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

Inflammatory bowel disease

Anti-TNF therapy in IBD exerts its therapeutic effect through macrophage IL-10 signalling J Koelink1, Felicia M Bloemendaal1,2, Bofeng Li3, Liset Westera1, Esther W M Vogels1, Manon van Roest1, Anouk K Gloudemans4, Angelique B van ‘t Wout4, Hannelie Korf5, Séverine Vermeire5, Anje A te Velde1, Cyriel Y Ponsioen6, Geert RAM D’Haens6, J Sjef Verbeek7, Terrence L Geiger3, Manon E Wildenberg1,6, Gijs R van den Brink1,6,8
1Tytgat Insitute for Liver & Intestinal Research, Amsterdam UMC, Amsterdam, The Netherlands

2Department of Gastroenterology and hepatology, Amsterdam UMC, Amsterdam, The Netherlands

3Department of Pathology, St. Jude Children’s Research Hospital, Memphis, Tennessee, USA

4Janssen Prevention Center of Janssen Vaccines & Prevention BV, Janssen Pharmaceutical Companies of Johnson & Johnson, Leiden, The Netherlands

5Translational Research Center for Gastrointestinal Disorders (TARGID), Department of Clinical and Experimental Medicine, KU Leuven, Leuven, Belgium

6Gastroenterology & Hepatology, Amsterdam UMC, Amsterdam, The Netherlands

7Human Genetics, Leids Universitair Medisch Centrum, Leiden, The Netherlands

8Roche Innovation Center Basel, F Hoffmann-La Roche AG, Basel, Switzerland
Correspondence to
Dr Pim J Koelink, Tytgat Insitute for Liver & Intestinal Research, Amsterdam UMC, Amsterdam 1105 BK, The Netherlands; p.j.koelink{at}

AbstractObjective 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+CD45Rbhigh 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+CD45Rbhigh 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.IBD basic researchTNFantibody targeted therapyinfliximabinterleukins is an open access article distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited, appropriate credit is given, any changes made indicated, and the use is non-commercial. See: Full Text

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IBD basic researchTNFantibody targeted therapyinfliximabinterleukinsSignificance of this studyWhat is already known on this subject?Intestinal immune tolerance depends on interleukin (IL)-10 signalling in lamina propria macrophages to maintain a regulatory CD206+ regulatory phenotype.The effect of anti-tumour necrosis factor (TNF) therapy in IBD depends on Fcγ-receptor signalling.Fcγ-receptor (FcγR) signalling induces IL-10 production in macrophages.What are the new findings?Anti-TNF therapy induces FcγR-dependent IL-10 production in mouse and human macrophages in vitro.Anti-TNF therapy polarises human and mouse macrophages towards CD206+ macrophages in an IL-10-dependent manner in vitro.The efficacy of anti-TNF therapy in the T-cell transfer model of colitis depends on IL-10 signalling, specifically in macrophages and not T cells.Anti-TNF therapy induces IL-10 production and polarises macrophages towards CD206+ regulatory macrophages in intestinal biopsies of patients with Crohn’s disease in vivo, specifically in anti-TNF responders in contrast to non-responders.How might it impact on clinical practice in the foreseeable future ?Our results point to a central role for IL-10-induced repolarisation of macrophages to a regulatory phenotype in the therapy of patients with IBD.A reduced response to IL-10 contributes to primary non-response to anti-TNF in IBD and might be used to identify such patients.IntroductionDespite the considerable efficacy of antitumour necrosis factor (TNF)-α in the treatment of IBD, up to 30% of patients with IBD do not respond, and it is unknown what causes this primary non-reponse.1 This uncertainty is in part related to the fact that the mechanism of action of anti-TNF therapy in IBD has not been fully elucidated.2 The mechanism of action of anti-TNF therapy in IBD cannot solely be attributed to the neutralisation of TNF alone, as some TNF-neutralising drugs, such as etanercept, are successful in the treatment of rheumatoid arthritis3 4 but do not show efficacy in IBD.5 6 Recently, it has been shown that high oncostatin M (OSM) levels are associated with the non-response to anti-TNF in IBD.7
Interleukin (IL)-10 is a cytokine with convincing evidence for a role in the pathogenesis of IBD. IL-10 is linked to the aetiology of IBD through multiple genome-wide association studies (GWAS), and both mice and humans develop spontaneous IBD when IL-10 or its receptors are genetically disrupted.8–11 It has previously been established that the role of IL-10 in IBD depends on IL-10 signalling in macrophages. Here IL-10 acts to maintain lamina propria macrophages in a regulatory CD206+ phenotype that is critical to maintain intestinal immune tolerance.12 13 Interestingly, it has previously been described that IL-10 mutant mice are refractory to anti-TNF therapy, whereas they are highly responsive to antibodies to IL-12/IL-23p40.14 It is currently not known why IL-10 mutant mice are refractory to anti-TNF. Perhaps IL-10 mutant mice display a type of inflammation that develops independently of an intervention with anti-TNF. Alternatively and more intriguingly, there is a possibility that IL-10 signalling is required for the mechanism of action of anti-TNF.We have previously shown that full monoclonal antibodies (mAbs) against TNF engage Fcγ receptors (FcγRs) and that this Fc–FcγR interaction is necessary for the therapeutic efficacy of anti-TNF in a preclinical model of IBD. Mice without activating FcγRs completely lost response to anti-TNF therapy in the T-cell transfer colitis model, and conversely, a hypofucosylated anti-TNF antibody with increased Fc-binding affinity showed improved efficacy.15 16 Additionally, we have shown that anti-TNF skews human monocytes towards CD206+ regulatory macrophages in an Fc-dependent manner in vitro17 and in vivo,16 and that the response to anti-TNF therapy in both mice and humans was accompanied by the formation of these CD206+ regulatory macrophages in the intestine.16 18 However, the exact signalling mechanism of the induction of these macrophages and their precise role in the resolution of intestinal inflammation on anti-TNF therapy are currently unknown.Interestingly, it has previously been shown that FcγR signalling in macrophages increases the secretion of IL-10.19–21 Given the crucial role of macrophage IL-10 signalling in specifying an M2-like CD206+ regulatory macrophage phenotype to prevent intestinal inflammation,12 13 the dependence of response to anti-TNF on FcγR signalling16 and the observation that IL-10 mutant mice are refractory to anti-TNF, we decided to examine the role of IL-10 signalling in the therapeutic response to anti-TNF.Here we show that anti-TNF mAb therapy increases IL-10 production in monocytes/macrophages in an FcγR-dependent manner in vitro, which skews them towards a CD206+ regulatory phenotype. Using cell type-specific mutant mice, we find that IL-10 signalling in macrophages is absolutely required for anti-TNF-induced macrophage skewing to a CD206+ regulatory phenotype as well as therapeutic response.Materials and methodsFor information on mice strains and materials used, in situ hybridisation, immunohistochemistry, flow cytometry, in vitro experiments and the quantification of proteins by western blot or ELISA, see online supplementary information.Mouse experimentsThe T-cell transfer model was performed as previously described.22 Colitis was induced by injecting 3–5×105 CD4+CD45RBhigh T-cells intraperitoneally, with mice without a transfer as controls. Il10KO animals were treated with anti-mouse TNF, anti-mouse IL-12/23-p40 or isotype control mAbs, 300 µg, two times per week intraperitoneally from 8 to 14 weeks of age. The anti-TNF dose titration was described previously.23 The effective dosage of 100 µg anti-TNF twice a week intraperitoneally was used for all further experiments from 3 weeks after the T-cell transfer, when animals began to exhibit clinical signs of colitis, until the end of the experiments. The T-cell transfer experiment with the FcgRKO/Rag1KO animals was described previously.16 The anti-IL-10Rα antibody was administered from 2 weeks after the T-cell transfer, 250 µg two times a week intraperitoneally until the end of the experiment. The Mouse Colitis Endoscopy Index (MCEI) and the Mouse Colitis Histology Index (MCHI) were determined as recently described.24 All experiments were approved by the Academic Medical Center (AMC)Cohort studies of patients with Crohn’s disease (CD)Intestinal biopsies were collected from patients with CD before and during anti-TNF treatment (online supplementary table 1). Seven responders and seven non-responders, with response defined as a decrease of>25% Crohn’s Disease Endoscopic Index for severity (CDEI) or Simple Endoscopic Score for Crohn’s Disease (SES-CD), were included. The study protocols were approved by the medical ethical committee of the Amsterdam UMC and of the University Hospital in Leuven, and all participants provided written informed consent.Statistical analysisData are presented as bar–scatter plots with bars indicating median and dots representing individual values/animals, or bar graphs representing mean+SEM. Data were analysed by GraphPad Prism V.7.02 (GraphPad Software, La Jolla, CA, USA) with statistical tests specified in the figure legends. Values of p
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