Phosphatase Shp2 exacerbates intestinal inflammation by disrupting macrophage responsiveness to interleukin-10

Abstract

Inflammatory cytokines produced by activated macrophages largely contribute to the pathological signs of inflammatory bowel disease (IBD). Interleukin-10 (IL-10) is the predominant anti-inflammatory cytokine in the intestine, and its therapeutic efficacy for IBD has been clinically tested. Nevertheless, how the function of IL-10 is regulated in the intestinal microenvironment remains unknown, which largely hinders the further development of IL-10-based therapeutic strategies. Here, we found that the expression of phosphatase Shp2 was increased in colonic macrophages and blood monocytes from IBD patients compared with those from healthy controls. Shp2 deficiency in macrophages protects mice from colitis and colitis-driven colon cancer. Mechanistically, Shp2 disrupts IL-10-STAT3 signaling and its dependent anti-inflammatory response in human and mouse macrophages. Furthermore, a Shp2-inducing role of TNF-α is unveiled in our study. Collectively, our work identifies Shp2 as a detrimental factor for intestinal immune homeostasis and hopefully will be helpful in the future exploitation of IL-10 immunotherapy for IBD.

Figure 1.

Loss of Shp2 in macrophages decreases the susceptibility of mouse colitis. Mice were challenged with 2.5% DSS in drinking water for 7 d. (A) Body weight loss, diarrhea, and rectal bleeding were monitored daily. Data are accumulated from three independent experiments with n = 4–9 mice/group in each experiment. (B) Colon length was measured at day 7. (C) Colon sections were analyzed for histological damage. Bars, 100 µm. (D) Periodic acid–Schiff staining of colon sections. Bars, 100 µm. (E) The expression of bacterial 16S rRNA in spleens was determined by qPCR. (F) Mice were challenged with 4% DSS for 7 d, and the survival of mice was monitored. n = 7–13. Data are mean ± SEM and are representative of three independent experiments. *, P < 0.05; **, P < 0.01; two-tailed unpaired Student’s t test (A–C and E) and log-rank test (F).

Figure 2.

Loss of Shp2 in macrophages attenuates innate intestinal inflammation. Mice were challenged with 2.5% DSS and were sacrificed at day 7. (A and B) Single-cell suspensions of CLP were analyzed for the frequencies of different cell populations by flow cytometry (gated on CD45⁺ cells). (C and D) The levels of TNF-α, IL-6, and IL-1β in serum (C) and culture supernatant from colon tissues (D) were determined by ELISA. n = 5 for DSS cohorts and n = 3 for H₂O cohorts. (E) Colon tissues were pooled from three mice, CD11b⁺ cells were purified by magnetic-activated cell sorting, and the levels of TNF-α, IL-6, and IL-1β were evaluated by qPCR. (F) The levels of IFN-γ and IL-17A in culture supernatant from colon tissues were determined by ELISA. (G) The mRNA levels of T cell–related cytokines and transcriptional factors in mLN and spleen were determined by qPCR. n = 4–7. u.d., undetectable. Data are mean ± SEM and are representative of three independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001; two-tailed unpaired Student’s t test.

Figure 3.

Shp2 deficiency in macrophages inhibits colitis-associated colon cancer. (A) Experimental design of AOM/DSS-induced colon cancer. (B) Colonic tumors were photographed (left) and counted (right). n = 9–11. Data are mean ± SEM and are compiled from two independent experiments. (C) Representative H&E staining of colon tissues. Bar, 100 µm. (D) The frequencies of different cell populations in CLP were analyzed by flow cytometry. Representative plots showed the myeloid populations. (E) The expression of indicated cytokines in CLP was determined by qPCR. Data are mean ± SEM and are representative of two independent experiments. *, P < 0.05; **, P < 0.01; two-tailed unpaired Student’s t test.

Figure 4.

Macrophage production of inflammatory mediators is not obviously affected by Shp2 deficiency. (A–C) Peritoneal macrophages (PMs) from Shp2^M WT and Shp2^M KO mice were stimulated with LPS (100 ng/ml; A), PGN (2 µg/ml; B), or heat-killed E. coli (multiplicity of infection = 0.1; C), the expression of TNF-α, IL-6, and IL-1β was determined by qPCR. (D–F) PMs were stimulated with LPS (100 ng/ml), surface expression of CD80, CD86, and MHC-II (D), or intracellular production of ROS (E) or iNOS (F) were detected by flow cytometry. Data are mean ± SEM and are representative of three independent experiments. ***, P < 0.001; two-tailed unpaired Student’s t test.

Figure 5.

Loss of Shp2 enhances the inhibitory function of IL-10 in macrophages. (A) IL-10 concentration was measured in culture supernatant from colon tissues. (B) Shp2^M WT and Shp2^M KO PMs were stimulated with LPS (100 ng/ml) in the absence or presence of IL-10 (10 ng/ml) or anti–IL-10 (αIL-10) for indicated time, the levels of TNF-α and IL-6 in culture supernatant were determined by ELISA. (C) PMs were stimulated with LPS (100 ng/ml) in the absence or presence of indicated concentrations of IL-10 for 12 h; the level of TNF-α in culture supernatant was determined by ELISA. Results were presented as relative cytokine values (value_{LPS + IL-10}/value_{LPS alone}). (D) PMs were stimulated with PGN or E. coli in the absence or presence of IL-10 for 12 h, relative TNF-α, and IL-6 levels were determined by ELISA. (E) PMs were stimulated with LPS in the absence or presence of IL-10 for 3 h; relative TNF-α mRNA level was determined by qPCR. (F) PMs were stimulated with IL-10 (10 ng/ml) for indicated time, the phosphorylation of STAT3, ERK, and p38 was determined by Western blot. Data are mean ± SEM and are representative of at least three independent experiments. (G) CLPMs isolated from IL-10^−/−Shp2^M WT and IL-10^−/−Shp2^M KO mice were stimulated with LPS in the absence or presence of IL-10 (5 ng/ml) for 12 h; the levels of TNF-α and IL-6 in culture supernatant were determined by ELISA. (H) CLPMs were stimulated with IL-10 (5 ng/ml) for 30 min; the phosphorylation of STAT3 was determined by Western blot. (I) CLPMs were stimulated with IL-10 (5 ng/ml) for 2 h; the level of SOCS3 was determined by qPCR. Data are mean ± SEM and are representative of two independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001; two-tailed unpaired Student’s t test.

Figure 6.

The colitis-protective effect in Shp2^M KO mice is abrogated in the absence of IL-10 function. (A–C) DKO and control mice were fed with 1.5% DSS. (A) Body weight loss, diarrhea, and rectal bleeding were monitored daily. (B) Colon length was measured at day 7. (C) The levels of inflammatory cytokines were measured in colon homogenates by ELISA. n = 5–6. (D–G) Mice were injected intraperitoneally with 20, 40, and 40 µg anti–IL-10 or IgG isotype at days 1, 3, and 5 after DSS challenge, respectively. (D) Body weight loss, diarrhea, and rectal bleeding were monitored daily. (E and F) Colon length and histological changes were measured at day 7. (G) The levels of TNF-α, IL-6, and MCP-1 in culture supernatant from colon tissues were measured by ELISA. n = 5–8. (H) CLPMs were treated with culture supernatant from coCM in the presence of anti–IL-10 or IgG isotype; the phosphorylation of STAT3 was evaluated by Western blot. Data are mean ± SEM and are representative of two independent experiments. *, &, and #, P < 0.05; **, P < 0.01; two-tailed unpaired Student’s t test. n.s., not significant.

Figure 7.

The clinical relevance of macrophage-expressed Shp2 in IBD patients. (A) The expression of Shp2 in CD68⁺ colonic macrophages were examined in mucosal biopsy specimens by immunofluorescence staining. Bars, 20 µm. Data are representative of three independent experiments. (B) The percentage of CD68⁺Shp2^hi cells was quantified. Each group contained three individuals. Data are mean ± SEM and are compiled from three independent experiments. (C) The expression level of Shp2 in peripheral monocytes was evaluated by qPCR. (D) Association between mRNA levels of Shp2 and TNF-α or IL-6 was analyzed by Spearman’s rank correlation test. Data are mean ± SEM and are from one independent experiment. (E–H) THP-1 monocytes (E and F) or THP-1–differentiated macrophages (G and H) were transduced with Shp2-knockdown lentivirus (Lv-Shp2) or control lentivirus (Lv-scr), followed by stimulation with IL-10 for 30 min. The phosphorylation of STAT3 was determined by Western blot (E and G). IL-10 inhibition of TNF-α was measured by ELISA (F and H). (I) GST-tagged STAT3 and myc-tagged Shp2 full length or truncations were overexpressed in HEK293T cells. The interaction between STAT3 and various Shp2 domains was detected by co-IP. (J and K) THP-1–differentiated macrophages were stimulated with TNF-α (20 ng/ml); the level of Shp2 was evaluated by Western blot (J) or flow cytometry (K). Data are mean ± SEM and are representative of at least two independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001; two-tailed unpaired Student’s t test (B, C, F, H, and K) and Spearman’s rank correlation test (D).

Figure 8.

Working model. When colitis occurs, colonic macrophages activated by invading microbes produce inflammatory cytokines, but Shp2 does not play a prominent role during this process. On the other hand, loss of Shp2 potentiates IL-10–STAT3 signaling and its dependent deactivating programs in macrophages, thus decreasing their production of inflammatory cytokines and reducing the severity of colitis. Furthermore, TNF-α has the ability to up-regulate Shp2 expression in macrophages, suggesting that IL-10 supplementation may achieve higher anti-inflammatory efficacy when used in combination with anti–TNF-α antibody.