RESUMO
Th17 cells are enriched in the gut mucosa and play a critical role in maintenance of the mucosal barrier and host defense against extracellular bacteria and fungal infections. During chronic human immunodeficiency virus (HIV) infection, Th17 cells were more depleted compared to Th1 cells, even when the patients had low or undetectable viremia. To investigate the differential effects of HIV infection on Th17 and Th1 cells, a culture system was used in which CCR6(+) CD4(+) T cells were sorted from healthy human peripheral blood and activated in the presence of interleukin 1ß (IL-1ß) and IL-23 to drive expansion of Th17 cells while maintaining Th1 cells. HIV infection of these cultures had minimal effects on Th1 cells but caused depletion of Th17 cells. Th17 loss correlated with greater levels of virus-infected cells and cell death. In identifying cellular factors contributing to higher susceptibility of Th17 cells to HIV, we compared Th17-enriched CCR6(+) and Th17-depleted CCR6(-) CD4 T cell cultures and noted that Th17-enriched CCR6(+) cells expressed higher levels of α4ß7 and bound HIV envelope in an α4ß7-dependent manner. The cells also had greater expression of CD4 and CXCR4, but not CCR5, than CCR6(-) cells. Moreover, unlike Th1 cells, Th17 cells produced little CCR5 ligand, and transfection with one of the CCR5 ligands, MIP-1ß (CCL4), increased their resistance against HIV. These results indicate that features unique to Th17 cells, including higher expression of HIV receptors and lack of autocrine CCR5 ligands, are associated with enhanced permissiveness of these cells to HIV.
Assuntos
Infecções por HIV/virologia , HIV-1/patogenicidade , Receptores CCR5/metabolismo , Receptores CCR6/metabolismo , Receptores Virais/metabolismo , Células Th1/virologia , Células Th17/virologia , Apoptose , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD4-Positivos/virologia , Células Cultivadas , Quimiocina CCL3/metabolismo , Quimiocina CCL4/metabolismo , Quimiocina CCL5/metabolismo , Citocinas/metabolismo , Infecções por HIV/imunologia , Infecções por HIV/metabolismo , Humanos , Interleucina-17/metabolismo , Receptores CXCR4/metabolismo , Células Th1/imunologia , Células Th1/metabolismo , Células Th17/imunologia , Células Th17/metabolismo , Viremia/metabolismo , Viremia/patologia , Internalização do Vírus , Replicação ViralRESUMO
BACKGROUND: Inflammatory bowel diseases (IBD) are believed to be driven by dysregulated interactions between the host and the gut microbiota. Our goal is to characterize and infer relationships between mucosal T cells, the host tissue environment, and microbial communities in patients with IBD who will serve as basis for mechanistic studies on human IBD. METHODS: We characterized mucosal CD4 T cells using flow cytometry, along with matching mucosal global gene expression and microbial communities data from 35 pinch biopsy samples from patients with IBD. We analyzed these data sets using an integrated framework to identify predictors of inflammatory states and then reproduced some of the putative relationships formed among these predictors by analyzing data from the pediatric RISK cohort. RESULTS: We identified 26 predictors from our combined data set that were effective in distinguishing between regions of the intestine undergoing active inflammation and regions that were normal. Network analysis on these 26 predictors revealed SAA1 as the most connected node linking the abundance of the genus Bacteroides with the production of IL17 and IL22 by CD4 T cells. These SAA1-linked microbial and transcriptome interactions were further reproduced with data from the pediatric IBD RISK cohort. CONCLUSIONS: This study identifies expression of SAA1 as an important link between mucosal T cells, microbial communities, and their tissue environment in patients with IBD. A combination of T cell effector function data, gene expression and microbial profiling can distinguish between intestinal inflammatory states in IBD regardless of disease types.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Microbioma Gastrointestinal/imunologia , Doenças Inflamatórias Intestinais/genética , Doenças Inflamatórias Intestinais/imunologia , Proteína Amiloide A Sérica/fisiologia , Adulto , Biópsia , Estudos de Casos e Controles , Criança , Colo/imunologia , Colo/microbiologia , Colo/patologia , Expressão Gênica , Humanos , Imunidade Celular , Doenças Inflamatórias Intestinais/patologia , Interleucina-17/biossíntese , Interleucinas/biossíntese , Mucosa Intestinal/imunologia , Mucosa Intestinal/microbiologia , Mucosa Intestinal/patologia , Células Th17/imunologia , Interleucina 22RESUMO
BACKGROUND: Experiential learning in medical education, as exemplified by objective structured clinical examinations (OSCEs), is a well-validated approach for improving trainee performance. Furthermore, the Accreditation Council for Graduate Medical Education has identified OSCEs as an ideal method for assessing the core competency of interpersonal and communication skills. Here, we describe a novel educational tool, the inflammatory bowel disease OSCE (IBD OSCE), to assess and improve this clinical skill set in Gastroenterology fellows. METHODS: We developed a 4-station IBD OSCE that assessed shared decision making, physician-physician communication, and physician-patient consultative skills specifically related to the care of patients with IBD. Each station was videotaped and observed live by faculty gastroenterologists. Behaviorally anchored checklists were scored independently by a faculty observer and the standardized patient/physician, who both provided feedback to the fellow immediately after each case. Post-OSCE, fellows attended a debriefing session on patient communication and were surveyed to assess their perspective on the examination's educational value. RESULTS: Twelve second-year gastroenterology fellows from 5 fellowship programs participated in the IBD OSCE. Fellows performed well in all measured domains and rated the experience highly for its educational value. Fellows cited IBD as an area of relative deficiency in their education compared with other knowledge areas within gastroenterology. CONCLUSIONS: To our knowledge, this is the first OSCE designed specifically for the evaluation of skills as they relate to IBD management. Using OSCEs for IBD education provides an opportunity to robustly assess core competencies and the role of the physician as an educator.
Assuntos
Competência Clínica , Gastroenterologia/educação , Doenças Inflamatórias Intestinais , Aprendizagem Baseada em Problemas/métodos , Tomada de Decisões , Bolsas de Estudo , Humanos , Doenças Inflamatórias Intestinais/diagnóstico , Doenças Inflamatórias Intestinais/terapia , Relações Médico-Paciente , Projetos PilotoRESUMO
Much of our understanding of gut-microbial interactions has come from mouse models. Intestinal immunity is complex and a combination of host genetics and environmental factors play a significant role in regulating intestinal immunity. Due to this complexity, no mouse model to date gives a complete and accurate representation of human intestinal diseases, such as inflammatory bowel diseases. However, intestinal tissue from patients undergoing bowel resection reflects a condition of severe disease that has failed treatment; hence a more dynamic perspective of varying inflammatory states in IBD could be obtained through the analyses of pinch biopsy material. Here we describe our protocol for analyzing mucosal pinch biopsies collected predominantly during colonoscopies. We have optimized flow cytometry panels to analyze up to 8 cytokines produced by CD4+ and CD8+ cells, as well as for characterizing nuclear proteins and transcription factors such as Ki67 and Foxp3. Furthermore, we have optimized approaches to analyze the production of cytokines, including TGF-beta from direct ex vivo cultures of pinch biopsies and LPMCs isolated from biopsies. These approaches are part of our workflow to try and understand the role of the gut microbiota in complex and dynamic human intestinal diseases.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Colite Ulcerativa/imunologia , Doença de Crohn/imunologia , Citocinas/metabolismo , Biópsia , Linfócitos T CD4-Positivos/citologia , Linfócitos T CD8-Positivos/citologia , Colo/citologia , Colo/imunologia , Colonoscopia , Fatores de Transcrição Forkhead/metabolismo , Humanos , Mucosa Intestinal/citologia , Mucosa Intestinal/imunologia , Antígeno Ki-67/metabolismo , Microbiota/imunologia , Fator de Crescimento Transformador beta/metabolismoRESUMO
The mucosal microbiota lives in close proximity with the intestinal epithelium and may interact more directly with the host immune system than the luminal/fecal bacteria. The availability of nutrients in the mucus layer of the epithelium is also very different from the gut lumen environment. Inferred metagenomic analysis for microbial function of the mucosal microbiota is possible by PICRUSt. We recently found that by using this approach, actively inflamed tissue of ulcerative colitis (UC) patients have mucosal communities enriched for genes involved in lipid and amino acid metabolism, and reduced for carbohydrate and nucleotide metabolism. Here, we find that the same bacterial taxa (e.g. Acinetobacter) and predicted microbial pathways enriched in actively inflamed colitis tissue are also enriched in the mucosa of subjects undergoing routine screening colonoscopies, when compared with paired samples of luminal/fecal bacteria. These results suggest that the mucosa of healthy individuals may be a reservoir of aerotolerant microbial communities expanded during colitis.
Assuntos
Biota , Colite/microbiologia , Fezes/microbiologia , Mucosa Intestinal/microbiologia , Redes e Vias Metabólicas/genética , Humanos , Masculino , Pessoa de Meia-IdadeRESUMO
BACKGROUND: Inflammation during inflammatory bowel disease may alter nutrient availability to adherent mucosal bacteria and impact their metabolic function. Microbial metabolites may regulate intestinal CD4 T-cell homeostasis. We investigated the relationship between inflammation and microbial function by inferred metagenomics of the mucosal microbiota from colonic pinch biopsies of patients with inflammatory bowel disease. METHODS: Paired pinch biopsy samples of known inflammation states were analyzed from ulcerative colitis (UC) (23), Crohn's disease (CD) (21), and control (24) subjects by 16S ribosomal sequencing, histopathologic assessment, and flow cytometry. PICRUSt was used to generate metagenomic data and derive relative Kyoto Encyclopedia of Genes and Genomes Pathway abundance information. Leukocytes were isolated from paired biopsy samples and analyzed by multicolor flow cytometry. Active inflammation was defined by neutrophil infiltration into the epithelium. RESULTS: Carriage of metabolic pathways in the mucosal microbiota was relatively stable among patients with inflammatory bowel disease, despite large variations in individual bacterial community structures. However, microbial function was significantly altered in inflamed tissue of UC patients, with a reduction in carbohydrate and nucleotide metabolism in favor of increased lipid and amino acid metabolism. These differences were not observed in samples from CD patients. In CD, microbial lipid, carbohydrate, and amino acid metabolism tightly correlated with the frequency of CD4Foxp3 Tregs, whereas in UC, these pathways correlated with the frequency of CD4IL-22 (TH22) cells. CONCLUSIONS: Metabolic pathways of the mucosal microbiota in CD do not vary as much as UC with inflammation state, indicating a more systemic perturbation of host-bacteria interactions in CD compared with more localized dysfunction in UC.
Assuntos
Aminoácidos/metabolismo , Metabolismo dos Carboidratos/fisiologia , Colite Ulcerativa , Doença de Crohn , Metabolismo dos Lipídeos/fisiologia , Microbiota/fisiologia , Nucleotídeos/metabolismo , Contagem de Linfócito CD4 , Colite Ulcerativa/microbiologia , Colo/microbiologia , Doença de Crohn/microbiologia , Humanos , Inflamação/microbiologia , Mucosa Intestinal/microbiologia , Redes e Vias Metabólicas , Metagenômica , Linfócitos T ReguladoresRESUMO
A 38-year-old man with a history of HIV infection virologically suppressed on antiretroviral therapy presents to his gastroenterologist for evaluation of iron deficiency anemia and weight loss. A diagnostic colonoscopy demonstrates a two-centimeter ulcerated mass in the cecum. Biopsies of the lesion return moderately differentiated adenocarcinoma that is wild type for the KRAS mutation by real-time PCR.
Assuntos
Antirretrovirais/administração & dosagem , Trato Gastrointestinal/imunologia , Prebióticos , Probióticos/administração & dosagem , Síndrome de Imunodeficiência Adquirida dos Símios/imunologia , Síndrome de Imunodeficiência Adquirida dos Símios/terapia , Animais , HumanosRESUMO
The epidemiology of autoimmune diseases and helminth infections led to suggestions that helminths could improve inflammatory conditions, which was then tested using animal models. This has translated to clinical investigations aimed at the safe and controlled reintroduction of helminthic exposure to patients suffering from autoimmune diseases (so-called 'helminthic therapy') in an effort to mitigate the inflammatory response. In this review, we summarize the results of recent clinical trials of helminthic therapy, with particular attention to mechanisms of action. Whereas previous reviews have emphasized immune regulatory mechanisms activated by helminths, we propose that enhancement of mucosal barrier function may have an equally important role in improving conditions of inflammatory bowel diseases.
Assuntos
Doenças Autoimunes/terapia , Helmintíase/imunologia , Helmintos/imunologia , Mucosa/imunologia , Terapia com Helmintos , Animais , Doenças Autoimunes/parasitologia , Ensaios Clínicos como Assunto , Humanos , Resultado do TratamentoRESUMO
TGF-ß1 and VEGF, both angiogenesis inducers, have opposing effects on vascular endothelial cells. TGF-ß1 induces apoptosis; VEGF induces survival. We have previously shown that TGF-ß1 induces endothelial cell expression of VEGF, which mediates TGF-ß1 induction of apoptosis through activation of p38 mitogen-activated protein kinase (MAPK). Because VEGF activates p38(MAPK) but protects the cells from apoptosis, this finding suggested that TGF-ß1 converts p38(MAPK) signaling from prosurvival to proapoptotic. Four isoforms of p38(MAPK) -α, ß, γ, and δ-have been identified. Therefore, we hypothesized that different p38(MAPK) isoforms control endothelial cell apoptosis or survival, and that TGF-ß1 directs VEGF activation of p38(MAPK) from a prosurvival to a proapoptotic isoform. Here, we report that cultured endothelial cells express p38α, ß, and γ. VEGF activates p38ß, whereas TGF-ß1 activates p38α. TGF-ß1 treatment rapidly induces p38α activation and apoptosis. Subsequently, p38α activation is downregulated, p38ß is activated, and the surviving cells become refractory to TGF-ß1 induction of apoptosis and proliferate. Gene silencing of p38α blocks TGF-ß1 induction of apoptosis, whereas downregulation of p38ß or p38γ expression results in massive apoptosis. Thus, in endothelial cells p38α mediates apoptotic signaling, whereas p38ß and p38γ transduce survival signaling. TGF-ß1 activation of p38α is mediated by VEGF, which in the absence of TGF-ß1 activates p38ß. Therefore, these results show that TGF-ß1 induces endothelial cell apoptosis by shifting VEGF signaling from the prosurvival p38ß to the proapoptotic p38α.
Assuntos
Apoptose/genética , Sobrevivência Celular/genética , Proteína Quinase 11 Ativada por Mitógeno/metabolismo , Proteína Quinase 12 Ativada por Mitógeno/metabolismo , Proteína Quinase 14 Ativada por Mitógeno/metabolismo , Animais , Bovinos , Regulação Neoplásica da Expressão Gênica , Células Endoteliais da Veia Umbilical Humana , Humanos , Proteína Quinase 11 Ativada por Mitógeno/genética , Proteína Quinase 12 Ativada por Mitógeno/genética , Proteína Quinase 14 Ativada por Mitógeno/genética , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , RNA Interferente Pequeno , Transdução de Sinais , Fator de Crescimento Transformador beta1/administração & dosagem , Fator de Crescimento Transformador beta1/metabolismo , Fator A de Crescimento do Endotélio Vascular/administração & dosagem , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
There is increasing evidence that dysregulation of CD4(+) T cell populations leads to intestinal inflammation, but the regional distribution of these populations throughout the intestinal tract in healthy individuals remains unclear. Here, we show that T(H)17, T(H)22 and T(Reg) cells are enriched in the healthy human cecum compared to the terminal ileum and sigmoid colon, whereas T(H)1 and T(H)2 cells do not significantly vary by location. Transcriptional profiling analysis of paired pinch biopsies from different regions of the intestine identified significant differences in the metabolic state of the terminal ileum, cecum, and sigmoid colon. An increased proportion of T(H)17 cells was positively associated with expression of resistin (RETN) and negatively associated with expression of trefoil factor 1 (TFF1). These results suggest that CD4(+) T helper cells that are important in maintaining mucosal barrier function may be enriched in the cecum as a result of metabolic differences of the surrounding microenvironment.
Assuntos
Ceco/citologia , Subpopulações de Linfócitos T/citologia , Linfócitos T Reguladores/citologia , Células Th17/citologia , Biópsia , Colo/citologia , Feminino , Citometria de Fluxo , Humanos , Intestino Delgado/citologia , Masculino , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase em Tempo RealRESUMO
A hallmark of the immune system is the ability to ignore self-antigens. In attempts to bypass normal immune tolerance, a post-translational protein modification was introduced into self-antigens to break T and B cell tolerance. We demonstrate that immune tolerance is bypassed by immunization with a post-translationally modified melanoma antigen. In particular, the conversion of an aspartic acid to an isoaspartic acid within the melanoma antigen tyrosinase-related protein (TRP)-2 peptide-(181-188) makes the otherwise immunologically ignored TRP-2 antigen immunogenic. Tetramer analysis of iso-Asp TRP-2 peptide-immunized mice demonstrated that CD8+ T cells not only recognized the isoaspartyl TRP-2 peptide but also the native TRP-2 peptide. These CD8+ T cells functioned as cytotoxic T lymphocytes, as they effectively lysed TRP-2 peptide-pulsed targets both in vitro and in vivo. Potentially, post-translational protein modification can be utilized to trigger strong immune responses to either tumor proteins or potentially weakly immunogenic pathogens.
Assuntos
Linfócitos B/imunologia , Ácido Isoaspártico/metabolismo , Processamento de Proteína Pós-Traducional , Linfócitos T/imunologia , Vacinação , Animais , Antígenos de Neoplasias/imunologia , Linfócitos T CD8-Positivos/imunologia , Linhagem Celular Tumoral , Técnicas In Vitro , Ácido Isoaspártico/química , Melanoma Experimental/imunologia , Melanoma Experimental/patologia , Camundongos , Camundongos Endogâmicos C57BL , Estrutura MolecularRESUMO
CD19 is a B cell-surface molecule that participates as an important regulatory signaling complex for antigen bound at the surface by Ig. Triggering of CD19 through its linkage with CD21 amplifies signals transduced through the Src family kinases and modulates B cell differentiation in response to antigen. This study examines the kinetics of antigen uptake and processing of antigen directly targeted to the CD19 protein on purified B cells. We have demonstrated that the antigen internalized within minutes through CD19 forms a cap at the B cell surface and can be found within lysosomes in the cytoplasm in 90 min. B cells acquiring antigen via CD19 express elevated levels of B7-1 and B7-2 co-stimulatory molecules. Moreover, antigen-anti-CD19 complexes administered intravenously bind B cells in vivo and activate antigen-specific T cells more efficiently than non-specific uptake and in a manner similar to antigen taken up through surface IgM on B cells. This work illustrates an important and previously unrecognized mechanism for targeting proteins to B lymphocytes for antigen presentation and activation of CD4 T cells.