IL-27 has been shown to play a suppressive role in experimental autoimmune encephalomyelitis (EAE) as demonstrated by more severe disease in IL-27R-deficient (WSX-1−/−) mice. However, whether IL-27 influences the induction or effector phase of EAE is unknown. This is an important question as therapies for autoimmune diseases are generally started after autoreactive T cells have been primed. In this study, we demonstrate maximal gene expression of IL-27 subunits and its receptor in the CNS at the effector phases of relapsing-remitting EAE including disease peak and onset of relapse. We also show that activated astrocyte cultures secrete IL-27p28 protein which is augmented by the endogenous factor, IFN-γ. To investigate functional significance of a correlation between gene expression and disease activity, we examined the effect of IL-27 at the effector phase of disease using adoptive transfer EAE. Exogenous IL-27 potently suppressed the ability of encephalitogenic lymph node and spleen cells to transfer EAE. IL-27 significantly inhibited both nonpolarized and IL-23-driven IL-17 production by myelin-reactive T cells thereby suppressing their encephalitogenicity in adoptive transfer EAE. Furthermore, we demonstrate a strong suppressive effect of IL-27 on active EAE in vivo when delivered by s.c. osmotic pump. IL-27-treated mice had reduced CNS inflammatory infiltration and, notably, a lower proportion of Th17 cells. Together, these data demonstrate the suppressive effect of IL-27 on primed, autoreactive T cells, particularly, cells of the Th17 lineage. IL-27 can potently suppress the effector phase of EAE in vivo and, thus, may have therapeutic potential in autoimmune diseases such as multiple sclerosis.

Our results indicate that diminished Th17 and Th1/17 responses, rather than Th1 responses, are particularly relevant to the abrogation of new relapsing disease activity observed in this cohort of patients with aggressive MS following chemoablation and HSCT.

Experimental autoimmune encephalomyelitis is a well-characterized model of cell-mediated autoimmunity. TLRs expressed on APCs recognize microbial components and induce innate immune responses, leading to the elimination of invading infectious agents. Certain TLR agonists have been reported to have adjuvant properties in CNS autoimmune inflammatory demyelination. We report in this study that TLR3 stimulation by polyinosinic-polycytidylic acid, a double-stranded RNA analog, suppresses relapsing demyelination in a murine experimental autoimmune encephalomyelitis model. Disease suppression is associated with the induction of endogenous IFN-β and the peripheral induction of the CC chemokine CCL2. These data indicate that a preferential activation of the MyD88-independent, type I IFN-inducing TLR pathway has immunoregulatory potential in this organ-specific autoimmune disease.

In autoimmunity, the balance of different helper T (Th) cell subsets can influence the tissue damage caused by autoreactive T cells. Pro-inflammatory Th1 and Th17 T cells are implicated as mediators of several human autoimmune conditions such as multiple sclerosis (MS). Autologous hematopoietic stem cell transplantation (aHSCT) has been tested in phase 2 clinical trials for MS patients with aggressive disease. Abrogation of new clinical relapses and brain lesions can be seen after ablative aHSCT, accompanied by significant reductions in Th17, but not Th1, cell populations and activity. The cause of this selective decrease in Th17 cell responses following ablative aHSCT is not completely understood. We identified an increase in the kinetics of natural killer (NK) cell reconstitution, relative to CD4+ T cells, in MS patients post-aHSCT, resulting in an increased NK cell:CD4+ T cell ratio that correlated with the degree of decrease in Th17 responses. Ex vivo removal of NK cells from post-aHSCT peripheral blood mononuclear cells resulted in higher Th17 cell responses, indicating that NK cells can regulate Th17 activity. NK cells were also found to be cytotoxic to memory Th17 cells, and this toxicity is mediated through NKG2D-dependent necrosis. Surprisingly, NK cells induced memory T cells to secrete more IL-17A. This was preceded by an early rise in T cell expression of RORC and IL17A mRNA, and could be blocked with neutralizing antibodies against CD58, a costimulatory receptor expressed on NK cells. Thus, NK cells provide initial co-stimulation that supports the induction of a Th17 response, followed by NKG2D-dependent cytotoxicity that limits these cells. Together these data suggest that rapid reconstitution of NK cells following aHSCT contribute to the suppression of the re-emergence of Th17 cells. This highlights the importance of NK cells in shaping the reconstituting immune system following aHSCT in MS patients.

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Multiple sclerosis (MS) is characterized by inflammation within the CNS. This inflammatory response is associated with production of nitric oxide (NO) and NO-related species that nitrosylate thiols. We postulated that MS patients would exhibit an antibody (Ab) response directed against proteins containing S-nitrosocysteine (SNO-cysteine) and showed that anti-NO-cysteine Abs of the IgM isotype are in fact present in the sera of some MS patients (Boullerne et al., 1995). We report here the presence of a seemingly identical Ab response directed against SNO-cysteine in an acute model of MS, experimental autoimmune encephalomyelitis (EAE) induced in Lewis rats with the 68-84 peptide of guinea pig myelin basic protein (MBP(68-84)). Serum levels of anti-SNO-cysteine Abs peaked 1 week before the onset of clinical signs and well before the appearance of anti-MBP(68-84) Abs. The anti-SNO-cysteine Ab peak titer correlated with the extent of subsequent CNS demyelination, suggesting a link between Ab level and CNS lesion formation. In relapsing-remitting MS patients, we found elevated anti-SNO-cysteine Ab at times of relapse and normal values in most patients judged to be in remission. Two-thirds of patients with secondary progressive MS had elevated anti-SNO-cysteine Ab levels, including those receiving interferon beta-1b. The data show that a rise in circulating anti-SNO-cysteine Ab levels precedes onset of EAE. Anti-SNO-cysteine Abs are also elevated at times of MS attacks and in progressive disease, suggesting a possible role for these Abs, measurable in blood, as a biological marker for clinical activity.

The Bowman-Birk inhibitor (BBI) is a soybean-derived serine protease inhibitor. BBI concentrate (BBIC) is an extract enriched with BBI, but predominantly contains other ingredients including several protease inhibitors. We previously found that BBIC administration to Lewis rats with experimental autoimmune encephalomyelitis (EAE) significantly suppresses disease. In the present study we determined whether BBI mediates the suppressive effects of BBIC in EAE, evaluated its potential neuroprotective effects, and investigated mechanisms of BBI action. We tested effects of purified BBI on clinical and histopathological parameters of EAE in two models (relapsing/remitting EAE in SJL/J mice and chronic EAE in C57BL/6 mice). Effects of BBI were compared to BBIC in relapsing/remitting EAE, and effects of BBI on neuronal survival were examined during acute optic neuritis. Treatment with BBI in both EAE models significantly improved EAE disease parameters (onset, severity, weight loss, inflammation and demyelination). BBI significantly reduced the incidence of optic neuritis and prevented loss of retinal ganglion cells. In most experiments proliferation of immune cells derived from BBI-treated mice was significantly lower relative to control groups. Using Boyden's chamber assay we found that BBI inhibited invasiveness of activated splenocytes through the matrigel barrier. BBI also induced higher production of EAE-suppressive cytokine IL-10 by immune cells. These results demonstrate that BBI is the active component of BBIC that ameliorates clinical EAE. BBI reduces inflammation and attenuates neuronal loss, making it an excellent candidate for oral therapy in MS. BBI likely ameliorates EAE by inhibiting multiple pathways involved in disease pathogenesis.

In multiple sclerosis (MS) the mechanisms of injury caused by peroxynitrite remain uncertain. To study histological, ultra structural and molecular alterations caused by peroxynitrite in brain, the peroxynitrite donor 3-morpholinosydnonimine was injected in rat corpus callosum. Peroxynitrite induces strong primary axonal damage with characteristics of primary acute axonopathy, together with severe myelin alteration, myelin vacuolation and demyelination, and nitrotyrosine formation as confirmed by detection of nitrosated target proteins. Administration of the peroxynitrite scavenger uric acid inhibited these effects. In vivo, peroxynitrite leads to a disorganisation of myelin and to axonal damage presenting some similarities to the formation of MS lesions. Understanding the action of peroxynitrite in this process will open new therapeutic strategies by specific inhibition of peroxynitrite formation and action.