RESUMEN
Escalated innate immunity plays a critical role in SARS-CoV-2 pathology; however, the molecular mechanism is incompletely understood. Thus, we aim to characterize the molecular mechanism by which SARS-CoV-2 Spike protein advances human macrophage (MÏ´) inflammatory and glycolytic phenotypes and uncover novel therapeutic strategies. We found that human MÏ´s exposed to Spike protein activate IRAK4 phosphorylation. Blockade of IRAK4 in Spike protein-stimulated MÏ´s nullifies signaling of IRAK4, AKT, and baseline p38 without affecting ERK and NF-κB activation. Intriguingly, IRAK4 inhibitor (IRAK4i) rescues the SARS-CoV-2-induced cytotoxic effect in ACE2+HEK 293 cells. Moreover, the inflammatory reprogramming of MÏ´s by Spike protein was blunted by IRAK4i through IRF5 and IRF7, along with the reduction of monokines, IL-6, IL-8, TNFα, and CCL2. Notably, in Spike protein-stimulated MÏ´s, suppression of the inflammatory markers by IRAK4i was coupled with the rebalancing of oxidative phosphorylation over metabolic activity. This metabolic adaptation promoted by IRAK4i in Spike protein-activated MÏ´s was shown to be in part through constraining PFKBF3, HIF1α, cMYC, LDHA, lactate expression, and reversal of citrate and succinate buildup. IRAK4 knockdown could comparably impair Spike protein-enhanced inflammatory and metabolic imprints in human MÏ´s as those treated with ACE2, TLR2, and TLR7 siRNA. Extending these results, in murine models, where human SARS-CoV-2 Spike protein was not recognized by mouse ACE2, TLRs were responsible for the inflammatory and glycolytic responses instigated by Spike protein and were dysregulated by IRAK4i therapy. In conclusion, IRAK4i may be a promising strategy for severe COVID-19 patients by counter-regulating ACE2 and TLR-mediated MÏ´ hyperactivation. IRAK4i therapy counteracts MÏ´ inflammatory and glycolytic reprogramming triggered by Spike protein. This study illustrates that SARS-CoV-2 Spike protein activates IRAK4 signaling via ACE2 as well as TLR2 and TLR7 sensing in human MÏ´s. Remarkably, IRAK4i treatment can dysregulate both ACE-dependent and independent (via TLR sensing) SARS-CoV-2 Spike protein-activated inflammatory and metabolic imprints.
Asunto(s)
COVID-19 , Glicoproteína de la Espiga del Coronavirus , Enzima Convertidora de Angiotensina 2 , Animales , Células HEK293 , Humanos , Factores Reguladores del Interferón/metabolismo , Factores Reguladores del Interferón/farmacología , Quinasas Asociadas a Receptores de Interleucina-1/genética , Quinasas Asociadas a Receptores de Interleucina-1/metabolismo , Macrófagos/metabolismo , Ratones , SARS-CoV-2 , Glicoproteína de la Espiga del Coronavirus/genética , Glicoproteína de la Espiga del Coronavirus/metabolismo , Receptor Toll-Like 2/metabolismo , Receptor Toll-Like 7/metabolismoRESUMEN
Recurrent waves of COVID19 remain a major global health concern. Repurposing either FDA-approved or clinically advanced drug candidates can save time and effort required for validating the safety profile and FDA approval. However, the selection of appropriate screening approaches is key to identifying novel candidate drugs with a higher probability of clinical success. Here, we report a rapid, stratified two-step screening approach using pseudovirus entry inhibition assay followed by an infectious prototypic SARS CoV2 cytotoxic effect inhibition assay in multiple cell lines. Using this approach, we screened a library of FDA-approved and clinical-stage drugs and identified four compounds, apilimod, berbamine, cepharanthine and (S)-crizotinib which potently inhibited SARS CoV2-induced cell death. Importantly, these drugs exerted similar inhibitory effect on the delta and omicron variants although they replicated less efficiently than the prototypic strain. Apilimod is currently under clinical trial (NCT04446377) for COVID19 supporting the validity and robustness of our screening approach.
Asunto(s)
Tratamiento Farmacológico de COVID-19 , Humanos , SARS-CoV-2RESUMEN
Class-switched antinuclear autoantibodies produced by T follicular helper (TFH) cell-dependent germinal center (GC) B cell response play an essential pathogenic role in lupus nephritis (LN). The role of T follicular regulatory (TFR) cells, an effector subset of CD4+Foxp3+ T regulatory cells (Tregs), which are specialized in suppressing TFH-GC response and Ab production, remains elusive in LN. Contrasting reports have shown increased/reduced circulating TFR cells in human lupus that might not accurately reflect their presence in the GCs of relevant lymphoid organs. In this study, we report a progressive reduction in TFR cells and decreased TFR/TFH ratio despite increased Tregs in the renal lymph nodes of NZBWF1/j mice, which correlated with increased GC-B cells and proteinuria onset. Cotreatment with soluble OX40L and Jagged-1 (JAG1) proteins increased Tregs, TFR cells, and TFR/TFH ratio, with a concomitant reduction in TFH cells, GC B cells, and anti-dsDNA IgG Ab levels, and suppressed LN onset. Mechanistic studies showed attenuated TFH functions and diminished GC events such as somatic hypermutation and isotype class-switching in OX40L-JAG1-treated mice. RNA sequencing studies revealed inhibition of hypoxia-inducible factor 1-α (HIF-1a) and STAT3 signaling in T conventional cells from OX40L-JAG1-treated mice, which are critical for the glycolytic flux and differentiation into TFH cell lineage. Therefore, the increased TFR/TFH ratio seen in OX40L-JAG1-treated mice could involve both impaired differentiation of TFH cells from T conventional cells and expansion of TFR cells. We show a key role for GC-TFR/TFH imbalance in LN pathogenesis and how restoring homeostatic balance can suppress LN.
Asunto(s)
Nefritis Lúpica , Animales , Centro Germinal , Nefritis Lúpica/metabolismo , Ratones , Células T Auxiliares Foliculares , Linfocitos T Colaboradores-Inductores , Linfocitos T ReguladoresRESUMEN
TNF receptor superfamily comprises many T-cell costimulatory receptors, including TNFRSF1, TNFRSF2, TNFRSF4 (OX40), TNFRSF9 (4-1BB), TNFRSF18 (GITR), and TNFRSF7 (CD27). Signaling through these costimulatory stimulatory receptors can promote conventional T-cell (Tconv) proliferation, and effector functions in an antigen-dependent manner. Thus, agonistic antibodies and ligands for OX40, 4-1BB, GITR, and CD27 have been tested for inducing T-cell-mediated antitumor responses in several cancers. However, recently emerging reports show critical role for TNFR signaling in regulatory T-cell (Treg) differentiation and expansion, which might suppress effector T-cell proliferation and functions. Here, we show preferential over expression of TNFR2, OX40, 4-1BB, and GITR in Treg cells over Tconv cells, and the ability of OX40L and GITRL to induce selective proliferation of Treg cells, but not Tconv cells, in an antigen-independent manner. We describe the standard protocols used for Affymetrix gene expression profiling, T-cell isolation, and Cell Trace Violet-based cell proliferation assay.
Asunto(s)
Antígenos/inmunología , Activación de Linfocitos/inmunología , Ligando OX40/metabolismo , Linfocitos T Reguladores/inmunología , Linfocitos T Reguladores/metabolismo , Factores de Necrosis Tumoral/metabolismo , Animales , Biomarcadores , Biología Computacional/métodos , Perfilación de la Expresión Génica , Inmunofenotipificación , Ligandos , Activación de Linfocitos/genética , Ratones , Ratones Transgénicos , Familia de Multigenes , Factores de Necrosis Tumoral/genéticaRESUMEN
Type-1 Diabetes (T1D) is the major autoimmune disease affecting the juvenile population in which insulin-producing pancreatic ß-cells are destroyed by self-reactive T-cells and B-cells. Emerging studies have identified the presence of autoantibodies and altered T-cell reactivity against several autoantigens in individuals who are at risk of developing T1D even before the clinical onset of diabetes. Whilst these findings could lead to the development of predictive biomarkers for early diagnosis, growing evidence on the generation of neoepitopes, epitope spreading and diverse antigen repertoire in T1D poses a major challenge for developing approaches to induce antigen-specific tolerance. Mechanisms of neoepitope generation include post-translational modifications of existing epitopes, aberrant translational products, peptide fusion, and differences in MHC binding registers. Here, we focus our discussion on how post-translational modifications can give rise to immunogenic neoepitopes in T1D and present our perspective on how it could affect the development of therapeutic approaches to induce antigen-specific tolerance.
Asunto(s)
Diabetes Mellitus Tipo 1/inmunología , Diabetes Mellitus Tipo 1/metabolismo , Epítopos/inmunología , Epítopos/metabolismo , Procesamiento Proteico-Postraduccional , Animales , Variación Antigénica , Autoantígenos/inmunología , Autoantígenos/metabolismo , Biomarcadores , Diabetes Mellitus Tipo 1/patología , Diabetes Mellitus Tipo 1/terapia , Manejo de la Enfermedad , Progresión de la Enfermedad , Susceptibilidad a Enfermedades , Estrés del Retículo Endoplásmico , Humanos , Tolerancia Inmunológica , Insulina/metabolismo , Células Secretoras de Insulina/inmunología , Células Secretoras de Insulina/metabolismo , Estrés Oxidativo , Transducción de SeñalRESUMEN
Regulatory T-cells (Tregs) can facilitate immune evasion by tumor cells by dampening anti-tumor immunity. Reduced Teff/Treg ratio and enhanced Treg functional activity have been observed in patients suffering from different types of cancers, and attenuated Treg numbers/functions can serve as prognostic indicators. Normally, Tregs play an essential role in the maintenance of immune tolerance and prevention of autoimmunity. The most common immune checkpoint blockers (ICB) targeting co-inhibitory receptors such as anti-CTLA4 (ipilimumab and tremelimumab) and anti-PD1 (pembrolizumab and nivolumab)/anti-PD-L1 (atezolizumab) have achieved unprecedented success in cancer treatment by facilitating an effective anti-tumor immune response, at least in part, by blocking Treg mediated immunosuppression. While ICBs have shown remarkable success in cancer immunotherapy, immune-related adverse events (IRAEs) arising from ICB have forced consideration of ways to maintain immune homeostasis post ICB treatment. Preclinical models of IRAEs have shown a negative correlation between Treg numbers and IRAEs. Therefore, understanding the "ying-yang" role of Tregs in the regulation of autoimmunity and anti-tumor immunity is critical to provoking an effective anti-tumor response while maintaining immune homeostasis. Studies aimed at developing effective approaches to minimize IRAEs without compromising anti-tumor immunity are underway. Herein, we discuss 1) the critical role of key co-inhibitory receptors on Treg homeostasis and tumor tolerance; 2) how co-receptor blockade by cancer immunotherapy can lead to autoimmune adverse events; and 3) recently emerging management strategies to minimize autoimmune adverse events arising from ICB.
Asunto(s)
Antineoplásicos Inmunológicos/efectos adversos , Enfermedades Autoinmunes/etiología , Inmunoterapia/efectos adversos , Neoplasias/tratamiento farmacológico , Linfocitos T Reguladores/inmunología , Microambiente Tumoral/inmunología , Animales , Enfermedades Autoinmunes/patología , Antígeno B7-H1/antagonistas & inhibidores , Antígeno CTLA-4/antagonistas & inhibidores , Homeostasis , Humanos , Neoplasias/inmunología , Neoplasias/patologíaRESUMEN
Foxp3+T regulatory cells (Tregs) control autoimmune response by suppressing proliferation and effector functions of self-reactive Foxp3-CD4+/CD8+ T cells and thereby maintain the critical balance between self-tolerance and autoimmunity. Earlier, we had shown that OX40L-JAG1 cosignaling mediated through their cognate receptors OX40 and Notch3 preferentially expressed on murine Tregs can selectively induce their proliferation in the absence of TCR stimulation. However, the differential molecular mechanisms regulating TCR-independent versus TCR-dependent Treg proliferation and lineage stability of the expanded Tregs remained unknown. In this study, we show that OX40L-JAG1 treatment induced TCR-independent proliferation of Tregs in the thymus and periphery. The use of Src kinase inhibitor permitted us to demonstrate selective inhibition of TCR-dependent T cell proliferation with little to no effect on OX40L-JAG1-induced TCR-independent Treg expansion in vitro, which was critically dependent on noncanonical NF-κB signaling. OX40L-JAG1-expanded Tregs showed sustained lineage stability as indicated by stable demethylation marks in Treg signature genes such as Foxp3, Il2ra, Ctla4, Ikzf2, and Ikzf4. Furthermore, OX40L-JAG1 treatment significantly increased CTLA4+ and TIGIT+ Tregs and alleviated experimental autoimmune thyroiditis in mice. Relevance of our findings to humans became apparent when human OX40L and JAG1 induced TCR-independent selective expansion of human Tregs in thymocyte cultures and increased human Tregs in the liver tissue of humanized NSG mice. Our findings suggest that OX40L-JAG1-induced TCR-independent Treg proliferation is a conserved mechanism that can be used to expand lineage-stable Tregs to treat autoimmune diseases.
Asunto(s)
Proteína Jagged-1/metabolismo , FN-kappa B/metabolismo , Ligando OX40/metabolismo , Transducción de Señal , Linfocitos T Reguladores/inmunología , Linfocitos T Reguladores/metabolismo , Animales , Enfermedades Autoinmunes/etiología , Enfermedades Autoinmunes/metabolismo , Autoinmunidad/inmunología , Biomarcadores , Linaje de la Célula , Femenino , Activación de Linfocitos/inmunología , Ratones , Receptores de Antígenos de Linfocitos T/metabolismo , Familia-src Quinasas/metabolismoRESUMEN
Anaplastic Thyroid Cancer (ATC) is an aggressive malignancy with limited therapeutic options and dismal patient survival. We have previously shown MADD to be differentially overexpressed in multiple cancer histologies and to contribute to tumor cell growth and survival. Therefore, we targeted MADD by gene silencing, explored its effect on cellular proliferation and metastases and examined its therapeutic potential in an orthotopic ATC model in athymic nude mice. When compared to untreated control and scramble siRNA, MADD siRNA treatment inhibited the proliferative capacity of 8505C, C643 and HTH7 cells in vitro and 8505C-derived-orthotopic tumor growth in vivo. MADD ablation caused a significant reduction in cellular migration and invasion potential; clonogenic capacity; as well as, mitochondrial length and potential in vitro. This MADD siRNA-induced anti-migratory/invasive effect corresponded with inhibition of epithelial-mesenchymal transition (EMT) and Wnt signaling. Mechanistically, MADD siRNA inhibited TNFα induced activation of pERK, pGSK3ß and ß-catenin, suggesting that MADD knockdown might exert its anti-migratory/invasive effects, by blocking TNFα/ERK/GSK3ß axis. MADD siRNA can inhibit ß-catenin nuclear translocation and consequently, the expression of its target genes in ATC cells. In in vivo experiments, along with tumor regression, MADD siRNA treatment also decreased evidence of lung metastases. Immunohistochemically, MADD siRNA-treated tumor tissues exhibited a reduction in Ki67 and N-Cadherin expression, and an increase in E-Cadherin expression. In conclusion, we show the crucial role of MADD in ATC tumorigenesis and metastasis and its potential implications as a molecular target for ATC therapy.
Asunto(s)
Proteínas Adaptadoras de Señalización del Receptor del Dominio de Muerte/biosíntesis , Factores de Intercambio de Guanina Nucleótido/biosíntesis , Carcinoma Anaplásico de Tiroides/metabolismo , Neoplasias de la Tiroides/metabolismo , Animales , Puntos de Control del Ciclo Celular/fisiología , Línea Celular Tumoral , Movimiento Celular/fisiología , Proliferación Celular/fisiología , Proteínas Adaptadoras de Señalización del Receptor del Dominio de Muerte/deficiencia , Proteínas Adaptadoras de Señalización del Receptor del Dominio de Muerte/genética , Técnicas de Silenciamiento del Gen , Factores de Intercambio de Guanina Nucleótido/deficiencia , Factores de Intercambio de Guanina Nucleótido/genética , Xenoinjertos , Humanos , Ratones , Ratones Desnudos , Carcinoma Anaplásico de Tiroides/genética , Carcinoma Anaplásico de Tiroides/patología , Neoplasias de la Tiroides/genética , Neoplasias de la Tiroides/patología , TransfecciónRESUMEN
Self-tolerance, the state of unresponsiveness to self-tissues/antigens, is maintained through central and peripheral tolerance mechanisms, and a breach of these mechanisms leads to autoimmune diseases. Foxp3â¯+â¯T-regulatory cells (Tregs) play an essential role in suppressing autoimmune response directed against self-antigens and thereby regulate self-tolerance. Natural Tregs are differentiated in the thymus on the basis of their higher TCR-affinity to self-antigens and migrate to the periphery where they maintain peripheral tolerance. In addition, extra-thymic differentiation of induced Tregs can occur in the periphery which can control abrupt immune responses under inflammatory conditions. A defect in Treg cell numbers and/or function is found to be associated with the development of autoimmune disease in several experimental models and human autoimmune diseases. Moreover, augmentation of Tregs has been shown to be beneficial in treating autoimmunity in preclinical models, and Treg based cellular therapy has shown initial promise in clinical trials. However, emerging studies have identified an unstable subpopulation of Tregs which expresses pro-inflammatory cytokines under both homeostatic and autoimmune conditions, as well as in ex vivo cultures. In addition, clinical translation of Treg cellular therapy is impeded by limitations such as lack of easier methods for selective expansion of Tregs and higher cost associated with GMP-facilities required for cell sorting, ex vivo expansion and infusion of ex vivo expanded Tregs. Here, we discuss the recent advances in molecular mechanisms regulating Treg differentiation, Foxp3 expression and lineage stability, the role of Tregs in the prevention of various autoimmune diseases, and critically review their clinical utility for treating human autoimmune diseases.
Asunto(s)
Enfermedades Autoinmunes/inmunología , Autoinmunidad/inmunología , Autotolerancia/inmunología , Linfocitos T Reguladores/inmunología , Animales , Diferenciación Celular/inmunología , HumanosRESUMEN
Regulatory T cells (Tregs) play a pivotal role in immune-tolerance, and loss of Treg function can lead to the development of autoimmunity. Natural Tregs generated in the thymus substantially contribute to the Treg pool in the periphery, where they suppress self-reactive effector T cells (Teff) responses. Recently, we showed that OX40L (TNFSF4) is able to drive selective proliferation of peripheral Tregs independent of canonical antigen presentation (CAP-independent) in the presence of low-dose IL-2. Therefore, we hypothesized that OX40 signaling might be integral to the TCR-independent phase of murine and human thymic Treg (tTreg) development. Development of tTregs is a two-step process: Strong T-cell receptor (TCR) signals in combination with co-signals from the TNFRSF members facilitate tTreg precursor selection, followed by a TCR-independent phase of tTreg development in which their maturation is driven by IL-2. Therefore, we investigated whether OX40 signaling could also play a critical role in the TCR-independent phase of tTreg development. OX40-/- mice had significantly reduced numbers of CD25-Foxp3low tTreg precursors and CD25+Foxp3+ mature tTregs, while OX40L treatment of WT mice induced significant proliferation of these cell subsets. Relative to tTeff cells, OX40 was expressed at higher levels in both murine and human tTreg precursors and mature tTregs. In ex vivo cultures, OX40L increased tTreg maturation and induced CAP-independent proliferation of both murine and human tTregs, which was mediated through the activation of AKT-mTOR signaling. These novel findings show an evolutionarily conserved role for OX40 signaling in tTreg development and proliferation, and might enable the development of novel strategies to increase Tregs and suppress autoimmunity.
Asunto(s)
Activación de Linfocitos/inmunología , Receptores de Antígenos de Linfocitos T/inmunología , Receptores OX40/metabolismo , Subgrupos de Linfocitos T/inmunología , Linfocitos T Reguladores/inmunología , Timocitos/inmunología , Animales , Proliferación Celular , Células Cultivadas , Citocinas/metabolismo , Femenino , Factores de Transcripción Forkhead/metabolismo , Humanos , Ratones , Ratones Endogámicos C57BL , Transducción de Señal , Timocitos/citologíaRESUMEN
We have previously shown GM-CSF derived bone-marrow dendritic cells (G-BMDCs) can induce the selective expansion of Tregs through the surface-bound molecule OX40L; however, the physiological role of this ex vivo derived DC subset remained to be elucidated. We determined GM-CSF administration to mice induced the generation of in vivo derived OX40L+ DCs, phenotypically similar to ex vivo OX40L+G-BMDCs, in the spleen, brachial lymph nodes and liver. The generation of OX40L+ DCs correlated with increased percentages of functionally suppressive Tregs in the spleen, brachial lymph nodes, and liver of GM-CSF treated mice. DCs from GM-CSF treated mice expanded Tregs in CD4+ T-cell co-cultures in an OX40L dependent manner, suggesting OX40L+ DCs may play a role in peripheral Treg homeostasis. Furthermore, comparing the transcriptome data of OX40L+ DCs to that of all immune cell types revealed OX40L+ DCs to be distinct from steady-state immune cells and, microarray analysis of OX40L+G-BMDCs and OX40L-G-BMDCs revealed higher expression of molecules that are associated with tolerogenic phenotype and could play important roles in the function of OX40L+ DCs. These findings suggest that OX40L+ DCs may represent a unique DC subset induced under inflammatory conditions that may play an essential role in maintaining Treg homeostasis.
Asunto(s)
Células Dendríticas/inmunología , Glicoproteínas de Membrana/inmunología , Linfocitos T Reguladores/inmunología , Factores de Necrosis Tumoral/inmunología , Animales , Células Cultivadas , Células Dendríticas/metabolismo , Femenino , Citometría de Flujo , Inflamación/genética , Inflamación/inmunología , Glicoproteínas de Membrana/análisis , Glicoproteínas de Membrana/genética , Ratones Endogámicos C57BL , Ligando OX40 , Linfocitos T Reguladores/metabolismo , Transcriptoma , Factores de Necrosis Tumoral/análisis , Factores de Necrosis Tumoral/genéticaRESUMEN
The immune system ensures optimum T-effector (Teff) immune responses against invading microbes and tumor antigens while preventing inappropriate autoimmune responses against self-antigens with the help of T-regulatory (Treg) cells. Thus, Treg and Teff cells help maintain immune homeostasis through mutual regulation. While Tregs can contribute to tumor immune evasion by suppressing anti-tumor Teff response, loss of Treg function can result in Teff responses against self-antigens leading to autoimmune disease. Thus, loss of homeostatic balance between Teff/Treg cells is often associated with both cancer and autoimmunity. Co-stimulatory and co-inhibitory receptors, collectively known as co-signaling receptors, play an indispensable role in the regulation of Teff and Treg cell expansion and function and thus play critical roles in modulating autoimmune and anti-tumor immune responses. Over the past three decades, considerable efforts have been made to understand the biology of co-signaling receptors and their role in immune homeostasis. Mutations in co-inhibitory receptors such as CTLA4 and PD1 are associated with Treg dysfunction, and autoimmune diseases in mice and humans. On the other hand, growing tumors evade immune surveillance by exploiting co-inhibitory signaling through expression of CTLA4, PD1 and PDL-1. Immune checkpoint blockade (ICB) using anti-CTLA4 and anti-PD1 has drawn considerable attention towards co-signaling receptors in tumor immunology and created renewed interest in studying other co-signaling receptors, which until recently have not been as well studied. In addition to co-inhibitory receptors, co-stimulatory receptors like OX40, GITR and 4-1BB have also been widely implicated in immune homeostasis and T-cell stimulation, and use of agonistic antibodies against OX40, GITR and 4-1BB has been effective in causing tumor regression. Although ICB has seen unprecedented success in cancer treatment, autoimmune adverse events arising from ICB due to loss of Treg homeostasis poses a major obstacle. Herein, we comprehensively review the role of various co-stimulatory and co-inhibitory receptors in Treg biology and immune homeostasis, autoimmunity, and anti-tumor immunity. Furthermore, we discuss the autoimmune adverse events arising upon targeting these co-signaling receptors to augment anti-tumor immune responses.
Asunto(s)
Autoinmunidad , Homeostasis/inmunología , Neoplasias/inmunología , Transducción de Señal/inmunología , Linfocitos T Reguladores/inmunología , Animales , Antineoplásicos Inmunológicos/uso terapéutico , Antígeno B7-H1/genética , Antígeno B7-H1/inmunología , Antígeno CTLA-4/genética , Antígeno CTLA-4/inmunología , Regulación de la Expresión Génica , Proteína Relacionada con TNFR Inducida por Glucocorticoide/genética , Proteína Relacionada con TNFR Inducida por Glucocorticoide/inmunología , Humanos , Ratones , Neoplasias/tratamiento farmacológico , Neoplasias/genética , Neoplasias/patología , Receptor de Muerte Celular Programada 1/genética , Receptor de Muerte Celular Programada 1/inmunología , Receptores OX40/genética , Receptores OX40/inmunología , Linfocitos T Citotóxicos/inmunología , Escape del Tumor , Miembro 9 de la Superfamilia de Receptores de Factores de Necrosis Tumoral/genética , Miembro 9 de la Superfamilia de Receptores de Factores de Necrosis Tumoral/inmunologíaRESUMEN
A high-fat diet can induce inflammation and metabolic diseases such as diabetes and atherosclerosis. The receptor for advanced glycation endproducts (RAGE) plays a critical role in metabolic disease pathophysiology and the soluble form of the receptor (sRAGE) can mitigate these effects. However, little is known about RAGE in the postprandial condition and the effect of exercise in this context. Thus, we aimed to determine the effects of a single high-fat meal (HFM) with and without prior exercise on peripheral blood mononuclear cell (PBMC) RAGE biology. Healthy males (n = 12) consumed a HFM on two occasions, one without prior exercise and one 16-18 hours following acute aerobic exercise. Total soluble RAGE (sRAGE) and endogenous secretory RAGE (esRAGE) were determined via ELISA and cleaved RAGE (cRAGE) was calculated as the difference between the two. Isolated PBMCs were analyzed for RAGE, ADAM10, TLR4, and MyD88 protein expression and ADAM10 activity. The HFM significantly (P < 0.01) attenuated sRAGE, esRAGE, and cRAGE by 9.7%, 6.9%, and 10.5%, respectively. Whereas, the HFM increased PBMC RAGE protein expression by 10.3% (P < 0.01), there was no meal effect on PBMC TLR4, MYD88, or ADAM10 protein expression, nor ADAM10 activity. There was also no exercise effect on any experimental outcomes. These findings suggest that PBMC RAGE and soluble RAGE may be important in the postprandial response to a HFM, and that prior aerobic exercise does not alter these processes in young healthy adult males. The mechanisms by which a HFM induces RAGE expression and reduces circulating soluble RAGE isoforms requires further study.
Asunto(s)
Dieta Alta en Grasa/efectos adversos , Ejercicio Físico , Productos Finales de Glicación Avanzada/metabolismo , Periodo Posprandial , Proteína ADAM10/metabolismo , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Células Cultivadas , Productos Finales de Glicación Avanzada/sangre , Humanos , Masculino , Comidas , Proteínas de la Membrana/metabolismo , Monocitos/metabolismo , Factor 88 de Diferenciación Mieloide/metabolismo , Receptor Toll-Like 4/metabolismo , Adulto JovenRESUMEN
We have previously shown that OX40L/OX40 interaction is critical for TCR-independent selective proliferation of Foxp3+ Tregs, but not Foxp3- effector T-cells (Teff), when CD4+ T-cells are co-cultured with GM-CSF derived bone marrow dendritic cells (G-BMDCs). Events downstream of OX40L/OX40 interaction in Tregs responsible for this novel mechanism are not understood. Earlier, OX40L/OX40 interaction has been shown to stimulate CD4+ T-cells through the formation of a signalosome involving TRAF2/PKC-Ѳ leading to NF-kB activation. In this study, using CD4+ T-cells from WT and OX40-/- mice we first established that OX40 mediated activation of NF-kB was critical for this Treg proliferation. Although CD4+ T-cells from PKC-Ѳ-/- mice were also defective in G-BMDC induced Treg proliferation ex vivo, this defect could be readily corrected by adding exogenous IL-2 to the co-cultures. Furthermore, by treating WT, OX40-/-, and PKC-Ѳ-/- mice with soluble OX40L we established that OX40L/OX40 interaction was required and sufficient to induce Treg proliferation in vivo independent of PKC-Ѳ status. Although PKC-Ѳ is dispensable for TCR-independent Treg proliferation per se, it is essential for optimum IL-2 production by Teff cells. Finally, our findings suggest that OX40L binding to OX40 likely results in recruitment of TRAF1 for downstream signalling.
Asunto(s)
Proliferación Celular , Interleucina-2/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteína Quinasa C-theta/metabolismo , Receptores OX40/metabolismo , Linfocitos T Reguladores/fisiología , Factores de Necrosis Tumoral/metabolismo , Animales , Ratones , Ratones Noqueados , FN-kappa B/metabolismo , Ligando OX40 , Proteína Quinasa C-theta/deficiencia , Receptores OX40/deficienciaRESUMEN
Regulatory T-cells (Tregs) play a pivotal role in maintaining peripheral tolerance. Increasing Treg numbers/functions has been shown to ameliorate autoimmune diseases. However, common Treg expansion approaches use T-Cell Receptor (TCR)-mediated stimulation which also causes proliferation of effector T-cells (Teff). To overcome this limitation, purified patient-specific Tregs are expanded ex vivo and transfused. Although promising, this approach is not suitable for routine clinical use. Therefore, an alternative approach to selectively expand functional Tregs in vivo is highly desired. We report a novel TCR-independent strategy for the selective proliferation of Foxp3+Tregs (without Teff proliferation), by co-culturing CD4+ T-cells with OX40 L+Jagged(JAG)-1+ bone marrow-derived DCs differentiated with GM-CSF or treating them with soluble OX40 L and JAG1 in the presence of exogenous IL-2. Tregs expanded using soluble OX40 L and JAG1 were of suppressive phenotype and delayed the onset of diabetes in NOD mice. Ligation of OX40 L and JAG1 with their cognate-receptors OX40 and Notch3, preferentially expressed on Tregs but not on Teff cells, was required for selective Treg proliferation. Soluble OX40L-JAG1-induced NF-κB activation as well as IL-2-induced STAT5 activation were essential for the proliferation of Tregs with sustained Foxp3 expression. Altogether, these findings demonstrate the utility of soluble OX40 L and JAG1 to induce TCR-independent Treg proliferation.
Asunto(s)
Células Dendríticas/inmunología , Ligando OX40/metabolismo , Linfocitos T Reguladores/fisiología , Proliferación Celular , Células Cultivadas , Técnicas de Cocultivo , Factores de Transcripción Forkhead/metabolismo , Humanos , Interleucina-2/metabolismo , Proteína Jagged-1/metabolismo , FN-kappa B/metabolismo , Receptor Notch3/metabolismo , Receptores de Antígenos de Linfocitos T/metabolismo , Receptores OX40/metabolismo , Transducción de SeñalRESUMEN
Earlier, we have shown that GM-CSF derived bone marrow (BM) dendritic cells (G-BMDCs) can expand Foxp3(+) regulatory T-cells (Tregs) through a TCR-independent, but IL-2 dependent mechanism that required OX40L/OX40 interaction. While some reports have shown suppression of autoimmunity upon treatment with an OX40 agonist, others have shown exacerbation of autoimmune disease instead. To better understand the basis for these differing outcomes, we compared the effects of OX40L treatment in 6-week-old pre-diabetic and 12-week-old near diabetic NOD mice. Upon treatment with OX40L, 6-week-old NOD mice remained normoglycemic and showed a significant increase in Tregs in their spleen and lymph nodes, while 12-week-old NOD mice very rapidly developed hyperglycemia and failed to show Treg increase in spleen or LN. Interestingly, OX40L treatment increased Tregs in the thymus of both age groups. However, it induced Foxp3(+)CD103(+)CD38(-) stable-phenotype Tregs in the thymus and reduced the frequency of autoreactive Teff cells in 6-week-old mice; while it induced Foxp3(+)CD103(-)CD38(+) labile-phenotype Tregs in the thymus and increased autoreactive CD4(+) T cells in the periphery of 12-week-old mice. This increase in autoreactive CD4(+) T cells was likely due to either a poor suppressive function or conversion of labile Tregs into Teff cells. Using ex vivo cultures, we found that the reduction in Treg numbers in 12-week-old mice was likely due to IL-2 deficit, and their numbers could be increased upon addition of exogenous IL-2. The observed divergent effects of OX40L treatment were likely due to differences in the ability of 6- and 12-week-old NOD mice to produce IL-2.
Asunto(s)
Ligando de CD40/metabolismo , Diabetes Mellitus/etiología , Diabetes Mellitus/metabolismo , Traslado Adoptivo , Factores de Edad , Animales , Glucemia , Antígenos CD40/metabolismo , Ligando de CD40/administración & dosificación , Citocinas/genética , Citocinas/metabolismo , Células Dendríticas/efectos de los fármacos , Células Dendríticas/inmunología , Células Dendríticas/metabolismo , Expresión Génica , Factor Estimulante de Colonias de Granulocitos y Macrófagos/farmacología , Mediadores de Inflamación/metabolismo , Ganglios Linfáticos/inmunología , Activación de Linfocitos/efectos de los fármacos , Activación de Linfocitos/inmunología , Ratones , Ratones Endogámicos NOD , Unión Proteica , Bazo/inmunología , Subgrupos de Linfocitos T/inmunología , Subgrupos de Linfocitos T/metabolismoRESUMEN
GM-CSF was originally identified as a colony stimulating factor (CSF) because of its ability to induce granulocyte and macrophage populations from precursor cells. Multiple studies have demonstrated that GM-CSF is also an immune-modulatory cytokine, capable of affecting not only the phenotype of myeloid lineage cells, but also T-cell activation through various myeloid intermediaries. This property has been implicated in the sustenance of several autoimmune diseases like arthritis and multiple sclerosis. In contrast, several studies using animal models have shown that GM-CSF is also capable of suppressing many autoimmune diseases such as Crohn's disease, Type-1 diabetes, Myasthenia gravis and experimental autoimmune thyroiditis. Knockout mouse studies have suggested that the role of GM-CSF in maintaining granulocyte and macrophage populations in the physiological steady state is largely redundant. Instead, its immune-modulatory role plays a significant role in the development or resolution of autoimmune diseases. This is mediated either through the differentiation of precursor cells into specialized non-steady state granulocytes, macrophages and dendritic cells, or through the modulation of the phenotype of mature myeloid cells. Thus, outside of myelopoiesis, GM-CSF has a profound role in regulating the immune response and maintaining immunological tolerance.
Asunto(s)
Enfermedades Autoinmunes/inmunología , Autoinmunidad/inmunología , Factor Estimulante de Colonias de Granulocitos y Macrófagos/inmunología , Tolerancia Inmunológica/inmunología , Células Mieloides/citología , Animales , Diferenciación Celular/inmunología , Células Dendríticas/citología , Células Dendríticas/inmunología , Granulocitos/citología , Granulocitos/inmunología , Humanos , Activación de Linfocitos/inmunología , Macrófagos/citología , Macrófagos/inmunología , Ratones , Linfocitos T Reguladores/inmunologíaRESUMEN
The emergence of Th17 cells as a unique sub-population of CD4(+) T cells has revolutionized the current understanding of adaptive immune system and autoimmune diseases. Th17 cells are characterized by the expression of effector cytokines IL-17A, IL-17F, IL-21 and IL-22, and lineage specific transcription factor ROR-C in human and ROR-γt in mice. Generation and differentiation of Th17 cells from naive CD4(+) T cells is driven by transforming growth factor (TGF)-ß, IL-6, IL-23, IL-1ß and IL-21. Recent studies suggest that the pathogenicity of Th17 cells is determined by the presence of IL-23 and TGF-ß3 in local micro-environment. Emerging reports highlight the importance of T-helper cell plasticity in pathogenesis of various autoimmune diseases. Th17 cells exhibit significant plasticity and converted to Th1-like cells under pathogenic conditions. Albeit growing body of evidences stating a pathogenic role for Th17 cells in autoimmune diabetes, conflicting reports also state an indifferent or protective role for Th17 cells. The operating mechanisms modulating Th17 immune response in autoimmune diabetes remain elusive. This review discusses recent advances in the understanding of transcriptional and post-transcriptional mechanisms of Th17 polarization, factors influencing pathogenicity of Th17 cells, molecular mechanisms of Th17/Th1 and Treg/Th17 plasticity and implications of these phenomena in autoimmune diabetes.
Asunto(s)
Diabetes Mellitus Tipo 1/inmunología , Diabetes Mellitus Tipo 1/metabolismo , Células Th17/citología , Animales , Autoinmunidad , Diferenciación Celular , Linaje de la Célula , Metilación de ADN , Modelos Animales de Enfermedad , Epigénesis Genética , Factores de Transcripción Forkhead/metabolismo , Humanos , Ratones , MicroARNs/metabolismo , Biosíntesis de Proteínas , Procesamiento Postranscripcional del ARN , Células TH1/citología , Transcripción GenéticaRESUMEN
High glucose is an independent risk factor that alters the expression pattern of cytokines/chemokine leading to leukocyte activation in diabetes. Fluctuation of cytokine milieu in lymphocytes may lead to differentiation into a particular subset. Our objectives were to profile high glucose induced inflammatory gene expression in lymphocytes, to examine in vivo relevance in diabetes and to identify the key transcription factors and signaling pathways involved. Cytokine gene arrays and T-helper (Th1/Th2/Th17) cytokine profiler RT(2)-PCR arrays used for cytokine expression profiling followed by validation using Real Time-qPCR and relative RT-PCR in Jurkat T-lymphocytes, peripheral blood lymphocytes (PBLCs) from normal and diabetes subjects. Luciferase reporter plasmid, pharmacological inhibitors and mutant plasmids were used for promoter activation and signaling pathway studies. High glucose induced gene profiling in Jurkat T-lymphocytes showed significantly increased expression of 64 proinflammatory genes including IL-6 and IL-17A and most of these genes were Nuclear Factor (NF)-κB and AP-1 regulated. RT(2)-PCR array results suggested the transcriptional activation of IL-17 and its downstream signaling in Jurkat T-lymphocytes upon high glucose treatment. Candidate genes like Interleukin (IL)-17A, IL-17E IL-17F and IL-6 were up-regulated in both Jurkat T-lymphocytes and PBLCs from normal and diabetes subjects. This high glucose induced cytokine expression was due to promoter activation. Pharmacology inhibitor studies showed the involvement of NF-κB, protein kinase-C, p38 Mitogen activated protein kinase; Janus activated kinase-signal transducer and activator of transcription and extracellular regulated kinase signaling pathways. Further, high glucose treatment increased the adhesion of lymphocytes to human umbilical vein endothelial cells. These results show that IL-17 cytokines are induced by high glucose via key signaling pathways leading to lymphocyte activation and relevant to the pathogenesis of diabetic complications like atherosclerosis.
Asunto(s)
Glucosa/farmacología , Interleucina-17/sangre , Interleucina-6/sangre , Estrés Oxidativo/efectos de los fármacos , Aterosclerosis/patología , Adhesión Celular/efectos de los fármacos , Quimiocina CCL11/sangre , Diabetes Mellitus/genética , Células Endoteliales de la Vena Umbilical Humana/inmunología , Humanos , Interleucina-17/biosíntesis , Interleucina-17/genética , Interleucina-6/biosíntesis , Interleucina-6/genética , Interleucina-6/inmunología , Células Jurkat/inmunología , Activación de Linfocitos/genética , FN-kappa B/biosíntesis , Regiones Promotoras Genéticas , ARN Mensajero/biosíntesis , Edulcorantes/farmacología , Células Th17/citología , Células Th17/inmunología , Factor de Transcripción AP-1/biosíntesisRESUMEN
HYPOTHESIS: ATP Binding Cassette Transporter (ABC) A1 is one of the key regulators of HDL synthesis and reverse cholesterol transport. Activation of Receptors for Advanced Glycation End products (RAGE) is involved in the pathogenesis of diabetes, and its complications. The aim of the present study is to examine the effect of RAGE ligand S100B on ABCA1 expression. METHODS: S100B mediated regulation of LXR target genes like ABCA1, ABCG1, ABCG8, LXR-α and LXR-ß in THP-1 cells was analyzed by real-time PCR, RT-PCR and western blots. ABCA1 mRNA expression in monocytes from diabetic patients was studied. Effect of LXR ligand on S100B induced changes in LXR target genes was also studied. Luciferase reporter assay was used for S100B induced ABCA1 promoter regulation. RESULTS: S100B treatment resulted in a significant 2-3 fold reduction (p<0.01) in ABCA1 and ABCG1 mRNA in dose and time dependent manner in THP1 cells. ABCA1 protein level was also significantly (p<0.01) reduced. S100B-induced reduction on ABCA1 mRNA expression was blocked by treating THP-1 cell with anti-RAGE antibody. Reduced ABCA1 mRNA levels seen in peripheral blood monocytes from diabetes patients showed the in-vivo relevance of our in-vitro results. Effect of S100B on ABCA1 and ABCG1 expression was reversed by LXR ligand treatment. S100B treatment showed significant 2 fold (p<0.01) decrease in T1317 induced ABCA1 promoter activation. CONCLUSIONS: These results show for the first time that ligation of RAGE with S100B can attenuate the expression of ABCA1 and ABCG1 through the LXRs. This could reduce ApoA-I-mediated cholesterol efflux from monocytes.