Rachel R. Caspi

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.

Cutting Edge: NKT Cells Constitutively Express IL-23 Receptor and RORγt and Rapidly Produce IL-17 upon Receptor Ligation in an IL-6-Independent Fashion

Th17 cells require IL-6 and TGFβ for lineage commitment and IL-23 for maintenance. Unexpectedly, naive IL-6−/− splenocytes stimulated with anti-CD3 and IL-23 produced normal amounts of IL-17 during the first 24 h of culture. These rapid IL-6-independent IL-17 producers were identified as predominantly DX5+ TCRβ+ NKT cells, and a comparable response could be found using the invariant NKT-specific ligand α-galactosylceramide. Human NKT cells also produced IL-17. NKT cells constitutively expressed IL-23R and RORγt. Ligation of either TCR or IL-23R triggered IL-17 production and both together had a synergistic effect, suggesting independent but convergent pathways. IL-17 production was not restricted to a particular subset of NKT cells but they were NK1.1 negative. Importantly, in vivo administration of α-galactosylceramide triggered a rapid IL-17 response in the spleen. These data suggest an important biological role for innate IL-17 production by NKT cells that is rapid and precedes the adaptive IL-17 response.

The Rd8 Mutation of the Crb1 Gene Is Present in Vendor Lines of C57BL/6N Mice and Embryonic Stem Cells, and Confounds Ocular Induced Mutant Phenotypes

PURPOSE We noted an unexpected inheritance pattern of lesions in several strains of gene-manipulated mice with ocular phenotypes. The lesions, which appeared at various stages of backcross to C57BL/6, bore resemblance to the rd8 retinal degeneration phenotype. We set out to examine the prevalence of this mutation in induced mutant mouse lines, vendor C57BL/6 mice and in widely used embryonic stem cells. METHODS Ocular lesions were evaluated by fundus examination and histopathology. Detection of the rd8 mutation at the genetic level was performed by PCR with appropriate primers. Data were confirmed by DNA sequencing in selected cases. RESULTS Analysis of several induced mutant mouse lines with ocular disease phenotypes revealed that the disease was associated 100% with the presence of the rd8 mutation in the Crb1 gene rather than with the gene of interest. DNA analysis of C57BL/6 mice from common commercial vendors demonstrated the presence of the rd8 mutation in homozygous form in all C57BL/6N substrains, but not in the C57BL/6J substrain. A series of commercially available embryonic stem cells of C57BL/6N origin and C57BL/6N mouse lines used to generate ES cells also contained the rd8 mutation. Affected mice displayed ocular lesions typical of rd8, which were detectable by funduscopy and histopathology as early as 6 weeks of age. CONCLUSIONS These findings identify the presence of the rd8 mutation in the C57BL/6N mouse substrain used widely to produce transgenic and knockout mice. The results have grave implications for the vision research community who develop mouse lines to study eye disease, as presence of rd8 can produce significant disease phenotypes unrelated to the gene or genes of interest. It is suggested that researchers screen for rd8 if their mouse lines were generated on the C57BL/6N background, bear resemblance to the rd8 phenotype, or are of indeterminate origin.

Th1 and Th17 cells

Autoreactive effector CD4+ T cells have been associated with the pathogenesis of autoimmune disorders. Early studies implicated the interferon (IFN)‐γ‐producing T helper (Th)1 subset of CD4+ cells as the causal agents in the pathogenesis of autoimmunity. However, further studies have suggested a more complex story. In models thought to be driven by Th1 cells, mice lacking the hallmark Th1 cytokine IFN‐γ were not protected but tended to have enhanced susceptibility to disease. Identification of the IL‐17‐producing CD4+ effector cell lineage (Th17) has helped shed light on this issue. Th17 effector cells are induced in parallel to Th1, and, like Th1, polarized Th17 cells have the capacity to cause inflammation and autoimmune disease. This, together with the finding that deficiency of the Th17‐related cytokine IL‐23 but not the Th1‐related cytokine IL‐12 causes resistance, led to the notion that Th17 cells are the chief contributors to autoimmune tissue inflammation. Nevertheless, mice lacking IL‐17 are not protected from disease and display elevated numbers of IFN‐γ‐producing CD4+ T cells, and, in some cases, lack of IFN‐γ does confer resistance. Recent studies report overlapping as well as differential roles of these cells in tissue inflammation, which suggests the existence of a more complex relationship between these two effector T‐cell subsets than has hitherto been suspected. This review will attempt to bring together current information regarding interaction, balance, and collaborative potential between the Th1 and Th17 effector lineages.

Eosinophil-derived neurotoxin acts as an alarmin to activate the TLR2–MyD88 signal pathway in dendritic cells and enhances Th2 immune responses

Eosinophil-derived neurotoxin (EDN) is an eosinophil granule–derived secretory protein with ribonuclease and antiviral activity. We have previously shown that EDN can induce the migration and maturation of dendritic cells (DCs). Here, we report that EDN can activate myeloid DCs by triggering the Toll-like receptor (TLR)2–myeloid differentiation factor 88 signaling pathway, thus establishing EDN as an endogenous ligand of TLR2. EDN activates TLR2 independently of TLR1 or TLR6. When mice were immunized with ovalbumin (OVA) together with EDN or with EDN-treated OVA-loaded DCs, EDN enhanced OVA-specific T helper (Th)2-biased immune responses as indicated by predominant production of OVA-specific interleukin (IL)-5, IL-6, IL-10, and IL-13, as well as higher levels of immunoglobulin (Ig)G1 than IgG2a. Based on its ability to serve as a chemoattractant and activator of DCs, as well as the capacity to enhance antigen-specific immune responses, we consider EDN to have the properties of an endogenous alarmin that alerts the adaptive immune system for preferential enhancement of antigen-specific Th2 immune responses.

Spontaneous autoimmunity prevented by thymic expression of a single self-antigen

The expression of self-antigen in the thymus is believed to be responsible for the deletion of autoreactive T lymphocytes, a critical process in the maintenance of unresponsiveness to self. The Autoimmune regulator (Aire) gene, which is defective in the disorder autoimmune polyglandular syndrome type 1, has been shown to promote the thymic expression of self-antigens. A clear link, however, between specific thymic self-antigens and a single autoimmune phenotype in this model has been lacking. We show that autoimmune eye disease in aire-deficient mice develops as a result of loss of thymic expression of a single eye antigen, interphotoreceptor retinoid-binding protein (IRBP). In addition, lack of IRBP expression solely in the thymus, even in the presence of aire expression, is sufficient to trigger spontaneous eye-specific autoimmunity. These results suggest that failure of thymic expression of selective single self-antigens can be sufficient to cause organ-specific autoimmune disease, even in otherwise self-tolerant individuals.

A look at autoimmunity and inflammation in the eye

Autoimmune and inflammatory uveitis are a group of potentially blinding intraocular inflammatory diseases that arise without a known infectious trigger and are often associated with immunological responses to unique retinal proteins. In the United States, about 10% of the cases of severe visual handicap are attributed to this group of disorders. As I discuss here, experimental models of ocular autoimmunity targeting retinal proteins have brought about a better understanding of the basic immunological mechanisms involved in the pathogenesis of uveitis and are serving as templates for the development of novel therapies.

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.

Pathology of experimental autoimmune uveoretinitis in mice

The histopathology and immunopathology of murine experimental autoimmune uveoretinitis (EAU) following active immunization with the interphotoreceptor retinoid-binding protein (IRBP) were studied. The methods used included conventional light microscopy and immunoperoxidase staining. Lesions were located mainly in the uvea and the retina and were characteristically focal. The prominent histopathologic findings in the retina were vasculitis, granuloma, retinal fold, focal serous detachment, and loss of photoreceptors. Granulomas, formation of Dalen-Fuchs nodules, inflammatory cellular infiltration and increase in the thickness of the choroid and ciliary body were frequent findings. Subretinal neovascularization occurred in 10% of the experimental animals. Mild to moderate inflammation was also noted in the vitreous. The predominant infiltrating cells in the retinal and uveal granuloma and the Dalen-Fuchs nodules were macrophages. In contrast, the predominant infiltrating cell types in the vitreous were T helper/inducer lymphocytes. T suppressor/cytotoxic cells were rarely seen. Expression of Ia antigens on the ocular cells was confined to the immediate area of the inflammatory sites. The kinetics of histopathology showed two peaks at the 5th and 10th week after immunization, suggesting a relapsing course of the disease.

Treatment of Uveitis by Oral Administration of Retinal Antigens: Results of a Phase I/II Randomized Masked Trial

PURPOSE To evaluate the effect and safety of the oral administration of retinal antigens as a treatment of ocular inflammation. METHODS In a phase I/II randomized masked trial, patients with endogenous uveitis who were dependent on immunosuppressive agents were randomly assigned to receive either retinal S antigen alone (10 patients), retinal S antigen and a mixture of soluble retinal antigens (10 patients), a mixture of soluble retinal antigens alone (10 patients), or placebo (15 patients). An attempt was then made to taper patients completely off their standard immunosuppressive therapy over an 8 week period. The primary study endpoint was time to ocular inflammatory attack. The secondary study endpoint was the ability to taper patients completely off their immunosuppressive or cytotoxic medication within 8 weeks. RESULTS Time to development of the main study endpoint was not statistically significantly different for any of the four treatment groups. However, the group receiving the purified S antigen alone appeared to be tapered off their immunosuppressive medication more successfully compared with patients given placebo (P = .08), whereas all the other groups appeared to do worse than did those receiving placebo. No toxic effects attributable to any treatment were observed. CONCLUSIONS This phase I/II study is the first to test the use of orally administered S antigen in the treatment of uveitis. Although not statistically significant, patients given S antigen were more likely to be tapered off their chronically administered systemic immunosuppressive therapy than were the other groups tested.