RESUMEN
Intestinal intraepithelial lymphocytes (IELs) reside in the gut epithelial layer, where they help in maintaining intestinal homeostasis. Peripheral CD4+ T cells can develop into CD4+CD8αα+ IELs upon arrival at the gut epithelium via the lamina propria (LP). Although this specific differentiation of T cells is well established, the mechanisms preventing it from occurring in the LP remain unclear. Here, we show that chemokine receptor 9 (CCR9) expression is low in epithelial CD4+CD8αα+ IELs, but CCR9 deficiency results in CD4+CD8αα+ over-differentiation in both the epithelium and the LP. Single-cell RNA sequencing shows an enriched precursor cell cluster for CD4+CD8αα+ IELs in Ccr9-/- mice. CD4+ T cells isolated from the epithelium of Ccr9-/- mice also display increased expression of Cbfß2, and the genomic occupancy modification of Cbfß2 expression reveals its important function in CD4+CD8αα+ differentiation. These results implicate a link between CCR9 downregulation and Cbfb2 splicing upregulation to enhance CD4+CD8αα+ IEL differentiation.
Asunto(s)
Linfocitos Intraepiteliales , Receptores CCR , Animales , Ratones , Diferenciación Celular , Regulación hacia Abajo , Epitelio , Regulación hacia Arriba , Receptores CCR/metabolismoRESUMEN
Loss of antimicrobial proteins such as REG3 family members compromises the integrity of the intestinal barrier. Here, we demonstrate that overproduction of REG3 proteins can also be detrimental by reducing a protective species in the microbiota. Patients with inflammatory bowel disease (IBD) experiencing flares displayed heightened levels of secreted REG3 proteins that mediated depletion of Enterococcus faecium (Efm) from the gut microbiota. Efm inoculation of mice ameliorated intestinal inflammation through activation of the innate immune receptor NOD2, which was associated with the bacterial DL-endopeptidase SagA that generates NOD2-stimulating muropeptides. NOD2 activation in myeloid cells induced interleukin-1ß (IL-1ß) secretion to increase the proportion of IL-22-producing CD4+ T helper cells and innate lymphoid cells that promote tissue repair. Finally, Efm was unable to protect mice carrying a NOD2 gene variant commonly found in IBD patients. Our findings demonstrate that inflammation self-perpetuates by causing aberrant antimicrobial activity that disrupts symbiotic relationships with gut microbes.
Asunto(s)
Antiinfecciosos , Enterococcus faecium , Enfermedades Inflamatorias del Intestino , Animales , Ratones , Inmunidad Innata , Linfocitos , InflamaciónRESUMEN
Loss of antimicrobial proteins such as REG3 family members compromises the integrity of the intestinal barrier. Here, we demonstrate that overproduction of REG3 proteins can also be detrimental by reducing a protective species in the microbiota. Patients with inflammatory bowel disease (IBD) experiencing flares displayed heightened levels of secreted REG3 proteins that mediated depletion of Enterococcus faecium ( Efm ) from the gut microbiota. Efm inoculation of mice ameliorated intestinal inflammation through activation of the innate immune receptor NOD2, which was associated with the bacterial DL-endopeptidase SagA. Microbiota sensing by NOD2 in myeloid cells mediated IL-1ß secretion and increased the proportion of IL-22-producing CD4 + T helper cells and innate lymphoid cells. Finally, Efm was unable to protect mice carrying a NOD2 gene variant commonly found in IBD patients. Our findings demonstrate that inflammation self-perpetuates by causing aberrant antimicrobial activity that disrupts symbiotic relationships with gut microbes.
RESUMEN
The microbiome contributes to the development and maturation of the immune system. In response to commensal bacteria, intestinal CD4+ T lymphocytes differentiate into functional subtypes with regulatory or effector functions. The development of small intestine intraepithelial lymphocytes that coexpress CD4 and CD8αα homodimers (CD4IELs) depends on the microbiota. However, the identity of the microbial antigens recognized by CD4+ T cells that can differentiate into CD4IELs remains unknown. We identified ß-hexosaminidase, a conserved enzyme across commensals of the Bacteroidetes phylum, as a driver of CD4IEL differentiation. In a mouse model of colitis, ß-hexosaminidase-specific lymphocytes protected against intestinal inflammation. Thus, T cells of a single specificity can recognize a variety of abundant commensals and elicit a regulatory immune response at the intestinal mucosa.
Asunto(s)
Bacteroidetes , Linfocitos T CD4-Positivos , Colitis , Mucosa Intestinal , beta-N-Acetilhexosaminidasas , Animales , Bacteroidetes/enzimología , Bacteroidetes/inmunología , Linfocitos T CD4-Positivos/inmunología , Antígenos CD8/inmunología , Colitis/inmunología , Colitis/microbiología , Modelos Animales de Enfermedad , Mucosa Intestinal/inmunología , Mucosa Intestinal/microbiología , Ratones , Ratones Endogámicos C57BL , beta-N-Acetilhexosaminidasas/inmunologíaRESUMEN
The intestinal epithelium comprises the body's largest surface exposed to viruses. Additionally, the gut epithelium hosts a large population of intraepithelial T lymphocytes, or IELs, although their role in resistance against viral infections remains elusive. By fate-mapping T cells recruited to the murine intestine, we observed an accumulation of newly recruited CD4+ T cells after infection with murine norovirus CR6 and adenovirus type-2 (AdV), but not reovirus. CR6- and AdV-recruited intraepithelial CD4+ T cells co-expressed Ly6A and chemokine receptor CCR9, exhibited T helper 1 and cytotoxic profiles, and conferred protection against AdV in vivo and in an organoid model in an IFN-γ-dependent manner. Ablation of the T cell receptor (TCR) or the transcription factor ThPOK in CD4+ T cells prior to AdV infection prevented viral control, while TCR ablation during infection did not impact viral clearance. These results uncover a protective role for intraepithelial Ly6A+CCR9+CD4+ T cells against enteric adenovirus.
Asunto(s)
Intestino Delgado , Virosis , Animales , Antígenos Ly , Linfocitos T CD4-Positivos , Mucosa Intestinal , Proteínas de la Membrana , Ratones , Receptores de QuimiocinaRESUMEN
Mesenteric lymph node (mLN) T cells undergo tissue adaptation upon migrating to intestinal lamina propria and epithelium, ensuring appropriate balance between tolerance and resistance. By combining mouse genetics with single-cell and chromatin analyses, we uncovered the molecular imprinting of gut epithelium on T cells. Transcriptionally, conventional and regulatory (Treg) CD4+ T cells from mLN, lamina propria and intestinal epithelium segregate based on the gut layer they occupy; trajectory analysis suggests a stepwise loss of CD4 programming and acquisition of an intraepithelial profile. Treg cell fate mapping coupled with RNA sequencing and assay for transposase-accessible chromatin followed by sequencing revealed that the Treg cell program shuts down before an intraepithelial program becomes fully accessible at the epithelium. Ablation of CD4-lineage-defining transcription factor ThPOK results in premature acquisition of an intraepithelial lymphocyte profile by mLN Treg cells, partially recapitulating epithelium imprinting. Thus, coordinated replacement of the circulating lymphocyte program with site-specific transcriptional and chromatin changes is necessary for tissue imprinting.
Asunto(s)
Diferenciación Celular , Ensamble y Desensamble de Cromatina , Impresión Genómica , Mucosa Intestinal/metabolismo , Linfocitos Intraepiteliales/metabolismo , Ganglios Linfáticos/metabolismo , Linfocitos T Reguladores/metabolismo , Transcripción Genética , Animales , Linaje de la Célula , Células Cultivadas , Perfilación de la Expresión Génica , Regulación del Desarrollo de la Expresión Génica , Mucosa Intestinal/inmunología , Linfocitos Intraepiteliales/inmunología , Ganglios Linfáticos/inmunología , Ratones Noqueados , Fenotipo , RNA-Seq , Análisis de la Célula Individual , Linfocitos T Reguladores/inmunología , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , TranscriptomaRESUMEN
The gut epithelium is populated by intraepithelial lymphocytes (IELs), a heterogeneous T cell population with cytotoxic and regulatory properties, which can be acquired at the epithelial layer. However, the role of T cell receptor (TCR) in this process remains unclear. Single-cell transcriptomic analyses revealed distinct clonal expansions between cell states, with CD4+CD8αα+ IELs being one of the least diverse populations. Conditional deletion of TCR on differentiating CD4+ T cells or of major histocompatibility complex (MHC) class II on intestinal epithelial cells prevented CD4+CD8αα+ IEL differentiation. However, TCR ablation on differentiated CD4+CD8αα+ IELs or long-term cognate antigen withdraw did not affect their maintenance. TCR re-engagement of antigen-specific CD4+CD8αα+ IELs by Listeria monocytogenes did not alter their state but correlated with reduced bacterial invasion. Thus, local antigen recognition is an essential signal for differentiation of CD4+ T cells at the epithelium, yet differentiated IELs are able to preserve an effector program in the absence of TCR signaling.
Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/metabolismo , Mucosa Intestinal/inmunología , Mucosa Intestinal/metabolismo , Linfocitos Intraepiteliales/inmunología , Linfocitos Intraepiteliales/metabolismo , Receptores de Antígenos de Linfocitos T/metabolismo , Animales , Diferenciación Celular/genética , Diferenciación Celular/inmunología , Evolución Clonal/genética , Evolución Clonal/inmunología , Antígenos de Histocompatibilidad Clase II/genética , Antígenos de Histocompatibilidad Clase II/inmunología , Inmunofenotipificación , Ratones , Receptores de Antígenos de Linfocitos T alfa-beta/metabolismo , Transducción de Señal , Análisis de la Célula Individual , Subgrupos de Linfocitos T/inmunología , Subgrupos de Linfocitos T/metabolismoRESUMEN
RNase H2 has two distinct functions: initiation of the ribonucleotide excision repair (RER) pathway by cleaving ribonucleotides (rNMPs) incorporated during DNA replication and processing the RNA portion of an R-loop formed during transcription. An RNase H2 mutant lacking RER activity but supporting R-loop removal revealed that rNMPs in DNA initiate p53-dependent DNA damage response and early embryonic arrest in mouse. However, an RNase H2 AGS-related mutant with residual RER activity develops to birth. Estimations of the number of rNMPs in DNA in these two mutants define a ribonucleotide threshold above which p53 induces apoptosis. Below the threshold, rNMPs in DNA trigger an innate immune response. Compound heterozygous cells, containing both defective enzymes, retain rNMPs above the threshold, indicative of competition for RER substrates between active and inactive enzymes, suggesting that patients with compound heterozygous mutations in RNASEH2 genes may not reflect the properties of recombinantly expressed proteins.
Asunto(s)
Desarrollo Embrionario , Mutación/genética , Ribonucleasa H/genética , Ribonucleótidos/metabolismo , Animales , ADN/metabolismo , Daño del ADN , Reparación del ADN/efectos de los fármacos , Pérdida del Embrión/patología , Embrión de Mamíferos/anomalías , Desarrollo Embrionario/efectos de los fármacos , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Interferones/farmacología , Proteínas de la Membrana/metabolismo , Ratones Noqueados , Proteínas Mutantes/metabolismo , Estabilidad del ARN/efectos de los fármacos , Ribonucleasa H/metabolismo , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
Foxp3(+) regulatory T cells in peripheral tissues (pT(regs)) are instrumental in limiting inflammatory responses to nonself antigens. Within the intestine, pT(regs) are located primarily in the lamina propria, whereas intraepithelial CD4(+) T cells (CD4(IELs)), which also exhibit anti-inflammatory properties and depend on similar environmental cues, reside in the epithelium. Using intravital microscopy, we show distinct cell dynamics of intestinal T(regs) and CD4(IELs) Upon migration to the epithelium, T(regs) lose Foxp3 and convert to CD4(IELs) in a microbiota-dependent manner, an effect attributed to the loss of the transcription factor ThPOK. Finally, we demonstrate that pT(regs) and CD4(IELs) perform complementary roles in the regulation of intestinal inflammation. These results reveal intratissue specialization of anti-inflammatory T cells shaped by discrete niches of the intestine.
Asunto(s)
Animales , Movimiento Celular , Rastreo Celular , Colitis , Factor Nuclear 3-gamma del Hepatocito , Mucosa Intestinal , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Microbiota , Linfocitos T Reguladores , Factores de TranscripciónRESUMEN
Oral tolerance prevents pathological inflammatory responses to innocuous foreign antigens by peripheral regulatory T cells (pT(reg) cells). However, whether a particular subset of antigen-presenting cells (APCs) is required during dietary antigen exposure for the 'instruction' of naive CD4(+) T cells to differentiate into pT(reg) cells has not been defined. Using myeloid lineage-specific APC depletion in mice, we found that monocyte-derived APCs were dispensable, while classical dendritic cells (cDCs) were critical, for pT(reg) cell induction and oral tolerance. CD11b(-) cDCs from the gut-draining lymph nodes efficiently induced pT(reg) cells and, conversely, loss of transcription factor IRF8-dependent CD11b(-) cDCs impaired their polarization, although oral tolerance remained intact. These data reveal the hierarchy of cDC subsets in the induction of pT(reg) cells and their redundancy during the development of oral tolerance.
Asunto(s)
Antígenos/inmunología , Células Dendríticas/inmunología , Tolerancia Inmunológica/inmunología , Linfocitos T Reguladores/inmunología , Animales , Células Presentadoras de Antígenos/inmunología , Células Presentadoras de Antígenos/metabolismo , Antígeno CD11b/inmunología , Antígeno CD11b/metabolismo , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/metabolismo , Diferenciación Celular/genética , Diferenciación Celular/inmunología , Células Cultivadas , Técnicas de Cocultivo , Células Dendríticas/metabolismo , Dieta , Citometría de Flujo , Tolerancia Inmunológica/genética , Inmunización/métodos , Factores Reguladores del Interferón/genética , Factores Reguladores del Interferón/inmunología , Factores Reguladores del Interferón/metabolismo , Ganglios Linfáticos/inmunología , Ganglios Linfáticos/metabolismo , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Linfocitos T Reguladores/metabolismo , Transcriptoma/genética , Transcriptoma/inmunologíaRESUMEN
BACKGROUND: CD4 T cells with features of both T-helper-type 1 (Th1) and 17 (Th17) cells have been implicated in several autoimmune diseases suggesting that plasticity among CD4 T-cell lineages is potentially pathogenic. However, the factors that regulate T-cell lineage stability are largely unknown. Retinoic acid (RA) is synthesised at sites of inflammation. We hypothesised that retinoic acid, a profound epigenetic modifier, could regulate T-cell lineage stability. METHODS: We used a mouse model in which retinoic acid signalling is specifically ablated within the T-cell compartment through overexpression of a dominant negative retinoic acid receptor α (RARα) (dnRARα mice) to investigate its role in the regulation of Th1 lineage stability. Genome-wide ChIP-seq analysis was done to identify RARα targets. In parallel, we performed global mapping of regulatory regions, termed enhancers, to gain mechanistic insight into retinoic acid regulation of T-cell fate. The in-vivo relevance of our findings was determined in a model of oral antigen-induced intestinal inflammation. FINDINGS: We found that retinoic acid is crucial for maintenance of the Th1 lineage. Abrogation of retinoic acid signalling in Th1 cells resulted in loss of T-bet expression and STAT4 activity. Th1 cells from dnRARα mice showed enhanced plasticity with the emergence of hybrid Th1-Th17 and Th17 effector cells. Global analysis of RARα binding and enhancer mapping revealed that RA-RARα directly regulated enhancer activity at Th1 lineage defining genes while repressing genes that regulate Th17 cell fate. Retinoic acid inhibition of Th1 plasticity was essential for maintaining appropriate Th cell responses in vivo and preventing autoimmune intestinal inflammation. INTERPRETATION: Our study has identified RA-RARα as a key component of the regulatory network governing maintenance and plasticity of Th1 cells and defines a new pathway for the development of pathogenic Th17 cells. Retinoids might be novel therapeutic agents for Th17-associated autoimmune diseases. FUNDING: Wellcome Trust.
RESUMEN
CD4(+) T cells differentiate into phenotypically distinct T helper cells upon antigenic stimulation. Regulation of plasticity between these CD4(+) T-cell lineages is critical for immune homeostasis and prevention of autoimmune disease. However, the factors that regulate lineage stability are largely unknown. Here we investigate a role for retinoic acid (RA) in the regulation of lineage stability using T helper 1 (Th1) cells, traditionally considered the most phenotypically stable Th subset. We found that RA, through its receptor RARα, sustains stable expression of Th1 lineage specifying genes, as well as repressing genes that instruct Th17-cell fate. RA signaling is essential for limiting Th1-cell conversion into Th17 effectors and for preventing pathogenic Th17 responses in vivo. Our study identifies RA-RARα as a key component of the regulatory network governing maintenance and plasticity of Th1-cell fate and defines an additional pathway for the development of Th17 cells.
Asunto(s)
Linaje de la Célula/efectos de los fármacos , Receptores de Ácido Retinoico/genética , Linfocitos T Colaboradores-Inductores/efectos de los fármacos , Células TH1/efectos de los fármacos , Células Th17/efectos de los fármacos , Tretinoina/farmacología , Animales , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/inmunología , Linaje de la Célula/inmunología , Regulación de la Expresión Génica , Redes Reguladoras de Genes , Homeostasis/efectos de los fármacos , Homeostasis/inmunología , Integrasas/genética , Integrasas/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Receptores de Ácido Retinoico/inmunología , Receptor alfa de Ácido Retinoico , Transducción de Señal , Linfocitos T Colaboradores-Inductores/citología , Linfocitos T Colaboradores-Inductores/inmunología , Células TH1/citología , Células TH1/inmunología , Células Th17/citología , Células Th17/inmunología , Tretinoina/inmunologíaRESUMEN
An engineered variant of the protease subtilisin from Bacillus amyloliquefaciens, in which the D32A mutation renders the enzyme's activity dependent on the presence of certain small anions such as fluoride or azide, has been produced. This modified enzyme has applications as an azide or fluoride-triggered expression-purification tool. We report activity measurements showing that the enzyme is activated more than 3000-fold by azide and describe the 1.8 A resolution structure of an inactive form (by replacing the catalytic nucleophile Ser 221 with alanine) of the protease, in complex with azide and with a substrate that spans the active site. Both enzyme and substrate have been engineered to increase their stability and the affinity of their interaction. The substrate is based on a stabilized subtilisin prodomain, extended across the active site by the addition of four residues at its C-terminus. In the crystal structure, the substrate is well-ordered across the active site, and the azide anion is observed bound adjacent to Ala 32. The structures of the substrate complex in three different crystals (anion-free, fluoride-soaked, and azide-soaked) are compared. These structures provide extensive information for understanding subtilisin's substrate binding and catalytic mechanism, and for the development of biotechnology tools based on anion-activated proteolysis. The mechanism of anion-dependent proteolysis appears to be a slight modification of the accepted charge-relay mechanism for serine proteases.