RESUMO
OBJECTIVE: Mucosal T cells play a major role in inflammatory bowel disease (IBD). However, their immunometabolism during intestinal inflammation is poorly understood. Due to its impact on cellular metabolism and proinflammatory immune cell function, we here focus on the enzyme ATP citrate lyase (ACLY) in mucosal T cell immunometabolism and its relevance for IBD. DESIGN: ACLY expression and its immunometabolic impact on colitogenic T cell function were analysed in mucosal T cells from patients with IBD and in two experimental colitis models. RESULTS: ACLY was markedly expressed in colon tissue under steady-state conditions but was significantly downregulated in lamina propria mononuclear cells in experimental dextran sodium sulfate-induced colitis and in CD4+ and to a lesser extent in CD8+ T cells infiltrating the inflamed gut in patients with IBD. ACLY-deficient CD4+ T cells showed an impaired capacity to induce intestinal inflammation in a transfer colitis model as compared with wild-type T cells. Assessment of T cell immunometabolism revealed that ACLY deficiency dampened the production of IBD-relevant cytokines and impaired glycolytic ATP production but enriched metabolites involved in the biosynthesis of phospholipids and phosphatidylcholine. Interestingly, the short-chain fatty acid butyrate was identified as a potent suppressor of ACLY expression in T cells, while IL-36α and resolvin E1 induced ACLY levels. In a translational approach, in vivo administration of the butyrate prodrug tributyrin downregulated mucosal infiltration of ACLYhigh CD4+ T cells and ameliorated chronic colitis. CONCLUSION: ACLY controls mucosal T cell immunometabolism and experimental colitis. Therapeutic modulation of ACLY expression in T cells emerges as a novel strategy to promote the resolution of intestinal inflammation.
Assuntos
Colite , Doenças Inflamatórias Intestinais , Linfócitos Intraepiteliais , Humanos , Animais , Linfócitos Intraepiteliais/metabolismo , ATP Citrato (pro-S)-Liase/metabolismo , Linfócitos T CD8-Positivos/metabolismo , Colite/metabolismo , Inflamação/metabolismo , Butiratos , Mucosa Intestinal/metabolismo , Sulfato de Dextrana , Modelos Animais de DoençasRESUMO
OBJECTIVE: The anti-α4ß7 integrin antibody vedolizumab is administered at a fixed dose for the treatment of IBDs. This leads to a wide range of serum concentrations in patients and previous studies had suggested that highest exposure levels are associated with suboptimal clinical response. We aimed to determine the mechanisms underlying these non-linear exposure-efficacy characteristics of vedolizumab. DESIGN: We characterised over 500 samples from more than 300 subjects. We studied the binding of vedolizumab to T cells and investigated the functional consequences for dynamic adhesion, transmigration, gut homing and free binding sites in vivo. Employing single-cell RNA sequencing, we characterised α4ß7 integrin-expressing T cell populations 'resistant' to vedolizumab and validated our findings in vitro and in samples from vedolizumab-treated patients with IBD. We also correlated our findings with a post-hoc analysis of the Gemini II and III studies. RESULTS: Regulatory T (TReg) cells exhibited a right-shifted vedolizumab binding profile compared with effector T (TEff) cells. Consistently, in a certain concentration range, the residual adhesion, transmigration, homing of and availability of functional α4ß7 on TReg cells in vivo was higher than that of/on TEff cells. We identified a vedolizumab-'resistant' α4ß7-expressing ß1+PI16+ TReg cell subset with pronounced regulatory properties as the substrate for this effect. Our observations correlated with exposure-efficacy data from Gemini II and III trials. CONCLUSION: Completely blocking TEff cell trafficking with vedolizumab, while simultaneously permitting residual homing of powerful TReg cells in an optimal 'therapeutic window' based on target exposure levels might be a strategy to optimise treatment outcomes in patients with IBD.
Assuntos
Fármacos Gastrointestinais , Doenças Inflamatórias Intestinais , Anticorpos Monoclonais Humanizados , Proteínas de Transporte , Fármacos Gastrointestinais/farmacologia , Fármacos Gastrointestinais/uso terapêutico , Glicoproteínas/metabolismo , Glicoproteínas/uso terapêutico , Humanos , Doenças Inflamatórias Intestinais/tratamento farmacológico , Integrinas , Linfócitos T Reguladores/metabolismoRESUMO
OBJECTIVE: To study the role of α4ß7 integrin for gut homing of monocytes and to explore the biological consequences of therapeutic α4ß7 inhibition with regard to intestinal wound healing. DESIGN: We studied the expression of homing markers on monocyte subsets in the peripheral blood and on macrophage subsets in the gut of patients with IBD and controls with flow cytometry and immunohistochemistry. Integrin function was addressed with dynamic adhesion assays and in vivo gut homing assays. In vivo wound healing was studied in mice deficient for or depleted of α4ß7 integrin. RESULTS: Classical and non-classical monocytes were clearly dichotomous regarding homing marker expression including relevant expression of α4ß7 integrin on human and mouse non-classical monocytes but not on classical monocytes. Monocyte-expressed α4ß7 integrin was functionally important for dynamic adhesion to mucosal vascular addressin cell adhesion molecule 1 and in vivo gut homing. Impaired α4ß7-dependent gut homing was associated with reduced (effect size about 20%) and delayed wound healing and suppressed perilesional presence of wound healing macrophages. Non-classical monocytes in the peripheral blood were increased in patients with IBD under clinical treatment with vedolizumab. CONCLUSION: In addition to reported effects on lymphocytes, anti-α4ß7 therapy in IBD also targets non-classical monocytes. Impaired gut homing of such monocytes might lead to a reduction of wound healing macrophages and could potentially explain increased rates of postoperative complications in vedolizumab-treated patients, which have been observed in some studies.
Assuntos
Doenças Inflamatórias Intestinais/patologia , Integrinas/fisiologia , Intestinos/patologia , Monócitos/fisiologia , Cicatrização/fisiologia , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Anticorpos Monoclonais Humanizados/farmacologia , Estudos de Casos e Controles , Adesão Celular/efeitos dos fármacos , Adesão Celular/fisiologia , Quimiotaxia de Leucócito/fisiologia , Feminino , Fármacos Gastrointestinais/farmacologia , Humanos , Doenças Inflamatórias Intestinais/sangue , Doenças Inflamatórias Intestinais/fisiopatologia , Integrinas/antagonistas & inibidores , Integrinas/sangue , Mucosa Intestinal/metabolismo , Intestinos/fisiologia , Masculino , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Monócitos/metabolismo , Cicatrização/efeitos dos fármacos , Adulto JovemRESUMO
BACKGROUND & AIMS: It is not clear how regulation of T-cell function is altered during development of inflammatory bowel diseases (IBD). We studied the mechanisms by which geranylgeranyltransferase-mediated prenylation controls T-cell localization to the intestine and chronic inflammation. METHODS: We generated mice with T-cell-specific disruption of the geranylgeranyltransferase type I, beta subunit gene (Pggt1b), called Pggt1bΔCD4 mice, or the ras homolog family member A gene (Rhoa), called RhoaΔCD4 mice. We also studied mice with knockout of CDC42 or RAC1 and wild-type mice (controls). Intestinal tissues were analyzed by histology, multiphoton and confocal microscopy, and real-time polymerase chain reaction. Activation of CDC42, RAC1, and RHOA were measured with G-LISA, cell fractionation, and immunoblots. T cells and lamina propria mononuclear cells from mice were analyzed by flow cytometry or transferred to Rag1-/- mice. Mice were given injections of antibodies against integrin alpha4beta7 or gavaged with the RORC antagonist GSK805. We obtained peripheral blood and intestinal tissue samples from patients with and without IBD and analyzed them by flow cytometry. RESULTS: Pggt1bΔCD4 mice developed spontaneous colitis, characterized by thickening of the intestinal wall, edema, fibrosis, accumulation of T cells in the colon, and increased expression of inflammatory cytokines. Compared with control CD4+ T cells, PGGT1B-deficient CD4+ T cells expressed significantly higher levels of integrin alpha4beta7, which regulates their localization to the intestine. Inflammation induced by transfer of PGGT1B-deficient CD4+ T cells to Rag1-/- mice was blocked by injection of an antibody against integrin alpha4beta7. Lamina propria of Pggt1bΔCD4 mice had increased numbers of CD4+ T cells that expressed RORC and higher levels of cytokines produced by T-helper 17 cells (granulocyte-macrophage colony-stimulating factor, interleukin [IL]17A, IL17F, IL22, and tumor necrosis factor [TNF]). The RORC inverse agonist GSK805, but not antibodies against IL17A or IL17F, prevented colitis in Pggt1bΔCD4 mice. PGGT1B-deficient CD4+ T cells had decreased activation of RHOA. RhoAΔCD4 mice had a similar phenotype to Pggt1bΔCD4 mice, including development of colitis, increased numbers of CD4+ T cells in colon, increased expression of integrin alpha4beta7 by CD4+ T cells, and increased levels of IL17A and other inflammatory cytokines in lamina propria. T cells isolated from intestinal tissues from patients with IBD had significantly lower levels of PGGT1B than tissues from individuals without IBD. CONCLUSION: Loss of PGGT1B from T cells in mice impairs RHOA function, increasing CD4+ T-cell expression of integrin alpha4beta7 and localization to colon, resulting in increased expression of inflammatory cytokines and colitis. T cells isolated from gut tissues from patients with IBD have lower levels of PGGT1B than tissues from patients without IBD.
Assuntos
Alquil e Aril Transferases/deficiência , Quimiotaxia de Leucócito , Colite/enzimologia , Colo/enzimologia , Integrinas/metabolismo , Linfócitos T/enzimologia , Proteínas rho de Ligação ao GTP/metabolismo , Imunidade Adaptativa , Alquil e Aril Transferases/genética , Animais , Estudos de Casos e Controles , Células Cultivadas , Colite/genética , Colite/imunologia , Colite/patologia , Colo/imunologia , Colo/patologia , Citocinas/metabolismo , Modelos Animais de Doenças , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Humanos , Imunidade Inata , Mediadores da Inflamação/metabolismo , Ativação Linfocitária , Camundongos Knockout , Neuropeptídeos/genética , Neuropeptídeos/metabolismo , Transdução de Sinais , Linfócitos T/imunologia , Linfócitos T/patologia , Proteína cdc42 de Ligação ao GTP/genética , Proteína cdc42 de Ligação ao GTP/metabolismo , Proteínas rac1 de Ligação ao GTP/genética , Proteínas rac1 de Ligação ao GTP/metabolismo , Proteínas rho de Ligação ao GTP/deficiência , Proteínas rho de Ligação ao GTP/genética , Proteína rhoA de Ligação ao GTPRESUMO
BACKGROUND: Age-related macular degeneration (AMD) is the leading cause of vision loss in Western societies with a strong genetic component. Candidate gene studies as well as genome-wide association studies strongly implicated genetic variations in complement genes to be involved in disease risk. So far, no association of AMD with complement component 4 (C4) was reported probably due to the complex nature of the C4 locus on chromosome 6. METHODS: We used multiplex ligation-dependent probe amplification (MLPA) to determine the copy number of the C4 gene as well as of both relevant isoforms, C4A and C4B, and assessed their association with AMD using logistic regression models. RESULTS: Here, we report on the analysis of 2645 individuals (1536 probands and 1109 unaffected controls), across three different centers, for multiallelic copy number variation (CNV) at the C4 locus. We find strong statistical significance for association of increased copy number of C4A (OR 0.81 (0.73; 0.89);P = 4.4 × 10(-5)), with the effect most pronounced in individuals over 78 years (OR 0.67 (0.55; 0.81)) and females (OR 0.77 (0.68; 0.87)). Furthermore, this association is independent of known AMD-associated risk variants in the nearby CFB/C2 locus, particularly in females and in individuals over 78 years. CONCLUSIONS: Our data strengthen the notion that complement dysregulation plays a crucial role in AMD etiology, an important finding for early intervention strategies and future therapeutics. In addition, for the first time, we provide evidence that multiallelic CNVs are associated with AMD pathology.
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Complemento C4a/genética , Dosagem de Genes , Predisposição Genética para Doença/genética , Degeneração Macular/genética , Alelos , Estudos de Coortes , Feminino , Estudo de Associação Genômica Ampla , Genótipo , Humanos , Masculino , Reação em Cadeia da Polimerase MultiplexRESUMO
Generalized pustular psoriasis is a severe psoriatic subtype characterized by epidermal neutrophil infiltration. Although variants in IL36RN and MPO have been shown to affect immune cells, a systematic analysis of neutrophils and PBMC subsets and their differential gene expression dependent on MPO genotypes was not performed yet. We assessed the transcriptomes of MPO-deficient patients using single-cell RNA sequencing of PBMCs and RNA sequencing of neutrophils in a stable disease state. Cell-type annotation by multimodal reference mapping of single-cell RNA-sequencing data was verified by flow cytometry of surface and intracellular markers; the proportions of CD4+ cytotoxic T lymphocytes and other CD4+ effector cells were increased in generalized pustular psoriasis, whereas the frequencies of naïve CD4+ T cells were significantly lower. The expression of FGFBP2 marking CD4+ cytotoxic T lymphocytes and CD8+ effector memory T cells was elevated in patients with generalized pustular psoriasis with disease-contributing variants compared with that in noncarriers (P = 0.0015). In neutrophils, differentially expressed genes were significantly enriched in genes of the classical complement activation pathway. Future studies assessing affected cell types and pathways will show their contribution to generalized pustular psoriasis's pathogenesis and indicate whether findings can be transferred to the acute epidermal situation and whether depletion or inactivation of CD4+ cytotoxic T lymphocytes may be a reasonable therapeutic approach.
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Peroxidase , Psoríase , Dermatopatias Vesiculobolhosas , Transcriptoma , Doença Aguda , Linfócitos T CD4-Positivos/patologia , Doença Crônica , Humanos , Leucócitos Mononucleares/patologia , Peroxidase/deficiência , Psoríase/patologia , Dermatopatias Vesiculobolhosas/patologia , Linfócitos T CitotóxicosRESUMO
The occurrence of epithelial defects in the gut relevantly contributes to the pathogenesis of inflammatory bowel diseases (IBD), whereby the impairment of intestinal epithelial barrier integrity seems to represent a primary trigger as well as a disease amplifying consequence of the chronic inflammatory process. Besides epithelial cell intrinsic factors, accumulated and overwhelmingly activated immune cells and their secretome have been identified as critical modulators of the pathologically altered intestinal epithelial cell (IEC) function in IBD. In this context, over the last 10 years increasing levels of attention have been paid to the group of innate lymphoid cells (ILCs). This is in particular due to a preferential location of these rather newly described innate immune cells in close proximity to mucosal barriers, their profound capacity to secrete effector cytokines and their numerical and functional alteration under chronic inflammatory conditions. Aiming on a comprehensive and updated summary of our current understanding of the bidirectional mucosal crosstalk between ILCs and IECs, this review article will in particular focus on the potential capacity of gut infiltrating type-1, type-2, and type-3 helper ILCs (ILC1s, ILC2s, and ILC3s, respectively) to impact on the survival, differentiation, and barrier function of IECs. Based on data acquired in IBD patients or in experimental models of colitis, we will discuss whether the different ILC subgroups could serve as potential therapeutic targets for maintenance of epithelial integrity and/or mucosal healing in IBD.
RESUMO
During the last decade, group-2 innate lymphoid cells (ILC2s) have been discovered and successfully established as crucial mediators of lung allergy, airway inflammation and fibrosis, thus affecting the pathogenesis and clinical course of many respiratory diseases, like for instance asthma, cystic fibrosis and chronic rhinosinusitis. As an important regulatory component in this context, the local pulmonary milieu at inflammatory tissue sites does not only determine the activation status of lung-infiltrating ILC2s, but also influences their motility and migratory behavior. In general, many data collected in recent murine and human studies argued against the former concept of a very strict tissue residency of innate lymphoid cells (ILCs) and instead pointed to a context-dependent homing capacity of peripheral blood ILC precursors and the inflammation-dependent capacity of specific ILC subsets for interorgan trafficking. In this review article, we provide a comprehensive overview of the so far described molecular mechanisms underlying the pulmonary migration of ILC2s and thereby the numeric regulation of local ILC2 pools at inflamed or fibrotic pulmonary tissue sites and discuss their potential to serve as innovative therapeutic targets in the treatment of inflammatory lung diseases.
Assuntos
Quimiotaxia de Leucócito , Imunidade Inata , Pulmão/imunologia , Linfócitos/imunologia , Pneumonia/imunologia , Animais , Humanos , Mediadores da Inflamação/metabolismo , Pulmão/metabolismo , Pulmão/patologia , Linfócitos/metabolismo , Fenótipo , Pneumonia/metabolismo , Pneumonia/patologia , Transdução de SinaisRESUMO
Since their identification as a unique cell population, innate lymphoid cells (ILCs) have revolutionized our understanding of immune responses, leaving their impact on multiple inflammatory and fibrotic pathologies without doubt. Thus, a tightly controlled regulation of local ILC numbers and their activity is of crucial importance. Even though this has been extensively studied in murine ILCs in the last few years, our knowledge of human ILCs is still lagging behind. Our review article will therefore summarize recent insights into the function of human ILCs and will particularly focus on their regulation under inflammatory conditions. The quality and intensity of ILC involvement into local immune responses at mucosal sites of the human body can potentially be modulated via three different axes: (1) activation of tissue-resident mature ILCs, (2) plasticity and local transdifferentiation of specific ILC subsets, and (3) tissue migration and accumulation of peripheral ILCs. Despite a still ongoing scientific effort in this field, already existing data on the fate of human ILCs under different pathologic conditions clearly indicate that all three of these mechanisms are of relevance for the clinical course of chronic inflammatory and autoimmune diseases and might likewise provide new target structures for future therapeutic strategies.
Assuntos
Imunidade Inata , Imunidade nas Mucosas , Inflamação/imunologia , Linfócitos/imunologia , Diferenciação Celular/imunologia , Movimento Celular/imunologia , Citocinas/imunologia , Humanos , Imunomodulação , Inflamação/patologia , Subpopulações de Linfócitos/imunologia , Subpopulações de Linfócitos/patologia , Linfócitos/patologia , Modelos ImunológicosRESUMO
Cystic fibrosis patients suffer from a progressive, often fatal lung disease, which is based on a complex interplay between chronic infections, locally accumulating immune cells and pulmonary tissue remodeling. Although group-2 innate lymphoid cells (ILC2s) act as crucial initiators of lung inflammation, our understanding of their involvement in the pathogenesis of cystic fibrosis remains incomplete. Here we report a marked decrease of circulating CCR6+ ILC2s in the blood of cystic fibrosis patients, which significantly correlated with high disease severity and advanced pulmonary failure, strongly implicating increased ILC2 homing from the peripheral blood to the chronically inflamed lung tissue in cystic fibrosis patients. On a functional level, the CCR6 ligand CCL20 was identified as potent promoter of lung-directed ILC2 migration upon inflammatory conditions in vitro and in vivo using a new humanized mouse model with light-sheet fluorescence microscopic visualization of lung-accumulated human ILC2s. In the lung, blood-derived human ILC2s were able to augment local eosinophil and neutrophil accumulation and induced a marked upregulation of pulmonary type-VI collagen expression. Studies in primary human lung fibroblasts additionally revealed ILC2-derived IL-4 and IL-13 as important mediators of this type-VI collagen-inducing effect. Taken together, the here acquired results suggest that pathologically increased CCL20 levels in cystic fibrosis airways induce CCR6-mediated lung homing of circulating human ILC2s. Subsequent ILC2 activation then triggers local production of type-VI collagen and might thereby drive extracellular matrix remodeling potentially influencing pulmonary tissue destruction in cystic fibrosis patients. Thus, modulating the lung homing capacity of circulating ILC2s and their local effector functions opens new therapeutic avenues for cystic fibrosis treatment.
Assuntos
Fibrose Cística/sangue , Imunidade Inata , Pulmão/imunologia , Ativação Linfocitária , Linfócitos/imunologia , Receptores CCR6/metabolismo , Insuficiência Respiratória/imunologia , Adolescente , Adulto , Idoso , Animais , Artrite Reumatoide/sangue , Movimento Celular/imunologia , Quimiocina CCL20/metabolismo , Modelos Animais de Doenças , Feminino , Humanos , Doenças Inflamatórias Intestinais/sangue , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Adulto JovemRESUMO
Overwhelming tissue accumulation of highly activated immune cells represents a hallmark of various chronic inflammatory diseases and emerged as an attractive therapeutic target in the clinical management of affected patients. In order to further optimize strategies aiming at therapeutic regulation of pathologically imbalanced tissue infiltration of pro-inflammatory immune cells, it will be of particular importance to achieve improved insights into disease- and organ-specific homing properties of peripheral lymphocytes. The here described experimental protocol allows to monitor lung accumulation of fluorescently labeled and adoptively transferred human lymphocytes in the context of papain-induced pulmonary inflammation. In contrast to standard in vitro assays frequently used for the analysis of immune cell migration and chemotaxis, the now introduced in vivo setting takes into account lung-specific aspects of tissue organization and the influence of the complex inflammatory scenario taking place in the living murine organism. Moreover, three-dimensional cross-sectional light-sheet fluorescence microscopic imaging does not only provide quantitative data on infiltrating immune cells, but also depicts the pattern of immune cell localization within the inflamed lung. Overall, we are able to introduce an innovative technique of high value for immunological research in the field of chronic inflammatory lung diseases, which can be easily applied by following the provided step-by-step protocol.
Assuntos
Diagnóstico por Imagem , Hipersensibilidade/diagnóstico , Inflamação/diagnóstico , Pulmão/patologia , Linfócitos/patologia , Microscopia/métodos , Animais , Estudos Transversais , Modelos Animais de Doenças , Humanos , Hipersensibilidade/complicações , Inflamação/complicações , Inflamação/patologia , Camundongos Endogâmicos C57BL , Gravação em VídeoRESUMO
Group 2 innate lymphoid cells (ILC2s) possess indispensable roles during type 2-mediated inflammatory diseases. Although their physiological and detrimental immune functions seem to depend on the anatomical compartment they reside, their tissue tropism and the molecular and immunological processes regulating the self-renewal of the local pool of ILC2s in the context of inflammation or infection are incompletely understood. Here, we analyzed the role of the CC-chemokine receptor CCR8 for the biological functions of ILC2s. In vitro and in vivo experiments indicated that CCR8 is in comparison to the related molecule CCR4 less important for migration of these cells. However, we found that activated mouse and human ILC2s produce the CCR8 ligand CCL1 and are a major source of CCL1 in vivo. CCL1 signaling to ILC2s regulates their proliferation and supports their capacity to protect against helminthic infections. In summary, we identify a novel chemokine receptor-dependent mechanism by which ILC2s are regulated during type 2 responses.