators of obesity-associated inflammation and PPARα and -γ agonism in macrophages has anti-inflammatory effects. In this study, we tested the efficacy with which liposomal delivery could target the PPARα/γ dual agonist tesaglitazar to macrophages while reducing drug action in common sites of drug toxicity: the liver and kidney, and whether tesaglitazar had anti-inflammatory effects in an in vivo model of obesity-associated dysmetabolism. Methods: Male leptin-deficient (ob/ob) mice were administered tesaglitazar or vehicle for one week in a standard oral formulation or encapsulated in liposomes. Following the end of treatment, circulating metabolic parameters were measured and pro-inflammatory adipose tissue macrophage populations were quantified by flow cytometry. Cellular uptake of liposomes in tissues was assessed using immunofluorescence and a broad panel of cell subset markers by flow cytometry. Finally, PPARα/γ gene target expression levels in the liver, kidney, and sorted macrophages were quantified to determine levels of drug targeting to and drug action in these tissues and cells. Results: Administration of a standard oral formulation of tesaglitazar effectively treated symptoms of obesity-associated dysmetabolism and reduced the number of pro-inflammatory adipose tissue macrophages. Macrophages are the major cell type that took up liposomes with many other immune and stromal cell types taking up liposomes to a lesser extent. Liposome delivery of tesaglitazar did not have effects on inflammatory macrophages nor did it improve metabolic parameters to the extent of a standard oral formulation. Liposomal delivery did, however, attenuate effects on liver weight and liver and kidney expression of PPARα and -γ gene targets compared to oral delivery. Conclusions: These findings reveal for the first time that tesaglitazar has anti-inflammatory effects on adipose tissue macrophage populations in vivo. These data also suggest that while nanoparticle delivery reduced off-target effects, yet the lack of tesaglitazar actions in non-targeted cells such (as hepatocytes and adipocytes) and the uptake of drug-loaded liposomes in many other cell types, albeit to a lesser extent, may have impacted overall therapeutic efficacy. This fulsome analysis of cellular uptake of tesaglitazar-loaded liposomes provides important lessons for future studies of liposome drug delivery.

ApoE attenuates unresolvable inflammation by complex formation with activated C1q.

Apolipoprotein-E (ApoE) has been implicated in Alzheimer's disease, atherosclerosis, and other unresolvable inflammatory conditions but a common mechanism of action remains elusive. We found in ApoE-deficient mice that oxidized lipids activated the classical complement cascade (CCC), resulting in leukocyte infiltration of the choroid plexus (ChP). All human ApoE isoforms attenuated CCC activity via high-affinity binding to the activated CCC-initiating C1q protein (KD~140-580 pM) in vitro, and C1q-ApoE complexes emerged as markers for ongoing complement activity of diseased ChPs, Aß plaques, and atherosclerosis in vivo. C1q-ApoE complexes in human ChPs, Aß plaques, and arteries correlated with cognitive decline and atherosclerosis, respectively. Treatment with small interfering RNA (siRNA) against C5, which is formed by all complement pathways, attenuated murine ChP inflammation, Aß-associated microglia accumulation, and atherosclerosis. Thus, ApoE is a direct checkpoint inhibitor of unresolvable inflammation, and reducing C5 attenuates disease burden.

Publisher Correction: ApoE attenuates unresolvable inflammation by complex formation with activated C1q.

In the version of this article originally published, a sentence was erroneously included in the author contributions, and information regarding second shared authorship was missing from the author contributions. The following should not have been included in the author contributions: "C.W. and A.J.R.H. supervised the work presented in Figs. 1, 2, 5, 6; P.Z. and C.S. supervised the work presented in Figs. 3, 4." Additionally, this sentence should have appeared at the beginning of the author contributions: "These authors contributed equally: C.W., P.F.Z., C.S., and A.J.R.H." The errors have been corrected in the print, PDF and HTML versions of the article.