RESUMEN
Regulatory T cell (Treg) therapy is a promising strategy to treat inflammatory bowel disease (IBD). Data from animal models has shown that Tregs specific for intestinal antigens are more potent than polyclonal Tregs at inhibiting colitis. Flagellins, the major structural proteins of bacterial flagella, are immunogenic antigens frequently targeted in IBD subjects, leading to the hypothesis that flagellin-specific Tregs could be an effective cell therapy for IBD. We developed a novel chimeric antigen receptor (CAR) specific for flagellin derived from Escherichia coli H18 (FliC). We used this CAR to confer FliC-specificity to human Tregs and investigated their therapeutic potential. FliC-CAR Tregs were activated by recombinant FliC protein but not a control flagellin protein, demonstrating CAR specificity and functionality. In a humanized mouse model, expression of the FliC-CAR drove preferential migration to the colon and expression of the activation marker PD1. In the presence of recombinant FliC protein in vitro, FliC-CAR Tregs were significantly more suppressive than control Tregs and promoted the establishment of colon-derived epithelial cell monolayers. These results demonstrate the potential of FliC-CAR Tregs to treat IBD and more broadly show the therapeutic potential of CARs targeting microbial-derived antigens.
Asunto(s)
Enfermedades Inflamatorias del Intestino , Receptores Quiméricos de Antígenos , Animales , Ratones , Humanos , Receptores Quiméricos de Antígenos/genética , Receptores Quiméricos de Antígenos/metabolismo , Flagelina/metabolismo , Proteínas Recombinantes/metabolismo , Enfermedades Inflamatorias del Intestino/terapia , Enfermedades Inflamatorias del Intestino/metabolismo , Linfocitos T ReguladoresRESUMEN
Dendritic cells are sentinels of the immune system and represent a key cell in the activation of the adaptive immune response. Hypoxia-inducible factor 1 alpha (HIF-1α)-a crucial oxygen sensor stabilized during hypoxic conditions-has been shown to have both activating and inhibitory effects in immune cells in a context- and cell-dependent manner. Previous studies have demonstrated that in some immune cell types, HIF-1α serves a pro-inflammatory role. Genetic deletion of HIF-1α in macrophages has been reported to reduce their pro-inflammatory function. In contrast, loss of HIF-1α enhanced the pro-inflammatory activity of dendritic cells in a bacterial infection model. In this study, we aimed to further clarify the effects of HIF-1α in dendritic cells. Constitutive expression of HIF-1α resulted in diminished immunostimulatory capacity of dendritic cells in vivo, while conditional deletion of HIF-1α in dendritic cells enhanced their ability to induce a cytotoxic T cell response. HIF-1α-expressing dendritic cells demonstrated increased production of inhibitory mediators including IL-10, iNOS and VEGF, which correlated with their reduced capacity to drive effector CD8+ T cell function. Altogether, these data reveal that HIF-1α can promote the anti-inflammatory functions of dendritic cells and provides insight into dysfunctional immune responses in the context of HIF-1α activation.
Asunto(s)
Biomarcadores/metabolismo , Linfocitos T CD8-positivos/inmunología , Células Dendríticas/citología , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Animales , Células Cultivadas , Células Dendríticas/metabolismo , Técnicas de Inactivación de Genes , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Interleucina-10/metabolismo , Ratones , Óxido Nítrico Sintasa de Tipo II/metabolismo , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
BACKGROUND & AIMS: Bacterial flagellin is an important antigen in inflammatory bowel disease, but the role of flagellin-specific CD4+ T cells in disease pathogenesis remains unclear. Also unknown is how changes in intestinal microbiome intersect with those in microbiota-specific CD4+ T cells. We aimed to quantify and characterize flagellin-specific CD4+ T cells in Crohn's disease (CD) and ulcerative colitis (UC) patients and study their relationship with intestinal microbiome diversity. METHODS: Blood was collected from 3 cohorts that included CD patients, UC patients, and healthy controls. Flow cytometry analyzed CD4+ T cells specific for Lachnospiraceae-derived A4-Fla2 and Escherichia coli H18 FliC flagellins, or control vaccine antigens. Serum antiflagellin IgG and IgA antibodies were detected by enzyme-linked immunosorbent assay and stool samples were collected and subjected to 16S ribosomal DNA sequencing. RESULTS: Compared with healthy controls, CD and UC patients had lower frequencies of vaccine-antigen-specific CD4+ T cells and, as a proportion of vaccine-specific cells, higher frequencies of flagellin-specific CD4+ T cells. The proportion of flagellin-specific CD4+ T cells that were CXCR3negCCR4+CCR6+ Th17 cells was reduced in CD and UC patients, with increased proportions of CD39+, PD-1+, and integrin ß7+ cells. Microbiome analysis showed differentially abundant bacterial species in patient groups that correlated with immune responses to flagellin. CONCLUSIONS: Both CD and UC patients have relative increases in the proportion of circulating Fla2-specific CD4+ T cells, which may be associated with changes in the intestinal microbiome. Evidence that the phenotype of these cells strongly correlate with disease severity provides insight into the potential roles of flagellin-specific CD4+ T cells in inflammatory bowel disease.
Asunto(s)
Anticuerpos Antibacterianos/sangre , Colitis Ulcerosa/inmunología , Enfermedad de Crohn/inmunología , Disbiosis/complicaciones , Proteínas de Escherichia coli/inmunología , Flagelina/inmunología , Inmunidad Adaptativa , Adolescente , Adulto , Anciano , Anticuerpos Antibacterianos/inmunología , Antígenos Bacterianos/inmunología , Linfocitos T CD4-Positivos/inmunología , Clostridiales/genética , Clostridiales/inmunología , Colitis Ulcerosa/sangre , Colitis Ulcerosa/microbiología , Enfermedad de Crohn/sangre , Enfermedad de Crohn/microbiología , Estudios Transversales , ADN Bacteriano/aislamiento & purificación , Disbiosis/diagnóstico , Disbiosis/inmunología , Disbiosis/microbiología , Ensayo de Inmunoadsorción Enzimática , Heces/microbiología , Femenino , Microbioma Gastrointestinal/inmunología , Voluntarios Sanos , Humanos , Masculino , Persona de Mediana Edad , ARN Ribosómico 16S/genética , Subgrupos de Linfocitos T/inmunología , Adulto JovenAsunto(s)
Acidosis/terapia , Antídotos/uso terapéutico , Errores Diagnósticos , Errores Médicos , Metanol/sangre , Intoxicación/terapia , Pirazoles/uso terapéutico , Diálisis Renal , Revelación de la Verdad , Acidosis/inducido químicamente , Acidosis/diagnóstico , Alcoholismo/complicaciones , Femenino , Fomepizol , Humanos , Metanol/envenenamiento , Persona de Mediana Edad , Intoxicación/complicaciones , Intoxicación/diagnóstico , Solventes/envenenamientoRESUMEN
FOXP3-expressing T regulatory cells (Tregs) can be divided into two distinct subsets: naturally occurring Tregs (nTregs) that develop in the thymus, and induced Tregs (iTregs) that differentiate in peripheral tissues upon exposure to Ag in a tolerogenic environment. Recently it has been proposed that expression of Helios, an Ikaros family transcription factor, may specifically identify nTregs, allowing specific tracking of Tregs from different origins in health and disease. Surprisingly, we found that Helios(-) cells can be readily identified within naive (CD45RA(+)CD31(+)CCR7(+)CD62L(+)) FOXP3(+) Tregs, a finding inconsistent with the notion that lack of Helios expression identifies Ag-experienced iTregs that should express memory markers. To investigate the phenotype and function of naive Helios(+) and Helios(-) Tregs within the nTreg population, we isolated single-cell clones from each subset. We found that both Helios(+) and Helios(-) nTreg clones have a similar suppressive capacity, as well as expression of FOXP3 and cell surface proteins, including CD39 and CTLA-4. Helios(-) nTregs, however, produced significantly more CCL3 and IFN-γ compared with Helios(+) nTregs. Despite increased cytokine/chemokine production, Helios(-) FOXP3(+) nTreg clones were demethylated at the FOXP3 Treg-specific demethylated region, indicative of Treg lineage stability. When cultured under Th1-polarizing conditions, Helios(+) and Helios(-) nTreg clones had an equal ability to produce IFN-γ. Collectively, these data show that a lack of Helios expression does not exclusively identify human iTregs, and, to our knowledge, the data provide the first evidence for the coexistence of Helios(+) and Helios(-) nTregs in human peripheral blood.
Asunto(s)
Linaje de la Célula/inmunología , Factores de Transcripción Forkhead/genética , Factor de Transcripción Ikaros/genética , Linfocitos T Reguladores/citología , Timo/citología , Antígenos CD/genética , Antígenos CD/inmunología , Apirasa/genética , Apirasa/inmunología , Antígeno CTLA-4/genética , Antígeno CTLA-4/inmunología , Diferenciación Celular , Quimiocina CCL3/genética , Quimiocina CCL3/inmunología , Células Clonales , Citometría de Flujo , Factores de Transcripción Forkhead/inmunología , Expresión Génica , Humanos , Factor de Transcripción Ikaros/inmunología , Inmunofenotipificación , Interferón gamma/genética , Interferón gamma/inmunología , Especificidad de Órganos , Linfocitos T Reguladores/inmunología , Linfocitos T Reguladores/metabolismo , Timo/inmunología , Timo/metabolismoRESUMEN
T regulatory (Treg) cells are critical for maintaining immune homeostasis and establishing tolerance to foreign, non-pathogenic antigens including those found in commensal bacteria and food. Because of their multiple suppressive mechanisms, Tregs represent a promising strategy for engineering tolerance to self and non-self antigens in chronic inflammatory diseases. Already in clinical trials in the transplantation setting, the question remains whether this therapy would be effective for the treatment of mucosal inflammatory diseases that do not pose an immediate threat to life. In this review we will discuss evidence from both animal models and patients suggesting that Treg therapy would be beneficial in the context of inflammatory bowel disease (IBD). We will examine the role of T-cell versus Treg dysfunction in IBD and discuss the putative antigens that could be potential targets of antigen-directed Treg therapy. Finally, the challenges of using Treg therapy in IBD will be discussed, with a specific emphasis on the role that the microbiota may play in the outcome of this treatment. As Treg therapy becomes a bedside reality in the field of transplantation, there is great hope that it will soon also be deployed in the setting of IBD and ultimately prove more effective than the current non-specific immunosuppressive therapies.
Asunto(s)
Enfermedades Inflamatorias del Intestino/terapia , Linfocitos T Reguladores/inmunología , Linfocitos T Reguladores/trasplante , Animales , Modelos Animales de Enfermedad , Humanos , Enfermedades Inflamatorias del Intestino/inmunología , Enfermedades Inflamatorias del Intestino/microbiología , Mucosa Intestinal/inmunología , Mucosa Intestinal/microbiología , RatonesRESUMEN
One of the defining features of the majority of FOXP3(+) Tregs is their inability to produce typical T-cell-derived cytokines. Little is known, however, about their capacity to produce chemokines. As Tregs are constitutively present in, and rapidly traffic to, non-lymphoid tissues, we hypothesized that they may produce chemokines to direct the composition of cells that infiltrate inflamed tissues. Surprisingly, we found that Tregs produce high amounts of CXCL8 (IL-8), a potent neutrophil chemoattractant. Tregs also produced other CC and CXC family chemokines, including CCL2-5, CCL7, and CXCL10. Whereas ectopic expression of FOXP3 suppressed cytokine production, it significantly induced CXCL8. Moreover, supernatants from Tregs attracted neutrophils via a CXCL8-dependent mechanism. These data provide the first evidence that although classical Tregs are defined by their lack of proinflammatory cytokine production, they secrete significant quantities of chemokines and thus may have an unappreciated role in directing the recruitment of immune cells.
Asunto(s)
Quimiotaxis/inmunología , Factores de Transcripción Forkhead/metabolismo , Interleucina-8/metabolismo , Neutrófilos/inmunología , Linfocitos T Reguladores/inmunología , Linfocitos T Reguladores/metabolismo , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/farmacología , Quimiocinas CC/metabolismo , Quimiocinas CXC/metabolismo , Quimiotaxis/efectos de los fármacos , Medios de Cultivo Condicionados/farmacología , Factores de Transcripción Forkhead/genética , Regulación de la Expresión Génica/inmunología , Humanos , Subunidad alfa del Receptor de Interleucina-2/metabolismo , Interleucina-8/genética , Interleucina-8/inmunología , Neutrófilos/citología , Neutrófilos/efectos de los fármacos , Transducción GenéticaRESUMEN
BACKGROUND: Although intestinal epithelial cells (IECs) are continually exposed to commensal microbes, under healthy conditions they contribute to intestinal homeostasis while keeping inflammatory responses in check. In response to invading pathogens, however, IECs respond vigorously by producing inflammatory mediators. To better understand the signals that regulate the inflammatory responses of IECs, we investigated whether the danger signal ATP (which is released from injured cells) could alter responses to bacterial products. METHODS: We measured chemokine production from Caco-2 cells stimulated with the Toll-like receptor 5 agonist flagellin with or without ATP. ATP increased flagellin-induced IL-8 secretion but reduced CCL20 secretion via distinct signaling pathways. RESULTS: ATP-enhanced IL-8 production was only partly blocked by the P(2) receptor antagonist suramin and required activation of NF-κB while ATP-mediated reduction of CCL20 was completely blocked by suramin and required activation of ERK1/2. The effects of ATP on both chemokines required extracellular calcium but not phospholipase C, implicating P(2) X receptor involvement. To investigate how ATP alters IEC responses to bacterial products in vivo, mice receiving dextran sodium sulfate were given intrarectal flagellin with or without ATP. Addition of ATP to flagellin caused greater weight loss and increased antiflagellin antibody titers, as well as decreased colonic interferon gamma (IFN-γ) and higher antiflagellin IgG1/IgG2 ratios, which indicate decreased Th1 polarization. CONCLUSIONS: Together, these data indicate that stress, in the form of extracellular ATP, reshapes both the inflammatory response of flagellin-stimulated IECs and downstream adaptive immunity, representing a possible strategy by which these cells differentiate between commensal and pathogenic bacteria.
Asunto(s)
Adenosina Trifosfato/farmacología , Colitis/inmunología , Células Epiteliales/efectos de los fármacos , Células Epiteliales/inmunología , Flagelina/metabolismo , Intestinos/efectos de los fármacos , Intestinos/inmunología , Animales , Células CACO-2 , Quimiocina CCL20/genética , Quimiocina CCL20/metabolismo , Quimiocinas/genética , Quimiocinas/metabolismo , Colitis/metabolismo , Colitis/patología , Modelos Animales de Enfermedad , Ensayo de Cambio de Movilidad Electroforética , Células Epiteliales/metabolismo , Flagelina/antagonistas & inhibidores , Humanos , Interleucina-8/metabolismo , Mucosa Intestinal/metabolismo , Masculino , Ratones , Ratones Endogámicos C3H , FN-kappa B/genética , FN-kappa B/metabolismo , ARN Mensajero/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal , Receptor Toll-Like 5/genética , Receptor Toll-Like 5/metabolismoRESUMEN
Intestinal epithelial cells act as innate immune sentinels, as the first cells that encounter diarrheal pathogens. They use pattern recognition molecules such as the Toll-like receptors (TLRs) to identify molecular signals found on microbes but not host cells or food components. TLRs cannot generally distinguish the molecular signals on pathogenic bacteria from those found in commensals, yet under healthy conditions epithelial immune responses are kept in check. We hypothesized that, in the setting of tissue damage or stress, intestinal epithelial cells would upregulate their responses to TLR ligands to reflect the greater need for immediate protection against pathogens. We treated Caco-2 cells with the TLR5 agonist flagellin in the presence or absence of H(2)O(2) and measured chemokine production and intracellular signaling pathways. H(2)O(2) increased flagellin-induced IL-8 (CXCL8) production in a dose-dependent manner. This was associated with synergistic phosphorylation of p38 MAP kinase and with prolonged I-kappaB degradation and NF-kappaB activation. The H(2)O(2)-mediated potentiation of IL-8 production required the activity of p38, tyrosine kinases, phospholipase Cgamma, and intracellular calcium, but not protein kinase C or protein kinase D. H(2)O(2) prolonged and augmented NF-kappaB activation by flagellin. In contrast to IL-8, CCL20 (MIP3alpha) production by flagellin was reduced by H(2)O(2), and this effect was not calcium dependent. Oxidative stress biases intestinal epithelial responses to flagellin, leading to increased production of IL-8 and decreased production of CCL20. This suggests that epithelial cells are capable of sensing the extracellular environment and adjusting their antimicrobial responses accordingly.
Asunto(s)
Quimiocina CCL20/metabolismo , Células Epiteliales/efectos de los fármacos , Flagelina/farmacología , Interleucina-8/metabolismo , Estrés Oxidativo/fisiología , Células CACO-2 , Calcio/metabolismo , Quelantes/farmacología , Quimiocina CCL20/genética , Ácido Egtácico/análogos & derivados , Ácido Egtácico/farmacología , Células Epiteliales/metabolismo , Regulación de la Expresión Génica/fisiología , Humanos , Peróxido de Hidrógeno/farmacología , Interleucina-8/genética , Mucosa Intestinal/citología , FN-kappa B/antagonistas & inhibidores , Nitrilos/farmacología , Oxidantes/farmacología , Transducción de Señal , Sulfonas/farmacologíaRESUMEN
BACKGROUND: The two forms of human inflammatory bowel disease, Crohn's disease (CD) and ulcerative colitis (UC), are both associated with loss of tolerance to gut microbial antigens. The dominant antigen recognized by antibody and T-cell responses in patients with CD is bacterial flagellin. Flagellin is also the only known ligand for Toll-like receptor 5 (TLR5), a key protein in innate immunity. Although flagellin activates TLR5 to produce inflammatory responses in many cell types in the gut, there is conflicting evidence as to whether TLR5 is harmful or protective in CD and murine colitis models. A recent study found that administration of flagellin enemas to mice along with dextran sodium sulfate (DSS) made their colitis worse. METHODS: We sought to determine whether this exacerbation was due to TLR5 ligation, or to TLR5-independent adaptive immune responses to flagellin as an antigen, by using a transposon insertional mutant of the Escherichia coli H18 flagellin, 2H3, which lacks TLR5 stimulatory activity. RESULTS: We found that flagellin enemas produced only a mild exacerbation of DSS colitis, and that 2H3 was equivalent to or worse than wildtype flagellin. Moreover, we found that DSS colitis was more severe in TLR5(-/-) mice than wildtype C57BL/6 mice. CONCLUSIONS: Together, these results suggest that flagellin-mediated exacerbation of colitis is independent of TLR5.
Asunto(s)
Colitis/inducido químicamente , Colitis/inmunología , Flagelina/inmunología , Receptor Toll-Like 5/inmunología , Receptor Toll-Like 5/metabolismo , Animales , Anticuerpos Antibacterianos/sangre , Colitis/mortalidad , Elementos Transponibles de ADN , Sulfato de Dextran/toxicidad , Enema , Escherichia coli/genética , Flagelina/genética , Flagelina/farmacología , Células HeLa , Humanos , Inmunoglobulina G/metabolismo , Interleucina-12/metabolismo , Mucosa Intestinal/inmunología , Mucosa Intestinal/metabolismo , Masculino , Ratones , Ratones Endogámicos C3H , Ratones Endogámicos C57BL , Ratones Mutantes , Salmonella typhimurium/genética , Receptor Toll-Like 5/genética , Pérdida de PesoRESUMEN
Two related chronic inflammatory diseases, Crohn's disease and ulcerative colitis, are together often referred to as inflammatory bowel disease (IBD). Current treatment options are not curative, and patients face lifelong therapy and debilitation. IBD is thought to be the product of a combination of genetic and environmental factors that result in the abnormal regulation of immune responses. Experimental models have demonstrated that normal CD4+ T-regulatory (Treg) cell responses and commensal bacteria are required for the maintenance of gut immune homeostasis. Recent evidence that CD4+ T cells express Toll-like receptors (TLRs) and respond directly to TLR ligands, suggests that signals from commensal bacteria may directly affect T-cell responses in the gut. In this review, we focus on evidence that defects in Treg cells may underlie IBD in humans. In addition, we discuss evidence that direct signaling via TLRs to T cells can affect IBD and that T-cell-dependent responses to bacterial proteins, such as flagellin, are central to the aetiology of this disease.
Asunto(s)
Enfermedades Inflamatorias del Intestino/inmunología , Linfocitos T Reguladores/inmunología , Receptores Toll-Like/inmunología , Animales , Antígenos Bacterianos/inmunología , Flagelina/inmunología , Humanos , Recuento de Linfocitos , Ratones , Modelos InmunológicosRESUMEN
SUMMARY: T-regulatory cells (Tregs) have a fundamental role in the establishment and maintenance of peripheral tolerance. There is now compelling evidence that deficits in the numbers and/or function of different types of Tregs can lead to autoimmunity, allergy, and graft rejection, whereas an over-abundance of Tregs can inhibit anti-tumor and anti-pathogen immunity. Experimental models in mice have demonstrated that manipulating the numbers and/or function of Tregs can decrease pathology in a wide range of contexts, including transplantation, autoimmunity, and cancer, and it is widely assumed that similar approaches will be possible in humans. Research into how Tregs can be manipulated therapeutically in humans is most advanced for two main types of CD4(+) Tregs: forkhead box protein 3 (FOXP3)(+) Tregs and interleukin-10-producing type 1 Tregs (Tr1 cells). The aim of this review is to highlight current information on the characteristics of human FOXP3(+) Tregs and Tr1 cells that make them an attractive therapeutic target. We discuss the progress and limitations that must be overcome to develop methods to enhance Tregs in vivo, expand or induce them in vitro for adoptive transfer, and/or inhibit their function in vivo. Although many technical and theoretical challenges remain, the next decade will see the first clinical trials testing whether Treg-based therapies are effective in humans.