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

项目摘要 艰难梭菌是一种粘膜相关病原体，可导致危及生命的疾病。细菌 是医院获得性GI感染的主要来源，但在健康人群中出现。无症状 定植是常见的，并且仅在某些个体中导致生产性感染，并且这些个体的子集 病人会有复发的更严重的疾病。C.艰难梭菌分泌毒素B（TcdB），一种小的 诱导上皮细胞死亡的GTP酶类。这种毒素有助于C。difficile建立它需要的利基 生产性感染C.艰难梭菌需要微生物群系的扰动来引发疾病。虽然最近 研究已经揭示了它如何克服寄生虫的一些方法，我们仍然不完全了解C。 艰难梭菌建立感染或引发再感染。宿主的免疫反应可能发挥了作用。我们的长期 目的是确定细胞因子生物学如何在治疗上靶向缓解初始C。菌感染或 高风险患者的复发。第3组先天性淋巴细胞（ILC 3）是罕见的免疫细胞，经常在淋巴细胞中发现。 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剂。IL-22是 对维持健康和疾病中的肠道内稳态很重要。通过维持上皮细胞 尽管IL-22在C.艰难梭菌感染尚不清楚。 在我们研究毒素和ILC 3之间相互作用的研究中，我们检查了TcdB对IL-22的影响。 我们的初步数据显示TcdB以GTP酶依赖性方式诱导ILC 3中的IL-22。药理 抑制表明IL-22的上调是通过Cdc 42。这些数据构成了我们假设的前提 梭艰难梭菌调节宿主免疫反应。本应用程序的总体目标是了解 TcdB可通过其调节ILC 3的IL-22产生并确定TcdB的益处的信号传导途径 IL-22升高至C.在感染期间，在目标1中，我们将研究如何小GTdc 42可以 调节活化的ILC 3中的IL-22产生。通过ILC 3中Cdc 42的遗传或siRNA介导的缺失， 我们将检查这个小的GT3是否是ILC 3激活的负调节因子，以及检查是否有 Cdc 42和其他信号通路之间的联系，包括STAT 3和MAPK。在目标2中，我们将研究 C.艰难梭菌和TcdB，以及IL-22和GI组织。我们将研究IL-1的功能。 22号信令在C.艰难感染使用简化方法与结肠类器官。研究将检查，如果 IL-22保护TcdB介导的细胞凋亡并鉴定哪些IL-22诱导因子有助于改变 niche体内C. difficile感染模型将补充体外实验。这项研究可以确定 参与IL-22调节的新信号传导途径，这对感染性疾病和 慢性炎症区。我们还将更全面地了解IL-22水平的增加 调节炎症环境以有利于C.与胃肠道中的其他细菌相比，这 如何操纵IL-22生物学在预防原发性或复发性C.艰难感染

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.