IL-1β mediates chronic intestinal inflammation by promoting the accumulation of IL-17A secreting innate lymphoid cells and CD4(+) Th17 cells

Abstract

Although very high levels of interleukin (IL)-1β are present in the intestines of patients suffering from inflammatory bowel diseases (IBD), little is known about the contribution of IL-1β to intestinal pathology. Here, we used two complementary models of chronic intestinal inflammation to address the role of IL-1β in driving innate and adaptive pathology in the intestine. We show that IL-1β promotes innate immune pathology in Helicobacter hepaticus-triggered intestinal inflammation by augmenting the recruitment of granulocytes and the accumulation and activation of innate lymphoid cells (ILCs). Using a T cell transfer colitis model, we demonstrate a key role for T cell-specific IL-1 receptor (IL-1R) signals in the accumulation and survival of pathogenic CD4(+) T cells in the colon. Furthermore, we show that IL-1β promotes Th17 responses from CD4(+) T cells and ILCs in the intestine, and we describe synergistic interactions between IL-1β and IL-23 signals that sustain innate and adaptive inflammatory responses in the gut. These data identify multiple mechanisms through which IL-1β promotes intestinal pathology and suggest that targeting IL-1β may represent a useful therapeutic approach in IBD.

Figure 1.

H. hepaticus–induced innate immune driven typhlocolitis is associated with IL-1β secretion. 129SvEv Rag2^−/− mice were infected with H. hepaticus and sacrificed >8 wk after infection. (A) IL-1β secretion from organ explants from colon, cecum, and ileum incubated overnight in complete medium. Results are shown as mean ± SEM (n = 3 for uninfected control and n = 14 for H. hepaticus–infected mice, pooled from 2 independent experiments). (B) IL-1β levels in the supernatants of cLPLs cultured overnight in complete media. Results are shown as mean ± SEM (n = 7 for uninfected controls and n = 22 for H. hepaticus-infected mice, pooled from 3 separate experiments). **, P < 0.01.

Figure 2.

IL-1β drives innate immune colitis. 129SvEv Rag2^−/− mice were infected with H. hepaticus, treated weekly with 1 mg of αIL-1β antibody or isotype control (i.p.), and sacrificed after 8 wk of infection. (A) Scores of colon inflammation. Each symbol represents an individual mouse, and data are pooled from four independent experiments. Bars represent the mean histological score for each group. (B) Spleen weights and splenocyte numbers. (C) Representative photomicrographs of mid-colon and liver sections. Bars, 200 µm. (D) Colonic lamina propria leukocytes were isolated from the described mice groups and cytokine secretion after overnight culture in complete media was determined. Data are shown as mean ± SEM from 3 pooled independent experiments (n = 9–16). (E) Cytokine concentration in blood serum (mean ± SEM, n = 6–10 from 2 pooled independent experiments). ND, not detectable; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

Figure 3.

Blocking IL-1β impairs the accumulation of proinflammatory CD11b⁺Gr1^Hi granulocytes in the inflamed colon. 129SvEv Rag2^−/− mice were infected with H. hepaticus, treated weekly with 1 mg of αIL-1β antibody or isotype control (i.p.), and sacrificed after 8 wk of infection. (A) Leukocytes were isolated from the colon and spleen, and the frequency of CD45⁺CD11b⁺Gr1^Hi cells was assessed by flow cytometry (mean ± SEM from 3 pooled independent experiments, n = 9–16). (B) Representative FACS plots from the data presented in A (gated on CD45⁺ cells). (C) Colon chemokine expression as evaluated by qRT-PCR on total colon homogenates (mean ± SEM, n = 6–12 from 2 pooled independent experiments). *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Figure 4.

αIL-1β treatment reduces the accumulation of IL-17A–producing ILCs in the colon. (A) IL-1R1 expression by Sca1⁺Thy1.2^Hi ILCs and rest of large intestine lamina propria cells. 129SvEv Rag2^−/− mice were infected with H. hepaticus and sacrificed after >8 wk of infection. LPLs were isolated from the large intestine, Sca1⁺Thy1.2^Hi ILCs were FACS sorted, and Il1r1 expression was evaluated by qRT-PCR (mean ± SEM, n = 2 from 2 independent experiments; 8–10 mice were pooled in each experiment). (B–E) 129SvEv Rag2^−/− mice were infected with H. hepaticus and treated weekly with 1 mg of αIL-1β antibody or isotype control (i.p.). After 8 wk, mice were sacrificed and cLPLs were isolated. (B) Total numbers of Sca1⁺ Thy1.2^Hi ILCs in the colon lamina propria as evaluated by FACS analysis. (C) Total numbers of IL-17A– or IFN-γ–producing ILCs from the colon of indicated mice groups. (D) Cytokine production by cLPLs after overnight culture in complete medium alone (n/a) or in the presence of 10 ng/ml IL-23. Data are represented as mean ± SEM from 2 pooled independent experiments (n = 6–11). (E) Il23r expression by cLPLs as evaluated by qRT-PCR. Data are normalized on Hprt expression. (F and G) 129SvEv Rag2^−/− mice were infected with H. hepaticus for 8 wk and CD45⁺lin⁻Sca1⁺Thy1.2^Hi ILCs were FACS sorted from the colon. (F) ILCs were cultured overnight in complete medium alone (n/a) or in the presence of 10 ng/ml IL-1β. Il23r and Rorc expression levels were evaluated by qRT-PCR and normalized on Hprt expression. Data are shown as mean ± SEM from 2 pooled independent experiments. In each experiment, 10–18 mice were pooled. (G) ILCs were cultured overnight in complete medium alone (n/a) or in the presence of 10 ng/ml IL-23. Il1r1 expression was evaluated by qRT-PCR and normalized on Hprt expression. Data are shown as mean ± SEM from two pooled independent experiments. In each experiment, 10–18 mice were pooled. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

Figure 5.

T cell–driven intestinal inflammation is attenuated in the absence of IL-1R signaling. (A) C57BL/6 Rag1^−/− mice were transferred with 4 × 10⁵ CD4⁺ CD25⁻ CD45RB^Hi T cells from C57BL/6 WT mice and culled at the development of clinical signs of disease. cLPLs were isolated from the colon and cultured overnight in complete medium. IL-1β secretion in the supernatants was evaluated by FlowCytoMix kit (eBioscience; mean ± SEM; n = 5 for C57BL/6 Rag1^−/− mice and n = 10 for recipients of CD4⁺CD45Rb^Hi T cells from 2 independent experiments). (B–G) C57BL/6 Rag1^−/− mice were transferred with 4 × 10⁵ CD4⁺ CD25⁻ CD45RB^Hi T cells from C57BL/6 WT or Il1r1^−/− mice. (B) Representative weight loss curve, shown as percentage of initial weight (data are shown as mean ± SEM; n = 8 mice/group, 1 representative of 3 independent experiments). (C and D) Intestinal inflammation scores for the colon (C) and the cecum (D) from three pooled independent experiments. Bars show the mean score. (E) Representative photomicrographs of colon and cecum (bar, 200 µm). (F) Spleen weight and spleen cell number. 1 representative experiment out of 3 is shown (n = 3 for untreated controls and n = 8 for naive CD4⁺ T cell recipients). (G) Cytokine secretion by colonic lamina propria lymphocytes from C57BL/6 Rag1^−/− mice transferred with WT or Il1r1^−/− naive CD4⁺ T cells. Data are shown as mean ± SEM (n = 10–14 from 2 pooled independent experiments). *, P < 0.05; **, P < 0.01; ***, P < 0.001; ND not detectable.

Figure 6.

Impaired Th17 accumulation in the gut in the absence of IL-1R1 signaling. (A) C57BL/6 Rag1^−/− mice were transferred with 4 × 10⁵ CD4⁺ CD25⁻ CD45RB^Hi T cells from C57BL/6 WT or Il1r1^−/− mice. At sacrifice, CD4⁺ T cells in the colon, MLN and spleen were evaluated by FACS analysis. Data are shown as mean ± SEM (n = 20–21 from 3 pooled independent experiments). (B) Total CD4⁺ T cells were isolated from the spleen of WT or Il1r1^−/− mice and cultured in the presence of 2.5 ng/ml TGF-β and 50 ng/ml IL-6, plus plate-bound αCD3 (2 µg/ml) and αCD28 (1 µg/ml). After 3 d, the frequency of IL-17A–producing cells was analyzed by intracellular staining. Data are shown as mean ± SD from 2 pooled independent experiments (n = 4), together with representative FACS plots (gated on CD4⁺TCRβ⁺ cells). (C–E) C57BL/6 Rag1^−/− mice were transferred with 4 × 10⁵ CD4⁺ CD25⁻ CD45RB^Hi T cells from C57BL/6 WT or Il1r1^−/− mice. (C and D) At sacrifice, colonic lamina propria leukocytes were isolated and analyzed by intracellular FACS analysis. (C) Frequency of indicated T helper subsets among CD4⁺ T cells. In the representative FACS plot shown on the right, data are gated on CD4⁺TCRβ⁺ cells. (D) Accumulation of effector CD4⁺ T cell populations in the colon. Data are shown as mean ± SEM (n = 20–21 from 3 pooled independent experiments). (E) CD4⁺ T cells were FACS sorted from the colon of recipients of Il1r1^−/− or WT naive CD4⁺ T cells and restimulated overnight with PMA and ionomycin. Cytokine secretion in the supernatants was assessed by FlowCytomix (n = 12–14 from 3 pooled independent experiments). (F) C57BL/6 Rag1^−/− mice were transferred with 4 × 10⁵ CD4⁺ CD25⁻ CD45RB^Hi T cells from C57BL/6 WT or csf2^−/− mice and culled at development of clinical signs of disease. Colonic inflammation score was assessed histologically (n = 7–10). *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Figure 7.

IL-1R signaling modulates CD4⁺ T cell apoptosis in vivo and in vitro. (A) C57BL/6 Rag1^−/− mice were transferred with 4 × 10⁵ CD4⁺ CD25⁻ CD45RB^Hi T cells from C57BL/6 WT or Il1r1^−/− mice. At sacrifice, cLPLs were isolated and Ki67 expression by cLPLs CD4⁺ T cells was assessed by intracellular staining. Data represent mean ± SEM (n = 20–21 from 3 pooled independent experiments). Representative FACS plots (gated on CD4⁺ T cells) and quantitation are shown. (B) C57BL/6 Rag1^−/− mice were transferred with 2 × 10⁶ CD4⁺ CD25⁻ CD45Rb^Hi T cells from C57BL/6 WT or Il1r1^−/− mice and sacrificed 2 wk after transfer. Total number of CD4⁺ T cells and Th17 cells from the colonic lamina propria were assessed by FACS. Data are shown as mean ± SEM (n = 5 mice/group). (C and D) C57BL/6 Rag1^−/− mice were transferred with 4 × 10⁵ CD4⁺ CD25⁻ CD45RB^Hi T cells from C57BL/6 WT or Il1r1^−/− mice. Mice were sacrificed when recipients of WT naive CD4⁺ T cells developed clinical signs of colitis, and CD4⁺ T cells were FACS sorted from cLPL preparations. (C) Expression levels of indicated chemokine receptors as revealed by qRT-PCR analysis. (D) Bcl-2 and Bcl-XL expression by WT or Il1r1^−/− CD4⁺ T cells upon ex vivo restimulation with 0.1 µg/ml PMA and 1 µg/ml ionomycin overnight in complete medium (n = 5 mice/group, 1 representative experiment out of 2 is shown). (E) Purified CD4⁺ T cells from WT or Il1r1^−/− mice were cultured for 3 d in the presence of plate-bound αCD3 and αCD28 mAb at the indicated concentrations. After staining with the phosphatidylserine-binding protein Annexin V and the vital dye 7AAD, Annexin V⁺ 7AAD⁻ cells were identified as early apoptotic events. The average frequency of Annexin V⁺ 7AAD⁻ cells in WT cells was set as a baseline (100%), and data were expressed as relative increase over baseline (n = 10 from 4 pooled independent experiments). Representative FACS plots (gated on CD4⁺ T cells) and quantitation are shown. (F) 129SvEv Rag2^−/− mice were infected with H. hepaticus for 8 wk. CD45⁺lin⁻ Sca1⁺Thy1.2^Hi ILCs were FACS sorted from the colon and cultured overnight in complete medium alone (n/a) or in the presence of IL-1β (10ng/ml). Bcl2 expression was evaluated by qRT-PCR and normalized on Hprt expression. Data are shown as mean ± SEM from two pooled independent experiments. In each experiment, 10–18 mice were pooled.

Figure 8.

IL-1R1 expression by colonic CD4⁺ T cells is dependent on IL-23 stimulation. (A) C57BL/6 Rag1^−/− mice were transferred with 4 × 10⁵ CD4⁺ CD25⁻ CD45RB^Hi T cells from C57BL/6 WT or Il1r1^−/− mice. Mice were sacrificed when recipient of WT naive CD4⁺ T cells developed clinical signs of colitis. CD4⁺ T cells were FACS sorted from cLPL preparations and Il23r expression was evaluated by qRT-PCR (n = 5 mice/group, 1 out of 2 representative experiments shown). (B) C57BL/6 mice were transferred with 1:1 mixtures of CD45.1⁺ (WT) and CD45.2⁺ (Il23r^−/−) CD4⁺CD45RB^hi T cells. Mice were sacrificed upon development of clinical signs of inflammation (∼6 wk) and WT or Il23r^−/− populations of T cells were FACS sorted based on the expression of CD45.2. Il1r1 and Rorc expression levels were evaluated by qRT-PCR (data are shown as mean ± SEM; n = 6 from 2 independent experiments). (C) CD62L^Hi CD44^low CD25⁻ naive CD4⁺ T cells isolated from WT or Il23r^−/− mice were cultured under Th17-polarizing conditions. At the indicated time points, Il1r1 expression was evaluated by qRT-PCR (data are shown as mean ± SD; n = 2 from 2 pooled independent experiments). *, P < 0.05; **, P < 0.01.