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Background Accumulating evidence indicate that B cells can exhibit pro- or anti-inflammatory activities. Similar to interleukin (IL)-10–competent B cells, we recently showed that transforming growth factor (TGF)-β1-producing regulatory B cells limit the induction of autoimmune neuroinflammation in mice, making them potentially important in maintaining peripheral immune tolerance in central nervous system inflammatory demyelinating disorders such as multiple sclerosis. Methods In this study, we compared B cell production of TGF-β1 and IL-10, the two most studied regulatory cytokines, and the pro-inflammatory B cell-derived IL-6 and tumor necrosis factor cytokines under basal conditions and following polyclonal stimulation with dual B cell receptor (BCR) cross-linking and Toll-like receptor (TLR)9 engagement. Results We showed that resting TGF-β1–producing B cells fall within both the naïve (CD27−) and memory (CD27+) B cell compartments. We found no spontaneous B cell-derived IL-10, IL-6 or tumor necrosis factor (TNF) production. Human B cell activation with anti-Ig antibodies plus CPG-B leads to only modest IL-10 production by memory CD19+CD27+ B cells while expression levels of IL-6 and TNF by both naive and memory B cells were strongly induced. Remarkably, stimulated B cells showed significantly reduced capacity to produce TGF-β1. Conclusions These findings indicate that B cell activation may facilitate the development of excessive immune responses and autoimmunity by restricting B cell-derived TGF-β1 production by resting B cells and favoring in turns the proinflammatory actions of activated cytokine-producing B cells. Electronic supplementary material The online version of this article (doi:10.1186/s12974-017-0798-5) contains supplementary material, which is available to authorized users.

Studies in experimental autoimmune encephalomyelitis (EAE), a murine model of multiple sclerosis (MS), have shown that regulatory B cells modulate the course of the disease via the production of suppressive cytokines. While data indicate a role for transforming growth factor (TGF)-β1 expression in regulatory B cell functions, this mechanism has not yet been tested in autoimmune neuroinflammation. Transgenic mice deficient for TGF-β1 expression in B cells (B–TGF-β1−/−) were tested in EAE induced by recombinant mouse myelin oligodendrocyte glycoprotein (rmMOG). In this model, B–TGF-β1−/− mice showed an earlier onset of neurologic impairment compared to their littermate controls. Exacerbated EAE susceptibility in B–TGF-β1−/− mice was associated with augmented CNS T helper (Th)1/17 responses. Moreover, selective B cell TGF-β1–deficiency increased the frequencies and activation of myeloid dendritic cells, potent professional antigen-presenting cells (APCs), suggesting that B cell-derived TGF-β1 can constrain Th1/17 responses through inhibition of APC activity. Collectively our data suggest that B cells can down-regulate the function of APCs, and in turn encephalitogenic Th1/17 responses, via TGF-β1, findings that may be relevant to B cell-targeted therapies.

CD8(+) cytotoxic T lymphocytes (CTLs) are critical mediators of anti-tumor immunity, and controlling the mechanisms that govern CTL functions could be crucial for enhancing patient outcome. Previously, we reported that hepatocyte growth factor (HGF) limits effective murine CTL responses via antigen-presenting cells. Here, we show that a fraction of murine effector CTLs expresses the HGF receptor c-Met (c-Met(+) CTLs). Phenotypic and functional analysis of c-Met(+) CTLs reveals that they display enhanced cytolytic capacities compared to their c-Met(-) CTL counterparts. Furthermore, HGF directly restrains the cytolytic function of c-Met(+) CTLs in cell-mediated cytotoxicity reactions in vitro and in vivo and abrogates T-cell responses against metastatic melanoma in vivo Finally, we establish in three murine tumor settings and in human melanoma tissues that c-Met(+) CTLs are a naturally occurring CD8(+) T-cell population. Together, our findings suggest that the HGF/c-Met pathway could be exploited to control CD8(+) T-cell-mediated anti-tumor immunity.

sIL-1Ra production induced by IFN-β in human monocytes requires the activation of MEK2 upstream of PI3Kδ, MEK1, and ERK1/2 activation being dispensable. IFN-β and sIL-1Ra play crucial roles in the regulation of innate immunity and inflammation. IFN-β, which is widely used to improve the course of relapsing, remitting multiple sclerosis, induces the production of sIL-1Ra in human monocytes through mechanisms that remain largely unknown. In this study, we identified PI3Kδ and MEK2 as key elements that control sIL-1Ra production in isolated human monocytes activated by IFN-β. Blockade of MEK2, but not of MEK1, by inhibitors and siRNA prevented IFN-β-induced PI3Kδ recruitment to the membrane, Akt phosphorylation, and sIL-1Ra production, suggesting that MEK2 acted upstream of PI3Kδ. Furthermore, ERK1/2, the only identified substrates of MEK1/2 to date, are dispensable for sIL-1Ra production in response to IFN-β stimulation. Upon IFN-β activation, MEK2 and PI3Kδ are translocated to monocyte membranes. These data suggest that MEK1 and MEK2 display different, nonredundant functions in IFN-β signaling. That neither MEK1 nor ERK1/2 play a part in this mechanism is also an unexpected finding that gives rise to a better understanding of the MAPK signaling network. Together, these findings demonstrate that IFN-β triggers an atypical MEK2/PI3Kδ signaling cascade to regulate sIL-1Ra expression in monocytes. The premise that MEK1 and MEK2 play a part in the induction of the proinflammatory cytokine, IL-1β in human monocytes provides a rationale for an alternative, IFN-β-mediated pathway to induce/enhance sIL-1Ra production and thus, to dampen inflammation.

Objective Clinical studies indicate that anti-CD20 B cell depletion may be an effective multiple sclerosis therapy. We investigated mechanisms of its immune modulation using two paradigms of experimental autoimmune encephalomyelitis (EAE).

Glatiramer acetate (GA), an immunomodulator used in multiple sclerosis (MS) therapy, induces the production of secreted IL-1 receptor antagonist (sIL-1Ra), a natural inhibitor of IL-1beta, in human monocytes, and in turn enhances sIL-1Ra circulating levels in MS patients. GA is a mixture of peptides with random Glu, Lys, Ala, and Tyr sequences of high polarity and hydrophilic nature that is unlikely to cross the blood-brain barrier. In contrast, sIL-1Ra crosses the blood-brain barrier and, in turn, may mediate GA anti-inflammatory activities within the CNS by counteracting IL-1beta activities. Here we identify intracellular signaling pathways induced by GA that control sIL-1Ra expression in human monocytes. By using kinase knockdown and specific inhibitors, we demonstrate that GA induces sIL-1Ra production via the activation of PI3Kdelta, Akt, MEK1/2, and ERK1/2, demonstrating that both PI3Kdelta/Akt and MEK/ERK pathways rule sIL-1Ra expression in human monocytes. The pathways act in parallel upstream glycogen synthase kinase-3alpha/beta (GSK3alpha/beta), the knockdown of which enhances sIL-1Ra production. Together, our findings demonstrate the existence of signal transduction triggered by GA, further highlighting the mechanisms of action of this drug in MS.

Deregulated production of cytokines, including IL-1beta, IL-6 and TNF plays an important role in chronic inflammation. Relevant to this condition, direct cellular contact with stimulated T cells is a potent inducer of cytokine production in human monocytes/macrophages. We previously demonstrated that PI3Ks regulate differential production of IL-1beta and its specific inhibitor secreted IL-1 receptor antagonist (sIL-1Ra) by human monocytes. Here we show that in contrast with PI3Kalpha, beta and gamma, PI3Kdelta accounts for most of the PI3K-dependent signaling ruling the production of IL-1beta, IL-6, TNF and sIL-1Ra in monocytes activated by cellular contact with stimulated T cells (mimicked by CHAPS-solubilized membranes of stimulated T cells, CE sHUT) and lipopolysaccharides (LPS); the latter stimuli being relevant to chronic/sterile and acute/infectious inflammation, respectively. Interestingly, PI3Kdelta activity dampened the production of pro-inflammatory cytokines in LPS-activated monocytes, but induced it in CE sHUT-activated cells. In both CE sHUT- and LPS-activated monocytes PI3Kdelta regulated cytokine transcript expression through the phosphorylation/inactivation of glycogen synthase kinase-3beta (GSK3beta). The blockade of GSK3beta displayed inverse effects to those of PI3Kdelta blockade. Thus, by displaying opposite functions in conditions mimicking chronic/sterile and acute/infectious inflammation, i.e., by repressing pro-inflammatory cytokine expression in LPS-activated monocytes but inducing such mediators in T cell contact-activated monocytes, PI3Kdelta represents a potential therapeutic target specific to chronic/sterile inflammatory conditions.

The unbalanced production of IL-1β and its natural, specific inhibitor, the secreted IL-1R antagonist (sIL-1Ra), plays an important role in chronic/sterile inflammation. Relevant to this condition is direct cellular contact with stimulated T cells which is a potent inducer of cytokine production in human monocytes/macrophages. We previously demonstrated that activation of PI3Ks is a prerequisite of the transcription of the sIL-1Ra gene in human monocytes activated by IFN-β. In this study, we addressed the question of PI3K involvement in the production of IL-1β and sIL-1Ra in monocytes activated by cellular contact with stimulated T cells (mimicked by CHAPS-solubilized membranes of stimulated T cells (CEsHUT)), and a crude preparation of LPS, to compare stimuli relevant to chronic/sterile and acute/infectious inflammation, respectively. In monocytes activated by either CEsHUT or LPS, the inhibition of PI3Ks abrogated sIL-1Ra transcript expression and sIL-1Ra production, demonstrating that PI3Ks control the induction of sIL-1Ra gene transcription. In contrast, PI3K inhibition increased the production of IL-1β protein in both CEsHUT- and LPS-activated monocytes, the enhancement being drastically higher in the former. This was not due to changes in IL-1β mRNA steady-state levels or transcript stability, but to the involvement of PI3Ks in the repression of IL-1β secretion. The downstream PI3K effector, Akt, was implicated in this process. The present results demonstrate that PI3Ks are involved in the inhibition of IL-1β secretion and in the induction of sIL-1Ra production in human blood monocytes by controlling different mechanisms in conditions mimicking chronic/sterile (CEsHUT) and acute/infectious (LPS) inflammation.

An imbalance in cytokine homeostasis is considered to play a major part in the pathogenesis of immuno-inflammatory diseases. Since the identification and cloning of cytokines and their receptors, therapeutic approaches have been developed with the purpose of impeding the interaction between the ligand (cytokine) and its specific receptor, or interactions that involve the use of anti-inflammatory cytokines to switch off inflammation. Although some diseases have been treated successfully with cytokines or anticytokines (i.e., anti-TNF, and to a lesser extent recombinant IL-1 receptor antagonist, in rheumatoid arthritis; IFN-beta in multiple sclerosis), the fact remains that these therapies do not abrogate the concomitant use of steroids or immunosuppressive drugs, and that a significant percentage of patients do not respond to such therapies; these are important limitations. The identification of signalling pathways preferentially used in inflammatory conditions has boosted approaches that target these intracellular mechanisms. This review examines the different therapeutic approaches that may be considered for the treatment of immuno-inflammatory diseases, and discusses the advantages and disadvantages of targeting extracellular or intracellular mechanisms.

IFN-β induces the production of secreted IL-1R antagonist (sIL-1Ra) without triggering synthesis of the agonist IL-1β in human monocytes. This might account for its anti-inflammatory properties. Canonically, IFN-β signals through activation of JAK/STAT pathway, although PI3K and MAPK have also been involved. In this study, the role of PI3K, MEK1, and STAT1 in IFN-β-induced sIL-1Ra production is investigated in freshly isolated human blood monocytes. PI3K, but not MEK1 activation is essential for sIL-1Ra production in monocytes treated with IFN-β, as demonstrated by using the respective inhibitors of PI3K and MEK1, Ly294002 and PD98059. The use of cycloheximide and actinomycin D shows that sIL-1Ra was an immediate early gene induced by IFN-β and that PI3K was controlling sIL-1Ra gene transcription. Although both inhibitors of PI3K and MEK1 diminished the Ser727 phosphorylation of STAT1 induced by IFN-β, only Ly294002 inhibited sIL-1Ra production. Furthermore, the inhibition of STAT1-Ser727 phosphorylation by Ly294002 did not affect STAT1 translocation, suggesting that STAT1 was not involved in sIL-1Ra gene induction. This was confirmed in monocytes that were transfected with small interfering RNA specifically targeting STAT1. Indeed, monocytes in which effective STAT1 gene knockdown was achieved were fully responsive to IFN-β in terms of sIL-1Ra production. Taken together, the present data demonstrate that the induction of sIL-1Ra transcription and production by IFN-β in human monocytes involved PI3K, but not STAT1 activation.

Multiple sclerosis (MS) is an immune-mediated disease improved by interferon-beta (IFNbeta) therapy. IFNbeta may owe its anti-inflammatory property to its ability to induce interleukin-1 receptor antagonist (IL-1Ra) without triggering IL-1beta synthesis in human monocytes. Furthermore, we recently demonstrated that IFNbeta inhibits the production of IL-1beta and tumor necrosis factor-alpha (TNF) in human monocytes activated by cellular contact with stimulated T cells, a mechanism which we suspected of playing an important part in the pathogenesis of chronic inflammatory diseases including MS. Here we compare modulatory effects of IFNbeta on the production of proinflammatory cytokines (IL-1beta, IL-1alpha, TNF, and IL-6) and IL-1Ra in human monocytes stimulated by lipopolysaccharides (LPS) and isolated plasma membranes of stimulated T cells (msHUT), which are likely to reflect monocyte activation in acute and chronic inflammation, respectively. In monocytes activated by either LPS or msHUT, IFNbeta did not modulate the secretion of IL-1alpha and IL-6, but it enhanced the production of IL-1Ra in a dose-dependent manner. However, in monocytes activated by msHUT, the expression of cell-associated and intracellular IL-1alpha was inhibited by IFNbeta, correlating with the inhibition of IL-1alpha transcript. IFNbeta inhibited the expression (mRNA) and production (protein) of IL-1beta and TNF, while enhancing those of IL-1Ra in monocytes activated by msHUT. In contrast, in monocytes activated by LPS, IFNbeta enhanced the expression and production of IL-1beta, TNF, and IL-1Ra, suggesting that it did not display anti-inflammatory properties in these conditions. This study demonstrates that IFNbeta displays opposite effects depending on the type of activation of human monocytes, suggesting that it may affect different pathogenic mechanisms in opposite ways.

Hepatocyte growth factor (HGF) is a pleiotropic cytokine that has been extensively studied over several decades, but was only recently recognized as a key player in mediating protection of many types of inflammatory and autoimmune diseases. HGF was reported to prevent and attenuate disease progression by influencing multiple pathophysiological processes involved in inflammatory and immune response, including cell migration, maturation, cytokine production, antigen presentation, and T cell effector function. In this review, we discuss the actions and mechanisms of HGF in inflammation and immunity and the therapeutic potential of this factor for the treatment of inflammatory and autoimmune diseases.

OBJECTIVE: Glatiramer acetate (GA; Copaxone), a disease-modifying therapy for multiple sclerosis (MS), promotes development of anti-inflammatory (M2, type II) monocytes that can direct differentiation of regulatory T cells. We investigated the innate immune signaling pathways that participate in GA-mediated M2 monocyte polarization. METHODS: Monocytes were isolated from myeloid differentiation primary response gene 88 (MyD88)-deficient, Toll-IL-1 receptor domain-containing adaptor inducing interferon (IFN)-β (TRIF)-deficient, IFN-α/β receptor subunit 1 (IFNAR1)-deficient, and wild-type (WT) mice and human peripheral blood. GA-treated monocytes were stimulated with Toll-like receptor ligands, then evaluated for activation of kinases and transcription factors involved in innate immunity, and secretion of proinflammatory cytokines. GA-treated mice were evaluated for cytokine secretion and susceptibility to experimental autoimmune encephalomyelitis. RESULTS: GA-mediated inhibition of proinflammatory cytokine production by monocytes occurred independently of MyD88 and nuclear factor-κB, but was blocked by TRIF deficiency. Furthermore, GA did not provide clinical benefit in TRIF-deficient mice. GA inhibited activation of p38 mitogen-activated protein kinase, an upstream regulator of activating transcription factor (ATF)-2, and c-Jun N-terminal kinase 1, which regulates IFN regulatory factor 3 (IRF3). Consequently, nuclear translocation of ATF-2 and IRF3, components of the IFN-β enhanceosome, was impaired. Consistent with these observations, GA inhibited production of IFN-β in vivo in WT mice, but did not modulate proinflammatory cytokine production by monocytes from IFNAR1-deficient mice. CONCLUSION: Our results demonstrate that GA inhibits the type I IFN pathway in M2 polarization of monocytes independently of MyD88, providing an important mechanism connecting innate and adaptive immune modulation in GA therapy and valuable insight regarding its potential use with other MS treatments. peerReviewed

Hepatocyte growth factor (HGF) limits mouse autoimmune neuroinflammation by promoting the development of tolerogenic dendritic cells (DCs). Given the role played by DCs in the establishment of immunological tolerance, agents that coerce DCs to adopt a protolerogenic function are currently under investigation for multiple sclerosis (MS) therapy. Here, we studied the immunomodulatory effects of HGF on DCs derived from human monocytes. DCs differentiated in the presence of HGF adopt a protolerogenic phenotype with increased ability to generate regulatory T cells, a property that might be exploited therapeutically in T cell-mediated immune disorders such as MS.

Antigen presentation, but not antibody secretion, by B cells drives CNS autoimmunity induced by immunization with human MOG., Whether B cells serve as antigen-presenting cells (APCs) for activation of pathogenic T cells in the multiple sclerosis model experimental autoimmune encephalomyelitis (EAE) is unclear. To evaluate their role as APCs, we engineered mice selectively deficient in MHC II on B cells (B–MHC II−/−), and to distinguish this function from antibody production, we created transgenic (Tg) mice that express the myelin oligodendrocyte glycoprotein (MOG)–specific B cell receptor (BCR; IgHMOG-mem) but cannot secrete antibodies. B–MHC II−/− mice were resistant to EAE induced by recombinant human MOG (rhMOG), a T cell– and B cell–dependent autoantigen, and exhibited diminished Th1 and Th17 responses, suggesting a role for B cell APC function. In comparison, selective B cell IL-6 deficiency reduced EAE susceptibility and Th17 responses alone. Administration of MOG-specific antibodies only partially restored EAE susceptibility in B–MHC II−/− mice. In the absence of antibodies, IgHMOG-mem mice, but not mice expressing a BCR of irrelevant specificity, were fully susceptible to acute rhMOG-induced EAE, also demonstrating the importance of BCR specificity. Spontaneous opticospinal EAE and meningeal follicle–like structures were observed in IgHMOG-mem mice crossed with MOG-specific TCR Tg mice. Thus, B cells provide a critical cellular function in pathogenesis of central nervous system autoimmunity independent of their humoral involvement, findings which may be relevant to B cell–targeted therapies.

BACKGROUND: Interleukin (IL)-6 is recognised as an important cytokine involved in inflammatory diseases of the central nervous system (CNS). OBJECTIVE: To perform a large retrospective study designed to test cerebrospinal fluid (CSF) IL-6 levels in the context of neurological diseases, and evaluate its usefulness as a biomarker to help discriminate multiple sclerosis (MS) from other inflammatory neurological diseases (OIND). PATIENTS AND METHODS: We analyzed 374 CSF samples for IL-6 using a quantitative enzyme-linked immunosorbent assay. Groups tested were composed of demyelinating diseases of the CNS (DD, n = 117), including relapsing-remitting MS (RRMS, n = 65), primary progressive MS (PPMS, n = 11), clinically isolated syndrome (CIS, n = 11), optic neuritis (ON, n = 30); idiopathic transverse myelitis (ITM, n = 10); other inflammatory neurological diseases (OIND, n = 35); and non-inflammatory neurological diseases (NIND, n = 212). Differences between groups were analysed using Kruskal-Wallis test and Mann-Whitney U-test. RESULTS: CSF IL-6 levels exceeded the positivity cut-off of 10 pg/ml in 18 (51.4%) of the 35 OIND samples, but in only three (3.9%) of the 76 MS samples collected. CSF IL-6 was negative for all NIND samples tested (0/212). IL-6 cut-off of 10 pg/ml offers 96% sensitivity to exclude MS. CONCLUSION: CSF IL-6 may help to differentiate MS from its major differential diagnosis group, OIND.

Interferon-β is a first-line therapy used to prevent relapses in relapsing-remitting multiple sclerosis. The clinical benefit of interferon-β in relapsing-remitting multiple sclerosis is attributed to its immunomodulatory effects on inflammatory mediators and T cell reactivity. Here, we evaluated the production of hepatocyte growth factor, a neuroprotective and neuroinflammation-suppressive mediator, by peripheral blood mononuclear cells collected from interferon-β--treated relapsing-remitting multiple sclerosis patients, relapsing remitting multiple sclerosis patients not treated with interferon-β, and healthy volunteers. Using intracellular flow cytometry analysis, increased production of hepatocyte growth factor was observed in circulating CD14(+) monocytes from patients undergoing long-term treatment with interferon-β versus untreated patients. Complementary in vitro studies confirmed that treatment with interferon-β induced rapid and transient transcription of the hepatocyte growth factor gene in CD14(+) monocytes and that intracellular and secreted monocytic hepatocyte growth factor protein levels were markedly stimulated by interferon-β treatment. Additional exploration revealed that "pro-inflammatory" (CD14(+)CD16(+)) monocytes produced similar levels of hepatocyte growth factor in response to interferon-β as "classical" (CD14(+)CD16(-)) monocytes, and that CD14(+) monocytes but not CD4(+) T cells express the hepatocyte growth factor receptor c-Met. Our findings suggest that interferon-β may mediate some of its therapeutic effects in relapsing-remitting multiple sclerosis through the induction of hepatocyte growth factor by blood monocytes by coupling immune regulation and neuroprotection.

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.

Neurological deficit in experimental autoimmune encephalomyelitis (EAE) and multiple sclerosis is widely considered to be a consequence of synergistic T and B cell responses to central nervous system (CNS) antigens. We show that mice immunized with encephalitogenic myelin oligodendrocyte glycoprotein (MOG(35-55)) peptide develop significant serum levels of anti-MOG antibodies in parallel with disease progression. Furthermore, EAE mice developed antibodies against DNA and RNA, a serological hallmark observed in autoimmune diseases such as systemic lupus erythematosus. The presence of anti-nucleic responsive B cells and antibodies during EAE may highlight a previously unappreciated mechanism in the pathogenesis of CNS autoimmunity.

Clinical studies indicate that anti-CD20 B-cell depletion may be an effective multiple sclerosis (MS) therapy. We investigated mechanisms of anti-CD20-mediated immune modulation using 2 paradigms of experimental autoimmune encephalomyelitis (EAE).Murine EAE was induced by recombinant myelin oligodendrocyte glycoprotein (rMOG), a model in which B cells are considered to contribute pathogenically, or MOG peptide (p)35-55, which does not require B cells.In EAE induced by rMOG, B cells became activated and, when serving as antigen-presenting cells (APCs), promoted differentiation of proinflammatory MOG-specific Th1 and Th17 cells. B-cell depletion prevented or reversed established rMOG-induced EAE, which was associated with less central nervous system (CNS) inflammation, elimination of meningeal B cells, and reduction of MOG-specific Th1 and Th17 cells. In contrast, in MOG p35-55-induced EAE, B cells did not become activated or efficiently polarize proinflammatory MOG-specific T cells, similar to naive B cells. In this setting, anti-CD20 treatment exacerbated EAE, and did not impede development of Th1 or Th17 cells. Irrespective of the EAE model used, B-cell depletion reduced the frequency of CD4(+)CD25(+)Foxp3(+) regulatory T cells (Treg), and increased the proinflammatory polarizing capacity of remaining myeloid APCs.Our study highlights distinct roles for B cells in CNS autoimmunity. Clinical benefit from anti-CD20 treatment may relate to inhibition of proinflammatory B cell APC function. In certain clinical settings, however, elimination of unactivated B cells, which participate in regulation of T cells and other APC, may be undesirable. Differences in immune responses to MOG protein and peptide may be important considerations when choosing an EAE model for testing novel B cell-targeting agents for MS.

Mechanisms of action as well as cellular targets of glatiramer acetate (GA) in multiple sclerosis (MS) are still not entirely understood. IL-1beta is present in CNS-infiltrating macrophages and microglial cells and is an important mediator of inflammation in experimental autoimmune encephalitis (EAE), the MS animal model. A natural inhibitor of IL-1beta, the secreted form of IL-1 receptor antagonist (sIL-1Ra) improves EAE disease course. In this study we examined the effects of GA on the IL-1 system. In vivo, GA treatment enhanced sIL-1Ra blood levels in both EAE mice and patients with MS, whereas IL-1beta levels remained undetectable. In vitro, GA per se induced the transcription and production of sIL-1Ra in isolated human monocytes. Furthermore, in T cell contact-activated monocytes, a mechanism relevant to chronic inflammation, GA strongly diminished the expression of IL-1beta and enhanced that of sIL-1Ra. This contrasts with the effect of GA in monocytes activated upon acute inflammatory conditions. Indeed, in LPS-activated monocytes, IL-1beta and sIL-1Ra production were increased in the presence of GA. These results demonstrate that, in chronic inflammatory conditions, GA enhances circulating sIL-1Ra levels and directly affects monocytes by triggering a bias toward a less inflammatory profile, increasing sIL-1Ra while diminishing IL-1beta production. This study sheds light on a mechanism that is likely to participate in the therapeutic effects of GA in MS.

Imbalance in cytokine homeostasis plays an important part in the pathogenesis of chronic inflammatory diseases such as multiple sclerosis and rheumatoid arthritis. We demonstrated that T cells might exert a pathological effect through direct cellular contact with human monocytes/macrophages, inducing a massive up-regulation of the prototypical proinflammatory cytokines IL-1β and TNF. This mechanism that might be implicated in chronic inflammation is specifically inhibited by high-density lipoproteins (HDL). Like many other stimuli, besides proinflammatory cytokines, the contact-mediated activation of monocytes induces the production of cytokine inhibitors such as the secreted form of the IL-1 receptor antagonist (sIL-1Ra). The present study demonstrates that stimulated T cells generate microparticles (MP) that induce the production of TNF, IL-1β, and sIL-1Ra in human monocytes; the production of TNF and IL-1β but not that of sIL-1Ra is inhibited in the presence of HDL. The results were similar when monocytes were stimulated by whole membranes of T cells or soluble extracts of the latter. This suggests that MP carry similar monocyte-activating factors to cells from which they originate. Thus, by releasing MP, T cells might convey surface molecules similar to those involved in the activation of monocytes by cellular contact. By extension, MP might affect the activity of cells, which are usually not in direct contact with T cells at the inflammatory site. Furthermore, this study demonstrates that HDL exert an anti-inflammatory effect in nonseptic activation of human monocytes, not only by inhibiting the production of IL-1β and TNF but also, by leaving sIL-1Ra production unchanged.

IL-21 is a key T-cell growth factor (TCGF) involved in innate and adaptive immune response. It contributes to the proliferation of naive, but not memory T lymphocytes. However, the full spectrum of IL-21 activity on T cells remains unclear. Here, we demonstrate that IL-21 primarily maintains the expression of specific naive cell surface markers such as CD45RA, CD27, CD62L and CCR7 on human CD4(+) T lymphocytes and that the expression of CCR7 induces cell migration by means of CCL21 chemoattraction. These effects contrast with those of IL-2 which induced the marked proliferation of CD4(+) T lymphocytes, leading to an activated-memory phenotype. Nevertheless, IL-21 maintained cell cycle activation and expression of proliferation markers, including proliferating cell nuclear antigen and Ki-67, and triggered T-cell proliferation via TCR and co-stimulation pathways. Unlike IL-2, IL-21 decreased the expression of the anti-apoptotic Bcl-2 protein, which correlated with the absence of activation of the phosphatidylinositol 3'-kinase/Akt signaling pathway. Thus, IL-21 is a TCGF whose function is the preservation of a pool of CD4(+) T lymphocytes in a naive phenotype, with a low proliferation rate but with the persistence of cell cycling proteins and cell surface expression of CCR7. These findings strongly suggest that IL-21 plays a part in innate and adaptive immune response owing to homeostasis of T cells and their homing to secondary lymphoid organs.

Cellular contact with stimulated T cells potently induces cytokine production in monocytes, a mechanism that is likely to be relevant to chronic inflammation. Although the identity of surface molecules involved in this process remains elusive, CD40 and its ligand, CD40L, are thought to be implicated, considering that they are expressed at the inflammatory site. To ascertain the involvement of CD40L, we compared the activation of three different types of human monocytic cells, i.e., freshly isolated monocytes, monocytes primed with IFN-γ (IFN-γ-macrophages), and THP-1 cells. These cells were activated by either membranes isolated from stimulated T cells (HUT-78 or T lymphocytes) to mimic cellular contact, soluble extracts from isolated membranes, or CD40L trimer (CD40LT). The production of TNF and IL-1β was induced by membranes of stimulated T cells in the three types of target cells, whereas CD40LT induced TNF production in IFN-γ-macrophages only. Similar results were obtained with soluble extracts of T cell membranes, demonstrating that the difference between membranes and CD40LT was not due to the particulate form of membranes. CD40LT induced neither transcript nor protein of cytokines in monocytes, whereas in IFN-γ-macrophages, IL-1β and TNF mRNA were observed, but only TNF was measured in cell supernatants. Finally, anti-CD40L Abs failed to inhibit TNF and IL-1β production induced in IFN-γ-macrophages by solubilized membranes, whereas TNF production induced by CD40LT was inhibited. These results demonstrate that CD40L is not required in monocyte activation by direct cellular contact with stimulated T cells, although soluble CD40LT induces the production of TNF in IFN-γ-macrophages.

Objective: To evaluate native full-length MOG for novel T cell epitopes. Background Amino acid (aa) residues 35-55 within the extracellular domain 1-117 of myelin oligodendrocyte glycoprotein (MOG) contain its only known encephalitogenic T cell determinant for C57BL/6 (H-2^b) mice, a strain frequently studied in the multiple sclerosis (MS) model, experimental autoimmune encephalomyelitis (EAE). Based upon studies showing that processing of native MOG is required for presentation of 35-55, we predicted that MOG contains additional T cell epitopes. Design/Methods: C57BL/6 mice were immunized with full-length (218 aa) murine (self) MOG or overlapping peptides encompassing its entire sequence in complete Freund9s adjuvant. Proliferation was measured by 3^H-thymidine incorporation. ELISPOT was used to measure frequency of epitope-specific IFN-γ-producing T cells that were generated by in vivo priming with full-length MOG. Results: Three novel T cell epitopes were discovered, an encephalitogenic determinant within transmembrane domain residues 119-132, and two discrete nonencephalitogenic determinants, p181-195 and p186-200, in the cytoplasmic domain. Clinical EAE, CNS inflammation and demyelination induced by p119-132 were equal, or greater, in severity than with p35-55. T cell recognition of MOG p119-132 was restricted by MHC II (I-A^b) molecules and p119-132 induced EAE in other H-2^b strains, but not in mice of other haplotypes. MOG p119-132-primed T cells, which produced higher levels of pathogenic Th1 and Th17 cytokines than p35-55-primed T cells, adoptively transferred EAE to naive recipients. Frequency of T cells that responded to p119-132 after immunization with full-length MOG was greater than to p35-55 or the non-encephalitogenic determinants, demonstrating that p119-132 is the immunodominant determinant. Conclusions: MOG p119-132, not p35-55, is the immunodominant encephalitogenic determinant of MOG. Not all T cell epitopes of a self antigen are pathogenic. Recognizing that MOG contains multiple encephalitogenic and non-encephalitogenic determinants permits testing tolerogenic approaches that target individual non-pathogenic or pathogenic T cell epitopes. Supported by: NIH and Boehringer-Ingelheim. Disclosure: Dr. Shetty has received research support from Boehringer Ingelheim. Dr. Gupta has received research support from Boehringer Ingelheim Pharmaceuticals, Inc. Dr. Weber has received personal compensation for activities with Hoffmann-LaRoche, Genentech, Inc., Novartis, Bayer Pharmaceuticals Corporation, Merck Serono, Biogen Idec and Teva Neuroscience as a speaker and/or participant on an advisory board. Dr. Weber has received research support from Teva Neuroscience. Dr. Molnarfi has nothing to disclose. Dr. Forsthuber has received research support from the National Institutes of Health. Dr. Sobel has received research support from the NIH. Dr. Bernard has nothing to disclose. Dr. Slavin has received personal compensation for activities with Boehringer Ingelheim as an employee. Dr. Slavin holds stock and/or stock options in Boehringer Ingelheim. Dr. Slavin has received research support from Boehringer Ingelheim. Dr. Zamvil has received personal compensation for activities with Biogen Idec, Teva Neuroscience and EMD Serono-Pfizer. Dr. Zamvil has received research support from the National Institutes of Health, National Multiple Sclerosis Society, the Maisin Foundation, the Guthy Jackson Charitable Foundation, Teva Neuroscience, and Boehringer Ingelheim Pharmaceuticals, Inc.

Objective: We investigated whether T cell immune modulation by laquinimod was mediated through its effects on myeloid antigen presenting cells (APC) or directly on T cells. Background Laquinimod is a novel oral drug that is being evaluated for treatment of RR multiple sclerosis (MS). In the MS model, EAE, laquinimod has been shown to reduce CNS infiltration and demyelination, as well as promote T cell immune modulation. Design/Methods: The effect on the function of myeloid APC was tested by using in vivo laquinimod-treated cells as APC for meylin-specific T cells and by transferring laquinimod treated APC into mice with established EAE. Results: Oral laquinimod treatment reversed established RR-EAE, and was associated with reduced 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 and an elevation of CD11bhiGr1hi monocytes. CD11b+ cells from these mice exhibited anti-inflammatory type II (M2) phenotype characterized by reduced STAT1 phosphorylation, decreased production of IL-6, IL-12/23 and TNF, and increased IL-10. In adoptive transfer, these donor type II monocytes suppressed clinical and histologic signs in recipients with established EAE. We examined whether T cell immune modulation occurred as a direct effect of laquinimod, or as a consequence of altered APC function. Inhibition of Th1 and Th17 differentiation was observed only when type II monocytes from laquinimod-treated mice were used as APC, regardless of whether myelin-specific T cells were obtained from laquinimod-treated or untreated mice. Conclusions: Our results show that Laquinimod does not act directly on T cells, but modulates adaptive T cell immune responses via its effect on myeloid APC. Supported by: Support was provided to S.S.Z. by NIH (RO1 AI073737 and NS063008), the NMSS (RG 4124), Teva Pharmaceuticals, and the Maisin Foundation. Disclosure: Dr. Schulze Topphoff has nothing to disclose. Dr. Shetty has received research support from Boehringer Ingelheim. Dr. Varrin-Doyer has nothing to disclose. Dr. Molnarfi has nothing to disclose. Dr. Sagan has received research support from the Guthy Jackson Charitable Foundation. Dr. Sobel has received research support from the NIH. Dr. Nelson has nothing to disclose. Dr. Zamvil has received personal compensation for activities with Biogen Idec, Teva Neuroscience and EMD Serono-Pfizer. Dr. Zamvil has received research support from the National Institutes of Health, National Multiple Sclerosis Society, the Maisin Foundation, the Guthy Jackson Charitable Foundation, Teva Neuroscience, and Boehringer Ingelheim Pharmaceuticals, Inc.

Studies in experimental autoimmune encephalomyelitis (EAE), a mouse model of multiple sclerosis, have shown that B cells markedly influence the course of the disease, although whether their effects are protective or pathological is a matter of debate. EndoS hydrolysis of the IgG glycan has profound effects on IgG effector functions, such as complement activation and Fc receptor binding, suggesting that the enzyme could be used as an immunomodulatory therapeutic agent against IgG-mediated diseases. We demonstrate here that EndoS has a protective effect in myelin oligodendrocyte glycoprotein peptide amino acid 35–55 (MOG35-55)-induced EAE, a chronic neuroinflammatory demyelinating disorder of the central nervous system (CNS) in which humoral immune responses are thought to play only a minor role. EndoS treatment in chronic MOG35-55-EAE did not impair encephalitogenic T cell priming and recruitment into the CNS of mice, consistent with a primary role of EndoS in controlling IgG effector functions. In contrast, reduced EAE severity coincided with poor serum complement activation and deposition within the spinal cord, suggesting that EndoS treatment impairs B cell effector function. These results identify EndoS as a potential therapeutic agent against antibody-mediated CNS autoimmune disorders.

Human myelin oligodendrocyte glycoprotein (MOG) induces experimental autoimmune encephalomyelitis (EAE) in a B cell-dependent manner, and is associated with MOG-specific antibodies (Abs). Whether B cells serve as antigen presenting cells (APC) for activation of encephalitogenic MOG-specific T cells in EAE induced by MOG is unknown. To distinguish the role of APC function by MOG-specific B cells from MOG-specific Abs, we created MOG-specific B cell receptor (BCR) transgenic (Tg) mice containing B cells that express membrane MOG-specific Ig only (MOGmIg), but cannot secrete Abs. EAE was compared in MOGmIg mice, MOG-specific BCR knock-in (Th) mice that can secrete all isotypes, in B cell-deficient and wild-type (WT) mice. B cell-deficient mice were resistant, confirming the requirement for B cells in human MOG-induced EAE. MOG induced EAE in MOG BCR knock-in mice, WT mice and MOGmIg Tg mice, indicating that B cells provide an accessory (i.e. APC) function, independent of Ab secretion in MOG-induced EAE. To further evaluate B cell function, mixed bone marrow (bm) chimera mice were created that contained B cells that were selectively MHC II-deficient. Mixed bm chimera mice that contained MHC II+ B cells were susceptible to MOG-induced EAE, whereas mice containing MHC II- B cells were resistant, despite MHC II expression on other APC. Our results conclusively demonstrate that independent of their humoral role, B cells provide a critical cellular function in EAE pathogenesis.

Background Accumulating evidence suggests a deleterious role for CD8+ T cells in multiple sclerosis (MS) pathogenesis. We have recently reported that hepatocyte growth factor (HGF), a potent neuroprotective factor, limits CD4+ T cell-mediated autoimmune neuroinflammation by promoting tolerogenic dendritic cells (DCs) and subsequently regulatory T cells. Whether HGF modulates cell-mediated immunity driven by MHC class I-restricted CD8+ T cells remains to be determined. Methods Here we examined whether HGF regulates antigen-specific CD8+ T cell responses using an established model of murine cytotoxic T lymphocyte (CTL)-mediated killing. Results We found that HGF treatment of gp100-pulsed DCs reduced the activation of gp100-specific T cell receptor (Pmel-1) CD8+ T cells and subsequent MHC class I-restricted CTL-mediated cytolysis of gp100-pulsed target cells. The levels of perforin, granzyme B, IFN-γ, and the degranulation marker CD107a as well as Fas ligand were decreased among CD8+ T cells, suggestive of a dual inhibitory effect of HGF on the perforin/granzyme B- and Fas-based lytic pathways in cell-mediated cytotoxicity. Treatment of CD8+ T cells with concanamycin A, a potent inhibitor of the perforin-mediated cytotoxic pathway, abrogated CTL cytotoxicity indicating that blockade of the perforin-dependent killing is a major mechanism by which HGF diminished cytolysis of gp100-pulsed target cells. Moreover, HGF suppressed the generation of effector memory CTLs. Conclusions Our findings indicate that HGF treatment limits both the generation and activity of effector CTL from naïve CD8+ T cells. Complementary to its impact on CD4+ T-cell CNS autoimmunity and myelin repair, our findings further suggest that HGF treatment could be exploited to control CD8+ T-cell-mediated, MHC I-restricted autoimmune dysfunctions such as MS.