Endogenous interleukin-10 constrains Th17 cells in patients with inflammatory bowel disease

Abstract

Background: Th17 cells play a role in inflammation. Interleukin (IL)-10 is a potent anti-inflammatory cytokine. However, it is poorly understood whether and how endogenous IL-10 impacts the development of Th17 cells in human pathologies.

Materials and methods: We examined the relationship between IL-10 and Th17 cells in patients with Crohn's disease and in IL-10-deficient (IL-10-/-) mice. Th17 cells and dendritic cells (DCs) were defined by flow cytometry and evaluated by functional studies.

Results: We detected elevated levels of IL-17 and Th17 cells in the intestinal mucosa of patients with Crohn's disease. Intestinal DCs from Crohn's patients produced more IL-1β than controls and were superior to blood DCs in Th17 induction through an IL-1-dependent mechanism. Furthermore, IL-17 levels were negatively associated with those of IL-10 and were positively associated those of IL-1β in intestinal mucosa. These data point toward an in vivo cellular and molecular link among endogenous IL-10, IL-1, and Th17 cells in patients with Crohn's disease. We further investigated this relationship in IL-10(-/-) mice. We observed a systemic increase in Th17 cells in IL-10(-/-) mice when compared to wild-type mice. Similar to the intestinal DCs in patients with Crohn's disease, murine IL-10-/- DCs produced more IL-1β than their wild-type counterparts and promoted Th17 cell development in an IL-1-dependent manner. Finally, in vivo blockade of IL-1 receptor signaling reduced Th17 cell accumulation and inflammation in a mouse model of chemically-induced colitis.

Conclusions: Endogenous IL-10 constrains Th17 cell development through the control of IL-1 production by DCs, and reaffirms the crucial anti-inflammatory role of IL-10 in patients with chronic inflammation.

Figure 1

Th17 cells and IL-17 are increased in CD patient tissue. a) Lamina propria mononuclear cells from control colon tissues or CD colons were cultured for two days with IL-1 and IL-23. Supernatants were subjected to ELISA for analysis of IL-17 protein. b) Serum from healthy or CD patients was subjected to ELISA for IL-17 quantification. n = 16. *P < 0.0001. c) Healthy blood or control colon tissue or CD colon tissue cells were stained with antibodies to CD3, CD4, IL-17, and FoxP3 and antigen expression was analyzed via FACS. Results are expressed as the percent of IL-17⁺ or Foxp3⁺ T cells in the CD4⁺ T cells. The gates were on CD4⁺CD3⁺ T cells for both Th17 and Treg cells. n = 16.

Figure 2

Relationships among IL-17, IL-1, and IL-10 in CD patients. a-c) IL-17, IL-10, and IL-1β message from fresh CD mononuclear lamina propria cells were quantified via real-time PCR. Chi-squared (χ²) test. P < 0.0001 for the following comparisons: IL-17 vs IL-10 (a), IL-1β vs IL-10 (b), and IL-17 vs IL-1β (c). n = 16. d) Crohn's DCs expressed high levels of IL-1β. Crohn's DCs and macrophages, and normal colon DCs were cultured for 40 hours with or without LPS. Normal control DCs were isolated from normal colon tissues at least 5 cm away from colon cancer. IL-1β protein was quantified via ELISA. n = 6. *, P < 0.005. e) Crohn's DCs induced high levels of T cell-derived IL-17 production through IL-1. CD4⁺ T cells were cultured for 48 hours with Crohn's myeloid DCs or macrophages in the presence anti-IL-1R. IL-17 protein was detected via ELISA in supernatants. n = 6. *, P < 0.005.

Figure 3

Crohn's disease T cell-derived IL-17 stimulates the production of inflammatory cytokines. "Normal" colon tissue cells were cultured for 40 hours with medium alone or CD T cell supernatants with or without anti-IL-17R. IL-1β (a), IL-6 (b), and IL-8 (c) were quantified in supernatant. n = 5. *, P < 0.05 and **, P < 0.01.

Figure 4

Murine IL-10^-/- DCs are powerful Th17 inducers. a, b) Increased Th17 cells in IL-10^-/- mice. Mononuclear cells from unchallenged IL-10^+/+ and IL-10^-/- mouse organs were analyzed via FACS. a) Representative FACS plots. b) Results are expressed as the percentage of Th17 cells in CD4⁺ T cell populations ± SEM. 6 mice/group, *P < 0.05. c, d). IL-10^-/- spleen T cells were susceptible to Th17 polarization. IL-10^+/+ and IL-10^-/- spleen T cells were polarized with Th17-polarizing cytokines for 6 days. Th17 cells were analyzed by FACS. c) Results are expressed as the percentage of Th17 cells in CD4⁺ T cells. d) Supernatant from Th17 cultures was collected on day 3 and analyzed via ELISA for levels of IL-17. *P < 0.05, average of three experiments. e) IL-10^-/- DCs were potent Th17 inducers. IL-10^+/+ CD4⁺ T cells were cultured with IL-10^-/- or IL-10^+/+ splenic DCs in a ratio of 5:1 for 5 days. Th17 cells were analyzed by FACS. Results are expressed as the percentage of Th17 cells in CD4⁺ T cells. n = 6, *P < 0.05. Y axis showed SSC and the dot plots were gated on CD4⁺CD3⁺ T cells (a, c, e).

Figure 5

Mouse IL-10^-/- DCs induce Th17 cells through the IL-1/IL-1R signaling pathway. a) Reduced Th17 cells in DC-stimulated IL-1R^-/- CD4⁺ T cell cultures. IL-1R^-/- or IL-1R^+/+ CD4⁺ T cells were stimulated with wild-type DCs for 5 days. The cells were analyzed for Th17. n = 3. b, c)IL-10^-/- DCs expressed high levels of IL-1. IL-10^+/+ or IL-10^-/- DCs were cultured with or without LPS for 8 (b) or 48 hours (c). IL-1α and IL-1β message was quantified via real-time PCR (b). IL-1β protein was detected in culture supernatant via ELISA (c). *P < 0.05, average of 3 experiments. d, e) Blockade of IL-1R reduced DC-mediated Th17 induction. IL-10^-/- or IL-10^+/+ DCs were cultured in a ratio of 1:5 with IL-10^+/+ CD4⁺ T cells with or without anti-IL-1R. The cells were analyzed on day 5 for Th17. Results are expressed as the percentage of Th17 cells in CD4⁺ T cells (d). IL-17 was detected in culture supernatants on day 3 via ELISA. Results are expressed as the mean values ± SEM (e). n = 4. *, P < 0.05. Y axis showed SSC and the dot plots were gated on CD4⁺CD3⁺ T cells (a, d).

Figure 6

In vivo IL-1R blockade reduces Th17 cells and inflammation. a) In vivo IL-1R blockade decreased homeostatic Th17 cells. Mice were treated with Anakinra or PBS for 7 days. Single-cell suspensions from different organs were analyzed for Th17 cells. Results are expressed as the percentage of Th17 cells in CD4⁺ T cells ± SEM. *P < 0.05, 5 mice/group. b) In vivo IL-1R blockade reduced DSS-induced Th17 cells. Mice were treated with DSS to induce inflammation and given Anakinra throughout the second cycle of treatment. Single-cell suspensions from different organs were analyzed for Th17 cells. Results are shown as the percentage of Th17 cells in mensenteric lymph node CD4⁺ T cells in mice treated with Anakinra or PBS. Y axis showed SSC and the dot plots were gated on CD4⁺CD3⁺ T cells. n = 4. c-e) In vivo IL-1R blockade prevented polyp formation and ameliorated inflammation induced by DSS. Colon polyps were counted in DSS-challenged IL-10^-/- mice treated with PBS or Anakinra (IL-1Ra). n = 8 mice per group, *P < 0.05 (c). Bloody and polyp-carrying colon sections from untreated mice (top) are shown, but were not observed in IL-1Ra-treated mice (bottom) (d). H&E staining showed heavy cellular infiltration in colon tissue from untreated mice (top), but not in colon tissue from IL-1Ra-treated mice (bottom) (e).