Consequences of chronic Interferon-gamma expression on the host

我们正在利用慢性IFN-γ表达的小鼠模型来确定IFN-γ的表达。 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 已经使用生物信息学方法来鉴定3 '非翻译的保守区域， 干扰素-γ mRNA的一部分。据信这些保守区域代表了 基因遗传结构中的重要调控元件，因为没有固有的 这是通过进化来保护的原因，除非mRNA的非编码区提供了 一些进化优势基于这种分析，我们靶向了小鼠的160-bp区域， 干扰素-γ 3 ′非翻译区缺失，因为该区域富含AUUA序列， 并且这些区域先前已经显示在细胞因子基因的调节中是重要的 mRNA不稳定性在C57 BL/6上成功地建立了敲除（KO）小鼠 遗传背景和我们的数据表明，这只老鼠产生了显着更多的 与野生型小鼠相比，在基础水平和在刺激后的干扰素-γ。 此外，淋巴结、脾和胸腺的结构被破坏， 有慢性炎症的迹象T细胞的稳态已经被破坏， 存在CD4+和CD8 + T细胞，并且小鼠中的T reg细胞可能具有更有效的免疫调节作用。 抑制活性也有增加的TH1反应和减少的TH2反应， 抗原刺激B细胞群也发生了改变， 是扭曲的在胸腺中也观察到B细胞的频率增加，因此表明 IFN-γ可以改变B细胞的运输。除了表型的后果，B 随着IgM和Ig2a ab应答的增加，细胞对抗原的应答也被破坏 IgG 1应答降低。强抗DNA和抗核抗原抗体 也观察到了反应，提示慢性IFN-γ表达可能在 狼疮的发展雌性小鼠也会发生原发性胆道胆管炎， 病因不明这是第一个重现人类疾病的小鼠模型 关于性别偏见，因为90%的人类病例是女性。PBC的发展可以 通过CD4 + T细胞转移到受体RAG小鼠中。此外，雄性小鼠还发育出一种 运动后明显的心脏缺陷，他们表现出高水平的乳酸， 他们的血清经过一段时间的锻炼。.此外，小鼠已被证明是 易患黑色素瘤，这可能是IL-27基因表达水平升高的结果， 现在已经被观察到了。IFN-γ和IL-27水平直接相关， IL-27受体基因导致小鼠自身免疫状态的改善。额外 新的研究发现，雌性小鼠可能表现出卵巢功能衰竭的迹象， 综合征，从而使其成为研究这种疾病的非常独特的模型。雌性老鼠也 在卵巢和子宫中表现出增加的CD8 + T细胞和减少的NK细胞。此外 雌性小鼠显示出强烈的抗卵巢抗体，进一步证实这是一种新的模型， 自身免疫性卵巢衰竭总之，我们对阐明多重 干扰素-γ生物学中涉及的机制证明了 干扰素-γ基因表达改变宿主体内平衡。因此，我们现在已经开发出一种小鼠， 模型的理解和阐明系统生物学效应的长期慢性 IFN-γ基因表达，导致宿主慢性炎症， 多种自身免疫性疾病在过去的一年里，实验室一直致力于获得和 监督样品质量，并提交用于代谢组学和RNAseq分析的多个 来自ARE小鼠的器官及其与IL-27受体敲低（IL-27r-/-）的遗传交叉， Ifnar基因敲减（Ifnar-/-）小鼠。已生成原始数据，并进行了深入协调 数据分析正在进行中。特别是，我们的分析集中在低估肾脏 自身免疫性疾病为了支持这一多组学工作，我们还完成了 通过ARE小鼠中的电子显微镜检查评估肾脏疾病， 与IL-27r-/-和ifnar-/-小鼠杂交。后者小鼠显示IL-27r的缺失 或ifnar转移疾病的严重程度，并针对过滤系统的不同组件， 肾脏总之，这些数据使我们能够在以下条件下对肾脏疾病进行完整的概述： 类似人类狼疮疾病状态的不同自身免疫景观。

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 deletion 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. The female mice also exhibit increased CD8+ T cells and decreased NK cells in the ovaries and uteri. Furthermore the female mice show strong anti-ovary antibodies further validating this as a new model for autoimmune ovarian failure. In summary, our approach towards elucidating the multiple mechanisms involved in the biology of interferon-gamma demonstrates the complexity by which interferon-gamma gene expression alters host homeostasis. Thus, we now have developed 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. 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.