Microcirculatory changes and oxidative stress have long been associated with acute kidney injury. Despite substantial progress made by two-photon microscopy of microvascular responses to acute kidney injury in rodent models, little is known about the underlying changes in blood oxygen delivery and tissue oxygen metabolism. To fill this gap, we developed a label-free kidney imaging technique based on photoacoustic microscopy, which enables simultaneous quantification of hemoglobin concentration, oxygen saturation of hemoglobin, and blood flow in peritubular capillaries in vivo. Based on these microvascular parameters, microregional oxygen metabolism was quantified. We demonstrated the utility of this technique by studying kidney hemodynamic and oxygen-metabolic responses to acute kidney injury in mice subject to lipopolysaccharide-induced sepsis. Dynamic photoacoustic microscopy of the peritubular capillary function and tissue oxygen metabolism revealed that sepsis induced an acute and significant reduction in peritubular capillary oxygen saturation of hemoglobin, concomitant with a marked reduction in kidney ATP levels and contrasted with nominal changes in peritubular capillary flow and plasma creatinine. Thus, our technique opens new opportunities to study microvascular and metabolic dysfunction in acute and chronic kidney diseases.

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.

Progressive tubulointerstitial fibrosis may occur after acute kidney injury due to persistent inflammation. Purinergic signaling by 5'-ectonucleotidase, CD73, an enzyme that converts AMP to adenosine on the extracellular surface, can suppress inflammation. The role of CD73 in progressive kidney fibrosis has not been elucidated. We evaluated the effect of deletion of CD73 from kidney perivascular cells (including pericytes and/or fibroblasts of the Foxd1+ lineage) on fibrosis. Perivascular cell expression of CD73 was necessary to suppress inflammation and prevent kidney fibrosis in Foxd1CreCD73fl/fl mice evaluated 14 days after unilateral ischemia-reperfusion injury or folic acid treatment (250 mg/kg). Kidneys of Foxd1CreCD73fl/fl mice had greater collagen deposition, expression of proinflammatory markers (including various macrophage markers), and platelet-derived growth factor recepetor-β immunoreactivity than CD73fl/fl mice. Kidney dysfunction and fibrosis were rescued by administration of soluble CD73 or by macrophage deletion. Isolated CD73-/- kidney pericytes displayed an activated phenotype (increased proliferation and α-smooth muscle actin mRNA expression) compared with wild-type controls. In conclusion, CD73 in perivascular cells may act to suppress myofibroblast transformation and influence macrophages to promote a wound healing response. These results suggest that the purinergic signaling pathway in the kidney interstitial microenvironment orchestrates perivascular cells and macrophages to suppress inflammation and prevent progressive fibrosis.

B1 cells exert protective effects in atherosclerosis through production of anti-inflammatory IgM antibodies that recognize oxidation-specific epitopes (OSE). Factors mediating B1 IgM production are currently unclear. Quantification of chemokine receptor expression on B1 cells in human subjects undergoing coronary artery assessment by intravascular ultrasound demonstrates that expression of the chemokine receptor CXCR4 on circulating B1 cells associates with increased plasma levels of anti-OSE IgM antibodies (p=0.0009) and decreased plaque burden (p=0.0002). Mice with B cell-specific loss of CXCR4 on the atherogenic ApoE -/- background (CXCR4 BKO ) contain fewer B1a cells (n=6-8,p<0.001) and IgM antibody-secreting cells (ASC) (n=6,p<0.01) in the bone marrow, and reduced serum IgM levels (n=6-8,p<0.05), relative to littermate controls (CXCR4 WT ). Absence of CXCR4 results in diminished B1a cell migration to the bone marrow (n=3, p<0.01) and reduced IL-5R expression on B1a cells in the bone marrow (n=9, p<0.01), but not spleen. To determine the atheroprotective role of CXCR4 on B1a cells, we adoptively transferred CXCR4 WT or CXCR4 BKO B1a cells into lymphocyte-deficient Rag1 -/- ApoE -/- mice. After 16 weeks of Western diet feeding, recipients given CXCR4 BKO B1a cells demonstrate reduced plasma IgM levels (n=7,p<0.001), and fewer donor IgM ASC in the bone marrow and spleen (n=11,p<0.001) compared to recipients given CXCR4 WT B1a cells. However, Rag1 -/- ApoE -/- recipients displayed no differences in number of donor IgM ASC in the perivascular adipose tissue or omental fat, nor in IgM+ area within atherosclerotic lesions in the aortic root. Moreover, B1a cell transfer reduced plasma cholesterol levels in recipient mice independently of CXCR4. Overall, this resulted in equivalent protection from atherosclerosis in Rag1 -/- ApoE -/- recipients given CXCR4 WT and CXCR4 BKO B1a cells compared to control mice receiving PBS. Finally, retroviral-mediated overexpression of CXCR4 on B-1a cells in vivo associated with increased B-1a localization to the bone marrow (p<0.01) and increased circulating levels of anti-OSE IgM antibodies (p<0.05). In sum, these data indicate that CXCR4 is an important regulator of bone marrow B1a maintenance and IgM production.

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.

B1 cells exert protective effects in atherosclerosis through production of anti-inflammatory IgM antibodies recognizing oxidation-specific epitopes, such as MDA-LDL, present in diseased arteries. However, factors mediating B1 IgM production are currently unclear. We evaluated MDA-LDL binding and chemokine receptor expression on human B1 cells in a cohort of subjects undergoing intravascular ultrasound (IVUS) for coronary artery assessment. Results demonstrate that a subset of human B1 cells (~35%) is able to bind MDA-LDL. Moreover, expression of the chemokine receptor CXCR4 on circulating B1 cells associates with increased plasma levels of anti-MDA-LDL IgM antibodies (p=0.0009), and decreased plaque burden in coronary arteries (p=0.0002). Mice with B cell-specific loss of CXCR4 on the atherogenic ApoE -/- background (CXCR4 BKO ) demonstrate fewer B1a cells (n=6-8,p<0.0001) and IgM antibody-secreting cells (n=6,p<0.01) in the bone marrow, and reduced plasma IgM levels (n=6-8,p<0.05), relative to littermate controls (CXCR4 WT ). Furthermore, retroviral-mediated overexpression of CXCR4 on B1a cells in vivo is associated with increased B1a localization to the bone marrow (p<0.01) and increased circulating levels of anti-MDA-LDL IgM antibodies (p<0.05). To determine the atheroprotective role of CXCR4 on the B1a cell subset, we adoptively transferred CXCR4 WT or CXCR4 BKO B1a cells into lymphocyte-deficient Rag1 -/- ApoE -/- mice. After 16 weeks of Western diet feeding, recipients given CXCR4 BKO B1a cells demonstrate reduced plasma IgM levels (n=7,p<0.001), and fewer donor B1a cells in the bone marrow and spleen (n=7,p<0.05) compared to recipients given CXCR4 WT B1a cells. Intriguingly, B1a transfer reduces plasma cholesterol levels in mice regardless of CXCR4 expression (n=7,p<0.05). However, CXCR4 further strengthens the atheroprotective ability of B1a cells, as recipients given CXCR4 WT B1a cells have reduced aortic lesion area compared to PBS controls (n=7,p<0.01) while recipients given CXCR4 BKO B1a cells did not attain the same level of protection. Overall, these data suggest that CXCR4 is an important regulator of IgM production and B1a-mediated atheroprotection.

The proximal tubule epithelium relies on mitochondrial function for energy, rendering the kidney highly susceptible to ischemic AKI. Dynamin-related protein 1 (DRP1), a mediator of mitochondrial fission, regulates mitochondrial function; however, the cell-specific and temporal role of DRP1 in AKI in vivo is unknown. Using genetic murine models, we found that proximal tubule-specific deletion of Drp1 prevented the renal ischemia-reperfusion-induced kidney injury, inflammation, and programmed cell death observed in wild-type mice and promoted epithelial recovery, which associated with activation of the renoprotective β-hydroxybutyrate signaling pathway. Loss of DRP1 preserved mitochondrial structure and reduced oxidative stress in injured kidneys. Lastly, proximal tubule deletion of DRP1 after ischemia-reperfusion injury attenuated progressive kidney injury and fibrosis. These results implicate DRP1 and mitochondrial dynamics as an important mediator of AKI and progression to fibrosis and suggest that DRP1 may serve as a therapeutic target for AKI.

Background Pannexin1 (Panx1), an ATP release channel, is present in most mammalian tissues, but the role of Panx1 in health and disease is not fully understood. Panx1 may serve to modulate AKI; ATP is a precursor to adenosine and may function to block inflammation, or ATP may act as a danger-associated molecular pattern and initiate inflammation.Methods We used pharmacologic and genetic approaches to evaluate the effect of Panx1 on kidney ischemia-reperfusion injury (IRI), a mouse model of AKI.Results Pharmacologic inhibition of gap junctions, including Panx1, by administration of carbenoxolone protected mice from IRI. Furthermore, global deletion of Panx1 preserved kidney function and morphology and diminished the expression of proinflammatory molecules after IRI. Analysis of bone marrow chimeric mice revealed that Panx1 expressed on parenchymal cells is necessary for ischemic injury, and both proximal tubule and vascular endothelial Panx1 tissue-specific knockout mice were protected from IRI. In vitro, Panx1-deficient proximal tubule cells released less and retained more ATP under hypoxic stress.Conclusions Panx1 is involved in regulating ATP release from hypoxic cells, and reducing this ATP release may protect kidneys from AKI.

Mechanisms that govern transcriptional regulation of inflammation in atherosclerosis remain largely unknown. Here, we identify the nuclear transcription factor c-Myb as an important mediator of atherosclerotic disease in mice. Atherosclerosis-prone animals fed a diet high in cholesterol exhibit increased levels of c-Myb in the bone marrow. Use of mice that either harbor a c-Myb hypomorphic allele or where c-Myb has been preferentially deleted in B cell lineages revealed that c-Myb potentiates atherosclerosis directly through its effects on B lymphocytes. Reduced c-Myb activity prevents the expansion of atherogenic B2 cells yet associates with increased numbers of IgM-producing antibody-secreting cells (IgM-ASCs) and elevated levels of atheroprotective oxidized low-density lipoprotein (OxLDL)-specific IgM antibodies. Transcriptional profiling revealed that c-Myb has a limited effect on B cell function but is integral in maintaining B cell progenitor populations in the bone marrow. Thus, targeted disruption of c-Myb beneficially modulates the complex biology of B cells in cardiovascular disease.

Assessment of structural and functional changes of mitochondria is vital for biomedical research as mitochondria are the power plants essential for biological processes and tissue/organ functions. Others and we have developed a novel reporter gene, pMitoTimer, which codes for a redox sensitive mitochondrial targeted protein that switches from green fluorescence protein (GFP) to red fluorescent protein (DsRed) when oxidized. It has been shown in transfected cells, transgenic C. elegans and Drosophila m., as well as somatically transfected adult skeletal muscle that this reporter gene allows quantifiable assessment of mitochondrial structure, oxidative stress, and lysosomal targeting of mitochondria-containing autophagosomes. Here, we generated CAG-CAT-MitoTimer transgenic mice using a transgene containing MitoTimer downstream of LoxP-flanked bacterial chloramphenicol acetyltransferase (CAT) gene with stop codon under the control of the cytomegalovirus (CMV) enhancer fused to the chicken β-actin promoter (CAG). When CAG-CAT-MitoTimer mice were crossbred with various tissue-specific (muscle, adipose tissue, kidney, and pancreatic tumor) or global Cre transgenic mice, the double transgenic offspring showed MitoTimer expression in tissue-specific or global manner. Lastly, we show that hindlimb ischemia-reperfusion caused early, transient increases of mitochondrial oxidative stress, mitochondrial fragmentation and lysosomal targeting of autophagosomes containing mitochondria as well as a later reduction of mitochondrial content in skeletal muscle along with mitochondrial oxidative stress in sciatic nerve. Thus, we have generated conditional MitoTimer mice and provided proof of principle evidence of their utility to simultaneously assess mitochondrial structure, oxidative stress, and mitophagy in vivo in a tissue-specific, controllable fashion.

Progressive fibrosis, a hallmark of chronic kidney disease (CKD), can occur following an acute insult such as ischemia-reperfusion injury (IRI). Sphingosine kinase 2 (Sphk2) deletion in CD4+ T cells protects from folic acid-induced fibrosis; an effect mediated IFNγ. Mitochondrial metabolism has emerged to be important in CD4+ T cell function. We hypothesized that Sphk2 regulates mitochondrial function and IFNγ production in CD4+ T cells in protection against IRI induced fibrosis. CD4+ T cells were isolated from WT, Sphk2−/− or Sphk2−/− Ifng−/− mice and adoptively transferred to WT mice prior to unilateral IRI. After 14 days, plasma creatinine (PCr) was quantified as a measure of kidney function and kidney transcript levels of a fibrosis marker αSMA (Acta2) was quantified by RT-qPCR. Mitochondrial function was assessed by Seahorse and TMRE uptake by flow cytometry. WT mice that received Sphk2−/− CD4+ T cells had better kidney function indicated by a reduced PCr level (0.68 +/− 0.01, n = 3) compared to vehicle (0.99 +/− 0.07, n = 4), WT (0.86 +/− 0.04, n = 3), and Sphk2−/− Ifng−/− (1.16 +/− 0.06, n = 4) CD4+ T cells. Additionally, Sphk2−/− CD4+ T cell recipients had reduced transcript levels of Acta2. Naïve and effector Sphk2−/− CD4+ T cells had increased basal oxygen consumption rate compared to WT CD4+ T cells. Effector Sphk2−/− CD4+ T cells had increased TMRE fluorescence compared to WT control CD4+ T cells. Sphk2−/− CD4+ T cells protect against IRI induced decline in kidney function and fibrosis, an effect mediated by production of IFNγ. Additionally, deletion of Sphk2 may promote mitochondrial function and effector CD4+ T cell function. Thus, targeting the Sphk2 pathway in T cell adoptive therapy may be a therapeutic strategy in the treatment of CKD.

Kidney disease affects millions of people worldwide and it is now widely accepted that many pathological processes may persist after acute kidney injury that can cause the progression to CKD. Tubulointerstitial fibrosis manifests soon after injury and while many cellular and molecular components of kidney fibrosis have been discovered, largely in animal models, new therapeutic strategies are still desperately needed. The renal endothelium has emerged as important in progression of fibrosis through regulation of hypoxia, inflammation and cellular crosstalk. This review aims to highlight our current understanding of the role of the endothelium in interstitial fibrosis and to identify potential therapeutic targets.

Epithelial and endothelial injury and a cascade of immune and interstitial cell activation in the kidney lead to AKI. After mild to moderate AKI, the epithelium can regenerate and restore kidney function, yet little is known about the endothelium during these repair processes. Sphingosine 1-phosphate receptor 1 (S1P1), a G protein–coupled receptor, is necessary for vascular homeostasis. Here, we used an inducible genetic approach in a mouse model of AKI, ischemia–reperfusion injury (IRI), to determine the temporal effects of endothelial S1P1 during AKI. Deletion of endothelial S1P1 before IRI exacerbated kidney injury and inflammation, and the delayed deletion of S1P1 after IRI prevented kidney recovery, resulting in chronic inflammation and progressive fibrosis. Specifically, S1P1 directly suppressed endothelial activation of leukocyte adhesion molecule expression and inflammation. Altogether, the data indicate activation of endothelial S1P1 is necessary to protect from IRI and permit recovery from AKI. Endothelial S1P1 may be a therapeutic target for the prevention of early injury as well as prevention of progressive kidney fibrosis after AKI.

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.

Macrophage colony-stimulating factor (CSF-1 or M-CSF) is important for kidney repair after acute kidney injury (AKI). CSF-1 is upregulated in tubule epithelial cells in response to kidney injury stimuli and binds to its sole receptor, CSF1R, in an autocrine and paracrine manner. Wang and colleagues used a genetic approach to constitutively delete Csf1 in proximal tubules to establish that proximal tubule production of CSF-1 is important for polarizing and skewing macrophages toward an M2 phenotype, and for recovery from AKI.

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 due to 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 (OSE) on LDL both in vitro and in vivo. Additionally, we demonstrate that B1b cells provide atheroprotection after adoptive transfer into B and T cell deficient (Rag1-/-Apoe-/-) hosts. We implicate Id3 in the regulation of B-1b cells as B cell-specific Id3 knockout mice (Id3BKOApoe-/-) have increased numbers of B-1b cells systemically, increased titers of OSE-reactive IgM antibodies, and significantly reduced diet-induced atherosclerosis compared to Id3WTApoe-/controls. Finally, we report that the presence of a homozygous SNP in ID3 in humans that attenuates Id3 function is associated with an increased percentage of circulating B-1 cells and anti-MDA-LDL IgM suggesting clinical relevance. Conclusions: These results provide novel evidence that B-1b cells produce atheroprotective OSE-reactive IgM antibodies and protect against atherosclerosis in mice, and suggest that similar mechanisms may occur in humans.

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.

Aims: We previously identified association between the ID3 SNP rs11574 and carotid intima-media thickness in the Diabetes Heart Study, a predominantly White diabetic population. The nonsynonymous SNP rs11574 results in an amino acid substitution in the C-terminal region of ID3, attenuating the dominant negative function of ID3 as an inhibitor of basic HLH factor E12-mediated transcription. In the current investigation, we characterize the association between the functionally significant polymorphism in ID3, rs11574, with human coronary pathology.Methods and Results: The Multi-Ethnic Study of Atherosclerosis (MESA) is a longitudinal study of subclinical cardiovascular disease, including non-Hispanic White (n = 2,588), African American (n = 2,560) and Hispanic (n = 2,130) participants with data on coronary artery calcium (CAC). The Coronary Assessment in Virginia cohort (CAVA) included 71 patients aged 30-80 years, undergoing a medically necessary cardiac catheterization and intravascular ultrasound (IVUS) at the University of Virginia. ID3 SNP rs11574 risk allele was associated with the presence of CAC in MESA Whites (P = 0.017). In addition, the risk allele was associated with greater atheroma burden and stenosis in the CAVA cohort (P = 0.003, P = 0.04 respectively). The risk allele remained predictive of atheroma burden in multivariate analysis (Model 1: covariates age, gender, and LDL, regression coefficient = 9.578, SE = 3.657, p = 0.0110; Model 2: covariates Model 1, presence of hypertension, presence of diabetes, regression coefficient = 8.389, SE = 4.788, p = 0.0163).Conclusions: We present additional cohorts that demonstrate association of ID3 SNP rs11574 directly with human coronary artery pathology as measured by CAC and IVUS: one a multiethnic, relatively healthy population with low levels of diabetes and the second a predominantly White population with a higher incidence of T2DM referred for cardiac catheterization.

Innate B-1 B cells can protect against inflammatory disease through production of natural IgM antibodies, but little is known regarding their role in obesity-induced metabolic dysfunction. In this study, we explore the role murine B-1 B cells play in regulating diet-induced glucose intolerance. In addition, we examine bariatric surgery samples for the presence of B cells in human adipose tissue and circulating natural IgM antibodies. We show that mice with increased visceral adipose tissue B-1b B cells due to B cell specific deletion of Id3 (Id3 BcellKO ) have attenuated high-fat diet-induced glucose intolerance compared to littermate controls. Omental visceral adipose tissue from Id3 BcellKO mice had enhanced local natural IgM antibody secretion (49.0 ± 5.9 vs. 17.5 ± 4.2 U/mg fat), and demonstrated attenuated HFD-induced secretion of TNFa (2.5 ± 0.3 vs. 6.7 ± 1.3 pg/mg fat) and IFNg (0.10 ± 0.02 vs. 0.48 ± 0.14 pg/mg fat). Adoptive transfer of B-1b B cells null for Id3 protected against diet-induced glucose intolerance in Rag1 -/- hosts, while B-1b B cells unable to secrete IgM had no effect. Additional studies in humans undergoing bariatric surgery showed CD20+CD27+CD43+ B cells, previously shown to have B-1-like characteristics, within omental adipose tissue. A correlation was found between their presence and serum natural IgM levels (r=0.52). In addition, natural IgM levels were inversely associated with the inflammatory chemokine, MCP-1 (r=-0.21). Finally, IgM, but not IgG, natural antibodies were inversely associated with insulin resistance (r=0.32). Together, our findings provide the first evidence that B-1b B cells protect against diet-induced glucose intolerance in an IgM-dependent manner in mice, and suggest that anti-inflammatory natural IgM antibodies may modulate the inflammatory and metabolic consequences of obesity in humans.

Introduction: B cells have been shown to have subset specific functions in atherosclerosis. B1a B cells, which contribute to innate immunity, are atheroprotective due to the spontaneous generation of protective natural antibodies. B1b B cells, the sister population to B1a B cells, also produce natural antibodies and unlike B1a B cells, have memory and inducible properties. B1b B cells have not been studied in atherosclerosis. Our lab has demonstrated that the helix-loop-helix transcription factor Id3 is important for B cell mediated atheroprotection. However, how Id3 in B cells regulates atheroprotection is incompletely understood. Hypothesis: Loss of Id3 specifically in B cells regulates an atheroprotective subset. Methods: To determine the impact of loss of Id3 specifically in B cells, we generated B cell specific Id3 knockout mice on the Apoe -/- background (Id3 fl/fl Apoe -/- CD19 cre/+ ). To measure atherosclerosis, 8 week old male Id3 fl/fl Apoe -/- CD19 cre/+ mice were fed Western diet for 16 weeks (n=7). These mice developed significantly less atherosclerosis in the aortic sinus compared to litter matched wildtype controls (Id3 fl/fl Apoe -/- CD19 +/+ , n=10) suggesting loss of Id3 in B cells was atheroprotective. Additionally, immunohistochemistry of atherosclerotic plaques demonstrated decreased intraplaque macrophage accumulation and fewer apoptotic bodies compared to wildtype. Immunophenotyping of Id3 fl/fl Apoe -/- CD19 cre/+ mice by flow cytometry revealed a specific increase of B1b B cells in the peritoneal cavity, spleen, and in circulation compared to wildtype (n=12-19). Additionally, 8 week old Id3 fl/fl Apoe -/- CD19 cre/+ mice (n=8) were shown by ELISA to have significantly greater circulating IgM and, after 16 weeks of Western diet feeding (n=10), significantly greater circulating natural antibodies compared to wildtype (n= 9 & 11 respectively). Conclusion: B cell-specific loss of Id3 enhances the B1b B cell pool, natural antibody production, and atheroprotection suggesting B1b B cells are important in defending against atherosclerosis in an antibody dependent manner.