Michel Varrin-Doyer

MHC class II-dependent B cell APC function is required for induction of CNS autoimmunity independent of myelin-specific antibodies

Antigen presentation, but not antibody secretion, by B cells drives CNS autoimmunity induced by immunization with human MOG.

Aquaporin 4-Specific T Cells in Neuromyelitis Optica Exhibit a Th17 Bias and Recognize Clostridium ABC Transporter

Aquaporin 4 (AQP4)‐specific autoantibodies in neuromyelitis optica (NMO) are immunoglobulin (Ig)G1, a T cell‐dependent Ig subclass, indicating that AQP4‐specific T cells participate in NMO pathogenesis. Our goal was to identify and characterize AQP4‐specific T cells in NMO patients and healthy controls (HC).

Proinflammatory role of aquaporin-4 in autoimmune neuroinflammation

Aquaporin‐4 (AQP4) deficiency in mice reduces neuroinflammation in experimental autoimmune encephalomyelitis (EAE) produced by active immunization with myelin oligodendrocyte glycoprotein peptide (MOG). Potential mechanisms for the protective effect of AQP4 deficiency were investigated, including AQP4‐dependent leukocyte and microglia cell function, immune cell entry in the central nervous system (CNS), intrinsic neuroinflammation, and humoral immune response. As we found with active‐immunization EAE, neuroinflammation was greatly reduced in AQP4‐knockout mice in adoptive‐transfer EAE. AQP4 was absent in immune cells, including activated T lymphocytes. The CNS migration of fluo‐rescently labeled, MOG‐sensitized T lymphocytes was comparable in wild‐type and AQP4‐knockout mice. Microglia did not express AQP4. Serum anti‐AQP4 antibodies were absent in EAE. Remarkably, intracerebral injection of LPS produced much greater neuroinflammation in wild‐type than in AQP4‐knockout mice, and cytokine (TNF‐α and IL‐6) secretion was reduced in astrocyte cultures from AQP4‐knockout mice. Ade‐novirus‐mediated expression of AQP4, or of an unrelated aquaporin, AQP1, increased cytokine secretion in astrocyte and nonastrocyte cell cultures, supporting the involvement of aquaporin water permeability in cyto‐kine secretion. Our data suggest an intrinsic proinflammatory role of AQP4 involving AQP4‐dependent astrocyte swelling and cytokine release. Reduction in AQP4 water transport may be protective in neuroinflammatory CNS diseases.—Li, L., Zhang, H., Varrin‐Doyer, M., Zamvil, S. S., Verkman, A. S. Proinflammatory role of aquaporin‐4 in autoimmune neuroinflammation. FASEB J. 25, 1556–1566 (2011). www.fasebj.org

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 CD11bhiGr1hi 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.

Dimethyl fumarate treatment induces adaptive and innate immune modulation independent of Nrf2

Significance Dimethyl fumarate (DMF) (BG-12, Tecfidera), a fumaric acid ester (FAE), is a commonly prescribed oral therapy for multiple sclerosis (MS), a CNS autoimmune inflammatory demyelinating disease that may result in sustained neurologic damage. It is thought that the benefit of DMF in MS therapy is mediated through activation of the antioxidative transcription factor nuclear factor (erythroid-derived 2)-like 2 (Nrf2) pathway. However, the role of Nrf2 in the antiinflammatory effects of DMF has not been fully elucidated. Here, we investigated the role of Nrf2 in DMF treatment of the MS model, experimental autoimmune encephalomyelitis (EAE), and demonstrated DMF can modulate T cells, B cells, and antigen-presenting cells, and reduce clinical and histologic EAE, independent of Nrf2. Dimethyl fumarate (DMF) (BG-12, Tecfidera) is a fumaric acid ester (FAE) that was advanced as a multiple sclerosis (MS) therapy largely for potential neuroprotection as it was recognized that FAEs are capable of activating the antioxidative transcription factor nuclear factor (erythroid-derived 2)-like 2 (Nrf2) pathway. However, DMF treatment in randomized controlled MS trials was associated with marked reductions in relapse rate and development of active brain MRI lesions, measures considered to reflect CNS inflammation. Here, we investigated the antiinflammatory contribution of Nrf2 in DMF treatment of the MS model, experimental autoimmune encephalomyelitis (EAE). C57BL/6 wild-type (WT) and Nrf2-deficient (Nrf2−/−) mice were immunized with myelin oligodendrocyte glycoprotein (MOG) peptide 35–55 (p35–55) for EAE induction and treated with oral DMF or vehicle daily. DMF protected WT and Nrf2−/− mice equally well from development of clinical and histologic EAE. The beneficial effect of DMF treatment in Nrf2−/− and WT mice was accompanied by reduced frequencies of IFN-γ and IL-17–producing CD4+ cells and induction of antiinflammatory M2 (type II) monocytes. DMF also modulated B-cell MHC II expression and reduced the incidence of clinical disease in a B-cell–dependent model of spontaneous CNS autoimmunity. Our observations that oral DMF treatment promoted immune modulation and provided equal clinical benefit in acute EAE in Nrf2−/− and WT mice, suggest that the antiinflammatory activity of DMF in treatment of MS patients may occur through alternative pathways, independent of Nrf2.

Immunodominant T Cell Determinants of Aquaporin-4, the Autoantigen Associated with Neuromyelitis Optica

Autoantibodies that target the water channel aquaporin-4 (AQP4) in neuromyelitis optica (NMO) are IgG1, a T cell-dependent Ig subclass. However, a role for AQP4-specific T cells in this CNS inflammatory disease is not known. To evaluate their potential role in CNS autoimmunity, we have identified and characterized T cells that respond to AQP4 in C57BL/6 and SJL/J mice, two strains that are commonly studied in models of CNS inflammatory diseases. Mice were immunized with either overlapping peptides or intact hAQP4 protein encompassing the entire 323 amino acid sequence. T cell determinants identified from examination of the AQP4 peptide (p) library were located within AQP4 p21-40, p91-110, p101-120, p166-180, p231-250 and p261-280 in C57BL/6 mice, and within p11-30, p21-40, p101-120, p126-140 and p261-280 in SJL/J mice. AQP4-specific T cells were CD4+ and MHC II-restricted. In recall responses to immunization with intact AQP4, T cells responded primarily to p21-40, indicating this region contains the immunodominant T cell epitope(s) for both strains. AQP4 p21-40-primed T cells secreted both IFN-γ and IL-17. The core immunodominant AQP4 21-40 T cell determinant was mapped to residues 24-35 in C57BL/6 mice and 23-35 in SJL/J mice. Our identification of the AQP4 T cell determinants and characterization of its immunodominant determinant should permit investigators to evaluate the role of AQP4-specific T cells in vivo and to develop AQP4-targeted murine NMO models.

Gut microbiome analysis in neuromyelitis optica reveals overabundance of Clostridium perfringens

T cells from neuromyelitis optica (NMO) patients, which recognize the immunodominant epitope of aquaporin‐4, exhibit Th17 polarization and cross‐react with a homologous sequence of a Clostridium perfringens adenosine triphosphate‐binding cassette transporter. Therefore, this commensal microbe might participate in NMO pathogenesis. We examined the gut microbiome by PhyloChip G3 from 16 NMO patients, 16 healthy controls (HC), and 16 multiple sclerosis patients. A significant difference in the abundance of several microbial communities was observed between NMO and HC (Adonis test, p = 0.001). Strikingly, C. perfringens was overrepresented in NMO (p = 5.24 × 10−8). These observations support a potential role for C. perfringens in NMO pathogenesis. Ann Neurol 2016;80:443–447

Phosphorylation of collapsin response mediator protein 2 on Tyr-479 regulates CXCL12-induced T lymphocyte migration.

In the central nervous system, collapsin response mediator protein 2 (CRMP2) is a transducer protein that supports the semaphorin-induced guidance of axons toward their cognate target. However, we previously showed that CRMP2 is also expressed in immune cells and plays a crucial role in T lymphocyte migration. Here we further investigated the molecular mechanisms underlying CRMP2 function in chemokine-directed T-cell motility. Examining Jurkat T-cells treated with the chemokine CXCL12, we found that 1) CXCL12 induces a dynamic re-localization of CRMP2 to uropod, the flexible structure of migrating lymphocyte, and increases its binding to the cytoskeletal protein vimentin; 2) CXCL12 decreases phosphorylation of the glycogen synthase kinase-3β-targeted residues CRMP2-Thr-509/514; and 3) tyrosine Tyr-479 is a new phosphorylation CRMP2 residue and a target for the Src-family kinase Yes. Moreover, phospho-Tyr-479 increased under CXCL12 signaling while phospho-Thr-509/514 decreased. The functional importance of this tyrosine phosphorylation was demonstrated by Y479F mutation that strongly reduced CXCL12-mediated T-cell polarization and motility as tested in a transmigration model and on neural tissue. We propose that differential phosphorylation by glycogen synthase kinase-3β and Yes modulates the contribution of CRMP2 to cytoskeletal reorganization during chemokine-directed T-cell migration. In addition to providing a novel mechanism for T lymphocyte motility, our findings reveal CRMP2 as a transducer of chemokine signaling.

Drug Binding Assays do not Reveal Specific Binding of Lacosamide to Collapsin Response Mediator Protein 2 (CRMP-2)

Aims: Lacosamide (LCM; SPM 927, Vimpat®) is an antiepileptic drug (AED) used as adjunctive treatment for adults with partial‐onset seizures. LCM has a different mode of action from traditional sodium channel blocking AEDs in that it selectively enhances slow inactivation of sodium channels without affecting fast inactivation. Initial investigations suggested that LCM might have an additional mode of action by binding to the collapsin response mediator protein 2 (CRMP‐2), which is further investigated here. Methods: LCM binding to native and cloned human CRMP‐2 was determined using radioligand binding experiments and surface plasmon resonance measurements. Results: No specific binding of [3H]LCM (free concentration 100–1450 nM) to isolated or membrane bound human CRMP‐2 expressed in mammalian cell systems and bacteria was observed. Surface plasmon resonance analysis also showed that LCM, over a concentration range of 0.39–100 μM, does not specifically bind to human CRMP‐2. Conclusion: The diverse drug binding methods employed here are well suited to detect specific binding of LCM to CRMP‐2 in the micromolar range, yet the results obtained were all negative. Results of this study suggest that LCM does not specifically bind to CRMP‐2.

Tob1 plays a critical role in the activation of encephalitogenic T cells in CNS autoimmunity

Loss of antiproliferative gene TOB1 results in more severe EAE driven by augmented pathogenic T cell responses.