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.
Abstract
Interleukin-2 (IL-2) is critical for regulatory T cell (Treg) function and homeostasis. At low doses, IL-2 can suppress immune pathologies by expanding Tregs that constitutively express the ...high affinity IL-2Rα subunit. However, even low dose IL-2, signaling through the IL2-Rβ/γ complex, may lead to the activation of proinflammatory, non-Treg T cells, so improving specificity toward Tregs may be desirable. Here we use messenger RNAs (mRNA) to encode a half-life-extended human IL-2 mutein (HSA-IL2m) with mutations promoting reliance on IL-2Rα. Our data show that IL-2 mutein subcutaneous delivery as lipid-encapsulated mRNA nanoparticles selectively activates and expands Tregs in mice and non-human primates, and also reduces disease severity in mouse models of acute graft versus host disease and experimental autoimmune encephalomyelitis. Single cell RNA-sequencing of mouse splenic CD4
+
T cells identifies multiple Treg states with distinct response dynamics following IL-2 mutein treatment. Our results thus demonstrate the potential of mRNA-encoded HSA-IL2m immunotherapy to treat autoimmune diseases.
Dimethyl fumarate (DMF), recently approved as an oral immunomodulatory treatment for relapsing-remitting multiple sclerosis (MS), metabolizes to monomethyl fumarate (MMF) which crosses the ...blood–brain barrier and has demonstrated neuroprotective effects in experimental studies. We postulated that MMF exerts neuroprotective effects through modulation of microglia activation, a critical component of the neuroinflammatory cascade that occurs in neurodegenerative diseases such as MS. To ascertain our hypothesis and define the mechanistic pathways involved in the modulating effect of fumarates, we used real-time PCR and biochemical assays to assess changes in the molecular and functional phenotype of microglia, quantitative Western blotting to monitor activation of postulated pathway components, and ex vivo whole-cell patch clamp recording of excitatory post-synaptic currents in corticostriatal slices from mice with experimental autoimmune encephalomyelitis (EAE), a model for MS, to study synaptic transmission. We show that exposure to MMF switches the molecular and functional phenotype of activated microglia from classically activated, pro-inflammatory type to alternatively activated, neuroprotective one, through activation of the hydroxycarboxylic acid receptor 2 (HCAR2). We validate a downstream pathway mediated through the AMPK–Sirt1 axis resulting in deacetylation, and thereby inhibition, of NF-κB and, consequently, of secretion of pro-inflammatory molecules. We demonstrate through ex vivo monitoring of spontaneous glutamate-mediated excitatory post-synaptic currents of single neurons in corticostriatal slices from EAE mice that the neuroprotective effect of DMF was exerted on neurons at pre-synaptic terminals by modulating glutamate release. By exposing control slices to untreated and MMF-treated activated microglia, we confirm the modulating effect of MMF on microglia function and, thereby, its indirect neuroprotective effect at post-synaptic level. These findings, whereby DMF-induced activation of a new HCAR2-dependent pathway on microglia leads to the modulation of neuroinflammation and restores synaptic alterations occurring in EAE, represent a possible novel mechanism of action for DMF in MS.
Neutrophils are prominent participants in the joint inflammation of human rheumatoid arthritis (RA) patients, but the extent of their role in the inductive phase of joint inflammation is unknown. In ...the K/BxN mouse RA model, transfer of autoreactive Ig from the K/BxN mouse into mice induces a rapid and profound joint-specific inflammatory response reminiscent of human RA. We observed that after K/BxN serum transfer, the earliest clinical signs of inflammation in the ankle joint correlated with the presence of neutrophils in the synovial regions of recipient mouse ankle joints. In this study, we investigated the role of neutrophils in the early inflammatory response to transferred arthritogenic serum from the K/BxN transgenic mouse. Mice were treated with a neutrophil-depleting mAb before and following transfer of arthritogenic serum and scored for clinical indications of inflammation and severity of swelling in ankle joints and front paws. In the absence of neutrophils, mice were completely resistant to the inflammatory effects of K/BxN serum. Importantly, depletion of neutrophils in diseased recipient mice up to 5 days after serum transfer reversed the inflammatory reaction in the joints. Transfer of serum into mice deficient in the generation of nitrogen or oxygen radicals (inducible NO synthase 2 or gp91(phox) genes, respectively) gave normal inflammatory responses, indicating that neither pathway is essential for disease induction. These studies have identified a critical role for neutrophils in initiating and maintaining inflammatory processes in the joint.
In traumatic brain injury (TBI), a diversity of brain resident and peripherally derived myeloid cells have the potential to worsen damage and/or to assist in healing. We define the heterogeneity of ...microglia and macrophage phenotypes during TBI in wild-type (WT) mice and Ccr2−/− mice, which lack macrophage influx following TBI and are resistant to brain damage. We use unbiased single-cell RNA sequencing methods to uncover 25 microglia, monocyte/macrophage, and dendritic cell subsets in acute TBI and normal brains. We find alterations in transcriptional profiles of microglia subsets in Ccr2−/− TBI mice compared to WT TBI mice indicating that infiltrating monocytes/macrophages influence microglia activation to promote a type I IFN response. Preclinical pharmacological blockade of hCCR2 after injury reduces expression of IFN-responsive gene, Irf7, and improves outcomes. These data extend our understanding of myeloid cell diversity and crosstalk in brain trauma and identify therapeutic targets in myeloid subsets.
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•TBI elevates distinct phenotypes of microglia, macrophages, and dendritic cells•Ccr2 deficiency alters cell proportions and reduces ISG expression in microglia•TBI induces crosstalk between microglia and circulating monocytes•Preclinical translational studies to target human CCR2 after TBI improves outcomes
By single-cell RNA sequencing of traumatically injured and normal brains from wild-type and Ccr2−/− mice, Somebang et al. define microglia, macrophage, and dendritic cell phenotypes in TBI. Targeting mouse and/or human CCR2 reduces specific TBI brain CNS myeloid compartments, dampens type I interferon responses, and improves cognition after TBI.
Restoring numbers and function of regulatory T cells (Tregs) is a novel therapeutic strategy for neurodegenerative disorders. Whether Treg function is boosted by adoptive cell transfer, ...pharmaceuticals, or immune modulators, the final result is a robust anti-inflammatory and neuronal sparing response. Herein, a newly developed lipid nanoparticle (LNP) containing mRNA encoding granulocyte-macrophage colony-stimulating factor (Gm-csf mRNA) was developed to peripherally induce Tregs and used for treatment in preclinical Parkinson's disease (PD) models. Administration of Gm-csf mRNA to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated mice and rats overexpressing alpha-synuclein produced dose-dependent increases in plasma GM-CSF levels and peripheral CD4+CD25+FoxP3+ Treg populations. This upregulation paralleled nigrostriatal neuroprotection, upregulated immunosuppression-associated mRNAs that led to the detection of a treatment-induced CD4+ T cell population, and decreased reactive microgliosis. The current findings strengthen prior works utilizing immune modulation by harnessing Gm-csf mRNA to augment adaptive immune function by employing a new delivery platform to treat PD and potentially other neurodegenerative disorders.
Myelin is composed primarily of lipids and diseases affecting myelin are associated with alterations in its lipid composition. However, correlation of the spatial (in situ) distribution of lipids ...with the disease-associated compositional and morphological changes is not well defined. Herein we applied high resolution matrix-assisted laser desorption ionization imaging mass spectrometry (MALDI-IMS), immunohistochemistry (IHC), and liquid chromatography–electrospray ionization–mass spectrometry (LC-ESI-MS) to evaluate brain lipid alterations in the dysmyelinating shiverer (Shi) mouse and cuprizone (Cz) mouse model of reversible demyelination. MALDI-IMS revealed a decrease in the spatial distribution of sulfatide (SHexCer) species, SHexCer (d42:2), and a phosphatidylcholine (PC) species, PC (36:1), in white matter regions like corpus callosum (CC) both in the Shi mouse and Cz mouse model. Changes in these lipid species were restored albeit not entirely upon spontaneous remyelination after demyelination in the Cz mouse model. Lipid distribution changes correlated with the local morphological changes as confirmed by IHC. LC-ESI-MS analyses of CC extracts confirmed the MALDI-IMS derived reductions in SHexCer and PC species. These findings highlight the role of SHexCer and PC in preserving the normal myelin architecture and our experimental approaches provide a morphological basis to define lipid abnormalities relevant to myelin diseases.
Summary
Interleukin‐2 (IL‐2) is a critical regulator of immune homeostasis through its non‐redundant role in regulatory T (Treg) cell biology. There is major interest in therapeutic modulation of the ...IL‐2 pathway to promote immune activation in the context of tumour immunotherapy or to enhance immune suppression in the context of transplantation, autoimmunity and inflammatory diseases. Antibody‐mediated targeting of the high‐affinity IL‐2 receptor α chain (IL‐2Rα or CD25) offers a direct mechanism to target IL‐2 biology and is being actively explored in the clinic. In mouse models, the rat anti‐mouse CD25 clone PC61 has been used extensively to investigate the biology of IL‐2 and Treg cells; however, there has been controversy and conflicting data on the exact in vivo mechanistic function of PC61. Engineering antibodies to alter Fc/Fc receptor interactions can significantly alter their in vivo function. In this study, we re‐engineered the heavy chain constant region of an anti‐CD25 monoclonal antibody to generate variants with highly divergent Fc effector function. Using these anti‐CD25 Fc variants in multiple mouse models, we investigated the in vivo impact of CD25 blockade versus depletion of CD25+ Treg cells on immune homeostasis. We report that immune homeostasis can be maintained during CD25 blockade but aberrant T‐cell activation prevails when CD25+ Treg cells are actively depleted. These results clarify the impact of PC61 on Treg cell biology and reveal an important distinction between CD25 blockade and depletion of CD25+ Treg cells. These findings should inform therapeutic manipulation of the IL‐2 pathway by targeting the high‐affinity IL‐2R.
In the K/BxN mouse model of arthritis, autoantibodies against glucose-6-phosphate isomerase cause joint-specific inflammation and destruction. We have shown using micro-positron emission tomography ...that these glucose-6-phosphate isomerase-specific autoantibodies rapidly localize to distal joints of mice. In this study we used micro-positron emission tomography to delineate the stages involved in the development of arthritis. Localization of Abs to the joints depended upon mast cells, neutrophils, and FcRs, but not on C5. Surprisingly, anti-type II collagen Abs alone did not accumulate in the distal joints, but could be induced to do so by coinjection of irrelevant preformed immune complexes. Control Abs localized to the joint in a similar manner. Thus, immune complexes are essential initiators of arthritis by sequential activation of neutrophils and mast cells to allow Abs access to the joints, where they must bind a target Ag to initiate inflammation. Our findings support a four-stage model for the development of arthritis and identify checkpoints where the disease is reversible.
In the K/BxN mouse model of rheumatoid arthritis, the transfer of autoantibodies specific for glucose-6-phosphate isomerase (GPI) into naïve mice rapidly induces joint-specific inflammation similar ...to that seen in human rheumatoid arthritis. The ubiquitous expression of GPI and the systemic circulation of anti-GPI immunoglobulin G (IgG) seem incongruous with the tissue specificity of this disease. By using PET (positron emission tomography), we show here that purified anti-GPI IgG localizes specifically to distal joints in the front and rear limbs within minutes of intravenous injection, reaches saturation by 20 min and remains localized for at least 24 h. In contrast, control IgG does not localize to joints or cause inflammation. The rapid kinetics of anti-GPI IgG joint localization supports a model in which autoantibodies bind directly to pre-existing extracellular GPI in normal healthy mouse joints.
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