RESUMEN
The ocular surface is a mucosal barrier tissue colonized by commensal microbes, which tune local immunity by eliciting IL-17 from conjunctival γδ T cells to prevent pathogenic infection. The commensal Corynebacterium mastitidis (C. mast) elicits protective IL-17 responses from conjunctival Vγ4 T cells through a combination of γδ TCR ligation and IL-1 signaling. Here, we identify Vγ6 T cells as a major C. mast-responsive subset in the conjunctiva and uncover its unique activation requirements. We demonstrate that Vγ6 cells require not only extrinsic (via dendritic cells) but also intrinsic TLR2 stimulation for optimal IL-17A response. Mechanistically, intrinsic TLR2 signaling was associated with epigenetic changes and enhanced expression of genes responsible for metabolic shift to fatty acid oxidation to support Il17a transcription. We identify one key transcription factor, IκBζ, which is upregulated by TLR2 stimulation and is essential for this program. Our study highlights the importance of intrinsic TLR2 signaling in driving metabolic reprogramming and production of IL-17A in microbiome-specific mucosal γδ T cells.
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Roughly one-half of mice with partial defects in two immune tolerance pathways (AireGW/+Lyn-/- mice) spontaneously develop severe damage to their retinas due to T cell reactivity to Aire-regulated interphotoreceptor retinoid-binding protein (IRBP). Single-cell T cell receptor (TCR) sequencing of CD4+ T cells specific for a predominate epitope of IRBP showed a remarkable diversity of autoantigen-specific TCRs with greater clonal expansions in mice with disease. TCR transgenic mice made with an expanded IRBP-specific TCR (P2.U2) of intermediate affinity exhibited strong but incomplete negative selection of thymocytes. This negative selection was absent in IRBP-/- mice and greatly defective in AireGW/+ mice. Most P2.U2+/- mice and all P2.U.2+/-AireGW/+ mice rapidly developed inflammation of the retina and adjacent uvea (uveitis). Aire-dependent IRBP expression in the thymus also promoted Treg differentiation, but the niche for this fate determination was small, suggesting differences in antigen presentation leading to negative selection vs. thymic Treg differentiation and a stronger role for negative selection in preventing autoimmune disease in the retina.
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Presentación de Antígeno , Receptores de Antígenos de Linfocitos T , Animales , Ratones , Autoantígenos , Modelos Animales de Enfermedad , Ratones Endogámicos , Ratones TransgénicosRESUMEN
Purpose: Using the model of experimental autoimmune uveitis (EAU) induced by immunization with a retinal antigen, two studies have reported contradictory results on disease development following oral antibiotic treatment (ABX). We showed that long-term ABX did not affect EAU, but another study showed that short-term ABX was protective. We therefore studied the effects of ABX on EAU, gut microbiota, and host immune responses as a function of treatment duration. Methods: EAU-susceptible mice were treated orally with broad-spectrum antibiotics starting at least 10 weeks (long-term) or 1 week (short-term) before immunization until termination of the experiment. Gut microbiota were characterized by 16S amplicon sequencing, and host gut immune elements were studied phenotypically and functionally. Results: Long-term ABX had no effect, whereas short-term ABX delayed EAU, as previously reported by us and others, respectively. Microbial sequencing revealed progressive reduction of gut microbiota that showed some differences in the two ABX groups. Interestingly, duration of ABX was associated with a gradual disappearance of the CD4+ and CD4+CD8+ subset of gut intraepithelial lymphocytes (IELs). This IEL subset is microbiota dependent and is absent in germ-free mice. Relative abundance of Lactobacillus reuteri correlated with the frequencies of CD4+CD8+ IELs. IELs suppressed antigen-specific activation of autoreactive T cells in culture. Conclusions: Gut microbiota may play dual roles in uveitis development: They promote EAU development but also help maintain IEL populations that have regulatory function against autoreactive T cells. We propose that the progressive loss of this population during long-term ABX reverses the EAU-ameliorating effects of microbiota depletion.
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Microbioma Gastrointestinal , Microbiota , Uveítis , Animales , Ratones , Inmunización , Administración Oral , Antibacterianos , Uveítis/prevención & controlRESUMEN
OBJECTIVES: To test the hypothesis that ROSAH (retinal dystrophy, optic nerve oedema, splenomegaly, anhidrosis and headache) syndrome, caused by dominant mutation in ALPK1, is an autoinflammatory disease. METHODS: This cohort study systematically evaluated 27 patients with ROSAH syndrome for inflammatory features and investigated the effect of ALPK1 mutations on immune signalling. Clinical, immunologic and radiographical examinations were performed, and 10 patients were empirically initiated on anticytokine therapy and monitored. Exome sequencing was used to identify a new pathogenic variant. Cytokine profiling, transcriptomics, immunoblotting and knock-in mice were used to assess the impact of ALPK1 mutations on protein function and immune signalling. RESULTS: The majority of the cohort carried the p.Thr237Met mutation but we also identified a new ROSAH-associated mutation, p.Tyr254Cys.Nearly all patients exhibited at least one feature consistent with inflammation including recurrent fever, headaches with meningeal enhancement and premature basal ganglia/brainstem mineralisation on MRI, deforming arthritis and AA amyloidosis. However, there was significant phenotypic variation, even within families and some adults lacked functional visual deficits. While anti-TNF and anti-IL-1 therapies suppressed systemic inflammation and improved quality of life, anti-IL-6 (tocilizumab) was the only anticytokine therapy that improved intraocular inflammation (two of two patients).Patients' primary samples and in vitro assays with mutated ALPK1 constructs showed immune activation with increased NF-κB signalling, STAT1 phosphorylation and interferon gene expression signature. Knock-in mice with the Alpk1 T237M mutation exhibited subclinical inflammation.Clinical features not conventionally attributed to inflammation were also common in the cohort and included short dental roots, enamel defects and decreased salivary flow. CONCLUSION: ROSAH syndrome is an autoinflammatory disease caused by gain-of-function mutations in ALPK1 and some features of disease are amenable to immunomodulatory therapy.
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Enfermedades Autoinflamatorias Hereditarias , FN-kappa B , Proteínas Quinasas/genética , Amiloidosis , Animales , Estudios de Cohortes , Mutación con Ganancia de Función , Enfermedades Autoinflamatorias Hereditarias/genética , Humanos , Inflamación/genética , Ratones , Mutación , FN-kappa B/genética , FN-kappa B/metabolismo , Proteínas Quinasas/metabolismo , Calidad de Vida , Proteína Amiloide A Sérica , Síndrome , Inhibidores del Factor de Necrosis TumoralRESUMEN
Interleukin-27 is constitutively secreted by microglia in the retina or brain, and upregulation of IL-27 during neuroinflammation suppresses encephalomyelitis and autoimmune uveitis. However, while IL-35 is structurally and functionally similar to IL-27, the intrinsic roles of IL-35 in CNS tissues are unknown. Thus, we generated IL-35/YFP-knock-in reporter mice (p35-KI) and demonstrated that photoreceptor neurons constitutively secrete IL-35, which might protect the retina from persistent low-grade inflammation that can impair photoreceptor functions. Furthermore, the p35-KI mouse, which is hemizygous at the il12a locus, develops more severe uveitis because of reduced IL-35 expression. Interestingly, onset and exacerbation of uveitis in p35-KI mice caused by extravasation of proinflammatory Th1/Th17 lymphocytes into the retina were preceded by a dramatic decrease of IL-35, attributable to massive death of photoreceptor cells. Thus, while inflammation-induced death of photoreceptors and loss of protective effects of IL-35 exacerbated uveitis, our data also suggest that constitutive production of IL-35 in the retina might have housekeeping functions that promote sterilization immunity in the neuroretina and maintain ocular immune privilege.
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Enfermedades Autoinmunes , Interleucinas , Uveítis , Animales , Modelos Animales de Enfermedad , Regulación de la Expresión Génica , Privilegio Inmunológico , Inflamación/metabolismo , Interleucina-27/metabolismo , Interleucinas/genética , Interleucinas/metabolismo , Ratones , Ratones Endogámicos C57BL , Células Fotorreceptoras/metabolismo , Retina/metabolismo , Células Th17 , Uveítis/metabolismoRESUMEN
Regulatory B cells (Breg cells) that secrete IL-10 or IL-35 (i35-Breg) play key roles in regulating immunity in tumor microenvironment or during autoimmune and infectious diseases. Thus, loss of Breg function is implicated in development of autoimmune diseases while aberrant elevation of Breg prevents sterilizing immunity, exacerbates infectious diseases, and promotes cancer metastasis. Breg cells identified thus far are largely antigen-specific and derive mainly from B2-lymphocyte lineage. Here, we describe an innate-like IL-27-producing natural regulatory B-1a cell (i27-Breg) in peritoneal cavity and human umbilical cord blood. i27-Bregs accumulate in CNS and lymphoid tissues during neuroinflammation and confers protection against CNS autoimmune disease. i27-Breg immunotherapy ameliorated encephalomyelitis and uveitis through up-regulation of inhibitory receptors (Lag3, PD-1), suppression of Th17/Th1 responses, and propagating inhibitory signals that convert conventional B cells to regulatory lymphocytes that secrete IL-10 and/or IL-35 in eye, brain, or spinal cord. Furthermore, i27-Breg proliferates in vivo and sustains IL-27 secretion in CNS and lymphoid tissues, a therapeutic advantage over administering biologics (IL-10, IL-35) that are rapidly cleared in vivo. Mutant mice lacking irf4 in B cells exhibit exaggerated increase of i27-Bregs with few i35-Bregs, while mice with loss of irf8 in B cells have abundance of i35-Bregs but defective in generating i27-Bregs, identifying IRF8/BATF and IRF4/BATF axis in skewing B cell differentiation toward i27-Breg and i35-Breg developmental programs, respectively. Consistent with its developmental origin, disease suppression by innate i27-Bregs is neither antigen-specific nor disease-specific, suggesting that i27-Breg would be effective immunotherapy for a wide spectrum of autoimmune diseases.
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Enfermedades Autoinmunes/inmunología , Enfermedades del Sistema Nervioso Central/inmunología , Interleucina-27/metabolismo , Enfermedades Neuroinflamatorias/inmunología , Animales , Linfocitos B Reguladores/inmunología , Diferenciación Celular , Encefalitis , Factores Reguladores del Interferón , Interleucina-10 , Ratones , Uveítis/inmunologíaRESUMEN
STAT3 activates transcription of genes that regulate cell growth, differentiation, and survival of mammalian cells. Genetic deletion of Stat3 in T cells has been shown to abrogate Th17 differentiation, suggesting that STAT3 is a potential therapeutic target for Th17-mediated diseases. However, a major impediment to therapeutic targeting of intracellular proteins such as STAT3 is the lack of efficient methods for delivering STAT3 inhibitors into cells. In this study, we developed a novel antibody (SBT-100) comprised of the variable (V) region of a STAT3-specific heavy chain molecule and demonstrate that this 15 kDa STAT3-specific nanobody enters human and mouse cells, and induced suppression of STAT3 activation and lymphocyte proliferation in a concentration-dependent manner. To investigate whether SBT-100 would be effective in suppressing inflammation in vivo, we induced experimental autoimmune uveitis (EAU) in C57BL/6J mice by active immunization with peptide from the ocular autoantigen, interphotoreceptor retinoid binding protein (IRBP651-670). Analysis of the retina by fundoscopy, histological examination, or optical coherence tomography showed that treatment of the mice with SBT-100 suppressed uveitis by inhibiting expansion of pathogenic Th17 cells that mediate EAU. Electroretinographic (ERG) recordings of dark and light adapted a- and b-waves showed that SBT-100 treatment rescued mice from developing significant visual impairment observed in untreated EAU mice. Adoptive transfer of activated IRBP-specific T cells from untreated EAU mice induced EAU, while EAU was significantly attenuated in mice that received IRBP-specific T cells from SBT-100 treated mice. Taken together, these results demonstrate efficacy of SBT-100 in mice and suggests its therapeutic potential for human autoimmune diseases.
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Enfermedades Autoinmunes/prevención & control , Factor de Transcripción STAT3/inmunología , Células Th17/inmunología , Uveítis/prevención & control , Traslado Adoptivo , Animales , Autoantígenos/inmunología , Autoantígenos/metabolismo , Enfermedades Autoinmunes/inmunología , Proliferación Celular/efectos de los fármacos , Citocinas/metabolismo , Modelos Animales de Enfermedad , Electrorretinografía , Proteínas del Ojo/inmunología , Proteínas del Ojo/metabolismo , Humanos , Ratones , Ratones Endogámicos C57BL , Proteínas de Unión al Retinol/inmunología , Proteínas de Unión al Retinol/metabolismo , Factor de Transcripción STAT3/metabolismo , Células Th17/patología , Uveítis/inmunologíaRESUMEN
Dysregulated Th17 cell responses underlie multiple inflammatory and autoimmune diseases, including autoimmune uveitis and its animal model, EAU. However, clinical trials targeting IL-17A in uveitis were not successful. Here, we report that Th17 cells were regulated by their own signature cytokine, IL-17A. Loss of IL-17A in autopathogenic Th17 cells did not reduce their pathogenicity and instead elevated their expression of the Th17 cytokines GM-CSF and IL-17F. Mechanistic in vitro studies revealed a Th17 cell-intrinsic autocrine loop triggered by binding of IL-17A to its receptor, leading to activation of the transcription factor NF-κB and induction of IL-24, which repressed the Th17 cytokine program. In vivo, IL-24 treatment ameliorated Th17-induced EAU, whereas silencing of IL-24 in Th17 cells enhanced disease. This regulatory pathway also operated in human Th17 cells. Thus, IL-17A limits pathogenicity of Th17 cells by inducing IL-24. These findings may explain the disappointing therapeutic effect of targeting IL-17A in uveitis.
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Citocinas/metabolismo , Interleucina-17/metabolismo , Células Th17/patología , Uveítis/patología , Adulto , Animales , Citocinas/genética , Modelos Animales de Enfermedad , Femenino , Factor Estimulante de Colonias de Granulocitos y Macrófagos/metabolismo , Humanos , Interleucina-17/genética , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , FN-kappa B/metabolismo , Células Th17/inmunología , Uveítis/inmunología , Adulto JovenRESUMEN
IFN-γ and IL-17A can each elicit ocular autoimmunity independently of the other. Since absence of IFN-γ or IL-17A individually failed to abolish pathology of experimental autoimmune uveitis (EAU), we examined EAU development in the absence of both these cytokines. Ifng-/-Il17a-/- mice were fully susceptible to EAU with a characteristic eosinophilic ocular infiltrate, as opposed to a mononuclear infiltrate in WT mice. Retinal pathology in double-deficient mice was ameliorated when eosinophils were genetically absent or their migration was blocked, supporting a pathogenic role for eosinophils in EAU in the concurrent absence of IFN-γ and IL-17A. In EAU-challenged Ifng-/-Il17a-/- mice, ocular infiltrates contained increased GM-CSF-producing CD4+ T cells, and supernatants of retinal antigen-stimulated splenocytes contained enhanced levels of GM-CSF that contributed to activation and migration of eosinophils in vitro. Systemic or local blockade of GM-CSF ameliorated EAU in Ifng-/-Il17a-/- mice, reduced eosinophil peroxidase levels in the eye and in the serum and decreased eosinophil infiltration to the eye. These results support the interpretation that, in the concurrent absence of IFN-γ and IL-17A, GM-CSF takes on a major role as an inflammatory effector cytokine and drives an eosinophil-dominant pathology. Our findings may impact therapeutic strategies aiming to target IFN-γ and IL-17A in autoimmune uveitis.
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Autoinmunidad , Eosinofilia/patología , Factor Estimulante de Colonias de Granulocitos y Macrófagos/metabolismo , Interferón gamma/metabolismo , Interleucina-17/metabolismo , Retinitis/etiología , Retinitis/metabolismo , Animales , Enfermedades Autoinmunes/etiología , Enfermedades Autoinmunes/metabolismo , Enfermedades Autoinmunes/patología , Modelos Animales de Enfermedad , Susceptibilidad a Enfermedades/inmunología , Eosinófilos/inmunología , Eosinófilos/metabolismo , Eosinófilos/patología , Interferón gamma/genética , Interleucina-17/genética , Ratones , Ratones Noqueados , Retinitis/patología , Subgrupos de Linfocitos T/inmunología , Subgrupos de Linfocitos T/metabolismoRESUMEN
Corticosteroids are effective therapy for autoimmune diseases but serious adverse effects preclude their prolonged use. However, immune-suppressive biologics that inhibit lymphoid proliferation are now in use as corticosteroid sparing-agents but with variable success; thus, the need to develop alternative immune-suppressive approaches including cell-based therapies. Efficacy of ex-vivo-generated IL-35-producing regulatory B-cells (i35-Bregs) in suppressing/ameliorating encephalomyelitis or uveitis in mouse models of multiple sclerosis or uveitis, respectively, is therefore a promising therapeutic approach for CNS autoimmune diseases. However, i35-Breg therapy in human uveitis would require producing autologous Bregs from each patient to avoid immune-rejection. Because exosomes exhibit minimal toxicity and immunogenicity, we investigated whether i35-Bregs release exosomes that can be exploited therapeutically. Here, we demonstrate that i35-Bregs release exosomes that contain IL-35 (i35-Exosomes). In this proof-of-concept study, we induced experimental autoimmune uveitis (EAU), monitored EAU progression by fundoscopy, histology, optical coherence tomography and electroretinography, and investigated whether i35-Exosomes treatment would suppress uveitis. Mice treated with i35-Exosomes developed mild EAU with low EAU scores and disease protection correlated with expansion of IL-10 and IL-35 secreting Treg cells with concomitant suppression of Th17 responses. In contrast, significant increase of Th17 cells in vitreous and retina of control mouse eyes was accompanied by severe choroiditis, massive retinal-folds, and photoreceptor cell damage. These hallmark features of severe uveitis were absent in exosome-treated mice and visual impairment detected by ERG was modest compared to control mice. Absence of toxicity or alloreactivity associated with exosomes thus makes i35-Exosomes attractive therapeutic option for delivering IL-35 into CNS tissues.
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Enfermedades Autoinmunes/inmunología , Linfocitos B Reguladores/inmunología , Exosomas/metabolismo , Inmunoterapia Adoptiva/métodos , Interleucinas/metabolismo , Inflamación Neurogénica/inmunología , Uveítis/inmunología , Animales , Enfermedades Autoinmunes/terapia , Linfocitos B Reguladores/trasplante , Células Cultivadas , Modelos Animales de Enfermedad , Humanos , Tolerancia Inmunológica , Inmunomodulación , Interleucina-10/metabolismo , Ratones , Ratones Endogámicos C57BL , Inflamación Neurogénica/terapia , Linfocitos T Reguladores/inmunologíaRESUMEN
The eicosanoid lipoxin A4 (LXA4) has emerging roles in lymphocyte-driven diseases. We identified reduced LXA4 levels in posterior segment uveitis patients and investigated the role of LXA4 in the pathogenesis of experimental autoimmune uveitis (EAU). Immunization for EAU with a retinal self-antigen caused selective downregulation of LXA4 in lymph nodes draining the site of immunization, while at the same time amplifying LXA4 in the inflamed target tissue. T cell effector function, migration and glycolytic responses were amplified in LXA4-deficient mice, which correlated with more severe pathology, whereas LXA4 treatment attenuated disease. In vivo deletion or supplementation of LXA4 identified modulation of CC-chemokine receptor 7 (CCR7) and sphingosine 1- phosphate receptor-1 (S1PR1) expression and glucose metabolism in CD4+ T cells as potential mechanisms for LXA4 regulation of T cell effector function and trafficking. Our results demonstrate the intrinsic lymph node LXA4 pathway as a significant checkpoint in the development and severity of adaptive immunity.
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Autoinmunidad/fisiología , Ojo/inmunología , Lipoxinas/fisiología , Ganglios Linfáticos/fisiología , Retina/inmunología , Animales , Enfermedades Autoinmunes/inmunología , Linfocitos T CD4-Positivos/inmunología , Estudios de Casos y Controles , Homeostasis , Humanos , Lipoxinas/biosíntesis , Lipoxinas/genética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Receptores CCR7/metabolismo , Receptores de Esfingosina-1-Fosfato/metabolismo , Uveítis/inmunologíaRESUMEN
IL-22 has opposing effects in different tissues, from pro-inflammatory (skin, joints) to protective (liver, intestine) but little is known about its effects on neuroinflammation. We examined the effect of IL-22 on retinal tissue by using the model of experimental autoimmune uveitis (EAU) in IL-22-/- mice, as well as by intraocular injections of recombinant IL-22 or anti-IL-22 antibodies in wild type animals. During EAU, IL-22 was produced in the eye by CD4+ eye-infiltrating T cells. EAU-challenged IL-22-/- mice, as well as WT mice treated systemically or intraocularly with anti-IL-22 antibodies during the expression phase of disease, developed exacerbated retinal damage. Furthermore, IL-22-/- mice were more susceptible than WT controls to glutamate-induced neurotoxicity, whereas local IL-22 supplementation was protective, suggesting direct or indirect neuroprotective effects. Mechanistic studies revealed that retinal glial Müller cells express IL-22rα1 in vivo, and in vitro IL-22 enhanced their ability to suppress proliferation of effector T cells. Finally, IL-22 injected into the eye concurrently with IL-1, inhibited the (IL-1-induced) expression of multiple proinflammatory and proapoptotic genes in retinal tissue. These findings suggest that IL-22 can function locally within the retina to reduce inflammatory damage and provide neuroprotection by affecting multiple molecular and cellular pathways.
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Autoinmunidad , Sistema Nervioso Central/inmunología , Sistema Nervioso Central/metabolismo , Susceptibilidad a Enfermedades , Interleucinas/metabolismo , Animales , Enfermedades Autoinmunes/etiología , Enfermedades Autoinmunes/metabolismo , Enfermedades Autoinmunes/patología , Autoinmunidad/genética , Sistema Nervioso Central/patología , Citocinas/metabolismo , Modelos Animales de Enfermedad , Encefalomielitis Autoinmune Experimental/etiología , Encefalomielitis Autoinmune Experimental/metabolismo , Encefalomielitis Autoinmune Experimental/patología , Células Ependimogliales/inmunología , Células Ependimogliales/metabolismo , Células Ependimogliales/patología , Perfilación de la Expresión Génica , Regulación de la Expresión Génica/efectos de los fármacos , Interleucinas/genética , Interleucinas/farmacología , Activación de Linfocitos/inmunología , Ratones , Ratones Noqueados , Enfermedades del Sistema Nervioso/etiología , Enfermedades del Sistema Nervioso/metabolismo , Enfermedades del Sistema Nervioso/patología , Neuroprotección/genética , Índice de Severidad de la Enfermedad , Linfocitos T/inmunología , Linfocitos T/metabolismo , Uveítis/etiología , Uveítis/metabolismo , Uveítis/patología , Interleucina-22RESUMEN
AS101 is an organotellurium compound with multifaceted immunoregulatory properties that is remarkable for its lack of toxicity. We tested the therapeutic effect of AS101 in experimental autoimmune uveitis (EAU), a model for human autoimmune uveitis. Unexpectedly, treatment with AS101 elicited Treg generation in vivo in otherwise unmanipulated mice. Mice immunized for EAU with the retinal antigen IRBP and treated with AS101 developed attenuated disease, as did AS101-treated recipients of retina-specific T cells activated in vitro. In both settings, eye-infiltrating effector T cells were decreased, whereas regulatory T (Treg) cells in the spleen were increased. Mechanistic studies in vitro revealed that AS101 restricted polarization of retina-specific T cells towards Th1 or Th17 lineage by repressing activation of their respective lineage-specific transcription factors and downstream signals. Retina-specific T cells polarized in vitro towards Th1 or Th17 in the presence of AS101 had impaired ability to induce EAU in naïve recipients. Finally, AS101 promoted differentiation of retina-specific T cells to Tregs in vitro independently of TGF-ß. We conclude that AS101 modulates autoimmune T cells by inhibiting acquisition and expression of effector function and by promoting Treg generation, and suggest that AS101 could be useful as a therapeutic approach for autoimmune uveitis.
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Enfermedades Autoinmunes/tratamiento farmacológico , Etilenos/farmacología , Linfocitos T Reguladores/inmunología , Células TH1/inmunología , Células Th17/inmunología , Uveítis/tratamiento farmacológico , Animales , Enfermedades Autoinmunes/genética , Enfermedades Autoinmunes/inmunología , Enfermedades Autoinmunes/patología , Modelos Animales de Enfermedad , Ratones , Ratones Transgénicos , Linfocitos T Reguladores/patología , Células TH1/patología , Células Th17/patología , Uveítis/genética , Uveítis/inmunología , Uveítis/patologíaRESUMEN
During chronic inflammation, tertiary lymphoid tissue (TLT) can form within an inflamed organ, including the CNS. However, little is known about TLT formation in the neuroretina. In a novel spontaneous autoimmune mouse model of uveitis (R161H), we identified well-organized lymphoid aggregates in the retina and examined them for TLT characteristics. Presence of immune cells, tissue-specific markers, and gene expression patterns typically associated with germinal centers and T follicular helper cells were examined using immunohistochemistry and gene analysis of laser capture microdissected retina. Our data revealed the retinal lymphoid structures contained CD4(+) T cells and B cells in well-defined zonal areas that expressed classic germinal center markers, peanut lectin (agglutinin) and GL-7. Gene expression analysis showed upregulation of T follicular helper cell markers, most notably CXCR5 and its ligand CXCL13, and immunohistochemical analysis confirmed CXCR5 expression, typically associated with CD4(+) T follicular helper cells. Highly organized stromal cell networks, a hallmark of organized lymphoid tissue, were also present. Positive staining for phospho-Zap70 in retina-specific T cells indicated CD4(+) T cells were being activated within these lymphoid structures. CD138(+)/B220(+) plasma cells were detected, suggesting the retinal lymphoid aggregates give rise to functional germinal centers, which produce Abs. Interestingly, eyes with lymphoid aggregates exhibited lower inflammatory scores by fundus examination and a slower initial rate of loss of visual function by electroretinography, compared with eyes without these structures. Our findings suggest that the lymphoid aggregates in the retina of R161H mice represent organized TLT, which impact the course of chronic uveitis.
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Enfermedades Autoinmunes/patología , Tejido Linfoide/patología , Uveítis/patología , Animales , Enfermedades Autoinmunes/inmunología , Modelos Animales de Enfermedad , Electrorretinografía , Ensayo de Inmunoadsorción Enzimática , Inmunohistoquímica , Captura por Microdisección con Láser , Tejido Linfoide/inmunología , Ratones , Ratones Transgénicos , Microscopía Confocal , Reacción en Cadena de la Polimerasa , Transcriptoma , Uveítis/inmunología , Visión Ocular/fisiologíaRESUMEN
BACKGROUND: Grafting has been widely practiced for centuries in the propagation and production of many vegetable and fruit species. However, the underlying molecular and genetic mechanisms for how the graft partners interact with each other to produce a successful graft remain largely unknown. We hypothesized that genome-wide mRNA exchanges, which were recently documented in grafted model plant species, are a general phenomenon widely present in grafted plants, including those in vegetable and fruit species, and have specific genotype- and environment-dependent characteristics modulating plant performance. METHODS: Using diagnostic SNPs derived from high throughput genome sequencing, we identified and characterized the patterns of genome-wide mRNA exchanges across graft junctions in grafted grapevines grown in the in vitro and field conditions. RESULTS: We identified more than 3000 genes transporting mRNAs across graft junctions. These genes were involved in diverse biological processes and those involved in basic cellular, biosynthetic, catabolic, and metabolic activities, as well as responses to stress and signal transduction, were highly enriched. Field-grown mature grafts had much fewer genes transmitting mRNAs than the in vitro young grafts (987 vs. 2679). These mobile mRNAs could move directionally or bi-directionally between scions and rootstocks. The mRNA transmission rates of these genes were generally low, with 65% or more having transmission rates lower than 0.01. Furthermore, genotypes, graft combinations and growth environments had impact on the directions of mRNA movement as well as the numbers and species of mRNAs being exchanged. Moreover, we found evidence for the presences of both passive and selective mechanisms underlying long distance mRNA trafficking in grafted grapevines. CONCLUSIONS: We extended the studies of mRNA exchanges in model species to grapevines and demonstrated that genomic-scale mRNA exchange across graft junctions occurred in grapevines in a passive or genotype and environment-dependent manner.
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Raíces de Plantas/genética , Tallos de la Planta/genética , Transporte de ARN , Vitis/genética , Secuencia de Bases , Genotipo , Datos de Secuencia Molecular , Transporte de ARN/genética , ARN Mensajero/genética , ARN Mensajero/metabolismoRESUMEN
IFN-γ is a pathogenic cytokine involved in inflammation. Paradoxically, its deficiency exacerbates experimental autoimmune encephalomyelitis, uveitis, and arthritis. Here, we demonstrate using IFN-γ(-/-) mice repleted with IFN-γ +/+: NK cells that innate production of IFN-γ from NK cells is necessary and sufficient to trigger an endogenous regulatory circuit that limits autoimmunity. After immunization, DCs recruited IFN-γ-producing NK cells to the draining lymph node and interacted with them in a CXCR3-dependent fashion. The interaction caused DCs to produce IL-27, which in turn enhanced IFN-γ production by NK cells, forming a self-amplifying positive feedback loop. IL-10, produced by the interacting cells themselves, was able to limit this process. The NK-DC-dependent IL-27 inhibited development of the adaptive pathogenic IL-17 response and induced IL-10-producing Tr1-like cells, which ameliorated disease in an IL-10-dependent manner. Our data reveal that an early NK-DC interaction controls the adaptive Th17 response and limits tissue-specific autoimmunity through an innate IFN-γ-IL-27 axis.
Asunto(s)
Células Dendríticas/inmunología , Interferón gamma/inmunología , Interleucinas/inmunología , Células Asesinas Naturales/inmunología , Células Th17/inmunología , Uveítis/inmunología , Animales , Enfermedades Autoinmunes/inmunología , Retroalimentación Fisiológica , Interferón gamma/genética , Interferón gamma/metabolismo , Interleucina-10/metabolismo , Interleucinas/metabolismo , Células Asesinas Naturales/metabolismo , Ratones Endogámicos C57BL , Ratones Noqueados , Receptores CXCR3/genética , Receptores CXCR3/inmunología , Receptores CXCR3/metabolismo , Uveítis/patologíaRESUMEN
PURPOSE: Experimental autoimmune uveitis (EAU) induced in mice using the retinal antigen interphotoreceptor retinoid binding protein (IRBP) is an animal model for posterior uveitis in humans. However, EAU induced by native IRBP protein or its widely used epitope amino acid residues 1 to 20 of human IRBP (hIRBP1-20) is inconsistent, often showing low scores and incidence. We found an urgent need to identify a better pathogenic epitope for the C57BL/6 strain. METHODS: Mice were immunized with uveitogenic peptides or with native bovine IRBP. Clinical and histological disease and associated immunological responses were evaluated. Truncated and substituted peptides, as well as bioinformatic analyses, were used to identify critical major histocompatibility complex (MHC)/T cell receptor (TCR) contact residues and the minimal core epitope. RESULTS: The new uveitogenic epitope of IRBP, amino acid residues 651 to 670 of human IRBP (LAQGAYRTAVDLESLASQLT [hIRBP651-670]) is uveitogenic for mice of the H-2b haplotype and elicits EAU with a higher severity and incidence in C57BL/6 mice than the previously characterized hIRBP1-20 epitope. Using truncated and substituted peptides, as well as bioinformatic analysis, we identified the critical contact residues with MHC/TCR and defined the minimal core epitope. This made it possible to design MHC tetramers and use them to detect epitope-specific T cells in the uveitic eye and in lymphoid organs of hIRBP651-670-immunized mice. CONCLUSIONS: Data suggest that hIRBP651-670 is an epitope naturally processed from a conserved region of native IRBP, potentially explaining its relatively high uveitogenicity. This epitope should be useful for basic and preclinical studies of uveitis in the C57BL/6 model and gives access to genetically engineered mice available on this background.
Asunto(s)
Enfermedades Autoinmunes/inmunología , Proteínas del Ojo/inmunología , Antígenos de Histocompatibilidad Clase II/inmunología , Inmunidad Celular , Retinitis/inmunología , Proteínas de Unión al Retinol/inmunología , Linfocitos T/inmunología , Uveítis Posterior/inmunología , Animales , Enfermedades Autoinmunes/metabolismo , Enfermedades Autoinmunes/patología , Bovinos , Células Cultivadas , Modelos Animales de Enfermedad , Epítopos de Linfocito T/inmunología , Proteínas del Ojo/metabolismo , Haplotipos , Antígenos de Histocompatibilidad Clase II/metabolismo , Humanos , Activación de Linfocitos , Ratones , Ratones Endogámicos C57BL , Retinitis/metabolismo , Retinitis/patología , Proteínas de Unión al Retinol/metabolismo , Índice de Severidad de la Enfermedad , Uveítis Posterior/metabolismo , Uveítis Posterior/patologíaRESUMEN
Activated retina-specific T cells that have acquired the ability to break through the blood-retinal barrier are thought to be causally involved in autoimmune uveitis, a major cause of human blindness. It is unclear where these autoreactive T cells first become activated, given that their cognate antigens are sequestered within the immune-privileged eye. We demonstrate in a novel mouse model of spontaneous uveitis that activation of retina-specific T cells is dependent on gut commensal microbiota. Retina-specific T cell activation involved signaling through the autoreactive T cell receptor (TCR) in response to non-cognate antigen in the intestine and was independent of the endogenous retinal autoantigen. Our findings not only have implications for the etiology of human uveitis, but also raise the possibility that activation of autoreactive TCRs by commensal microbes might be a more common trigger of autoimmune diseases than is currently appreciated.
Asunto(s)
Intestinos/inmunología , Microbiota/inmunología , Retina/inmunología , Linfocitos T/inmunología , Uveítis/inmunología , Animales , Antígenos Bacterianos/administración & dosificación , Autoantígenos/inmunología , Autoinmunidad , Barrera Hematorretinal/inmunología , Células Cultivadas , Modelos Animales de Enfermedad , Proteínas del Ojo/genética , Proteínas del Ojo/inmunología , Proteínas del Ojo/metabolismo , Tolerancia Inmunológica , Intestinos/microbiología , Activación de Linfocitos , Ratones Endogámicos , Ratones Noqueados , Receptores de Antígenos de Linfocitos T/metabolismo , Proteínas de Unión al Retinol/genética , Proteínas de Unión al Retinol/inmunología , Proteínas de Unión al Retinol/metabolismo , Uveítis/microbiologíaRESUMEN
Experimental autoimmune uveitis (EAU) induced in mice by immunization with the retinal Ag interphotoreceptor retinoid-binding protein (IRBP) is a model of human autoimmune uveitis. We examined whether T regulatory cells (Tregs) found in uveitic eyes are IRBP specific, functionally suppressive, and play a role in natural resolution of disease and in maintenance of remission. Progressive increase of Foxp3(+) Treg to T effector cell (Teff) ratio in uveitic eyes correlated with resolution of disease. At peak disease, up to 20% of Tregs (CD4(+)Foxp3(+)) and up to 60% of Teffs (CD4(+)Foxp3(-)) were IRBP specific, whereas in lymphoid organs retina-specific T cells were undetectable. Tregs isolated from eyes of mice with EAU efficiently suppressed IRBP-specific responses of Teffs from the same eyes. Importantly, systemic depletion of Tregs at peak disease delayed resolution of EAU, and their depletion after resolution triggered a relapse. This could be partially duplicated by depletion of Tregs locally within the eye. Thus, the T cell infiltrate in uveitic eyes of normal mice with a polyclonal T cell repertoire is highly enriched in IRBP-specific Tregs and Teffs. Unlike what has been reported for Tregs in other inflammatory sites, Tregs from uveitic eyes appear unimpaired functionally. Finally, Foxp3(+) Tregs play a role in the natural resolution of uveitis and in the maintenance of remission, which occurs at least in part through an effect that is local to the eye.