PTPN2 Regulates Inflammasome Activation and Controls Onset of Intestinal Inflammation and Colon Cancer

Abstract

Variants in the gene locus encoding protein tyrosine phosphatase non-receptor type 2 (PTPN2) are associated with inflammatory disorders, including inflammatory bowel diseases, rheumatoid arthritis, and type 1 diabetes. The anti-inflammatory role of PTPN2 is highlighted by the fact that PTPN2-deficient mice die a few weeks after birth because of systemic inflammation and severe colitis. However, the tissues, cells, and molecular mechanisms that contribute to this phenotype remain unclear. Here, we demonstrate that myeloid cell-specific deletion of PTPN2 in mice (PTPN2-LysMCre) promotes intestinal inflammation but protects from colitis-associated tumor formation in an IL-1β-dependent manner. Elevated levels of mature IL-1β production in PTPN2-LysMCre mice are a consequence of increased inflammasome assembly due to elevated phosphorylation of the inflammasome adaptor molecule ASC. Thus, we have identified a dual role for myeloid PTPN2 in directly regulating inflammasome activation and IL-1β production to suppress pro-inflammatory responses during colitis but promote intestinal tumor development.

Keywords: IBD; TC-PTP; colitis; inflammasome; inflammatory bowel disease; interleukin-1-alpha.

Figure 1.. Loss of PTPN2 in Myeloid Cells Promotes Acute Intestinal Inflammation

Acute colitis was induced in WT and PTPN2-LysMCre mice. (A–G) Shown are weight change (A), colon length (B), representative pictures from colonoscopy (C) and respective statistical analysis (D), myeloperoxidase (MPO) activity (E), spleen weight (F), and representative pictures and scoring of epithelial damage and inflammatory infiltration in the terminal colon (G). Depicted are pooled results from two independent experiments with four to six mice per group. Asterisks denote significant differences (*p < 0.05, **p < 0.01, and ***p < 0.001; Mann-Whitney U test with Bonferroni correction). Scale bars represent 100 μm. Error bars represent means ± SD. See Figures S1–S3 for characterization of PTPN2-LysMCre mice.

Figure 2.. IL-10^–/– Mice Lacking PTPN2 in Myeloid Cells Develop More Severe Colitis

(A) IL-10^–/– mice lacking PTPN2 in myeloid cells (IL-10xPTPN2-LysMCre) and their IL-10^–/– littermates were monitored for 200 days for prolapse incidence. At 50 and 150 days, mice that did not develop prolapses were analyzed for colitis severity parameters. (B–F) Shown are representative pictures from colonoscopy and respective statistical analysis (B), myeloperoxidase (MPO) activity (C), spleen weight (D), colon length (E), and representative pictures and scoring of epithelial damage and inflammatory infiltration in the terminal colon (F). Depicted are results from seven or eight mice per group. Asterisks denote significant differences (*p < 0.05 and **p < 0.01; Mann-Whitney U test with Bonferroni correction). Scale bars represent 100 mm. Error bars represent means ± SD.

Figure 3.. PTPN2-LysMCre Mice Are Protected from Colitis-Associated Tumor Induction

(A) Chronic colitis was induced in WT and PTPN2-LysMCre mice, and sections from the terminal colon were stained for the proliferation marker Ki67. (B–F) Colitis-associated tumors were induced in WT and PTPN2-LysMCre mice (B–D) or mice lacking PTPN2 in intestinal epithelial cells (PTPN2-VillinCre mice; E and F). Depicted are representative pictures of colonoscopy and histology of the terminal colon (B and E), observed tumor number in dissected colon (C and F), and (D) average tumor size in each mouse. Data are representative of one of two independent experiments with three to five mice per group. Asterisks denote statistical significance (*p < 0.05; Mann-Whitney U test with Bonferroni correction). Scale bars represent 100 μm. Error bars represent means ± SD. See Figures S4 and S5 for cytokine levels and T cells in PTPN2-LysMCre mice in the DSS and DSS/AOM models.

Figure 4.. Loss of PTPN2 Promotes ASC-Dependent Inflammasome Activation

(A) Colon pieces from WT and PTPN2-LysMCre mice with acute DSS colitis were analyzed by western blot for NLRP3, IL-1β, caspase-1, and IL-18 expression and maturation. (B) BMDMs from WT and PTPN2-LysMCre mice were treated with indicated inflammasome activators and cell culture supernatants analyzed for IL-1β and IL-18 secretion. (C) Serum samples from control (non-IBD) patients (n = 15) or CD patients homozygous either for the PTPN2 WT allele (PTPN2 TT, n = 8) or the CD-associated PTPN2 allele (PTPN2 CC, n = 8) were analyzed for presence of IL-1β and IL-18. (D) Intestinal biopsies from non-inflamed and inflamed regions were taken from the same patients as in (C) and analyzed for IL1b and IL18 mRNA expression. (E) BMDMs from WT, PTPN2-LysMCre, and ASC^–/– mice were left untreated or activated with MSU for 2 hr. ASC was precipitated and analyzed for tyrosine phosphorylation and co-precipitation of PTPN2. Results are representative of at least two different experiments, each with three to five mice per group (A) or three or four biological replicates(B and E). Asterisks denote significant different results (*p < 0.05 and **p < 0.01; Student’s t test with Bonferroni correction). Error bars represent means ± SD. See also Figure S6.

Figure 5.. Inhibition of JNK Prevents Inflammasome Activation in PTPN2-LysMCre Mice

(A) BMDMs from WT, PTPN2-LysMCre, and ASC^–/– mice were left untreated or activated with MSU for 2 hr and lysates analyzed for JNK and Syk phosphorylation and expression of PTPN2, ASC, and NLRP3. Each lane represents an individual mouse. (B and C) BMDMs from WT and PTPN2-LysMCre (KO) mice were treated with Syk, JNK, or p38 inhibitors 1 hr prior to activation with MSU. Analysis of the cell culture supernatant for IL-1β secretion (B) and maturation (C) by ELISA and western blot, respectively. (D) Precipitation of ASC from the cell lysate and analysis for tyrosine phosphorylation and co-precipitated NLRP3 and PTPN2. Asterisks denote significant differences (*p < 0.05 and **p < 0.01; Student’s t test with Bonferroni correction). Error bars represent means ± SD. See also Figure S7.

Figure 6.. Inhibition of IL-1 b Rescues PTPN2-LysMCre Mice from Increased Colitis

WT and PTPN72-LysMCre(KO) mice were immunized with IL-1β-coupled Qβ virus-like particles (IL-1β VLP) or with control Qβ virus-like particles 5,3, and 1 week prior to start of colitis/tumor induction. (A–C) Weight development (A), representative pictures of H&E-stained pieces of the terminal colon (B), and results of histological scoring of intestinal pathology (C) from mice with acute DSS colitis. (D–F) Representative pictures from colonoscopy (D), statistical analysis of histopathology (E), and fibrin score from mice with chronic DSS colitis (F). (G–I) Representative pictures of colonoscopy (G), observed tumor number in dissected colon (H), and average tumor size (I) in each mouse from mice subjected to AOM/DSS treatment. Results are representative of one of two independent experiments with three to five mice per group. Asterisks denote statistical significance (*p < 0.05, **p < 0.01, and *** = p< 0.001; Mann-Whitney U test with Bonferroni correction). Scale bars represent 100 mm. Error bars represent means ± SD. See also Figures S8 and S9.

Figure 7.. Inhibition of IL-1α Protects WT Mice from Tumor Induction

(A) Serum from WT and PTPN92-LysMCre mice with chronic DSS colitis was analyzed for IL-1α levels. Dashed line, detection limit of the ELISA. (B and C) Peritoneal macrophages and intestinal epithelial cells (lECs) from WT and PTPN2-LysMCre mice were analyzed for IL1a surface expression (B) and calpain mRNA levels (C). (D–F) WT and PTPN2-LysMCre mice were immunized with IL-1α-coupled Qβ virus-like particles (IL-1α VLP) or with control Qβ virus-like particles 5,3, and 1 week prior to start of colitis and/or tumor induction. (D) Weight development of mice in acute colitis, (E) representative pictures of H&E-stained slides, and (F) quantification of histology score of terminal colon pieces from WT and PTPN2-LysMCre mice subjected to chronic colitis. (G-I) Representative pictures of colonoscopy (G), observed tumor number in dissected colon (H), and average tumor size (I) in each mouse in the AOM/DSS tumor model. Results are representative of one of two independent experiments with three to five mice per group. Asterisks denote statistical significance (*p < 0.05; MannWhitney U test with Bonferroni correction). Scale bars represent 100 mm. Error bars represent means ± SD.