Protein tyrosine phosphatase non-receptor type 22 modulates colitis in a microbiota-dependent manner

Abstract

The gut microbiota is crucial for our health, and well-balanced interactions between the host's immune system and the microbiota are essential to prevent chronic intestinal inflammation, as observed in inflammatory bowel diseases (IBD). A variant in protein tyrosine phosphatase non-receptor type 22 (PTPN22) is associated with reduced risk of developing IBD, but promotes the onset of autoimmune disorders. While the role of PTPN22 in modulating molecular pathways involved in IBD pathogenesis is well studied, its impact on shaping the intestinal microbiota has not been addressed in depth. Here, we demonstrate that mice carrying the PTPN22 variant (619W mice) were protected from acute dextran sulfate sodium (DSS) colitis, but suffered from pronounced inflammation upon chronic DSS treatment. The basal microbiota composition was distinct between genotypes, and DSS-induced dysbiosis was milder in 619W mice than in WT littermates. Transfer of microbiota from 619W mice after the first DSS cycle into treatment-naive 619W mice promoted colitis, indicating that changes in microbial composition enhanced chronic colitis in those animals. This indicates that presence of the PTPN22 variant affects intestinal inflammation by modulating the host's response to the intestinal microbiota.

Keywords: Gastroenterology; Inflammatory bowel disease.

Figure 1. Presence of the 619W variant promotes chronic colitis.

Chronic colitis was induced in PTPN22^–/– and 619W mice as well as in their respective WT littermates via administration of 4 cycles of DSS (1.5% DSS in the drinking water for 7 days, followed by 10 days recovery, each cycle). (A–D) Weight development (A), representative pictures from colonoscopy (original magnification, ×10) and MEICS (B), quantification of colitis severity (C), and representative pictures from H&E-stained colon sections (original magnification, ×20) at day 85 (D) reveal that 619W mice show enhanced colitis from the second DSS cycle onward. Depicted are mean values and SEM (A–C). Data are representative of 1 of 2 independent experiments with n = 3–6 per experimental group, each experiment. (B–D) Each dot represents 1 mouse. Graph in A shows weight development from the same individuals as in B–D. *P < 0.05, **P < 0.01, Kruskal-Wallis test.

Figure 2. Presence of the 619W variant in PTPN22 affects inflammasome activation and IL-18 secretion.

Colitis was induced in WT, 619W, and PTPN22^–/– mice by (A) administration of DSS for 7 days; and (B) administration of 4 DSS cycles. Colon pieces were analyzed by Western blot and ELISA for IL-1β and IL-18, respectively. Each lane in the Western blot represents 1 individual mouse, and each dot in the graph represents 1 individual. Data are representative of 1 of 2 independent experiments with n = 3–6 per experimental group. *P < 0.05, **P < 0.01, ***P < 0.001, Kruskal-Wallis test. See complete unedited blots in the supplemental material.

Figure 3. Cohousing transfers the phenotype from 619W and PTPN22^–/– mice to WT littermates.

(A–C) Chronic colitis was induced in WT mice that were cohoused since birth with 619W or PTPN22^–/– mice as indicated (A); PTPN22^–/– mice that were cohoused since birth and throughout the experiment with WT mice, or mice that were housed only with PTPN22^–/– mice after weaning and throughout the experiment (B); or 619W mice that were cohoused since birth and throughout the experiment with WT mice, or mice that were housed only with 619W mice after weaning and throughout the experiment (C). (D) Representative pictures (original magnification, ×10) and scoring of histologic colitis severity in H&E-stained sections of the terminal colon from mice in A–C. Data are representative of 1 of 2 independent experiments. Weight curves show mean ± SEM for each group; n = 3 for H₂O group and n = 5 for all other groups. The other graphs show values and SEM, and each dot represents 1 individual mouse. –/–, PTPN22^–/–. *P < 0.05, **P < 0.01, Kruskal-Wallis test.

Figure 4. 619W and PTPN22^–/– genotypes are associated with a distinct gut microbiota.

Relative to matched WT controls, both 619W and PTPN22^–/– mice showed baseline differences in microbiota taxa richness (A) and overall community composition (B; principal coordinates analysis [PCoA] of Bray-Curtis dissimilarities) that were conferred to some extent to cohoused WT littermates, but not vice versa. See Supplemental Table 1 for full data and individual statistical test results.

Figure 5. Associations of individual taxa with 619W and PTPN22^–/– genotypes.

Operational taxonomic units (OTUs) from various taxonomic groups showed distinct univariate trends of enrichment or depletion relative to WT controls, concordant or discordant between PTPN22^–/– and 619W genotypes. See Supplemental Table 1 for full data and individual statistical test results.

Figure 6. Genotype-specific effects of DSS treatment on the gut microbiota.

(A) Distinct compositional shifts in each genotype relative to H₂O-treated controls were observed upon 1 cycle of DSS treatment (acute colitis), following a 10-day recovery phase, and upon 4 DSS cycles (chronic colitis). (B) These shifts were conferred to WT mice cohoused with PTPN22^–/– or 619W mice, but not vice versa. Note that mice in A were not cohoused, whereas in B, WT mice were cohoused with PTPN22^–/– or 619W, and vice versa. (C) Compositional shifts could be differentially attributed to the factors genotype, treatment, and time point, as indicated by R² values in PERMANOVA tests on data subsets. Note that for illustration purposes, only 1 of several replicate experiments is shown in A; the total number of mice per group used in statistical tests is reported on the right and in Supplemental Table 2.

Figure 7. Univariate associations of individual taxa with genotype and DSS treatment.

Average relative abundances (left) and relative fold changes (right) of selected taxa associated with genotype and DSS treatment response at various time points. See Supplemental Table 2 for full data on individual associations.

Figure 8. Phylogenetic representation of univariate genotype-specific taxa associations with DSS treatment.

Responders to DSS treatment (enrichment or depletion) across genotypes and time points were phylogenetically distributed, although some clades showed specific patterns consistent (or discordant) among genotypes (see main text and Figure 7). See Supplemental Table 2 for full data on individual associations.

Figure 9. Transfer of stool after the first DSS cycle recapitulates the phenotype in chronic colitis.

Cecum content was collected from WT, PTPN22^–/–, and 619W mice that were treated with DSS for 7 days and allowed to recover for 10 additional days (= day 18 DSS). Cecum content was then transferred into WT, PTPN22^–/–, or 619W mice that had been treated with an antibiotic cocktail for 1 week to deplete the microbiota. (A) Schematic overview of the experimental setup. (B–D) Weight development, representative pictures (original magnification, ×10) from H&E-stained terminal colon sections, and scoring of histological damage of 619W mice that received cecum content of treatment-naive 619W mice, WT mice upon colitis induction, or 619W mice after colitis induction (B); PTPN22^–/– mice that received cecum content of treatment-naive PTPN22^–/– mice, WT mice upon colitis induction, or PTPN22^–/– mice after colitis induction (C); and WT mice that received cecum content of treatment-naive WT mice or WT, 619W, or PTPN22^–/– mice after colitis induction (D). Depicted are mean values and SEM; n = 5 for each experimental group. Data are representative of 1 of 2 independent experiments. Abx, antibiotic treatment. *P < 0.05, **P < 0.01, Kruskal-Wallis test.

Figure 10. PTPN22 affects expression of antimicrobial peptides in an IL-18–dependent manner.

(A) Reg3g mRNA expression in WT, PTPN22^–/–, and 619W mice after the first DSS cycle (day 8), after recovery (day 18), or at the end of chronic colitis induction (day 85). (B) MC-38 cells were grown on inserts and cocultured with MDP-activated bone marrow–derived macrophages (BMDMs) or anti-CD3/CD28–activated T cells from WT, PTPN22^–/–, or 619W mice, and mRNA levels of Reg3g were analyzed by quantitative PCR. (C and D) MC-38 cells were cocultured with MDP-activated BMDMs from WT, PTPN22^–/–, or 619W mice in the presence of an isotype control or an anti–IL-18 antibody and analyzed for mRNA expression of Reg3g (C) and Defa5 (D). Data are representative of 1 of 2 independent experiments. Depicted are mean values and SEM, and each dot represents 1 individual mouse or experimental sample. *P < 0.05, **P < 0.01, ***P < 0.001, Kruskal-Wallis test.