Pertussis Toxin Inhibits Encephalitogenic T-Cell Infiltration and Promotes a B-Cell-Driven Disease during Th17-EAE.

Pertussis toxin (PTX) is a required co-adjuvant for experimental autoimmune encephalomyelitis (EAE) induced by immunization with myelin antigen. However, PTX's effects on EAE induced by the transfer of myelin-specific T helper cells is not known. Therefore, we investigated how PTX affects the Th17 transfer EAE model (Th17-EAE). We found that PTX significantly reduced Th17-EAE by inhibiting chemokine-receptor-dependent trafficking of Th17 cells. Strikingly, PTX also promoted the accumulation of B cells in the CNS, suggesting that PTX alters the disease toward a B-cell-dependent pathology. To determine the role of B cells, we compared the effects of PTX on Th17-EAE in wild-type (WT) and B-cell-deficient (µMT) mice. Without PTX treatment, disease severity was equivalent between WT and µMT mice. In contrast, with PTX treatment, the µMT mice had significantly less disease and a reduction in pathogenic Th17 cells in the CNS compared to the WT mice. In conclusion, this study shows that PTX inhibits the migration of pathogenic Th17 cells, while promoting the accumulation of pathogenic B cells in the CNS during Th17-EAE. These data provide useful methodological information for adoptive-transfer Th17-EAE and, furthermore, describe another important experimental system to study the pathogenic mechanisms of B cells in multiple sclerosis.

Loss of Nrf2 exacerbates the visual deficits and optic neuritis elicited by experimental autoimmune encephalomyelitis.

PURPOSE: Optic neuritis, inflammation of the optic nerve, is experienced by most patients with multiple sclerosis (MS) and is typically characterized by episodes of acute, monocular vision loss. These episodes of inflammation can lead to damage or degeneration of the retinal ganglion cells (RGCs), the axons of which comprise the optic nerve. Experimental autoimmune encephalomyelitis (EAE) is a well-established model of MS in which mice are immunized to produce a neuroautoimmunity that recapitulates the cardinal hallmarks of human disease, namely, inflammation, demyelination, and neurodegeneration of the brain, spinal cord, and optic nerve. Inflammation-associated oxidative stress plays a key role in promoting spinal cord damage in EAE. However, the role of oxidative stress in optic neuritis and the associated visual deficits has not been studied. To address this gap in research, we sought to determine how a deficiency in the master antioxidant transcription factor (using nuclear factor-E2-related factor [Nrf2]-deficient mice) affects visual pathology in the EAE model. METHODS: EAE was induced in 8-week-old wild-type (WT) and Nrf2 knockout (KO) mice by immunization against the myelin oligodendrocyte glycoprotein (MOG) peptide antigen. Motor deficits were monitored daily, as was visual acuity using the established functional optokinetic tracking (OKT) assay. Mice were euthanized 21 days post-immunization for histological analyses. The optic nerves were paraffin-embedded and stained with hematoxylin and eosin (H&E) or immune cell type-specific antibodies to analyze inflammatory infiltrates. The retinas were flatmounted and stained with an RGC-specific antibody, and the RGCs were counted to assess neurodegeneration. T-helper (Th) cell-associated cytokines were measured in spleens with enzyme-linked immunosorbent assay (ELISA). Immune analyses of healthy, non-EAE mice were characterized with flow cytometry to assess the baseline immune cell profiles. RESULTS: Female Nrf2 KO mice exhibited more severe EAE-induced motor deficits compared with female WT mice. In both genders, EAE elicited more severe visual acuity deficits, inflammation of the optic nerve, and RGC degeneration in KO mice compared with their strain- and age-matched WT counterparts. Visual acuity deficits were primarily present in (and only exacerbated in) one eye of each mouse. Excess inflammatory cells within the optic nerves of the KO mice were primarily comprised of T-cells, and greater RGC degeneration in the KO mice was most prevalent in the central retina compared with the peripheral retina. Nrf2 KO spleens exhibited an increased Th1- but not Th17-associated immune response. This enhanced pathology in the KO mice was not due to global differences in immune system development between the two genotypes. CONCLUSIONS: This is the first study to report that genetic ablation of Nrf2 exacerbates visual deficits, inflammation of the optic nerve, and RGC degeneration in a murine model of MS, suggesting that Nrf2 plays a neuro- and cytoprotective role in EAE-associated optic neuritis.

Biomarker panel increases accuracy for identification of an MS relapse beyond sNfL.

BACKGROUND: For relapsing-remitting multiple sclerosis (RRMS), there is a need for biomarker development beyond clinical manifestations and MRI. Soluble neurofilament light chain (sNfL) has emerged as a biomarker for inflammatory activity in RRMS. However, there are limitations to the accuracy of sNfL in identifying relapses. Here, we sought to identify a panel of biomarkers that would increase the precision of distinguishing patients in relapse compared to sNfL alone. METHODS: We used a multiplex approach to measure levels of 724 blood proteins in two distinct RRMS cohorts. Multiple t-tests with covariate correction determined biomarkers that were differentially regulated in relapse and remission. Logistic regression models determined the accuracy of biomarkers to distinguish relapses from remission. RESULTS: The discovery cohort identified 37 proteins differentially abundant in active RRMS relapse compared to remission. The verification cohort confirmed four proteins, including sNfL, were altered in active RRMS relapse compared to remission. Logistic regression showed that the 4-protein panel identified active relapse with higher accuracy (AUC = 0.87) than sNfL alone (AUC = 0.69). CONCLUSION: Our studies confirmed that sNfL is elevated during relapses in RRMS patients. Furthermore, we identified three other blood proteins, uPA, hK8 and DSG3 that were altered during relapse. Together, these four biomarkers could be used to monitor disease activity in RRMS patients.

Interferon-ß Intensifies Interleukin-23-Driven Pathogenicity of T Helper Cells in Neuroinflammatory Disease.

Interferon (IFN)-ß is a popular therapy for multiple sclerosis (MS). However, 25-40% of patients are nonresponsive to this therapy, and it worsens neuromyelitis optica (NMO), another neuroinflammatory disease. We previously identified, in both NMO patients and in mice, that IFN-ß treatment had inflammatory effects in T Helper (TH) 17-induced disease through the production of the inflammatory cytokine IL-6. However, other studies have shown that IFN-ß inhibits the differentiation and function of TH17 cells. In this manuscript, we identified that IFN-ß had differential effects on discrete stages of TH17 development. During early TH17 development, IFN-ß inhibits IL-17 production. Conversely, during late TH17 differentiation, IFN-ß synergizes with IL-23 to promote a pathogenic T cell that has both TH1 and TH17 characteristics and expresses elevated levels of the potent inflammatory cytokines IL-6 and GM-CSF and the transcription factor BLIMP. Together, these findings help resolve a paradox surrounding IFN-ß and TH17-induced disease and illuminate the pathways responsible for the pathophysiology of NMO and MS patients who are IFN-ß nonresponders.

CNS Autoimmune Responses in BCMA-Deficient Mice Provide Insight for the Failure of Atacicept in MS.

OBJECTIVE: B cells have emerged as a therapeutic target for MS. Anti-CD20 antibodies, which deplete B cells, are effective therapies for MS. However, atacicept (TACI-Fc), which blocks BAFF and APRIL and reduces B cells, unexpectedly exacerbates MS. We tested the hypothesis that B cell maturation antigen (BCMA), a receptor for BAFF and APRIL, plays a role in the paradoxical effects of anti-CD20 antibody and TACI-Fc using experimental autoimmune encephalomyelitis (EAE). METHODS: EAE was induced in wild-type (BCMA+/+) and BCMA-deficient (BCMA-/-) mice with an immunization of rodent myelin oligodendrocyte glycoprotein (MOG)35-55 peptide. Treatment with anti-CD20 antibody, TACI-Fc, and isotype controls was administered by intraperitoneal injections. CNS infiltration was evaluated by histology; immune cell phenotypes were evaluated by flow cytometry; MOG-specific antibodies were determined by ELISA. Mixed bone marrow chimeras and cell culture assays were used to identify the specific subsets of immune cells affected by BCMA deficiency. RESULTS: First, we found that BCMA-/- mice had more severe EAE compared with BCMA+/+ mice and the increased disease was associated with elevated anti-MOG B-cell responses. Second, we found that anti-CD20 therapy attenuated EAE in BCMA-/- mice but not in BCMA+/+ mice. Third, TACI-Fc attenuated EAE in BCMA+/+ mice but not in BCMA-/- mice. Mixed bone marrow chimeric and cell culture experiments demonstrated that BCMA deficiency elevates inflammatory B-cell responses but inhibits inflammatory responses in macrophages. CONCLUSIONS: BCMA has multifaceted roles during inflammation that affects therapeutic efficacies of anti-CD20 and TACI-Fc in EAE. Our results from BCMA-deficient mice provide insights into the failure of atacicept in MS.

B cell function impacts the efficacy of IFN-ß therapy in EAE.

Recent studies identified that interferon beta (IFN-ß) treatment skews B-cells towards a regulatory phenotype in multiple sclerosis. To assess B cell involvement during IFN-ß therapy, we compared IFN-ß treatment in a B cell-independent model and a B cell-dependent model of experimental autoimmune encephalomyelitis (EAE). We show that in B cell-independent EAE, IFN-ß ameliorates neuroinflammation. Conversely, in B cell-dependent EAE, IFN-ß has no effect on disease. Effective IFN-ß therapy in B cell-independent EAE was associated with reduced inflammatory T cells in the CNS and skewed splenic B cells towards an immature population and away from a germinal center population. These immune cell populations were unchanged in B cell-dependent EAE. Finally, we found that IFN-ß increased marginal zone B cells in both EAE models. These findings indicate that B cell function impacts IFN-ß efficacy during neuroinflammation.

Transcriptomics and proteomics reveal a cooperation between interferon and T-helper 17 cells in neuromyelitis optica.

Type I interferon (IFN-I) and T helper 17 (TH17) drive pathology in neuromyelitis optica spectrum disorder (NMOSD) and in TH17-induced experimental autoimmune encephalomyelitis (TH17-EAE). This is paradoxical because the prevalent theory is that IFN-I inhibits TH17 function. Here we report that a cascade involving IFN-I, IL-6 and B cells promotes TH17-mediated neuro-autoimmunity. In NMOSD, elevated IFN-I signatures, IL-6 and IL-17 are associated with severe disability. Furthermore, IL-6 and IL-17 levels are lower in patients on anti-CD20 therapy. In mice, IFN-I elevates IL-6 and exacerbates TH17-EAE. Strikingly, IL-6 blockade attenuates disease only in mice treated with IFN-I. By contrast, B-cell-deficiency attenuates TH17-EAE in the presence or absence of IFN-I treatment. Finally, IFN-I stimulates B cells to produce IL-6 to drive pathogenic TH17 differentiation in vitro. Our data thus provide an explanation for the paradox surrounding IFN-I and TH17 in neuro-autoimmunity, and may have utility in predicting therapeutic response in NMOSD.

Role of TFH Cells in Promoting T Helper 17-Induced Neuroinflammation.

Both T cells and B cells are implicated in the pathology of multiple sclerosis (MS), but how these cells cooperate to drive disease remains unclear. Recent studies using experimental autoimmune encephalomyelitis (EAE) demonstrated that the TH17 pathway is correlated with increased numbers of ectopic B-cell follicles in the central nervous system (CNS). As follicular T helper (TFH) cells are regulators of B cell responses, we sought to examine the role of TFH cells in EAE induced by the transfer of myelin-specific TH17 cells (TH17-EAE). In this study, we first confirmed previous reports that B-cells are a major cell type infiltrating the CNS during TH17-EAE. In addition, we found that B cells contribute to the severity of TH17-EAE. Class-switched B-cells in the CNS were positively correlated with disease and, strikingly, the severity TH17-EAE was diminished in B cell deficient mice. We next focused on the role TFH cells play in TH17-EAE. We found substantial numbers of CXCR5+PD1+CD4+ TFH cells in the CNS tissue of TH17-EAE mice and that at the peak of disease, the number of infiltrating TFHs was correlated with the number of infiltrating B-cells. Using congenic CD45.1+ donor mice and CD45.2+ recipient mice, we determined that the TFH cells were recipient-derived, whereas IL-17+ cells were donor-derived. We assessed whether myelin-specific TFH cells are capable of inducing EAE in recipient mice and found that transferring TFH cells failed to induce EAE. Finally, we tested the effects of blocking TFH trafficking in TH17-EAE using an antagonistic antibody against CXCL13, the chemokine ligand for CXCR5 on TFH cells. We found anti-CXCL13 treatment significantly reduced TH17-EAE disease. This treatment blocked CD4+ T cells from entering the CNS, but had no effect on infiltration of B cells. Strikingly, this antibody treatment had no measurable effect on TH17 disease in B cell-deficient mice. These data demonstrate that infiltrating TFH cells are a key cell type that contributes to an inflammatory B cell response in TH17-EAE and provide evidence for targeting TFH cells as a treatment for neuro-autoimmune diseases like MS.

Identification of the YfgF MASE1 domain as a modulator of bacterial responses to aspartate.

Complex 3'-5'-cyclic diguanylic acid (c-di-GMP) responsive regulatory networks that are modulated by the action of multiple diguanylate cyclases (DGC; GGDEF domain proteins) and phosphodiesterases (PDE; EAL domain proteins) have evolved in many bacteria. YfgF proteins possess a membrane-anchoring domain (MASE1), a catalytically inactive GGDEF domain and a catalytically active EAL domain. Here, sustained expression of the Salmonella enterica spp. Enterica ser. Enteritidis YfgF protein is shown to mediate inhibition of the formation of the aspartate chemotactic ring on motility agar under aerobic conditions. This phenomenon was c-di-GMP-independent because it occurred in a Salmonella strain that lacked the ability to synthesize c-di-GMP and also when PDE activity was abolished by site-directed mutagenesis of the EAL domain. YfgF-mediated inhibition of aspartate chemotactic ring formation was impaired in the altered redox environment generated by exogenous p-benzoquinone. This ability of YfgF to inhibit the response to aspartate required a motif, (213)Lys-Lys-Glu(215), in the predicted cytoplasmic loop between trans-membrane regions 5 and 6 of the MASE1 domain. Thus, for the first time the function of a MASE1 domain as a redox-responsive regulator of bacterial responses to aspartate has been shown.