Phyllis B. Silver

Either a Th17 or a Th1 effector response can drive autoimmunity: conditions of disease induction affect dominant effector category

Experimental autoimmune uveitis (EAU) represents autoimmune uveitis in humans. We examined the role of the interleukin (IL)-23–IL-17 and IL-12–T helper cell (Th)1 pathways in the pathogenesis of EAU. IL–23 but not IL-12 was necessary to elicit disease by immunization with the retinal antigen (Ag) interphotoreceptor retinoid-binding protein (IRBP) in complete Freunds adjuvant. IL-17 played a dominant role in this model; its neutralization prevented or reversed disease, and Th17 effector cells induced EAU in the absence of interferon (IFN)-γ. In a transfer model, however, a polarized Th1 line could induce severe EAU independently of host IL-17. Furthermore, induction of EAU with IRBP-pulsed mature dendritic cells required generation of an IFN-γ–producing effector response, and an IL-17 response by itself was insufficient to elicit pathology. Finally, genetic deficiency of IL-17 did not abrogate EAU susceptibility. Thus, autoimmune pathology can develop in the context of either a Th17 or a Th1 effector response depending on the model. The data suggest that the dominant effector phenotype may be determined at least in part by conditions present during initial exposure to Ag, including the quality/quantity of Toll-like receptor stimulation and/or type of Ag-presenting cells. These data also raise the possibility that the nonredundant requirement for IL-23 in EAU may extend beyond its role in promoting the Th17 effector response and help provide a balance in the current Th1 versus Th17 paradigm.

Calcitriol Suppresses Antiretinal Autoimmunity through Inhibitory Effects on the Th17 Effector Response

Experimental autoimmune uveitis (EAU) serves as a model for human autoimmune uveitis and for cell-mediated autoimmunity in general. EAU induced in mice by immunization with the retinal Ag interphotoreceptor retinoid-binding protein in CFA is driven by the Th17 response. Oral calcitriol (1,25-dihydroxyvitamin D3) prevented as well as partly reversed disease and suppressed immunological responses. In vitro, calcitriol directly suppressed IL-17 induction in purified naive CD4+ T cells without inhibiting Th17 lineage commitment, as reflected by unaltered RORγt, STAT3, and FoxP3 expression. In contrast, in vivo treatment with calcitriol of mice challenged for EAU impaired commitment to the Th17 lineage, as judged by reduction of both RORγt and IL-17 in CD4+ T cells. Innate immune response parameters in draining lymph nodes of treated mice were suppressed, as was production of IL-1, IL-6, TNF-α, and IL-12/IL-23p40, but not IL-10, by explanted splenic dendritic cells (DC). Finally, supernatants of calcitriol-conditioned bone marrow-derived DC had reduced ability to support Th17 polarization of naive CD4+ T cells in vitro and in vivo. Thus, calcitriol appears to suppress autoimmunity by inhibiting the Th17 response at several levels, including the ability of DC to support priming of Th17 cells, the ability of CD4+ T cells to commit to the Th17 lineage, and the ability of committed Th17 T cells to produce IL-17.

Essential Role of the MyD88 Pathway, but Nonessential Roles of TLRs 2, 4, and 9, in the Adjuvant Effect Promoting Th1-Mediated Autoimmunity

Induction of tissue-specific experimental autoimmune diseases involves an obligatory adjuvant effect to trigger an innate response of a type that will drive a Th1-biased adaptive response. This is achieved by use of CFA containing mycobacteria (Mycobacterium tuberculosis), whose recognition by cells of the innate immune system depends on TLRs that signal through the adaptor molecule MyD88. We examined the role of selected components of the MyD88 pathway in promoting experimental autoimmune uveitis (EAU). Mice deficient in MyD88, TLR2, TLR4, or TLR9 were immunized with the retinal Ag interphotoreceptor retinoid-binding protein in CFA, and their EAU scores and associated immunological responses were examined. MyD88−/− mice were completely resistant to EAU and had a profound defect in Th1, but not Th2, responses to autoantigen challenge. Surprisingly, TLR2−/−, TLR4−/−, and TLR9−/− mice were fully susceptible to EAU and had unaltered adaptive responses to interphotoreceptor retinoid-binding protein. Examination of IL-1R family members, which share the common adaptor MyD88 with the TLR family, revealed that IL-1R-deficient mice, but not IL-18-deficient mice, are resistant to EAU and have profoundly reduced Th1 and Th2 responses. These data are compatible with the interpretation that TLR9, TLR4, and TLR2 signaling is either not needed, or, more likely, redundant in the adjuvant effect needed to induce EAU. In contrast, signaling through the IL-1R plays a necessary and nonredundant role in EAU and can by itself account for the lack of EAU development in MyD88 mice.

Rodent Models of Experimental Autoimmune Uveitis

The model of experimental autoimmune uveitis (EAU) in mice and in rats is described. EAU targets immunologically privileged retinal antigens and serves as a model of autoimmune uveitis in humans as well as a model for autoimmunity in a more general sense. EAU is a well-characterized, robust, and reproducible model that is easily followed and quantitated. It is inducible with synthetic peptides derived from retinal autoantigens in commonly available strains of rats and mice. The ability to induce EAU in various gene-manipulated, including HLA-transgenic, mouse strains makes the EAU model suitable for the study of basic mechanisms as well as in clinically relevant interventions.

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.

Autoimmune Uveitis Elicited with Antigen-Pulsed Dendritic Cells Has a Distinct Clinical Signature and Is Driven by Unique Effector Mechanisms: Initial Encounter with Autoantigen Defines Disease Phenotype

Human autoimmune uveitis is a heterogeneous group of potentially blinding ocular diseases in which most patients who exhibit immunity recognize the same retinal Ag. It is represented by the model of experimental autoimmune uveitis (EAU) induced in mice by immunization with retinal Ag in CFA. Murine EAU is characterized by a Th1/Th17 response pattern, which may not represent all types of human uveitis. We report in this study a new model of EAU induced by injection of matured dendritic cells loaded with a uveitogenic retinal peptide. Dendritic cell-induced EAU demonstrated unique characteristics compared with traditional EAU in terms of clinical manifestations, the nature of the inflammatory infiltrating cells, the cytokine response profile, and a strict requirement for IFN-γ, whereas IL-17 appeared to play a minor role. Disease was self-limiting, but could be reinduced with the same Ag in CFA, albeit with reduced severity, suggesting postrecovery resistance. Our study demonstrates in a disease setting that the context in which the same autoantigen is initially presented to the immune system precipitates distinct forms of pathology via a distinct pathogenic pathway on the same genetic background. These findings may shed new light on the complex biology and the heterogeneous nature of human uveitis, and provide an alternative model for uveitic diseases of immune origin.

An Immunologically Privileged Retinal Antigen Elicits Tolerance Major Role for Central Selection Mechanisms

Immunologically privileged retinal antigens can serve as targets of experimental autoimmune uveitis (EAU), a model for human uveitis. The tolerance status of susceptible strains, whose target antigen is not expressed in the thymus at detectable levels, is unclear. Here, we address this issue directly by analyzing the consequences of genetic deficiency versus sufficiency of a uveitogenic retinal antigen, interphotoreceptor retinoid-binding protein (IRBP). IRBP-knockout (KO) and wild-type (WT) mice on a highly EAU-susceptible background were challenged with IRBP. The KO mice had greatly elevated responses to IRBP, an altered recognition of IRBP epitopes, and their primed T cells induced exacerbated disease in WT recipients. Ultrasensitive immunohistochemical staining visualized sparse IRBP-positive cells, undetectable by conventional assays, in thymi of WT (but not of KO) mice. IRBP message was PCR amplified from these cells after microdissection. Thymus transplantation between KO and WT hosts demonstrated that this level of expression is functionally relevant and sets the threshold of immune (and autoimmune) reactivity. Namely, KO recipients of WT thymi generated reduced IRBP-specific responses, and WT recipients of KO thymi developed enhanced responses and a highly exacerbated disease. Repertoire culling and thymus-dependent CD25+ T cells were implicated in this effect. Thus, uveitis-susceptible individuals display a detectable and functionally significant tolerance to their target antigen, in which central mechanisms play a prominent role.

A humanized model of experimental autoimmune uveitis in HLA class II transgenic mice

Experimental autoimmune uveitis (EAU) is a disease of the neural retina induced by immunization with retinal antigens, such as interphotoreceptor retinoid-binding protein (IRBP) and arrestin (retinal soluble antigen, S-Ag). EAU serves as a model for human autoimmune uveitic diseases associated with major histocompatibility complex (HLA) genes, in which patients exhibit immunological responses to retinal antigens. Here we report the development of a humanized EAU model in HLA transgenic (TG) mice. HLA-DR3, -DR4, -DQ6, and -DQ8 TG mice were susceptible to IRBP-induced EAU. Importantly, HLA-DR3 TG mice developed severe EAU with S-Ag, to which wild-type mice are highly resistant. Lymphocyte proliferation was blocked by anti-HLA antibodies, confirming that antigen is functionally presented by the human MHC molecules. Disease could be transferred by immune cells with a Th1-like cytokine profile. Antigen-specific T cell repertoire, as manifested by responses to overlapping peptides derived from S-Ag or IRBP, differed from that of wild-type mice. Interestingly, DR3 TG mice, but not wild-type mice, recognized an immunodominant S-Ag epitope between residues 291 and 310 that overlaps with a region of S-Ag recognized by uveitis patients. Thus, EAU in HLA TG mice offers a new model of uveitis that should represent human disease more faithfully than currently existing models.

T cell mechanisms in experimental autoimmune uveoretinitis: Susceptibility is a function of the cytokine response profile

This study addresses the question whether susceptibility versus resistance to experimental autoimmune uveoretinitis (EAU) is connected to a Th1-type (interferon-gamma high, interleukin-4 low), versus a Th2-type (IFN-γ low, IL-4 high) response. Primed lymph node cells of susceptible Lewis rats produced IFN-γ in response to antigen in culture and transferred EAU to syngeneic recipients, whereas lymph node cells of resistant F344 rats made no IFN-γ and did not transfer disease. Reversal of the disease pattern, by treatment of F344 rats with B. pertussis toxin and immunisation of Lewis rats with antigen in incomplete Freunds adjuvant, resulted in a parallel reversal of these response patterns. Neither strain produced significant IL-4 responses. A study of the response patterns in mice confirmed that high Th1 responders were susceptible, whereas low Th1 responders and Th2 responders were resistant. We conclude that susceptibility to EAU is connected with a Th1-dominant response, but resistance can involve either a ‘null’, F344-like response (Th1-low/Th2-low) or a Th2-dominant response.

Endogenous IRBP can be dispensable for generation of natural CD4+CD25+ regulatory T cells that protect from IRBP-induced retinal autoimmunity

Susceptibility to experimental autoimmune uveitis (EAU), a model for human uveitis induced in mice with the retinal antigen interphotoreceptor retinoid-binding protein (IRBP), is controlled by “natural” CD4+CD25+ regulatory T (T reg) cells. To examine whether endogenous expression of IRBP is necessary to generate these T reg cells, we studied responses of IRBP knockout (KO) versus wild-type (WT) mice. Unexpectedly, not only WT but also IRBP KO mice immunized with a uveitogenic regimen of IRBP in complete Freunds adjuvant (CFA) exhibited CD25+ regulatory cells that could be depleted by PC61 treatment, which suppressed development of uveitogenic effector T cells and decreased immunological responses to IRBP. These EAU-relevant T reg cells were not IRBP specific, as their activity was not present in IRBP KO mice immunized with IRBP in incomplete Freunds adjuvant (IFA), lacking mycobacteria (whereas the same mice exhibited normal T reg cell activity to retinal arrestin in IFA). We propose that mycobacterial components in CFA activate T reg cells of other specificities to inhibit generation of IRBP-specific effector T cells in a bystander fashion, indicating that effective T reg cells can be antigen nonspecific. Our data also provide the first evidence that generation of specific T reg cells to a native autoantigen in a mouse with a diverse T cell repertoire requires a cognate interaction.