Microbiota-Dependent Activation of an Autoreactive T Cell Receptor Provokes Autoimmunity in an Immunologically Privileged Site.

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.

Single particle cryo-EM of the complex between interphotoreceptor retinoid-binding protein and a monoclonal antibody.

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.

Cyclosporine A-Loaded Nanocarriers for Topical Treatment of Murine Experimental Autoimmune Uveoretinitis.

In the present study, tissue distribution and the therapeutic effect of topically applied cyclosporine A (CsA)-loaded methoxy-poly(ethylene-glycol)-hexyl substituted poly(lactic acid) (mPEGhexPLA) nanocarriers (ApidSOL) on experimental autoimmune uveitis (EAU) were investigated. The CsA-loaded mPEGhexPLA nanocarrier was tolerated well locally and showed no signs of immediate toxicity after repeated topical application in mice with EAU. Upon unilateral CsA treatment, CsA accumulated predominantly in the corneal and sclera-choroidal tissue of the treated eye and in lymph nodes (LN). This regimen reduced EAU severity in treated eyes compared to PBS-treated controls. This improvement was accompanied by reduced T-cell count, T-cell proliferation, and IL-2 secretion of cells from ipsilateral LN. In conclusion, topical treatment with CsA-loaded mPEGhexPLA nanocarriers significantly improves the outcome of EAU.

Regulation of Adenosine Deaminase on Induced Mouse Experimental Autoimmune Uveitis.

Adenosine is an important regulator of the immune response, and adenosine deaminase (ADA) inhibits this regulatory effect by converting adenosine into functionally inactive molecules. Studies showed that adenosine receptor agonists can be anti- or proinflammatory. Clarification of the mechanisms that cause these opposing effects should provide a better guide for therapeutic intervention. In this study, we investigated the effect of ADA on the development of experimental autoimmune uveitis (EAU) induced by immunizing EAU-prone mice with a known uveitogenic peptide, IRBP1-20. Our results showed that the effective time to administer a single dose of ADA to suppress induction of EAU was 8-14 d postimmunization, shortly before EAU expression; however, ADA treatment at other time points exacerbated disease. ADA preferentially inhibited Th17 responses, and this effect was γδ T cell dependent. Our results demonstrated that the existing immune status strongly influences the anti- or proinflammatory effects of ADA. Our observations should help to improve the design of ADA- and adenosine receptor-targeted therapies.

TLR7 Engagement on Dendritic Cells Enhances Autoreactive Th17 Responses via Activation of ERK.

In this study, we showed that TLR7 activation significantly promoted interphotoreceptor retinoid-binding protein (IRBP)-specific Th17 responses by upregulating RORγt, IL-17, GM-CSF, and IL-23R expression in experimental autoimmune uveitis mice. In vivo administration of CL097 activated dendritic cells (DCs) and endowed them with an increased ability to activate IRBP-specific Th17 cells. CL097-treated DCs (CL097-DCs) formed a cytokine milieu that favored the generation and maintenance of Th17 cells by stimulating IL-1ß, IL-6, and IL-23 expression. Furthermore, IRBP-specific T cells from immunized mice injected with CL097-DCs produced more IL-17 and transferred more severe experimental autoimmune uveitis than did those from mice injected with DCs. The enhanced immunostimulatory activities of CL097-DCs depended on JNK, ERK, and p38 activation. Blockade of ERK, but not p38 or JNK, completely abolished the Th17 responses induced by CL097-DCs. Collectively, our findings suggest that CL097 treatment significantly promotes autoreactive IL-17+ T cell responses through enhancing DC activation, which is mediated, at least in part, via the activation of ERK signaling.

Retina-specific T regulatory cells bring about resolution and maintain remission of autoimmune uveitis.

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.

Anti-inflammatory or proinflammatory effect of an adenosine receptor agonist on the Th17 autoimmune response is inflammatory environment-dependent.

Adenosine is a key endogenous signaling molecule that regulates a wide range of physiological functions, including immune system function and inflammation. Studies have shown that adenosine receptor (AR) agonists can be either anti-inflammatory or proinflammatory in immune responses and in inflammation, and the clarification of the mechanisms causing these opposing effects should provide a better guide for therapeutic intervention. Whereas previous studies mostly examined the effects of AR agonists on Th1-type immune responses, in this study, we compared their effect on Th17 and Th1 autoimmune responses in experimental autoimmune uveitis, a mouse model of human uveitis induced by immunization with the human interphotoreceptor retinoid-binding protein peptides 1-20. We showed that injection of mice with a nonselective AR agonist, 5'-N-ethylcarboxamidoadenosine (NECA), at an early stage after immunization had an inhibitory effect on both Th1 and Th17 responses, whereas injection of the same amount of NECA at a late stage inhibited the Th1 response but had an enhancing effect on the Th17 response. We also showed that the effects of NECA on Th1 and Th17 responses were completely dissociated, that the enhancing effect of NECA on Th17 responses was modulated by γδ T cells, and that the response of γδ T cells to NECA was determined by their activation status. We conclude that the inflammatory environment has a strong impact on converting the effect of AR agonist on the Th17 autoimmune response from anti-inflammatory to proinflammatory. Our observation should help in the designing of better AR-targeted therapies.

A novel pathogenic RBP-3 peptide reveals epitope spreading in persistent experimental autoimmune uveoretinitis.

Experimental autoimmune uveoretinitis (EAU) in the C57BL/6J mouse is a model of non-infectious posterior segment intraocular inflammation that parallels clinical features of the human disease. The purpose of this study was to analyse the immune response to the four murine subunits of retinol binding protein-3 (RBP-3) to identify pathogenic epitopes to investigate the presence of intramolecular epitope spreading during the persistent inflammation phase observed in this model of EAU. Recombinant murine subunits of the RBP-3 protein were purified and used to immunize C57BL/6J mice to induce EAU. An overlapping peptide library was used to screen RBP-3 subunit 3 for immunogenicity and pathogenicity. Disease phenotype and characterization of pathogenic subunits and peptides was undertaken by topical endoscopic fundal imaging, immunohistochemistry, proliferation assays and flow cytometry. RBP-3 subunits 1, 2 and 3 induced EAU in the C57BL/6J mice, with subunit 3 eliciting the most destructive clinical disease. Within subunit 3 we identified a novel uveitogenic epitope, 629-643. The disease induced by this peptide was comparable to that produced by the uveitogenic 1-20 peptide. Following immunization, peptide-specific responses by CD4(+) and CD8(+) T-cell subsets were detected, and cells from both populations were present in the retinal inflammatory infiltrate. Intramolecular epitope spreading between 629-643 and 1-20 was detected in mice with clinical signs of disease. The 629-643 RBP-3 peptide is a major uveitogenic peptide for the induction of EAU in C57BL/6J mice and the persistent clinical disease induced with one peptide leads to epitope spreading.

Annexin-A1 restricts Th17 cells and attenuates the severity of autoimmune disease.

Annexin-A1 (Anx-A1) is an endogenous anti-inflammatory molecule and while described as a repressor of innate immune responses, the role of Anx-A1 in adaptive immunity, and in particular in T helper (Th) cell responses, remains controversial. We have used a T-cell mediated mouse model of retinal autoimmune disease to unravel the role of Anx-A1 in the development of autoreactive Th cell responses and pathology. RBP1-20-immunized C57BL/6 Anx-A1(-/-) mice exhibit significantly enhanced retinal inflammation and pathology as a result of an uncontrolled proliferation and activation of Th17 cells. This is associated with a limited capacity to induce SOCS3, resulting in un-restricted phosphorylation of STAT3. RBP1-20-specific CD4(+) cells from immunized Anx-A1(-/-) animals generated high levels of Th17 cells-associated cytokines. Following disease induction, daily systemic administration of human recombinant Anx-A1 (hrAnx-A1), during the afferent phase of disease, restrained autoreactive CD4(+) cell proliferation, reduced expression of pro-inflammatory cytokines IL-17, IFN-γ and IL-6 and attenuated autoimmune retinal inflammatory disease. Furthermore, in man, Anx-A1 serum levels when measured in active uveitis patient sera were low and associated with the detection of IgM and IgG anti-Anx-A1 antibodies when compared to healthy individuals. This data supports Anx-A1 as an early and critical regulator of Th17 cell driven autoimmune diseases such as uveitis.

Caffeic acid phenethyl ester lessens disease symptoms in an experimental autoimmune uveoretinitis mouse model.

Experimental autoimmune uveoretinitis (EAU) is an autoimmune disease that models human uveitis. Caffeic acid phenethyl ester (CAPE), a phenolic compound isolated from propolis, possesses anti-inflammatory and immunomodulatory properties. CAPE demonstrates therapeutic potential in several animal disease models through its ability to inhibit NF-κB activity. To evaluate these therapeutic effects in EAU, we administered CAPE in a model of EAU that develops after immunization with interphotoreceptor retinal-binding protein (IRBP) in B10.RIII and C57BL/6 mice. Importantly, we found that CAPE lessened the severity of EAU symptoms in both mouse strains. Notably, treated mice exhibited a decrease in the ocular infiltration of immune cell populations into the retina; reduced TNF-α, IL-6, and IFN-γ serum levels: and inhibited TNF-α mRNA expression in retinal tissues. Although CAPE failed to inhibit IRBP-specific T cell proliferation, it was sufficient to suppress cytokine, chemokine, and IRBP-specific antibody production. In addition, retinal tissues isolated from CAPE-treated EAU mice revealed a decrease in NF-κB p65 and phospho-IκBα. The data identify CAPE as a potential therapeutic agent for autoimmune uveitis that acts by inhibiting cellular infiltration into the retina, reducing the levels of pro-inflammatory cytokines, chemokine, and IRBP-specific antibody and blocking NF-κB pathway activation.

Characterization of microRNA expression profiling in peripheral blood lymphocytes in rats with experimental autoimmune uveitis.

OBJECTIVE AND DESIGN: We aimed to investigate the alterations of microRNA (miRNA) genomics in peripheral blood lymphocytes in experimental autoimmune uveitis (EAU) rats versus control samples. MATERIALS/METHODS: Six Lewis rats received interphotoreceptor retinoid-binding protein (IRBP) emulsion to induce EAU. On day 12, peripheral blood lymphocytes were isolated, and total RNAs were extracted. Using microarray analysis, we analyzed the aberrant miRNAs, validated the relevant expression of differentially expressed miRNAs, and predicted the possible miRNA targets and signaling pathways. RESULTS: The results indicated that 36 miRNAs were upregulated and 31 miRNAs were downregulated in EAU rats versus normal samples. Real-time quantitative PCR substantiated a high degree of confidence for the differentially expressed miRNAs, and miRNA analyses showed the differentially expressed miRNA targets were involved not only in the multicellular organismal process and developmental process, but also in T cell receptor signaling pathway, B cell receptor signaling pathway and so on. CONCLUSIONS: Our findings show that the differentially expressed miRNAs in EAU rats were closely associated with immune signaling pathways and may be applied in early prevention, prognosis and possible therapy in uveitis, indicating that miRNAs play an important role in the development of uveitis.

Both MC5r and A2Ar are required for protective regulatory immunity in the spleen of post-experimental autoimmune uveitis in mice.

The ocular microenvironment uses a poorly defined mela5 receptor (MC5r)-dependent pathway to recover immune tolerance following intraocular inflammation. This dependency is seen in experimental autoimmune uveoretinitis (EAU), a mouse model of endogenous human autoimmune uveitis, with the emergence of autoantigen-specific regulatory immunity in the spleen that protects the mice from recurrence of EAU. In this study, we found that the MC5r-dependent regulatory immunity increased CD11b(+)F4/80(+)Ly-6C(low)Ly-6G(+)CD39(+)CD73(+) APCs in the spleen of post-EAU mice. These MC5r-dependent APCs require adenosine 2A receptor expression on T cells to activate EAU-suppressing CD25(+)CD4(+)Foxp3(+) regulatory T cells. Therefore, in the recovery from autoimmune disease, the ocular microenvironment induces tolerance through a melanocortin-mediated expansion of Ly-6G(+) regulatory APCs in the spleen that use the adenosinergic pathway to promote activation of autoantigen-specific regulatory T cells.

Detection of an autoreactive T-cell population within the polyclonal repertoire that undergoes distinct autoimmune regulator (Aire)-mediated selection.

The autoimmune regulator (Aire) plays a critical role in central tolerance by promoting the display of tissue-specific antigens in the thymus. To study the influence of Aire on thymic selection in a physiological setting, we used tetramer reagents to detect autoreactive T cells specific for the Aire-dependent tissue-specific antigen interphotoreceptor retinoid-binding protein (IRBP), in the polyclonal repertoire. Two class II tetramer reagents were designed to identify T cells specific for two different peptide epitopes of IRBP. Analyses of the polyclonal T-cell repertoire showed a high frequency of activated T cells specific for both IRBP tetramers in Aire(-/-) mice, but not in Aire(+/+) mice. Surprisingly, although one tetramer-binding T-cell population was efficiently deleted in the thymus in an Aire-dependent manner, the second tetramer-binding population was not deleted and could be detected in both the Aire(-/-) and Aire(+/+) T-cell repertoires. We found that Aire-dependent thymic deletion of IRBP-specific T cells relies on intercellular transfer of IRBP between thymic stroma and bone marrow-derived antigen-presenting cells. Furthermore, our data suggest that Aire-mediated deletion relies not only on thymic expression of IRBP, but also on proper antigen processing and presentation of IRBP by thymic antigen-presenting cells.

Retinal antigen-specific regulatory T cells protect against spontaneous and induced autoimmunity and require local dendritic cells.

BACKGROUND: We previously reported that the peripheral regulatory T cells (pTregs) generated 'on-demand' in the retina were crucial to retinal immune privilege, and in vitro analysis of retinal dendritic cells (DC) showed they possessed antigen presenting cell (APC) activity that promoted development of the Tregs and effector T cells (Teffs). Here, we expanded these findings by examining whether locally generated, locally acting pTregs were protective against spontaneous autoimmunity and autoimmunity mediated by interphotoreceptor retinoid-binding protein (IRBP). We also examined the APC capacity of retinal DC in vivo. METHODS: Transgenic (Tg) mice expressing diphtheria toxin receptor (DTR) and/or green fluorescent protein (GFP) under control of the endogenous FoxP3 promoter (GFP only in FG mice, GFP and DTR in FDG mice) or the CD11c promoter (GFP and DTR in CDG mice) were used in conjunction with Tg mice expressing beta-galactosidase (ßgal) as retinal neo-self antigen and ßgal-specific TCR Tg mice (BG2). Retinal T cell responses were assayed by flow cytometry and retinal autoimmune disease assessed by histological examination. RESULTS: Local depletion of the Tregs enhanced actively induced experimental autoimmune uveoretinitis to the highly expressed retinal self-antigen IRBP in FDG mice and spontaneous autoimmunity in ßgal-FDG-BG2 mice, but not in mice lacking autoreactive T cells or their target antigen in the retina. The presence of retinal ßgal downregulated the generation of antigen-specific Teffs and pTregs within the retina in response to local ßgal challenge. Retinal DC depletion prevented generation of Tregs and Teffs within retina after ßgal injection. Microglia remaining after DC depletion did not make up for loss of DC-dependent antigen presentation. CONCLUSIONS: Our results suggest that local retinal Tregs protect against spontaneous organ-specific autoimmunity and that T cell responses within the retina require the presence of local DC.

Ex-vivo tolerogenic F4/80⁺ antigen-presenting cells (APC) induce efferent CD8⁺ regulatory T cell-dependent suppression of experimental autoimmune uveitis.

It is known that inoculation of antigen into the anterior chamber (a.c.) of a mouse eye induces a.c.-associated immune deviation (ACAID), which is mediated in part by antigen-specific local and peripheral tolerance to the inciting antigen. ACAID can also be induced in vivo by intravenous (i.v.) inoculation of ex-vivo-generated tolerogenic antigen-presenting cells (TolAPC). The purpose of this study was to test if in-vitro-generated retinal antigen-pulsed TolAPC suppressed established experimental autoimmune uveitis (EAU). Retinal antigen-pulsed TolAPC were injected i.v. into mice 7 days post-induction of EAU. We observed that retinal antigen-pulsed TolAPC suppressed the incidence and severity of the clinical expression of EAU and reduced the expression of associated inflammatory cytokines. Moreover, extract of whole retina efficiently replaced interphotoreceptor retinoid-binding protein (IRBP) in the preparation of TolAPC used to induce tolerance in EAU mice. Finally, the suppression of EAU could be transferred to a new set of EAU mice with CD8⁺ but not with CD4⁺ regulatory T cells (T(reg)). Retinal antigen-pulsed TolAPC suppressed ongoing EAU by inducing CD8⁺ T(reg) cells that, in turn, suppressed the effector activity of the IRBP-specific T cells and altered the clinical symptoms of autoimmune inflammation in the eye. The ability to use retinal extract for the antigen raises the possibility that retinal extract could be used to produce autologous TolAPC and then used as therapy in human uveitis.

The living eye "disarms" uncommitted autoreactive T cells by converting them to Foxp3(+) regulatory cells following local antigen recognition.

Immune privilege is used by the eye, brain, reproductive organs, and gut to preserve structural and functional integrity in the face of inflammation. The eye is arguably the most vulnerable and, therefore, also the most "privileged" of tissues; paradoxically, it remains subject to destructive autoimmunity. It has been proposed, although never proven in vivo, that the eye can induce T regulatory cells (Tregs) locally. Using Foxp3-GFP reporter mice expressing a retina-specific TCR, we now show that uncommitted T cells rapidly convert in the living eye to Foxp3(+) Tregs in a process involving retinal Ag recognition, de novo Foxp3 induction, and proliferation. This takes place within the ocular tissue and is supported by retinoic acid, which is normally present in the eye because of its function in the chemistry of vision. Nonconverted T cells showed evidence of priming but appeared restricted from expressing effector function in the eye. Pre-existing ocular inflammation impeded conversion of uncommitted T cells into Tregs. Importantly, retina-specific T cells primed in vivo before introduction into the eye were resistant to Treg conversion in the ocular environment and, instead, caused severe uveitis. Thus, uncommitted T cells can be disarmed, but immune privilege is unable to protect from uveitogenic T cells that have acquired effector function prior to entering the eye. These findings shed new light on the phenomenon of immune privilege and on its role, as well as its limitations, in actively controlling immune responses in the tissue.

Breakdown of immune privilege and spontaneous autoimmunity in mice expressing a transgenic T cell receptor specific for a retinal autoantigen.

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⁺ 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⁺ T cells displayed a naïve phenotype, but proliferated in vitro in response to IRBP and elicited uveitis upon adoptive transfer. In contrast, CD4⁺ 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⁺ 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.

[Effects of exogenous interleukin-2, interleukin-23 on differentiation of IRBP 1-20-specific T cells toward Th1 and Th17 and comparison of the pathogenicity of IRBP 1-20-specific T cells].

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.

Ag@BSA core/shell microspheres as an electrochemical interface for sensitive detection of urinary retinal-binding protein.

The level of urinary retinol-binding protein (RBP) can be estimated as a significant index of renal tubular injury. In this work, we used Ag@BSA microspheres as a sensing interface to cross-link RBP monoclonal antibody (RBP mAb) via glutaraldehyde for sensitive detection of RBP. The Ag@BSA microspheres covered on a Au electrode could provide a larger surface area and multifunctional substrate for the effective immobilization of RBP mAb, and the outside BSA layer acted as a biocompatible support to maintain the bioactivity and stability of immobilized immunogen. Electrochemical measurements containing electrochemical impedance spectroscopy (EIS) and differential pulse voltammetry (DPV) were employed to evaluate the analytical performance of the fabricated immunosensor and a higher detection sensitivity was obtained by DPV attributed to the excellent electrical conductivity of Ag@BSA which could enhance the peak current response. This immunosensor had a best detection limit (DL) of 18 ng mL(-1) and a linear response range between 50 and 4500 ng mL(-1). The proposed approach showed high specificity for RBP detection, acceptable reproducibility with an RSD of 5.6%, and good precision with the RSD of 4.5% and 6.3% at the RBP concentrations of 500 and 1500 ng mL(-1). Compared with the ELISA method by analyzing real urine samples from a patient, this immunosensor revealed acceptable accuracy with a relative deviation lower than 6.5%, indicating a potential alternative method for RBP detection in clinical diagnosis.

Invariant NKT cells regulate experimental autoimmune uveitis through inhibition of Th17 differentiation.

Although NKT cells have been implicated in diverse immunomodulatory responses, the effector mechanisms underlying the NKT cell-mediated regulation of pathogenic T helper cells are not well understood. Here, we show that invariant NKT cells inhibited the differentiation of CD4(+) T cells into Th17 cells both in vitro and in vivo. The number of IL-17-producing CD4(+) T cells was reduced following co-culture with purified NK1.1(+) TCR(+) cells from WT, but not from CD1d(-/-) or Jα18(-/-) , mice. Co-cultured NKT cells from either cytokine-deficient (IL-4(-/-) , IL-10(-/-) , or IFN-γ(-/-) ) or WT mice efficiently inhibited Th17 differentiation. The contact-dependent mechanisms of NKT cell-mediated regulation of Th17 differentiation were confirmed using transwell co-culture experiments. On the contrary, the suppression of Th1 differentiation was dependent on IL-4 derived from the NKT cells. The in vivo regulatory capacity of NKT cells on Th17 cells was confirmed using an experimental autoimmune uveitis model induced with human IRBP(1-20) (IRBP, interphotoreceptor retinoid-binding protein) peptide. NKT cell-deficient mice (CD1d(-/-) or Jα18(-/-) ) demonstrated an increased disease severity, which was reversed by the transfer of WT or cytokine-deficient (IL-4(-/-) , IL-10(-/-) , or IFN-γ(-/-) ) NKT cells. Our results indicate that invariant NKT cells inhibited autoimmune uveitis predominantly through the cytokine-independent inhibition of Th17 differentiation.