RESUMEN
T-bet is essential for natural regulatory T cells (nTreg) to regulate Th1 inflammation, but whether T-bet controls other Treg functions after entering the inflammatory site is unknown. In an islet allograft model, T-bet(-/-) nTreg, but not induced Treg, failed to prolong graft survival as effectively as wild-type Treg. T-bet(-/-) nTreg had no functional deficiency in vitro but failed to home from the graft to draining lymph nodes (dLN) as efficiently as wild type. T-bet regulated expression of adhesion- and migration-related molecules, influencing nTreg distribution in tissues, so that T-bet(-/-) nTreg remained in the grafts rather than migrating to lymphatics and dLN. In contrast, both wild-type and T-bet(-/-) CD4(+) conventional T cells and induced Treg migrated normally toward afferent lymphatics. T-bet(-/-) nTreg displayed instability in the graft, failing to suppress Ag-specific CD4(+) T cells and prevent their infiltration into the graft and dLN. Thus, T-bet regulates nTreg migration into afferent lymphatics and dLN and consequently their suppressive stability in vivo.
Asunto(s)
Quimiotaxis de Leucocito/inmunología , Tolerancia Inmunológica/inmunología , Proteínas de Dominio T Box/inmunología , Linfocitos T Reguladores/inmunología , Traslado Adoptivo , Animales , Modelos Animales de Enfermedad , Citometría de Flujo , Trasplante de Islotes Pancreáticos/inmunología , Ganglios Linfáticos/inmunología , Vasos Linfáticos/inmunología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
Endotoxin tolerance reprograms Toll-like receptor (TLR) 4-mediated macrophage responses by attenuating induction of proinflammatory cytokines while retaining expression of anti-inflammatory and antimicrobial mediators. We previously demonstrated deficient TLR4-induced activation of IL-1 receptor-associated kinase (IRAK) 4, IRAK1, and TANK-binding kinase (TBK) 1 as critical hallmarks of endotoxin tolerance, but mechanisms remain unclear. In this study, we examined the role of the E3 ubiquitin ligase Pellino-1 in endotoxin tolerance and TLR signaling. LPS stimulation increased Pellino-1 mRNA and protein expression in macrophages from mice injected with saline and in medium-pretreated human monocytes, THP-1, and MonoMac-6 cells, whereas endotoxin tolerization abrogated LPS inducibility of Pellino-1. Overexpression of Pellino-1 in 293/TLR2 and 293/TLR4/MD2 cells enhanced TLR2- and TLR4-induced nuclear factor κB (NF-κB) and expression of IL-8 mRNA, whereas Pellino-1 knockdown reduced these responses. Pellino-1 ablation in THP-1 cells impaired induction of myeloid differentiation primary response protein (MyD88), and Toll-IL-1R domain-containing adapter inducing IFN-ß (TRIF)-dependent cytokine genes in response to TLR4 and TLR2 agonists and heat-killed Escherichia coli and Staphylococcus aureus, whereas only weakly affecting phagocytosis of heat-killed bacteria. Co-expressed Pellino-1 potentiated NF-κB activation driven by transfected MyD88, TRIF, IRAK1, TBK1, TGF-ß-activated kinase (TAK) 1, and TNFR-associated factor 6, whereas not affecting p65-induced responses. Mechanistically, Pellino-1 increased LPS-driven K63-linked polyubiquitination of IRAK1, TBK1, TAK1, and phosphorylation of TBK1 and IFN regulatory factor 3. These results reveal a novel mechanism by which endotoxin tolerance re-programs TLR4 signaling via suppression of Pellino-1, a positive regulator of MyD88- and TRIF-dependent signaling that promotes K63-linked polyubiquitination of IRAK1, TBK1, and TAK1.
Asunto(s)
Proteínas Nucleares/fisiología , Receptor Toll-Like 2/metabolismo , Receptor Toll-Like 4/metabolismo , Ubiquitina-Proteína Ligasas/fisiología , Animales , Células HEK293 , Humanos , Quinasas Asociadas a Receptores de Interleucina-1/metabolismo , Lipopolisacáridos/farmacología , Quinasas Quinasa Quinasa PAM/metabolismo , Ratones Endogámicos C57BL , Monocitos/inmunología , Monocitos/metabolismo , FN-kappa B/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Transducción de Señal , Factor 6 Asociado a Receptor de TNF/metabolismo , Activación Transcripcional/inmunología , UbiquitinaciónRESUMEN
Asp(299)Gly (D299G) and, to a lesser extent, Thr(399)Ile (T399I) TLR4 polymorphisms have been associated with gram-negative sepsis and other infectious diseases, but the mechanisms by which they affect TLR4 signaling are unclear. In this study, we determined the impact of the D299G and T399I polymorphisms on TLR4 expression, interactions with myeloid differentiation factor 2 (MD2), LPS binding, and LPS-mediated activation of the MyD88- and Toll/IL-1R resistance domain-containing adapter inducing IFN-ß (TRIF) signaling pathways. Complementation of human embryonic kidney 293/CD14/MD2 transfectants with wild-type (WT) or mutant yellow fluorescent protein-tagged TLR4 variants revealed comparable total TLR4 expression, TLR4-MD2 interactions, and LPS binding. FACS analyses with anti-TLR4 Ab showed only minimal changes in the cell-surface levels of the D299G TLR4. Cells transfected with D299G TLR4 exhibited impaired LPS-induced phosphorylation of p38 and TANK-binding kinase 1, activation of NF-κB and IFN regulatory factor 3, and induction of IL-8 and IFN-ß mRNA, whereas T399I TLR4 did not cause statistically significant inhibition. In contrast to WT TLR4, expression of the D299G mutants in TLR4(-/-) mouse macrophages failed to elicit LPS-mediated induction of TNF-α and IFN-ß mRNA. Coimmunoprecipitation revealed diminished LPS-driven interaction of MyD88 and TRIF with the D299G TLR4 species, in contrast to robust adapter recruitment exhibited by WT TLR4. Thus, the D299G polymorphism compromises recruitment of MyD88 and TRIF to TLR4 without affecting TLR4 expression, TLR4-MD2 interaction, or LPS binding, suggesting that it interferes with TLR4 dimerization and assembly of intracellular docking platforms for adapter recruitment.
Asunto(s)
Proteínas Adaptadoras del Transporte Vesicular/inmunología , Macrófagos/inmunología , Factor 88 de Diferenciación Mieloide/inmunología , Polimorfismo Genético/inmunología , Transducción de Señal/inmunología , Receptor Toll-Like 4/inmunología , Proteínas Adaptadoras del Transporte Vesicular/genética , Proteínas Adaptadoras del Transporte Vesicular/metabolismo , Animales , Células Cultivadas , Células HEK293 , Humanos , Factor 3 Regulador del Interferón/genética , Factor 3 Regulador del Interferón/inmunología , Factor 3 Regulador del Interferón/metabolismo , Lipopolisacáridos/farmacología , Antígeno 96 de los Linfocitos/genética , Antígeno 96 de los Linfocitos/inmunología , Antígeno 96 de los Linfocitos/metabolismo , Macrófagos/citología , Macrófagos/metabolismo , Ratones , Ratones Noqueados , Factor 88 de Diferenciación Mieloide/genética , Factor 88 de Diferenciación Mieloide/metabolismo , FN-kappa B/genética , FN-kappa B/inmunología , FN-kappa B/metabolismo , Fosforilación/efectos de los fármacos , Fosforilación/genética , Fosforilación/inmunología , Proteínas Quinasas/genética , Proteínas Quinasas/inmunología , Proteínas Quinasas/metabolismo , Multimerización de Proteína/genética , Multimerización de Proteína/inmunología , Transducción de Señal/genética , Receptor Toll-Like 4/genética , Receptor Toll-Like 4/metabolismoRESUMEN
The R753Q polymorphism in the Toll-IL-1 receptor domain of Toll-like receptor 2 (TLR2) has been linked to increased incidence of tuberculosis and other infectious diseases, but the mechanisms by which it affects TLR2 functions are unclear. Here, we studied the impact of the R753Q polymorphism on TLR2 expression, hetero-dimerization with TLR6, tyrosine phosphorylation, and recruitment of myeloid differentiation primary response protein (MyD) 88 and MyD88 adapter-like (Mal). Complementation of HEK293 cells with transfected WT or R753Q TLR2 revealed their comparable total levels and only minimal changes in cell surface expression of the mutant species. Notably, even a 100-fold increase in amounts of transfected R753Q TLR2 versus WT variant did not overcome the compromised ability of the mutant TLR2 to activate nuclear factor κB (NF-κB), indicating that a minimal decrease in cell surface levels of the R753Q TLR2 cannot account for the signaling deficiency. Molecular modeling studies suggested that the R753Q mutation changes the electrostatic potential of the DD loop and results in a discrete movement of the residues critical for protein-protein interactions. Confirming these predictions, biochemical assays demonstrated that R753Q TLR2 exhibits deficient agonist-induced tyrosine phosphorylation, hetero-dimerization with TLR6, and recruitment of Mal and MyD88. These proximal signaling deficiencies correlated with impaired capacities of the R753Q TLR2 to mediate p38 phosphorylation, NF-κB activation, and induction of IL-8 mRNA in transfected HEK293 cells challenged with inactivated Mycobacterium tuberculosis or mycobacterial components. Thus, the R753Q polymorphism renders TLR2 signaling-incompetent by impairing its tyrosine phosphorylation, dimerization with TLR6, and recruitment of Mal and MyD88.
Asunto(s)
Factor 88 de Diferenciación Mieloide/metabolismo , Polimorfismo Genético , Receptor Toll-Like 2/genética , Receptor Toll-Like 2/metabolismo , Receptor Toll-Like 6/metabolismo , Sustitución de Aminoácidos/genética , Sustitución de Aminoácidos/inmunología , Animales , Células Cultivadas , Expresión Génica , Células HEK293 , Humanos , Immunoblotting , Interleucina-8/genética , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Ratones , Ratones Noqueados , Microscopía Confocal , Modelos Moleculares , Mycobacterium tuberculosis/inmunología , Factor 88 de Diferenciación Mieloide/genética , FN-kappa B/metabolismo , Fosforilación , Multimerización de Proteína , Estructura Terciaria de Proteína , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal/genética , Transducción de Señal/inmunología , Receptor Toll-Like 2/química , Receptor Toll-Like 6/química , Receptor Toll-Like 6/genética , Tirosina/metabolismoRESUMEN
PURPOSE OF REVIEW: The mechanisms of tolerance induction and maintenance remain incompletely understood and have yet to be translated to clinical practice. Advances in imaging techniques have allowed precise examination of cell interactions in the lymph node, often in real time. Herein we review evidence that lymph node structure is dynamic and controls the character of the immune response in a multistep, multiplayer dance. T-cell responses in particular can be initiated or influenced in regions beyond the canonical T-cell zone. We propose that the cortical ridge is one such region required for induction and maintenance of tolerance. RECENT FINDINGS: Lymph node domains are more complex than T-cell and B-cell zones. Different domains are important for different types of immune responses. These domains are in part defined by dynamic, malleable physical structures that guide cell interactions and influence immune outcomes. SUMMARY: Further probing as to how lymph node stromal cells and fibers interact with and determine the character of immune responses should yield fundamental insights into tolerance and immunity. Manipulation of lymph node structure and associated unique cell types and molecules may allow therapeutic interventions in the tolerogenic process.
Asunto(s)
Linfocitos B/inmunología , Tolerancia Inmunológica/fisiología , Ganglios Linfáticos/inmunología , Linfocitos T/inmunología , Animales , Comunicación Celular , HumanosRESUMEN
Antigen-specific tolerance is a key goal of experimental immunotherapies for autoimmune disease and allograft rejection. This outcome could selectively inhibit detrimental inflammatory immune responses without compromising functional protective immunity. A major challenge facing antigen-specific immunotherapies is ineffective control over immune signal targeting and integration, limiting efficacy and causing systemic non-specific suppression. Here we use intra-lymph node injection of diffusion-limited degradable microparticles that encapsulate self-antigens with the immunomodulatory small molecule, rapamycin. We show this strategy potently inhibits disease during pre-clinical type 1 diabetes and allogenic islet transplantation. Antigen and rapamycin are required for maximal efficacy, and tolerance is accompanied by expansion of antigen-specific regulatory T cells in treated and untreated lymph nodes. The antigen-specific tolerance in type 1 diabetes is systemic but avoids non-specific immune suppression. Further, microparticle treatment results in the development of tolerogenic structural microdomains in lymph nodes. Finally, these local structural and functional changes in lymph nodes promote memory markers among antigen-specific regulatory T cells, and tolerance that is durable. This work supports intra-lymph node injection of tolerogenic microparticles as a powerful platform to promote antigen-dependent efficacy in type 1 diabetes and allogenic islet transplantation.
Asunto(s)
Diabetes Mellitus Tipo 1 , Trasplante de Islotes Pancreáticos , Humanos , Tolerancia Inmunológica , Autoantígenos , Ganglios Linfáticos/patología , SirolimusRESUMEN
Fibroblastic reticular cells (FRCs) play important roles in tolerance by producing laminin α4 (Lama4) and altering lymph node (LN) structure and function. The present study revealed the specific roles of extracellular matrix Lama4 in regulating LN conduits using FRC-specific KO mouse strains. FRC-derived Lama4 maintained conduit fiber integrity, as its depletion altered conduit morphology and structure and reduced homeostatic conduit flow. Lama4 regulated the lymphotoxin ß receptor (LTßR) pathway, which is critical for conduit and LN integrity. Depleting LTßR in FRCs further reduced conduits and impaired reticular fibers. Lama4 was indispensable for FRC generation and survival, as FRCs lacking Lama4 displayed reduced proliferation but upregulated senescence and apoptosis. During acute immunization, FRC Lama4 deficiency increased antigen flow through conduits. Importantly, adoptive transfer of WT FRCs to FRC Lama4-deficient mice rescued conduit structure, ameliorated Treg and chemokine distribution, and restored transplant allograft acceptance, which were all impaired by FRC Lama4 depletion. Single-cell RNA sequencing analysis of LN stromal cells indicated that the laminin and collagen signaling pathways linked crosstalk among FRC subsets and endothelial cells. This study demonstrated that FRC Lama4 is responsible for maintaining conduits by FRCs and can be harnessed to potentiate FRC-based immunomodulation.
Asunto(s)
Células Endoteliales , Laminina , Ratones , Animales , Laminina/genética , Laminina/metabolismo , Ganglios Linfáticos , Transducción de Señal , Quimiocinas/metabolismoRESUMEN
Endotoxin tolerance reprograms Toll-like receptor 4 responses by impairing LPS-elicited production of pro-inflammatory cytokines without inhibiting expression of anti-inflammatory or anti-microbial mediators. In septic patients, Toll-like receptor tolerance is thought to underlie decreased pro-inflammatory cytokine expression in response to LPS and increased incidence of microbial infections. The impact of endotoxin tolerance on recruitment, post-translational modifications and signalosome assembly of IL-1 receptor-associated kinase (IRAK) 4, IRAK1, TNF receptor-associated factor (TRAF) 6, TGF-ß-activated kinase (TAK) 1, and IκB kinase (IKK) γ is largely unknown. We report that endotoxin tolerization of THP1 cells and human monocytes impairs LPS-mediated receptor recruitment and activation of IRAK4, ablates K63-linked polyubiquitination of IRAK1 and TRAF6, compromises assembly of IRAK1-TRAF6 and IRAK1-IKKγ platforms, and inhibits TAK1 activation. Deficiencies in these signaling events in LPS-tolerant cells coincided with increased expression of A20, an essential deubiquitination enzyme, and sustained A20-IRAK1 associations. Overexpression of A20 inhibited LPS-induced activation of NF-κB and ablated NF-κB reporter activation driven by ectopic expression of MyD88, IRAK1, IRAK2, TRAF6, and TAK1/TAB1, while not affecting the responses induced by IKKß and p65. A20 shRNA knockdown abolished LPS tolerization of THP1 cells, mechanistically linking A20 and endotoxin tolerance. Thus, deficient LPS-induced activation of IRAK4 and TAK1, K63-linked polyubiquitination of IRAK1 and TRAF6, and disrupted IRAK1-TRAF6 and IRAK1-IKKγ assembly associated with increased A20 expression and A20-IRAK1 interactions are new determinants of endotoxin tolerance.
Asunto(s)
Resistencia a Medicamentos/efectos de los fármacos , Regulación de la Expresión Génica/efectos de los fármacos , Quinasa I-kappa B/metabolismo , Quinasas Asociadas a Receptores de Interleucina-1/metabolismo , Lipopolisacáridos/farmacología , Quinasas Quinasa Quinasa PAM/metabolismo , Monocitos/metabolismo , Proteínas Nucleares/biosíntesis , Factor 6 Asociado a Receptor de TNF/metabolismo , Ubiquitinación/efectos de los fármacos , Línea Celular , Proteínas de Unión al ADN , Humanos , Quinasa I-kappa B/genética , Quinasas Asociadas a Receptores de Interleucina-1/genética , Péptidos y Proteínas de Señalización Intracelular/genética , Quinasas Quinasa Quinasa PAM/genética , Factor 88 de Diferenciación Mieloide/genética , Factor 88 de Diferenciación Mieloide/metabolismo , Proteínas Nucleares/genética , Factor 6 Asociado a Receptor de TNF/genética , Proteína 3 Inducida por el Factor de Necrosis Tumoral alfaRESUMEN
Regulatory T cell (Treg) lymphatic migration is required for resolving inflammation and prolonging allograft survival. Focusing on Treg interactions with lymphatic endothelial cells (LECs), we dissect mechanisms and functional consequences of Treg transendothelial migration (TEM). Using three genetic mouse models of pancreatic islet transplantation, we show that Treg lymphotoxin (LT) αß and LEC LTß receptor (LTßR) signaling are required for efficient Treg migration and suppressive function to prolong allograft survival. Inhibition of LT signaling increases Treg conversion to Foxp3loCD25lo exTregs. In a transwell-based model of TEM across polarized LECs, non-migrated Tregs become exTregs. Such conversion is regulated by LTßR nuclear factor κB (NF-κB) signaling in LECs, which increases interleukin-6 (IL-6) production and drives exTreg conversion. Migrating Tregs are ectonucleotidase CD39hi and resist exTreg conversion in an adenosine-receptor-2A-dependent fashion. Human Tregs migrating across human LECs behave similarly. These molecular interactions can be targeted for therapeutic manipulation of immunity and suppression.
Asunto(s)
Células Endoteliales , Linfocitos T Reguladores , Adenosina , Animales , Factores de Transcripción Forkhead/genética , Linfotoxina beta , Ratones , FN-kappa BRESUMEN
Lymph node (LN) fibroblastic reticular cells (FRCs) define LN niches and regulate lymphocyte homeostasis through producing diverse extracellular matrix (ECM) components. We examined the role of ECM laminin α4 (Lama4) using FRC-Lama4 conditional KO Pdgfrb-Cre-/- × Lama4fl/fl mice. Single-cell RNA-sequencing (scRNA-Seq) data showed the promoter gene Pdgfrb was exclusively expressed in FRCs. Depleting FRC-Lama4 reduced Tregs and dendritic cells, decreased high endothelial venules, impaired the conduit system, and downregulated T cell survival factors in LNs. FRC-Lama4 depletion impaired the homing of lymphocytes to LNs in homeostasis and after allografting. Alloantigen-specific T cells proliferated, were activated to greater degrees in LNs lacking FRC-Lama4, and were more prone to differentiate into effector phenotypes relative to the Treg phenotype. In murine cardiac transplantation, tolerogenic immunosuppression was not effective in FRC-Lama4 recipients, which produced more alloantibodies than WT. After lung transplantation, FRC-Lama4-KO mice had more severe graft rejection with fewer Tregs in their LNs. Overall, FRC-Lama4 critically contributes to a tolerogenic LN niche by supporting T cell migration, constraining T cell activation and proliferation, and promoting Treg differentiation. Hence, it serves as a therapeutic target for immunoengineering.
Asunto(s)
Laminina , Ganglios Linfáticos , Reticulina , Linfocitos T , Animales , Laminina/inmunología , Ganglios Linfáticos/inmunología , Ratones , Receptor beta de Factor de Crecimiento Derivado de Plaquetas , Reticulina/inmunología , Linfocitos T/inmunología , Linfocitos T Reguladores/inmunología , Inmunología del TrasplanteRESUMEN
Regulatory T cells (Tregs) express high levels of cell surface lymphotoxin alpha beta (LTα1ß2) to activate the LT beta receptor (LTßR) on the lymphatic endothelial cells (LECs), modulating LEC adhesion molecules, intercellular junctions, and chemokines. We demonstrate a role for Tregs through this pathway to condition the permissiveness of lymphatic endothelia for transendothelial migration (TEM), thus gating leukocyte traffic. Human Tregs share the same property with murine Tregs. Activation of TLR2 on Tregs during inflammation specifically augments LTα1ß2-LTßR signaling, which further enhances the permissiveness of LECs to facilitate TEM. The conditioning of endothelia may promote the resolution of inflammation by directing leukocytes out of tissues to lymphatic vessels and draining lymph nodes (dLNs). Thus, Tregs interact with lymphatic endothelia under homeostasis and inflammation and dictate endothelial permissiveness and gating mechanisms for subsequent leukocyte migration through endothelial barriers.
Asunto(s)
Movimiento Celular/inmunología , Endotelio Linfático/metabolismo , Inflamación/metabolismo , Linfocitos T Reguladores/metabolismo , Receptor Toll-Like 2/metabolismo , Migración Transendotelial y Transepitelial/inmunología , Animales , Linfocitos T CD4-Positivos/metabolismo , Cadherinas/metabolismo , Línea Celular , Movimiento Celular/efectos de los fármacos , Quimiocina CCL21/metabolismo , Endotelio Linfático/efectos de los fármacos , Humanos , Inflamación/inmunología , Inflamación/patología , Interleucina-2/farmacología , Islotes Pancreáticos/metabolismo , Ganglios Linfáticos/inmunología , Ganglios Linfáticos/metabolismo , Receptor beta de Linfotoxina/metabolismo , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Sistema de Señalización de MAP Quinasas/genética , Sistema de Señalización de MAP Quinasas/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , FN-kappa B/antagonistas & inhibidores , FN-kappa B/metabolismo , Protocadherinas , Receptores de Interleucina-2/metabolismo , Transducción de Señal/efectos de los fármacos , Transducción de Señal/inmunología , Linfocitos T Reguladores/efectos de los fármacos , Receptor Toll-Like 2/inmunología , Migración Transendotelial y Transepitelial/efectos de los fármacos , Molécula 1 de Adhesión Celular Vascular/metabolismoRESUMEN
Lymph node stromal cells (LNSCs) regulate immunity through constructing lymphocyte niches. LNSC-produced laminin α5 (Lama5) regulates CD4+ T cells but the underlying mechanisms of its functions are poorly understood. Here we show that depleting Lama5 in LNSCs resulted in decreased Lama5 protein in the LN cortical ridge (CR) and around high endothelial venules (HEVs). Lama5 depletion affected LN structure with increased HEVs, upregulated chemokines, and cell adhesion molecules, and led to greater numbers of Tregs in the T cell zone. Mouse and human T cell transendothelial migration and T cell entry into LNs were suppressed by Lama5 through the receptors α6 integrin and α-dystroglycan. During immune responses and allograft transplantation, depleting Lama5 promoted antigen-specific CD4+ T cell entry into the CR through HEVs, suppressed T cell activation, and altered T cell differentiation to suppressive regulatory phenotypes. Enhanced allograft acceptance resulted from depleting Lama5 or blockade of T cell Lama5 receptors. Lama5 and Lama4/Lama5 ratios in allografts were associated with the rejection severity. Overall, our results demonstrated that stromal Lama5 regulated immune responses through altering LN structures and T cell behaviors. This study delineated a stromal Lama5-T cell receptor axis that can be targeted for immune tolerance modulation.
Asunto(s)
Laminina/inmunología , Ganglios Linfáticos/inmunología , Tolerancia al Trasplante/inmunología , Animales , Distroglicanos/metabolismo , Humanos , Integrina alfa6/metabolismo , Laminina/genética , Laminina/metabolismo , Ganglios Linfáticos/citología , Ganglios Linfáticos/metabolismo , Vasos Linfáticos/citología , Vasos Linfáticos/inmunología , Vasos Linfáticos/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Células del Estroma/citología , Células del Estroma/inmunología , Células del Estroma/metabolismo , Linfocitos T/citología , Linfocitos T/inmunología , Linfocitos T/metabolismo , Linfocitos T Reguladores/inmunología , Migración Transendotelial y Transepitelial/inmunologíaRESUMEN
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
RESUMEN
Sphingosine 1-phosphate (S1P) and S1P receptors (S1PRs) regulate migration of lymphocytes out of thymus to blood and lymph nodes (LNs) to efferent lymph, whereas their role in other tissue sites is not known. Here, we investigated the question of how these molecules regulate leukocyte migration from tissues through afferent lymphatics to draining LNs (dLNs). S1P, but not other chemokines, selectively enhanced human and murine CD4 T cell migration across lymphatic endothelial cells (LECs). T cell S1PR1 and S1PR4, and LEC S1PR2, were required for migration across LECs and into lymphatic vessels and dLNs. S1PR1 and S1PR4 differentially regulated T cell motility and vascular cell adhesion molecule-1 (VCAM-1) binding. S1PR2 regulated LEC layer structure, permeability, and expression of the junction molecules VE-cadherin, occludin, and zonulin-1 through the ERK pathway. S1PR2 facilitated T cell transcellular migration through VCAM-1 expression and recruitment of T cells to LEC migration sites. These results demonstrated distinct roles for S1PRs in comodulating T cell and LEC functions in migration and suggest previously unknown levels of regulation of leukocytes and endothelial cells during homeostasis and immunity.
Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Movimiento Celular/inmunología , Células Endoteliales/inmunología , Vasos Linfáticos/inmunología , Receptores de Esfingosina-1-Fosfato/inmunología , Animales , Linfocitos T CD4-Positivos/fisiología , Línea Celular , Células Endoteliales/fisiología , Humanos , Lisofosfolípidos/inmunología , Sistema de Señalización de MAP Quinasas , Ratones Endogámicos C57BL , Ratones Transgénicos , Esfingosina/análogos & derivados , Esfingosina/inmunología , Receptores de Esfingosina-1-Fosfato/genética , Proteínas de Uniones Estrechas/inmunología , Molécula 1 de Adhesión Celular Vascular/inmunologíaRESUMEN
Sphingosine 1-phosphate (S1P) and lysophosphatidic acid (LPA) activate G protein-coupled receptors (GPCRs) to regulate biological processes. Using a genome-wide CRISPR/dCas9-based GPCR signaling screen, LPAR1 was identified as an inducer of S1PR1/ß-arrestin coupling while suppressing Gαi signaling. S1pr1 and Lpar1-positive lymphatic endothelial cells (LECs) of lymph nodes exhibit constitutive S1PR1/ß-arrestin signaling, which was suppressed by LPAR1 antagonism. Pharmacological inhibition or genetic loss of function of Lpar1 reduced the frequency of punctate junctions at sinus-lining LECs. Ligand activation of transfected LPAR1 in endothelial cells remodeled junctions from continuous to punctate structures and increased transendothelial permeability. In addition, LPAR1 antagonism in mice increased lymph node retention of adoptively transferred lymphocytes. These data suggest that cross-talk between LPAR1 and S1PR1 promotes the porous junctional architecture of sinus-lining LECs, which enables efficient lymphocyte trafficking. Heterotypic inter-GPCR coupling may regulate complex cellular phenotypes in physiological milieu containing many GPCR ligands.
Asunto(s)
Células Endoteliales/metabolismo , Ganglios Linfáticos/metabolismo , Receptor Cross-Talk , Animales , Proteína 9 Asociada a CRISPR , Sistemas CRISPR-Cas , Técnica del Anticuerpo Fluorescente , Edición Génica , Células HEK293 , Células Endoteliales de la Vena Umbilical Humana , Humanos , Lisofosfolípidos/metabolismo , Ratones , Reacción en Cadena en Tiempo Real de la Polimerasa , Receptores Acoplados a Proteínas G/metabolismo , Receptores del Ácido Lisofosfatídico/metabolismo , Esfingosina/análogos & derivados , Esfingosina/metabolismo , Receptores de Esfingosina-1-Fosfato/metabolismoRESUMEN
We hypothesized that the gut microbiota influences survival of murine cardiac allografts through modulation of immunity. Antibiotic pretreated mice received vascularized cardiac allografts and fecal microbiota transfer (FMT), along with tacrolimus immunosuppression. FMT source samples were from normal, pregnant (immune suppressed), or spontaneously colitic (inflammation) mice. Bifidobacterium pseudolongum (B. pseudolongum) in pregnant FMT recipients was associated with prolonged allograft survival and lower inflammation and fibrosis, while normal or colitic FMT resulted in inferior survival and worse histology. Transfer of B. pseudolongum alone resulted in reduced inflammation and fibrosis. Stimulation of DC and macrophage lines with B. pseudolongum induced the antiinflammatory cytokine IL-10 and homeostatic chemokine CCL19 but induced lesser amounts of the proinflammatory cytokines TNFα and IL-6. In contrast, LPS and Desulfovibrio desulfuricans (D. desulfuricans), more abundant in colitic FMT, induced a more inflammatory cytokine response. Analysis of mesenteric and peripheral lymph node structure showed that B. pseudolongum gavage resulted in a higher laminin α4/α5 ratio in the lymph node cortical ridge, indicative of a suppressive environment, while D. desulfuricans resulted in a lower laminin α4/α5 ratio, supportive of inflammation. Discrete gut bacterial species alter immunity and may predict graft outcomes through stimulation of myeloid cells and shifts in lymph node structure and permissiveness.
Asunto(s)
Microbioma Gastrointestinal/inmunología , Rechazo de Injerto/inmunología , Trasplante de Corazón/efectos adversos , Inmunidad Innata , Ganglios Linfáticos/inmunología , Aloinjertos/inmunología , Aloinjertos/patología , Animales , Antibacterianos/administración & dosificación , Línea Celular Tumoral , Colitis/inmunología , Citocinas/inmunología , Citocinas/metabolismo , Modelos Animales de Enfermedad , Trasplante de Microbiota Fecal , Femenino , Microbioma Gastrointestinal/efectos de los fármacos , Rechazo de Injerto/patología , Rechazo de Injerto/prevención & control , Supervivencia de Injerto/efectos de los fármacos , Supervivencia de Injerto/inmunología , Humanos , Inmunosupresores/administración & dosificación , Ratones , Miocardio/patología , Embarazo , Células RAW 264.7 , Tacrolimus/administración & dosificación , Resultado del TratamientoRESUMEN
Lymphotoxin-beta receptor (LTßR) signaling in lymphatic endothelial cells (LEC) regulates leukocyte afferent lymphatic transendothelial migration (TEM). The function of individual signaling pathways for different leukocyte subsets is currently unknown. Here, we show that LTßR signals predominantly via the constitutive and ligand-driven non-classical NIK pathway. Targeting LTßR-NIK by an LTßR-derived decoy peptide (nciLT) suppresses the production of chemokines CCL21 and CXCL12, and enhances the expression of classical NFκB-driven VCAM-1 and integrin ß4 to retain T cells on LEC and precludes T cell and dendritic cell TEM. nciLT inhibits contact hypersensitivity (CHS) at both the sensitization and elicitation stages, likely by inhibiting leukocyte migration. By contrast, targeting LTßR-classical NFκB signaling during the elicitation and resolution stages attenuates CHS, possibly by promoting leukocyte egress. These findings demonstrate the importance of LTßR signaling in leukocyte migration and LEC and lymphatic vessel function, and show that antagonist peptides may serve as lead compounds for therapeutic applications.
Asunto(s)
Movimiento Celular , Vasos Linfáticos/metabolismo , Receptor beta de Linfotoxina/metabolismo , FN-kappa B/metabolismo , Transducción de Señal , Linfocitos T/citología , Animales , Anticuerpos Monoclonales/farmacología , Movimiento Celular/efectos de los fármacos , Dermatitis por Contacto/inmunología , Dermatitis por Contacto/patología , Células Endoteliales/efectos de los fármacos , Células Endoteliales/metabolismo , Perfilación de la Expresión Génica , Inflamación/patología , Ratones Endogámicos C57BL , Péptidos/metabolismo , Linfocitos T/efectos de los fármacosRESUMEN
Here, we report the complete genome sequence of Bifidobacterium pseudolongum strain UMB-MBP-01, isolated from the feces of C57BL/6J mice. This strain was identified in microbiome profiling studies and associated with improved transplant outcome in a murine model of cardiac heterotypic transplantation.
RESUMEN
Trans-endothelial migration (TEM) is essential for leukocyte circulation. While much is known about trans-blood endothelial migration, far less is known about trans-lymphatic endothelial migration. We established an in vitro system to evaluate lymphatic TEM for various cell types across primary mouse and human lymphatic endothelial cells (LEC), and validated the model for the murine LEC cell line SVEC4-10. T cells exhibited enhanced unidirectional migration from the basal (abluminal) to the apical (luminal) surface across LEC, whereas for blood endothelial cells (BEC) they migrated similarly in both directions. This preferential, vectorial migration was chemotactic toward many different chemoattractants and dose-dependent. Stromal protein fibers, interstitial type fluid flow, distribution of chemokines in the stromal layer, and inflammatory cytokines influenced LEC phenotype and leukocyte TEM. Activated and memory CD4 T cells, macrophages, and dendritic cell (DC) showed chemoattractantΔdriven vectorial migration, while CD8 T cell migration across LEC was not. The system was further validated for studying cancer cell transmigration across lymphatic endothelium. This model for lymphatic TEM for various migrating and endothelial cell types possesses the capacity to be high-throughput, highly reproducible and integrate the complexities of lymphatic biology, stromal variability, chemoattractant distribution, and fluid flow.
Asunto(s)
Movimiento Celular , Células Endoteliales/citología , Endotelio Linfático/citología , Modelos Biológicos , Animales , Linfocitos T CD4-Positivos/citología , Moléculas de Adhesión Celular/metabolismo , Línea Celular Tumoral , Microambiente Celular , Quimiocinas/metabolismo , Quimiotaxis , Humanos , Inflamación/patología , Ratones Endogámicos C57BL , FenotipoRESUMEN
Regulatory T cells (Tregs) are essential to suppress unwanted immunity or inflammation. After islet allo-transplant Tregs must migrate from blood to allograft, then via afferent lymphatics to draining LN to protect allografts. Here we show that Tregs but not non-Treg T cells use lymphotoxin (LT) during migration from allograft to draining LN, and that LT deficiency or blockade prevents normal migration and allograft protection. Treg LTαß rapidly modulates cytoskeletal and membrane structure of lymphatic endothelial cells; dependent on VCAM-1 and non-canonical NFκB signalling via LTßR. These results demonstrate a form of T-cell migration used only by Treg in tissues that serves an important role in their suppressive function and is a unique therapeutic focus for modulating suppression.