Growth Factor and Th2 Cytokine Signaling Pathways Converge at STAT6 to Promote Arginase Expression in Progressive Experimental Visceral Leishmaniasis

Host arginase 1 (arg1) expression is a significant contributor to the pathogenesis of progressive visceral leishmaniasis (VL), a neglected tropical disease caused by the intracellular protozoan Leishmania donovani. Previously we found that parasite-induced arg1 expression in macrophages was dependent on STAT6 activation. Arg1 expression was amplified by, but did not require, IL-4, and required de novo synthesis of unknown protein(s). To further explore the mechanisms involved in arg1 regulation in VL, we screened a panel of kinase inhibitors and found that inhibitors of growth factor signaling reduced arg1 expression in splenic macrophages from hamsters with VL. Analysis of growth factors and their signaling pathways revealed that the Fibroblast Growth Factor Receptor 1 (FGFR-1) and Insulin-like Growth Factor 1 Receptor (IGF-1R) and a number of downstream signaling proteins were activated in splenic macrophages isolated from hamsters infected with L. donovani. Recombinant FGF-2 and IGF-1 increased the expression of arg1 in L. donovani infected hamster macrophages, and this induction was augmented by IL-4. Inhibition of FGFR-1 and IGF-1R decreased arg1 expression and restricted L. donovani replication in both in vitro and ex vivo models of infection. Inhibition of the downstream signaling molecules JAK and AKT also reduced the expression of arg1 in infected macrophages. STAT6 was activated in infected macrophages exposed to either FGF-2 or IGF-1, and STAT6 was critical to the FGFR-1- and IGF-1R-mediated expression of arg1. The converse was also true as inhibition of FGFR-1 and IGF-1R reduced the activation of STAT6 in infected macrophages. Collectively, these data indicate that the FGFR/IGF-1R and IL-4 signaling pathways converge at STAT6 to promote pathologic arg1 expression and intracellular parasite survival in VL. Targeted interruption of these pathological processes offers an approach to restrain this relentlessly progressive disease.     

HDL Induces the Expression of the M2 Macrophage Markers Arginase 1 and Fizz-1 in a STAT6-Dependent Process

Our lab has previously shown in a mouse model that normalization of a low HDL level achieves atherosclerotic plaque regression. This included the shift from a pro (“M1”) to an anti-inflammatory (“M2”) phenotypic state of plaque macrophages. Whether HDL can directly cause this phenotypic change and, if so, what the signaling mechanism is, were explored in the present studies. Murine primary macrophages treated with HDL showed increased gene expression for the M2 markers Arginase-1 (Arg-1) and Fizz-1, which are classically induced by IL-4. HDL was able to potentiate the IL-4-induced changes in Arg-1, and tended to do the same for Fizz-1, while suppressing the expression of inflammatory genes in response to IFNγ. The effects of either IL-4 or HDL were suppressed when macrophages were from STAT6^-/- mice, but inhibitor studies suggested differential utilization of JAK isoforms by IL-4 and HDL to activate STAT6 by phosphorylation. Overall, our results describe a new function of HDL, namely its ability to directly enrich macrophages in markers of the M2, anti-inflammatory, state in a process requiring STAT6.     

IFN-gamma-induced SOCS-1 regulates STAT6-dependent eotaxin production triggered by IL-4 and TNF-alpha

The production of eotaxin, which is a critical mediator for airway inflammation, is inhibited by IFN-gamma. Here, we investigated the precise mechanisms underlying IFN-gamma-dependent inhibition of eotaxin production using mouse embryonic fibroblasts (MEF). MEF produced high levels of eotaxin in STAT6-dependent manner when they were cultured with both IL-4 and TNF-alpha. However, the eotaxin production by MEF was strongly inhibited by addition of IFN-gamma. Western-blotting analysis demonstrated that IFN-gamma downmodulated STAT6 phosphorylation induced by IL-4 and TNF-alpha. Moreover, IFN-gamma did not exhibit its inhibitory effect on both STAT6-phosphorylation and eotaxin production in MEF obtained from deficient mice in STAT1, a key molecule of IFN-gamma signaling. We also demonstrated that SOCS-1, a potent inhibitory molecule of IL-4 signaling, was induced by IFN-gamma in STAT1-dependent manner. This indicated that SOCS-1 might be involved in IFN-gamma-mediated STAT1-dependent inhibition of eotaxin production. In SOCS-1(-/-) MEF, IFN-gamma inhibited neither STAT6 phosphorylation nor eotaxin production induced by IL-4 and TNF-alpha. Conversely, retroviral transduction of SOCS-1 into MEF inhibited STAT6 phosphorylation and eotaxin production induced by IL-4 and TNF-alpha, in the absence of IFN-gamma. Thus, we demonstrated that IFN-gamma-induced inhibition of STAT6 phosphorylation and eotaxin production were mediated by SOCS-1 induced in STAT1-dependent manner.     

Human Immunodeficiency Virus Type 1 Replication Is Modulated by Host Cyclophilin A Expression Levels

Human immunodeficiency virus type 1 (HIV-1) Gag and the cellular protein cyclophilin A form an essential complex in the virion core: virions produced by proviruses encoding Gag mutants with decreased cyclophilin A affinity exhibit attenuated infectivity, as do virions produced in the presence of the competitive inhibitor cyclosporine. The A224E Gag mutant has no effect on cyclophilin A affinity but renders HIV-1 replication cyclosporine resistant in Jurkat T cells. In contrast, A224E mutant virus is dead in H9 T cells, although replication is rescued by cyclosporine or by expression in cis of a Gag mutant that decreases cyclophilin A-affinity. The observation that disruption of the Gag-cyclophilin A interaction rescues A224E mutant replication in H9 cells prompted experiments which revealed that, relative to Jurkat cells, H9 cells express greater quantities of cyclophilin A. The resulting larger quantity of cyclophilin A shown to be packaged into virions produced by H9 cells is presumably disruptive to the A224E mutant virion core. Further evidence that increased cyclophilin A expression in H9 cells is of functional relevance was provided by the finding that Gag mutants with decreased cyclophilin A affinity are dead in Jurkat cells but capable of replication in H9 cells. Similarly, cyclosporine concentrations which inhibit wild-type HIV-1 replication in Jurkat cells stimulate HIV-1 replication in H9 cells. These results suggest that HIV-1 virion infectivity imposes narrow constraints upon cyclophilin A stoichiometry in virions and that infectivity is finely tuned by host cyclophilin A expression levels.     

卵黄蛋白原1(vtg1)启动子调控绿色荧光蛋白表达的转基因斑马鱼的构建

环境雌激素严重危害人类的健康.为了简便直观地检测水环境中的雌激素污染,构建了一种转基因斑马鱼,在这种鱼的体内,利用卵黄蛋白原1(vitellogenin1,vtg1)的启动子调控报告基因绿色荧光蛋白(enhancedgreenfluorescentprotein,EGFP)的表达.用1ng/L的17!-炔雌醇(17"-ethynylestradiol,EE2)诱导4天后,仔鱼肝脏中出现绿色荧光.RT-PCR和整体原位杂交实验证实,仔鱼体内EGFP和vtg1的时空表达模式相同.通过在显微镜下观察转基因鱼的绿色荧光,可以直观判断水环境中是否含有雌激素活性物质.该研究为环境雌激素的监测提供了一种简便直观的新型工具.     

Visceral Endoderm Expression of Yin-Yang1 (YY1) Is Required for VEGFA Maintenance and Yolk Sac Development

Mouse embryos lacking the polycomb group gene member Yin-Yang1 (YY1) die during the peri-implantation stage. To assess the post-gastrulation role of YY1, a conditional knock-out (cKO) strategy was used to delete YY1 from the visceral endoderm of the yolk sac and the definitive endoderm of the embryo. cKO embryos display profound yolk sac defects at 9.5 days post coitum (dpc), including disrupted angiogenesis in mesoderm derivatives and altered epithelial characteristics in the visceral endoderm. Significant changes in both cell death and proliferation were confined to the YY1-expressing yolk sac mesoderm indicating that loss of YY1 in the visceral endoderm causes defects in the adjacent yolk sac mesoderm. Production of Vascular Endothelial Growth Factor A (VEGFA) by the visceral endoderm is essential for normal growth and development of the yolk sac vasculature. Reduced levels of VEGFA are observed in the cKO yolk sac, suggesting a cause for the angiogenesis defects. Ex vivo culture with exogenous VEGF not only rescued angiogenesis and apoptosis in the cKO yolk sac mesoderm, but also restored the epithelial defects observed in the cKO visceral endoderm. Intriguingly, blocking the activity of the mesoderm-localized VEGF receptor, FLK1, recapitulates both the mesoderm and visceral endoderm defects observed in the cKO yolk sac. Taken together, these results demonstrate that YY1 is responsible for maintaining VEGF in the developing visceral endoderm and that a VEGF-responsive paracrine signal, originating in the yolk sac mesoderm, is required to promote normal visceral endoderm development.