Loss of STAT3 in CD4+ T cells prevents development of experimental autoimmune diseases

Th17 cells are implicated in CNS autoimmune diseases. We show that mice with targeted-deletion of Stat3 in CD4(+) T cells (CD4(Stat3)(-/-)) do not develop experimental autoimmune uveoretinitis (EAU) or experimental autoimmune encephalomyelitis. Defective Th17 differentiation noted in CD4(Stat3)(-/-) mice is compensated by exaggerated increases in Foxp3-, IL-10-, IL-4-, and IFN-gamma-expressing T cells, suggesting critical roles of STAT3 in shaping Ag-specific CD4(+) T cell repertoire. In mice with EAU, a high percentage of IL-17-expressing T cells in their peripheral lymphoid organs also secrete IFN-gamma while these double-expressors are absent in CD4(Stat3)(-/-) and wild-type mice without EAU, raising the intriguing possibility that uveitis maybe mediated by Th17 and IL-17-expressing Th1 cells. Resistance of Stat3-deficient mice to EAU derives in part from an inability of uveitogenic Th17 and Th1 cells to enter eyes or brain of the CD4(Stat3)(-/-) mouse because of the reduction in the expression of activated alpha4/beta1 integrins on CD4(Stat3)(-/-) T cells. Adoptive transfer of activated interphotoreceptor retinoid-binding protein-specific uveitogenic T cells induced in CD4(Stat3)(-/-) mice a severe EAU characterized by development of retinal folds, infiltration of inflammatory cells into the retina, and destruction of retinal architecture, underscoring our contention that the loss of STAT3 in CD4(+) T cells results in an intrinsic developmental defect that renders CD4(Stat3)(-/-) resistant to CNS inflammatory diseases. STAT3 requirement for IL-17 production by Th17, generation of double positive T cells expressing IL-17 and IFN-gamma, and for T cell trafficking into CNS tissues suggests that STAT3 may be a therapeutic target for modulating uveitis, sceritis, or multiple sclerosis.     

Novel IL27p28/IL12p40 Cytokine Suppressed Experimental Autoimmune Uveitis by Inhibiting Autoreactive Th1/Th17 Cells and Promoting Expansion of Regulatory T Cells

IL-12 family cytokines are important in host immunity. Whereas some members (IL-12, IL-23) play crucial roles in pathogenesis of organ-specific autoimmune diseases by inducing the differentiation of Th1 and Th17 lymphocytes, others (IL-27 and IL-35) suppress inflammatory responses and limit tissue injury induced by these T cell subsets. In this study, we have genetically engineered a novel IL27p28/IL12p40 heterodimeric cytokine (p28/p40) that antagonizes signaling downstream of the gp130 receptor. We investigated whether p28/p40 can be used to ameliorate uveitis, a CNS inflammatory disease. Experimental autoimmune uveitis (EAU) is the mouse model of human uveitis and is mediated by Th1 and Th17 cells. We show here that p28/p40 suppressed EAU by inhibiting the differentiation and inflammatory responses of Th1 and Th17 cells while promoting expansion of IL-10⁺- and Foxp3⁺-expressing regulatory T cells. Lymph node cells from mice treated with p28/p40 blocked adoptive transfer of EAU to naïve syngeneic mice by immunopathogenic T cells and suppressive effects of p28/p40 derived in part from antagonizing STAT1 and STAT3 pathways induced by IL-27 and IL-6. Interestingly, IL27p28 also suppressed EAU, but to a lesser extent than p28/p40. The inhibition of uveitogenic lymphocyte proliferation and suppression of EAU by p28/p40 and IL27p28 establish efficacy of single chain and heterodimeric IL-12 family cytokines in treatment of a CNS autoimmune disease. Creation of the biologically active p28/p40 heterodimeric cytokine represents an important proof-of-concept experiment, suggesting that cytokines comprising unique IL-12 α- and β-subunit pairing may exist in nature and may constitute a new class of therapeutic cytokines.     

Exogenous nitric oxide inhibits experimental autoimmune uveoretinitis development in Lewis rats by modulation of the Th1-dependent immune response.

Experimental autoimmune uveoretinitis (EAU) is a T cell-mediated autoimmune disease of posterior uvea that closely resembles a human disease. The uveitogenic effector T cell has a Th1-like phenotype [high interferon-gamma (IFN-gamma), low interleukin-4 (IL-4)], and genetic susceptibility to EAU that is associated with an elevated Th1 response. Suppression of CD4+ Th1 cells for the treatment of autoimmune disease is an attractive potential therapeutic approach. Nitric oxide (NO) has been implicated in the pathogenesis of several inflammatory and autoimmune diseases. In this study, we investigated the potential role of NO as an immunoregulator to alter Th1/Th2 cytokine production, as well as to inhibit the interphotoreceptor retinoid binding protein (IRBP)-induced EAU, a CD4+ Th1 cell-mediated autoimmune disease. Injection of IRBP (100 microg) into two footpads resulted in severe EAU. The beginning peak of the disease was days 12 to 15 after immunization. Oral treatment with molsidomine (MSDM), a NO donor, began 24 h before IRBP immunization to the end of the experiments, which resulted in a significant inhibition of the disease by clinical and histopathological criteria. When MSDM was administered until day 21, a complete reduction of incidence and severity of EAU was observed. To investigate the cytokine alterations from Th1 to Th2 cytokines by MSDM, the cytokines were assayed in a culture medium of IRBP-stimulated inguinal lymphocytes. IRBP-immunized rats secreted a high concentration of IFN-gamma and a low concentration of IL-10. In contrast, MSDM treatment enhanced IL-10 secretion and tended to decrease IFN-gamma secretion. In conclusion, we show that the administration of NO suppresses EAU by altering the Th1/Th2 balance of inflammatory immune responses. We suggest that NO may be useful in the therapeutic control of autoimmune uveitis.     

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-gamma, 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 post-recovery 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.     

Dynamics of Intraocular IFN-γ, IL-17 and IL-10-Producing Cell Populations during Relapsing and Monophasic Rat Experimental Autoimmune Uveitis

A major limitation of most animal models of autoimmune diseases is that they do not reproduce the chronic or relapsing-remitting pattern characteristic of many human autoimmune diseases. This problem has been overcome in our rat models of experimentally induced monophasic or relapsing-remitting autoimmune uveitis (EAU), which depend on the inducing antigen peptides from retinal S-Antigen (monophasic EAU) or interphotoreceptor retinoid-binding protein (relapsing EAU). These models enable us to compare autoreactive and regulatory T cell populations. Intraocular, but not peripheral T cells differ in their cytokine profiles (IFN-γ, IL-17 and IL-10) at distinct time points during monophasic or relapsing EAU. Only intraocular T cells concomitantly produced IFN-γ, IL-17 and/or IL-10. Monophasic EAU presented rising numbers of cells expressing IFN-γ and IL-17 (Th1/Th17) and cells expressing IL-10 or Foxp3. During relapsing uveitis an increase of intraocular IFN-γ+ cells and a concomitant decrease of IL-17+ cells was detected, while IL-10+ populations remained stable. Foxp3+ cells and cells expressing IL-10, even in combination with IFN-γ or IL-17, increased during the resolution of monophasic EAU, suggesting a regulatory role for these T cells. In general, cells producing multiple cytokines increased in monophasic and decreased in relapsing EAU. The distinct appearance of certain intraocular populations with characteristics of regulatory cells points to a differential influence of the ocular environment on T cells that induce acute and monophasic or relapsing disease. Here we provide evidence that different autoantigens can elicit distinct and differently regulated immune responses. IFN-γ, but not IL-17 seems to be the key player in relapsing-remitting uveitis, as shown by increased, synchronized relapses after intraocular application of IFN-γ. We demonstrated dynamic changes of the cytokine pattern during monophasic and relapsing-remitting disease with strongly increasing IL-10 expression in intraocular T cells during monophasic uveitis.     

Modulation of innate and adaptive immune responses by tofacitinib (CP-690,550)

Inhibitors of the JAK family of nonreceptor tyrosine kinases have demonstrated clinical efficacy in rheumatoid arthritis and other inflammatory disorders; however, the precise mechanisms by which JAK inhibition improves inflammatory immune responses remain unclear. In this study, we examined the mode of action of tofacitinib (CP-690,550) on JAK/STAT signaling pathways involved in adaptive and innate immune responses. To determine the extent of inhibition of specific JAK/STAT-dependent pathways, we analyzed cytokine stimulation of mouse and human T cells in vitro. We also investigated the consequences of CP-690,550 treatment on Th cell differentiation of naive murine CD4(+) T cells. CP-690,550 inhibited IL-4-dependent Th2 cell differentiation and interestingly also interfered with Th17 cell differentiation. Expression of IL-23 receptor and the Th17 cytokines IL-17A, IL-17F, and IL-22 were blocked when naive Th cells were stimulated with IL-6 and IL-23. In contrast, IL-17A production was enhanced when Th17 cells were differentiated in the presence of TGF-β. Moreover, CP-690,550 also prevented the activation of STAT1, induction of T-bet, and subsequent generation of Th1 cells. In a model of established arthritis, CP-690,550 rapidly improved disease by inhibiting the production of inflammatory mediators and suppressing STAT1-dependent genes in joint tissue. Furthermore, efficacy in this disease model correlated with the inhibition of both JAK1 and JAK3 signaling pathways. CP-690,550 also modulated innate responses to LPS in vivo through a mechanism likely involving the inhibition of STAT1 signaling. Thus, CP-690,550 may improve autoimmune diseases and prevent transplant rejection by suppressing the differentiation of pathogenic Th1 and Th17 cells as well as innate immune cell signaling.     

Cutting edge: loss of α4 integrin expression differentially affects the homing of Th1 and Th17 cells

The neutralization of α4 integrin is currently used as treatment in several autoimmune diseases and is thought to prevent the entry of most immune cells in target tissues. In this study, we showed that selective deletion of α4 integrin in T cells did not prevent but delayed the development of experimental autoimmune encephalomyelitis. Whereas both Th1 and Th17 cells infiltrate the CNS of wild-type mice, T cells present in the CNS of mice lacking α4 integrin were mainly enriched in Th17 cells, suggesting that this T cell subset uses other integrins to access the CNS. In contrast, α4 integrin expression is important for Th1 cells to enter the CNS and for the stability of their Th1-associated genetic program. Therefore, our data suggest that anti-α4 integrin Ab treatment may be more efficient in the treatment of Th1- rather than Th17-mediated disease.     

Autoreactive memory CD4+ T lymphocytes that mediate chronic uveitis reside in the bone marrow through STAT3-dependent mechanisms

Organ-specific autoimmune diseases are usually characterized by repeated cycles of remission and recurrent inflammation. However, where the autoreactive memory T cells reside in between episodes of recurrent inflammation is largely unknown. In this study, we have established a mouse model of chronic uveitis characterized by progressive photoreceptor cell loss, retinal degeneration, focal retinitis, retinal vasculitis, multifocal choroiditis, and choroidal neovascularization, providing for the first time to our knowledge a useful model for studying long-term pathological consequences of chronic inflammation of the neuroretina. We show that several months after inception of acute uveitis, autoreactive memory T cells specific to retinal autoantigen, interphotoreceptor retinoid-binding protein (IRBP), relocated to bone marrow (BM). The IRBP-specific memory T cells (IL-7Rα(High)Ly6C(High)CD4(+)) resided in BM in resting state but upon restimulation converted to IL-17/IFN-γ-expressing effectors (IL-7Rα(Low)Ly6C(Low)CD4(+)) that mediated uveitis. We further show that T cells from STAT3-deficient (CD4-STAT3KO) mice are defective in α4β1 and osteopontin expression, defects that correlated with inability of IRBP-specific memory CD4-STAT3KO T cells to traffic into BM. We adoptively transferred uveitis to naive mice using BM cells from wild-type mice with chronic uveitis but not BM cells from CD4-STAT3KO, providing direct evidence that memory T cells that mediate uveitis reside in BM and that STAT3-dependent mechanism may be required for migration into and retention of memory T cells in BM. Identifying BM as a survival niche for T cells that cause uveitis suggests that BM stromal cells that provide survival signals to autoreactive memory T cells and STAT3-dependent mechanisms that mediate their relocation into BM are attractive therapeutic targets that can be exploited to selectively deplete memory T cells that drive chronic inflammation.     

Estrogen receptor α signaling in T lymphocytes is required for estradiol-mediated inhibition of Th1 and Th17 cell differentiation and protection against experimental autoimmune encephalomyelitis

Estrogen treatment exerts a protective effect on experimental autoimmune encephalomyelitis (EAE) and is under clinical trial for multiple sclerosis therapy. Estrogens have been suspected to protect from CNS autoimmunity through their capacity to exert anti-inflammatory as well as neuroprotective effects. Despite the obvious impacts of estrogens on the pathophysiology of multiple sclerosis and EAE, the dominant cellular target that orchestrates the anti-inflammatory effect of 17β-estradiol (E2) in EAE is still ill defined. Using conditional estrogen receptor (ER) α-deficient mice and bone marrow chimera experiments, we show that expression of ERα is critical in hematopoietic cells but not in endothelial ones to mediate the E2 inhibitory effect on Th1 and Th17 cell priming, resulting in EAE protection. Furthermore, using newly created cell type-specific ERα-deficient mice, we demonstrate that ERα is required in T lymphocytes, but neither in macrophages nor dendritic cells, for E2-mediated inhibition of Th1/Th17 cell differentiation and protection from EAE. Lastly, in absence of ERα in host nonhematopoietic tissues, we further show that ERα signaling in T cells is necessary and sufficient to mediate the inhibitory effect of E2 on EAE development. These data uncover T lymphocytes as a major and nonredundant cellular target responsible for the anti-inflammatory effects of E2 in Th17 cell-driven CNS autoimmunity.     

CXCR3 antagonist AMG487 suppresses rheumatoid arthritis pathogenesis and progression by shifting the Th17/Treg cell balance

Rheumatoid arthritis (RA) is an autoimmune disease that is characterized by uncontrolled joint inflammation and damage to bone and cartilage. Previous studies have shown that chemokine receptors have important roles in RA development, and that blocking these receptors effectively inhibits RA progression. Our study was undertaken to investigate the role of AMG487, a selective CXCR3 antagonist, in DBA/1J mice bearing collagen-induced arthritis (CIA). Following induction of CIA, animals were treated with 5 mg/kg AMG487 intraperitoneally every 48 h, starting from day 21 until day 41 and evaluated for clinical score, and histological hallmarks of arthritic inflammation. We further investigated the effect of AMG487 on Th1 (T-bet), Th17 (IL-17A, RORγt, STAT3), Th22 (IL-22), and T regulatory (Treg; Foxp3 and IL-10) cells in splenic CXCR3⁺ and CD4⁺ T cells using flow cytometry. We also assessed the effect of AMG487 on T-bet, RORγt, IL-17A, IL-22, Foxp3, and IL-10 at both mRNA and protein levels using RT-PCR and Western blot analyses of knee samples. The severity of clinical scores, and histological inflammatory damage decreased significantly in AMG487-treated compared with CIA control mice. Moreover, the percentage of Th1, Th17, and Th22 cells decreased significantly and that of Treg cells increased in AMG487-treated mice. We further observed that AMG487-treatment downregulated T-bet, IL-17A, RORγt, and IL-22, whereas it upregulated Foxp3 and IL-10 mRNA and protein levels. This study demonstrates the antiarthritic effects of AMG487 in CIA animal model and supports the development of CXCR3 antagonists as a novel strategy for the treatment of inflammatory and arthritic conditions.     

Cutting Edge: Suppression of GM-CSF Expression in Murine and Human T Cells by IL-27

GM-CSF is a potent proinflammatory cytokine that plays a pathogenic role in the CNS inflammatory disease experimental autoimmune encephalomyelitis. As IL-27 alleviates experimental autoimmune encephalomyelitis, we hypothesized that IL-27 suppresses GM-CSF expression by T cells. We found that IL-27 suppressed GM-CSF expression in CD4(+) and CD8(+) T cells in splenocyte and purified T cell cultures. IL-27 suppressed GM-CSF in Th1, but not Th17, cells. IL-27 also suppressed GM-CSF expression by human T cells in nonpolarized and Th1- but not Th17-polarized PBMC cultures. In vivo, IL-27p28 deficiency resulted in increased GM-CSF expression by CNS-infiltrating T cells during Toxoplasma gondii infection. Although in vitro suppression of GM-CSF by IL-27 was independent of IL-2 suppression, IL-10 upregulation, or SOCS3 signaling, we observed that IL-27-driven suppression of GM-CSF was STAT1 dependent. Our findings demonstrate that IL-27 is a robust negative regulator of GM-CSF expression in T cells, which likely inhibits T cell pathogenicity in CNS inflammation.