Consequences of chronic Interferon-gamma expression on the host

我们正在利用慢性干扰素-γ表达的小鼠模型来确定对宿主的生物学后果以及这种表型与人类疾病的相关性。我们使用生物信息学的方法来确定干扰素-伽马基因3‘端非翻译部分的保守区。人们认为，这些保守的区域代表了基因遗传结构中的重要调控元件，因为除非mRNA的非编码区提供了一些进化优势，否则不存在通过进化来进行保守的内在原因。基于这一分析，我们将小鼠干扰素-γ3‘非翻译区的160bp区域作为删除目标，因为该区域含有丰富的AUUA序列，并且这些区域先前已被证明在调节细胞因子基因mRNA的不稳定性方面具有重要作用。基因敲除(KO)小鼠已经成功地建立在C57BL/6的遗传背景上，我们的数据表明，与野生型小鼠相比，该小鼠在基础水平和刺激下产生的干扰素-γ显著更多。此外，淋巴结、脾和胸腺的结构被破坏，肝脏显示出慢性炎症的迹象。随着CD4+和CD8+T细胞的增加，T细胞的动态平衡被破坏，小鼠体内的T细胞可能具有更强的抑制活性。对抗原刺激的TH1反应也增加，而TH2反应降低。B细胞群也会发生变化，基线抗体的产生也会发生偏差。B细胞也在胸腺中以更高的频率被观察到，因此表明干扰素-γ可能改变B细胞的运输。除了表型后果外，B细胞对抗原的反应也被破坏，因为IgM和Ig2aab反应增加，而IgG1反应减少。同时观察到较强的抗DNA和抗核抗原抗体反应，提示慢性干扰素-γ的表达可能在狼疮的发生发展中起一定作用。雌性小鼠还会患上原发性胆管炎，这是一种病因不明的疾病。这是第一个在性别偏见方面概括人类疾病的小鼠模型，因为90%的人类病例是在女性身上。PBC的发育可以通过CD4+T细胞转移到受体RAG小鼠体内。此外，雄性小鼠在运动后出现明显的心脏缺陷，并在短暂的运动后表现出较高的血清乳酸水平。。此外，小鼠已被证明对黑色素瘤敏感，这可能是现在观察到的IL-27基因表达水平增加的结果。干扰素-γ和IL-27水平直接相关，IL-27受体基因的消除会改善小鼠的自身免疫状态。更多的新研究发现，雌性小鼠可能表现出卵巢衰竭综合征的证据，从而使这一模型成为研究这种疾病的非常独特的模型。我们报道了在慢性血浆干扰素-γ水平和全身自身免疫的小鼠模型中，激活的CD8+T细胞在卵巢中的扩张。我们证明，这些小鼠经历了黄体化缺陷和胚胎植入失败。黄体黄体化缺陷发生在催乳素缺乏、黄体生成素受体下调、卵巢CD8+T细胞增多的情况下。体内研究证实，CD8+T细胞针对卵巢，子宫退行性变是卵巢炎症的继发性病变。综上所述，我们的数据揭示了免疫激活在黄体黄体功能缺陷和胚胎植入失败的发病机制中的基础作用，这与人类植入失败的慢性炎症有关。此外，雌性小鼠表现出强大的抗卵巢抗体，进一步证实了这是一种新的自身免疫性卵巢衰竭模型。在过去的一年里，该实验室一直专注于获取和监督样本质量，并提交来自ARE小鼠及其与IL-27受体基因敲除(IL27R-/-)和Ifnar基因敲除(Ifnar-/-)小鼠的多个器官的代谢组学和RNAseq分析。已生成原始数据，并正在协调进行深入数据分析。特别是，我们的分析集中在自身免疫环境中低估肾脏疾病的情况。为了支持这种多组学的努力，我们还完成了在ARE小鼠以及分别与IL-27R-/-和IFNAR-/-小鼠杂交的电子显微镜下对肾脏疾病的评估。后一组小鼠表明，IL-27r或ifnar的缺失改变了疾病的严重程度，并针对肾脏滤过系统的不同组件。总而言之，这些数据使我们能够在类似于人类狼疮疾病状态的不同自身免疫环境下收集肾脏疾病的完整概述。因此，我们现在有了一个小鼠模型来理解和阐明长期慢性干扰素-γ基因表达的系统生物学效应，导致宿主的慢性炎症和多种自身免疫性疾病的出现。这些研究的结果可能会导致新的治疗方法来治疗在这些小鼠身上发展起来的自身免疫性疾病。

We are utilizing a mouse model of chronic IFN-gamma expression to determine the biological consequences to the host and the relevance of this phenotype to human disease. We have used a bioinformatics approach to identify conserved regions of the 3' untranslated portion of the interferon-gamma mRNA. It is believed that these conserved regions represent important regulatory elements in the gene genetic structure, as there would be no inherent reason for conservation through evolution unless the non-coding regions of the mRNA provided some evolutionary advantage. Based on this analysis, we targeted a 160-bp region of the murine interferon-gamma 3' untranslated region for removal as this region is rich in AUUA sequences, and such regions have been previously shown to be important in the regulation of cytokine gene mRNA instability. The knockout (KO) mouse has been successfully created on the C57 BL/6 genetic background and our data indicates that this mouse produces significantly more interferon-gamma at a basal level and upon stimulation as compared to the wild-type mouse. Furthermore, the architecture of lymph nodes, spleen, and thymus is disrupted and the liver exhibits signs of chronic inflammation. T cell homeostasis has been disrupted as increased CD4+ and CD8+ T cells are present and the T reg cells in the mouse may have more potent suppressor activity. There is also an increased TH1 response and a decreased TH2 response to antigenic stimulation. The B cell population is also altered and baseline antibody production is skewed. B cells are also observed in the thymus at increased frequency, thus indicating that IFN-gamma may alter B cell trafficking. In addition to the phenotypic consequences, the B cell response to antigen is also disrupted as increased IgM and Ig2a ab responses are seen with a decrease in the IgG1 response. Strong anti-DNA and anti-nuclear antigen antibody responses are also observed suggesting that chronic IFN-gamma expression may play a role in the development of lupus. The female mice also develop primary biliary cholangitis, a disease that has no known etiology. This is the first mouse model to recapitulate the human disease with respect to sex bias as 90% of the human cases are in women. The development of PBC can be transferred into recipient RAG mice by CD4+ T cells. Additionally the male mice develop a heart defect that is apparent after exercise and they exhibit high levels of lactic acid in their serum following a brief period of exercise. . Furthermore the mice have proven to be susceptible to melanoma that may be a result of increased levels of IL-27 gene expression that has now been observed. IFN-gamma and IL-27 levels directly correlate and elimination of the IL-27 receptor gene results in improvement of the autoimmune status of the mice. Additional new studies have found that the female mice may be exhibiting evidence of ovarian failure syndrome, thus making this a very unique model to study this disease. We report an expansion of activated CD8+T cells in the ovaries of a mouse model with chronic plasma IFN-gamma levels and systemic autoimmunity. We demonstrate that these mice experience defective luteinization and embryo implantation failure. The defective corpus luteum luteinization occurred in the presence of prolactin deficiency, luteinizing hormone receptor downregulation, and expanded activated CD8+ T cells in the ovaries. In vivo studies confirmed that the CD8+ T cells specifically targeted the ovaries, and that the uterine degeneration was secondary to the ovarian inflammation. Collectively, our data revealed a fundamental role for immune activation in the pathogenesis of corpus luteal luteinization defect and embryo implantation failure, which show relevance for chronic inflammation in human implantation failure. Furthermore the female mice show strong anti-ovary antibodies further validating this as a new model for autoimmune ovarian failure. During the past year, the lab has been focused on obtaining and supervising sample quality and submission for metabolomics and RNAseq analysis of multiple organs from ARE mice and its genetic crossings with IL-27 receptor knockdown (IL27r-/-) and Ifnar knockdown (Ifnar -/-) mice. Raw data has been generated and coordination for in depth data analysis is underway. Particularly, our analysis is focused on understating kidney disease in autoimmune landscapes. To support this multi-omics effort, we have also completed the assessment of kidney disease by electron microscopy in the ARE mice and respective crossings with IL-27r-/- and ifnar-/- mice. The latter mice showed that the deletion of IL-27r or ifnar shifts disease severity and targets different components of the filtration system in kidneys. Together, these data allow us to assemble a complete overview of kidney disease under different autoimmune landscapes resembling human lupus disease states. Thus, we now have a mouse model for understanding and elucidating the systems biology effects of long term chronic IFN-gamma gene expression, resulting in chronic inflammation in the host and the appearance of multiple autoimmune diseases. The results of these studies may lead to new treatments for the autoimmune diseases that develop in these mice.