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，结肠炎的发作时间、发病率和严重程度在也携带TLR-4突变的IL-10-/-小鼠中加剧。通过与其在抗原呈递细胞（APC）的先天免疫信号传导中的作用明显无关的机制，CD 4 + T细胞的TLR-4信号传导调节对随后TCR活化的应答。当直接离体测量或过继转移到Rag 1-/-受体后测量时，TLR-4-/- x IL-10-/-（DKO）小鼠的CD 4 + T细胞表现出增强的促炎性结肠炎性效应子应答。此外，从TLR-4-/-小鼠中分离的Foxp 3 + T细胞分泌IL-10的能力受损。因此，CD 4 + T细胞中的TLR-4信号传导增强Treg功能并抑制炎性T效应子功能;这两种作用可能协同作用，以预防健康小鼠的肠道炎症。然而，在DKO小鼠中，Foxp 3 + T细胞聚集在发炎的固有层（LP）中，分泌IFN-？和IL-17，并未能控制疾病。MyD 88依赖性TLR-4介导的信号在肠上皮屏障的稳态维持中的作用已经很好地确立。我们发现，显然来自肠上皮细胞的凋亡残留物在螺杆菌阳性（Hh+）TLR-4-/-小鼠的结肠LP（在没有炎症的情况下）以及IL-10-/-和DKO小鼠的发炎LP中积累。在WT C57 Bl/6小鼠或无螺杆菌（Hh-）TLR-4-/-或DKO小鼠的结肠LP中未检测到凋亡细胞。TLR-4介导的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，凋亡上皮细胞积累在大肠杆菌定殖的DKO小鼠的结肠LP，并诱导炎症性Th 17反应。IFN-？也在CD 4+效应T细胞中缺乏紧张性TLR-4信号传导的情况下被诱导。细胞凋亡诱导的IL-17应答导致炎症细胞更早和更多地募集到肠中，最终导致慢性结肠炎。在Helicobacter-colonized TLR-4-/-小鼠中，这种慢性炎症反应受到IL-10的免疫调节作用的抑制。然而，在DKO小鼠中，T细胞活化在TLR-4信号传导不存在的情况下失调，从而增强炎性T效应子应答。Treg功能和/或稳定性在DKO小鼠中也受到损害，并且Treg本身成为致病性效应细胞。在目的1中，将使用体外和体内方法来检查在结肠LP中由APC吞噬的螺杆菌诱导的凋亡上皮残余物是否诱导产生IL-17的Foxp 3-和Foxp 3 + T细胞并驱动DKO小鼠中的肠道炎症。在目的2中，我们将进一步表征DKO小鼠的LP中的推定致病性Treg和使该Treg亚群不稳定或转化的触发物。我们还将研究这些细胞的功能抑制和/或效应能力。 公共卫生相关性：我们将使用一种新的自发性肠道炎症小鼠模型来研究导致保护健康个体免受疾病影响的反应失败的触发因素。更清楚地了解调节这种保护的机制可能会为开发新的免疫方法来治疗或预防炎症性肠病（IBD）提供信息。

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).