T‐cell Protein Tyrosine Phosphatase is a Key Regulator of Paneth Cell Function

Abstract

Loss‐of‐function single nucleotide polymorphisms (SNPs) in the Protein Tyrosine Phosphatase non‐receptor Type 2 (PTPN2) gene, are associated with increased susceptibility to Inflammatory Bowel Disease (IBD). PTPN2 encodes the T Cell Protein Tyrosine Phosphatase (TCPTP) which is a negative regulator of intracellular signaling pathways including JAK‐STAT. We previously presented that Paneth and goblet cell numbers are reduced in Ptpn2 knockout (KO) mice, consistent with altered differentiation of these intestinal epithelial cell (IEC) types in IBD. Alterations in IEC populations are detrimental for intestinal homeostasis and increase susceptibility to microbial infection.GoalTo identify whether Ptpn2‐KO mice display altered IEC differentiation and Paneth cell antimicrobial peptide (AMP) production.MethodsIntestinal tissues (ileum, cecum, proximal and distal colon) from 21 day old constitutive Ptpn2‐KO mice and control littermates, and from adult tamoxifen‐inducible intestinal epithelial‐specific Ptpn2ΔIEC mice (Vil‐Cre/Ptpn2), were harvested and processed for imaging, western blot, flow cytometry and qPCR.ResultsConstitutive Ptpn2‐KO mice display reduced numbers of Paneth cells and loss of the AMP lysozyme (p=0.0158; n=5). This was confirmed by Western blots of isolated IECs (~67% reduction; n=4). Expression of Paneth cell markers and differentiation factors (Mmp7, Defa5 and Sox9) were not altered in KO mice (n=10). Interestingly, Reg3‐γ, which like lysozyme also targets gram‐(+) bacteria and is secreted by Paneth cells and enterocytes, was increased in KO mice vs. control littermates (p=0.0066; n=10). Next, we identified by immunofluorescence that loss of Ptpn2 only in intestinal epithelium (Ptpn2ΔIEC) dramatically reduced colocalization of lysozyme and another Paneth cell marker, UEA‐1 (n=6). Decreased lysozyme expression in Ptpn2ΔIEC ileum was confirmed by western blot (p=0.0315; n=6). Since Paneth cells reside in the stem cell compartment, we investigated whether Ptpn2 deficiency affects the intestinal stem cells. Gene expression of Lgr5, an intestinal stem cell marker, Ephb3, a marker of the stem cell compartment, and Ascl2, a gene that controls stem cell renewal in the crypts, was unchanged in Ptpn2‐KO mice (n=10). In addition, expression of early IEC differentiation factors, Hes1 and Math1, were not altered (n=10). Il‐22 and IFN‐γ are known to stimulate secretion of AMPs by Paneth cells. Interestingly, flow cytometry showed increased abundance of Il‐22+ T cells (p=0.0187; n=5) and IFN‐γ+ T cells (p=0.0277; n=5) in the Ileum of constitutive KO mice, indicating that the defect in AMP production is likely not due to a lack of immune cell‐derived stimuli.ConclusionPtpn2‐deficiency decreases Paneth cell number and has opposing effects on their production of specific AMPs. These effects on Paneth cell number and function may contribute to the increased susceptibility to infection and the dysbiotic microbiota in Ptpn2‐deficient mice.Support or Funding InformationSupported by NIH 2R01DK091281 (DFM) and the Crohn’s and Colitis Foundation (DFM)