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.

Neither the African-Centric S47 Nor P72 Variant of TP53 Is Associated With Reduced Risk of Febrile Malaria in a Malian Cohort Study.

BACKGROUND: TP53 has been shown to play a role in inflammatory processes, including malaria. We previously found that p53 attenuates parasite-induced inflammation and predicts clinical protection to Plasmodium falciparum infection in Malian children. Here, we investigated whether p53 codon 47 and 72 polymorphisms are associated with differential risk of P. falciparum infection and uncomplicated malaria in a prospective cohort study of malaria immunity. METHODS: p53 codon 47 and 72 polymorphisms were determined by sequencing TP53 exon 4 in 631 Malian children and adults enrolled in the Kalifabougou cohort study. The effects of these polymorphisms on the prospective risk of febrile malaria, incident parasitemia, and time to fever after incident parasitemia over 6 months of intense malaria transmission were assessed using Cox proportional hazards models. RESULTS: Confounders of malaria risk, including age and hemoglobin S or C, were similar between individuals with or without p53 S47 and R72 polymorphisms. Relative to their respective common variants, neither S47 nor R72 was associated with differences in prospective risk of febrile malaria, incident parasitemia, or febrile malaria after parasitemia. CONCLUSIONS: These findings indicate that p53 codon 47 and 72 polymorphisms are not associated with protection against incident P. falciparum parasitemia or uncomplicated febrile malaria.

CD200 Limits Monopoiesis and Monocyte Recruitment in Atherosclerosis.

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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.

2019 Russell Ross Memorial Lecture in Vascular Biology: B Lymphocyte-Mediated Protective Immunity in Atherosclerosis.

Atherosclerosis-the major underlying pathology of cardiovascular disease-is characterized by accumulation and subsequent oxidative modification of lipoproteins within the artery wall, leading to inflammatory cell infiltration and lesion formation that can over time result in arterial stenosis, ischemia, and downstream adverse events. The contribution of innate and adaptive immunity to atherosclerosis development is well established, and B cells have emerged as important modulators of both pro- and anti-inflammatory effects in atherosclerosis. Murine B cells can broadly be divided into 2 subsets: (1) B-2 cells, which are bone marrow derived and include conventional follicular and marginal zone B cells, and (2) B-1 cells, which are largely fetal liver derived and persist in adults through self-renewal. B-cell subsets are developmentally, functionally, and phenotypically distinct with unique subset-specific contributions to atherosclerosis development. Mechanisms whereby B cells regulate vascular inflammation and atherosclerosis will be discussed with a particular emphasis on B-1 cells. B-1 cells have a protective role in atherosclerosis that is mediated in large part by IgM antibody production. Accumulating evidence over the last several years has pointed to a previously underappreciated heterogeneity in B-1 cell populations, which may have important implications for understanding atherosclerosis development and potential targeted therapeutic approaches. This heterogeneity within atheroprotective innate B-cell subsets will be highlighted.

Subclinical Inflammation in Asymptomatic Schoolchildren With Plasmodium falciparum Parasitemia Correlates With Impaired Cognition.

BACKGROUND: Subclinical inflammation and cognitive deficits have been separately associated with asymptomatic Plasmodium falciparum infections in schoolchildren. However, whether parasite-induced inflammation is associated with worse cognition has not been addressed. We conducted a cross-sectional pilot study to better assess the effect of asymptomatic P. falciparum parasitemia and inflammation on cognition in Kenyan schoolchildren. METHODS: We enrolled 240 children aged 7-14 years residing in high malaria transmission in Western Kenya. Children performed five fluid cognition tests from a culturally adapted NIH toolbox and provided blood samples for blood smears and laboratory testing. Parasite densities and plasma concentrations of 14 cytokines were determined by quantitative PCR and multiplex immunoassay, respectively. Linear regression models were used to determine the effects of parasitemia and plasma cytokine concentrations on each of the cognitive scores as well as a composite cognitive score while controlling for age, gender, maternal education, and an interaction between age and P. falciparum infection status. RESULTS: Plasma concentrations of TNF, IL-6, IL-8, and IL-10 negatively correlated with the composite score and at least one of the individual cognitive tests. Parasite density in parasitemic children negatively correlated with the composite score and measures of cognitive flexibility and attention. In the adjusted model, parasite density and TNF, but not P. falciparum infection status, independently predicted lower cognitive composite scores. By mediation analysis, TNF significantly mediated ~29% of the negative effect of parasitemia on cognition. CONCLUSIONS: Among schoolchildren with PCR-confirmed asymptomatic P. falciparum infections, the negative effect of parasitemia on cognition could be mediated, in part, by subclinical inflammation. Additional studies are needed to validate our findings in settings of lower malaria transmission and address potential confounders that could affect both inflammation and cognitive performance.

A protective role for B-1 cells and oxidation-specific epitope IgM in lung fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a morbid fibrotic lung disease with limited treatment options. The pathophysiology of IPF remains poorly understood, and elucidation of the cellular and molecular mechanisms of IPF pathogenesis is key to the development of new therapeutics. B-1 cells are an innate B cell population which play an important role linking innate and adaptive immunity. B-1 cells spontaneously secrete natural IgM and prevent inflammation in several disease states. One class of these IgM recognize oxidation-specific epitopes (OSE), which have been shown to be generated in lung injury and to promote fibrosis. A main B-1 cell reservoir is the pleural space, adjacent to the typical distribution of fibrosis in IPF. In this study, we demonstrate that B-1 cells are recruited to the lung during injury where they secrete IgM to OSE (IgM OSE ). We also show that the pleural B-1 cell reservoir responds to lung injury through regulation of the chemokine receptor CXCR4. Mechanistically we show that the transcription factor Id3 is a novel negative regulator of CXCR4 expression. Using mice with B-cell specific Id3 deficiency, a model of increased B-1b cells, we demonstrate decreased bleomycin-induced fibrosis compared to littermate controls. Furthermore, we show that mice deficient in secretory IgM ( sIgM -/- ) have higher mortality in response to bleomycin-induced lung injury, which is partially mitigated through airway delivery of the IgM OSE E06. Additionally, we provide insight into potential mechanisms of IgM in attenuation of fibrosis through RNA sequencing and pathway analysis, highlighting complement activation and extracellular matrix deposition as key differentially regulated pathways.

Intra-tumoral T cells in pediatric brain tumors display clonal expansion and effector properties.

Brain tumors in children are a devastating disease in a high proportion of patients. Owing to inconsistent results in clinical trials in unstratified patients, the role of immunotherapy remains unclear. We performed an in-depth survey of the single-cell transcriptomes and clonal relationship of intra-tumoral T cells from children with brain tumors. Our results demonstrate that a large fraction of T cells in the tumor tissue are clonally expanded with the potential to recognize tumor antigens. Such clonally expanded T cells display enrichment of transcripts linked to effector function, tissue residency, immune checkpoints and signatures of neoantigen-specific T cells and immunotherapy response. We identify neoantigens in pediatric brain tumors and show that neoantigen-specific T cell gene signatures are linked to better survival outcomes. Notably, among the patients in our cohort, we observe substantial heterogeneity in the degree of clonal expansion and magnitude of T cell response. Our findings suggest that characterization of intra-tumoral T cell responses may enable selection of patients for immunotherapy, an approach that requires prospective validation in clinical trials.

Innate immune activation restricts priming and protective efficacy of the radiation-attenuated PfSPZ malaria vaccine.

A systems analysis was conducted to determine the potential molecular mechanisms underlying differential immunogenicity and protective efficacy results of a clinical trial of the radiation-attenuated whole-sporozoite PfSPZ vaccine in African infants. Innate immune activation and myeloid signatures at prevaccination baseline correlated with protection from P. falciparum parasitemia in placebo controls. These same signatures were associated with susceptibility to parasitemia among infants who received the highest and most protective PfSPZ vaccine dose. Machine learning identified spliceosome, proteosome, and resting DC signatures as prevaccination features predictive of protection after highest-dose PfSPZ vaccination, whereas baseline circumsporozoite protein-specific (CSP-specific) IgG predicted nonprotection. Prevaccination innate inflammatory and myeloid signatures were associated with higher sporozoite-specific IgG Ab response but undetectable PfSPZ-specific CD8+ T cell responses after vaccination. Consistent with these human data, innate stimulation in vivo conferred protection against infection by sporozoite injection in malaria-naive mice while diminishing the CD8+ T cell response to radiation-attenuated sporozoites. These data suggest a dichotomous role of innate stimulation for malaria protection and induction of protective immunity by whole-sporozoite malaria vaccines. The uncoupling of vaccine-induced protective immunity achieved by Abs from more protective CD8+ T cell responses suggests that PfSPZ vaccine efficacy in malaria-endemic settings may be constrained by opposing antigen presentation pathways.

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.

Longitudinal IgG antibody responses to Plasmodium vivax blood-stage antigens during and after acute vivax malaria in individuals living in the Brazilian Amazon.

BACKGROUND: To make progress towards malaria elimination, a highly effective vaccine targeting Plasmodium vivax is urgently needed. Evaluating the kinetics of natural antibody responses to vaccine candidate antigens after acute vivax malaria can inform the design of serological markers of exposure and vaccines. METHODOLOGY/PRINCIPAL FINDINGS: The responses of IgG antibodies to 9 P. vivax vaccine candidate antigens were evaluated in longitudinal serum samples from Brazilian individuals collected at the time of acute vivax malaria and 30, 60, and 180 days afterwards. Antigen-specific IgG correlations, seroprevalence, and half-lives were determined for each antigen using the longitudinal data. Antibody reactivities against Pv41 and PVX_081550 strongly correlated with each other at each of the four time points. The analysis identified robust responses in terms of magnitude and seroprevalence against Pv41 and PvGAMA at 30 and 60 days. Among the 8 P. vivax antigens demonstrating >50% seropositivity across all individuals, antibodies specific to PVX_081550 had the longest half-life (100 days; 95% CI, 83-130 days), followed by PvRBP2b (91 days; 95% CI, 76-110 days) and Pv12 (82 days; 95% CI, 64-110 days). CONCLUSION/SIGNIFICANCE: This study provides an in-depth assessment of the kinetics of antibody responses to key vaccine candidate antigens in Brazilians with acute vivax malaria. Follow-up studies are needed to determine whether the longer-lived antibody responses induced by natural infection are effective in controlling blood-stage infection and mediating clinical protection.

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.

Retroviral Overexpression of CXCR4 on Murine B-1a Cells and Adoptive Transfer for Targeted B-1a Cell Migration to the Bone Marrow and IgM Production.

As cell function is influenced by niche-specific factors in the cellular microenvironment, methods to dissect cell localization and migration can provide further insight on cell function. B-1a cells are a unique B cell subset in mice that produce protective natural IgM antibodies against oxidation-specific epitopes that arise during health and disease. B-1a cell IgM production differs depending on B-1a cell location, and therefore it becomes useful from a therapeutic standpoint to target B-1a localization to niches supportive of high antibody production. Here we describe a method to target B-1a cell migration to the bone marrow by retroviral-mediated overexpression of the C-X-C motif chemokine receptor 4 (CXCR4). Gene induction in primary murine B cells can be challenging and typically yields low transfection efficiencies of 10-20% depending on technique. Here we demonstrate that retroviral transduction of primary murine B-1a cells results in 30-40% transduction efficiency. This method utilizes adoptive cell transfer of transduced B-1a cells into B cell-deficient recipient mice so that donor B-1a cell migration and localization can be visualized. This protocol can be modified for other retroviral constructs and can be used in diverse functional assays post-adoptive transfer, including analysis of donor cell or host cell phenotype and function, or analysis of soluble factors secreted post B-1a cell transfer. The use of distinct donor and recipient mice differentiated by CD45.1 and CD45.2 allotype and the presence of a GFP reporter within the retroviral plasmid could also enable detection of donor cells in other, immune-sufficient mouse models containing endogenous B cell populations.

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.

c-Myb Exacerbates Atherosclerosis through Regulation of Protective IgM-Producing Antibody-Secreting Cells.

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.

Regulation of Transcription Factors by Reactive Oxygen Species and Nitric Oxide in Vascular Physiology and Pathology.

SIGNIFICANCE: Cardiovascular diseases are the main cause of death worldwide and pose an immense economical burden. In most cases, the underlying problem is vascular occlusion by atherosclerotic plaques. Importantly, different cell types of the vascular wall and the immune system play crucial roles in atherosclerosis at different stages of the disease. Furthermore, atherosclerosis and conditions recognized as risk factors are characterized by a reduced availability of the vasoprotective molecule nitric oxide and an increase in reactive oxygen species, so-called oxidative stress. Transcription factors function as intracellular signal integrators and relays and thus, play a central role in cellular responses to changing conditions. Recent Advances: Work on specific transcriptional regulators has uncovered many of their functions and the upstream pathways modulating their activity in response to reactive oxygen and nitrogen species. Here, we have reviewed for a few selected examples how this can contribute not only to protection against atherosclerosis development but also to disease progression and the occurrence of clinical manifestations, such as plaque rupture. CRITICAL ISSUES: Transcription factors have pleiotropic outputs and often also divergent functions in different cell types and tissues. Thus, in light of potential severe adverse side effects, a global activation or inhibition of particular transcriptions factors does not seem a feasible therapeutic option. FUTURE DIRECTIONS: A further in-depth characterization of the cell- and stage-specific actions and regulation of transcription factors in atherosclerosis with respect to protein-protein interactions and target genes could open up new avenues for prevention or therapeutic interventions in this vascular disease. Antioxid. Redox Signal. 26, 679-699.

Perivascular Adipose Tissue Harbors Atheroprotective IgM-Producing B Cells.

Adipose tissue surrounding major arteries (Perivascular adipose tissue or PVAT) has long been thought to exist to provide vessel support and insulation. Emerging evidence suggests that PVAT regulates artery physiology and pathology, such as, promoting atherosclerosis development through local production of inflammatory cytokines. Yet the immune subtypes in PVAT that regulate inflammation are poorly characterized. B cells have emerged as important immune cells in the regulation of visceral adipose tissue inflammation and atherosclerosis. B cell-mediated effects on atherosclerosis are subset-dependent with B-1 cells attenuating and B-2 cells aggravating atherosclerosis. While mechanisms whereby B-2 cells aggravate atherosclerosis are less clear, production of immunoglobulin type M (IgM) antibodies is thought to be a major mechanism whereby B-1 cells limit atherosclerosis development. B-1 cell-derived IgM to oxidation specific epitopes (OSE) on low density lipoproteins (LDL) blocks oxidized LDL-induced inflammatory cytokine production and foam cell formation. However, whether PVAT contains B-1 cells and whether atheroprotective IgM is produced in PVAT is unknown. Results of the present study provide clear evidence that the majority of B cells in and around the aorta are derived from PVAT. Interestingly, a large proportion of these B cells belong to the B-1 subset with the B-1/B-2 ratio being 10-fold higher in PVAT relative to spleen and bone marrow. Moreover, PVAT contains significantly greater numbers of IgM secreting cells than the aorta. ApoE-/- mice with B cell-specific knockout of the gene encoding the helix-loop-helix factor Id3, known to have attenuated diet-induced atherosclerosis, have increased numbers of B-1b cells and increased IgM secreting cells in PVAT relative to littermate controls. Immunostaining of PVAT on human coronary arteries identified fat associated lymphoid clusters (FALCs) harboring high numbers of B cells, and flow cytometry demonstrated the presence of T cells and B cells including B-1 cells. Taken together, these results provide evidence that murine and human PVAT harbor B-1 cells and suggest that local IgM production may serve to provide atheroprotection.

Increased serum CXCL1 and CXCL5 are linked to obesity, hyperglycemia, and impaired islet function.

Proinflammatory cytokines are thought to play a significant role in the pathogenesis of type 2 diabetes (T2D) and are elevated in the circulation even before the onset of the disease. However, the full complement of cytokines involved in the development of T2D is not known. In this study, 32 serum cytokines were measured from diabetes-prone BKS.Cg-m+/+Lepr(db)/J (db/db) mice and heterozygous age-matched control mice at 5 weeks (non-diabetic/non-obese), 6-7 weeks (transitional-to-diabetes), or 11 weeks (hyperglycemic/obese) and then correlated with body weight, blood glucose, and fat content. Among these 32 cytokines, C-X-C motif ligand 1 (CXCL1) showed the greatest increase (+78%) in serum levels between db/db mice that were hyperglycemic (blood glucose: 519±23 mg/dl, n=6) and those that were non-hyperglycemic (193±13 mg/dl, n=8). Similarly, increased CXCL1 (+68%) and CXCL5 (+40%) were associated with increased obesity in db/db mice; note that these effects could not be entirely separated from age. We then examined whether islets could be a source of these chemokines. Exposure to cytokines mimicking low-grade systemic inflammation (10 pg/ml IL1ß+20 pg/ml IL6) for 48 h upregulated islet CXCL1 expression by 53±3-fold and CXCL5 expression by 83±10-fold (n=4, P<0.001). Finally, overnight treatment with the combination of CXCL1 and CXCL5 at serum levels was sufficient to produce a significant decrease in the peak calcium response to glucose stimulation, suggesting reduced islet function. Our findings demonstrated that CXCL1 and CXCL5 i) are increased in the circulation with the onset of T2D, ii) are produced by islets under stress, and iii) synergistically affect islet function, suggesting that these chemokines participate in the pathogenesis of T2D.

Inhibition of serine palmitoyltransferase reduces Aß and tau hyperphosphorylation in a murine model: a safe therapeutic strategy for Alzheimer's disease.

The contribution of the autosomal dominant mutations to the etiology of familial Alzheimer's disease (AD) is well characterized. However, the molecular mechanisms contributing to sporadic AD are less well understood. Increased ceramide levels have been evident in AD patients. We previously reported that increased ceramide levels, regulated by increased serine palmitoyltransferase (SPT), directly mediate amyloid ß (Aß) levels. Therefore, we inhibited SPT in an AD mouse model (TgCRND8) through subcutaneous administration of L-cylcoserine. The cortical Aß42 and hyperphosphorylated tau levels were down-regulated with the inhibition of SPT/ceramide. Positive correlations were observed among cortical SPT, ceramide, and Aß42 levels. With no evident toxic effects observed, inhibition of SPT could be a safe therapeutic strategy to ameliorate the AD pathology. We previously observed that miR-137, -181c, -9, and 29a/b post-transcriptionally regulate SPT levels, and the corresponding miRNA levels in the blood sera are potential diagnostic biomarkers for AD. Here, we observe a negative correlation between cortical Aß42 and sera Aß42, and a positive correlation between cortical miRNA levels and sera miRNA levels suggesting their potential as noninvasive diagnostic biomarkers.