Abstract
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.
Highlights
Factors that regulate intestinal stem cell (ISC) proliferation and apoptosis are unknown
We show that the Lgr5-positive intestinal stem cells within the crypts of the small intestine in the newborn mouse express Toll-like receptor 4 (TLR4), that activation of TLR4 within the intestinal crypts results in reduced proliferation and increased apoptosis, that TIR-domain-containing adapter-inducing interferon- (TRIF) is required for the effects of TLR4 on crypt apoptosis, and that these events require the activation of p53up-regulated modulator of apoptosis (PUMA) in response to TLR4
In seeking to define the physiological relevance of these findings, we further show that TLR4-induced PUMA activation within the intestinal crypts of the newborn mice plays a key role in the development of necrotizing enterocolitis (NEC), a devastating disease of premature newborns that is characterized by exaggerated TLR4 signaling [19, 33]
Summary
Factors that regulate intestinal stem cell (ISC) proliferation and apoptosis are unknown. 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. Exaggerated TLR4 activation has been shown to lead to the development of necrotizing enterocolitis (NEC), a devastating disease of the premature intestine, that is characterized by TLR4-mediated reduction in proliferation and an induction of enterocyte apoptosis [18, 19] These findings raise the intriguing possibility that TLR4 itself may be expressed on the intestinal stem cells and regulate their function. In support of this hypothesis, using a variety of knockout and transgenic strains, we show that TLR4 is expressed on intestinal stem cells and that the activation of TLR4 leads to a loss of proliferation and increase in apoptosis in a mechanism that is dependent upon the activation of the p53-up-regulated modulator of apoptosis (PUMA) in the pathogenesis of NEC
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
