Regulation of C. difficile infection by the cytokine interleukin-22 (IL-22)

项目总结 艰难梭状芽胞杆菌是一种与粘膜相关的病原体，可导致危及生命的疾病。细菌 是医院获得性胃肠道感染的主要来源，但在健康人群中正在出现。无症状 殖民是常见的，只会导致某些人的生产性感染，而这些人中的一部分 患者会患上复发的更严重的疾病。艰难梭菌分泌B毒素(TcdB)，一种小分子的灭活剂 诱导上皮细胞死亡的GTP酶。这种毒素帮助艰难梭菌建立其所需的生态位 生殖性感染。艰难梭菌需要微生物群的扰动才能引发疾病。虽然是最近的 研究已经揭示了它如何克服共生的一些方法，我们仍然不完全理解C. 艰难梭菌会引起感染或再次感染。宿主免疫反应可能起到一定作用。我们的长期合作 目标是确定细胞因子生物学如何在治疗上针对缓解艰难梭菌最初的感染或 高危患者的复发。第3组先天淋巴细胞(ILC3)是一种罕见的免疫细胞，常见于 粘膜组织。它们产生高水平的IL-22，这是粘膜组织反应的关键调节因子。IL-22是 对于在健康和疾病中维持肠道内环境平衡很重要。通过对上皮细胞的维护 尽管IL-22在艰难梭菌感染中的作用尚不清楚，但该细胞因子在胃肠道感染中具有保护作用。 在我们研究毒素与ILC3相互作用的研究中，我们研究了TcdB对IL-22的影响。 我们的初步数据显示，TcdB以GTPase依赖的方式诱导ILC3中的IL-22。药理作用 抑制表明IL-22的上调是通过CDC42实现的。这些数据构成了我们假设的前提 艰难梭菌调节宿主的免疫反应。这个应用程序的总体目标是理解 TcdB调节IL-22产生ILC3的信号通路(S)及其益处 在感染期间，艰难梭菌的IL-22升高。在目标1中，我们将研究小GTP酶CDC42如何 调节激活的ILC3s中IL-22的产生。通过遗传或siRNA介导的ILC3s中CDC42的缺失， 我们将检查这个小的GTP酶是否是ILC3激活的负调节因子，以及是否存在 CDC42与其他信号通路之间的链接，包括STAT3和MAPK。在目标2中，我们将研究 艰难梭菌与TcdB、IL-22与胃肠道组织的相互作用。我们将研究IL-2的功能。 22在艰难梭菌感染中使用结肠类有机物的还原方法。研究将检查是否 IL-22对TcdB介导的细胞凋亡的保护作用并确定哪些IL-22诱导的因子有助于改变 利基市场。艰难梭菌体内感染模型将补充体外实验。这项研究可能会确定 新的信号通路参与调节IL-22，这与感染性疾病和 慢性炎症区。我们还将更全面地了解IL-22水平升高是如何 调节炎症环境，使艰难梭菌胜过胃肠道的其他细菌。这有 IL-22生物学在预防艰难梭菌原发或复发感染中的作用。

PROJECT SUMMARY Clostridioides difficile is a mucosal-associated pathogen that can cause life-threatening illness. The bacterium is a leading source of hospital-acquired GI infections, but is emerging in the healthy population. Asymptomatic colonization is common and only leads to productive infection in some individuals, and a subset of these patients will have a relapsing, more severe disease. C. difficile secretes toxin B (TcdB), an inactivator of small GTPases that induces epithelial cell death. This toxin helps C. difficile establish the niche it needs for productive infection. C. difficile requires perturbation in the microbiome to initiate disease. Although recent studies have revealed some means of how it overcomes commensals, we still do not fully understand how C. difficile establishes infection or initiates re-infection. Host immune responses likely play a role. Our long-term goal is to identify how cytokine biology may be therapeutically targeted to alleviate initial C. difficile infection or relapses in high-risk patients. Group 3 innate lymphocytes (ILC3s) are rare immune cells often found in mucosal tissues. They produce high levels of IL-22, a critical modulator of mucosal tissue responses. IL-22 is important for maintaining intestinal homeostasis in health and disease. Through maintenance of the epithelial barrier, the cytokine is protective in GI infections, although the role of IL-22 in C. difficile infection is not clear. In our studies investigating interactions between toxins and ILC3s, we examined the effects of TcdB on IL-22. Our preliminary data show that TcdB induces IL-22 in ILC3s in a GTPase-dependent manner. Pharmacological inhibition suggests that upregulation of IL-22 is through Cdc42. These data form the premise for our hypothesis that C. difficile modulates the host immune response. The overall objective for this application is to understand signaling pathway(s) through which TcdB may modulate ILC3 production of IL-22 and ascertain the benefits of elevated IL-22 to C. difficile during infection. In Aim 1 we will examine how the small GTPase Cdc42 may regulate IL-22 production in activated ILC3s. Through genetic or siRNA-mediated deletion of Cdc42 in ILC3s, we will examine if this small GTPase is a negative regulator of ILC3 activation as well as examine if there are links between Cdc42 and other signaling pathways, including STAT3 and MAPKs. In Aim 2, we will examine the interactions between C. difficile and TcdB, and IL-22 and the GI tissues. We will examine the function of IL- 22 signaling in C. difficile infection using a reductionist approach with colonic organoids. Studies will examine if IL-22 protects against TcdB-mediated apoptosis and identify which IL-22-inducible factors contribute to altering the niche. An in vivo C. difficile infection model will complement in vitro experiments. This study may identify new signaling pathways involved in regulation of IL-22, which is of interest to both the infectious disease and chronic inflammation fields. We will also have a more complete understanding of how increased IL-22 levels modulate the inflammatory environment to favor C. difficile over other bacteria in the GI tract. This has implications on how IL-22 biology may be manipulated in preventing primary or relapsing C. difficile infection.