The IBD-candidate gene, PTPN2, is a Key Regulator of Ileal Paneth Cell Function in Mice
Authors: Canale Vinicius , McCole Declan 
Published: May 2023

Loss-of-function variants in the Protein Tyrosine Phosphatase non-receptor Type 2 ( PTPN2) gene are associated with increased risk of Inflammatory Bowel Disease (IBD). PTPN2 encodes T Cell Protein Tyrosine Phosphatase (TCPTP), a negative regulator of intracellular signaling pathways including JAK-STAT. PTPN2 is critical for intestinal epithelial cell (IEC) barrier maintenance, IEC-macrophage crosstalk, and modulating the gut microbiome in mice, although the mechanism in which PTPN2 loss affects gut microbiome is unknown. IBD patient biopsies and studies in transgenic mice show that IBD-associated genes can modify IEC subtypes and their functional roles in intestinal homeostasis. Hypothesis: PTPN2 loss compromises modulation of gut microbiome by impairing number and function of IEC subtypes. Methods: To identify how loss of PTPN2 activity in vivo affects ileal IEC subtypes and their function, RNA and protein were isolated from ileal IECs of whole-body Ptpn2-wild-type (WT), heterozygous (Het) and KO mice. RNA was assayed by two separate Nanostring® panels: PanCancer and AutoImmune totaling >1500 targets. Dysregulated targets associated with IEC function were validated by Western blot, immunostaining, and transmission electron microscopy (TEM). Altered genes were then investigated in tamoxifen-inducible intestinal epithelial-specific Ptpn2 knockout mice ( Ptpn2 Δ IEC) and Ptpn2 fl/fl controls. Results: 1398 targets had detectable expression. Notably, Paneth cell (PC)-associated genes Lyz1, Defa6, Pla2g2a, and Fgfr3 were markedly downregulated (p<0.05 and FDR<0.1; n=4). Reduced PC numbers in whole-body Ptpn2-KO mice were confirmed by decreased Lysozyme and Cryptdin-1 staining (p<0.001; n=5), reduced Lysozyme protein levels by western blotting (p=0.004; n=5), and loss of PC phenotype by TEM (n=3). Ki-67 staining revealed elevated ileal crypt cell proliferation (p=0.02; n=3) and longer villi (p=0.03; n=9) in Ptpn2-KO mice vs. WT. However, unchanged cleaved caspase 3 and TUNEL staining indicated no increase in IEC apoptosis. Further, TEM analysis showed disrupted endoplasmic reticulum (ER) architecture in PCs of Ptpn2-KO mice (n=3) accompanied by higher protein levels of the ER stress marker CHOP (p=0.01; n=8). Conversely, Ptpn2 Δ IEC mice showed no change in ileal villus length (n=6), number of PCs (n=7), and TEM showed abundant cytosolic granules compared to Ptpn2 fl/fl (n=4). However, immunostaining (n=6) and Western blotting (p<0.05; n=6) showed reduced Lysozyme protein accompanied by elevated levels of unfolded protein response Xbp-1s in Ptpn2 Δ IEC mice (p<0.05; n=6). Conclusion: Ptpn2-deficiency affects Paneth cell viability by decreasing cell number, promoting ER stress, and impairing production of PC-associated antimicrobial peptides. This likely contributes to the increased susceptibility to infection and the dysbiotic microbiota in Ptpn2-deficient mice and may explain the association of PTPN2 variants with dysbiosis in clinical IBD.

Supported by NIH 2R01DK091281, 1R01AI153314-01, R21AI152017 and R01AI165490 (DFM)

This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.