Purpose:To investigate whether an anti-interleukin-6 (IL-6) receptor antibody (MR16-1) can prevent retinal and optic nerve dysfunction, as well as myelitis, in a mouse model of myelitis induced by immunization with aquaporin-4 (AQP4) peptide. Methods:Mice immunized with AQP4 peptide received MR16-1 or vehicle. Retinal function was assessed by electroretinography (ERG), including b-wave, a-wave, and positive scotopic threshold response (pSTR) amplitudes. Histologic analysis evaluated inflammatory cell infiltration and aberrant Müller cell activation (punctate pattern of glial fibrillary acidic protein staining). Blood-retinal barrier (BRB) integrity was assessed using Evans blue dye, IgG leakage, and transendothelial electrical resistance (TEER) in primary mouse retinal microvascular endothelial cells (mRMECs) exposed to mouse serum. Results:AQP4 peptide immunization led to reductions in ERG b-wave and pSTR amplitudes and increased inflammatory cell infiltration, Müller cell alteration, and retinal vascular permeability. MR16-1 treatment significantly mitigated these changes, suppressing reductions in pSTR and b-wave amplitude, preventing inflammation and aberrant Müller cell activation, and reducing vascular leakage. The improvement of a-wave amplitude was not significant. Serum from MR16-1-treated mice did not reduce mRMEC TEER, while serum from AQP4 peptide-immunized mice reduced it. Conclusions:IL-6 receptor blockade with MR16-1 has the potential to suppress AQP4 peptide immunization-related retinal and optic nerve dysfunction and pathology, likely by protecting BRB integrity and suppressing inflammation. These findings support the therapeutic potential of IL-6 inhibition in preserving visual function in neuromyelitis optica spectrum disorder (NMOSD). Translational Relevance:IL-6 receptor blockade may be a viable strategy for preventing visual impairment in patients with NMOSD.

BACKGROUND:Some patients with myasthenia gravis (MG) are refractory to available treatments, highlighting the need to further understand the pathogenesis of the disease. This study aimed to determine whether components in the serum from patients with acetylcholine receptor (AChR) antibody-positive MG affect myotubes, to explore their possible role in disease pathogenesis beyond the inhibition of acetylcholine signal transmission. METHODS:Serum was collected from 14 patients with AChR antibody-positive MG. The differentiated human myotubes were stimulated with 10% serum from healthy controls or patients with MG. After 24 h, ribonucleic acid extraction/sequencing was performed, and differentially expressed genes (DEGs) were extracted. Pathway analysis was completed using DEGs that were downregulated by stimulation with serum from patients with MG. Expression of genes important for muscle contraction was measured and myotube diameter was determined by immunostaining. RESULTS:Approximately 1200 DEGs were extracted by comparing gene expression in cultured human myotube cells stimulated with serum from healthy controls and patients with MG. Gene ontology terms linked with muscle function were suppressed in myotube cells stimulated with patient serum. Suppression of pathways associated with muscle atrophy/weakness, decreased expression of genes associated with muscle contraction, and smaller myotube diameter were confirmed in myotube cells stimulated with serum from patients versus healthy controls. CONCLUSION:Factors other than acetylcholine signal transmission inhibition may contribute to the pathogenesis of AChR antibody-positive MG. Further research is needed to clarify the pathways involved, potentially leading to more tailored pharmacotherapies.

Myasthenia gravis (MG) is an autoimmune disease characterized by autoantibodies targeting the acetylcholine receptor (AChR) or muscle-specific tyrosine kinase (MuSK). These autoantibodies inhibit ACh signal transmission at the neuromuscular junction, leading to muscle weakness and fatigue. Anti-MuSK antibody-positive MG (MuSK+MG) appears more rarely than anti-AChR antibody-positive MG but more frequently results in muscle atrophy. However, the underlying mechanism is unknown. In this study, we analyzed whether serum from MuSK+MG patients has any pathogenic effect on cultured myotube cells. Primary human skeletal muscle myoblasts were differentiated into myotubes, which were then treated with serum from healthy control individuals or MuSK+MG patients. After one day, RNA-seq analysis and Western blotting were performed and myotube diameters were measured. Calcium dynamics following caffeine stimulation was also assessed. Comparing myotube cells treated with healthy control serum with those treated with serum from MuSK+MG patients, RNA-seq analysis showed suppression of pathways associated with muscle function and Western blotting analysis revealed reduced expression of Type II myosin heavy chain. These changes are consistent with muscle atrophy and weakness, although myotube diameter remained unchanged. Caffeine stimulation induced higher cytoplasmic calcium levels. Expression of sarcomere components was significantly reduced. Serum from MuSK+MG patients directly affected gene and protein expression in cultured human myotube cells, leading to changes associated with muscle atrophy and weakness. These findings suggest that there are mechanisms in addition to impaired ACh signal transmission at the neuromuscular junction that can cause muscle weakness and fatigue in MG patients.

Purpose:To investigate functional and structural changes in the retinas and optic nerves of mice immunized with an aquaporin-4 (AQP4) peptide, which was previously shown to induce paralysis mimicking human neuromyelitis optica spectrum disorder (NMOSD). Methods:Electroretinography and histological analyses were used to evaluate retinal function, and inflammatory cell infiltration or glial fibrillary acidic protein immunoreactivity in the retinas and optic nerves of AQP4-immunized mice. Additionally, the blood-retinal barrier function was assessed by Evans blue dye injection to measure in vivo retinal vascular permeability and by in vitro transendothelial electrical resistance using mouse primary retinal microvascular endothelial cells exposed to serum from AQP4-immunized mice. Results:AQP4 immunization led to a significant reduction in b-wave and scotopic threshold response amplitudes, indicating impaired inner retinal function. Histological analysis revealed inflammatory cell infiltration at the optic nerve head. Whole-mounted retinal glial fibrillary acidic protein immunoreactivity showed aberrant Müller cell activation, particularly in the juxtapapillary region. AQP4-immunized mice exhibited increased retinal Evans blue dye leakage and mouse retinal microvascular endothelial cells exhibited reduced transendothelial electrical resistance, indicating blood-retinal barrier disruption. Conclusions:AQP4 immunization induced functional impairment, inflammatory cell infiltration, glial cell activation, and blood-retinal barrier disruption in the retinas and optic nerves in mice, which mimics human NMOSD-associated optic neuritis. Translational Relevance:Along with the previously reported development of paralysis, this study indicates that AQP4 peptide-immunized mice can be considered an animal model of NMOSD and a powerful tool for further understanding the pathophysiology of NMOSD-associated optic neuritis and for assessing the efficacy of drugs in its treatment.

Neuromyelitis optica spectrum disorder (NMOSD) is a rare autoimmune disease characterized by periods of remission and relapse; severe relapses often lead to permanent neurological disability. Satralizumab, an anti-interleukin-6 receptor (anti-IL-6R) antibody, has been proven in previous studies to reduce the frequency and severity of relapses in patients with NMOSD. There are several reports on the mechanisms through which anti-IL-6R antibodies are thought to suppress relapse. However, the mechanisms underlying how anti-IL-6R antibodies reduce the severity of myelitis have not been elucidated. We investigated the effect of an anti-IL-6R antibody (MR16-1) on the severity of myelitis in an AQP4 peptide-immunized mice model. This mouse model exhibits NMOSD-like pathological characteristics and the production of anti-AQP4 autoantibodies. Unlike the previously reported experimental protocol where antibody and peptide are administered simultaneously, we tested delayed administration of MR16-1 (9 days after peptide immunization). We found that delayed MR16-1 administration suppressed the clinical score of AQP4 peptide-immunized mice experiencing myelitis. Mice treated with MR16-1 showed a greater percentage of CD11c+ microglia in the spinal cord, along with upregulated expression of phagocytosis-related genes. Blockade of IL-6R by anti-IL-6R antibodies may suppress the severity of myelitis by increasing CD11c+ microglia and enhancing phagocytic function in AQP4 peptide-immunized mice.

Neuromyelitis optica spectrum disorder (NMOSD) is an autoimmune disease characterized by the production of autoantibodies against aquaporin-4 (AQP4). Treatment with prednisolone (PSL) or anti-IL-6 receptor (IL-6R) antibody can reduce the frequency of relapse in patients with AQP4 antibody-positive NMOSD. We previously established a mouse model of paralysis induced by intradermal immunization with AQP4 peptide. In this study, we investigated the effects of PSL and anti-IL-6R antibody treatment on paralysis and on bone fragility in this NMOSD mouse model. Prednisolone and anti-IL-6R antibody treatment each suppressed the clinical scores and incidence of paralytic symptoms in AQP4 peptide-immunized mice. High-PSL treatment induced thinning of cortical bone and reduction of tissue mineral density in the femoral shaft and a decrease in femoral bone strength, although it increased bone volume/tissue volume in the trabecular bone of the distal femur. In contrast, anti-IL-6R treatment showed no significant differences in bone strength or cortical thickness compared to the non-immunized naive group. Bone morphometric analysis showed that high-PSL treatment reduced the bone formation rate in both cortical and trabecular bone, with a predominance of bone resorption, whereas anti-IL-6R treatment demonstrated no notable effect on bone metabolism. These results suggest that anti-IL-6R antibody can prevent the development of paralytic symptoms in AQP4 peptide-immunized mice without reducing bone strength.

Myasthenia gravis (MG) is a chronic autoimmune disease characterized by muscle weakness and fatigue. It is caused by pathological autoantibodies against components expressed at neuromuscular junctions, such as acetylcholine receptor (AChR). Interleukin-6 (IL-6) has been suggested to play a role in the pathogenesis of MG, and IL-6 receptor (IL-6R) antibody treatment may provide a novel therapeutic option. In this study, we investigated the effects of IL-6R antibody treatment in an experimental autoimmune MG (EAMG) mouse model. We demonstrated that IL-6R antibody treatment improved muscle weakness, reduced IgG deposition at neuromuscular junctions, and the levels of AChR autoantibodies in serum. In addition, follicular helper T cells and Th17, plasma cells in lymph nodes were lower in IL-6R antibody treated mice. Our findings suggest that IL-6R blockade may be a novel and effective therapeutic strategy for the treatment of MG.

The objective of this study was to determine the effect of anti-IL-6 receptor antibody on muscle weakness in experimental autoimmune myasthenia gravis (EAMG) mice.

We challenged to create a mouse model of neuromyelitis optica spectrum disorder (NMOSD) induced by AQP4 peptide immunization. Intradermal immunization with AQP4 p201-220 peptide induced paralysis in C57BL/6J mice, but not in AQP4 KO mice. AQP4 peptide-immunized mice showed pathological features similar to NMOSD. Administration of anti-IL-6 receptor antibody (MR16-1) inhibited the induction of clinical signs and prevented the loss of GFAP/AQP4 and deposition of complement factors in AQP4 peptide-immunized mice. This novel experimental model may contribute to further understanding the pathogenesis of NMOSD, elucidating the mechanism of action of therapeutic agents, and developing new therapeutic approaches.

Neuromyelitis optica spectrum disorder (NMOSD) is an autoimmune astrocytopathy caused by antibodies against the aquaporin 4(AQP4) in end-feet of astrocytes. Breakdown of the blood–brain barrier (BBB) allowing ingress of AQP4 antibodies into the central nervous system (CNS) plays a key role in NMOSD. Although IL-6 blockade therapies such as satralizumab are effective in NMOSD, the therapeutic mechanism of IL-6 blockade, especially with respect to BBB disruption, are not fully understood because of the lack of the human models that are specialized to evaluate the BBB function. We constructed new in vitro human BBB models for evaluating continued barrier function, leukocyte transmigration and intracerebral transferability of IgGs utilizing the newly established triple co-culture system. In vitro and vivo experiments revealed that NMO-IgG increased intracerebral transferability of satralizumab, and that satralizumab suppressed the NMO-IgG-induced transmigration of T cells and barrier dysfunction. These results suggest that satralizumab, which can pass through the BBB in the presence of NMO-IgG, suppresses the barrier dysfunction and the disrupting controlled cellular infiltration at the BBB, leading to prevention of onset of NMOSD. One sentence summary Satralizumab and IL-6 blockade prevent lymphocyte migration and barrier dysfunction induced by NMO-IgG in EAE and novel triple co-culture BBB models.

BACKGROUND AND OBJECTIVES:To evaluate the pathophysiology of neuromyelitis optica spectrum disorder (NMOSD) and the therapeutic mechanism and levels of interleukin-6 (IL-6) blockade (satralizumab), especially with respect to blood-brain barrier (BBB) disruption with the new in vitro and ex vivo human BBB models and in vivo model.METHODS:We constructed new static in vitro and flow-based ex vivo models for evaluating continued barrier function, leukocyte transmigration, and intracerebral transferability of neuromyelitis optica-immunoglobulin G (NMO-IgG) and satralizumab across the BBB using the newly established triple coculture system that are specialized to closely mimic endothelial cell contact of pericytes and endfeet of astrocytes. In the in vivo study, we assessed the effects of an anti-IL-6 receptor antibody for mice (MR16-1) on in vivo BBB disruption in mice with experimental autoimmune encephalomyelitis in which IL-6 concentration in the spinal cord dramatically increases.RESULTS:In vitro and ex vivo experiments demonstrated that NMO-IgG increased intracerebral transferability of satralizumab and NMO-IgG and that satralizumab suppressed the NMO-IgG-induced transmigration of T cells and barrier dysfunction. In the in vivo study, the blockade of IL-6 signaling suppressed the migration of T cells into the spinal cord and prevented the increased BBB permeability.DISCUSSION:These results suggest that (1) our triple-cultured in vitro and in ex vivo BBB models are ideal for evaluating barrier function, leukocyte transmigration, and intracerebral transferability; (2) NMO-IgG increased the intracerebral transferability of NMO-IgG via decreasing barrier function and induced secretion of IL-6 from astrocytes causing more dysfunction of the barrier and disrupting controlled cellular infiltration; and (3) satralizumab, which can pass through the BBB in the presence of NMO-IgG, suppresses the BBB dysfunction and the infiltration of inflammatory cells, leading to prevention of onset of NMOSD.

Abstract Background Neuropathic pain in neuroimmunological disorders refers to pain caused by a lesion or disease of the somatosensory system such as multiple sclerosis (MS) and neuromyelitis optica spectrum disorder (NMOSD). MS and NMOSD are autoimmune disorders of the central nervous system, and ≥ 50% of patients with these disorders experience chronic neuropathic pain. The currently available medications for the management of neuropathic pain have limited effectiveness in patients with MS and NMOSD, and there is an unmet medical need to identify novel therapies for the management of chronic neuropathic pain in these patients. In this review article, we summarize the role of interleukin-6 (IL-6) in the pathogenesis of MS and NMOSD and the ameliorative effects of anti–IL-6 therapies in mouse models of experimental autoimmune encephalomyelitis (EAE). Main body Intraperitoneal injection of MR16-1, an anti–IL-6 receptor (IL-6R) antibody, reduced mechanical allodynia and spontaneous pain in EAE mice, which was attributed to a reduction in microglial activation and inhibition of the descending pain inhibitory system. The effect of anti–IL-6 therapies in ameliorating neuropathic pain in the clinical setting is controversial; a reduction in pain intensity has been reported with an anti–IL-6 antibody in four studies, namely a case report, a pilot study, a retrospective observational study, and a case series. Pain intensity was evaluated using a numerical rating scale (NRS), with a lower score indicating lesser pain. A reduction in the NRS score was reported in all four studies. However, in two randomized controlled trials of another anti–IL-6R antibody, the change in the visual analog scale pain score was not statistically significantly different when compared with placebo. This was attributed to the low mean pain score at baseline in both the trials and the concomitant use of medications for pain in one of the trials, which may have masked the effects of the anti–IL-6R antibody on neuropathic pain. Conclusion Thus, anti–IL-6 therapies might have a potential to reduce neuropathic pain, but further investigations are warranted to clarify the effect of inhibition of IL-6 signaling on neuropathic pain associated with MS and NMOSD.

Background: Although erythropoiesis-stimulating agents (ESAs) exert renoprotective effects in renal disease models, it has not been revealed whether the prolonged duration of action of ESAs contributes to their renoprotective effects. Objective: We examined whether the prolonged duration of ESAs’ action contributes to their renoprotective effects by comparing a divided administration of a short-acting ESA, epoetin beta (EPO), or a single administration of a long-acting ESA, epoetin beta pegol (continuous erythropoietin receptor activator; C.E.R.A.), to a single administration of EPO in chronic glomerulonephritis (GN) rats. Materials and Methods: Chronic GN was induced by intravenous injection of anti-Thy 1.1 antibody (0.6 mg/kg) into uninephrectomized rats (day 0). Chronic GN rats were intravenously injected once with vehicle (disease control; DC), EPO 5,000 IU/kg (single EPO), or C.E.R.A. 25 μg/kg (single C.E.R.A.) on day 1; or 3 times during the first week with EPO 1,667 IU/kg from day 1 (divided EPO; total 5,000 IU/kg). Hemoglobin (Hb) level and urinary total protein (U-TP) level which are the indexes of hematopoiesis and renoprotective effects, respectively, were measured several times over 8 weeks. Results: Divided EPO and single C.E.R.A. increased Hb levels more greatly than did single EPO. In all chronic GN rats, elevated U-TP levels decreased transiently 2 weeks after chronic GN induction and then flared again. Single EPO significantly suppressed this exacerbation of U-TP levels compared to DC. Divided EPO and single C.E.R.A. each significantly suppressed the exacerbation of U-TP levels compared to single EPO. Conclusion: Prolonged duration of ESAs’ action contributed significantly to their renoprotective effects.