S. Shaffiey1,2, H. Jia1, C. Sodhi1, K. Thadikona1, M. Good3, M. Neal2, Y. Yamaguchi1, S. Mielo1, T. Prindle1, D. J. Hackam1,2 1Children’s Hospital Of Pittsburgh Of UPMC,Pediatric Surgery,Pittsburgh, PA, USA 2University Of Pittsburg,General Surgery,Pittsburgh, PA, USA 3Children’s Hospital Of Pittsburgh Of UPMC,Neonatal Medicine,Pittsburgh, PA, USA
Introduction: Autophagy is an adaptive response to cellular stress important in the pathogenesis of many disease states. The stress response of intestinal stem cells (ISC) has been incompletely studied. Our lab has previously elucidated the importance of TLR4 signaling in both autophagy and intestinal stem cell proliferation. We now focus on linking the role of autophagy in the intestinal stem cells with TLR4 following stress conditions.
Methods: Enteroids were cultured following exposure to radiation or LPS in vivo or following two days of in vitro culturing from C57/Bl6 (WT), mice lacking the critical autophagy protein ATG7 selectively in the intestinal villi (ATG7ΔIEC), and mice lacking TLR4 selectively in labeled intestinal stem cells. Whole mount imaging was performed and mRNA isolated for qt-PCR. WT, ISC (LGR5 or Bmi1) labeled, ISC labeled cells lacking TLR4, TLR4ΔIEC and ATG7ΔIEC strains (n=8/group) of mice underwent endotoxemia and radiation models at different dosages. BrdU was injected at the time of injury to evaluate differentiation from the stem cell compartment following stress. Tissue was collected for immunohistochemistry and qt-PCR for ISC, autophagy, and proliferative markers.
Results: Improved proliferation was seen from ATG7ΔIEC, TLR4ΔIEC and labeled ISC missing TLR4 enteroids compared to WT under homeostatic conditions. However, autophagy deficient enteroids showed increased apoptosis following LPS or radiation treatment both when injury occurred in vivo or in vitro. Interestingly qt-PCR also showed an exaggerated cytokine response (iNOS, IL6) in these same groups. Imaging confirmed the inability to undergo autophagy with decreased LC3 staining in apoptotic enteroids. In vivo, stem cell and proliferative mRNA levels were markedly increased in TLR4ΔIEC and ATG7ΔIEC mice at baseline, but were unable to increase following endotoxemia models compared to WT mice. Following radiation models a robust increase in autophagy was noted in WT mice by mRNA levels but lacking in mice missing TLR4 from the intestinal stem cells and TLR4ΔIEC. BrdU staining showed impaired migration from the stem cell compartment in both endotoxemia and radiation models in TLR4ΔIEC, TLR4 missing from the ISC and ATG7ΔIEC mice compared to WT mice. Repeated models showed the effects to be dose dependent. Linage tracing showed following stress models ISC’s lacking TLR4 are unable to recapitulate the intestinal villi with decreased levels of LC3 staining in the stem cell compartment.
Conclusion: Autophagy through TLR4 signaling is an important mediator of the intestinal stem cell stress response. Our findings suggest that pharmacologic regulation of autophagy pathways may provide novel therapeutic approaches for diseases of impaired healing including radiation or bacterial injury.