RESUMO
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.
Assuntos
Linfócitos T CD4-Positivos , Mucosa Intestinal , Receptores CCR , Receptores de Dopamina D5 , Transdução de Sinais , Receptores CCR/metabolismo , Receptores CCR/genética , Humanos , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD4-Positivos/imunologia , Receptores de Dopamina D5/metabolismo , Receptores de Dopamina D5/genética , Mucosa Intestinal/metabolismo , Colo/metabolismo , Movimento Celular , Dopamina/metabolismo , Doenças Inflamatórias Intestinais/metabolismo , Doenças Inflamatórias Intestinais/patologia , Doenças Inflamatórias Intestinais/imunologiaRESUMO
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.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Trato Gastrointestinal/imunologia , Trato Gastrointestinal/patologia , Inflamação/imunologia , Multimerização Proteica , Receptores CCR/metabolismo , Receptores de Dopamina D5/metabolismo , Sequência de Aminoácidos , Animais , Movimento Celular , Proliferação de Células , Colite/imunologia , Colite/patologia , Humanos , Inflamação/patologia , Doenças Inflamatórias Intestinais/imunologia , Doenças Inflamatórias Intestinais/patologia , Integrina beta1/metabolismo , Células Jurkat , Sistema de Sinalização das MAP Quinases , Camundongos Endogâmicos C57BL , Modelos Biológicos , Peptídeos/química , Fosforilação , Receptores CCR/deficiência , Receptores de Dopamina D5/deficiência , Transdução de Sinais , TropismoRESUMO
Intrathymic T-cell differentiation is a key process for the development and maintenance of cell-mediated immunity, and occurs concomitantly to highly regulated migratory events. We have proposed a multivectorial model for describing intrathymic thymocyte migration. One of the individual vectors comprises interactions mediated by laminins (LMs), a heterotrimeric protein family of the extracellular matrix. Several LMs are expressed in the thymus, being produced by microenvironmental cells, particularly thymic epithelial cells (TECs). Also, thymocytes and epithelial cells express integrin-type LM receptors. Functionally, it has been reported that the dy/dy mutant mouse (lacking the LM isoform 211) exhibits defective thymocyte differentiation. Several data show haptotactic effects of LMs upon thymocytes, as well as their adhesion on TECs; both effects being prevented by anti-LM or anti-LM receptor antibodies. Interestingly, LM synergizes with chemokines to enhance thymocyte migration, whereas classe-3 semaphorins and B ephrins, which exhibit chemorepulsive effects in the thymus, downregulate LM-mediated migratory responses of thymocytes. More recently, we showed that knocking down the ITGA6 gene (which encodes the α6 integrin chain of LM receptors) in human TECs modulates a large number of cell migration-related genes and results in changes of adhesion pattern of thymocytes onto the thymic epithelium. Overall, LM-mediated interactions can be placed at the cross-road of the multivectorial process of thymocyte migration, with a direct influence per se, as well as by modulating other molecular interactions associated with the intrathymic-trafficking events.