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方法来鉴定干扰素γ mRNA的3'非翻译部分的保守区域。据认为，这些保守区域代表了基因遗传结构中重要的调控元件，因为除非mRNA的非编码区域提供了一些进化优势，否则通过进化就没有固有的保护原因。基于这一分析，我们针对小鼠干扰素- γ 3'非翻译区域的160 bp区域进行删除，因为该区域富含AUUA序列，并且该区域在之前被证明对细胞因子基因mRNA不稳定性的调节很重要。在C57 BL/6基因背景下成功构建了敲除（KO）小鼠，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更多的干扰素- γ。此外，淋巴结、脾脏和胸腺的结构被破坏，肝脏表现出慢性炎症的迹象。随着CD4+和CD8+ T细胞的增加，T细胞的稳态被破坏，小鼠体内的T细胞可能具有更强的抑制活性。对抗原刺激，TH1反应增加，TH2反应减少。B细胞群也被改变，基线抗体产生是倾斜的。在胸腺中也观察到B细胞的频率增加，从而表明ifn - γ可能改变B细胞的运输。除了表型后果外，由于IgM和Ig2a抗体反应增加而IgG1反应减少，B细胞对抗原的反应也被破坏。强烈的抗dna和抗核抗原抗体反应也被观察到，这表明慢性ifn - γ表达可能在狼疮的发展中起作用。雌性小鼠也会患上原发性胆管炎，这是一种病因不明的疾病。这是第一个概括人类疾病与性别偏见的小鼠模型因为90%的人类病例是女性。PBC的发育可通过CD4+ T细胞转移到受体RAG小鼠体内。此外，雄性小鼠在运动后会出现明显的心脏缺陷，在短暂的运动后，它们的血清中会出现高水平的乳酸。此外，小鼠已被证明易患黑色素瘤，这可能是目前已观察到的IL-27基因表达水平增加的结果。ifn - γ和IL-27水平直接相关，IL-27受体基因的消除导致小鼠自身免疫状态的改善。另外的新研究发现，雌性小鼠可能表现出卵巢衰竭综合征的证据，因此使其成为研究这种疾病的非常独特的模型。雌性小鼠卵巢和子宫的CD8+ T细胞增加，NK细胞减少。此外，雌性小鼠表现出强烈的抗卵巢抗体，进一步验证了这是自身免疫性卵巢衰竭的新模型。总之，我们阐明干扰素- γ生物学中涉及的多种机制的方法表明了干扰素- γ基因表达改变宿主稳态的复杂性。因此，我们现在已经建立了一个小鼠模型来理解和阐明长期慢性ifn - γ基因表达的系统生物学效应，导致宿主慢性炎症和多种自身免疫性疾病的出现。在过去的一年中，该实验室一直专注于获取和监督样品质量，并提交来自ARE小鼠及其与IL-27受体敲低（IL27r-/-）和Ifnar敲低（Ifnar -/-）小鼠的多器官代谢组学和RNAseq分析。原始数据已经生成，深入数据分析的协调工作正在进行中。特别地，我们的分析集中在自身免疫性景观中低估肾脏疾病。为了支持这项多组学研究，我们还通过电子显微镜完成了ARE小鼠肾脏疾病的评估，并分别与IL-27r-/-和ifnar-/-小鼠杂交。后一组小鼠表明，IL-27r或ifnar的缺失会改变疾病的严重程度，并针对肾脏过滤系统的不同组成部分。总之，这些数据使我们能够在不同的自身免疫性环境下，对类似于人类狼疮疾病状态的肾脏疾病进行完整的概述。

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.