RESUMEN
Inflammatory bowel diseases (IBDs) involve chronic inflammation of the gastrointestinal tract, where effector CD4+ T-cells play a central role. Thereby, the recruitment of T-cells into the colonic mucosa represents a key process in IBD. We recently found that CCR9 and DRD5 might form a heteromeric complex on the T-cell surface. The increase in CCL25 production and the reduction in dopamine levels associated with colonic inflammation represent a dual signal stimulating the CCR9:DRD5 heteromer, which promotes the recruitment of CD4+ T-cells into the colonic lamina propria. Here, we aimed to analyse the molecular requirements involved in the heteromer assembly as well as to determine the underlying cellular mechanisms involved in the colonic tropism given by the stimulation of the CCR9:DRD5 complex. The results show that dual stimulation of the CCR9:DRD5 heteromer potentiates the phosphorylation of the myosin light chain 2 (MLC2) and the migration speed in confined microchannels. Accordingly, disrupting the CCR9:DRD5 assembly induced a sharp reduction in the pMLC2 in vitro, decreased the migratory speed in confined microchannels, and dampened the recruitment of CD4+ T-cells into the inflamed colonic mucosa. Furthermore, in silico analysis confirmed that the interface of interaction of CCR9:DRD5 is formed by the transmembrane segments 5 and 6 from each protomer. Our findings demonstrated that the CCR9:DRD5 heteromeric complex plays a fundamental role in the migration of CD4+ T-cells into the colonic mucosa upon inflammation. Thereby, the present study encourages the design of strategies for disassembling the formation of the CCR9:DRD5 as a therapeutic opportunity to treat IBD.
Asunto(s)
Linfocitos T CD4-Positivos , Mucosa Intestinal , Receptores CCR , Receptores de Dopamina D5 , Transducción de Señal , Receptores CCR/metabolismo , Receptores CCR/genética , Humanos , Linfocitos T CD4-Positivos/metabolismo , Linfocitos T CD4-Positivos/inmunología , Receptores de Dopamina D5/metabolismo , Receptores de Dopamina D5/genética , Mucosa Intestinal/metabolismo , Colon/metabolismo , Movimiento Celular , Dopamina/metabolismo , Enfermedades Inflamatorias del Intestino/metabolismo , Enfermedades Inflamatorias del Intestino/patología , Enfermedades Inflamatorias del Intestino/inmunologíaRESUMEN
BACKGROUND: Recent evidence has shown dopamine as a major regulator of inflammation. Accordingly, dopaminergic regulation of immune cells plays an important role in the physiopathology of inflammatory disorders. Multiple sclerosis (MS) is an inflammatory disease involving a CD4+ T-cell-driven autoimmune response to central nervous system (CNS) derived antigens. Evidence from animal models has suggested that B cells play a fundamental role as antigen-presenting cells (APC) re-stimulating CD4+ T cells in the CNS as well as regulating T-cell response by mean of inflammatory or anti-inflammatory cytokines. Here, we addressed the role of the dopamine receptor D3 (DRD3), which displays the highest affinity for dopamine, in B cells in animal models of MS. METHODS: Mice harbouring Drd3-deficient or Drd3-sufficient B cells were generated by bone marrow transplantation into recipient mice devoid of B cells. In these mice, we compared the development of experimental autoimmune encephalomyelitis (EAE) induced by immunization with a myelin oligodendrocyte glycoprotein (MOG)-derived peptide (pMOG), a model that leads to CNS-autoimmunity irrespective of the APC-function of B cells, or by immunization with full-length human MOG protein (huMOG), a model in which antigen-specific activated B cells display a fundamental APC-function in the CNS. APC-function was assessed in vitro by pulsing B cells with huMOG-coated beads and then co-culturing with MOG-specific T cells. RESULTS: Our data show that the selective Drd3 deficiency in B cells abolishes the disease development in the huMOG-induced EAE model. Mechanistic analysis indicates that although DRD3-signalling did not affect the APC-function of B cells, DRD3 favours the CNS-tropism in a subset of pro-inflammatory B cells in the huMOG-induced EAE model, an effect that was associated with higher CXCR3 expression. Conversely, the results show that the selective Drd3 deficiency in B cells exacerbates the disease severity in the pMOG-induced EAE model. Further analysis shows that DRD3-stimulation increased the expression of the CNS-homing molecule CD49d in a B-cell subset with anti-inflammatory features, thus attenuating EAE manifestation in the pMOG-induced EAE model. CONCLUSIONS: Our findings demonstrate that DRD3 in B cells exerts a dual role in CNS-autoimmunity, favouring CNS-tropism of pro-inflammatory B cells with APC-function and promoting CNS-homing of B cells with anti-inflammatory features. Thus, these results show DRD3-signalling in B cells as a critical regulator of CNS-autoimmunity.
Asunto(s)
Autoinmunidad/fisiología , Linfocitos B/metabolismo , Dopamina/metabolismo , Encefalomielitis Autoinmune Experimental/metabolismo , Receptores de Dopamina D3/metabolismo , Secuencia de Aminoácidos , Animales , Linfocitos B/inmunología , Células Cultivadas , Sistema Nervioso Central/inmunología , Sistema Nervioso Central/metabolismo , Dopamina/inmunología , Encefalomielitis Autoinmune Experimental/genética , Encefalomielitis Autoinmune Experimental/inmunología , Femenino , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Receptores de Dopamina D3/genética , Receptores de Dopamina D3/inmunologíaRESUMEN
BACKGROUND AND AIMS: CD4+ T cells constitute central players in inflammatory bowel diseases (IBDs), driving inflammation in the gut mucosa. Current evidence indicates that CCR9 and the integrin α4ß7 are necessary and sufficient to imprint colonic homing on CD4+ T cells upon inflammation. Interestingly, dopaminergic signaling has been previously involved in leukocyte homing. Despite dopamine levels are strongly reduced in the inflamed gut mucosa, the role of dopamine in the gut homing of T cells remains unknown. Here, we study how dopaminergic signaling affects T cells upon gut inflammation. METHODS: Gut inflammation was induced by transfer of naïve T cells into Rag1-/- mice or by administration of dextran sodium sulfate. T cell migration and differentiation were evaluated by adoptive transfer of congenic lymphocytes followed by flow cytometry analysis. Protein interaction was studied by bioluminescence resonance energy transfer analysis, bimolecular fluorescence complementation, and in situ proximity ligation assays. RESULTS: We show the surface receptor providing colonic tropism to effector CD4+ T cells upon inflammation is not CCR9 but the complex formed by CCR9 and the dopamine receptor D5 (DRD5). Assembly of the heteromeric complex was demonstrated in vitro and in vivo using samples from mouse and human origin. The CCR9:DRD5 heteroreceptor was upregulated in the intestinal mucosa of IBD patients. Signaling assays confirmed that complexes behave differently than individual receptors. Remarkably, the disruption of CCR9:DRD5 assembly attenuated the recruitment of CD4+ T cells into the colonic mucosa. CONCLUSIONS: Our findings describe a key homing receptor involved in gut inflammation and introduce a new cell surface module in immune cells: macromolecular complexes formed by G protein-coupled receptors integrating the sensing of multiple molecular cues.
Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Tracto Gastrointestinal/inmunología , Tracto Gastrointestinal/patología , Inflamación/inmunología , Multimerización de Proteína , Receptores CCR/metabolismo , Receptores de Dopamina D5/metabolismo , Secuencia de Aminoácidos , Animales , Movimiento Celular , Proliferación Celular , Colitis/inmunología , Colitis/patología , Humanos , Inflamación/patología , Enfermedades Inflamatorias del Intestino/inmunología , Enfermedades Inflamatorias del Intestino/patología , Integrina beta1/metabolismo , Células Jurkat , Sistema de Señalización de MAP Quinasas , Ratones Endogámicos C57BL , Modelos Biológicos , Péptidos/química , Fosforilación , Receptores CCR/deficiencia , Receptores de Dopamina D5/deficiencia , Transducción de Señal , TropismoRESUMEN
Dendritic cells (DCs) promote T-cell mediated tolerance to self-antigens and induce inflammation to innocuous-antigens. This dual potential makes DCs fundamental players in inflammatory disorders. Evidence from inflammatory colitis mouse models and inflammatory bowel diseases (IBD) patients indicated that gut inflammation in IBD is driven mainly by T-helper-1 (Th1) and Th17 cells, suggesting an essential role for DCs in the development of IBD. Here we show that GSK-J4, a selective inhibitor of the histone demethylase JMJD3/UTX, attenuated inflammatory colitis by reducing the inflammatory potential and increasing the tolerogenic features of DCs. Mechanistic analyses revealed that GSK-J4 increased activating epigenetic signals while reducing repressive marks in the promoter of retinaldehyde dehydrogenase isoforms 1 and 3 in DCs, enhancing the production of retinoic acid. This, in turn, has an impact on regulatory T cells (Treg) increasing their lineage stability and gut tropism as well as potentiating their suppressive activity. Our results open new avenues for the treatment of IBD patients.
Asunto(s)
Benzazepinas/farmacología , Colitis/inmunología , Células Dendríticas/inmunología , Enfermedades Inflamatorias del Intestino/inmunología , Pirimidinas/farmacología , Tretinoina/inmunología , Familia de Aldehído Deshidrogenasa 1/genética , Familia de Aldehído Deshidrogenasa 1/inmunología , Animales , Colitis/tratamiento farmacológico , Colitis/genética , Colitis/patología , Células Dendríticas/patología , Enfermedades Inflamatorias del Intestino/tratamiento farmacológico , Enfermedades Inflamatorias del Intestino/genética , Enfermedades Inflamatorias del Intestino/patología , Ratones , Ratones Noqueados , Retinal-Deshidrogenasa/genética , Retinal-Deshidrogenasa/inmunología , Linfocitos T Reguladores/inmunología , Linfocitos T Reguladores/patología , Células TH1/inmunología , Células TH1/patología , Células Th17/inmunología , Células Th17/patologíaRESUMEN
A number of studies have shown pharmacologic evidence indicating that stimulation of type I dopamine receptor (DR), favors T-helper-17 (Th17)-mediated immunity involved in experimental autoimmune encephalomyelitis (EAE) and in some other inflammatory disorders. Nevertheless, the lack of drugs that might discriminate between DRD1 and DRD5 has made the pharmacological distinction between the two receptors difficult. We have previously shown genetic evidence demonstrating a relevant role of DRD5-signaling in dendritic cells (DCs) favoring the CD4+ T-cell-driven inflammation in EAE. However, the role of DRD5-signaling confined to CD4+ T-cells in the development of EAE is still unknown. Here, we analyzed the functional role of DRD5-signaling in CD4+ T-cell-mediated responses and its relevance in EAE by using a genetic approach. Our results show that DRD5-signaling confined to naive CD4+ T-cells exerts a pro-inflammatory effect promoting the development of EAE with a stronger disease severity. This pro-inflammatory effect observed for DRD5-signaling in naive CD4+ T-cells was related with an exacerbated proliferation in response to T-cell activation and to an increased ability to differentiate toward the Th17 inflammatory phenotype. On the other hand, quite unexpected, our results show that DRD5-signaling confined to Tregs strengthens their suppressive activity, thereby dampening the development of EAE manifestation. This anti-inflammatory effect of DRD5-signaling in Tregs was associated with a selective increase in the expression of glucocorticoid-induced tumor necrosis factor receptor-related protein (GITR), which has been described to play a critical role in the expansion of Tregs. Our findings here indicate a complex role for DRD5-signaling in CD4+ T-cells-driven responses potentiating early inflammation mediated by effector T-cells in EAE, but exacerbating suppressive activity in Tregs and thereby dampening disease manifestation in late EAE stages.
RESUMEN
The dual potential to promote tolerance or inflammation to self-antigens makes dendritic cells (DCs) fundamental players in autoimmunity. Previous results have shown that stimulation of dopamine receptor D5 (DRD5) in DCs potentiates their inflammatory behaviour, favouring the development of experimental autoimmune encephalomyelitis (EAE). Here, we aimed to decipher the underlying mechanism and to test its relevance in multiple sclerosis (MS) patients. Our data shows that DRD5-deficiency confined to DCs in EAE mice resulted in reduced frequencies of CD4+ T-cell subsets with inflammatory potential in the central nervous system, including not only Th1 and Th17 cells but also granulocyte-macrophage colony-stimulating factor producers. Importantly, ex vivo depletion of dopamine from DCs resulted in a dramatic reduction of EAE severity, highlighting the relevance of an autocrine loop promoting inflammation in vivo. Mechanistic analyses indicated that DRD5-signalling in both mouse DCs and human monocytes involves the attenuation of signal transducer and activator of transcription 3-activation, a transcription factor that limits the production of the inflammatory cytokines interleukin (IL)-12 and IL-23. Furthermore, we found an exacerbated expression of all dopamine receptors in peripheral blood pro-inflammatory monocytes obtained from MS patients. These findings illustrate a novel mechanism by which myeloid antigen-presenting cells may trigger the onset of their inflammatory behaviour promoting the development of autoimmunity.
Asunto(s)
Células Dendríticas/inmunología , Dopamina/inmunología , Encefalomielitis Autoinmune Experimental/inmunología , Monocitos/inmunología , Esclerosis Múltiple/inmunología , Factor de Transcripción STAT3/inmunología , Adulto , Animales , Citocinas/inmunología , Citocinas/metabolismo , Células Dendríticas/metabolismo , Dopamina/metabolismo , Encefalomielitis Autoinmune Experimental/genética , Encefalomielitis Autoinmune Experimental/metabolismo , Femenino , Células HEK293 , Humanos , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Persona de Mediana Edad , Monocitos/metabolismo , Esclerosis Múltiple/genética , Esclerosis Múltiple/metabolismo , Receptores de Dopamina D5/genética , Receptores de Dopamina D5/inmunología , Receptores de Dopamina D5/metabolismo , Factor de Transcripción STAT3/genética , Factor de Transcripción STAT3/metabolismo , Células TH1/inmunología , Células TH1/metabolismo , Células Th17/inmunología , Células Th17/metabolismoRESUMEN
Galectin-8 (Gal-8) is a member of a glycan-binding protein family that regulates the immune system, among other functions, and is a target of antibodies in autoimmune disorders. However, its role in multiple sclerosis (MS), an autoimmune inflammatory disease of the central nervous system (CNS), remains unknown. We study the consequences of Gal-8 silencing on lymphocyte subpopulations and the development of experimental autoimmune encephalitis (EAE), to then assess the presence and clinical meaning of anti-Gal-8 antibodies in MS patients. Lgals8/Lac-Z knock-in mice lacking Gal-8 expression have higher polarization toward Th17 cells accompanied with decreased CCR6+ and higher CXCR3+ regulatory T cells (Tregs) frequency. These conditions result in exacerbated MOG35-55 peptide-induced EAE. Gal-8 eliminates activated Th17 but not Th1 cells by apoptosis and ameliorates EAE in C57BL/6 wild-type mice. ß-gal histochemistry reflecting the activity of the Gal-8 promoter revealed Gal-8 expression in a wide range of CNS regions, including high expression in the choroid-plexus. Accordingly, we detected Gal-8 in human cerebrospinal fluid, suggesting a role in the CNS immune-surveillance circuit. In addition, we show that MS patients generate function-blocking anti-Gal-8 antibodies with pathogenic potential. Such antibodies block cell adhesion and Gal-8-induced Th17 apoptosis. Furthermore, circulating anti-Gal-8 antibodies associate with relapsing-remitting MS (RRMS), and not with progressive MS phenotypes, predicting clinical disability at diagnosis within the first year of follow-up. Our results reveal that Gal-8 has an immunosuppressive protective role against autoimmune CNS inflammation, modulating the balance of Th17 and Th1 polarization and their respective Tregs. Such a role can be counteracted during RRMS by anti-Gal-8 antibodies, worsening disease prognosis. Even though anti-Gal-8 antibodies are not specific for MS, our results suggest that they could be a potential early severity biomarker in RRMS.
Asunto(s)
Autoanticuerpos/inmunología , Encefalomielitis Autoinmune Experimental/inmunología , Galectinas/inmunología , Esclerosis Múltiple/inmunología , Linfocitos T Reguladores/inmunología , Células Th17/inmunología , Animales , Apoptosis/fisiología , Encéfalo/inmunología , Encéfalo/metabolismo , Adhesión Celular/fisiología , Encefalomielitis Autoinmune Experimental/genética , Encefalomielitis Autoinmune Experimental/metabolismo , Femenino , Galectinas/genética , Galectinas/metabolismo , Silenciador del Gen , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Esclerosis Múltiple/genética , Esclerosis Múltiple/metabolismo , Pronóstico , Linfocitos T Reguladores/metabolismo , Células Th17/metabolismoRESUMEN
As it has been established that demethylation of lysine 27 of histone H3 by the lysine-specific demethylase JMJD3 increases immune responses and thus elicits inflammation, we hypothesize that inhibition of JMJD3 may attenuate autoimmune disorders. We found that in vivo administration of GSK-J4, a selective inhibitor of JMJD3 and UTX, ameliorates the severity of experimental autoimmune encephalomyelitis (EAE). In vitro experiments revealed that the anti-inflammatory effect of GSK-J4 was exerted through an effect on dendritic cells (DCs), promoting a tolerogenic profile characterized by reduced expression of costimulatory molecules CD80/CD86, an increased expression of tolerogenic molecules CD103 and TGF-ß1, and reduced secretion of proinflammatory cytokines IL-6, IFN-γ, and TNF. Adoptive transfer of GSK-J4-treated DCs into EAE mice reduced the clinical manifestation of the disease and decreased the extent of inflammatory CD4+ T cells infiltrating the central nervous system. Notably, Treg generation, stability, and suppressive activity were all exacerbated by GSK-J4-treated DCs without affecting Th1 and Th17 cell production. Our data show that GSK-J4-mediated modulation of inflammation is achieved by a direct effect on DCs and that systemic treatment with GSK-J4 or adoptive transfer of GSK-J4-treated DCs ex vivo may be promising approaches for the treatment of inflammatory and autoimmune disorders.
Asunto(s)
Benzazepinas/farmacología , Células Dendríticas/efectos de los fármacos , Encefalomielitis Autoinmune Experimental/prevención & control , Histona Demetilasas con Dominio de Jumonji/antagonistas & inhibidores , Pirimidinas/farmacología , Traslado Adoptivo , Animales , Antígenos CD/inmunología , Antígenos CD/metabolismo , Antígeno B7-1/inmunología , Antígeno B7-1/metabolismo , Antígeno B7-2/inmunología , Antígeno B7-2/metabolismo , Western Blotting , Linfocitos T CD4-Positivos/inmunología , Citocinas/genética , Citocinas/inmunología , Citocinas/metabolismo , Células Dendríticas/inmunología , Células Dendríticas/metabolismo , Encefalomielitis Autoinmune Experimental/genética , Encefalomielitis Autoinmune Experimental/inmunología , Expresión Génica/efectos de los fármacos , Tolerancia Inmunológica/genética , Tolerancia Inmunológica/inmunología , Mediadores de Inflamación/inmunología , Mediadores de Inflamación/metabolismo , Cadenas alfa de Integrinas/inmunología , Cadenas alfa de Integrinas/metabolismo , Histona Demetilasas con Dominio de Jumonji/inmunología , Histona Demetilasas con Dominio de Jumonji/metabolismo , Ratones Endogámicos C57BL , Ratones Transgénicos , Fenotipo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factor de Crecimiento Transformador beta1/inmunología , Factor de Crecimiento Transformador beta1/metabolismoRESUMEN
T helper type 17 (Th17) lymphocytes, characterized by the production of interleukin-17 and other pro-inflammatory cytokines, are present in intestinal lamina propria and have been described as important players driving intestinal inflammation. Recent evidence, supporting the notion of a functional and phenotypic instability of Th17 cells, has shown that Th17 differentiate into type 1 regulatory (Tr1) T cells during the resolution of intestinal inflammation. Moreover, it has been suggested that the expression of CD39 ectonucleotidase endows Th17 cells with immunosuppressive properties. However, the exact role of CD39 ectonucleotidase in Th17 cells has not been studied in the context of intestinal inflammation. Here we show that Th17 cells expressing CD39 ectonucleotidase can hydrolyze ATP and survive to ATP-induced cell death. Moreover, in vitro-generated Th17 cells expressing the CD39 ectonucleotidase produce IL-10 and are less pathogenic than CD39 negative Th17 cells in a model of experimental colitis in Rag-/- mice. Remarkably, we show that CD39 activity regulates the conversion of Th17 cells to IL-10-producing cells in vitro, which is abrogated in the presence of ATP and the CD39-specific inhibitor ARL67156. All these data suggest that CD39 expression by Th17 cells allows the depletion of ATP and is crucial for IL-10 production and survival during the resolution of intestinal inflammation.