G2A Protects Mice against Sepsis by Modulating Kupffer Cell Activation: Cooperativity with Adenosine Receptor 2b.

G2A is a GPCR abundantly expressed in immune cells. G2A-/- mice showed higher lethality, higher plasma cytokines, and an impaired bacterial clearance in response to a murine model of sepsis (cecal ligation and puncture), which were blocked by GdCl3, an inhibitor of Kupffer cells. Anti-IL-10 Ab reversed the impaired bacterial clearance in G2A-/- mice. Indomethacin effectively blocked both the increased i.p. IL-10 levels and the impaired bacterial clearance, indicating that disturbed PG system is the proximal cause of these phenomena. Stimulation with LPS/C5a induced an increase in Escherichia coli phagocytosis and intracellular cAMP levels in G2A+/+ peritoneal macrophages but not G2A-/- cells, which showed more PGE2/nitrite release and intracellular reactive oxygen species levels. Heterologous coexpression of G2A and adenosine receptor type 2b (A2bAR) induced a synergistic increase in cAMP signaling in a ligand-independent manner, with the evidence of physical interaction of G2A with A2bAR. BAY 60-6583, a specific agonist for A2bAR, increased intracellular cAMP levels in Kupffer cells from G2A+/+ but not from G2A-/- mice. Both G2A and A2bAR were required for antiseptic action of lysophosphatidylcholine. These results show inappropriate activation of G2A-/- Kupffer cells to septic insults due to an impaired cAMP signaling possibly by lack of interaction with A2bAR.

Genetic deletion of 12/15 lipoxygenase promotes effective resolution of inflammation following myocardial infarction.

12/15 lipoxygenase (LOX) directs inflammation and lipid remodeling. However, the role of 12/15LOX in post-myocardial infarction (MI) left ventricular remodeling is unclear. To determine the role of 12/15LOX, 8-12 week-old C57BL/6 J wild-type (WT; n = 93) and 12/15LOX-/- (n = 97) mice were subjected to permanent coronary artery ligation and monitored at day (d)1 and d5 post-operatively. Post-MI d28 survival was measured in male and female mice. No-MI surgery mice were maintained as d0 naïve controls. 12/15LOX-/- mice exhibited higher survival rates with lower cardiac rupture and improved LV function as compared with WT post-MI. Compared to WT, neutrophils and macrophages in 12/15LOX-/- mice were polarized towards N2 and M2 phenotypes, respectively, with increased of expression mrc-1, ym-1, and arg-1 post-MI. 12/15LOX-/- mice exhibited lower levels of pro-inflammatory 12-(S)-hydroperoxyeicosatetraenoic acid (12(S)-HETE) and higher CYP2J-derived epoxyeicosatrienoic acids (EETs) levels. CYP2J-derived 5,6-, 8,9-, 11,12-, and 14,15-EETs activated macrophage-specific hemeoxygenase (HO)-1 marked with increases in F4/80+/Ly6Clow and F4/80+/CD206high cells at d5 post-MI in 12/15LOX-/- mice. In contrast, inhibition of HO-1 led to total mortality in 12/15LOX-/- mice by post-MI d5. 12/15LOX-/- mice exhibited reduced collagen density and lower α-smooth muscle actin (SMA) expression at d5 post-MI, indicating delayed or limited fibroblast-to-myofibroblast differentiation. In conclusion, genetic deletion of 12/15LOX reduces 12(S)-HETE and activates CYP2J-derived EETs to promote effective resolution of inflammation post-MI leading to reduced cardiac rupture, improved LV function, and better survival.

Cholesterol-Independent Suppression of Lymphocyte Activation, Autoimmunity, and Glomerulonephritis by Apolipoprotein A-I in Normocholesterolemic Lupus-Prone Mice.

Apolipoprotein (Apo)A-I, the major lipid-binding protein of high-density lipoprotein, can prevent autoimmunity and suppress inflammation in hypercholesterolemic mice by attenuating lymphocyte cholesterol accumulation and removing tissue-oxidized lipids. However, whether ApoA-I mediates immune-suppressive or anti-inflammatory effects under normocholesterolemic conditions and the mechanisms involved remain unresolved. We transferred bone marrow from systemic lupus erythematosus (SLE)-prone Sle123 mice into normal, ApoA-I-knockout (ApoA-I(-/-)) and ApoA-I-transgenic (ApoA-I(tg)) mice. Increased ApoA-I in ApoA-I(tg) mice suppressed CD4(+) T and B cell activation without changing lymphocyte cholesterol levels or reducing major ApoA-I-binding oxidized fatty acids. Unexpectedly, oxidized fatty acid peroxisome proliferator-activated receptor γ ligands 13- and 9-hydroxyoctadecadienoic acid were increased in lymphocytes of autoimmune ApoA-I(tg) mice. ApoA-I reduced Th1 cells independently of changes in CD4(+)Foxp3(+) regulatory T cells or CD11c(+) dendritic cell activation and migration. Follicular helper T cells, germinal center B cells, and autoantibodies were also lower in ApoA-I(tg) mice. Transgenic ApoA-I also improved SLE-mediated glomerulonephritis. However, ApoA-I deficiency did not have the opposite effects on autoimmunity or glomerulonephritis, possibly as the result of compensatory increases in ApoE on high-density lipoprotein. We conclude that, although compensatory mechanisms prevent the proinflammatory effects of ApoA-I deficiency in normocholesterolemic mice, increasing ApoA-I can attenuate lymphocyte activation and autoimmunity in SLE independently of cholesterol transport, possibly through oxidized fatty acid peroxisome proliferator-activated receptor γ ligands, and it can reduce renal inflammation in glomerulonephritis.

Early lipid changes in acute kidney injury using SWATH lipidomics coupled with MALDI tissue imaging.

Acute kidney injury (AKI) is one of the leading causes of in-hospital morbidity and mortality, particularly in critically ill patients. Although our understanding of AKI at the molecular level remains limited due to its complex pathophysiology, recent advances in both quantitative and spatial mass spectrometric approaches offer new opportunities to assess the significance of renal metabolomic changes in AKI models. In this study, we evaluated lipid changes in early ischemia-reperfusion (IR)-related AKI in mice by using sequential window acquisition of all theoretical spectra (SWATH)-mass spectrometry (MS) lipidomics. We found a significant increase in two abundant ether-linked phospholipids following IR at 6 h postinjury, a plasmanyl choline, phosphatidylcholine (PC) O-38:1 (O-18:0, 20:1), and a plasmalogen, phosphatidylethanolamine (PE) O-42:3 (O-20:1, 22:2). Both of these lipids correlated with the severity of AKI as measured by plasma creatinine. In addition to many more renal lipid changes associated with more severe AKI, PC O-38:1 elevations were maintained at 24 h post-IR, while renal PE O-42:3 levels decreased, as were all ether PEs detected by SWATH-MS at this later time point. To further assess the significance of this early increase in PC O-38:1, we used matrix-assisted laser desorption ionization imaging mass spectrometry (MALDI-IMS) to determine that it occurred in proximal tubules, a region of the kidney that is most prone to IR injury and also rich in the rate-limiting enzymes involved in ether-linked phospholipid biosynthesis. Use of SWATH-MS lipidomics in conjunction with MALDI-IMS for lipid localization will help in elucidating the role of lipids in the pathobiology of AKI.

Obesity superimposed on aging magnifies inflammation and delays the resolving response after myocardial infarction.

Polyunsaturated fatty acid (PUFA) intake has increased over the last 100 yr, contributing to the current obesogenic environment. Obesity and aging are prominent risk factors for myocardial infarction (MI). How obesity interacts with aging to alter the post-MI response, however, is unclear. We tested the hypothesis that obesity in aging mice would impair the resolution of post-MI inflammation. PUFA diet (PUFA aging group) feeding to 12-mo-old C57BL/6J mice for 5 mo showed higher fat mass compared with standard lab chow (LC)-fed young (LC young group; 3-5 mo old) or aging alone control mice (LC aging group). LC young, LC aging, and PUFA aging mice were subjected to coronary artery ligation to induce MI. Despite similar infarct areas post-MI, plasma proteomic profiling revealed higher VCAM-1 in the PUFA aging group compared with LC young and LC aging groups, leading to increased neutrophil infiltration in the PUFA aging group (P<0.05). Macrophage inflammatory protein-1γ and CD40 were also increased at day 1, and myeloperoxidase remained elevated at day 5, an observation consistent with delayed wound healing in the PUFA aging group. Lipidomic analysis showed higher levels of arachidonic acid and 12(S)-hydroxyeicosatetraenoic acid at day 1 post-MI in the PUFA aging group compared with the LC aging group (all P<0.05), thereby mediating neutrophil extravasation in the PUFA aging group. The inflammation-resolving enzymes 5-lipoxygenase, cyclooxygenase-2, and heme oxyegnase-1 were altered to delay wound healing post-MI in the PUFA aging group compared with LC young and LC aging groups. PUFA aging magnifies the post-MI inflammatory response and impairs the healing response by stimulating prolonged neutrophil trafficking and proinflammatory lipid mediators.

Calreticulin Regulates Neointima Formation and Collagen Deposition following Carotid Artery Ligation.

BACKGROUND/AIMS: The endoplasmic reticulum (ER) stress protein, calreticulin (CRT), is required for the production of TGF-ß-stimulated extracellular matrix (ECM) by fibroblasts. Since TGF-ß regulates vascular fibroproliferative responses and collagen deposition, we investigated the effects of CRT knockdown on vascular smooth-muscle cell (VSMC) fibroproliferative responses and collagen deposition. METHODS: Using a carotid artery ligation model of vascular injury, Cre-recombinase-IRES-GFP plasmid was delivered with microbubbles (MB) to CRT-floxed mice using ultrasound (US) to specifically reduce CRT expression in the carotid artery. RESULTS: In vitro, Cre-recombinase-mediated CRT knockdown in isolated, floxed VSMCs decreased the CRT transcript and protein, and attenuated the induction of collagen I protein in response to TGF-ß. TGF-ß stimulation of collagen I was partly blocked by the NFAT inhibitor 11R-VIVIT. Following carotid artery ligation, CRT staining was upregulated with enhanced expression in the neointima 14-21 days after injury. Furthermore, Cre-recombinase-IRES-GFP plasmid delivered by targeted US reduced CRT expression in the neointima of CRT-floxed mice and led to a significant reduction in neointima formation and collagen deposition. The neointimal cell number was also reduced in mice, with a local, tissue-specific knockdown of CRT. CONCLUSIONS: This work establishes a novel role for CRT in mediating VSMC responses to injury through the regulation of collagen deposition and neointima formation.

CD36, but not G2A, modulates efferocytosis, inflammation, and fibrosis following bleomycin-induced lung injury.

Macrophage G2A and CD36 lipid receptors are thought to mediate efferocytosis following tissue injury and thereby prevent excessive inflammation that could compromise tissue repair. To test this, we subjected mice lacking G2A or CD36 receptor to bleomycin-induced lung injury and measured efferocytosis, inflammation, and fibrosis. Loss of CD36 (but not G2A) delayed clearance of apoptotic alveolar cells (mean 78% increase in apoptotic cells 7 days postinjury), potentiated inflammation (mean 56% increase in lung neutrophils and 75% increase in lung KC levels 7 days postinjury, 51% increase in lung macrophages 14 days postinjury), and reduced lung fibrosis (mean 41% and 29% reduction 14 and 21 days postinjury, respectively). Reduced fibrosis in CD36(-/-) mice was associated with lower levels of profibrotic TH2 cytokines (IL-9, IL-13, IL-4), decreased expression of the M2 macrophage marker Arginase-1, and reduced interstitial myofibroblasts. G2A, on the other hand, was required for optimal clearance of apoptotic neutrophils during zymosan-induced peritoneal inflammation (50.3% increase in apoptotic neutrophils and 30.6% increase in total neutrophils 24 h following zymosan administration in G2A(-/-) mice). Thus, CD36 is required for timely removal of apoptotic cells in the context of lung injury and modulates subsequent inflammatory and fibrotic processes relevant to fibrotic lung disease.

Autoimmune-mediated reduction of high-density lipoprotein-cholesterol and paraoxonase 1 activity in systemic lupus erythematosus-prone gld mice.

OBJECTIVE: To characterize modifications of high-density lipoprotein (HDL) in autoimmune gld mice that may be relevant to premature atherosclerosis in systemic lupus erythematosus, and to assess their relationship to specific aspects of autoimmune disease. METHODS: HDL cholesterol (HDL-C), apolipoprotein A-I (Apo A-I), paraoxonase 1 (PON1) activity, hepatic gene expression, and HDL biogenesis were measured in aging female gld and wild-type congenic mice. Autoantibodies, lymphoid organs, and cytokines were analyzed by enzyme-linked immunosorbent assay, flow cytometry, and multiplex assay, respectively. RESULTS: Plasma HDL-C, HDL Apo A-I, and HDL-associated PON1 activity were reduced in aging gld mice in association with the development of autoimmunity, independent of changes in hepatic Apo A-I and PON1 expression or HDL biogenesis. Hepatic induction of the acute-phase reactant serum amyloid A1 resulted in its incorporation into HDL in gld mice. Deletion of the lipid-sensitive receptor G2A in gld mice (G2A-/- gld) attenuated reductions in HDL-C and PON1 activity without altering hepatic Apo A-I and PON1 expression, HDL biogenesis, or levels of acute-phase proinflammatory cytokines. Plasma anti-Apo A-I autoantibodies were elevated in aging gld mice commensurate with detectable increases in Apo A-I immune complexes. Autoantibody levels were lower in aging G2A-/- gld mice compared with gld mice, and anti-Apo A-I autoantibody levels were significantly related to HDL-C concentrations (r=-0.645, P<0.00004) and PON1 activity (r=-0.555, P<0.0007) among autoimmune gld and G2A-/- gld mice. CONCLUSION: Autoantibodies against Apo A-I contribute to reducing HDL-C and PON1 activity in autoimmune gld mice independently of hepatic HDL biogenesis, suggesting that functional impairment and premature clearance of HDL immune complexes may be principal mechanisms involved.

Positron emission tomography imaging analysis of G2A as a negative modifier of lymphoid leukemogenesis initiated by the BCR-ABL oncogene.

G2A is a lymphocyte-expressed G protein-coupled receptor whose genetic ablation results in the development of autoimmunity. Using HSV-TK reporter gene directed positron emission tomography (PET), we demonstrate that prior to any indication of the onset of illness, mice transplanted with BCR-ABL transduced G2A-deficient bone marrow harbor expanded populations of leukemic cells compared to recipients of wild-type bone marrow. The target cell type and anatomical locations of leukemia development are indistinguishable in animals transplanted with G2A+/+ or G2A-/- cells. Shorter disease latency in the G2A-deficient background is associated with an increased rate of cellular expansion. PET can be successfully applied to the temporal and spatial analysis of Bcr-Abl driven leukemic progression and should have utility for the study of other leukemias and lymphomas.

ApoE-dependent modulation of HDL and atherosclerosis by G2A in LDL receptor-deficient mice independent of bone marrow-derived cells.

OBJECTIVE: Deletion of the lysophospholipid-sensitive receptor, G2A, in low-density lipoprotein receptor knockout (LDLR(-/-)) mice elevates plasma high-density lipoprotein (HDL) cholesterol and suppresses atherosclerosis. However, chemotactic action of G2A in monocytes/macrophages, in addition to its modulatory effect on HDL, may contribute to the proatherogenic action of G2A. METHODS AND RESULTS: We determined that deletion of G2A in LDLR(-/-) mice increases the ApoA1, ApoE, and cholesterol content of plasma HDL fractions. Hepatocytes were shown to express G2A and hepatocytes from G2A-deficient LDLR(-/-) mice secreted more ApoA1 and ApoE in HDL fractions compared to their G2A-sufficient counterparts. The atheroprotective and HDL modulatory effects of G2A deficiency were dependent on the presence of ApoE, as deletion of G2A in ApoE(-/-) and ApoE(-/-)LDLR(-/-) mice failed to raise HDL and did not suppress atherosclerosis. G2A deficiency in bone marrow-derived cells of LDLR(-/-) mice had no effect on atherosclerosis or HDL, whereas G2A deficiency in resident tissues was sufficient to raise HDL and suppress atherosclerosis. CONCLUSIONS: These data demonstrate that the chemotactic function of G2A in bone marrow-derived monocytes does not modulate atherosclerosis in LDLR(-/-) mice and suggest an ApoE-dependent function for G2A in the control of hepatic HDL metabolism that might contribute to its proatherogenic action.

Deletion of the G2A receptor fails to attenuate experimental autoimmune encephalomyelitis.

Lysophosphatidylcholine (LPC) is a chemotactic lysolipid produced during inflammation by the hydrolytic action of phospholipase A(2) enzymes. LPC stimulates chemotaxis of T cells in vitro through activation of the G protein-coupled receptor, G2A. This has led to the proposition that G2A contributes to the recruitment of T cells to sites of inflammation and thus promotes chronic inflammatory autoimmune diseases associated with the generation and subsequent tissue infiltration of auto-antigen-specific effector T cells. However, one study suggests that G2A may negatively regulate T cell proliferative responses to antigen receptor engagement and thereby attenuates autoimmunity by reducing the generation of autoreactive T cells. To address the relative contribution of these G2A-mediated effects to the pathophysiology of T cell-mediated autoimmune disease, we examined the impact of G2A inactivation on the onset and severity of murine experimental autoimmune encephalomyelitis (EAE), a model of multiple sclerosis (MS). Wild type (G2A(+/+)) and G2A-deficient (G2A(-/-)) C57BL/6J mice exhibited a similar incidence and onset of disease following immunization with MOG(35-55) peptide. Disease severity was only moderately reduced in G2A(-/-) mice. Similar numbers of MOG(35-55) specific T cells were generated in secondary lymphoid organs of MOG(35-55)-immunized G2A(+/+) and G2A(-/-) mice. Comparable numbers of T cells were detected in spinal cords of G2A(+/+) and G2A(-/-) mice. We conclude that the proposed anti-proliferative and chemotactic functions of G2A are not manifested in vivo and therefore therapeutic targeting of G2A is unlikely to be beneficial in the treatment of MS.

G2A and LPC: regulatory functions in immunity.

The G2A receptor was originally identified by virtue of its transcriptional induction in murine B lymphoid cells in response to oncogenic transformation and treatment with various DNA-damaging agents. While preliminary characterization of cellular responses to G2A overexpression in fibroblastic cell lines suggested that this receptor may negatively regulate cell growth under conditions of proliferative and genotoxic stress, subsequent studies driven by the discovery of lysophosphatidylcholine (LPC) as a regulator of G2A signaling in immunoregulatory cells point to an important role for this receptor in innate and adaptive immunity.

Loss of the lysophosphatidylcholine effector, G2A, ameliorates aortic atherosclerosis in low-density lipoprotein receptor knockout mice.

OBJECTIVE: Lysophosphatidylcholine is a major product of low-density lipoprotein (LDL) oxidation and secretory phospholipase A2-mediated lipid hydrolysis within atherosclerotic lesions. The G2A receptor mediates chemotaxis of cultured macrophages and T cells to lysophosphatidylcholine, supporting a pro-atherogenic role for this receptor in vivo. We investigated the ability of G2A to modulate atherosclerosis in mice. METHODS AND RESULTS: We measured atherosclerosis in G2A+/+ and G2A-/- LDL receptor knockout (LDLR-/-) mice. Consistent with a previous study, early lesion size at the aortic sinus was unaffected by G2A deficiency. However, G2A deficiency attenuated lesion progression at this site (42% to 44% reduction in average lesion area) and led to robust suppression of atherosclerosis throughout the aorta after short and extended periods of diet intervention (reduction in aortic lesion coverage: 62% to 73% at 9 weeks, 75% to 84% at 20 weeks). In G2A-/- LDLR-/- mice, intimal macrophage accumulation at lesion-prone sites of the aorta was significantly reduced in the absence of any detectable effect on T cell recruitment. Examination of lipoprotein profiles revealed elevated levels of circulating high-density lipoprotein (HDL) cholesterol in G2A-/- LDLR-/- mice compared with their G2A+/+ LDLR-/- counterparts after extended periods of diet intervention (54% increase in mean HDL cholesterol concentration). CONCLUSIONS: G2A provides a pro-atherogenic stimulus in vivo consistent with its chemotactic action but to which a pleiotropy of effects, including modulation of lipoprotein metabolism, may also contribute.

Effect of n-3 and n-6 Polyunsaturated Fatty Acids on Microsomal P450 Steroidogenic Enzyme Activities and In Vitro Cortisol Production in Adrenal Tissue From Yorkshire Boars.

Dysregulation of adrenal glucocorticoid production is increasingly recognized to play a supportive role in the metabolic syndrome although the mechanism is ill defined. The adrenal cytochrome P450 (CYP) enzymes, CYP17 and CYP21, are essential for glucocorticoid synthesis. The omega-3 and omega-6 polyunsaturated fatty acids (PUFA) may ameliorate metabolic syndrome, but it is unknown whether they have direct actions on adrenal CYP steroidogenic enzymes. The aim of this study was to determine whether PUFA modify adrenal glucocorticoid synthesis using isolated porcine microsomes. The enzyme activities of CYP17, CYP21, 11ß-hydroxysteroid dehydrogenase type 1, hexose-6-phosphate dehydrogenase (H6PDH), and CYP2E1 were measured in intact microsomes treated with fatty acids of disparate saturated bonds. Cortisol production was measured in a cell-free in vitro model. Microsomal lipid composition after arachidonic acid (AA) exposure was determined by sequential window acquisition of all theoretical spectra-mass spectrometry. Results showed that adrenal microsomal CYP21 activity was decreased by docosapentaenoic acid (DPA), docosahexaenoic acid (DHA), eicosapentaenoic acid, α-linolenic acid, AA, and linoleic acid, and CYP17 activity was inhibited by DPA, DHA, eicosapentaenoic acid, and AA. Inhibition was associated with the number of the PUFA double bonds. Similarly, cortisol production in vitro was decreased by DPA, DHA, and AA. Endoplasmic enzymes with intraluminal activity were unaffected by PUFA. In microsomes exposed to AA, the level of AA or oxidative metabolites of AA in the membrane was not altered. In conclusion, these observations suggest that omega-3 and omega-6 PUFA, especially those with 2 or more double bonds (DPA, DHA, and AA), impede adrenal glucocorticoid production.

Training in metabolomics research. I. Designing the experiment, collecting and extracting samples and generating metabolomics data.

The study of metabolism has had a long history. Metabolomics, a systems biology discipline representing analysis of known and unknown pathways of metabolism, has grown tremendously over the past 20 years. Because of its comprehensive nature, metabolomics requires careful consideration of the question(s) being asked, the scale needed to answer the question(s), collection and storage of the sample specimens, methods for extraction of the metabolites from biological matrices, the analytical method(s) to be employed and the quality control of the analyses, how collected data are correlated, the statistical methods to determine metabolites undergoing significant change, putative identification of metabolites and the use of stable isotopes to aid in verifying metabolite identity and establishing pathway connections and fluxes. The National Institutes of Health Common Fund Metabolomics Program was established in 2012 to stimulate interest in the approaches and technologies of metabolomics. To deliver one of the program's goals, the University of Alabama at Birmingham has hosted an annual 4-day short course in metabolomics for faculty, postdoctoral fellows and graduate students from national and international institutions. This paper is the first part of a summary of the training materials presented in the course to be used as a resource for all those embarking on metabolomics research. The complete set of training materials including slide sets and videos can be viewed at http://www.uab.edu/proteomics/metabolomics/workshop/workshop_june_2015.php. Copyright © 2016 John Wiley & Sons, Ltd.

Training in metabolomics research. II. Processing and statistical analysis of metabolomics data, metabolite identification, pathway analysis, applications of metabolomics and its future.

Metabolomics, a systems biology discipline representing analysis of known and unknown pathways of metabolism, has grown tremendously over the past 20 years. Because of its comprehensive nature, metabolomics requires careful consideration of the question(s) being asked, the scale needed to answer the question(s), collection and storage of the sample specimens, methods for extraction of the metabolites from biological matrices, the analytical method(s) to be employed and the quality control of the analyses, how collected data are correlated, the statistical methods to determine metabolites undergoing significant change, putative identification of metabolites and the use of stable isotopes to aid in verifying metabolite identity and establishing pathway connections and fluxes. This second part of a comprehensive description of the methods of metabolomics focuses on data analysis, emerging methods in metabolomics and the future of this discipline. Copyright © 2016 John Wiley & Sons, Ltd.

Interferon-induced mechanosensing defects impede apoptotic cell clearance in lupus.

Systemic lupus erythematosus (SLE) is a severe autoimmune disease that is associated with increased circulating apoptotic cell autoantigens (AC-Ags) as well as increased type I IFN signaling. Here, we describe a pathogenic mechanism in which follicular translocation of marginal zone (MZ) B cells in the spleens of BXD2 lupus mice disrupts marginal zone macrophages (MZMs), which normally clear AC debris and prevent follicular entry of AC-Ags. Phagocytosis of ACs by splenic MZMs required the megakaryoblastic leukemia 1 (MKL1) transcriptional coactivator-mediated mechanosensing pathway, which was maintained by MZ B cells through expression of membrane lymphotoxin-α1ß2 (mLT). Specifically, type I IFN-induced follicular shuttling of mLT-expressing MZ B cells disengaged interactions between these MZ B cells and LTß receptor-expressing MZMs, thereby downregulating MKL1 in MZMs. Loss of MKL1 expression in MZMs led to defective F-actin polymerization, inability to clear ACs, and, eventually, MZM dissipation. Aggregation of plasmacytoid DCs in the splenic perifollicular region, follicular translocation of MZ B cells, and loss of MKL1 and MZMs were also observed in an additional murine lupus model and in the spleens of patients with SLE. Collectively, the results suggest that lupus might be interrupted by strategies that maintain or enhance mechanosensing signaling in the MZM barrier to prevent follicular entry of AC-Ags.

Lysophosphatidylcholine as a ligand for immunoregulation.

Despite the recognized effects of lysophosphatidylcholine upon cells of the immune system and its association with inflammatory processes, its mechanism of action has remained poorly characterized. Our recent identification of the first lysophosphatidylcholine receptor as an immunoregulatory G protein-coupled receptor named G2A whose genetic ablation results in the development of inflammatory autoimmune disease has, therefore, provided a new perspective on the role of this lysophospholipid as a modulator of immune responses. This commentary discusses the biological properties of lysophosphatidylcholine as an immunoregulatory ligand for cells of the innate and adaptive arms of the immune system. Although we focus primarily on ligand interactions with G2A, we also discuss the issue of possible functional redundancy with other receptors with recently established ligand specificities towards phosphorylcholine-containing lysolipids including lysophosphatidylcholine.

Increased sensitivity of Apolipoprotein E knockout mice to swainsonine dependent immunomodulation.

The mechanisms that mediate accelerated atherosclerosis in autoimmune diseases remain unclear. One common mechanism that has been documented in autoimmune diseases and atherosclerosis is formation of hypoglycosyalted N-glycans on the cell surface. In this study we tested the effects of swainsonine, a class II α-mannosidase inhibitor which results in formation of hypoglycosylated N-glycans, on atherogenesis and immune cell dynamics in the atheroprone and hypercholesterolemic ApoE -/- mouse. Wild type or ApoE-/- mice (8 weeks of age) were fed a normal chow diet and administered swainsonine via the drinking water for 8 weeks at which time, atherosclerosis, and systemic markers of markers of inflammation were evaluated. Interestingly, no change in the rate of atherosclerosis development was observed in ApoE -/- mice treated with swainsonine. However, swainsonine significantly increased the number of peripheral blood leukocytes in ApoE -/- mice, with trends toward similar increases in swainsonine treated wild type mice noted. Assessment of leukocyte subsets using specific markers of all major blood lineages indicated that the increase in circulating leukocytes was due to the elevated number of progenitor cells. Consistent with swainsonine having a greater effect in ApoE -/- vs. wild type mice, increases in circulating inflammatory markers (IgA, IgG and chemokines) were observed in the former. Collectively, these data demonstrate that predisposition of ApoE -/- mice to vascular disease is associated with sensitization to the immunomodulatory effects of swainsonine and indicate that changes in N-glycans may provide a mechanism linking autoimmunity to atherogenesis.

Iron-ion radiation accelerates atherosclerosis in apolipoprotein E-deficient mice.

Radiation exposure from a number of terrestrial sources is associated with an increased risk for atherosclerosis. Recently, concern over whether exposure to cosmic radiation might pose a similar risk for astronauts has increased. To address this question, we examined the effect of 2 to 5 Gy iron ions ((56)Fe), a particularly damaging component of cosmic radiation, targeted to specific arterial sites in male apolipoprotein E-deficient (apoE(-/-)) mice. Radiation accelerated the development of atherosclerosis in irradiated portions of the aorta independent of any systemic effects on plasma lipid profiles or circulating leukocytes. Further, radiation exposure resulted in a more rapid progression of advanced aortic root lesions, characterized by larger necrotic cores associated with greater numbers of apoptotic macrophages and reduced lesional collagen compared to sham-treated mice. Intima media thickening of the carotid arteries was also exacerbated. Exposure to (56)Fe ions can therefore accelerate the development of atherosclerotic lesions and promote their progression to an advanced stage characterized by compositional changes indicative of increased thrombogenicity and instability. We conclude that the potential consequences of radiation exposure for astronauts on prolonged deep-space missions are a major concern. Knowledge gained from further studies with animal models should lead to a better understanding of the pathophysiological effects of accelerated ion radiation to better estimate atherogenic risk and develop appropriate countermeasures to mitigate its damaging effects.