RESUMO
Defective autophagy is linked to diseases such as rheumatoid arthritis, lupus and inflammatory bowel disease (IBD). However, the mechanisms by which autophagy limits inflammation remain poorly understood. Here we found that loss of the autophagy-related gene Atg16l1 promoted accumulation of the adaptor TRIF and downstream signaling in macrophages. Multiplex proteomic profiling identified SQSTM1 and Tax1BP1 as selective autophagy-related receptors that mediated the turnover of TRIF. Knockdown of Tax1bp1 increased production of the cytokines IFN-ß and IL-1ß. Mice lacking Atg16l1 in myeloid cells succumbed to lipopolysaccharide-mediated sepsis but enhanced their clearance of intestinal Salmonella typhimurium in an interferon receptor-dependent manner. Human macrophages with the Crohn's disease-associated Atg16l1 variant T300A exhibited more production of IFN-ß and IL-1ß. An elevated interferon-response gene signature was observed in patients with IBD who were resistant to treatment with an antibody to the cytokine TNF. These findings identify selective autophagy as a key regulator of signaling via the innate immune system.
Assuntos
Proteínas Adaptadoras de Transporte Vesicular/imunologia , Autofagia/imunologia , Imunidade Inata/imunologia , Inflamação/imunologia , Animais , Proteínas Relacionadas à Autofagia/genética , Proteínas Relacionadas à Autofagia/imunologia , Doença de Crohn/imunologia , Feminino , Humanos , Macrófagos/imunologia , Masculino , Camundongos , Camundongos Transgênicos , Transdução de Sinais/imunologiaRESUMO
Small bowel strictures remain a debilitating consequence of Crohn's disease and contribute to poor outcomes for patients. Recently, TGFß has been identified as an important driver of intestinal fibrosis. We studied the localization of TGFß isoforms in ileal strictures of patients with Crohn's disease using in situ hybridization to understand TGFß's role in stricture formation. The mucosa of strictures was characterized by higher TGFß1 while the stricture submucosa showed higher TGFß3 compared to normal ileum from patients without Crohn's disease (p = 0.02 and p = 0.044, respectively). We correlated these findings with single-cell transcriptomics which demonstrated that TGFß3 transcripts overall are very rare, which may partially explain why its role in intestinal fibrosis has remained unclear to date. There were no significant differences in fibroblast or B cell TGFß1 and/or TGFß3 expression in inflamed vs. noninflamed ileum. We discuss the implications of these findings for therapeutic development strategies to treat patients with fibrostenotic Crohn's disease.
Assuntos
Doença de Crohn , Fibrose , Fator de Crescimento Transformador beta1 , Fator de Crescimento Transformador beta3 , Humanos , Doença de Crohn/metabolismo , Doença de Crohn/patologia , Doença de Crohn/complicações , Fator de Crescimento Transformador beta1/metabolismo , Fator de Crescimento Transformador beta3/metabolismo , Constrição Patológica/metabolismo , Fibrose/metabolismo , Masculino , Feminino , Adulto , Intestino Delgado/metabolismo , Intestino Delgado/patologia , Pessoa de Meia-Idade , Íleo/metabolismo , Íleo/patologiaRESUMO
BACKGROUND: The interleukin-22 cytokine (IL-22) has demonstrated efficacy in preclinical colitis models with non-immunosuppressive mechanism of action. Efmarodocokin alfa (UTTR1147A) is a fusion protein agonist that links IL-22 to the crystallisable fragment (Fc) of human IgG4 for improved pharmacokinetic characteristics, but with a mutation to minimise Fc effector functions. METHODS: This randomised, phase 1b study evaluated the safety, tolerability, pharmacokinetics and pharmacodynamics of repeat intravenous dosing of efmarodocokin alfa in healthy volunteers (HVs; n=32) and patients with ulcerative colitis (n=24) at 30-90 µg/kg doses given once every 2 weeks or monthly (every 4 weeks) for 12 weeks (6:2 active:placebo per cohort). RESULTS: The most common adverse events (AEs) were on-target, reversible, dermatological effects (dry skin, erythema and pruritus). Dose-limiting non-serious dermatological AEs (severe dry skin, erythema, exfoliation and discomfort) were seen at 90 µg/kg once every 2 weeks (HVs, n=2; patients, n=1). Pharmacokinetics were generally dose-proportional across the dose levels, but patients demonstrated lower drug exposures relative to HVs at the same dose. IL-22 serum biomarkers and IL-22-responsive genes in colon biopsies were induced with active treatment, and microbiota composition changed consistent with a reversal in baseline dysbiosis. As a phase 1b study, efficacy endpoints were exploratory only. Clinical response was observed in 7/18 active-treated and 1/6 placebo-treated patients; clinical remission was observed in 5/18 active-treated and 0/6 placebo-treated patients. CONCLUSION: Efmarodocokin alfa had an adequate safety and pharmacokinetic profile in HVs and patients. Biomarker data confirmed IL-22R pathway activation in the colonic epithelium. Results support further investigation of this non-immunosuppressive potential inflammatory bowel disease therapeutic. TRIAL REGISTRATION NUMBER: NCT02749630.
Assuntos
Colite Ulcerativa , Humanos , Colite Ulcerativa/tratamento farmacológico , Colite Ulcerativa/patologia , Voluntários Saudáveis , Administração Intravenosa , BiomarcadoresRESUMO
Targeting interactions between α4ß7 integrin and endothelial adhesion molecule MAdCAM-1 to inhibit lymphocyte migration to the gastrointestinal tract is an effective therapy in inflammatory bowel disease (IBD). Following lymphocyte entry into the mucosa, a subset of these cells expresses αEß7 integrin, which is expressed on proinflammatory lymphocytes, to increase cell retention. The factors governing lymphocyte migration into the intestinal mucosa and αE integrin expression in healthy subjects and IBD patients remain incompletely understood. We evaluated changes in factors involved in lymphocyte migration and differentiation within tissues. Both ileal and colonic tissue from active IBD patients showed upregulation of ICAM-1, VCAM-1, and MAdCAM-1 at the gene and protein levels compared with healthy subjects and/or inactive IBD patients. ß1 and ß7 integrin expression on circulating lymphocytes was similar across groups. TGF-ß1 treatment induced expression of αE on both ß7+ and ß7- T cells, suggesting that cells entering the mucosa independently of MAdCAM-1/α4ß7 can become αEß7+ ITGAE gene polymorphisms did not alter protein induction following TGF-ß1 stimulation. Increased phospho-SMAD3, which is directly downstream of TGF-ß, and increased TGF-ß-responsive gene expression were observed in the colonic mucosa of IBD patients. Finally, in vitro stimulation experiments showed that baseline ß7 expression had little effect on cytokine, chemokine, transcription factor, and effector molecule gene expression in αE+ and αE- T cells. These findings suggest cell migration to the gut mucosa may be altered in IBD and α4ß7-, and α4ß7+ T cells may upregulate αEß7 in response to TGF-ß once within the gut mucosa.
Assuntos
Antígenos CD/metabolismo , Doenças Inflamatórias Intestinais/imunologia , Cadeias alfa de Integrinas/metabolismo , Cadeias beta de Integrinas/metabolismo , Mucosa Intestinal/imunologia , Receptores de Retorno de Linfócitos/metabolismo , Linfócitos T/imunologia , Adulto , Idoso , Movimento Celular , Feminino , Humanos , Cadeias beta de Integrinas/genética , Masculino , Pessoa de Meia-Idade , Transdução de Sinais , Proteína Smad3/metabolismo , Fator de Crescimento Transformador beta1/metabolismoRESUMO
Interleukin-22 (IL-22) plays a role in epithelial barrier function and repair, and may provide benefits in conditions like inflammatory bowel disease. However, limited human data are available to assess the clinical effect of IL-22 administration. This study used a human intestinal cell line to identify an IL-22-dependent gene signature that could serve as a pharmacodynamic biomarker for IL-22 therapy. The response to IL-22Fc (UTTR1147A, an Fc-stabilized version of IL-22) was assessed in HT-29 cells by microarray, and the selected responsive genes were confirmed by qPCR. HT-29 cells demonstrated dose-dependent increases in STAT3 phosphorylation and multiple gene expression changes in response to UTTR1147A. Genes were selected that were upregulated by UTTR1147A, but to a lesser extent by IL-6, which also signals via STAT3. IL-1R1 was highly upregulated by UTTR1147A, and differential gene expression patterns were observed in response to IL-22Fc in the presence of IL-1ß. An IL-22-dependent gene signature was identified that could serve as a pharmacodynamic biomarker in intestinal biopsies to support the clinical development of an IL-22 therapeutic. The differential gene expression pattern in the presence of IL-1ß suggests that an inflammatory cytokine milieu in the disease setting could influence the clinical responses to IL-22.
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Anti-Inflamatórios/farmacologia , Imunoglobulina G/genética , Doenças Inflamatórias Intestinais/metabolismo , Interleucinas/genética , Transcriptoma/efeitos dos fármacos , Biomarcadores/metabolismo , Células HT29 , Humanos , Imunoglobulina G/metabolismo , Interleucinas/metabolismo , Receptores de Interleucina-1/genética , Receptores de Interleucina-1/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacologia , Fator de Transcrição STAT3/metabolismo , Interleucina 22RESUMO
Inflammatory bowel disease (IBD) is characterized by uncontrolled inflammation in the gastrointestinal tract. The underlying pathobiology of IBD includes an increase in infiltrating gut-homing lymphocytes. Although lymphocyte homing is typically a tightly regulated and stepwise process involving multiple integrins and adhesion molecules expressed on endothelial cells, the distinct roles of integrin-expressing immune cells is not fully understood in the pathology of IBD. In this review, we detail the involvement of integrins expressed on specific lymphocyte subsets in the pathogenesis of IBD and discuss the current status of approved and investigational integrin-targeted therapies.
Assuntos
Drogas em Investigação/uso terapêutico , Doenças Inflamatórias Intestinais/tratamento farmacológico , Doenças Inflamatórias Intestinais/patologia , Integrinas/metabolismo , Animais , Drogas em Investigação/farmacologia , Trato Gastrointestinal/patologia , Humanos , Doenças Inflamatórias Intestinais/metabolismo , Terapia de Alvo Molecular , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologiaRESUMO
OBJECTIVE: Both endoscopy and histology may be included in the definition of mucosal healing in UC. This study aimed to establish the association between patient-reported outcomes, specifically symptom measures, and the presence of inflammation as measured by endoscopy and histology in UC. DESIGN: Using patient data from an observational multicentre study of UC (n=103), rectal bleeding (RB) and stool frequency (SF) symptom subscores of the Mayo Clinic Score (MCS) were compared with the endoscopic subscore (MCSe) and histology. Faecal calprotectin and biopsy cytokine expression were also evaluated. RESULTS: When identifying UC patients with inactive disease, RB scores were superior to SF scores and the combination (sensitivity/specificity: MCSe=0/1, RB 77%/81%, SF 62%/95%, RB+SF 54%/95%; MCSe=0, RB 87%/66%, SF 76%/83%, RB+SF 68%/86%). Across different definitions of mucosal healing (MCSe≤1; 0; or 0 plus inactive histology), a larger subset of patients reported increased SF (39%, 25% and 27%, respectively) compared with RB (24%, 13% and 10%). Faecal calprotectin and inflammatory cytokine expression were higher in patients with active disease compared with patients with mucosal healing, but there were no differences between patients using increasingly stringent definitions of mucosal healing. CONCLUSIONS: Endoscopically inactive disease is associated with absence of RB but not with complete normalisation of SF. Achieving histological remission did not improve symptomatic relief. In addition, in these patients, higher inflammatory biomarker levels were not observed. These data suggest that non-inflammatory changes, such as bowel damage, may contribute to SF in UC.
Assuntos
Colite Ulcerativa/patologia , Colonoscopia , Medidas de Resultados Relatados pelo Paciente , Adulto , Biomarcadores/metabolismo , Biópsia , Citocinas/metabolismo , Fezes/química , Feminino , Humanos , Mucosa Intestinal/metabolismo , Complexo Antígeno L1 Leucocitário/metabolismo , Masculino , Estudos Retrospectivos , Sensibilidade e Especificidade , Cicatrização/fisiologiaRESUMO
BACKGROUND & AIMS: Etrolizumab is a humanized monoclonal antibody against the ß7 integrin subunit that has shown efficacy vs placebo in patients with moderate to severely active ulcerative colitis (UC). Patients with colon tissues that expressed high levels of the integrin αE gene (ITGAE) appeared to have the best response. We compared differences in colonic expression of ITGAE and other genes between patients who achieved clinical remission with etrolizumab vs those who did. METHODS: We performed a retrospective analysis of data collected from 110 patients with UC who participated in a phase 2 placebo-controlled trial of etrolizumab, as well as from 21 patients with UC or without inflammatory bowel disease (controls) enrolled in an observational study at a separate site. Colon biopsies were collected from patients in both studies and analyzed by immunohistochemistry and gene expression profiling. Mononuclear cells were isolated and analyzed by flow cytometry. We identified biomarkers associated with response to etrolizumab. In the placebo-controlled trial, clinical remission was defined as total Mayo Clinic Score ≤2, with no individual subscore >1, and mucosal healing was defined as endoscopic score ≤1. RESULTS: Colon tissues collected at baseline from patients who had a clinical response to etrolizumab expressed higher levels of T-cell-associated genes than patients who did not respond (P < .05). Colonic CD4(+) integrin αE(+) cells from patients with UC expressed higher levels of granzyme A messenger RNA (GZMA mRNA) than CD4(+) αE(-) cells (P < .0001); granzyme A and integrin αE protein were detected in the same cells. Of patients receiving 100 mg etrolizumab, a higher proportion of those with high levels of GZMA mRNA (41%) or ITGAE mRNA (38%) than those with low levels of GZMA (6%) or ITGAE mRNA (13%) achieved clinical remission (P < .05) and mucosal healing (41% GZMA(high) vs 19% GZMA(low) and 44% ITGAE(high) vs 19% ITGAE(low)). Compared with ITGAE(low) and GZMA(low) patients, patients with ITGAE(high) and GZMA(high) had higher baseline numbers of epithelial crypt-associated integrin αE(+) cells (P < .01 for both), but a smaller number of crypt-associated integrin αE(+) cells after etrolizumab treatment (P < .05 for both). After 10 weeks of etrolizumab treatment, expression of genes associated with T-cell activation and genes encoding inflammatory cytokines decreased by 40%-80% from baseline (P < .05) in patients with colon tissues expressing high levels of GZMA at baseline. CONCLUSIONS: Levels of GZMA and ITGAE mRNAs in colon tissues can identify patients with UC who are most likely to benefit from etrolizumab; expression levels decrease with etrolizumab administration in biomarker(high) patients. Larger, prospective studies of markers are needed to assess their clinical value.
Assuntos
Anti-Inflamatórios/uso terapêutico , Anticorpos Monoclonais Humanizados/uso terapêutico , Antígenos CD/metabolismo , Colite Ulcerativa/tratamento farmacológico , Colo/efeitos dos fármacos , Fármacos Gastrointestinais/uso terapêutico , Granzimas/metabolismo , Cadeias alfa de Integrinas/metabolismo , Antígenos CD/genética , Biópsia , Ensaios Clínicos Fase II como Assunto , Colite Ulcerativa/diagnóstico , Colite Ulcerativa/enzimologia , Colite Ulcerativa/genética , Colo/enzimologia , Colo/patologia , Perfilação da Expressão Gênica/métodos , Granzimas/genética , Humanos , Imuno-Histoquímica , Cadeias alfa de Integrinas/genética , Valor Preditivo dos Testes , RNA Mensageiro/metabolismo , Ensaios Clínicos Controlados Aleatórios como Assunto , Indução de Remissão , Estudos Retrospectivos , Fatores de Tempo , Resultado do Tratamento , Cicatrização/efeitos dos fármacosRESUMO
In this issue of Immunity, Chaturvedi et al. (2008) describe a mechanism for the bridging of innate and adaptive immune receptor functions. In their model, B cell-receptor signaling induces the fusion of Toll-like receptor 9 (TLR9)-containing endosomes with internalized signaling-competent BCR into autophagosomes.
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Fagossomos/metabolismo , Receptores de Antígenos de Linfócitos B/metabolismo , Transdução de Sinais , Receptor Toll-Like 9/metabolismo , Animais , Autoimunidade , Humanos , Ativação Linfocitária , Fagossomos/imunologia , Receptores de Antígenos de Linfócitos B/imunologia , Receptor Toll-Like 9/imunologiaRESUMO
Chronic inflammatory disorders are complex and characterized by significant heterogeneity in molecular, pathological, and clinical features. This heterogeneity poses challenges for the development of targeted molecular interventions for these disorders, as not all patients with a given clinical diagnosis have disease driven by a single dominant molecular pathway, hence not all patients will benefit equally from a given intervention. Biomarkers related to molecular manifestations of disease are increasingly being applied to enable stratified approaches to drug development. Biomarkers may be used to identify which patients are most likely to benefit from an intervention (predictive), identify patients at increased risk of disease progression (prognostic), and monitor biological responsiveness to an intervention (pharmacodynamic). Here we consider how biomarker-guided stratification of patients may increase benefit from targeted therapies for asthma, rheumatoid arthritis and inflammatory bowel diseases.
Assuntos
Artrite Reumatoide/metabolismo , Asma/metabolismo , Biomarcadores/metabolismo , Colite Ulcerativa/metabolismo , Adalimumab/uso terapêutico , Anti-Inflamatórios/uso terapêutico , Anticorpos Monoclonais/uso terapêutico , Artrite Reumatoide/diagnóstico , Artrite Reumatoide/tratamento farmacológico , Asma/diagnóstico , Asma/tratamento farmacológico , Colite Ulcerativa/diagnóstico , Colite Ulcerativa/tratamento farmacológico , Humanos , Terapia de Alvo Molecular/métodos , Resultado do TratamentoRESUMO
BACKGROUND: Etrolizumab is a humanised monoclonal antibody that selectively binds the ß7 subunit of the heterodimeric integrins α4ß7 and αEß7. We aimed to assess etrolizumab in patients with moderately-to-severely active ulcerative colitis. METHODS: In this double-blind, placebo-controlled, randomised, phase 2 study, patients with moderately-to-severely active ulcerative colitis who had not responded to conventional therapy were recruited from 40 referral centres in 11 countries. Eligible patients (aged 18-75 years; Mayo Clinic Score [MCS] of 5 of higher [or ≥6 in USA]; and disease extending 25 cm or more from anal verge) were randomised (1:1:1) to one of two dose levels of subcutaneous etrolizumab (100 mg at weeks 0, 4, and 8, with placebo at week 2; or 420 mg loading dose [LD] at week 0 followed by 300 mg at weeks 2, 4, and 8), or matching placebo. The primary endpoint was clinical remission at week 10, defined as MCS of 2 or less (with no individual subscore of >1), analysed in the modified intention-to-treat population (mITT; all randomly assigned patients who had received at least one dose of study drug, had at least one post-baseline disease-activity assessment, and had a centrally read screening endoscopic subscore of ≥2). This study is registered with ClinicalTrials.gov, number NCT01336465. FINDINGS: Between Sept 2, 2011, and July 11, 2012, 124 patients were randomly assigned, of whom five had a endoscopic subscore of 0 or 1 and were excluded from the mITT population, leaving 39 patients in the etrolizumab 100 mg group, 39 in the etrolizumab 300 mg plus LD group, and 41 in the placebo group for the primary analyses. No patients in the placebo group had clinical remission at week 10, compared with eight (21% [95% CI 7-36]) patients in the etrolizumab 100 mg group (p=0·0040) and four (10% [0·2-24]) patients in the 300 mg plus LD group (p=0·048). Adverse events occurred in 25 (61%) of 41 patients in the etrolizumab 100 mg group (five [12%] of which were regarded as serious), 19 (48%) of 40 patients in the etrolizumab 300 mg plus LD group (two [5%] serious), and 31 (72%) of 43 patients in the placebo group (five [12%] serious). INTERPRETATION: Etrolizumab was more likely to lead to clinical remission at week 10 than was placebo. Therefore, blockade of both α4ß7 and αEß7 might provide a unique therapeutic approach for the treatment of ulcerative colitis, and phase 3 studies have been planned. FUNDING: Genentech.
Assuntos
Anticorpos Monoclonais Humanizados/uso terapêutico , Anticorpos Monoclonais/uso terapêutico , Colite Ulcerativa/tratamento farmacológico , Adulto , Anticorpos Monoclonais/administração & dosagem , Anticorpos Monoclonais Humanizados/administração & dosagem , Método Duplo-Cego , Feminino , Humanos , Masculino , Indução de Remissão/métodos , Fatores de Tempo , Resultado do TratamentoRESUMO
OBJECTIVE: Etrolizumab (rhuMAb ß7, anti-ß7, PRO145223) is a humanised monoclonal antibody targeting the ß7 subunit of the heterodimeric integrins α4ß7 and αEß7, which are implicated in leucocyte migration and retention in ulcerative colitis (UC). This randomised phase I study evaluated the safety and pharmacology of etrolizumab in patients with moderate to severe UC. DESIGN: In the single ascending dose (SAD) stage, etrolizumab (0.3, 1.0, 3.0, 10 mg/kg intravenous, 3.0 mg/kg subcutaneous (SC) or placebo) was administered 4:1 (n=25) in each cohort. In the multiple dose (MD) stage, new patients received monthly etrolizumab (0.5 mg/kg SC (n=4), 1.5 mg/kg SC (n=5), 3.0 mg/kg SC (n=4), 4.0 mg/kg intravenous (n=5)) or placebo (n=5). The pharmacokinetics was studied and Mayo Clinic Score evaluated at baseline, day 29 (SAD), and days 43 and 71 (MD). RESULTS: In the SAD stage, there were no dose limiting toxicities, infusion or injection site reactions. Two impaired wound healing serious adverse events occurred in two patients receiving etrolizumab. In the MD stage, there were no dose limiting toxicities, and no infusion or injection site reactions. Headache was the most common adverse event, occurring more often in etrolizumab patients. Antietrolizumab antibodies were detected in two subjects. The duration of ß7 receptor full occupancy was dose related. A clinical response was observed in 12/18 patients, and clinical remission in 3/18 patients treated with etrolizumab in the MD stage, compared with 4/5 and 1/5 placebo patients, respectively. CONCLUSION: Etrolizumab is well tolerated in moderate to severe UC. Further investigation is warranted.
Assuntos
Anticorpos Monoclonais/administração & dosagem , Colite Ulcerativa/tratamento farmacológico , Fármacos Gastrointestinais/administração & dosagem , Adolescente , Adulto , Idoso , Anticorpos Monoclonais/efeitos adversos , Anticorpos Monoclonais/sangue , Anticorpos Monoclonais/uso terapêutico , Anticorpos Monoclonais Humanizados , Colite Ulcerativa/sangue , Relação Dose-Resposta a Droga , Método Duplo-Cego , Feminino , Fármacos Gastrointestinais/efeitos adversos , Fármacos Gastrointestinais/sangue , Fármacos Gastrointestinais/uso terapêutico , Humanos , Infusões Intravenosas , Injeções Subcutâneas , Masculino , Pessoa de Meia-Idade , Índice de Gravidade de Doença , Resultado do Tratamento , Adulto JovemRESUMO
BACKGROUND AND AIMS: The gut microbiota contributes to aberrant inflammation in inflammatory bowel disease, but the bacterial factors causing or exacerbating inflammation are not fully understood. Further, the predictive or prognostic value of gut microbial biomarkers for remission in response to biologic therapy is unclear. METHODS: We perform whole metagenomic sequencing of 550 stool samples from 287 ulcerative colitis patients from a large phase 3 head-to-head study of infliximab and etrolizumab. RESULTS: We identify several bacterial species in baseline and/or post-treatment samples that associate with clinical remission. These include previously described associations (Faecalibacterium prausnitzii_F) as well as new associations with remission to biologic therapy (Flavonifractor plautii). We build multivariate models and find that gut microbial species are better predictors for remission than clinical variables alone. Finally, we describe patient groups that differ in microbiome composition and remission rate after induction therapy, suggesting the potential utility of microbiome-based endotyping. CONCLUSIONS: In this large study of ulcerative colitis patients, we show that few individual species associate strongly with clinical remission, but multivariate models including microbiome can predict clinical remission and have better predictive power compared to clinical data alone.
RESUMO
Ubiquitination is a posttranslational modification that is crucial for the dynamic regulation of diverse signaling pathways. To enhance our understanding of ubiquitination-mediated signaling, we generated a new class of bispecific antibodies that combine recognition of ubiquitination substrates and specific polyubiquitin linkages. RIP1-K63 and RIP1-linear (Lin) linkage polyubiquitin bispecific antibodies detected linkage-specific ubiquitination of the proinflammatory kinase RIP1 in cells and in tissues and revealed RIP1 ubiquitination by immunofluorescence. Similarly, ubiquitination of the RIP1-related kinase RIP2 with K63 or linear linkages was specifically detected with the RIP2-K63 and RIP2-Lin bispecific antibodies, respectively. Furthermore, using the RIP2-K63 and RIP2-Lin bispecific antibodies, we found prominent K63-linked and linear RIP2 ubiquitination in samples from patients with ulcerative colitis and Crohn's disease. We also developed a bispecific antibody (K63-Lin) that simultaneously recognizes K63-linked and linear ubiquitination of components of various signaling pathways. Together, these bispecific antibodies represent a new class of reagents with the potential to be developed for the detection of inflammatory biomarkers.
Assuntos
Anticorpos Biespecíficos , Ubiquitina , Humanos , Anticorpos Biespecíficos/metabolismo , Poliubiquitina/metabolismo , Transdução de Sinais/fisiologia , Ubiquitina/metabolismo , UbiquitinaçãoRESUMO
BACKGROUND AND AIMS: This study aimed to identify microbial drivers of inflammatory bowel disease [IBD], by investigating mucosal-associated bacteria and their detrimental products in IBD patients. METHODS: We directly cultured bacterial communities from mucosal biopsies from paediatric gastrointestinal patients and examined for pathogenicity-associated traits. Upon identifying Clostridium perfringens as toxigenic bacteria present in mucosal biopsies, we isolated strains and further characterized toxicity and prevalence. RESULTS: Mucosal biopsy microbial composition differed from corresponding stool samples. C. perfringens was present in eight of nine patients' mucosal biopsies, correlating with haemolytic activity, but was not present in all corresponding stool samples. Large IBD datasets showed higher C. perfringens prevalence in stool samples of IBD adults [18.7-27.1%] versus healthy controls [5.1%]. In vitro, C. perfringens supernatants were toxic to cell types beneath the intestinal epithelial barrier, including endothelial cells, neuroblasts, and neutrophils, while the impact on epithelial cells was less pronounced, suggesting C. perfringens may be particularly damaging when barrier integrity is compromised. Further characterization using purified toxins and genetic insertion mutants confirmed perfringolysin O [PFO] toxin was sufficient for toxicity. Toxin RNA signatures were found in the original patient biopsies by PCR, suggesting intestinal production. C. perfringens supernatants also induced activation of neuroblast and dorsal root ganglion neurons in vitro, suggesting C. perfringens in inflamed mucosal tissue may directly contribute to abdominal pain, a frequent IBD symptom. CONCLUSIONS: Gastrointestinal carriage of certain toxigenic C. perfringens may have an important pathogenic impact on IBD patients. These findings support routine monitoring of C. perfringens and PFO toxins and potential treatment in patients.
Assuntos
Toxinas Bacterianas , Clostridium perfringens , Fezes , Doenças Inflamatórias Intestinais , Mucosa Intestinal , Humanos , Clostridium perfringens/isolamento & purificação , Clostridium perfringens/genética , Clostridium perfringens/patogenicidade , Criança , Mucosa Intestinal/microbiologia , Mucosa Intestinal/patologia , Doenças Inflamatórias Intestinais/microbiologia , Toxinas Bacterianas/genética , Fezes/microbiologia , Feminino , Masculino , Adolescente , Biópsia , Infecções por Clostridium/microbiologia , Proteínas HemolisinasRESUMO
OBJECTIVES: In Crohn's disease (CD), clinical symptoms correspond poorly to inflammatory disease activity. Biomarkers reflective of mucosal and bowel wall inflammation would be useful to monitor disease activity. The EMBARK study evaluated disease activity in patients with ulcerative colitis (UC) and CD, and used endoscopy with or without cross-sectional imaging for biomarker discovery. METHODS: UC (n=107) and CD (n=157) patients were characterized and underwent ileocolonoscopy (ICO). A subset of CD patients (n=66) also underwent computed tomography enterography (CTE). ICO and CTE were scored by a gastroenterologist and radiologist who incorporated findings of inflammation into a single score (ICO-CTE) for patients that underwent both procedures. Serum and fecal biomarkers were evaluated for association with the Mayo Clinic endoscopy score in UC patients and with ICO alone or ICO-CTE in CD patients. Individual biomarkers with a moderate degree of correlation (P≤0.3) were evaluated using multivariate analysis with model selection using a stepwise procedure. RESULTS: In UC, ordinal logistic regression using Mayo Clinic endoscopy subscore selected the combination of fecal calprotectin and serum matrix metalloproteinase 9 (MMP9; pseudo R(2)=0.353). In CD, we found that use of the ICO-CTE increased specificity of known biomarkers. Using ICO-CTE as the dependent variable for biomarker discovery, the selected biomarkers were the combination of fecal calprotectin, serum MMP9, and serum IL-22 (r=0.699). CONCLUSIONS: Incorporation of both ICO and CTE into a single measure increased biomarker performance in CD. Combinations of fecal calprotectin and serum MMP9 for UC, and combinations of fecal calprotectin, serum MMP9, and serum interleukin-22 in CD, demonstrated the strongest association with imaging/endoscopy-defined inflammation.
Assuntos
Biomarcadores/metabolismo , Doença de Crohn/metabolismo , Fezes/química , Interleucinas/sangue , Complexo Antígeno L1 Leucocitário/metabolismo , Metaloproteinase 9 da Matriz/sangue , Adolescente , Adulto , Idoso , Colonoscopia , Doença de Crohn/diagnóstico por imagem , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Tomografia Computadorizada por Raios X , Interleucina 22RESUMO
Programmed death-1 ligand 1 (PD-L1) is a coinhibitory molecule that negatively regulates multiple tolerance checkpoints. In the NOD mouse model, PD-L1 regulates the development of diabetes. PD-L1 has two binding partners, programmed death-1 and B7-1, but the significance of the PD-L1:B7-1 interaction in regulating self-reactive T cell responses is not yet clear. To investigate this issue in NOD mice, we have compared the effects of two anti-PD-L1 Abs that have different blocking activities. Anti-PD-L1 mAb 10F.2H11 sterically and functionally blocks only PD-L1:B7-1 interactions, whereas anti-PD-L1 mAb 10F.9G2 blocks both PD-L1:B7-1 and PD-L1:programmed death-1 interactions. Both Abs had potent, yet distinct effects in accelerating diabetes in NOD mice: the single-blocker 10F.2H11 mAb was more effective at precipitating diabetes in older (13-wk-old) than in younger (6- to 7-wk-old) mice, whereas the dual-blocker 10F.9G2 mAb rapidly induced diabetes in NOD mice of both ages. Similarly, 10F.2H11 accelerated diabetes in recipients of T cells from diabetic, but not prediabetic mice, whereas 10F.9G2 was effective in both settings. Both anti-PD-L1 mAbs precipitated diabetes in adoptive transfer models of CD4(+) and CD8(+) T cell-driven diabetes. Taken together, these data demonstrate that the PD-L1:B7-1 pathway inhibits potentially pathogenic self-reactive effector CD4(+) and CD8(+) T cell responses in vivo, and suggest that the immunoinhibitory functions of this pathway may be particularly important during the later phases of diabetogenesis.
Assuntos
Antígeno B7-1/fisiologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Diabetes Mellitus Tipo 1/imunologia , Diabetes Mellitus Tipo 1/prevenção & controle , Inibidores do Crescimento/fisiologia , Glicoproteínas de Membrana/fisiologia , Peptídeos/fisiologia , Transdução de Sinais/imunologia , Transferência Adotiva , Animais , Anticorpos Bloqueadores/administração & dosagem , Antígenos CD/fisiologia , Proteínas Reguladoras de Apoptose/fisiologia , Doenças Autoimunes/genética , Doenças Autoimunes/imunologia , Doenças Autoimunes/metabolismo , Antígeno B7-1/genética , Antígeno B7-1/imunologia , Antígeno B7-H1 , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD4-Positivos/transplante , Linfócitos T CD8-Positivos/metabolismo , Linfócitos T CD8-Positivos/transplante , Diabetes Mellitus Tipo 1/genética , Feminino , Ligantes , Glicoproteínas de Membrana/deficiência , Glicoproteínas de Membrana/imunologia , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Camundongos Transgênicos , Peptídeos/deficiência , Peptídeos/imunologia , Receptor de Morte Celular Programada 1 , Ligação Proteica/imunologia , Transdução de Sinais/genéticaRESUMO
BACKGROUND: IL-22 is induced by aryl hydrocarbon receptor (AhR) signaling and plays a critical role in gastrointestinal barrier function through effects on antimicrobial protein production, mucus secretion, and epithelial cell differentiation and proliferation, giving it the potential to modulate the microbiome through these direct and indirect effects. Furthermore, the microbiome can in turn influence IL-22 production through the synthesis of L-tryptophan (L-Trp)-derived AhR ligands, creating the prospect of a host-microbiome feedback loop. We evaluated the impact IL-22 may have on the gut microbiome and its ability to activate host AhR signaling by observing changes in gut microbiome composition, function, and AhR ligand production following exogenous IL-22 treatment in both mice and humans. RESULTS: Microbiome alterations were observed across the gastrointestinal tract of IL-22-treated mice, accompanied by an increased microbial functional capacity for L-Trp metabolism. Bacterially derived indole derivatives were increased in stool from IL-22-treated mice and correlated with increased fecal AhR activity. In humans, reduced fecal concentrations of indole derivatives in ulcerative colitis (UC) patients compared to healthy volunteers were accompanied by a trend towards reduced fecal AhR activity. Following exogenous IL-22 treatment in UC patients, both fecal AhR activity and concentrations of indole derivatives increased over time compared to placebo-treated UC patients. CONCLUSIONS: Overall, our findings indicate IL-22 shapes gut microbiome composition and function, which leads to increased AhR signaling and suggests exogenous IL-22 modulation of the microbiome may have functional significance in a disease setting. Video Abstract.
Assuntos
Microbioma Gastrointestinal , Humanos , Animais , Camundongos , Receptores de Hidrocarboneto Arílico/metabolismo , Interleucinas , Indóis , Interleucina 22RESUMO
Signal regulatory protein (SIRPα) is an immune inhibitory receptor expressed by myeloid cells to inhibit immune cell phagocytosis, migration, and activation. Despite the progress of SIRPα and CD47 antagonist antibodies to promote anti-cancer immunity, it is not yet known whether SIRPα receptor agonism could restrain excessive autoimmune tissue inflammation. Here, we report that neutrophil- and monocyte-associated genes including SIRPA are increased in inflamed tissue biopsies from patients with rheumatoid arthritis and inflammatory bowel diseases, and elevated SIRPA is associated with treatment-refractory ulcerative colitis. We next identify an agonistic anti-SIRPα antibody that exhibits potent anti-inflammatory effects in reducing neutrophil and monocyte chemotaxis and tissue infiltration. In preclinical models of arthritis and colitis, anti-SIRPα agonistic antibody ameliorates autoimmune joint inflammation and inflammatory colitis by reducing neutrophils and monocytes in tissues. Our work provides a proof of concept for SIRPα receptor agonism for suppressing excessive innate immune activation and chronic inflammatory disease treatment.
Assuntos
Colite , Neoplasias , Humanos , Fagocitose , Neoplasias/tratamento farmacológico , Neutrófilos/metabolismo , Inflamação/patologia , Colite/metabolismoRESUMO
Programmed death 1 (PD-1), an inhibitory receptor expressed on activated lymphocytes, regulates tolerance and autoimmunity. PD-1 has two ligands: PD-1 ligand 1 (PD-L1), which is expressed broadly on hematopoietic and parenchymal cells, including pancreatic islet cells; and PD-L2, which is restricted to macrophages and dendritic cells. To investigate whether PD-L1 and PD-L2 have synergistic or unique roles in regulating T cell activation and tolerance, we generated mice lacking PD-L1 and PD-L2 (PD-L1/PD-L2(-/-) mice) and compared them to mice lacking either PD-L. PD-L1 and PD-L2 have overlapping functions in inhibiting interleukin-2 and interferon-gamma production during T cell activation. However, PD-L1 has a unique and critical role in controlling self-reactive T cells in the pancreas. Our studies with bone marrow chimeras demonstrate that PD-L1/PD-L2 expression only on antigen-presenting cells is insufficient to prevent the early onset diabetes that develops in PD-L1/PD-L2(-/-) non-obese diabetic mice. PD-L1 expression in islets protects against immunopathology after transplantation of syngeneic islets into diabetic recipients. PD-L1 inhibits pathogenic self-reactive CD4+ T cell-mediated tissue destruction and effector cytokine production. These data provide evidence that PD-L1 expression on parenchymal cells rather than hematopoietic cells protects against autoimmune diabetes and point to a novel role for PD-1-PD-L1 interactions in mediating tissue tolerance.