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

开发小鼠模型来检查 IFNa 在生殖道衣原体感染发病机制中的具体影响

• 批准号: 10041346
• 负责人: WILBERT A DERBIGNY
• 金额: $ 6.76万
• 依托单位: MARIAN UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-08-19 至 2023-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10041346
• 关键词: Animal Model; Animals; Antibiotics; Bacteria; CRISPR/Cas technology; Case Study; Cell physiology; Chlamydia; Chlamydia Infections; Chlamydia muridarum; Chlamydia trachomatis; Chronic; Chronic Disease; Clinical; Clustered Regularly Interspaced Short Palindromic Repeats; Communities; Complex; Data; Defense Mechanisms; Disease; Ensure; Epithelial; Epithelial Cells; Epithelium; Exhibits; Female; Funding; Future; Gene Expression; Gene Proteins; Genes; Host Defense; Immune response; In Vitro; Inbreeding; Infection; Infertility; Inflammation; Inflammatory; Interferon Type I; Interferon-alpha; Interferon-beta; Interferons; Investigation; Knockout Mice; Literature; Mammalian Oviducts; Methodology; Mouse Strains; Mus; Outcome; Pathogenesis; Pathology; Pathology Report; Pelvic Inflammatory Disease; Play; Production; Publishing; Reagent; Research; Research Personnel; Role; Sexually Transmitted Diseases; Signal Pathway; Suggestion; TLR3 gene; Testing; United States; Uterus; Wild Type Mouse; Woman; adaptive immune response; autocrine; chemokine; cytokine; in vivo; mouse model; neutralizing antibody; overexpression; paracrine; pathogen; protein function; receptor; reproductive tract; response

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.

衣原体感染引起的生殖道病理是对存在的免疫反应的结果 女性生殖道内的衣原体。先天免疫反应和获得性免疫反应都是作为宿主的一部分诱导的 对感染的防御，以及特定的细胞因子和趋化因子在调节这些防御机制中发挥作用。 因为我们之前发表的数据显示，TLR3缺乏会导致干扰素-β的合成显著减少 在OE细胞和小鼠感染鼠隐孢子虫的过程中，我们推测干扰素-β可能不是一个促成因素。 TLR3基因缺陷小鼠生殖道病理性的增加。我们进一步假设干扰素-β 相反，它是对CM感染的保护性免疫反应的必要组成部分。为了检验这些假设，我们 用105IFU cm经阴道感染野生型和干扰素-βKO小鼠，初步表明干扰素-βKO小鼠 与WT小鼠相比，Cm感染小鼠具有更高的衣原体载量和更多的生殖道病变。 我们的数据挑战了1型干扰素在衣原体感染的宿主反应中有害的范式 由其他研究人员利用干扰素α/β受体(IFNAR)KO小鼠建立。然而，令人兴奋的结果之一 我们对衣原体-TLR3相互作用机制的体外研究中，TLR3缺乏导致 几种干扰素α亚型的过度表达。这一意想不到的发现揭示了TLR3对干扰素α和 干扰素-β，从而在CM感染OE细胞的过程中刺激诱导干扰素-β，同时下调 干扰素α的表达。干扰素α在TLR3缺乏症中的过度表达提示干扰素α(而不是干扰素-β)可能是 导致TLR3缺陷小鼠生殖道病理增加的因素，并支持一种类型- 1干扰素加重衣原体引起的生殖道病理，其他人使用IFNAR KO小鼠报告。这项研究 针对特定目标的计划#1描述了使用CRISPR-Cas9产生近亲交配的敲除小鼠品系，该品系在 干扰素α亚型2、4、12和13的基因表达和蛋白功能。由于这四种亚型干扰素α代表 只有在Cm感染小鼠生殖道上皮过程中诱导的干扰素α亚型，这只四联KO小鼠将成为 Cm感染过程中干扰素α参与生殖道病理的重要试剂 在体外和体内都有。