RESUMEN
Purpose: Tofacitinib is a pan-Janus kinase (JAK) inhibitor that suppresses cytokine signaling and in turn, the cells that participate in inflammatory immunopathogenic processes. We examined the capacity of tofacitinib to inhibit the induction of experimental autoimmune uveitis (EAU) and related immune responses. Methods: EAU was induced in B10.A mice with immunization with bovine interphotoreceptor retinoid-binding protein (IRBP), emulsified in complete Freund's adjuvant (CFA), and a simultaneous injection of pertussis toxin. Tofacitinib, 25 mg/kg, was administered daily, and the vehicle was used for control. EAU development was assessed by histological analysis of the mouse eyes, and related immune responses were assessed by (i) the levels of interferon (IFN)-γ and interleukin (IL)-17, secreted by spleen cells cultured with IRBP; (ii) flow cytometric analysis of intracellular expression by spleen, or eye-infiltrating CD4 or CD8 cells of IFN-γ, IL-17, and their transcription factors, T-bet and RORγt. In addition, the inflammation-related cell markers CD44 and CD62L and Ki67, a proliferation marker, were tested. The proportions of T-regulatory cells expressing FoxP3 were determined by flow cytometric intracellular staining, while levels of antibody to IRBP were measured with enzyme-linked immunosorbent assay (ELISA). Results: Treatment with tofacitinib significantly suppressed the development of EAU and reduced the levels of secreted IFN-γ, but not of IL-17. Further, treatment with tofacitinib reduced in the spleen and eye-infiltrating cells the intracellular expression of IFN-γ and its transcription factor T-bet. In contrast, treatment with tofacitinib had essentially no effect on the intracellular expression of IL-17 and its transcription factor, RORγt. The selective effect of tofacitinib treatment was particularly evident in the CD8 population. Treatment with tofacitinib also increased the population of CD44, but reduced the populations of cells producing CD62L and Ki67. Treatment with tofacitinib had no effect on the proportion of FoxP3 producing regulatory cells and on the antibody production to IRBP. Conclusions: Treatment with tofacitinib inhibited the development of EAU, reduced the production of IFN-γ, but had essentially no effect on the production of IL-17.
Asunto(s)
Ojo/metabolismo , Piperidinas/farmacología , Pirimidinas/farmacología , Células TH1/efectos de los fármacos , Células Th17/efectos de los fármacos , Uveítis/tratamiento farmacológico , Uveítis/inmunología , Animales , Antígenos CD4/sangre , Linfocitos T CD4-Positivos/efectos de los fármacos , Linfocitos T CD4-Positivos/inmunología , Antígenos CD8/sangre , Linfocitos T CD8-positivos/efectos de los fármacos , Linfocitos T CD8-positivos/inmunología , Ojo/efectos de los fármacos , Ojo/patología , Proteínas del Ojo/farmacología , Factores de Transcripción Forkhead/sangre , Receptores de Hialuranos/sangre , Terapia de Inmunosupresión , Interferón gamma/sangre , Interleucina-17/sangre , Antígeno Ki-67/sangre , Selectina L/sangre , Ratones , Piperidinas/administración & dosificación , Pirimidinas/administración & dosificación , Proteínas de Unión al Retinol/farmacología , Células TH1/inmunología , Células Th17/inmunologíaRESUMEN
Non-infectious uveitis, a common cause of blindness in man, is often mediated by autoimmunity, a process in which cytokines play major roles. The biosynthesis and secretion of pro-inflammatory cytokines are regulated in part by tristetraprolin (TTP), an endogenous anti-inflammatory protein that acts by binding directly to specific sequence motifs in the 3'-untranslated regions of target mRNAs, promoting their turnover, and inhibiting synthesis of their encoded proteins. We recently developed a TTP-overexpressing mouse (TTPΔARE) by deleting an AU-rich element (ARE) instability motif from the TTP mRNA, resulting in increased accumulation of TTP mRNA and protein throughout the animal. Here, we show that homozygous TTPΔARE mice are resistant to the induction of experimental autoimmune uveitis (EAU) induced by interphotoreceptor retinoid-binding protein (IRBP), an established model for human autoimmune (noninfectious) uveitis. Lymphocytes from TTPΔARE mice produced lower levels of the pro-inflammatory cytokines IFN-γ, IL-17, IL-6, and TNFα than wild type (WT) mice. TTPΔARE mice also produced lower titers of antibodies against the uveitogenic protein. In contrast, TTPΔARE mice produced higher levels of the anti-inflammatory cytokine IL-10, and had higher frequencies of regulatory T-cells, which, moreover, displayed a moderately higher per-cell regulatory ability. Heterozygous mice developed EAU and associated immunological responses at levels intermediate between homozygous TTPΔARE mice and WT controls. TTPΔARE mice were able, however, to develop EAU following adoptive transfer of activated WT T-cells specific to IRBP peptide 651-670, and naïve T-cells from TTPΔARE mice could be activated by antibodies to CD3/CD28. Importantly, TTPΔARE antigen presenting cells were significantly less efficient compared to WT in priming naïve T cells, suggesting that this feature plays a major role in the dampened immune responses of the TTPΔARE mice. Our observations demonstrate that elevated systemic levels of TTP can inhibit the pathogenic processes involved in EAU, and suggest the possible use of TTP-based treatments in humans with uveitis and other autoimmune conditions.
Asunto(s)
Enfermedades Autoinmunes/metabolismo , Enfermedad Autoinmune Experimental del Sistema Nervioso/metabolismo , Tristetraprolina/metabolismo , Uveítis/metabolismo , Animales , Enfermedades Autoinmunes/inmunología , Enfermedades Autoinmunes/patología , Femenino , Técnicas de Sustitución del Gen , Masculino , Ratones , Ratones Endogámicos C57BL , Enfermedad Autoinmune Experimental del Sistema Nervioso/inmunología , Enfermedad Autoinmune Experimental del Sistema Nervioso/patología , Tristetraprolina/inmunología , Uveítis/inmunología , Uveítis/patologíaRESUMEN
Type I interferons (IFNs) have therapeutic potential in CNS autoimmune diseases, such as uveitis, but the molecular mechanisms remain unclear. Using a T cell-transfer model of experimental autoimmune uveitis (EAU), we found that IFN-α/ß treatment inhibited the migration of IFN-γ-producing pathogenic CD4+ T cells to effector sites. IFN-α/ß upregulated the expression of the cognate ligands CXCL9, CXCL10, and CXCL11, causing ligand-mediated downregulation of CXCR3 expression and effector T cell retention in the spleen. Accordingly, type I IFN did not alter EAU progression in CXCR3-/- mice. In uveitis patients, disease exacerbations correlated with reduced serum IFN-α concentrations. IFN-α/ß reduced CXCR3 expression and migration by human effector T cells, and these parameters were associated with the therapeutic efficacy of IFN-α in uveitis patients. Our findings provide insight into the molecular basis of type I IFN therapy for CNS autoimmune diseases and identify CXCR3 as a biomarker for effective type I IFN immunotherapy.
Asunto(s)
Enfermedades Autoinmunes/genética , Inmunoterapia/métodos , Interferón Tipo I/metabolismo , Receptores CXCR3/antagonistas & inhibidores , Linfocitos T/metabolismo , Animales , Humanos , RatonesRESUMEN
Inflammatory intraocular eye diseases, grouped under the term uveitis are blinding conditions, believed to be mediated by pathogenic autoimmune processes that overcome the protective mechanisms of the immune privilege status of the eye. An animal model for these diseases, named experimental autoimmune uveitis (EAU), is induced by initiation of immunity against ocular-specific antigens, or it develops spontaneously in mice with T-cells that transgenically express TCR specific to the target eye antigen(s). T-Cells specific to ocular antigens are generated in the thymus and their majority are eliminated by exposure to their target antigen expressed in this organ. T-cells that escape this negative selection acquire pathogenicity by their activation with the target antigen. In spontaneous EAU, the microbiota play crucial roles in the acquisition of pathogenicity by providing both antigenic stimulation, by molecules that mimic the target ocular antigen, and an additional stimulation that allows invasion of tissues that harbor the target antigen. The pathogenic process is physiologically inhibited by the peripheral tolerance, composed of antigen-specific T-regulatory (Treg) lymphocytes. Deleting the Tregs enhances the ocular inflammation, whereas adoptively transferring them suppresses the pathogenic response. Potential usage of Treg cells for suppression of autoimmune diseases in humans is under intensive investigation.
Asunto(s)
Oftalmopatías/etiología , Oftalmopatías/metabolismo , Tolerancia Inmunológica , Animales , Ojo/inmunología , Ojo/metabolismo , Oftalmopatías/patología , Oftalmopatías/terapia , Humanos , Uveítis/etiología , Uveítis/metabolismo , Uveítis/patología , Uveítis/terapiaRESUMEN
Experimental autoimmune uveitis (EAU), an animal model for severe intraocular inflammatory eye diseases, is mediated by both Th1 and Th17 cells. Here, we examined the capacity of TMP778, a selective inhibitor of RORγt, to inhibit the development of EAU, as well as the related immune responses. EAU was induced in B10.A mice by immunization with interphotoreceptor retinoid-binding protein (IRBP). Treatment with TMP778 significantly inhibited the development of EAU, determined by histological examination. In addition, the treatment suppressed the cellular immune response to IRBP, determined by reduced production of IL-17 and IFN-γ, as well as lower percentages of lymphocytes expressing these cytokines, as compared to vehicle-treated controls. The inhibition of IFN-γ expression by TMP778 is unexpected in view of this compound being a selective inhibitor of RORγt. The observation was further confirmed by the finding of reduced expression of the T-bet (Tbx21) gene, the transcription factor for IFN-γ, by cells of TMP778-treated mice. Thus, these data demonstrate the capacity of TMP778 to inhibit pathogenic autoimmunity in the eye and shed new light on its mode of action in vivo.
Asunto(s)
Enfermedades Autoinmunes/tratamiento farmacológico , Compuestos Heterocíclicos de 4 o más Anillos/farmacología , Miembro 3 del Grupo F de la Subfamilia 1 de Receptores Nucleares/antagonistas & inhibidores , Células TH1/efectos de los fármacos , Células Th17/efectos de los fármacos , Uveítis/tratamiento farmacológico , Animales , Enfermedades Autoinmunes/metabolismo , Citocinas/metabolismo , Modelos Animales de Enfermedad , Proteínas del Ojo/metabolismo , Femenino , Interferón gamma/metabolismo , Interleucina-17/metabolismo , Ratones , Proteínas de Unión al Retinol/metabolismo , Proteínas de Dominio T Box/metabolismo , Células TH1/metabolismo , Células Th17/metabolismo , Uveítis/metabolismoRESUMEN
In this study we compared polarized mouse T-helper (Th) lymphocytes of four populations, sensitized against an ocular antigen, for their patterns of migration and induction of inflammatory processes in recipient mouse eyes expressing the target antigen. Th1, Th2, Th9 and Th17 cells transgenically expressing T-cell receptor (TCR) specific against hen egg lysozyme (HEL) were adoptively transferred to recipient mice expressing HEL in their eyes. Recipient eyes collected 4 or 7 days post injection were analyzed for histopathological changes. Th1 and Th17 cells induced moderate to severe intraocular inflammation in the recipient mouse eyes, but essentially did not migrate into the conjunctiva. In contrast, Th2 and Th9 cells invaded minimally the intraocular space of recipient eyes, but accumulated in the limbus and migrated into the conjunctiva of the recipient mice and initiated allergy-like inflammatory responses, as indicated by remarkable eosinophil involvement. These data thus shed new light on the differences between the migration patterns and ocular pathogenic processes mediated by Th1/Th17 and by Th2/Th9 populations.
Asunto(s)
Movimiento Celular , Conjuntiva/patología , Eosinofilia/patología , Limbo de la Córnea/parasitología , Linfocitos T Colaboradores-Inductores/inmunología , Animales , Modelos Animales de Enfermedad , Cristalino/metabolismo , Ratones , Muramidasa , Células TH1/inmunología , Células Th17/inmunología , Células Th2/inmunologíaAsunto(s)
Oftalmopatías/inmunología , Oftalmólogos/historia , Médicos/historia , Anticuerpos Monoclonales Humanizados/uso terapéutico , Ciclosporina/uso terapéutico , Daclizumab , Historia del Siglo XX , Historia del Siglo XXI , Humanos , Inmunoglobulina G/uso terapéutico , Inmunosupresores/uso terapéutico , National Eye Institute (U.S.)/organización & administración , Estados Unidos , Uveítis/tratamiento farmacológicoRESUMEN
Th cells sensitized against autoantigens acquire pathogenicity following two sequential events, namely activation by their target Ag and a process named "licensing." In this study, we analyzed these processes in a transgenic mouse system in which TCR-transgenic Th cells specific to hen egg lysozyme (HEL) are adoptively transferred to recipients and induce inflammation in eyes expressing HEL. Our data show that the notion that the lung is the organ where "licensing" for pathogenicity takes place is based on biased data collected with cells injected i.v., a route in which most transferred cells enter via the lung. Thus, we found that when donor cells were activated in vitro and injected intraperitoneally, or were activated in vivo, they migrated simultaneously to the lung, spleen, and other tested organs. In all, tested organs donor cells undergo "licensing" for pathogenicity, consisting of vigorous increase in number and changes in expression levels of inflammation-related genes, monitored by both flow cytometry and microarray analysis. After reaching peak numbers, around day 3, the "licensed" donor cells migrate to the circulation and initiate inflammation in the HEL-expressing recipient eyes. Importantly, the kinetics of increase in number and of changes in gene expression by the donor cells were similar in lung, spleen, and other tested organs of the recipient mice. Furthermore, the total numbers of donor cells in the spleen at their peaks were 10- to 100-fold larger in the spleen than in the lung, contradicting the notion that the lung is the organ where "licensing" takes place.
Asunto(s)
Enfermedades Autoinmunes/inmunología , Autoinmunidad/inmunología , Activación de Linfocitos/inmunología , Linfocitos T Colaboradores-Inductores/inmunología , Traslado Adoptivo , Animales , Autoantígenos/inmunología , Modelos Animales de Enfermedad , Citometría de Flujo , Pulmón/inmunología , Ratones , Ratones Transgénicos , Muramidasa/inmunología , Bazo/inmunologíaRESUMEN
PURPOSE: Digoxin, a major medication for heart disease, was recently reported to have immunosuppressive capacity. Here, we determined the immunosuppressive capacity of digoxin on the development of experimental autoimmune uveitis (EAU) and on related immune responses. METHODS: The B10.A mice were immunized with interphotoreceptor retinoid-binding protein (IRBP) and were treated daily with digoxin or vehicle control. On postimmunization day 14, the mouse eyes were examined histologically, while spleen cells were tested for cytokine production in response to IRBP and purified protein derivative. The immunosuppressive activity of digoxin was also tested in vitro, by its capacity to inhibit development of Th1 or Th17 cells. To investigate the degenerative effect of digoxin on the retina, naïve (FVB/N × B10.BR)F1 mice were similarly treated with digoxin and tested histologically and by ERG. RESULTS: Treatment with digoxin inhibited the development of EAU, as well as the cellular response to IRBP. Unexpectedly, treatment with digoxin suppressed the production of interferon-γ to a larger extent than the production of interleukin 17. Importantly, digoxin treatment induced severe retinal degeneration, determined by histologic analysis with thinning across all layers of the retina. Digoxin treatment also induced dose-dependent vision loss monitored by ERG on naïve mice without induction of EAU. CONCLUSIONS: Treatment of mice with digoxin inhibited the development of EAU and cellular immune response to IRBP. However, the treatment induced severe damage to the retina. Thus, the use of digoxin in humans should be avoided due to its toxicity to the retina.
Asunto(s)
Diabetes Mellitus Tipo 1 , Digoxina/farmacología , Inmunidad Celular/efectos de los fármacos , Degeneración Retiniana/prevención & control , Uveítis/tratamiento farmacológico , Animales , Modelos Animales de Enfermedad , Inhibidores Enzimáticos/farmacología , Femenino , Ratones , Degeneración Retiniana/etiología , Degeneración Retiniana/inmunología , Índice de Severidad de la Enfermedad , Uveítis/complicaciones , Uveítis/inmunologíaRESUMEN
BACKGROUND: Mutations in LRRK2 are related to certain forms of Parkinson's disease and, possibly, to the pathogenesis of Crohn's disease. In both these diseases inflammatory processes participate in the pathogenic process. LRRK2 is expressed in lymphoid cells and, interestingly, Lrrk2 (-/-) mice were reported to develop more severe experimental colitis than their wild type (WT) controls. Here, we examined the possible involvement of LRRK2 in the pathogenesis of experimental autoimmune uveitis (EAU), an animal model for human uveitis, by testing Lrrk2 (-/-) mice for their capacity to develop this experimental eye disease and related immune responses. METHODS: Lrrk2 (-/-) mice and their WT controls (C57Bl/6) were immunized with interphotoreceptor retinoid-binding protein (IRBP) and compared for their development of EAU, delayed type hypersensitivity (DTH) by skin tests, production of cytokines in culture, and expression of interferon (IFN)-γ, interleukin (IL)-17 and FoxP3 by spleen cells, using flow cytometry. Peritoneal macrophages were examined for their production of cytokines/chemokines in culture following stimulation with LPS or the oligodeoxynucleotide CpG. The Lrrk2 (-/-) and WT mice were also compared for their response to bovine serum albumin (BSA). RESULTS: The Lrrk2 (-/-) mice developed lower levels of EAU, DTH responses and cytokine production by lymphocytes than did their WT controls. Intracellular expression of IFN-γ and IL-17, by spleen cells, and secretion of cytokines/chemokines by activated peritoneal macrophages of Lrrk2 (-/-) mice trended toward diminished levels, although variabilities were noted. The expression levels of FoxP3 by Lrrk2 (-/-) spleen cells, however, were similar to those seen in WT controls. Consistent with their low response to IRBP, Lrrk2 (-/-) mice responded to BSA less vigorously than their WT controls. CONCLUSIONS: Lrrk2 deficiency in mice diminished the development of EAU and the related adaptive immune responses to IRBP as compared to the WT controls.
Asunto(s)
Enfermedades Autoinmunes/patología , Proteínas Serina-Treonina Quinasas/genética , Uveítis/patología , Inmunidad Adaptativa , Animales , Enfermedades Autoinmunes/metabolismo , Quimiocinas/metabolismo , Modelos Animales de Enfermedad , Proteínas del Ojo/inmunología , Factores de Transcripción Forkhead/metabolismo , Hipersensibilidad Tardía/etiología , Hipersensibilidad Tardía/inmunología , Interferón gamma/metabolismo , Interleucina-17/metabolismo , Proteína 2 Quinasa Serina-Treonina Rica en Repeticiones de Leucina , Macrófagos Peritoneales/inmunología , Macrófagos Peritoneales/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas Serina-Treonina Quinasas/deficiencia , Proteínas de Unión al Retinol/inmunología , Pruebas Cutáneas , Células TH1/citología , Células TH1/inmunología , Células TH1/metabolismo , Células Th17/citología , Células Th17/inmunología , Células Th17/metabolismo , Uveítis/metabolismoRESUMEN
Glucocorticoids remain the cornerstone of treatment for inflammatory conditions, but their utility is limited by a plethora of side effects. One of the key goals of immunotherapy across medical disciplines is to minimize patients' glucocorticoid use. Increasing evidence suggests that variations in the adaptive immune response play a critical role in defining the dose of glucocorticoids required to control an individual's disease, and Th17 cells are strong candidate drivers for nonresponsiveness [also called steroid resistance (SR)]. Here we use gene-expression profiling to further characterize the SR phenotype in T cells and show that Th17 cells generated from both SR and steroid-sensitive individuals exhibit restricted genome-wide responses to glucocorticoids in vitro, and that this is independent of glucocorticoid receptor translocation or isoform expression. In addition, we demonstrate, both in transgenic murine T cells in vitro and in an in vivo murine model of autoimmunity, that Th17 cells are reciprocally sensitive to suppression with the calcineurin inhibitor, cyclosporine A. This result was replicated in human Th17 cells in vitro, which were found to have a conversely large genome-wide shift in response to cyclosporine A. These observations suggest that the clinical efficacy of cyclosporine A in the treatment of SR diseases may be because of its selective attenuation of Th17 cells, and also that novel therapeutics, which target either Th17 cells themselves or the effector memory T-helper cell population from which they are derived, would be strong candidates for drug development in the context of SR inflammation.
Asunto(s)
Ciclosporina/química , Glucocorticoides/química , Células Th17/citología , Animales , Autoinmunidad , Linfocitos T CD4-Positivos/citología , Linfocitos T CD4-Positivos/inmunología , Calcineurina/química , Inhibidores de la Calcineurina/química , Núcleo Celular/metabolismo , Proliferación Celular , Modelos Animales de Enfermedad , Humanos , Inflamación , Interferón gamma/metabolismo , Interleucina-17/metabolismo , Ratones , Ratones Transgénicos , Fenotipo , Esteroides/químicaRESUMEN
The TNF family cytokine TL1A (Tnfsf15) costimulates T cells and type 2 innate lymphocytes (ILC2) through its receptor DR3 (Tnfrsf25). DR3-deficient mice have reduced T cell accumulation at the site of inflammation and reduced ILC2-dependent immune responses in a number of models of autoimmune and allergic diseases. In allergic lung disease models, immunopathology and local Th2 and ILC2 accumulation is reduced in DR3-deficient mice despite normal systemic priming of Th2 responses and generation of T cells secreting IL-13 and IL-4, prompting the question of whether TL1A promotes the development of other T cell subsets that secrete cytokines to drive allergic disease. In this study, we find that TL1A potently promotes generation of murine T cells producing IL-9 (Th9) by signaling through DR3 in a cell-intrinsic manner. TL1A enhances Th9 differentiation through an IL-2 and STAT5-dependent mechanism, unlike the TNF-family member OX40, which promotes Th9 through IL-4 and STAT6. Th9 differentiated in the presence of TL1A are more pathogenic, and endogenous TL1A signaling through DR3 on T cells is required for maximal pathology and IL-9 production in allergic lung inflammation. Taken together, these data identify TL1A-DR3 interactions as a novel pathway that promotes Th9 differentiation and pathogenicity. TL1A may be a potential therapeutic target in diseases dependent on IL-9.
Asunto(s)
Asma/inmunología , Diferenciación Celular/inmunología , Interleucina-9/inmunología , Miembro 25 de Receptores de Factores de Necrosis Tumoral/inmunología , Linfocitos T Colaboradores-Inductores/inmunología , Miembro 15 de la Superfamilia de Ligandos de Factores de Necrosis Tumoral/inmunología , Animales , Asma/genética , Asma/patología , Diferenciación Celular/genética , Inflamación/genética , Inflamación/inmunología , Inflamación/patología , Interleucina-13/genética , Interleucina-13/inmunología , Interleucina-4/genética , Interleucina-4/inmunología , Interleucina-9/genética , Ratones , Ratones Noqueados , Miembro 25 de Receptores de Factores de Necrosis Tumoral/genética , Transducción de Señal/genética , Transducción de Señal/inmunología , Linfocitos T Colaboradores-Inductores/patología , Miembro 15 de la Superfamilia de Ligandos de Factores de Necrosis Tumoral/genéticaRESUMEN
PURPOSE: The inflammatory process plays a major role in the pathogenesis of AMD, and recent data indicate the involvement of inflammasomes. Inflammasomes are intracellular structures that trigger inflammation by producing mature interleukin-(IL)-1ß and IL-18. This study examined the capacity of 7-ketocholesterol (7KCh), an oxysterol that accumulates in the retinal pigmented epithelium (RPE) and choroid, to initiate inflammasome formation in RPE and bone marrow-derived cells. METHODS: Tested cells included fetal human RPE (fhRPE), human ARPE-19 cells, primary human brain microglia cells, and human THP-1 monocyte cells. 7-Ketocholesterol and other compounds were added to the cell cultures, and their stimulatory effects were determined by quantitative PCR and release of cytokines, measured by ELISA and Western blotting. RESULTS: 7-Ketocholesterol efficiently induced inflammasome formation by all primed cell populations, but secreted cytokine levels were higher in cultures of bone marrow-derived cells (microglia and THP-1 cells) than in RPE cultures. Interestingly, inflammasomes formed in cells of the two populations differed strikingly in their preferential production of the two cytokines. Thus, whereas bone marrow-derived cells produced levels of IL-1ß that were higher than those of IL-18, the opposite was found with RPE cells, which secreted higher levels of IL-18. Importantly, Western blot analysis showed that IL-18, but not IL-1ß, was expressed constitutively by RPE cells. CONCLUSIONS: 7-Ketocholesterol efficiently stimulates inflammasome formation and is conceivably involved in the pathogenesis of AMD. In contrast to bone marrow-derived cells, RPE cells produced higher levels of IL-18 than IL-1ß. Further, IL-18, a multifunctional cytokine, was expressed constitutively by RPE cells. These observations provide new information about stimuli and cells and their products assumed to be involved in the pathogenesis of AMD.
Asunto(s)
Células de la Médula Ósea/efectos de los fármacos , Células de la Médula Ósea/fisiología , Inflamasomas/efectos de los fármacos , Inflamasomas/fisiología , Interleucina-18/metabolismo , Interleucina-1beta/metabolismo , Cetocolesteroles/farmacología , Epitelio Pigmentado de la Retina/efectos de los fármacos , Epitelio Pigmentado de la Retina/fisiología , Western Blotting , Células Cultivadas , Ensayo de Inmunoadsorción Enzimática , HumanosRESUMEN
Regulatory T-cells (Tregs) are responsible for homeostasis of the immune system, as well as for inhibition of pathogenic autoimmune processes. Induced-(i)-Tregs, can be generated in vitro by activation of CD4 cells in the presence of TGF-ß. A commonly used activation mechanism is by antibodies against CD3 and CD28. The physiological-like activation of T-cells, however, is with the specific target antigen presented by antigen-presenting cells (APC). The two modes of activation have been considered to yield the same populations of iTregs. Here, we compared between iTreg populations generated by either one of the two methods and found differences between their capacities to inhibit T-lymphocyte proliferative response, their expression of cell surface antigens and particularly, in their transcript expression profiles of certain chemokines and chemokine receptors. Our data thus indicate that iTregs generated by activation with anti-CD3/CD28 antibodies cannot be considered identical to iTregs generated by antigen/APC.
Asunto(s)
Técnicas In Vitro/métodos , Activación de Linfocitos/inmunología , Subgrupos de Linfocitos T/inmunología , Linfocitos T Reguladores/inmunología , Animales , Células Presentadoras de Antígenos/inmunología , Antígenos CD28/inmunología , Complejo CD3/inmunología , Citocinas/biosíntesis , Citometría de Flujo , Ratones , Ratones Transgénicos , Reacción en Cadena de la PolimerasaRESUMEN
T-helper (Th) lineages have been generated in vitro by activating CD4 cells with anti-CD3/CD28 antibodies during polarization. Physiologically, however, the generation of Th lineages is by activation with the specific antigen presented by antigen-presenting cells (APC). Here, we used T-cell receptor (TCR)-transgenic mice to compare the phenotypes of Th1, Th9 and Th17 lineages when generated by either one of the two activation modes. Lineage Th cells specific against hen egg lysozyme (HEL), were adoptively transferred into recipient mice transgenically expressing HEL in their lens. Remarkable differences were found between lineages of Th1, Th9 or Th17, generated by either one of the two modes in their capacities to migrate to and proliferate in the recipient spleen and, importantly, to induce inflammation in the recipient mouse eyes. Substantial differences were also observed between the lineage pairs in their transcript expression profiles of certain chemokines and chemokine receptors. Surprisingly, however, close similarities were observed between the transcript expression profiles of lineages of the three phenotypes, activated by the same mode. Furthermore, Th cell lineages generated by the two activation modes differed considerably in their pattern of gene expression, as monitored by microarray analysis, but exhibited commonality with lineages of other phenotypes generated by the same activation mode. This study thus shows that (i) Th lineages generated by activation with anti-CD3/CD28 antibodies differ from lineages generated by antigen/APC; and (ii) the mode of activation determines to a large extent the expression profile of major transcripts.
Asunto(s)
Anticuerpos/inmunología , Antígenos CD28/inmunología , Complejo CD3/inmunología , Linaje de la Célula/inmunología , Activación de Linfocitos/inmunología , Linfocitos T Colaboradores-Inductores/citología , Linfocitos T Colaboradores-Inductores/inmunología , Animales , Células Presentadoras de Antígenos/efectos de los fármacos , Células Presentadoras de Antígenos/inmunología , Biomarcadores/metabolismo , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Quimiocinas/genética , Quimiocinas/metabolismo , Pollos , Regulación de la Expresión Génica/efectos de los fármacos , Activación de Linfocitos/efectos de los fármacos , Ratones , Muramidasa/inmunología , Análisis de Secuencia por Matrices de Oligonucleótidos , Fenotipo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Receptores de Quimiocina/genética , Receptores de Quimiocina/metabolismo , Bazo/citología , Bazo/inmunologíaRESUMEN
PURPOSE: Ligands for aryl hydrocarbon receptor (AHR), such as dioxins, are highly toxic. One such ligand, TCDD, was found to exert potent immunosuppressive capacities in mice developing pathogenic autoimmune processes, including EAU, but its toxicity makes it unusable for humans. A recently identified endogenous AHR ligand, ITE, is also immunosuppressive, but is nontoxic and could therefore be useful for therapy in humans. Here, we tested ITE for its capacity to inhibit EAU and related immune responses. METHODS: EAU was induced in B10.A mice by immunization with interphotoreceptor retinoid-binding protein (IRBP; 40 µg) in CFA. Treatment with ITE was by daily intraperitoneal injection of 0.2 mg. Disease severity was assessed by both fundoscopy and histological examination. Draining lymph node cells were tested for proliferation by thymidine uptake and for cytokine production and release by ELISA. In addition, the intracellular expression of cytokines and Foxp3 was determined by flow cytometry. Serum antibodies were measured by ELISA. RESULTS: Treatment with ITE efficiently inhibited the development of EAU in mice, as well as the cellular immune responses against IRBP and PPD. ITE treatment inhibited the expansion of both Th1 and Th17 subpopulations, as well as their release of the signature cytokines, IFN-gamma and IL-17. The treatment moderately increased, however, the proportion of Foxp3 expressing T-regulatory cells. Antibody production was not affected by the treatment. CONCLUSIONS: ITE, an endogenous AHR ligand, efficiently inhibits EAU development and related cellular immune responses. Being nontoxic, ITE may be considered for treatment of pathogenic immunity in humans.
Asunto(s)
Enfermedades Autoinmunes/prevención & control , Proteínas del Ojo/metabolismo , Inmunidad Celular/inmunología , Proteínas de Unión al Retinol/metabolismo , Uveítis/prevención & control , Animales , Enfermedades Autoinmunes/inmunología , Enfermedades Autoinmunes/patología , Diferenciación Celular/inmunología , Citocinas/inmunología , Citocinas/metabolismo , Modelos Animales de Enfermedad , Ensayo de Inmunoadsorción Enzimática , Proteínas del Ojo/inmunología , Femenino , Citometría de Flujo , Ratones , Proteínas de Unión al Retinol/inmunología , Linfocitos T Reguladores/inmunología , Linfocitos T Reguladores/patología , Uveítis/inmunología , Uveítis/patologíaRESUMEN
DNAX-activation protein 12 (DAP12), a transmembrane adapter, plays a major role in transducing activation signals in natural killer cells and various myeloid cells. Quantitative RT-PCR detected in normal mouse eyes considerable levels of DAP12 and multiple DAP12-coupled receptors, in particular TREM-1, Clec5a and SIRPb1. The role of DAP12 and its receptors in experimental autoimmune diseases has been controversial. Here, we analysed the effect of DAP12 deficiency on the capacity of mice to mount immunopathogenic cellular responses to the uveitogenic ocular antigen and interphotoreceptor retinoid-binding protein (IRBP), and to develop experimental autoimmune uveitis (EAU). Surprisingly, sequential analysis of EAU in mice deficient in DAP12 in two different animal facilities at first revealed enhanced disease as compared with wild-type mice, but when these mice were re-derived into a second, cleaner, animal facility, the response of control mice was essentially unchanged, whereas the DAP12 null mice were markedly hyporesponsive relative to controls in the new facility. Accordingly, when stimulated in vitro with IRBP, lymphocytes from the DAP12-deficient mice housed in the two facilities proliferated and produced opposite profiles of pro-inflammatory and anti-inflammatory cytokines, compared with their controls. These findings therefore demonstrate that the effects of DAP12 deficiency on development of autoimmune disease are dramatically affected by environmental factors.