RESUMO
BACKGROUND AND AIMS: Regulatory macrophages play a critical role in tissue repair, and we have previously shown that anti-tumour necrosis factor [TNF] antibodies induce these macrophages in vitro and in vivo in IBD patients. The induction of regulatory macrophages can be potentiated using the combination of anti-TNF and thiopurines, consistent with the enhanced efficacy of this combination therapy described in clinical trials. As thiopurines are unfortunately associated with significant side effects, we here aimed to identify alternatives for combination therapy with anti-TNF, using the macrophage induction model as a screening tool. METHODS: Mixed lymphocyte reactions were treated with anti-TNF and a library of 1600 drug compounds. Induction of CD14+CD206+ macrophages was analysed by flow cytometry. Positive hits were validated in vitro and in the T cell transfer model of colitis. RESULTS: Among the 98 compounds potentiating the induction of regulatory macrophages by anti-TNF were six benzimidazoles, including albendazole. Albendazole treatment in the presence of anti-TNF resulted in alterations in the tubulin skeleton and signalling though AMPK, which was required for the enhanced induction. Combination therapy also increased expression levels of the immunoregulatory cytokine IL-10. In vivo, albendazole plus anti-TNF combination therapy was superior to monotherapy in a model of colitis, in terms of both induction of regulatory macrophages and improvement of clinical symptoms. CONCLUSIONS: Albendazole enhances the induction of regulatory macrophages by anti-TNF and potentiates clinical efficacy in murine colitis. Given its favourable safety profile, these data indicate that the repurposing of albendazole may be a novel option for anti-TNF combination therapy in IBD.
Assuntos
Albendazol/farmacologia , Colite/tratamento farmacológico , Infliximab/farmacologia , Ativação de Macrófagos/efeitos dos fármacos , Macrófagos/fisiologia , Moduladores de Tubulina/farmacologia , Proteínas Quinases Ativadas por AMP/metabolismo , Albendazol/uso terapêutico , Animais , Benzimidazóis/farmacologia , Células Cultivadas , Sinergismo Farmacológico , Feminino , Humanos , Infliximab/uso terapêutico , Interleucina-10/metabolismo , Lectinas Tipo C/metabolismo , Receptores de Lipopolissacarídeos/metabolismo , Macrófagos/metabolismo , Receptor de Manose , Lectinas de Ligação a Manose/metabolismo , Camundongos , Receptores de Superfície Celular/metabolismo , Transdução de Sinais/efeitos dos fármacos , Tubulina (Proteína)/metabolismo , Moduladores de Tubulina/uso terapêutico , Fator de Necrose Tumoral alfa/antagonistas & inibidoresRESUMO
BACKGROUND & AIMS: Although tumor necrosis factor (TNF) antagonists reduce many clinical features of inflammatory bowel disease, complete mucosal healing occurs in fewer than 50% of patients. The Fc-region of monoclonal antibodies against TNF has immunosuppressive properties via effects on macrophage polarization. We examined the interaction between the anti-TNF Fc-region and Fcγ receptors (FcγR), and whether the absence of the Fc core fucose (which increases binding to FcγRIIIa) increases the efficacy of anti-TNF in mice with colitis. METHODS: We generated Rag1-/- mice that lack all activating FcγRs (FcγRI, FcγRIII, and FcγRIV; called FcγR-/-Rag1-/- mice). We produced hypo-fucosylated antibodies against mouse and human TNF (adalimumab). Colitis was induced in mice by transfer of CD4+CD45RBhi to FcγR-/-Rag1-/- or Rag1-/- littermates; mice were given different antibodies against TNF or isotype (control) antibodies and disease activity index scores were determined. Colon tissues were collected and analyzed by histology. Human peripheral blood mononuclear cells (PBMCs) were isolated from blood of healthy donors. T-cell proliferation and proportions of CD206+ (immune regulatory) macrophages were measured in mixed lymphocyte reactions. Human PBMCs were genotyped for FCGR3A158 (the FcγRIIIa-158F allotype displays a lower Fc binding affinity) using the TaqMan single nucleotide polymorphism genotype assay. RESULTS: Rag1-/- mice with colitis given anti-TNF had near complete mucosal healing and Rag1-/- mice given an isotype control antibody developed severe colitis. In contrast, FcγR-/-Rag1-/- mice were refractory to the effects of anti-TNF: their histological colitis scores were as severe as those from FcγR-/-Rag1-/- mice given a control antibody. Colons from Rag1-/- mice that received anti-TNF had an increased number of CD206+ macrophages compared with Rag1-/- mice given control antibody; in FcγR-/-Rag1-/- mice given anti-TNF these numbers were as low as FcγR-/-Rag1-/- given the control antibody. In human PBMCs, anti-TNF increased the number of CD206+ macrophages: this required expression of FcγRIIIa; numbers of these cells were reduced in PBMCs with the low-affinity FcγRIIIa-158F genotype. A hypo-fucosylated form of adalimumab bound human FcγRIIIa with a higher affinity than control adalimumab. When hypo-fucosylated adalimumab was added to PBMCs, a larger number of CD206+ macrophages formed and T-cell proliferation was reduced, compared with addition of a control adalimumab. Hypo-fucosylated adalimumab increased the number of CD206+ macrophages in PMBCs that expressed the low-affinity FcγRIIIa. In mice with colitis, hypo-fucosylated anti-TNF significantly increased the number of CD206+ macrophages in the colon compared with control anti-TNF and was more effective in reducing colitis severity as measured by histology. CONCLUSIONS: In a study of the in vitro and in vivo mechanisms of anti-TNF, we found FcγR engagement by anti-TNF to be required for reduction of colitis in mice and development of CD206+ macrophages. A hypo-fucosylated form of anti-TNF binds FcγRIIIa with higher affinity and induces development of CD206+ macrophages in human PBMCs, especially PBMCs that express low-affinity FcγRIIIa. Hypo-fucosylated anti-TNF might be more effective in patients with inflammatory bowel disease.
Assuntos
Adalimumab/farmacologia , Anticorpos Monoclonais/farmacologia , Colite/tratamento farmacológico , Colo/efeitos dos fármacos , Imunossupressores/farmacologia , Mucosa Intestinal/efeitos dos fármacos , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Transferência Adotiva , Animais , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Colite/genética , Colite/imunologia , Colite/metabolismo , Colo/imunologia , Colo/metabolismo , Colo/patologia , Modelos Animais de Doenças , Predisposição Genética para Doença , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Mucosa Intestinal/imunologia , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patologia , Lectinas Tipo C/imunologia , Lectinas Tipo C/metabolismo , Ativação Linfocitária/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Macrófagos/metabolismo , Receptor de Manose , Lectinas de Ligação a Manose/imunologia , Lectinas de Ligação a Manose/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fenótipo , Receptores de Superfície Celular/imunologia , Receptores de Superfície Celular/metabolismo , Receptores de IgG/deficiência , Receptores de IgG/genética , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologia , Linfócitos T/metabolismo , Linfócitos T/transplante , Fatores de Tempo , Fator de Necrose Tumoral alfa/imunologia , Cicatrização/efeitos dos fármacosRESUMO
Several anti-tumour necrosis factor [TNF] blocking strategies have been evaluated in patients with Crohn's disease. Compounds that have been tested included the full monoclonal IgG1 antibodies infliximab and adalimumab, the pegylated anti-TNF F[ab']2 fragment certolizumab, an IgG4 anti-TNF CDP571 with reduced affinity for the Fc receptor, the soluble TNF receptor I onercept, and the TNF receptor II-Fc fusion protein etanercept. The endpoints of these studies suggest that not all methods of blocking TNF are equal. Here we will review the differences in the clinical, biochemical, and endoscopic endpoints of the major clinical studies. Collectively the data suggest that only IgG1 monoclonal antibodies have the ability to induce complete clinical, biochemical, and endoscopic remission. We discuss the potential multiple modes of action that may contribute to the response to full IgG1 anti-TNFs, focusing on the rapid induction of lamina propria T cell apoptosis and Fc receptor-dependent induction of M2-type wound-healing macrophages. We discuss how novel insights into the mechanism of action of anti-TNFs in Crohn's disease may contribute to the development of novel anti-TNFs with improved efficacy.
Assuntos
Adalimumab/farmacologia , Anti-Inflamatórios/farmacologia , Certolizumab Pegol/farmacologia , Doenças Inflamatórias Intestinais/tratamento farmacológico , Infliximab/farmacologia , Mucosa Intestinal/efeitos dos fármacos , Cicatrização/efeitos dos fármacos , Adalimumab/uso terapêutico , Anti-Inflamatórios/uso terapêutico , Apoptose/efeitos dos fármacos , Biomarcadores/metabolismo , Certolizumab Pegol/uso terapêutico , Endoscopia Gastrointestinal , Humanos , Doenças Inflamatórias Intestinais/diagnóstico por imagem , Doenças Inflamatórias Intestinais/metabolismo , Doenças Inflamatórias Intestinais/patologia , Infliximab/uso terapêutico , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patologia , Linfócitos T/metabolismo , Linfócitos T/patologia , Resultado do Tratamento , Fator de Necrose Tumoral alfa/antagonistas & inibidoresRESUMO
BACKGROUND AND AIMS: Anti-tumour necrosis factor [TNF] antibodies induce regulatory macrophages which display a phenotype resembling M2 type macrophages. Anti-TNF induced macrophages [MÏind] have immunosuppressive and wound healing properties. The factors that contribute to the induction of MÏind remain to be explored. Autophagy has been described as a factor that is important for the induction and function of M2 type macrophages. We studied the contribution of autophagy to the induction of MÏind. METHODS: We studied the effect of autophagy on MÏind in vitro using peripheral blood mononuclear cells. Interferon gamma [IFN-γ] induced macrophages [Mφ1] were generated by culturing monocytes in the presence of IFN-γ. MÏind were generated by performing mixed lymphocyte reactions [MLR] in the presence of anti-TNF antibodies; 28 healthy donors were genotyped for rs_2241880 [ATG16L1]. Cells were analysed by autophagy gene array, immunofluorescence, western blot, flowcytometry, 3H-thymidine incorporation and MTS assay. RESULTS: MÏind had a different expression profile of autophagy related transcripts with increased expression of 33/40 altered genes compared with Mφ1. In addition, autophagic activity was increased in MÏind compared with Mφ1. Induction of MÏind was positively correlated to the number of wild-type alleles for the ATG16L1 T300A risk allele present in the culture. Finally, the autophagy-related protein cathepsin S was highly expressed in Mφind and inhibition resulted in decreased viability as well as decreased expression of CD206. CONCLUSIONS: MÏind have increased levels of autophagy compared with inflammatory Mφ1, and the induction of these macrophages is impaired in donors carrying the T300A risk allele for the ATG16L1. Given the association between MÏind and clinical response, this suggests that an intact autophagy pathway may be important for an optimal response to anti-TNF therapy in inflammatory bowel disease.
Assuntos
Proteínas Relacionadas à Autofagia/genética , Autofagia/efeitos dos fármacos , Resistência a Medicamentos/genética , Fármacos Gastrointestinais/farmacologia , Infliximab/farmacologia , Macrófagos/efeitos dos fármacos , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Autofagia/genética , Autofagia/imunologia , Western Blotting , Citometria de Fluxo , Frequência do Gene , Marcadores Genéticos , Humanos , Macrófagos/imunologia , Fator de Necrose Tumoral alfa/imunologiaRESUMO
BACKGROUND AND AIMS: Anti-tumour necrosis factor [TNF] monoclonal antibodies [infliximab, adalimumab] induce complete mucosal healing in a proportion of patients with Crohn's disease whereas a TNF receptor fusion protein [etanercept] is not effective and the anti-TNF F[ab']2 fragment [certolizumab] shows a very low rate of complete mucosal healing. In contrast, all four TNF-neutralising drugs have demonstrated efficacy in the treatment of rheumatoid arthritis. These observations suggest that factors other than neutralisation of TNF may contribute to clinical outcomes in Crohn's disease. Here we tested the hypothesis that Fc receptor [FcR]-mediated effects may contribute to the therapeutic response of anti-TNF antibodies in inflammatory bowel disease. METHODS: We modified an IgG2c mouse anti-TNF antibody that binds the high-affinity FcRs to generate an IgG1 isotype with strongly diminished binding. We examined the therapeutic effects of both antibodies in the T cell transfer model of inflammatory bowel disease and the collagen-induced arthritis model. RESULTS: The IgG2c anti-TNF antibody prevented colonic inflammation in the T cell transfer model of colitis, whereas the IgG1 anti-TNF did not. Conversely, both the IgG2c and IgG1 anti-TNFs were similarly effective in reducing the severity of articular inflammation in mouse collagen-induced arthritis. CONCLUSION: These data support the concept that the mechanism of action for TNF-neutralising drugs may differ across immune-mediated diseases and, potentially, between therapeutics within a particular disease. Our data suggest a specific role of Fc-mediated immune regulation in the resolution of intestinal inflammation by anti-TNF monoclonal antibodies.
Assuntos
Anticorpos Monoclonais Humanizados/administração & dosagem , Doenças Inflamatórias Intestinais/tratamento farmacológico , Doenças Inflamatórias Intestinais/imunologia , Receptores Fc/efeitos dos fármacos , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Animais , Artrite Experimental/tratamento farmacológico , Artrite Experimental/imunologia , Artrite Experimental/patologia , Biomarcadores/metabolismo , Colite Ulcerativa/tratamento farmacológico , Colite Ulcerativa/imunologia , Colite Ulcerativa/patologia , Doença de Crohn/tratamento farmacológico , Doença de Crohn/imunologia , Doença de Crohn/patologia , Modelos Animais de Doenças , Feminino , Imuno-Histoquímica , Doenças Inflamatórias Intestinais/patologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos DBA , Camundongos SCID , Terapia de Alvo Molecular/métodos , Distribuição Aleatória , Receptores Fc/metabolismo , Sensibilidade e Especificidade , Fator de Necrose Tumoral alfa/administração & dosagemRESUMO
AIM: To investigate if azathioprine could reduce adenoma formation in Apc(Min/+) , a mouse model of sporadic intestinal tumorigenesis. METHODS: Azathioprine was administered via drinking water (estimated 6-20 mg/kg body weight per day) to Apc(Min/+) and wildtype mice. Control animals received vehicle only (DMSO) dissolved in drinking water. At 15 wk of age all mice were sacrificed and intestines of Apc(Min/+) were harvested for evaluation of polyp number. Azathioprine induced toxicity was investigated by immunohistochemical analysis on spleens. RESULTS: All azathioprine treated mice showed signs of drug-associated toxicity such as weight loss and development of splenic T-cell lymphomas. Although this suggests that the thiopurine concentration was clearly in the therapeutic range, it did not reduce tumor formation (48 ± 3.1 adenomas vs 59 ± 5.7 adenomas, P = 0.148). CONCLUSION: We conclude that in the absence of inflammation, azathioprine does not affect intestinal tumorigenesis.