Regulation of Intestinal inflammation by TLR4-mediated signals

描述(由申请人提供)：我们和其他人最近表明，在携带TLR-4突变的IL-10-/-小鼠中，结肠炎的发病时间、发病率和严重程度都会加剧。通过一种显然与其在抗原提呈细胞(APC)天然免疫信号中的作用无关的机制，CD4T细胞的TLR-4信号调节对随后TCR激活的反应。来自TLR-4-/-x IL-10-/-(DKO)小鼠的CD4T细胞在直接体外或过继转移到Rag1/-受体后显示出增强的促炎结肠炎效应反应。此外，从TLR-4-/-小鼠分离的Foxp3 Tregs分泌IL-10的能力受到损害。因此，CD4T细胞中的TLR-4信号增强了Treg功能，抑制了炎症T效应功能；这两个作用可能协同作用，以预防健康小鼠的肠道炎症。然而，在DKO小鼠中，Foxp3 Tregs聚集在炎症的固有层(LP)，分泌干扰素？和IL-17，并未能控制疾病。MyD88依赖的TLR-4介导的信号在维持肠上皮屏障的动态平衡中的作用已经得到了很好的证实。我们发现，明显来源于肠道上皮的凋亡残留物聚集在幽门螺杆菌阳性(HH)TLR-4-/-小鼠(在没有炎症的情况下)的结肠LP以及IL-10-/-和DKO小鼠的炎症LP中。WT C57BL/6小鼠、无幽门螺杆菌(HH-)TLR-4-/-或DKO小鼠的结肠LP中未检测到凋亡细胞。TLR-4介导信号的缺失如何加剧IL-10缺陷小鼠的肠道炎症？我们的工作假设是，凋亡的上皮细胞聚集在幽门螺杆菌定植的DKO小鼠的结肠LP中，并诱导炎性Th17反应。干扰素-？在缺乏紧张性TLR-4信号的情况下，也可以在CD4效应T细胞中诱导。细胞凋亡诱导的IL-17反应导致炎性细胞更早和更多地募集到肠道，最终导致慢性结肠炎。在幽门螺杆菌感染的TLR-4-/-小鼠中，IL-10的免疫调节作用抑制了这种慢性炎症反应。然而，在DKO小鼠中，在缺乏TLR-4信号的情况下，T细胞激活被失调，从而增强了炎性T效应反应。在DKO小鼠中，Treg的功能和/或稳定性也受到影响，Treg本身成为致病的效应细胞。目的1采用体外和体内实验方法，研究幽门螺杆菌诱导的结肠低密度脂蛋白中APC吞噬的凋亡上皮残留物是否诱导产生IL-17的Foxp3和Foxp3T细胞，从而促进DKO小鼠的肠道炎症。在目标2中，我们将进一步表征DKO小鼠LP中可能致病的Treg，以及破坏或转换这一Treg亚集的触发因素。我们还将研究这些细胞的功能抑制和/或效应器能力。 公共卫生相关性：我们将使用一种新的自发性肠道炎症的小鼠模型来检查导致保护健康个体免受疾病影响的反应失败的触发因素。更清楚地了解这种保护的调节机制可能有助于开发新的免疫治疗方法来治疗或预防炎症性肠病(IBD)。

DESCRIPTION (provided by applicant): We and others have recently shown that the time of onset, incidence and severity of colitis is exacerbated in IL-10-/- mice that also bear a mutation in TLR-4. By a mechanism apparently unrelated to its role in innate immune signaling by antigen presenting cells (APC), TLR-4 signaling by CD4+ T cells regulates the response to subsequent TCR activation. CD4+ T cells from TLR-4-/- x IL-10-/- (DKO) mice exhibit enhanced proinflammatory colitiogenic effector responses when measured either directly ex vivo or after adoptive transfer into Rag1-/- recipients. Moreover, Foxp3+ Tregs isolated from TLR-4-/- mice are impaired in their ability to secrete IL-10. TLR-4 signaling in CD4+ T cells therefore enhances Treg function and inhibits inflammatory T effector function; these two roles are likely to synergize to prevent intestinal inflammation in healthy mice. In DKO mice, however, Foxp3+ Tregs accumulate in the inflamed lamina propria (LP), secrete IFN-? and IL-17, and fail to control disease. A role for MyD88 dependent TLR-4 mediated signals in the homeostatic maintenance of the intestinal epithelial barrier is already well established. We found that apoptotic remnants apparently derived from the intestinal epithelium accumulate in the colonic LP of Helicobacter-positive (Hh+) TLR-4-/- mice (in the absence of inflammation) as well as in the inflamed LP of both IL-10-/- and DKO mice. Apoptotic cells were not detectable in the colonic LP of WT C57Bl/6 mice or Helicobacter free (Hh-) TLR-4-/- or DKO mice. How does the absence of TLR-4 mediated signals exacerbate intestinal inflammation in IL-10 deficient mice? Our working hypothesis is that apoptotic epithelial cells accumulate in the colonic LP of Helicobacter-colonized DKO mice and induce an inflammatory Th17 response. IFN-? is also induced in the absence of tonic TLR-4 signaling in CD4+ effector T cells. The apoptosis induced IL-17 response leads to an earlier and increased recruitment of inflammatory cells to the intestines that results ultimately in chronic colitis. In Helicobacter-colonized TLR-4-/- mice this chronic inflammatory response is held in check by the immunoregulatory effects of IL-10. In DKO mice, however, T cell activation is dysregulated in the absence of TLR-4 signaling, potentiating the inflammatory T effector response. Treg function and/or stability is also compromised in DKO mice and the Tregs themselves become pathogenic effector cells. In Aim 1, both in vitro and in vivo approaches will be used to examine whether Helicobacter induced apoptotic epithelial remnants phagocytosed by APC in the colonic LP induce IL-17 producing Foxp3- and Foxp3+ T cells and drive intestinal inflammation in DKO mice. In Aim 2 we will further characterize the putative pathogenic Tregs in the LP of DKO mice and the triggers that destabilize or convert this Treg subset. We will also examine the functional suppressive and/or effector capabilities of these cells. PUBLIC HEALTH RELEVANCE: We will use a novel murine model of spontaneous intestinal inflammation to examine the triggers that lead to the failure of the responses that protect healthy individuals from disease. A clearer understanding of the mechanisms regulating this protection may inform the development of new immunotherapeutic approaches to treat or prevent inflammatory bowel disease (IBD).