Experimental colitis in IL-10-deficient mice ameliorates in the absence of PTPN22

Abstract

Interleukin (IL)-10 plays a key role in controlling intestinal inflammation. IL-10-deficient mice and patients with mutations in IL-10 or its receptor, IL-10R, show increased susceptibility to inflammatory bowel diseases (IBD). Protein tyrosine phosphatase, non-receptor type 22 (PTPN22) controls immune cell activation and the equilibrium between regulatory and effector T cells, playing an important role in controlling immune homoeostasis of the gut. Here, we examined the role of PTPN22 in intestinal inflammation of IL-10-deficient (IL-10^-/- ) mice. We crossed IL-10^-/- mice with PTPN22^-/- mice to generate PTPN22^-/- IL-10^-/- double knock-out mice and induced colitis with dextran sodium sulphate (DSS). In line with previous reports, DSS-induced acute and chronic colitis was exacerbated in IL-10^-/- mice compared to wild-type (WT) controls. However, PTPN22^-/- IL-10^-/- double knock-out mice developed milder disease compared to IL-10^-/- mice. IL-17-promoting innate cytokines and T helper type 17 (Th17) cells were markedly increased in PTPN22^-/- IL-10^-/- mice, but did not provide a protctive function. CXCL1/KC was also increased in PTPN22^-/- IL-10^-/- mice, but therapeutic injection of CXCL1/KC in IL-10^-/- mice did not ameliorate colitis. These results show that PTPN22 promotes intestinal inflammation in IL-10-deficient mice, suggesting that therapeutic targeting of PTPN22 might be beneficial in patients with IBD and mutations in IL-10 and IL-10R.

Keywords: IL-10; PTPN22; colitis; inflammatory bowel diseases (IBD).

Figure 1

Protein tyrosine phosphatase, non‐receptor type 22 (PTPN22) promotes acute dextran sodium sulphate (DSS)‐induced experimental colitis in IL‐10‐deficient mice. (a) Weight was measured in DSS‐treated mice daily [wild‐type (WT), n = 15; PTPN22^–/–, n = 15; IL‐10^–/–, n = 17 and PTPN22^–/–IL‐10^–/–, n = 13]. Graph shows daily weight change (in percentage) compared to the initial weight. (b) Percentage of weight change from initial weight of DSS‐treated mice of (a) analysed at day 8. (c) Colon length among DSS‐treated mice at day 8 expressed in cm. (d,e) Colitis score and representative haematoxylin and eosin (H&E) colon sections from DSS‐treated mice of the indicated genotypes (n = 3/group). Scale bars: 200 μm, high‐power fields. (f) Spleen weight in grams analysed in DSS‐treated mice at day 8. Horizontal lines in (b,c,f) show the mean. Each symbol represents an individual mouse. Data are pooled from three experiments with similar results. Statistical significance was determined by Kruskal–Wallis analysis followed by Dunn’s post‐test [one‐way analysis of variance (anova) test] (*P < 0·05, **P < 0·01 and ***P < 0·001).

Figure 2

Protein tyrosine phosphatase, non‐receptor type 22 (PTPN22) promotes chronic dextran sodium sulphate (DSS)‐induced experimental colitis. (a) Weight was measured in DSS‐treated mice [wild‐type (WT), n = 7; PTPN22^–/–, n = 8; IL‐10^–/–, n = 7 and PTPN22^–/–IL‐10^–/–, n = 7). Graph shows weight change (in percentage) compared to the initial weight. The mice were followed until day 31. (b) Survival curve of mice exposed to DSS from days 1 to 31. The same mice as in (a) are included. Statistical analysis of weight change is summarized in Table 3.

Figure 3

Protein tyrosine phosphatase, non‐receptor type 22 (PTPN22) controls the number of forkhead box protein 3 (FoxP3)⁺ regulatory T cells (T_reg) and their expression of lymphocyte function‐associated antigen 1 (LFA‐1) in colitic IL‐10‐deficient mice. (a) Representative fluorescence activated cell sorter (FACS) plots show frequency of FoxP3⁺ and CD25⁺FoxP3⁺ T_reg cells in the mesenteric lymph nodes (MLN) of wild‐type (WT), PTPN22^–/–, IL‐10^–/– and PTPN22^–/–IL‐10^–/– mice treated with dextran sodium sulphate (DSS) for 8 days. One representative mouse per group is shown. (b,c) Graphs display T_reg frequencies in MLN (b) and large intestine (LI) (c) from multiple mice (n = 5–6/group). (d) Histogram overlay depicting LFA‐1 expression levels (% of max.) on FoxP3⁺ T_reg cells from MLNs. (e,f) Mean fluorescence intensity (gMFI) of LFA‐1 on FoxP3⁺ T_reg cells from MLN (e) and LI (f). Horizontal lines in (b,c) and (e,f) show mean. Each symbol represents an individual mouse. Data are pooled from two experiments with similar results. Statistical significance was determined by Kruskal–Wallis analysis followed by Dunn’s post‐test [one‐way analysis of variance (anova) test] (*P < 0·05, **P < 0·01 and ***P < 0·001).

Figure 4

Protein tyrosine phosphatase, non‐receptor type 22 (PTPN22) restricts interleukin (IL)‐17 production by CD4 T cells in IL‐10‐deficient mice treated with dextran sodium sulphate (DSS). Lamina propria mononuclear cells from WT, PTPN22^–/–, IL‐10^–/– and PTPN22^–/–IL‐10^–/– mice treated with DSS for 8 days were processed, fixed, permeabilized and stained to assess interferon (IFN)‐γ and IL‐17 by CD4⁺ T cells. (a) Representative fluorescence activated cell sorter (FACS) plots display frequency of IFN‐γ^–IL‐17⁺, IFN‐γ⁺IL‐17⁺ and IFN‐γ⁺IL‐17^– CD4 T cells from the mesenteric lymph nodes (MLN). A representative mouse from each strain is shown. (b,c) Graphs display frequency in MLN (b) and large intestine (LI) (c) from multiple mice (n = 3–5/group). Horizontal lines show mean. Each symbol represents an individual mouse. Data are pooled from two experiments with similar results. Statistical significance was determined by Kruskal–Wallis analysis followed by Dunn’s post‐test [one‐way analysis of variance (anova) test] (*P < 0·05, **P < 0·01 and ***P < 0·001).

Figure 5

Protein tyrosine phosphatase, non‐receptor type 22 (PTPN22) restricts interleukin (IL)‐17 production by CD8 T cells in IL‐10‐deficient mice treated with dextran sodium sulphate (DSS). Wild‐type (WT), PTPN22^–/–, IL‐10^–/– and PTPN22^–/–IL‐10^–/– mice were treated with DSS for 8 days and their mesenteric lymph node (MLN) and large intestine (LI) were processed, fixed, permeabilized and stained to assess interferon (IFN)‐γ and IL‐17 by CD8⁺ T cells. (a) Representative fluorescence activated cell sorter (FACS) plots display frequency of IFN‐γ^–IL‐17⁺, IFN‐γ⁺IL‐17⁺ and IFN‐γ⁺IL‐17^– CD8 T cells from the LI. A representative mouse from each strain is shown. (b,c) Graphs display frequency in MLN (b) and LI (c) from multiple mice (n = 5/group). Horizontal lines show mean. Each symbol represents an individual mouse. Data are pooled from two experiments with similar results. Statistical significance was determined by Kruskal–Wallis analysis followed by Dunn’s post‐test [one‐way analysis of variance (anova) test] (*P < 0·05, **P < 0·01 and ***P < 0·001).

Figure 6

Innate serum cytokine and chemokine analysis with Bio‐Plex. Serum was collected on day 3 after dextran sodium sulphate (DSS) treatment. (a) Plots show levels of between interleukin (IL)‐1b, IL‐6, IL‐12 (p40), IL‐2, macrophage inflammatory protein (MIP)‐1α, MIP‐1β, chemokine (C‐X‐C motif) ligand 1 (CXCL1) (KC), monocyte chemotactic protein (MCP)‐1 and regulated upon activation, normal T cell expressed, and secreted (RANTES) in WT (n = 5), protein tyrosine phosphatase, non‐receptor type 22 (PTPN22^–/–) (n = 5), IL‐10^–/– (n = 7) and PTPN22^–/–IL‐10^–/– (n = 7) mice. Statistical significance was determined by Kruskal–Wallis analysis followed by Dunn’s post‐test [one‐way analysis of variance (anova) test] (*P < 0·05, **P < 0·01 and ***P < 0·001). Each symbol represents an individual mouse.

Figure 7

Interleukin (IL)‐17 blockade in protein tyrosine phosphatase, non‐receptor type 22 (PTPN22^–/–)IL‐10^–/– mice does not increase susceptibility to dextran sodium sulphate (DSS)‐induced acute colitis. (a) Weight was measured in DSS‐treated mice daily (IL‐10^–/–, n = 6, PTPN22^–/–IL‐10^–/– n = 6 and PTPN22^–/–IL‐10^–/– + anti‐IL‐17, n = 4). Graph shows daily weight change (in percentage) compared to the initial weight. (b) Colon length among DSS‐treated mice at day 8 expressed in cm. (c) Weight was measured in DSS‐treated mice daily (IL‐10^–/–, n = 18, IL‐10^–/– +CXCL1/KC, n = 5). Graph shows daily weight change (in percentage) between IL‐10^–/– and IL‐10^–/– mice, treated with CXCL1/KC, compared to the initial weight.