Paneth cells (PCs) are located at the base of the intestinal crypt, where they support the intestinal stem cells (ISC) with growth factors and participate in innate immunity by secreting antimicrobial peptides (AMPs). Loss of or functional defects in PCs are seen in Crohn's disease, linking abnormalities in these cells to inflammatory bowel disease (IBD). We recently showed that activation of the neuregulin receptor ErbB4 protects PCs against injury. However, the role of ErbB3, the other neuregulin receptor, in PCs is unknown. In this study we tested the effects of ErbB3 activation or deletion on PC numbers and AMP production.METHODSIleal tissue and enteroid cultures derived from C57BL/6 (WT), ErbB4flox/flox, ErbB4flox/flox; Villin‐Cre (E4KOIE), ErbB3flox/flox, and ErbB3flox/flox; Villin‐Cre (E3KOIE) mice were used. Enteroids were given NRG1β (ErbB3/4 ligand; 10ng/ml) or NRG4 (ErbB4 ligand; 100ng/ml) for 24 h and RNA was extracted. Fixed tissue for immunofluorescence analysis, RNA for qPCR, and protein lysates for western blotting were collected from mice.RESULTSNRG1β treatment of WT enteroids for 24 h resulted in reduced expression of Lyz1 (by 29.47%, p=0.03) and Defa5 (by 46.60%, p= 0.001), and fewer lysozyme‐positive cells (by 18.03%, p=0.03). This effect of NRG1β was maintained in E4KOIE enteroids but was not recapitulated in NRG4‐treated WT enteroids, suggesting a specific role for ErbB3. Consistently, E3KOIE intestinal crypts displayed an increased number of lysozyme‐positive cells (by 25.36%, p=0.001) and MMP7/MUC2 double positive intermediate/precursor cells (by 16.24%, p=0.011). E3KOIE crypts also had increased Lyz1 (by 67.53%, p<0.0001) and Lgr5 (by 46.65%, p=0.012) RNA expression. This increase was not accompanied by differences in expression of other ErbB family members (Egfr, ErbB2, and ErbB4) or other secretory cell markers (Muc2 and Chga). Interestingly, E3KOIE mice had a significant increase in expression of the secretory regulator Atoh1 (by 59.20%, p=0.0009), which is required for PC development. Since MAPK signaling has been implicated in PC regulation, we performed western blot analysis for P‐Erk1/2 and P‐p38 on tissue lysates, but there was no difference between WT and E3KOIE mice. In contrast, E3KOIE mice had a lower level of P‐Akt (by 53.1%, p=0.02), suggesting potential involvement of the PI3K pathway.CONCLUSIONSThese results suggest that ErbB3 restricts PC numbers by a mechanism that may involve PI3K/Akt and Atoh1. Understanding the role of ErbB3 receptor could identify new therapeutic targets for regulating PCs and the ISC niche.Support or Funding InformationThis work was supported by a Senior Research Award from the Crohn's and Colitis Foundation of America and by NIH award R01DK095004.
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