RESUMEN
We specify the clinical features of a spontaneous experimental autoimmune uveitis (EAU) model, in which foreign hen-egg lysozyme (HEL) is expressed in the retina, controlled by the promoter for interphotoreceptor retinol binding protein (IRBP). We previously reported 100% P21 (post-partum day) IRBP:HEL single transgenic (sTg) mice, when crossed to transgenic T cell receptor mice (3A9) generating the double transgenic (dTg) genotype, develop EAU despite profound lymphopenia (thymic HEL-specific T cell deletion). In this work, we characterized the immune component of this model and found conventional dTg CD4+ T cells were less anergic than those from 3A9 controls. Furthermore, prior in vitro HEL-activation of 3A9 anergic T cells (Tan) rendered them uveitogenic upon adoptive transfer (Tx) to sTg mice, while antigen-experienced (AgX, dTg), but not naïve (3A9) T cells halted disease in P21 dTg mice. Flow cytometric analysis of the AgX cells elucidated the underlying pathology: FoxP3+CD25hiCD4+ T regulatory cells (Treg) comprised â¼18%, while FR4+CD73+FoxP3-CD25lo/-CD4+ Tan comprised â¼1.2% of total cells. Further Treg-enrichment (â¼80%) of the AgX population indicated FoxP3+CD25hiCD4+ Treg played a key role in EAU-suppression while FoxP3-CD25lo/-CD4+ T cells did not. Here we present the novel concept of dual immunological tolerance where spontaneous EAU is due to escape from anergy with consequent failure of Treg induction and subsequent imbalance in the [Treg:Teffector] cell ratio. The reduced numbers of Tan, normally sustaining Treg to prevent autoimmunity, are the trigger for disease, while immune homeostasis can be restored by supplementation with AgX, but not naïve, antigen-specific Treg.
Asunto(s)
Enfermedades Autoinmunes/inmunología , Inmunoterapia Adoptiva/métodos , Retina/patología , Linfocitos T Reguladores/inmunología , Uveítis/inmunología , Animales , Células Cultivadas , Modelos Animales de Enfermedad , Proteínas del Ojo/inmunología , Factores de Transcripción Forkhead/metabolismo , Humanos , Tolerancia Inmunológica , Subunidad alfa del Receptor de Interleucina-2/metabolismo , Ratones , Ratones Transgénicos , Proteínas de Unión al Retinol/inmunología , Linfocitos T Reguladores/trasplanteRESUMEN
Interphotoreceptor retinoid-binding protein (IRBP) is a highly expressed protein secreted by rod and cone photoreceptors that has major roles in photoreceptor homeostasis as well as retinoid and polyunsaturated fatty acid transport between the neural retina and retinal pigment epithelium. Despite two crystal structures reported on fragments of IRBP and decades of research, the overall structure of IRBP and function within the visual cycle remain unsolved. Here, we studied the structure of native bovine IRBP in complex with a monoclonal antibody (mAb5) by cryo-electron microscopy, revealing the tertiary and quaternary structure at sufficient resolution to clearly identify the complex components. Complementary mass spectrometry experiments revealed the structure and locations of N-linked carbohydrate post-translational modifications. This work provides insight into the structure of IRBP, displaying an elongated, flexible three-dimensional architecture not seen among other retinoid-binding proteins. This work is the first step in elucidation of the function of this enigmatic protein.
Asunto(s)
Proteínas del Ojo/química , Proteínas de Unión al Retinol/química , Animales , Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/inmunología , Complejo Antígeno-Anticuerpo/química , Bovinos , Microscopía por Crioelectrón , Proteínas del Ojo/inmunología , Femenino , Ratones , Ratones Endogámicos C57BL , Proteínas de Unión al Retinol/inmunología , Imagen Individual de MoléculaRESUMEN
Purpose: Previous studies have shown that parental abnormal physiological conditions such as inflammation, stress, and obesity can be transferred to offspring. The purpose of this study was to investigate the impact of parental uveitis on the development and susceptibility to experimental autoimmune uveitis (EAU) in offspring. Methods: Parental male and female B10RIII mice were immunized with interphotoreceptor retinoid binding protein (IRBP) 161-180 in complete Freund's adjuvant and were immediately allowed to mate. Gross examination of the offspring gestated with EAU was performed to determine the influence of parental uveitis on offspring development after birth. Gene expression profiles were analyzed in the affected eyes of offspring under EAU to identify differentially expressed genes (DEGs). Adult offspring were given 5, 25, and 50 µg IRBP161-180 to compare their susceptibility to EAU. Immunized mice were clinically and pathologically evaluated for the development of EAU. Ag-specific T-cell proliferation and IL-17 production from spleens and lymph nodes were evaluated on day 14 or 35 after immunization. Results: Hair loss, delay of eye opening, and swollen spleens in the offspring from parents with uveitis were observed from day 14 to 39 after birth. DEGs were involved in the immune system process, muscle system process, and cell development. The altered antigen processing and presentation, cell adhesion molecules, and phagosome in the eyes of the offspring from uveitis-affected parents were enriched. Offspring gestated with EAU showed a susceptibility to EAU and an earlier onset and higher severity of EAU compared to the control group mice. IRBP-specific lymphocyte proliferation and IL-17 production were observed in the EAU offspring with exposure to parental uveitis. Conclusions: The results suggest that mouse parents with uveitis can increase their offspring's susceptibility to EAU, probably through altering cell adhesion molecules and antigen processing and presentation related to the T-cell proliferation and Th17 response.
Asunto(s)
Enfermedades Autoinmunes/etiología , Uveítis/etiología , Animales , Autoantígenos/inmunología , Enfermedades Autoinmunes/genética , Enfermedades Autoinmunes/inmunología , Proliferación Celular , Modelos Animales de Enfermedad , Susceptibilidad a Enfermedades , Proteínas del Ojo/inmunología , Femenino , Perfilación de la Expresión Génica , Inmunización , Masculino , Herencia Materna/genética , Herencia Materna/inmunología , Intercambio Materno-Fetal/genética , Intercambio Materno-Fetal/inmunología , Ratones , Herencia Paterna/genética , Herencia Paterna/inmunología , Fragmentos de Péptidos/inmunología , Embarazo , Proteínas de Unión al Retinol/inmunología , Linfocitos T/inmunología , Linfocitos T/patología , Células Th17/inmunología , Uveítis/genética , Uveítis/inmunologíaRESUMEN
We investigated the effects of matairesinol (MAT) in the experimental autoimmune uveitis (EAU), a classical animal model of uveitis. We found that treatment with MAT could alleviate intraocular inflammation of EAU. Notably, Th17 cells in eyes of EAU mice could be predominantly restrained by MAT. Furthermore, MAT could inhibit Th17 differentiation in vitro. In addition, MAT inhibited the signaling of MAPK and ROR-γt, a pivotal transcription factor for Th17 cell differentiation in vitro and in vivo. Taken together, these results suggested that MAT had immune-suppressive effects on autoimmune inflammation through Th17 cells.
Asunto(s)
Enfermedades Autoinmunes/tratamiento farmacológico , Proteínas del Ojo/antagonistas & inhibidores , Furanos/uso terapéutico , Lignanos/uso terapéutico , Proteínas de Unión al Retinol/antagonistas & inhibidores , Células Th17/efectos de los fármacos , Uveítis/tratamiento farmacológico , Animales , Enfermedades Autoinmunes/inmunología , Enfermedades Autoinmunes/metabolismo , Proteínas del Ojo/inmunología , Proteínas del Ojo/metabolismo , Femenino , Adyuvante de Freund/toxicidad , Furanos/farmacología , Lignanos/farmacología , Ratones , Ratones Endogámicos C57BL , Proteínas de Unión al Retinol/inmunología , Proteínas de Unión al Retinol/metabolismo , Células Th17/inmunología , Células Th17/metabolismo , Uveítis/inmunología , Uveítis/metabolismoRESUMEN
BACKGROUND: Berberine (BBR) was reported to have immunoregulatory and anti-inflammatory properties. In this study, we investigated whether BBR could exert its effects on the development of experimental autoimmune uveitis (EAU), and if so, what was the underlying mechanism? METHODS: EAU was induced in B10R.III mice by immunization with IRBP 161-180, followed by 100 mg/kg/d BBR intragastric administration. Disease severity was assessed by evaluation of clinical and histopathological scores. Blood-retinal barrier (BRB) breakdown was tested by Evans blue. Effector and regulatory T (Treg) cell balance was evaluated by quantitative real-time PCR and flow cytometry. Spleen transcriptome was characterized by RNA sequencing (RNA-seq). Gut microbiota composition was investigated by 16S rRNA analysis. RESULTS: BBR treatment significantly blocked EAU as shown by the decrease of the clinical and histological scores, as well as the inhibition of BRB breakdown. The frequency of splenic Th1 and Th17 cells was decreased, whereas Treg cells were increased in the BBR-treated group. RNA-seq of the spleen revealed 476 differentially expressed genes (DEGs) between the EAU and EAU-BBR group. GO functional classification, as well as KEGG analysis demonstrated that BBR treatment markedly influences genes belonging to chromatin remodeling and immune-related pathways. Intervention with BBR modified the gut microbiome in EAU mice, increasing the number of bacteria with immunomodulatory capacity. Depletion of gut microbiota affected the efficacy of BBR on EAU. Moreover, the altered bacterial strains showed a significant correlation with the expression of histones. CONCLUSIONS: BBR inhibited IRBP induced EAU, which was associated with a significant change in the spleen transcriptome and intestinal microbial composition.
Asunto(s)
Antiinflamatorios/uso terapéutico , Enfermedades Autoinmunes/tratamiento farmacológico , Berberina/uso terapéutico , Microbioma Gastrointestinal/genética , ARN Ribosómico 16S/genética , Bazo/efectos de los fármacos , Células TH1/inmunología , Células Th17/inmunología , Uveítis/tratamiento farmacológico , Animales , Proteínas del Ojo/inmunología , Microbioma Gastrointestinal/efectos de los fármacos , Humanos , Ratones , Ratones Endogámicos , Modelos Animales , Proteínas de Unión al Retinol/inmunología , Análisis de Secuencia de ARN , Bazo/fisiología , TranscriptomaRESUMEN
Parasitic nematodes produce an unusual class of fatty acid and retinol (FAR)-binding proteins that may scavenge host fatty acids and retinoids. Two FARs from Brugia malayi (Bm-FAR-1 and Bm-FAR-2) were expressed as recombinant proteins, and their ligand binding, structural characteristics, and immunogenicities examined. Circular dichroism showed that rBm-FAR-1 and rBm-FAR-2 are similarly rich in α-helix structure. Unexpectedly, however, their lipid binding activities were found to be readily differentiated. Both FARs bound retinol and cis-parinaric acid similarly, but, while rBm-FAR-1 induced a dramatic increase in fluorescence emission and blue shift in peak emission by the fluorophore-tagged fatty acid (dansyl-undecanoic acid), rBm-FAR-2 did not. Recombinant forms of the related proteins from Onchocerca volvulus, rOv-FAR-1 and rOv-FAR-2, were found to be similarly distinguishable. This is the first FAR-2 protein from parasitic nematodes that is being characterized. The relative protein abundance of Bm-FAR-1 was higher than Bm-FAR-2 in the lysates of different developmental stages of B. malayi. Both FAR proteins were targets of strong IgG1, IgG3 and IgE antibody in infected individuals and individuals who were classified as endemic normal or putatively immune. In a B. malayi infection model in gerbils, immunization with rBm-FAR-1 and rBm-FAR-2 formulated in a water-in-oil-emulsion (®Montanide-720) or alum elicited high titers of antigen-specific IgG, but only gerbils immunized with rBm-FAR-1 formulated with the former produced a statistically significant reduction in adult worms (68%) following challenge with B. malayi infective larvae. These results suggest that FAR proteins may play important roles in the survival of filarial nematodes in the host, and represent potential candidates for vaccine development against lymphatic filariasis and related filarial infections.
Asunto(s)
Antígenos Helmínticos/inmunología , Brugia Malayi/inmunología , Proteínas de Unión a Ácidos Grasos/inmunología , Filariasis/prevención & control , Proteínas de Unión al Retinol/inmunología , Vacunas Sintéticas/inmunología , Adyuvantes Inmunológicos/administración & dosificación , Animales , Anticuerpos Antihelmínticos/sangre , Antígenos Helmínticos/química , Dicroismo Circular , Modelos Animales de Enfermedad , Proteínas de Unión a Ácidos Grasos/química , Femenino , Gerbillinae , Humanos , Inmunoglobulina E/sangre , Inmunoglobulina G/sangre , Masculino , Carga de Parásitos , Unión Proteica , Estructura Secundaria de Proteína , Proteínas de Unión al Retinol/química , Resultado del Tratamiento , Vacunas Sintéticas/administración & dosificación , Vacunas Sintéticas/aislamiento & purificación , Vitamina A/metabolismoRESUMEN
The purpose of the study was to investigate the anti-inflammatory efficiency of vorinostat, a histone deacetylase inhibitor, in experimental autoimmune uveitis (EAU). EAU was induced in female C57BL/6J mice immunized with interphotoreceptor retinoid-binding protein peptide. Vorinostat or the control treatment, phosphate-buffered saline, was administrated orally from 3 days before immunization until euthanasia at day 21 after immunization. The clinical and histopathological scores of mice were graded, and the integrity of the blood-retinal barrier was examined by Evans blue staining. T helper cell subsets were measured by flow cytometry, and the macrophage functions were evaluated with immunohistochemistry staining and immunofluorescence assays. The mRNA levels of tight junction proteins were measured by qRT-PCR. The expression levels of intraocular cytokines and transcription factors were examined by western blotting. Vorinostat relieved both clinical and histopathological manifestations of EAU in our mouse model, and the BRB integrity was maintained in vorinostat-treated mice, which had less vasculature leakage and higher mRNA and protein expressions of tight junction proteins than controls. Moreover, vorinostat repressed Th1 and Th17 cells and increased Th0 and Treg cells. Additionally, the INF-γ and IL-17A expression levels were significantly decreased, while the IL-10 level was increased by vorinostat treatment. Furthermore, due to the reduced TNF-α level, the macrophage activity was considerably inhibited in EAU mice. Finally, transcription factors, including STAT1, STAT3, and p65, were greatly suppressed by vorinostat treatment. Our data suggest that vorinostat might be a potential anti-inflammatory agent in the management of uveitis and other autoimmune inflammatory diseases.
Asunto(s)
Enfermedades Autoinmunes/tratamiento farmacológico , Inhibidores de Histona Desacetilasas/uso terapéutico , Ácidos Hidroxámicos/uso terapéutico , Activación de Macrófagos/efectos de los fármacos , Retinitis/tratamiento farmacológico , Subgrupos de Linfocitos T/efectos de los fármacos , Uveítis/tratamiento farmacológico , Animales , Autoantígenos/inmunología , Enfermedades Autoinmunes/inmunología , Barrera Hematorretinal , Citocinas/biosíntesis , Citocinas/genética , Evaluación Preclínica de Medicamentos , Proteínas del Ojo/inmunología , Femenino , Regulación de la Expresión Génica/efectos de los fármacos , Inhibidores de Histona Desacetilasas/farmacología , Ácidos Hidroxámicos/farmacología , Ratones , Ratones Endogámicos C57BL , ARN Mensajero/biosíntesis , Retinitis/inmunología , Proteínas de Unión al Retinol/inmunología , Organismos Libres de Patógenos Específicos , Subgrupos de Linfocitos T/inmunología , Uniones Estrechas/metabolismo , Factores de Transcripción/biosíntesis , Factores de Transcripción/genética , Factor de Necrosis Tumoral alfa/análisis , Uveítis/inmunología , VorinostatRESUMEN
AGEs are permanently modified macromolecule derivatives that form through nonenzymatic glycation of amino groups of proteins. Glycer-AGEs are highly toxic and play an important role in the pathogenesis of chronic inflammatory diseases. However, the contribution of glycer-AGEs to the pathogenesis of uveitis is unclear. In this study, we measured serum levels of glycer-AGEs in 100 patients with endogenous uveitis (22 with HLA-B27-associated uveitis, 20 with VKH disease, 14 with Behçet's disease, and 44 with sarcoidosis) and 33 healthy volunteers. We then examined the effect of the AGE inhibitor in a mouse model of human endogenous uveitis (EAU) by continuous oral administration of pyridoxamine at 200 or 400 mg/kg/day. Regardless of the etiology, serum glycer-AGE levels were significantly higher in patients with uveitis than in healthy subjects. Treatment with 400 mg/kg pyridoxamine significantly reduced the clinical and histological severity of EAU and was accompanied by a significant decrease in serum and retinal glycer-AGE levels and suppression of translocation of NF-κB p65 into the nucleus of retinal cells. Serum glycer-AGE levels may therefore serve as a biomarker of human uveitis, as well as systemic inflammation, and may contribute to the progression of uveitis, including diabetic iritis, via the activation of NF-κB.
Asunto(s)
Enfermedades Autoinmunes/tratamiento farmacológico , Productos Finales de Glicación Avanzada/antagonistas & inhibidores , Piridoxamina/uso terapéutico , Retinitis/tratamiento farmacológico , Uveítis/tratamiento farmacológico , Administración Oral , Adulto , Secuencia de Aminoácidos , Animales , Enfermedades Autoinmunes/sangre , Enfermedades Autoinmunes/patología , Síndrome de Behçet/sangre , Síndrome de Behçet/complicaciones , Modelos Animales de Enfermedad , Evaluación Preclínica de Medicamentos , Proteínas del Ojo/inmunología , Proteínas del Ojo/metabolismo , Proteínas del Ojo/toxicidad , Femenino , Antígeno HLA-B27/inmunología , Humanos , Masculino , Ratones , Persona de Mediana Edad , Datos de Secuencia Molecular , Fragmentos de Péptidos/inmunología , Fragmentos de Péptidos/toxicidad , Transporte de Proteínas/efectos de los fármacos , Piridoxamina/administración & dosificación , Piridoxamina/farmacología , Retina/metabolismo , Retinitis/sangre , Retinitis/etiología , Retinitis/patología , Proteínas de Unión al Retinol/inmunología , Proteínas de Unión al Retinol/toxicidad , Sarcoidosis/sangre , Sarcoidosis/complicaciones , Uveítis/sangre , Uveítis/etiología , Uveítis/patología , Síndrome Uveomeningoencefálico/sangre , Síndrome Uveomeningoencefálico/complicacionesRESUMEN
OBJECTIVES: To explore the effects of exogenous interleukin (IL)-2 and IL-23 on differentiation of IRBP 1-20-specific T cells originated from C57BL/6 mouse with experimental autoimmune uveitis (EAU) and to investigate the difference in pathogenicity. METHODS: Experimental study. Thirty C57BL/6 mice were immunized subcutaneously with 200 µl emulsion containing 200 µg interphotoreceptor retinoid-binding protein (IRBP) 1-20 and 0.8 mg mycobacterium tuberculosis in complete Freund's adjuvant, distributed over six spots at the tail base and on the flank. On postimmunization Day 13, spleens and draining lymph nodes were removed from mice, and a part of spleens, as the control group, was reserved for examining the expression of IFN-γ mRNA and IL-17 mRNA by RT-PCR. T cells were isolated from the rest of spleens and lymph nodes by passage through a nylon wool column, and then T cells were stimulated for 48 hours with IRBP 1-20 in the presence of antigen-presenting cell and mouse recombinant cytokine IL-2 or IL-23. The IRBP 1-20-specific T cells were separated by Ficoll gradient centrifugation, the expressions of IFN-γ mRNA and IL-17 mRNA were assessed by RT-PCR, and IFN-γ and IL-17 in T cells supernatant were detected using a commercial ELISA kit. The T cells were cultured for another 48 hours, and then the proportions of IL-17(+)γδ(+)T cells were analyzed by flow cytometry. EAU models were built in 30 C57BL/6 mice, T cells from which were randomly divided into two groups according to the methods mentioned above: IL-2 group and IL-23 group. Transfer EAU models were built in other 6 mice and divided into two groups, IL-2 group and IL-23 group, by intraperitoneal injection of 3.5×10(6) IRBP-special-T cells from IL-2 group or IL-23 group respectively. Clinical changes were observed by indirect ophthalmoscopy on postimmunization day 3, 7, 14. For histopathological evaluation, whole eyes were collected on postimmunization day 21. Rank sum test, one-way ANOVA and paired t test were used to analyze the results. A comparison of pathogenicity was made between Th1 and Th17 through clinical observation and histopathological evaluation of B6 mouse. RESULTS: Rosette formation was found among T cells on poststimulation day 2. There was a statistical difference in the expression of IFN-γ mRNA between the IL-2 group and normal group or IL-23 group (0.26 ± 0.02 vs. 0.12 ± 0.05 or 0.10 ± 0.00) (F = 80.51, P = 0.003); however, the expression of IFN-γ in the IL-2 group [(13 124.67 ± 107.73) µg/L] was higher than that of the IL-23 group [(3953.67 ± 117.34) µg/L] (t = 169.61, P = 0.000); and the expression of IL-17 mRNA in the IL-2 group [(588.67 ± 77.43) µg/L] was lower than that of the IL-23 group [(5038.33 ± 88.00) µg/L] (t = -361.71, P = 0.000). Flow cytometer showed that the concentration of IL-17 in the IL-2 group [(3.23 ± 0.28)%] was significantly lower than that in the IL-23 group [(9.93 ± 0.55)%] (t = -33.18, P = 0.001). There was a significant difference in the proportion of IL-17(+)γδ(+)T cells between the IL-23 group and IL-2 group (9.93 ± 0.55 vs. 3.23 ± 0.28) (t = -33.18, P = 0.001). Inflammatory response could not be detected in either group three days after injection of IRBP 1-20-specific T cells. Both groups of mice had the most severe inflammatory response on the 14 th day, of which that of the IL-23 group was significantly more severe. CONCLUSIONS: IL-23 facilitates IRBP 1-20-specific T cells to differentiate into Th17, whereas IL-2 inhibits this process and induces these cells to differentiate towards Th1. Further studies showed that the pathogenicity of Th17 cells was significantly higher than that of Th1 cells.
Asunto(s)
Enfermedades Autoinmunes/patología , Interleucina-23/efectos adversos , Interleucina-2/efectos adversos , Subgrupos de Linfocitos T/citología , Uveítis/patología , Animales , Diferenciación Celular , Modelos Animales de Enfermedad , Proteínas del Ojo/inmunología , Femenino , Inflamación , Interferón gamma/inmunología , Interleucina-17/inmunología , Ratones , Ratones Endogámicos C57BL , Proteínas de Unión al Retinol/inmunología , Subgrupos de Linfocitos T/efectos de los fármacos , Células TH1/inmunología , Células Th17/inmunologíaRESUMEN
Despite presence of circulating retina-specific T cells in healthy individuals, ocular immune privilege usually averts development of autoimmune uveitis. To study the breakdown of immune privilege and development of disease, we generated transgenic (Tg) mice that express a T cell receptor (TCR) specific for interphotoreceptor retinoid-binding protein (IRBP), which serves as an autoimmune target in uveitis induced by immunization. Three lines of TCR Tg mice, with different levels of expression of the transgenic R161 TCR and different proportions of IRBP-specific CD4⺠T cells in their peripheral repertoire, were successfully established. Importantly, two of the lines rapidly developed spontaneous uveitis, reaching 100% incidence by 2 and 3 months of age, respectively, whereas the third appeared "poised" and only developed appreciable disease upon immune perturbation. Susceptibility roughly paralleled expression of the R161 TCR. In all three lines, peripheral CD4⺠T cells displayed a naïve phenotype, but proliferated in vitro in response to IRBP and elicited uveitis upon adoptive transfer. In contrast, CD4⺠T cells infiltrating uveitic eyes mostly showed an effector/memory phenotype, and included Th1, Th17 as well as T regulatory cells that appeared to have been peripherally converted from conventional CD4⺠T cells rather than thymically derived. Thus, R161 mice provide a new and valuable model of spontaneous autoimmune disease that circumvents the limitations of active immunization and adjuvants, and allows to study basic mechanisms involved in maintenance and breakdown of immune homeostasis affecting immunologically privileged sites such as the eye.
Asunto(s)
Autoantígenos/inmunología , Autoinmunidad/inmunología , Receptores de Antígenos de Linfocitos T/inmunología , Retina/inmunología , Animales , Enfermedades Autoinmunes/inmunología , Linfocitos T CD4-Positivos/inmunología , Citocinas/inmunología , Proteínas del Ojo/inmunología , Humanos , Memoria Inmunológica/inmunología , Ratones , Ratones Transgénicos , Receptores de Antígenos de Linfocitos T/biosíntesis , Proteínas de Unión al Retinol/inmunología , Linfocitos T Reguladores/inmunología , Células TH1/inmunología , Células Th17/inmunología , Uveítis/inmunologíaRESUMEN
INTRODUCTION: Autoimmune uveitis is a group of HLA-associated inflammatory diseases of the eye, prevalent worldwide, that may cause blindness. It can be limited to the eye, or associated with a systemic syndrome. Furthermore, patients suffering from uveitis exhibit high serum and local nitric oxide (NO) levels as a consequence of cellular responses to immunologically privileged antigens within the eye such as interphotoreceptor retinoid binding protein (IRBP). To investigate NO production kinetics in autoimmune uveitis and its implication in mechanisms of ocular pathogenesis, we first attempted to develop an experimental model of autoimmune uveitis (EAU) on the Wistar rat, using the whole bovine retinal interphotoreceptor matrix extract (IPMe) and isolated IRBP. MATERIAL AND METHODS: Female Wistar rats (n=24) were divided into three experimental groups: "control rats" (n=3) consisting of non-immunized animals, "IRBP-immunized rats" (n=12) and "IPMe-immunized rats" (n=9), which received a subcutaneous injection, respectively, of 13 µg IRBP and 100 µg IPMe emulsified in complete Freund's adjuvant. On days 7, 14 and 21 post immunization, the rats were sacrificed. Nitrites were assessed in plasma and in homogenate of eyes using the Griess reaction. Meanwhile, eyes were collected for histological studies. RESULTS: Our results show the sensitivity of the Wistar strain to both IPMe and IRBP-induced EAU. In fact, we observed histological disorders affecting the retinal tissue in both models of EAU. On the other hand, a significantly increased production of NO in plasma and homogenate of eyes was also observed in comparison to the control group. Moreover, we noted with interest that maximal production of NO occurs prior to the alteration of retinal tissue. CONCLUSION: In summary, our results suggest the early involvement of NO in the mechanisms of pathogenesis of EAU. NO can be considered as a key bio-marker of poor prognosis in ocular autoimmune inflammation.
Asunto(s)
Enfermedades Autoinmunes/etiología , Óxido Nítrico/efectos adversos , Óxido Nítrico/fisiología , Proteínas de Unión al Retinol/fisiología , Uveítis/etiología , Animales , Enfermedades Autoinmunes/inducido químicamente , Enfermedades Autoinmunes/patología , Autoinmunidad/fisiología , Bovinos , Células Cultivadas , Modelos Animales de Enfermedad , Femenino , Inmunización , Óxido Nítrico/farmacología , Ratas , Ratas Wistar , Retina/química , Proteínas de Unión al Retinol/inmunología , Transducción de Señal/efectos de los fármacos , Factores de Tiempo , Extractos de Tejidos , Uveítis/inducido químicamente , Uveítis/patologíaRESUMEN
PURPOSE: Posttranslational modification of proteins plays an important role in cellular functions and is a key event in signal transduction pathways leading to oxidative stress and DNA damage. In this study, we used matrix-assisted laser desorption/ionization- time of flight (MALDI-TOF) to investigate the posttranslational modifications of the differentially expressed proteins in the retinal mitochondria during early experimental autoimmune uveitis (EAU). METHODS: EAU was induced in 18 B10RIII mice with 25 µg of inter-photoreceptor retinoid-binding protein (IRBP) emulsified with complete Freund's adjuvant (CFA); 18 mice treated with CFA without IRBP served as controls. Retinas were removed from the experimental and control groups on day 7 post immunization; mitochondrial fractions were extracted and subjected to 2 dimentional-difference in gel electrophoresis (2D-DIGE); and the protein spots indicating differential expression were subjected to MALDI-TOF for protein identification and indication of any posttranslational modifications. RESULTS: Of the 13 proteins found to be differentially expressed by 2D-DIGE (including upregulated aconitase, mitochondrial heat shock protein (mtHsp) 70, lamin-1, syntaxin-binding protein, αA crystallin, ßB2 crystallin, along with downregulated guanine nucleotide-binding protein and ATP synthase) nine were found to undergo posttranslational modification. Oxidation was a common modification found to occur on aconitase, mtHsp 70, ATP synthase, lamin-1, ßB2-crystallin, guanine nucleotide-binding protein, and manganese superoxide dismutase (MnSOD). In addition, aconitase hydratase, mtHsp 70, guanine nucleotide-binding protein, ATP synthase, syntaxin-binding protein, ßB2-crystallin, and lamin-1 were also modified by carbamidomethylation. αA-crystallin had a pyro-glu modification. CONCLUSIONS: Several proteins present in the retinal mitochondria are posttranslationally modified during early EAU, indicating the presence of oxidative stress and mitochondrial DNA damage. The most common modifications are oxidation and carbamidomethylation. A better understanding of the proteins susceptible to posttranslational modifications in the mitochondria at the early stage of the disease may serve to advance therapeutic interventions to attenuate disease progression.
Asunto(s)
Enfermedades Autoinmunes/genética , Proteínas del Ojo/inmunología , Mitocondrias/genética , Proteínas Mitocondriales/genética , Péptidos/inmunología , Procesamiento Proteico-Postraduccional , Retina/metabolismo , Proteínas de Unión al Retinol/inmunología , Uveítis/genética , Secuencia de Aminoácidos , Animales , Enfermedades Autoinmunes/inducido químicamente , Enfermedades Autoinmunes/inmunología , Enfermedades Autoinmunes/metabolismo , Enfermedades Autoinmunes/patología , Modelos Animales de Enfermedad , Proteínas del Ojo/administración & dosificación , Proteínas del Ojo/efectos adversos , Adyuvante de Freund/administración & dosificación , Expresión Génica , Perfilación de la Expresión Génica , Humanos , Ratones , Ratones Endogámicos , Mitocondrias/química , Mitocondrias/inmunología , Mitocondrias/metabolismo , Proteínas Mitocondriales/inmunología , Proteínas Mitocondriales/metabolismo , Datos de Secuencia Molecular , Estrés Oxidativo , Péptidos/administración & dosificación , Péptidos/efectos adversos , Procesamiento Proteico-Postraduccional/genética , Procesamiento Proteico-Postraduccional/inmunología , Retina/inmunología , Retina/patología , Proteínas de Unión al Retinol/administración & dosificación , Proteínas de Unión al Retinol/efectos adversos , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Electroforesis Bidimensional Diferencial en Gel , Uveítis/inducido químicamente , Uveítis/inmunología , Uveítis/metabolismo , Uveítis/patologíaRESUMEN
PURPOSE: To investigate the role of Foxp3-positive regulatory T cells in the development of experimental autoimmune uveoretinitis (EAU). METHODS: B10RIII mice were immunized with 50 microg IRBP(161-180) in complete Freund's adjuvant (CFA) to induce EAU. EAU was evaluated clinically and pathologically on days 0, 7, 14, 21, and 28. Foxp3 mRNA levels were detected using reverse transcription-PCR (RT-PCR) and the frequencies of CD4(+)Foxp3(+) T cells and CD4(+)CD25(+)Foxp3(+) T cells in splenocytes were assessed by flow cytometry at the aforementioned time points. RESULTS: The first clinical signs of EAU were observed on day 8-9, worsened up to day 14, and then gradually resolved. Histopathologic results showed that inflammatory signs occurred on day 7, reached their peak on day 14, and then gradually decreased. The levels of Foxp3 mRNA and the frequencies of CD4(+)Foxp3(+) T cells and CD4(+)CD25(+)Foxp3(+) T cells in splenocytes increased on day 7, reached a peak on day 14, and then maintained at a high level until day 28. CONCLUSION: An upregulation of Foxp3 expression is induced in EAU and paralleled with disease activity, suggesting a role for this lymphocyte subpopulation in the regression of this experimental uveitis model.
Asunto(s)
Enfermedades Autoinmunes/inmunología , Retinitis/inmunología , Bazo/inmunología , Linfocitos T Reguladores/inmunología , Uveítis/inmunología , Animales , Modelos Animales de Enfermedad , Factores de Transcripción Forkhead/inmunología , Subunidad alfa del Receptor de Interleucina-2/inmunología , Ratones , Fragmentos de Péptidos/inmunología , Proteínas de Unión al Retinol/inmunologíaRESUMEN
PURPOSE: Induction of tissue-specific experimental autoimmune diseases involves the use of complete Freund adjuvant containing Mycobacterium tuberculosis, whose recognition by the innate immune system depends on Toll-like receptors (TLRs) that signal through the adaptor molecule MyD88. The authors' previous study showed that MyD88(-/-) mice, but not TLR2(-/-), TLR4(-/-), or TLR9(-/-) mice, were resistant to experimental autoimmune uveitis (EAU). METHODS: The EAU induction in mice deficient in TLR3 or mice double deficient in TLR2+4, TLR2+9, and TLR4+9 was examined and the role of the TLR agonists in the adjuvant effect involved in the induction of EAU was assessed. RESULTS: TLR3-deficient and TLR2+4, TLR2+9, and TLR4+9 double-deficient mice were as susceptible to EAU as their control littermates. However, in mice immunized with a low-dose EAU regimen, TLR4 agonist lipopolysaccharide (LPS) enhanced EAU scores, delayed-type hypersensitivity responses, and antigen-specific T-cell proliferation. Antigen-specific IL-17 and IFN-gamma production by T lymphocytes was markedly increased in the LPS-treated group. The effects of LPS on EAU were abolished by treatment with an LPS deactivator polymyxin B. Inclusion of agonists for TLR2, TRL3, or TRL9 in immunization also enhanced EAU scores. CONCLUSIONS: These results suggest that signaling of TLR2, TRL3, TRL4, and TRL9 is highly redundant in the adjuvant effect needed to induce EAU and that diverse microbial infections may contribute to the pathogenesis of diseases such as uveitis.
Asunto(s)
Enfermedades Autoinmunes/inmunología , Autoinmunidad/inmunología , Modelos Animales de Enfermedad , Factor 88 de Diferenciación Mieloide/fisiología , Receptores Toll-Like/fisiología , Uveítis/inmunología , Animales , Autoantígenos/inmunología , Proteínas del Ojo/inmunología , Femenino , Silenciador del Gen/fisiología , Hipersensibilidad Tardía/inmunología , Interferón gamma/metabolismo , Interleucina-17/metabolismo , Lipopolisacáridos/farmacología , Activación de Linfocitos/inmunología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas de Unión al Retinol/inmunología , Transducción de Señal/fisiología , Linfocitos T/inmunología , Receptor Toll-Like 2/fisiología , Receptor Toll-Like 3/fisiología , Receptor Toll-Like 4/fisiología , Receptor Toll-Like 9/fisiología , Receptores Toll-Like/agonistasRESUMEN
PURPOSE: Intracameral (anterior chamber) injection of antigen inhibits the development of delayed-type hypersensitivity, a phenomenon known as anterior chamber-associated immune deviation (ACAID). The authors investigated the effect of intracameral injection of interphotoreceptor retinoid-binding protein (IRPB) peptides on the development of IFN-gamma(+) and IL-17(+) pathogenic T cells. METHODS: A uveitogenic (IRBP1-20) or nonuveitogenic (IRBP161-180) peptide was injected into the anterior chamber (AC) of B6 mice. Seven days later, the mice were primed with a pathogenic dose of IRBP1-20 in adjuvant. Thirteen days later, the pathogenic activity of the T cells isolated from the spleens of treated and untreated mice were compared, and the numbers of Th1 and Th17 T cells were assessed by intracellular staining. Regulatory T-cell activity was assessed by antibody staining and functional assays. The authors also compared the effect of inhibition on EAU of ocular injection to various sites, including the AC, the vitreous cavity, and the subretinal space. RESULTS: Intraocular injection of the uveitogenic peptide (IRBP1-20), but not the nonuveitogenic peptide (IRBP161-180), inhibited the generation of IFN-gamma(+) and IL-17(+) uveitogenic T cells and the development of experimental autoimmune uveitis (EAU). AC administration of IRBP1-20, but not IRBP161-180, significantly decreased the number of circulating gammadelta T cells after subsequent systemic immunization with IRBP1-20. Absence of the gammadelta T-cell population prohibited the development of ACAID. CONCLUSIONS: Injection of a uveitogenic peptide into the AC inhibited the development of EAU by regulation of Th1 and Th17 IRBP-specific T cells. The circulating gammadelta T-cell population was reduced and was associated with decreased activation of IL-17(+) uveitogenic T cells.
Asunto(s)
Cámara Anterior/inmunología , Proteínas del Ojo/inmunología , Interleucina-17/inmunología , Proteínas de Unión al Retinol/inmunología , Subgrupos de Linfocitos T/inmunología , Células TH1/inmunología , Animales , Células Presentadoras de Antígenos/inmunología , Enfermedades Autoinmunes/inmunología , Enfermedades Autoinmunes/prevención & control , Técnicas de Cocultivo , Ensayo de Inmunoadsorción Enzimática , Femenino , Citometría de Flujo , Interferón gamma/metabolismo , Activación de Linfocitos/inmunología , Ratones , Ratones Endogámicos C57BL , Fragmentos de Péptidos/inmunología , Receptores de Antígenos de Linfocitos T gamma-delta , Organismos Libres de Patógenos Específicos , Bazo/inmunología , Uveítis Anterior/inmunología , Uveítis Anterior/prevención & controlRESUMEN
PURPOSE: The neuropeptide, alpha-melanocyte stimulating hormone (alpha-MSH), is an endogenous antagonist of inflammation. Injections of alpha-MSH peptide into inflamed tissues have been found to be very effective in suppressing autoimmune and endotoxin mediated diseases. We evaluated the potential to suppress ocular autoimmune disease (uveitis) by augmenting the expression of alpha-MSH through subconjunctival injections of naked adrenocorticotropic hormone amino acids 1-17 (ACTH1-17) plasmid. METHODS: We clinically scored the uveitis over time in B10.RIII, C57BL/6, and melanocortin 5 receptor knock-out (MC5r((-/-))) mice with experimental autoimmune uveitis (EAU) that were conjunctively injected with a naked DNA plasmid encoding ACTH1-17 at the time of EAU onset and three days later. The post-EAU retina histology of plasmid injected eyes was examined, and post-EAU concentrations of alpha-MSH in aqueous humor was assayed by ELISA. RESULTS: The subconjunctival injection of ACTH1-17 plasmid augmented the concentration of alpha-MSH in the aqueous humor of all post-EAU mice. The injection of ACTH1-17 suppressed the severity of EAU in the B10.RIII and C57BL/6 mice but the MC5r((-/-)) mice. In all the models of EAU, the ACTH1-17 injection helped to preserve the structural integrity of the retina; however, post-EAU aqueous humor was not immunosuppressive. CONCLUSIONS: The subconjunctival injection of the alpha-MSH expression vector ACTH1-17 plasmid is effective in suppressing EAU. The suppressive activity is dependent on MC5r expression, and possibly works though alpha-MSH antagonism of inflammation than on alpha-MSH directly modulating immune cells. The results suggest that an effective therapy for uveitis could include a gene therapy approach based on delivering alpha-MSH.
Asunto(s)
Enfermedades Autoinmunes/inmunología , Terapia Genética , Terapia de Inmunosupresión , Uveítis/inmunología , alfa-MSH/metabolismo , Hormona Adrenocorticotrópica/genética , Animales , Antiinflamatorios/administración & dosificación , Humor Acuoso , Enfermedades Autoinmunes/genética , Enfermedades Autoinmunes/terapia , Proteínas del Ojo/inmunología , Adyuvante de Freund , Humanos , Inyecciones , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Modelos Animales , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/inmunología , Plásmidos , Ingeniería de Proteínas , Receptores de Melanocortina/deficiencia , Retina/inmunología , Retina/metabolismo , Retina/patología , Proteínas de Unión al Retinol/inmunología , Uveítis/genética , Uveítis/terapia , Vacunas de ADN/administración & dosificación , alfa-MSH/genética , alfa-MSH/inmunologíaRESUMEN
Experimental autoimmune uveitis (EAU) in animals serves as a model of human uveitis. EAU can be induced in mice by immunization with the retinal antigen interphotoreceptor retinoid binding protein (IRBP) in complete Freund's adjuvant (CFA) or by IRBP-pulsed mature dendritic cells, and can be driven either by a Th17 or a Th1 effector response, depending on the model. The direction of the response is affected by conditions present during the exposure to antigen, including the quality/quantity of innate receptor stimulation and/or type of APC. IL-17 and IFN-gamma production by innate cells such as NKT may also affect the disease process. If exposure to antigen is via a hydrodynamic DNA vaccination with an IRBP-encoding plasmid, the response is directed to a regulatory phenotype, and disease is ameliorated or prevented. Our data shed light on effector and regulatory responses in autoimmune disease, provide balance to the Th1/Th17 paradigm and help to explain the clinical heterogeneity of human uveitis, which occurs in the face of responses to the same ocular antigen(s).
Asunto(s)
Enfermedades Autoinmunes/inmunología , Linfocitos T Reguladores/inmunología , Linfocitos T/inmunología , Uveítis/inmunología , Animales , Enfermedades Autoinmunes/metabolismo , Enfermedades Autoinmunes/patología , Proteínas del Ojo/inmunología , Humanos , Interferón gamma/metabolismo , Interleucina-17/metabolismo , Proteínas de Unión al Retinol/inmunología , Linfocitos T/metabolismo , Células TH1/inmunología , Células TH1/metabolismo , Uveítis/metabolismo , Uveítis/patologíaRESUMEN
PURPOSE: Results in previous reports have demonstrated that immunization of the EAU-prone B6 mouse activates both CD4 and CD8 IRBP-specific T cells. The purpose of this study was to investigate structural and functional differences between CD4 and CD8 autoreactive T cells activated by the uveitogenic peptide. METHODS: Purified CD4 and CD8 isolated from B6 mice immunized with an uveitogenic peptide, interphotoreceptor retinoid-binding protein (IRBP)1-20, were stimulated in vitro with various doses of immunizing peptide. The activated T cells were determined for cytokine production, expression of Foxp3, and suppressor activity. RESULTS: CD4 autoreactive T cells underwent full activation when stimulated with high or medium concentrations of immunizing peptide, whereas a high dose of antigenic peptide resulted in only modest activation of CD8 autoreactive T cells. When stimulated by a low dose (<0.1 microg/mL) of antigen or by of a high dose of antigen and a small amount of TGF-beta1, the minimally activated CD8 T cells expressed a high level of Foxp3 and gained suppressor function. CONCLUSIONS: Minimally activated CD8 autoreactive T cells can be functionally suppressive and may neutralize the tissue-damaging effect of the CD4 autoreactive T cells.
Asunto(s)
Linfocitos T CD8-positivos/fisiología , Proteínas del Ojo/inmunología , Factores de Transcripción Forkhead/metabolismo , Activación de Linfocitos/inmunología , Proteínas de Unión al Retinol/inmunología , Linfocitos T Reguladores/fisiología , Uveítis Posterior/inmunología , Animales , Linfocitos T CD4-Positivos/fisiología , Antígenos CD8/inmunología , Ensayo de Inmunoadsorción Enzimática , Femenino , Citometría de Flujo , Interferón gamma/metabolismo , Interleucina-2/metabolismo , Activación de Linfocitos/efectos de los fármacos , Ratones , Ratones Endogámicos C57BL , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Organismos Libres de Patógenos Específicos , Factor de Crecimiento Transformador beta1/farmacologíaRESUMEN
AIMS: To investigate whether supplementation of natural CD4+CD25+ regulatory T cells ameliorates mouse experimental autoimmune uveoretinitis (EAU) induced by CD4+ T cell-dependent interphotoreceptor retinoid-binding protein (IRBP). METHODS: C57BL/6 mice were immunised with human interphotoreceptor retinoid-binding protein peptide 1-20 (IRBP(1-20)), and IRBP(1-20)-sensitised T cells were obtained. CD4+CD25+ T cells derived from naive mice were cocultured with IRBP(1-20)-sensitised T cells, and their proliferation responses and cytokine production were measured. In addition, CD4+CD25+ T cells were transferred intravenously into mice 7 or 15 days after immunisation with IRBP(1-20), and the severity of EAU and T cell proliferation responses were evaluated. RESULTS: CD4+CD25+ regulatory T cells effectively inhibited both the proliferation of, and interleukin (IL)2, IL5 and interferon (IFN)gamma production by, IRBP(1-20)-sensitised T cells. Adoptive transfer of CD4+CD25+ regulatory T cells to IRBP(1-20)-immunised mice conferred considerable protection from EAU development and inhibition of T cell proliferation responses to IRBP(1-20). CONCLUSION: These findings show that natural CD4+CD25+ regulatory T cells possess the ability to inhibit activation of IRBP-reactive T cells that have been already sensitised in vivo, and adoptive transfer of these cells ameliorates EAU even in the effector phase. Supplementation of natural CD4+CD25+ regulatory T cells may have therapeutic potential for effective treatment of uveitis.
Asunto(s)
Enfermedades Autoinmunes/inmunología , Retinitis/inmunología , Linfocitos T Reguladores/inmunología , Uveítis/inmunología , Traslado Adoptivo/métodos , Animales , División Celular/inmunología , Proteínas del Ojo/inmunología , Femenino , Factores de Transcripción Forkhead/inmunología , Interferón gamma/inmunología , Interleucina-2/inmunología , Subunidad alfa del Receptor de Interleucina-2/inmunología , Interleucina-5/inmunología , Activación de Linfocitos/inmunología , Ratones , Ratones Endogámicos C57BL , Proteínas de Unión al Retinol/inmunología , Células TH1/inmunologíaRESUMEN
PURPOSE: To identify H-2 Kb/Db-binding immunogenic peptides derived from retinal proteins. METHODS: Computer-based prediction was used to identify potentially H-2 Kb/Db-binding peptides derived from the interphotoreceptor retinol-binding protein (IRBP), soluble retinal antigen (S-antigen), recoverin, phosducin, and pigment epithelium-derived factor (PEDF). The affinity of the peptides was analyzed by their abilities to upregulate the expression of major histocompatibility complex (MHC) class I on TAP-deficient cells (RMA-S cells) with flow cytometry. C57BL/6 mice were immunized subcutaneously, with individual peptides in incomplete Freund's adjuvant (IFA). Eight days after immunization, splenocytes were isolated for cytotoxic T-lymphocyte (CTL) analysis. A 51chromium-release assay was used to detect specific CTL reactivity generated in the cultures. Eyes were enucleated for histopathological analysis on day 21 after immunization with IRBP or IRBP and the immunogenic peptides. RESULTS: All the 21 predicted peptides were found to upregulate expression of H-2 Kb/Db on RMA-S cells. Five peptides, the two IRBP-derived peptides IRBP89-96 and IRBP(101-108), and the three PEDF-derived peptides, PEDF389-397, PEDF139-147, and PEDF272-279, induced specific CTL responses in vivo, whereas the remaining 16 peptides, including 5 IRBP-derived peptides, 5 S-antigen-derived peptides, 1 recoverin-derived peptide, 1 phosducin-derived peptide, and 4 PEDF-derived peptides, did not induce specific CTL reactivity. The immunogenic peptides alone did not induce inflammation in the eyes, but they could enhance severity of uveitis induced by IRBP. CONCLUSIONS: Five of 21 H-2 Kb/Db-binding retinal protein-derived peptides were found to be immunogenic, suggesting that these peptides could function as autoantigenic epitopes in the development of inflammatory eye diseases, such as uveitis.