Anti-TNF therapy in IBD exerts its therapeutic effect through macrophage IL-10 signalling

Abstract

Objective: Macrophage interleukin (IL)-10 signalling plays a critical role in the maintenance of a regulatory phenotype that prevents the development of IBD. We have previously found that anti-tumour necrosis factor (TNF) monoclonal antibodies act through Fcγ-receptor (FcγR) signalling to promote repolarisation of proinflammatory intestinal macrophages to a CD206+ regulatory phenotype. The role of IL-10 in anti-TNF-induced macrophage repolarisation has not been examined.

Design: We used human peripheral blood monocytes and mouse bone marrow-derived macrophages to study IL-10 production and CD206+ regulatory macrophage differentiation. To determine whether the efficacy of anti-TNF was dependent on IL-10 signalling in vivo and in which cell type, we used the CD4+CD45Rb^high T-cell transfer model in combination with several genetic mouse models.

Results: Anti-TNF therapy increased macrophage IL-10 production in an FcγR-dependent manner, which caused differentiation of macrophages to a more regulatory CD206+ phenotype in vitro. Pharmacological blockade of IL-10 signalling prevented the induction of these CD206+ regulatory macrophages and diminished the therapeutic efficacy of anti-TNF therapy in the CD4+CD45Rb^high T-cell transfer model of IBD. Using cell type-specific IL-10 receptor mutant mice, we found that IL-10 signalling in macrophages but not T cells was critical for the induction of CD206+ regulatory macrophages and therapeutic response to anti-TNF.

Conclusion: The therapeutic efficacy of anti-TNF in resolving intestinal inflammation is critically dependent on IL-10 signalling in macrophages.

Keywords: IBD basic research; TNF; antibody targeted therapy; infliximab; interleukins.

Figure 1

Il10KO mice are anti-TNF refractory. Il10KO mice were treated with 300 µg anti-TNF (n=9), anti-IL-12/23 (n=9) or isotype control (n=10) two times per week for 6 weeks as described in the Materials and methods section (healthy control mice (n=10) were included in B–F. (A) Body weight, (B) colon density, (C) representative endoscopy images and (D) endoscopy score (MCEI). (E) Representative images of H&E-stained intestinal slides (E) and (F) histology score (MCHI). Significance was determined by Kruskal-Wallis followed by Dunn’s post hoc test with *p<0.05 and **p<0.01. IL, interleukin; MCEI, Mouse Colitis Endoscopy Index; MCHI, Mouse Colitis Histology Index; TNF, tumour necrosis factor.

Figure 2

The efficacy of anti-TNF is IL-10 dependent. Severe combined immunedeficient (SCID) animals received 3×10⁵ CD4+CD45Rb^high T cells and were treated with an anti-IL-10Rα (250 µg two times per week) from 2 weeks after the T-cell transfer, in combination with isotype or anti-TNF (100 µg two times per week) from 3 weeks after the T-cell transfer. Representative stills of endoscopy movies (A) scored for the MCEI in (B). Representative images of H&E-stained intestinal slides (C) scored for the MCHI in (D), the colon density in (E) and the bodyweight gain/loss during the experiment in (F). Intestinal expression levels of Il10, Ifng and Il1b relative to Gapdh and Il10/Ifng and Il10/Il1b ratios, determined on whole-colon mRNA, are shown (G). (H) Expression levels of Cd206, Cd163 and Nos2 relative to Gapdh and Cd206/Nos2 and Cd163/Nos2 ratios. All significance was determined by Kruskal-Wallis followed by Dunn’s post hoc test (n=11–13). Asterisks in (F) indicate comparison between anti-TNF and isotype (*, p<0.05) and between isotype +anti-IL10Rα and anti-TNF +anti-IL10Rα (****, p<0.0001). IL, interleukin; MCEI, Mouse Colitis Endoscopy Index; MCHI, Mouse Colitis Histology Index; TNF, tumour necrosis factor.*p<0.05, **p<0.01, ***p<0.001 and **** p<0.0001.

Figure 3

Anti-TNF therapy increases intestinal IL-10 levels. IL-10 levels in whole-colon homogenates of severe combined immunedeficient (SCID) mice that received a CD4+CD45Rb^high T-cell transfer and were treated with different dosages of anti-TNF (n=10–12) for 4 weeks from 3 weeks after the T-cell transfer. Animals treated with a therapeutic dosage of anti-TNF (100 μg) are shown in red (A). (B) Correlation between intestinal IL-10 levels and the histological score (MCHI) in all mice that received a T-cell transfer (n=53). The proportion of CD3-negative lamina propria cells expressing Il10 was determined by image analysis (C, n>10 images/condition, n=11 per group) as determined by fluorescent in situ hybridisation. (D) Representative Il10 in situ hybridisation (red) with F4/80 immunohistochemical staning (green) on a consectutive intestinal tissue slide of an anti-TNF-treated animal. Scale bar is 50 µm. Significance was determined by analysis of variance followed by Sidak’s post hoc in (A) and Mann-Whitney test in (D) with* p<0.05 and ***p<0.001. DAPI, 4',6-diamidino-2-fenylindool; IHC immunohistochemistry; IL, interleukin; ISH, in situ hybridisation; MCHI, Mouse Colitis Histology Index; TNF, tumour necrosis factor.

Figure 4

Anti-TNF induces IL-10-dependent M2 differentiation in BMDMs in vitro. (A) Intestinal Il10 expression level relative to Gapdh and Il10/Ifng and Il10/Il1b ratios of the Rag1 KO and FcgRKO/Rag1KO animals treated with anti-TNF. Relative Ifng and Il1b expression levels were reported in Bloemendaal et al. Significance was determined by Kruskal-Wallis followed by Dunn’s post hoc test, n=9–11. (B) IL-10 levels in medium of BMDMs stimulated with lipopolysacharide (LPS,100 ng/mL) and IFN-γ (20 ng/mL) in combination with the IgG isotype control antibody (10 µg mL), IgG immune complex (10 µg mL), the anti-TNF mAb (10 µg/mL) or Fab fragment (7.2 µg/mL) after 48 hours (B). (C) Levels of Il10 normalised for Gapdh expression in BMDMs after 24 hours. (D) IL-10 levels in medium of BMDMs (after 48 hours). (E) Western blot for pSTAT3, STAT3 and β-actin. (F) Levels of Cd206 normalised for Gapdh expression after 48 hours in (G) Representative histogram of flow cytometry for CD206, expressed as fold increase of the mean fluorescence intensity (MFI) in graph on BMDMs after 72 hours. Wild-type BMDMs are shown in black, wild-type BMDMs incubated with anti-IL-10Rα (10 µg/mL) in blue and Il10rbKO BMDMs in green. All data represent mean+SEM (n=3–6), representative of two to three independent experiments. Significance was determined by analysis of variance. followed by Sidak’s post hoc test (B, C) or unpaired t-test with *p<0.05, **p<0.01,***p<0.001 and ****p<0.0001. BMDM, bone marrow-derived macrophage; IFN, interferon; IL, interleukin; TNF, tumour necrosis factor.

Figure 5

Efficacy of anti-TNF is independent of IL-10 signalling in T-cells. Rag1KO animals received a 3×10⁵ wild type (grey) or 3×10⁵ Il10rbKO (green) CD4+CD45Rb^high T-cell transfer and were treated with isotype or anti-TNF. Control Rag1KO animals did not receive a T-cell transfer. Representative stills of endoscopy movies (A) scored for the MCEI in (B). Representative images of H&E-stained intestinal slides (C) scored for the MCHI in (D), the colon density (E) and the bodyweight gain/loss during the experiment in (F). (G) Intestinal expression levels of Il10, Ifng and Il1b relative to Gapdh and Il10/Ifng and Il10/Il1b ratios, determined on whole-colon mRNA. (H) Expression levels of Cd206, Cd163 and Nos2 relative to Gapdh and Cd206/Nos2 and Cd163/Nos2 ratios. All significance was determined by Kruskal-Wallis followed by Dunn’s post hoc test with *p<0.05, **p<0.01 and ***p<0.001 (n=10–12). IL, interleukin; MCEI, Mouse Colitis Endoscopy Index; MCHI, Mouse Colitis Histology Index; TNF, tumour necrosis factor.

Figure 6

The efficacy of anti-TNF is dependent on IL-10 signalling in macrophages in vivo. Rag1KO (grey) or LysMcreIl10ra^fl/flRag1KO (blue) animals received a CD4+CD45Rb^high T-cell transfer (5×10⁵ cells) and were treated with isotype or anti-TNF. Control animals (Rag1KO) did not receive a T-cell transfer. Representative stills of endoscopy movies (A) scored for the MCEI in (B). Representative images of H&E-stained intestinal slides (C) scored for the MCHI in (D), the colon density (E) and the bodyweight gain/loss during the experiment in (F). (G) Intestinal expression levels of Il10, Ifng and Il1b relative to Gapdh and Il10/Ifng and Il10/Il1b ratios, determined on whole-colon mRNA. Expression levels of Cd206, Cd163 and Nos2 relative to Gapdh and Cd206/Nos2 and Cd163/Nos2 ratios are shown (H). Flow cytometry analysis for CD206 and LY6C is shown in (I), after gating for colonic macrophages (DAPI⁻CD45⁺ CD11b⁺Ly6G⁻CD64⁺). All significance was determined by Kruskal-Wallis followed by Dunn’s post hoc test with *p<0.05, **p<0.01 and ***p<0.001, n=7–12. IL, interleukin; Mouse Colitis Endoscopy Index; MCHI, Mouse Colitis Histology Index; TNF, tumour necrosis factor.

Figure 7

Anti-TNF induces IL-10-dependent M2 differentiation in human monocytes in vitro. IL-10 levels in medium of human monocytes stimulated with lipopolysacharide (LPS,100 ng/mL) and IFN-γ (20 ng/mL) and the full monoclonal anti-TNF antibody (adalimumab, 10 µg/mL) or anti-TNF Fab fragment (certolizumab, 10 µg/mL) after 48 hours (A) and levels of CD206 normalised for Gapdh after 48 hours or flow cytometry for CD206 expressed as fold increase of MFI after 72 hours (C) in combination with and anti-IL-10 antibody (10 µg/mL) or isotype control (10 µg/mL). All graphs are mean+SEM representative of three independent donors with n=4–6 per condition. The IL10 and IFNG expression levels relative to GAPDH and IL10/IFNG ratios (D) and CD206 and NOS2 expression levels relative to GAPDH and CD206/NOS2 ratios (E) in intestinal biopsies before and after treatment in anti-TNF responding and non-responding patients with CD.Significance was determined by Mann Whitney in A, analysis of variance followed by Holm-Sidak's post hoc test in B andC, and Wilcoxon signed rank test in D and E with *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001. IFN, interferon; IL, interleukin.