Chanzyme TRPM7 Mediates the Ca2+ Influx Essential for Lipopolysaccharide-Induced Toll-Like Receptor 4 Endocytosis and Macrophage Activation.

Toll-like receptors (TLRs) sense pathogen-associated molecular patterns to activate the production of inflammatory mediators. TLR4 recognizes lipopolysaccharide (LPS) and drives the secretion of inflammatory cytokines, often contributing to sepsis. We report that transient receptor potential melastatin-like 7 (TRPM7), a non-selective but Ca2+-conducting ion channel, mediates the cytosolic Ca2+ elevations essential for LPS-induced macrophage activation. LPS triggered TRPM7-dependent Ca2+ elevations essential for TLR4 endocytosis and the subsequent activation of the transcription factor IRF3. In a parallel pathway, the Ca2+ signaling initiated by TRPM7 was also essential for the nuclear translocation of NFκB. Consequently, TRPM7-deficient macrophages exhibited major deficits in the LPS-induced transcriptional programs in that they failed to produce IL-1ß and other key pro-inflammatory cytokines. In accord with these defects, mice with myeloid-specific deletion of Trpm7 are protected from LPS-induced peritonitis. Our study highlights the importance of Ca2+ signaling in macrophage activation and identifies the ion channel TRPM7 as a central component of TLR4 signaling.

Cardiomyocyte PANX1 Controls Glycolysis and Neutrophil Recruitment in Hypertrophy.

BACKGROUND: PANX1 (pannexin 1), a ubiquitously expressed ATP release membrane channel, has been shown to play a role in inflammation, blood pressure regulation, and myocardial infarction. However, the possible role of PANX1 in cardiomyocytes in the progression of heart failure has not yet been investigated. METHOD: We generated a novel mouse line with constitutive deletion of PANX1 in cardiomyocytes (Panx1MyHC6). RESULTS: PANX1 deletion in cardiomyocytes had no effect on unstressed heart function but increased the glycolytic metabolism and resulting glycolytic ATP production, with a concurrent decrease in oxidative phosphorylation, both in vivo and in vitro. In vitro, treatment of H9c2 cardiomyocytes with isoproterenol led to PANX1-dependent release of ATP and Yo-Pro-1 uptake, as assessed by pharmacological blockade with spironolactone and siRNA-mediated knockdown of PANX1. To investigate nonischemic heart failure and the preceding cardiac hypertrophy, we administered isoproterenol, and we demonstrated that Panx1MyHC6 mice were protected from systolic and diastolic left ventricle volume increases as a result of cardiomyocyte hypertrophy. Moreover, we found that Panx1MyHC6 mice showed decreased isoproterenol-induced recruitment of immune cells (CD45+), particularly neutrophils (CD11b+, Ly6g+), to the myocardium. CONCLUSIONS: Together, these data demonstrate that PANX1 deficiency in cardiomyocytes increases glycolytic metabolism and protects against cardiac hypertrophy in nonischemic heart failure at least in part by reducing immune cell recruitment. Our study implies PANX1 channel inhibition as a therapeutic approach to ameliorate cardiac dysfunction in patients with heart failure.

Endocannabinoid biosynthetic enzymes regulate pain response via LKB1-AMPK signaling.

Diacylglycerol lipase-beta (DAGLß) serves as a principal 2-arachidonoylglycerol (2-AG) biosynthetic enzyme regulating endocannabinoid and eicosanoid metabolism in immune cells including macrophages and dendritic cells. Genetic or pharmacological inactivation of DAGLß ameliorates inflammation and hyper-nociception in preclinical models of pathogenic pain. These beneficial effects have been assigned principally to reductions in downstream proinflammatory lipid signaling, leaving alternative mechanisms of regulation largely underexplored. Here, we apply quantitative chemical- and phospho-proteomics to find that disruption of DAGLß in primary macrophages leads to LKB1-AMPK signaling activation, resulting in reprogramming of the phosphoproteome and bioenergetics. Notably, AMPK inhibition reversed the antinociceptive effects of DAGLß blockade, thereby directly supporting DAGLß-AMPK crosstalk in vivo. Our findings uncover signaling between endocannabinoid biosynthetic enzymes and ancient energy-sensing kinases to mediate cell biological and pain responses.

Efferocytosis induces a novel SLC program to promote glucose uptake and lactate release.

Development and routine tissue homeostasis require a high turnover of apoptotic cells. These cells are removed by professional and non-professional phagocytes via efferocytosis1. How a phagocyte maintains its homeostasis while coordinating corpse uptake, processing ingested materials and secreting anti-inflammatory mediators is incompletely understood1,2. Here, using RNA sequencing to characterize the transcriptional program of phagocytes actively engulfing apoptotic cells, we identify a genetic signature involving 33 members of the solute carrier (SLC) family of membrane transport proteins, in which expression is specifically modulated during efferocytosis, but not during antibody-mediated phagocytosis. We assessed the functional relevance of these SLCs in efferocytic phagocytes and observed a robust induction of an aerobic glycolysis program, initiated by SLC2A1-mediated glucose uptake, with concurrent suppression of the oxidative phosphorylation program. The different steps of phagocytosis2-that is, 'smell' ('find-me' signals or sensing factors released by apoptotic cells), 'taste' (phagocyte-apoptotic cell contact) and 'ingestion' (corpse internalization)-activated distinct and overlapping sets of genes, including several SLC genes, to promote glycolysis. SLC16A1 was upregulated after corpse uptake, increasing the release of lactate, a natural by-product of aerobic glycolysis3. Whereas glycolysis within phagocytes contributed to actin polymerization and the continued uptake of corpses, lactate released via SLC16A1 promoted the establishment of an anti-inflammatory tissue environment. Collectively, these data reveal a SLC program that is activated during efferocytosis, identify a previously unknown reliance on aerobic glycolysis during apoptotic cell uptake and show that glycolytic by-products of efferocytosis can influence surrounding cells.

Innate immune signaling in Drosophila shifts anabolic lipid metabolism from triglyceride storage to phospholipid synthesis to support immune function.

During infection, cellular resources are allocated toward the metabolically-demanding processes of synthesizing and secreting effector proteins that neutralize and kill invading pathogens. In Drosophila, these effectors are antimicrobial peptides (AMPs) that are produced in the fat body, an organ that also serves as a major lipid storage depot. Here we asked how activation of Toll signaling in the larval fat body perturbs lipid homeostasis to understand how cells meet the metabolic demands of the immune response. We find that genetic or physiological activation of fat body Toll signaling leads to a tissue-autonomous reduction in triglyceride storage that is paralleled by decreased transcript levels of the DGAT homolog midway, which carries out the final step of triglyceride synthesis. In contrast, Kennedy pathway enzymes that synthesize membrane phospholipids are induced. Mass spectrometry analysis revealed elevated levels of major phosphatidylcholine and phosphatidylethanolamine species in fat bodies with active Toll signaling. The ER stress mediator Xbp1 contributed to the Toll-dependent induction of Kennedy pathway enzymes, which was blunted by deleting AMP genes, thereby reducing secretory demand elicited by Toll activation. Consistent with ER stress induction, ER volume is expanded in fat body cells with active Toll signaling, as determined by transmission electron microscopy. A major functional consequence of reduced Kennedy pathway induction is an impaired immune response to bacterial infection. Our results establish that Toll signaling induces a shift in anabolic lipid metabolism to favor phospholipid synthesis and ER expansion that may serve the immediate demand for AMP synthesis and secretion but with the long-term consequence of insufficient nutrient storage.

Smooth muscle cell FTO regulates contractile function.

The fat mass and obesity gene (FTO) is a N6-methyladenosine RNA demethylase that was initially linked by Genome-wide association studies to increased rates of obesity. Subsequent studies have revealed multiple mass-independent effects of the gene, including cardiac myocyte contractility. We created a mouse with a conditional and inducible smooth muscle cell deletion of Fto (Myh11 Cre+ Ftofl/fl) and did not observe any changes in mouse body mass or mitochondrial metabolism. However, the mice had significantly decreased blood pressure (hypotensive), despite increased heart rate and sodium, and significantly increased plasma renin. Remarkably, the third-order mesenteric arteries from these mice had almost no myogenic tone or capacity to constrict to smooth muscle depolarization or phenylephrine. Microarray analysis from Fto-/--isolated smooth muscle cells demonstrated a significant decrease in serum response factor (Srf) and the downstream effectors Acta2, Myocd, and Tagln; this was confirmed in cultured human coronary arteries with FTO siRNA. We conclude Fto is an important component to the contractility of smooth muscle cells.NEW & NOTEWORTHY We show a key role for the fat mass obesity (FTO) gene in regulating smooth muscle contractility, possibly by methylation of serum response factor (Srf).

BAFF antagonism via the BAFF receptor 3 binding site attenuates BAFF 60-mer-induced classical NF-κB signaling and metabolic reprogramming of B cells.

Human recombinant B cell activating factor (BAFF) is secreted as 3-mers, which can associate to form 60-mers in culture supernatants. However, the presence of BAFF multimers in humans is still debated and it is incompletely understood how BAFF multimers activate the B cells. Here, we demonstrate that BAFF can exist as 60-mers or higher order multimers in human plasma. In vitro, BAFF 60-mer strongly induced the transcriptome of B cells which was partly attenuated by antagonism using a soluble fragment of BAFF receptor 3. Furthermore, compared to BAFF 3-mer, BAFF 60-mer strongly induced a transient classical and prolonged alternate NF-κB signaling, glucose oxidation by both aerobic glycolysis and oxidative phosphorylation, and succinate utilization by mitochondria. BAFF antagonism selectively attenuated classical NF-κB signaling and glucose oxidation. Altogether, our results suggest critical roles of BAFF 60-mer and its BAFF receptor 3 binding site in hyperactivation of B cells.

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.

Macrophage metabolic adaptation to heme detoxification involves CO-dependent activation of the pentose phosphate pathway.

Heme is an essential cofactor for numerous cellular functions, but release of free heme during hemolysis results in oxidative tissue damage, vascular dysfunction, and inflammation. Macrophages play a key protective role in heme clearance; however, the mechanisms that regulate metabolic adaptations that are required for effective heme degradation remain unclear. Here we demonstrate that heme loading drives a unique bioenergetic switch in macrophages, which involves a metabolic shift from oxidative phosphorylation toward glucose consumption. Metabolomic and transcriptional analysis of heme-loaded macrophages revealed that glucose is funneled into the pentose phosphate pathway (PPP), which is indispensable for efficient heme detoxification and is required to maintain redox homeostasis. We demonstrate that the metabolic shift to the PPP is controlled by heme oxygenase-dependent generation of carbon monoxide (CO). Finally, we show that PPP upregulation occurs in vivo in organ systems central to heme clearance and that PPP activity correlates with heme levels in mouse sickle cell disease (SCD). Together, our findings demonstrate that metabolic adaptation to heme detoxification in macrophages requires a shift to the PPP that is induced by heme-derived CO, suggesting pharmacologic targeting of macrophage metabolism as a novel therapeutic strategy to improve heme clearance in patients with hemolytic disorders.

Loss of Endothelial FTO Antagonizes Obesity-Induced Metabolic and Vascular Dysfunction.

RATIONALE: Increasing prevalence of obesity and its associated risk with cardiovascular diseases demands a better understanding of the contribution of different cell types within this complex disease for developing new treatment options. Previous studies could prove a fundamental role of FTO (fat mass and obesity-associated protein) within obesity; however, its functional role within different cell types is less understood. OBJECTIVES: We identify endothelial FTO as a previously unknown central regulator of both obesity-induced metabolic and vascular alterations. METHODS AND RESULTS: We generated endothelial Fto-deficient mice and analyzed the impact of obesity on those mice. While the loss of endothelial FTO did not influence the development of obesity and dyslipidemia, it protected mice from high-fat diet-induced glucose intolerance and insulin resistance by increasing AKT (protein kinase B) phosphorylation in endothelial cells and skeletal muscle. Furthermore, loss of endothelial FTO prevented the development of obesity-induced hypertension by preserving myogenic tone in resistance arteries. In Fto-deficient arteries, microarray analysis identified upregulation of L-Pgds with significant increases in prostaglandin D2 levels. Blockade of prostaglandin D2 synthesis inhibited the myogenic tone protection in resistance arteries of endothelial Fto-deficient mice on high-fat diet; conversely, direct addition of prostaglandin D2 rescued myogenic tone in high-fat diet-fed control mice. Myogenic tone was increased in obese human arteries with FTO inhibitors or prostaglandin D2 application. CONCLUSIONS: These data identify endothelial FTO as a previously unknown regulator in the development of obesity-induced metabolic and vascular changes, which is independent of its known function in regulation of obesity.

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.

Macrophage phenotype and bioenergetics are controlled by oxidized phospholipids identified in lean and obese adipose tissue.

Adipose tissue macrophages (ATMs) adapt their metabolic phenotype either to maintain lean tissue homeostasis or drive inflammation and insulin resistance in obesity. However, the factors in the adipose tissue microenvironment that control ATM phenotypic polarization and bioenergetics remain unknown. We have recently shown that oxidized phospholipids (OxPL) uniquely regulate gene expression and cellular metabolism in Mox macrophages, but the presence of the Mox phenotype in adipose tissue has not been reported. Here we show, using extracellular flux analysis, that ATMs isolated from lean mice are metabolically inhibited. We identify a unique population of CX3CR1neg/F4/80low ATMs that resemble the Mox (Txnrd1+HO1+) phenotype to be the predominant ATM phenotype in lean adipose tissue. In contrast, ATMs isolated from obese mice had characteristics typical of the M1/M2 (CD11c+CD206+) phenotype with highly activated bioenergetics. Quantifying individual OxPL species in the stromal vascular fraction of murine adipose tissue, using targeted liquid chromatography-mass spectrometry, revealed that high fat diet-induced adipose tissue expansion led to a disproportional increase in full-length over truncated OxPL species. In vitro studies showed that macrophages respond to truncated OxPL species by suppressing bioenergetics and up-regulating antioxidant programs, mimicking the Mox phenotype of ATMs isolated from lean mice. Conversely, full-length OxPL species induce proinflammatory gene expression and an activated bioenergetic profile that mimics ATMs isolated from obese mice. Together, these data identify a redox-regulatory Mox macrophage phenotype to be predominant in lean adipose tissue and demonstrate that individual OxPL species that accumulate in adipose tissue instruct ATMs to adapt their phenotype and bioenergetic profile to either maintain redox homeostasis or to promote inflammation.

Extracellular nucleotide signaling in solid organ transplantation.

The role of extracellular purine nucleotides, including adenosine triphosphate (ATP) and adenosine, as modulators of posttransplantation outcome and ischemia-reperfusion injury is becoming increasingly evident. Upon pathological release of ATP, binding and activation of P2 purinergic surface receptors promote tissue injury and inflammation, while the expression and activation of P1 receptors for adenosine have been shown to attenuate inflammation and limit ischemia-induced damage, which are central to the viability and long-term success of allografts. Here we review the current state of the transplant field with respect to the role of extracellular nucleotide signaling, with a focus on the sources and functions of extracellular ATP. The connection between ischemia reperfusion, purinergic signaling, and graft preservation, as well as the role of ATP and adenosine as driving factors in the promotion and suppression of posttransplant inflammation and allograft rejection, are discussed. We also examine novel therapeutic approaches that take advantage of the ischemia-reperfusion-responsive and immunomodulatory roles for purinergic signaling with the goal of enhancing graft viability, attenuating posttransplant inflammation, and minimizing complications including rejection, graft failure, and associated comorbidities.

Pannexin 1 Channels as an Unexpected New Target of the Anti-Hypertensive Drug Spironolactone.

RATIONALE: Resistant hypertension is a major health concern with unknown cause. Spironolactone is an effective antihypertensive drug, especially for patients with resistant hypertension, and is considered by the World Health Organization as an essential medication. Although spironolactone can act at the mineralocorticoid receptor (MR; NR3C2), there is increasing evidence of MR-independent effects of spironolactone. OBJECTIVE: Here, we detail the unexpected discovery that Panx1 (pannexin 1) channels could be a relevant in vivo target of spironolactone. METHODS AND RESULTS: First, we identified spironolactone as a potent inhibitor of Panx1 in an unbiased small molecule screen, which was confirmed by electrophysiological analysis. Next, spironolactone inhibited α-adrenergic vasoconstriction in arterioles from mice and hypertensive humans, an effect dependent on smooth muscle Panx1, but independent of the MR NR3C2. Last, spironolactone acutely lowered blood pressure, which was dependent on smooth muscle cell expression of Panx1 and independent of NR3C2. This effect, however, was restricted to steroidal MR antagonists as a nonsteroidal MR antagonist failed to reduced blood pressure. CONCLUSIONS: These data suggest new therapeutic modalities for resistant hypertension based on Panx1 inhibition.

Deficiency of Dab2 (Disabled Homolog 2) in Myeloid Cells Exacerbates Inflammation in Liver and Atherosclerotic Plaques in LDLR (Low-Density Lipoprotein Receptor)-Null Mice-Brief Report.

OBJECTIVE: Inflammatory macrophages promote the development of atherosclerosis. We have identified the adaptor protein Dab2 (disabled homolog 2) as a regulator of phenotypic polarization in macrophages. The absence of Dab2 in myeloid cells promotes an inflammatory phenotype, but the impact of myeloid Dab2 deficiency on atherosclerosis has not been shown. APPROACH AND RESULTS: To determine the role of myeloid Dab2 in atherosclerosis, Ldlr-/- mice were reconstituted with either Dab2-positive or Dab2-deficient bone marrow and fed a western diet. Consistent with our previous finding that Dab2 inhibits NFκB (nuclear factor κ-light-chain-enhancer of activated B cells) signaling in macrophages, Ldlr-/- mice reconstituted with Dab2-deficient bone marrow had increased systemic inflammation as evidenced by increased serum IL-6 (interleukin-6) levels and increased inflammatory cytokine expression levels in liver. Serum lipid levels were significantly lower in Ldlr-/- mice reconstituted with Dab2-deficient bone marrow, and further examination of livers from these mice revealed drastically increased inflammatory tissue damage and massive infiltration of immune cells. Surprisingly, the atherosclerotic lesion burden in Ldlr-/- mice reconstituted with Dab2-deficient bone marrow was decreased compared with Ldlr-/- mice reconstituted with wild-type bone marrow. Further analysis of aortic root sections revealed increased macrophage content and evidence of increased apoptosis in lesions from Ldlr-/- mice reconstituted with Dab2-deficient bone marrow but no difference in collagen or α-smooth muscle actin content. CONCLUSIONS: Dab2 deficiency in myeloid cells promotes inflammation in livers and atherosclerotic plaques in a mouse model of atherosclerosis. Nevertheless, decreased serum lipids as a result of massive inflammatory liver damage may preclude an appreciable increase in atherosclerotic lesion burden in mice reconstituted with Dab2-deficient bone marrow.

Novel Role of IL (Interleukin)-1ß in Neutrophil Extracellular Trap Formation and Abdominal Aortic Aneurysms.

OBJECTIVE: Neutrophils promote experimental abdominal aortic aneurysm (AAA) formation via a mechanism that is independent from MMPs (matrix metalloproteinases). Recently, we reported a dominant role of IL (interleukin)-1ß in the formation of murine experimental AAAs. Here, the hypothesis that IL-1ß-induced neutrophil extracellular trap formation (NETosis) promotes AAA was tested. APPROACH AND RESULTS: NETs were identified through colocalized staining of neutrophil, Cit-H3 (citrullinated histone H3), and DNA, using immunohistochemistry. NETs were detected in human AAAs and were colocalized with IL-1ß. In vitro, IL-1RA attenuated IL-1ß-induced NETosis in human neutrophils. Mechanistically, IL-1ß treatment of isolated neutrophils induced nuclear localization of ceramide synthase 6 and synthesis of C16-ceramide, which was inhibited by IL-1RA or fumonisin B1, an inhibitor of ceramide synthesis. Furthermore, IL-1RA or fumonisin B1 attenuated IL1-ß-induced NETosis. In an experimental model of murine AAA, NETs were detected at a very early stage-day 3 of aneurysm induction. IL-1ß-knockout mice demonstrated significantly lower infiltration of neutrophils to aorta and were protected from AAA. Adoptive transfer of wild-type neutrophils promoted AAA formation in IL-1ß-knockout mice. Moreover, treatment of wild-type mice with Cl-amidine, an inhibitor NETosis, significantly attenuated AAA formation, whereas, treatment with deoxyribonuclease, a DNA digesting enzyme, had no effect on AAA formation. CONCLUSIONS: Altogether, the results suggest a dominant role of IL-1ß-induced NETosis in AAA formation.

Myeloid P2Y2 receptor promotes acute inflammation but is dispensable for chronic high-fat diet-induced metabolic dysfunction.

The purinergic receptor P2Y2 binds ATP to control chemotaxis of myeloid cells, and global P2Y2 receptor knockout mice are protected in models of acute inflammation. Chronic inflammation mediated by macrophages and other immune cells in adipose tissue contributes to the development of insulin resistance. Here, we investigate whether mice lacking P2Y2 receptors on myeloid cells are protected against acute and chronic inflammation. Wild-type mice were transplanted with either wild-type or P2Y2 receptor null bone marrow and treated with a sublethal dose of endotoxin as a model of acute inflammation, or fed a high-fat diet to induce obesity and insulin resistance as a model of chronic inflammation. P2Y2-/- chimeric mice were protected against acute inflammation. However, high-fat diet feeding induced comparable inflammation and insulin resistance in both WT and P2Y2-/- chimeric mice. Of note, confocal microscopy revealed significantly fewer crown-like structures, assemblies of macrophages around adipocytes, in P2Y2-/- chimeric mice compared to WT chimeric mice. We conclude that P2Y2 receptors on myeloid cells are important in mediating acute inflammation but are dispensable for the development of whole body insulin resistance in diet-induced obese mice.

NKp46(+) natural killer cells attenuate metabolism-induced hepatic fibrosis by regulating macrophage activation in mice.

UNLABELLED: Nonalcoholic steatohepatitis (NASH) affects 3%-5% of the U.S. population, having severe clinical complications to the development of fibrosis and end-stage liver diseases, such as cirrhosis and hepatocellular carcinoma. A critical cause of NASH is chronic systemic inflammation promoted by innate immune cells, such as liver macrophages (Mϕ) and natural killer (NK) cells. However, little is known about how the crosstalk between Mϕ and NK cells contributes to regulate NASH progression to fibrosis. In this report, we demonstrate that NKp46(+) cells play an important role in preventing NASH progression to fibrosis by regulating M1/M2 polarization of liver Mϕ. Using a murine model of NASH, we demonstrate that DX5(+)NKp46(+) NK cells are increased during disease and play a role in polarizing Mϕ toward M1-like phenotypes. This NK's immunoregulatory function depends on the production of interferon-gamma (IFN-γ), but not by granzyme-mediated cytolytic activity. Notably, depletion of NKp46(+) cells promotes the development of fibrosis with increased expression of profibrogenic genes as well as skewed M2 Mϕ phenotypes in hepatic tissues. CONCLUSIONS: NK cell-derived IFN-γ may be essential for maintaining a balanced inflammatory environment that promotes tissue integrity and limiting NASH progression to fibrosis.

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.