TNFAIP8 is a central regulator of intestinal homeostasis and regeneration

Background After injury, intestinal epithelial cells can regenerate the epithelium and stem cell niche by de‐differentiating through a process recently defined as paligenosis. In the intestine, this process is marked by the YAP‐dependent‐induction of Sca‐1 + /Clu + regenerative stem cells, but other signaling pathways that regulate this regenerative program are unknown. Here we show that the protein TNFAIP8 (aka TIPE0) is a critical regulator of intestinal homeostasis and functions through the inhibition of basal microbiome‐dependent PI3K/Akt signaling. Results Loss of TIPE0 results in hyperactivation of the Akt pathway, leading to resistance to ischemia/reperfusion (Fig ) and radiation‐induced injuries. Similar results were recapitulated with enteroid cultures exposed to TNF or hypoxic conditions (Fig ). However, when the epithelium was disrupted through chemical means, the intestine was unable to regenerate. The loss of TNFAIP8 resulted in a baseline shift to more partially differentiated enterocytes (Fig ), with inappropriate baseline activation of the Sca‐1 + /Clu + regenerative program, but a lack of appropriate YAP/Sca‐1/Clu induction after injury (Fig ). Subsequent cellular signaling analysis demonstrated that PI3K/Akt signaling was enhanced upon loss of TIPE0 (Fig by IHC in mice, Fig in freshly isolated epithelium, and Fig in enteroid culture) and that this was responsible both for the resistance to injury and lack of regenerative function. TIPE0 was found to regulate PI3K/Akt signaling by extracting PIP from the plasma membrane, limiting the availability of PI3K to convert Ptdlns(4,5)P 2 to PIP 3 under basal conditions. Conclusions TNFAIP8 is a critical regulator of PI3K/Akt signaling, inhibiting commensal microbial stimulation by extracting Ptdlns(4,5)P 2 from the plasma membrane and inhibiting PIP 3 accumulation. TIPE0 is needed for intestinal homeostasis, and loss results in hyperactivation of PI3K/Akt‐mediated signaling, leading to altered differentiation as well as an inability to respond injury, with a gut that fails to regenerate but also resists some injuries. Support or Funding Information R01‐AI121166, R01‐AI136945, and R56‐AI132329 to Y.H.C.; grant F32‐DK116528 to J.R.G.; and grant P30‐DK050306 to Anil Rustgi. J.R.G was partially supported by NIH‐T32‐CA009140. TIPE0 controls intestinal injury responses and differentiation. A) Histological score of mice subjected to 60′ of ischemia and 90′ of reperfusion, with accompanying histology (B). Cell titer glo 3D assays were used to determine 1‐day survival of 7d old enteroids exposed to 100 ng/mL TNF (C) or hypoxic conditions (1% O 2 , D). Sc‐RNA‐Seq shows changes in enterocyte populations with loss of TIPE0 (E&F). G) The Sca‐1 + injury response program does not induce after injury with loss of TIPE0. Figure 1 Loss of TIPE0 results in basal increases in pAkt signaling. IHC staining for A) pAktS473, B) GSK3βS9, and C) β‐catenin in WT and Tipe0 − / − ileums with and without ischemia. D) Western blot of enterocytes isolated from healthy ileums. E) IF staining for pAktS473 and β‐catenin in 7d‐old enteroids. Figure 2

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