Division of Pediatric Surgery Children's Hospital of Pittsburgh and Department of Surgery, University of Pittsburgh School of Medicine, Pittsburgh, PA 15224, USA.
J Biol Chem. 2012 Oct 26;287(44):37296-308. doi: 10.1074/jbc.M112.375881. Epub 2012 Sep 6.
Factors regulating the proliferation and apoptosis of intestinal stem cells (ISCs) remain incompletely understood. Because ISCs exist among microbial ligands, immune receptors such as toll-like receptor 4 (TLR4) could play a role. We now hypothesize that ISCs express TLR4 and that the activation of TLR4 directly on the intestinal stem cells regulates their ability to proliferate or to undergo apoptosis. Using flow cytometry and fluorescent in situ hybridization for the intestinal stem cell marker Lgr5, we demonstrate that TLR4 is expressed on the Lgr5-positive intestinal stem cells. TLR4 activation reduced proliferation and increased apoptosis in ISCs both in vivo and in ISC organoids, a finding not observed in mice lacking TLR4 in the Lgr5-positive ISCs, confirming the in vivo significance of this effect. To define molecular mechanisms involved, TLR4 inhibited ISC proliferation and increased apoptosis via the p53-up-regulated modulator of apoptosis (PUMA), as TLR4 did not affect crypt proliferation or apoptosis in organoids or mice lacking PUMA. In vivo effects of TLR4 on ISCs required TIR-domain-containing adapter-inducing interferon-β (TRIF) but were independent of myeloid-differentiation primary response-gene 88 (MYD88) and TNFα. Physiological relevance was suggested, as TLR4 activation in necrotizing enterocolitis led to reduced proliferation and increased apoptosis of the intestinal crypts in a manner that could be reversed by inhibition of PUMA, both globally or restricted to the intestinal epithelium. These findings illustrate that TLR4 is expressed on ISCs where it regulates their proliferation and apoptosis through activation of PUMA and that TLR4 regulation of ISCs contributes to the pathogenesis of necrotizing enterocolitis.
调控肠干细胞（ISCs）增殖和凋亡的因素仍不完全清楚。由于 ISCs 存在于微生物配体中，免疫受体（如 Toll 样受体 4（TLR4））可能发挥作用。我们现在假设 ISCs 表达 TLR4，TLR4 对肠干细胞的直接激活调节它们增殖或凋亡的能力。我们通过流式细胞术和荧光原位杂交分析肠干细胞标志物 Lgr5，证明 TLR4 表达于 Lgr5 阳性 ISCs 上。TLR4 激活减少了体内和 ISC 类器官中 ISC 的增殖并增加了其凋亡，而在缺乏 TLR4 的 Lgr5 阳性 ISCs 中未观察到这种作用，证实了这种作用的体内意义。为了确定所涉及的分子机制，TLR4 通过 p53 上调凋亡调节剂（PUMA）抑制 ISC 增殖并增加凋亡，因为 TLR4 不影响类器官或缺乏 PUMA 的小鼠中隐窝的增殖或凋亡。TLR4 对 ISC 的体内作用需要 TIR 结构域包含衔接子诱导干扰素-β（TRIF），但不依赖于髓样分化初级反应基因 88（MYD88）和 TNFα。这表明存在生理相关性，因为坏死性小肠结肠炎中 TLR4 的激活以一种可通过抑制 PUMA 逆转的方式导致肠隐窝的增殖减少和凋亡增加，这种作用可以是全身性的或仅限于肠上皮。这些发现表明 TLR4 表达于 ISCs 上，通过激活 PUMA 调节其增殖和凋亡，并且 TLR4 对 ISC 的调节有助于坏死性小肠结肠炎的发病机制。
