Endocrine-immune-reproductive System Interactions

我们已经进行了几个系列的实验来检查内分泌和免疫系统之间的相互作用，特别是通过关注病毒感染，包括人类免疫缺陷综合征1型(HIV-1)、巨细胞病毒(CMV)或新城疫(NDV)病毒的感染。病毒被认为是宿主免疫和内分泌系统的强大激活剂和调节器，影响宿主组织中的荷尔蒙活动，如白细胞、脂肪组织和骨骼肌。这些作用可能进一步促进病毒的扩张和致病。事实上，我们证明了HIV-1辅助蛋白VPR增强了GR的转录活性，而它抑制了PPAR-γ和-Delta的活性，在典型的艾滋病相关脂营养不良和胰岛素抵抗综合征的发展中发挥了潜在的重要作用。这种病毒蛋白可以在艾滋病患者的血清中发现，并通过穿透细胞膜改变未感染HIV-1病毒的细胞的活性，如肝细胞和脂肪细胞。在本财年，我们发表了一篇手稿，展示了VPR对中间代谢的影响，方法是使用在脂肪组织和肝脏中特异表达VPR的转基因小鼠，以及植入ALZT泵的小鼠，ALZT泵将合成的VPR多肽释放到循环中。我们发现，两只小鼠都表现出全身脂肪分解加速，高血糖和高甘油三酯血症。在肝脏中，他们发生了严重的脂肪变性，PPAR反应基因的表达减弱，GR靶基因的表达增加。与人类HIV感染患者类似，VPR转基因小鼠的血清中循环存在VPR。VPR通过细胞周期停滞来阻止前脂肪细胞的分化，而在成熟脂肪细胞中，它通过相互改变PPAR和GR与各自靶基因启动子中识别DNA序列的结合来促进脂肪分解。这些结果表明，VPR在体内调节这些核受体的活性，很可能与艾滋病患者相关的脂营养不良和胰岛素抵抗综合征的发生有关。 在识别和呈递病毒抗原的树突状细胞(DC)中，CMV或NDV的感染，甚至HIV-1的感染，导致一组核激素受体(包括糖皮质激素和雌激素受体)以及几种转录辅助调节因子(包括p300和p160型组蛋白乙酰转移酶共激活物)的表达发生显著变化，可能改变这些细胞对干扰素和其他细胞因子的分泌和生产。由于Nor1是NR4A组核受体成员之一，是树突状细胞中对病毒感染调节最高的NR，因此我们从贝勒医学院的Connely博士那里获得了Nor1基因敲除小鼠，并检测了病原体感染对从Nor1基因敲除小鼠中纯化的DC作用的影响。与野生型小鼠相比，这些小鼠的DC在对病毒感染的反应中存在明显的缺陷，而NOR-1通过其DNA反应元件在报告实验中刺激了IL-12p40启动子的活性。NOR-1基因敲除小鼠在感染弓形虫时，IL-12的产生显著减少。弓形虫是一种原生动物，IL-12是抵抗宿主防御的重要成分。我们目前正在研究NOR-1在细胞和动物系统中对IL-12产生的分子调控细节。 在同一系列实验中，我们发现CMV和NDV刺激DC表达IL-10，糖皮质激素进一步增强病毒诱导的这种细胞因子的表达。由于IL-10抑制致炎细胞因子的合成，并具有抑制DC和单核细胞提呈抗原的能力，糖皮质激素协同激活IL-10可能解释了为什么应激暴露和随后HPA轴的激活增加了病毒感染的易感性，并可能导致病毒相关疾病的后续发展，如哮喘、动脉粥样硬化和癌症。我们发现，病毒感染激活了细胞外信号调节激酶(ERK)，ERK是丝裂原激活蛋白激酶家族的成员，它反过来使位于第211位的丝氨酸磷酸化人GR，并增强了GR在IL-10启动子上的转录活性。

We have performed several lines of experiments to examine the interactions between the endocrine and immune systems particularly by focusing on viral infection, including that by the human immunodeficiency syndrome type-1 (HIV-1), cytomegalovirus (CMV) or Newcastle disease (NDV) virus. Viruses are known as potent activators and modulators of the host immune and endocrine systems, influencing hormonal actions in host tissues, such as leukocytes, adipose tissue and skeletal muscles. These effects may further contribute to viral expansion and pathogenesis. Indeed, we demonstrated that HIV-1 accessory protein Vpr enhances GR transcriptional activity, whereas it suppresses PPAR-gamma and -delta activity, playing a potentially important role in the development of the characteristic AIDS-associated lipodystrophy and insulin-resistance syndrome. This viral protein can be found in sera of the AIDS patients and changes activity of the cells uninfected by the HIV-1 virus, such as hepatocytes and adipocytes, by penetrating their cell membrane. In this fiscal year, we published one manuscript demonstrating impact of Vpr on intermediary metabolism by employing transgenic mice specifically expressing Vpr in adipose tissue and liver, and those implanted with the Alzt pump, which releases a synthetic Vpr peptide into circulation. We found that both mice demonstrated accelerated whole-body lipolysis, hyperglycemia and hypertriglyceridemia. In the liver, they developed severe steatosis with blunted expression of the PPAR-responsive genes and increased expression of GR target genes. Similar to human HIV-infected patients, Vpr circulated in the serum of Vpr-transgenic mice. Vpr blocked differentiation in preadipocytes through cell cycle arrest, whereas in mature adipocytes, it increased lipolysis with reciprocally altered binding of PPARgamma and GR to their recognition DNA sequences in respective target gene promoters. These results thus indicate that Vpr regulates activities of these nuclear receptors in vivo, and most likely, contributes to the development of AIDS-associated lipodystrophy and insulin-resistance syndrome in AIDS patients. In dendritic cells (DCs), which play a central role in the recognition and presentation of viral antigens, infection of CMV or NDV, and perhaps HIV-1, causes dramatic changes in the expression of a group of nuclear hormone receptors, including the glucocorticoid and estrogen receptors, as well as of several transcriptional co-regulators, including p300 and p160-type histone acetyltransferase coactivators, possibly altering secretion/production of interferons and other cytokines by these cells. Since NOR1, one member of NR4A group nuclear receptors, was the most highly regulated NR upon viral infection in DCs, we obtained NOR1 knockout mice from Dr. Conneely, the Baylor Collage of Medicine, and have examined impact of pathogen infection to the action of DCs purified from NOR1 knockout mice. DCs from these mice showed a significant defect in the response of interleukin (IL)-12 to viral infection compared to those from wild type mice, while NOR-1 stimulated the IL-12 p40 promoter activity through its DNA response elements in reporter assays. NOR-1 knockout mice demonstrated significant reduction of IL-12 production against infection of Toxoplasma gondii, a protozoa against which IL-12 acts as an essential component for host defense. We are currently examining details of molecular regulation of NOR-1 on IL-12 production both in cellular and animal systems. In the same line of experiments, we found that CMV and NDV stimulated IL-10 expression in DCs, and glucocorticoids further potentiated such virus-induced expression of this cytokine. Since IL-10 inhibits synthesis of pro-inflammatory cytokines and has ability to suppress antigen presentation by DCs and monocytes, synergistic activation of IL-10 by glucocorticoids may explain why exposure to stress and subsequent activation of the HPA axis increases susceptibility to viral infection, and possibly, subsequent development of viral-associated disorders, such as asthma, atherosclerosis and cancers. We found that viral infection activated the extracellular signal-regulated kinase (ERK), a member of the mitogen-activated protein kinase family, which in turn phosphorylated the human GR at serine located at amino acid position 211 and enhanced the transcriptional activity of GR on the IL-10 promoter.