Developing a mouse model to examine the specific impact of IFNa in the pathogenesis of genital tract Chlamydia infection

由衣原体感染引起的生殖道病理是对衣原体存在的免疫应答的结果。 女性生殖道内的衣原体。先天性和适应性免疫反应都是作为宿主的一部分而被诱导的。 细胞因子和趋化因子在调节这些防御机制中发挥作用。 因为我们以前发表的数据表明，TLR 3缺乏导致IFN-β合成的显着减少， 在C.在OE细胞和小鼠中，我们假设IFN-β可能不是一个促成因素， 在TLR 3缺陷小鼠中观察到的生殖道病理学数量增加。我们进一步假设IFN-β 相反，它是对Cm感染的保护性免疫反应的必要组成部分。为了验证这些假设，我们 用105 IFU Cm阴道内感染野生型和IFN-β KO小鼠，我们的初步结果表明，IFN-β KO小鼠 与WT小鼠相比，在Cm感染期间具有更高的衣原体载量和遭受更多的生殖道病理。 我们的数据挑战了1型干扰素在宿主对衣原体感染的反应中是有害的这一范式， 由其他研究者使用干扰素α/β受体（IFNAR）KO小鼠建立。然而，令人兴奋的结果之一是， 我们对衣原体-TLR 3相互作用机制的体外研究表明，TLR 3缺乏导致 几种IFNα亚型的过表达。这一出乎意料的发现揭示了TLR 3对IFNα和IFN β的调节差异。 IFN-β，从而刺激OE细胞中Cm感染期间IFN-β的诱导，同时下调 IFNα的表达。在TLR 3缺乏期间IFNα的过度表达暗示IFNα（而不是IFN-β）可能是TLR 3缺乏的一个原因。 在TLR 3缺陷小鼠中观察到的生殖道病理学增加的贡献者，并将支持一种类型的叙述， 1 IFN加剧衣原体引起的生殖道病理学，其他人使用IFNAR KO小鼠报道。研究 Specific Aim #1的计划描述了使用CRISPR-Cas9来产生近交系敲除小鼠品系，该近交系敲除小鼠品系在CRISPR-Cas9中是缺陷的。 IFNα亚型2、4、12和13的基因表达和蛋白质功能。因为这四种IFNα亚型代表了 只有在小鼠生殖道上皮的Cm感染期间诱导IFNα亚型，这种四重KO小鼠将是一种 为我们检验α干扰素参与生殖道病理的假说提供了重要的试剂 无论是在体外还是在体内。

Reproductive tract pathology caused by Chlamydia infections is the result of the immune responses to the presence of Chlamydia within the female reproductive tract. Both innate and adaptive immune responses are induced as part of the host defense against infection, and specific cytokines and chemokines play a role in regulating these defense mechanisms. Because our previously published data show that TLR3 deficiency leads to a significant decrease in the synthesis of IFN-β during C. muridarum (Cm) infection in OE cells and mice, we hypothesized that IFN-β was likely not a contributing factor to the increased amount of genital tract pathology seen in the TLR3-deficient mice. We further hypothesized that IFN-β was instead a necessary component of the protective immune response to Cm infection. To test these hypotheses, we infected both wild-type and IFN-β KO mice intravaginally with 105 IFU Cm and our preliminary show that IFN-β KO mice had higher chlamydial loads and suffered more genital tract pathology during Cm infection when compared to WT mice. Our data challenge the paradigm that type-1 IFNs are detrimental in the host response to Chlamydia infection that was established by other investigators using interferon α/β receptor (IFNAR) KO mice. However, one of the exciting outcomes of our in vitro investigations into the mechanisms of the Chlamydia-TLR3 interaction was that TLR3-deficiency leads to an overexpression of several IFNα subtypes. This unexpected finding reveals that TLR3 differentially regulates IFNα and IFN-β, whereby it stimulates the induction of IFN-β during Cm infection in OE cells, while simultaneously down-regulating the expression of IFNα. The over-expression of IFNα during TLR3 deficiency implicates IFNα (and not IFN-β) as a possible contributor to the increased genital tract pathology seen in TLR3-deficient mice and would support the narrative of a type- 1 IFN exacerbating the Chlamydia-caused genital tract pathology reported by others using IFNAR KO mice. The research plan for Specific Aim #1 describes using CRISPR-Cas9 to generate an inbred knock-out mouse strain that is deficient in the gene expression and protein function of IFNα subtypes 2, 4, 12, and 13. Because these four subtypes of IFNα represent the only IFNα subtypes induced during Cm infection of murine genital tract epithelium, this quadruple KO mouse will be an important reagent for us to test the hypothesis that IFNα contributes to reproductive tract pathology during Cm infection both in vitro and in vivo.