Host T cells are the main producers of IL-17 within the central nervous system during initiation of experimental autoimmune encephalomyelitis induced by adoptive transfer of Th1 cell lines

Experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis, has long been thought to be mediated by Th1 CD4(+) T cells. Using adoptive transfer techniques, transfer of CNS specific Th1 T cells was sufficient to induce EAE in naive mice. However, recent studies found a vital role for IL-17 in induction of EAE. These studies suggested that a fraction of IL-17-producing T cells that contaminate Th1 polarized cell lines are largely responsible for initiation of EAE. In this study, we tracked the appearance and cytokine production capacity of adoptively transferred cells within the CNS of mice throughout EAE disease. IL-17-producing, adoptively transferred cells were not enriched over the low percentages present in vitro. Thus, there was no selective recruitment and/or preferential proliferation of adoptively transferred IL-17-producing cells during the induction of EAE. Instead a large number of CNS infiltrating host T cells in mice with EAE were capable of producing IL-17 following ex vivo stimulation. The IL-17-producing T cells contained both alphabeta and gammadelta TCR(+) T cells with a CD4(+)CD8(-) or CD4(-)CD8(-) phenotype. These cells concentrated within the CNS within 3 days of adoptive transfer, and appeared to play a role in EAE induction as adoptive transfer of Th1 lines derived from wild-type mice into IL-17-deficient mice induced reduced EAE clinical outcomes. This study demonstrates that an encephalitogenic Th1 cell line induces recruitment of host IL-17-producing T cells to the CNS during the initiation of EAE and that these cells contribute to the incidence and severity of disease.     

Mycobacterium bovis bacille Calmette-Guérin infection in the CNS suppresses experimental autoimmune encephalomyelitis and Th17 responses in an IFN-gamma-independent manner

Multiple sclerosis and an animal model resembling multiple sclerosis, experimental autoimmune encephalomyelitis (EAE), are inflammatory demyelinating diseases of the CNS that are suppressed by systemic mycobacterial infection in mice and BCG vaccination in humans. Host defense responses against Mycobacterium in mice are influenced by T lymphocytes and their cytokine products, particularly IFN-gamma, which plays a protective regulatory role in EAE. To analyze the counter-regulatory role of mycobacterial infection-induced IFN-gamma in the CNS on the function of the pathological Th17 cells and the clinical outcome of EAE, we induced EAE in mice that were intracerebrally infected with Mycobacterium bovis bacille Calmette-Guerin (BCG). In this study, we demonstrate that intracerebral (i.c.) BCG infection prevented inflammatory cell recruitment to the spinal cord and suppressed the development of EAE. Concomitantly, there was a significant decrease in the frequency of myelin oligodendrocyte glycoprotein-specific IFN-gamma-producing CD4(+) T cells in the CNS. IL-17(+)CD4(+) T cell responses were significantly suppressed in i.c. BCG-infected mice following EAE induction regardless of T cell specificity. The frequency of Foxp3(+)CD4(+) T cells in these mice was equivalent to that of control mice. Intracerebral BCG infection-induced protection of EAE and suppression of myelin oligodendrocyte glycoprotein-specific IL-17(+)CD4(+) T cell responses were similar in both wild-type and IFN-gamma-deficient mice. These data show that live BCG infection in the brain suppresses CNS autoimmunity. These findings also reveal that the regulation of Th17-mediated autoimmunity in the CNS can be independent of IFN-gamma-mediated mechanisms.     

Severe disease, unaltered leukocyte migration, and reduced IFN-gamma production in CXCR3-/- mice with experimental autoimmune encephalomyelitis

Experimental autoimmune encephalomyelitis (EAE) is a CD4(+) Th1 T cell-mediated disease of the CNS, used to study certain aspects of multiple sclerosis. CXCR3, the receptor for CXCL10, CXCL9, and CXCL11, is preferentially expressed on activated Th1 T cells and has been proposed to govern the migration of lymphocytes into the inflamed CNS during multiple sclerosis and EAE. Unexpectedly, CXCL10-deficient mice were susceptible to EAE, leaving uncertain what the role of CXCR3 and its ligands might play in this disease model. In this study, we report that CXCR3(-/-) mice exhibit exaggerated severity of EAE compared with wild-type (CXCR3(+/+)) littermate mice. Surprisingly, there were neither quantitative nor qualitative differences in CNS-infiltrating leukocytes between CXCR3(+/+) and CXCR3(-/-) mice with EAE. Despite these equivalent inflammatory infiltrates, CNS tissues from CXCR3(-/-) mice with EAE showed worsened blood-brain barrier disruption and more von Willebrand factor-immunoreactive vessels within inflamed spinal cords, as compared with CXCR3(+/+) mice. Spinal cords of CXCR3(-/-) mice with EAE demonstrated decreased levels of IFN-gamma, associated with reduced inducible NO synthase immunoreactivity, and lymph node T cells from CXCR3(-/-) mice primed with MOG(35-55) secreted less IFN-gamma in Ag-driven recall responses than cells from CXCR3(+/+) animals. CXCR3(-/-) lymph node T cells also showed enhanced Ag-driven proliferation, which was reduced by addition of IFN-gamma. Taken with prior findings, our data show that CXCL10 is the most relevant ligand for CXCR3 in EAE. CXCR3 does not govern leukocyte trafficking in EAE but modulates T cell IFN-gamma production and downstream events that affect disease severity.     

Kinetics and organ distribution of IL-17-producing CD4 cells in proteolipid protein 139-151 peptide-induced experimental autoimmune encephalomyelitis of SJL mice

In experimental autoimmune encephalomyelitis (EAE), the production of proinflammatory cytokines by neuroantigen-specific T cells is thought to initiate and maintain the inflammatory autoimmune pathology. Because gene knockout strategies have shown that IFN-gamma and TNF are not essential for EAE development, there is increasing interest in establishing the role of other proinflammatory cytokines, primarily IL-17 in EAE. We used an IL-17 ELISPOT assay to track the neuroantigen-specific IL-17-producing T cells at single-cell resolution in various organs of SJL mice undergoing PLP 139-151-induced EAE. Overall, the migration patterns and population kinetics of the PLP 139-151-specific IL-17-producing CD4 cells were reminiscent of the IFN-gamma-producing cells, with the exception of IL-17 producers far outnumbering the IFN-gamma and IL-2 producers in the inflamed CNS. The selective enrichment of IL-17-producing CD4 cells in the CNS is suggestive of the pathogenic role of an independent (non-Th1) IL-17-producing proinflammatory effector T cell class in EAE.     

Kit (W-sh) mice develop earlier and more severe experimental autoimmune encephalomyelitis due to absence of immune suppression

Mast cells (MCs) have been thought to play a pathogenic role in the development of autoimmune diseases, including experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis. However, an immunoregulatory function of these cells has recently been suggested. We investigated the role of MCs in EAE using the W(-sh) mouse strain, which is MC deficient. W(-sh) mice developed earlier and more severe clinical and pathological disease with extensive demyelination and inflammation in the CNS. The inflammatory cells were mainly composed of CD4(+) T cells, monocyte/macrophages, neutrophils, and dendritic cells. Compared with wild-type mice, MC-deficient mice exhibited an increased level of MCP-1/CCR2 and CD44 expression on CD4(+) T cells in addition to decreased production of regulatory T cells, IL-4, IL-5, IL-27, and IL-10. We also found that levels of IL-17, IFN-γ, and GM-CSF were significantly increased in peripheral lymphocytes from immunized W(-sh) mice compared with those in peripheral lymphocytes from wild-type mice. Reconstitution of W(-sh) mice downregulated susceptibility to EAE, which correlated with MC recruitment and regulatory T cell activation in the CNS. These findings indicate that responsiveness is not required in the pathogenesis of inflammatory demyelination in the CNS and that, in the absence of MCs, increased MCP-1, CCR2, IL-17, IFN-γ, CD44, and other inflammatory molecules may be responsible for increased severity of EAE.     

CD43 modulates severity and onset of experimental autoimmune encephalomyelitis

Experimental autoimmune encephalomyelitis (EAE) is a mouse model of multiple sclerosis characterized by infiltration of activated CD4(+) T lymphocytes into tissues of the CNS. This study investigated the role of CD43 in the induction and progression of EAE. Results demonstrate that CD43-deficient mice have reduced and delayed clinical and histological disease severity relative to CD43(+/+) mice. This reduction was characterized by decreased CD4(+) T cell infiltration of the CNS of CD43(-/-) mice but similar numbers of Ag-specific T cells in the periphery, suggesting a defect in T cell trafficking to the CNS. The absence of CD43 also affected cytokine production, as myelin oligodendrocyte glycoprotein (MOG) 35-55-specific CD43(-/-) CD4(+) T cells exhibited reduced IFN-gamma and increased IL-4 production. CD43(-/-) CD4(+) MOG-primed T cells exhibited reduced encephalitogenicity relative to CD43(+/+) cells upon adoptive transfer into naive recipients. These results suggest a role for CD43 in the differentiation and migration of MOG(35-55)-specific T cells in EAE, and identify it as a potential target for therapeutic intervention.     

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.     

CXCR3 signaling reduces the severity of experimental autoimmune encephalomyelitis by controlling the parenchymal distribution of effector and regulatory T cells in the central nervous system

The chemokine receptor CXCR3 promotes the trafficking of activated T and NK cells in response to three ligands, CXCL9, CXCL10, and CXCL11. Although these chemokines are produced in the CNS in multiple sclerosis and experimental autoimmune encephalomyelitis (EAE), their role in the pathogenesis of CNS autoimmunity is unresolved. We examined the function of CXCR3 signaling in EAE using mice that were deficient for CXCR3 (CXCR3(-/-)). The time to onset and peak disease severity were similar for CXCR3(-/-) and wild-type (WT) animals; however, CXCR3(-/-) mice had more severe chronic disease with increased demyelination and axonal damage. The inflammatory lesions in WT mice consisted of well-demarcated perivascular mononuclear cell infiltrates, mainly in the spinal cord and cerebellum. In CXCR3(-/-) mice, these lesions were more widespread throughout the CNS and were diffused and poorly organized, with T cells and highly activated microglia/macrophages scattered throughout the white matter. Although the number of CD4(+) and CD8(+) T cells infiltrating the CNS were similar in CXCR3(-/-) and WT mice, Foxp3(+) regulatory T cells were significantly reduced in number and dispersed in CXCR3(-/-) mice. The expression of various chemokine and cytokine genes in the CNS was similar in CXCR3(-/-) and WT mice. The genes for the CXCR3 ligands were expressed predominantly in and/or immediately surrounding the mononuclear cell infiltrates. We conclude that in EAE, CXCR3 signaling constrains T cells to the perivascular space in the CNS and augments regulatory T cell recruitment and effector T cell interaction, thus limiting autoimmune-mediated tissue damage.     

CD1-dependent regulation of chronic central nervous system inflammation in experimental autoimmune encephalomyelitis

The existence of T cells restricted for the MHC I-like molecule CD1 is well established, but the function of these cells is still obscure; one implication is that CD1-dependent T cells regulate autoimmunity. In this study, we investigate their role in experimental autoimmune encephalomyelitis (EAE), an animal model for multiple sclerosis, using CD1-deficient mice on a C57BL/6 background. We show that CD1-/- mice develop a clinically more severe and chronic EAE compared with CD1+/+ C57BL/6 mice, which was histopathologically confirmed with increased demyelination and CNS infiltration in CD1-/- mice. Autoantigen rechallenge in vitro revealed similar T cell proliferation in CD+/+ and CD1-/- mice but an amplified cytokine response in CD1-/- mice as measured by both the Th1 cytokine IFN-gamma and the Th2 cytokine IL-4. Investigation of cytokine production at the site of inflammation showed a CNS influx of TGF-beta1-producing cells early in the disease in CD1+/+ mice, which was absent in the CD1-/- mice. Passive transfer of EAE using an autoreactive T cell line induced equivalent disease in both groups, which suggested additional requirements for activation of the CD1-dependent regulatory pathway(s). When immunized with CFA before T cell transfer, the CD1-/- mice again developed an augmented EAE compared with CD1+/+ mice. We suggest that CD1 exerts its function during CFA-mediated activation, regulating development of EAE both through enhancing TGF-beta1 production and through limiting autoreactive T cell activation, but not necessarily via effects on the Th1/Th2 balance.     

Induction of experimental autoimmune encephalomyelitis in IL-12 receptor-beta 2-deficient mice: IL-12 responsiveness is not required in the pathogenesis of inflammatory demyelination in the central nervous system

IL-12 is thought to be involved in the susceptibility to experimental autoimmune encephalomyelitis (EAE), a Th1 cell-mediated autoimmune disorder of the CNS. IL-12 signals through a heterodimeric receptor (IL-12Rbeta1/IL-12Rbeta2), whose beta2-chain is up-regulated on activated, autoreactive Th1 cells. Contrary to the expectation that the absence of IL-12Rbeta2 would protect from EAE, we found that IL-12Rbeta2-deficient mice developed earlier and more severe disease, with extensive demyelination and CNS inflammation. The inflammatory cells were mainly comprised of CD4(+) T cells, monocyte/macrophages, and dendritic cells. Compared to wild-type mice, IL-12Rbeta2-deficient mice exhibited significantly increased autoantigen-induced proliferative response and increased production of TNF-alpha, GM-CSF, IL-17, IL-18/IL-18Ralpha, and NO. In addition, we found significantly increased levels of IL-23p19 mRNA expression in spleen cells from immunized IL-12Rbeta2(-/-) mice compared with wild-type mice. These findings indicate that IL-12 responsiveness is not required in the pathogenesis of inflammatory demyelination in the CNS, and that, in the absence of IL-12Rbeta2, increased IL-23 and other inflammatory molecules may be responsible for increased severity of EAE.     

Laquinimod, a quinoline-3-carboxamide, induces type II myeloid cells that modulate central nervous system autoimmunity

Laquinimod is a novel oral drug that is currently being evaluated for the treatment of relapsing-remitting (RR) multiple sclerosis (MS). Using the animal model for multiple sclerosis, experimental autoimmune encephalomyelitis (EAE), we examined how laquinimod promotes immune modulation. Oral laquinimod treatment reversed established RR-EAE and was associated with reduced central nervous system (CNS) inflammation, decreased Th1 and Th17 responses, and an increase in regulatory T cells (Treg). In vivo laquinimod treatment inhibited donor myelin-specific T cells from transferring EAE to naive recipient mice. In vivo laquinimod treatment altered subpopulations of myeloid antigen presenting cells (APC) that included a decrease in CD11c(+)CD11b(+)CD4(+) dendritic cells (DC) and an elevation of CD11b(hi)Gr1(hi) monocytes. CD11b(+) cells from these mice exhibited an anti-inflammatory type II phenotype characterized by reduced STAT1 phosphorylation, decreased production of IL-6, IL-12/23 and TNF, and increased IL-10. In adoptive transfer, donor type II monocytes from laquinimod-treated mice suppressed clinical and histologic disease in recipients with established EAE. As effects were observed in both APC and T cell compartments, we examined whether T cell immune modulation occurred as a direct effect of laquinimod on T cells, or as a consequence of altered APC function. Inhibition of Th1 and Th17 differentiation was observed only when type II monocytes or DC from laquinimod-treated mice were used as APC, regardless of whether myelin-specific T cells were obtained from laquinimod-treated or untreated mice. Thus, laquinimod modulates adaptive T cell immune responses via its effects on cells of the innate immune system, and may not influence T cells directly.     

Adjuvant immunotherapy of experimental autoimmune encephalomyelitis: immature myeloid cells expressing CXCL10 and CXCL16 attract CXCR3+CXCR6+ and myelin-specific T cells to the draining lymph nodes rather than the central nervous system

CFA is a strong adjuvant capable of stimulating cellular immune responses. Paradoxically, adjuvant immunotherapy by prior exposure to CFA or live mycobacteria suppresses the severity of experimental autoimmune encephalomyelitis (EAE) and spontaneous diabetes in rodents. In this study, we investigated immune responses during adjuvant immunotherapy of EAE. Induction of EAE in CFA-pretreated mice resulted in a rapid influx into the draining lymph nodes (dLNs) of large numbers of CD11b(+)Gr-1(+) myeloid cells, consisting of immature cells with ring-shaped nuclei, macrophages, and neutrophils. Concurrently, a population of mycobacteria-specific IFN-γ-producing T cells appeared in the dLNs. Immature myeloid cells in dLNs expressed the chemokines CXCL10 and CXCL16 in an IFN-γ-dependent manner. Subsequently, CD4(+) T cells coexpressing the cognate chemokine receptors CXCR3 and CXCR6 and myelin oligodendrocyte glycoprotein (MOG)-specific CD4(+) T cells accumulated within the chemokine-expressing dLNs, rather than within the CNS. Migration of CD4(+) T cells toward dLN cells was abolished by depleting the CD11b(+) cells and was also mediated by the CD11b(+) cells alone. In addition to altering the distribution of MOG-specific T cells, adjuvant treatment suppressed development of MOG-specific IL-17. Thus, adjuvant immunotherapy of EAE requires IFN-γ, which suppresses development of the Th17 response, and diverts autoreactive T cells away from the CNS toward immature myeloid cells expressing CXCL10 and CXCL16 in the lymph nodes.     

Delta-like ligand 4 regulates central nervous system T cell accumulation during experimental autoimmune encephalomyelitis

Experimental autoimmune encephalomyelitis (EAE) is a CD4(+) T cell-mediated inflammatory demyelinating disease of the CNS that serves as a model for multiple sclerosis. Notch receptor signaling in T lymphocytes has been shown to regulate thymic selection and peripheral differentiation. In the current study, we hypothesized that Notch ligand-receptor interaction affects EAE development by regulating encephalitogenic T cell trafficking. We demonstrate that CNS-infiltrating myeloid dendritic cells, macrophages, and resident microglia expressed Delta-like ligand 4 (DLL4) after EAE induction. Treatment of mice with a DLL4-specific blocking Ab significantly inhibited the development of clinical disease induced by active priming. Furthermore, the treatment resulted in decreased CNS accumulation of mononuclear cells in the CNS. Anti-DLL4 treatment did not significantly alter development of effector cytokine expression by Ag-specific T cells. In contrast, anti-DLL4 treatment reduced T cell mRNA and functional cell surface expression of the chemokine receptors CCR2 and CCR6. Adoptive transfer of Ag-specific T cells to mice treated with anti-DLL4 resulted in decreased clinical severity and diminished Ag-specific CD4(+) T cell accumulation in the CNS. These results suggest a role for DLL4 regulation of EAE pathogenesis through modulation of T cell chemokine receptor expression and migration to the CNS.     

Protein kinase Ctheta controls Th1 cells in experimental autoimmune encephalomyelitis

Molecules that regulate encephalitogenic T cells are of interest for multiple sclerosis. In this study we show that protein kinase Ctheta (PKCtheta) is critical for the development of Ag-specific Th1 cells in experimental allergic encephalomyelitis (EAE), a model of multiple sclerosis. PKCtheta-deficient mice immunized with myelin oligodendrocyte glycoprotein failed to develop cell infiltrates and Th1 cytokines in the CNS and were resistant to the development of clinical EAE. CD4 T cells became primed and accumulated in secondary lymphoid organs in the absence of PKCtheta, but had severely diminished IFN-gamma, TNF, and IL-17 production. Increasing Ag exposure and inflammatory conditions failed to induce EAE in PKCtheta-deficient mice, showing a profound defect in the myelin oligodendrocyte glycoprotein-reactive T cell population. These data provide evidence of a pivotal role for PKCtheta in the generation and effector function of autoimmune Th1 cells.     

IL-17 plays an important role in the development of experimental autoimmune encephalomyelitis

IL-17 is a proinflammatory cytokine that activates T cells and other immune cells to produce a variety of cytokines, chemokines, and cell adhesion molecules. This cytokine is augmented in the sera and/or tissues of patients with contact dermatitis, asthma, and rheumatoid arthritis. We previously demonstrated that IL-17 is involved in the development of autoimmune arthritis and contact, delayed, and airway hypersensitivity in mice. As the expression of IL-17 is also augmented in multiple sclerosis, we examined the involvement of this cytokine in these diseases using IL-17(-/-) murine disease models. We found that the development of experimental autoimmune encephalomyelitis (EAE), the rodent model of multiple sclerosis, was significantly suppressed in IL-17(-/-) mice; these animals exhibited delayed onset, reduced maximum severity scores, ameliorated histological changes, and early recovery. T cell sensitization against myelin oligodendrocyte glycoprotein was reduced in IL-17(-/-) mice upon sensitization. The major producer of IL-17 upon treatment with myelin digodendrocyte glycopritein was CD4+ T cells rather than CD8+ T cells, and adoptive transfer of IL-17(-/-) CD4+ T cells inefficiently induced EAE in recipient mice. Notably, IL-17-producing T cells were increased in IFN-gamma(-/-) cells, while IFN-gamma-producing cells were increased in IL-17(-/-) cells, suggesting that IL-17 and IFN-gamma mutually regulate IFN-gamma and IL-17 production. These observations indicate that IL-17 rather than IFN-gamma plays a crucial role in the development of EAE.