Neutrophil extracellular traps are involved in the occurrence of interstitial lung disease in a murine experimental autoimmune myositis model.

The excessive formation of neutrophil extracellular traps (NETs) has been demonstrated to be a pathogenic mechanism of idiopathic inflammatory myopathy (IIM)-associated interstitial lung disease (ILD). This study aimed to answer whether an experimental autoimmune myositis (EAM) model can be used to study IIM-ILD and whether NETs participate in the development of EAM-ILD. An EAM mouse model was established using skeletal muscle homogenate and pertussis toxin (PTX). The relationship between NETs and the ILD phenotype was determined via histopathological analysis. As NETs markers, serum cell-free DNA (cfDNA) and serum citrullinated histone 3 (Cit-H3)-DNA were tested. The healthy mouse was injected with PTX intraperitoneally to determine whether PTX intervention could induce NETs formation in vivo. Neutrophils isolated from the peripheral blood of healthy individuals were given different interventions to determine whether PTX and skeletal muscle homogenate can induce neutrophils to form NETs in vitro. EAM-ILD had three pathological phenotypes similar to IIM-ILD. Cit-H3, neutrophil myeloperoxidase, and neutrophil elastase were overexpressed in the lungs of EAM model mice. The serum cfDNA level and Cit-H3-DNA complex level were significantly increased in EAM model mice. Serum cfDNA levels were increased significantly in vivo intervention with PTX in mice. Both PTX and skeletal muscle homogenate-induced neutrophils to form NETs in vitro. EAM-ILD pathological phenotypes are similar to IIM-ILD, and NETs are involved in the development of ILD in a murine model of EAM. Thus, the EAM mouse model can be used as an ideal model targeting NETs to prevent and treat IIM-ILD.

Improved skeletal muscle fatigue resistance in experimental autoimmune myositis mice following high-intensity interval training.

BACKGROUND: Muscle weakness and decreased fatigue resistance are key manifestations of systemic autoimmune myopathies (SAMs). We here examined whether high-intensity interval training (HIIT) improves fatigue resistance in the skeletal muscle of experimental autoimmune myositis (EAM) mice, a widely used animal model for SAM. METHODS: Female BALB/c mice were randomly assigned to control (CNT) or EAM groups (n = 28 in each group). EAM was induced by immunization with three injections of myosin emulsified in complete Freund's adjuvant. The plantar flexor (PF) muscles of mice with EAM were exposed to either an acute bout or 4 weeks of HIIT (a total of 14 sessions). RESULTS: The fatigue resistance of PF muscles was lower in the EAM than in the CNT group (P < 0.05). These changes were associated with decreased activities of citrate synthase and cytochrome c oxidase and increased expression levels of the endoplasmic reticulum stress proteins (glucose-regulated protein 78 and 94, and PKR-like ER kinase) (P < 0.05). HIIT restored all these alterations and increased the peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) and the mitochondrial electron transport chain complexes (I, III, and IV) in the muscles of EAM mice (P < 0.05). CONCLUSIONS: HIIT improves fatigue resistance in a SAM mouse model, and this can be explained by the restoration of mitochondria oxidative capacity via inhibition of the ER stress pathway and PGC-1α-mediated mitochondrial biogenesis.

Activation of TLR4 induces inflammatory muscle injury via mTOR and NF-κB pathways in experimental autoimmune myositis mice.

Idiopathic inflammatory myopathy (IIM) is an autoimmune disease that invades skeletal muscle; however, the etiology of IIM is still poorly understood. Toll-like receptor (TLR) 4 has been widely reported to take part in the autoimmune inflammation of IIMs. The mammalian target of rapamycin, mTOR, is a key central substance which mediates immune responses and metabolic changes, and also has been confirmed to be involved in the pathogenesis of IIMs. However, the interconnectedness between TLR4 and mTOR in IIM inflammation has not been fully elucidated. We hypothesized that TLR4 may play an important role in IIM inflammatory muscle injury by regulating mTOR. Mice were divided into four groups: a normal control group, IIM animal model (experimental autoimmune myositis, EAM) group, TAK242 intervention group and rapamycin (RAPA) intervention group. The results of EAM mice showed that TLR4, mTOR, nuclear factor-kappa B (NF-κB) and inflammatory factors interleukin-17A (IL-17A) and interferon γ (IFN-γ) mRNA levels were significantly upregulated. These factors were positively correlated with the degree of muscle inflammatory injury. When EAM mice were given the antagonist TAK242 to inhibit the TLR4 pathway, the results demonstrated that both mTOR and NF-κB were downregulated in the muscle of the mice. Muscle staining showed that the inflammatory injury was alleviated and the EAM mouse muscle strength was improved. Then, RAPA was used to inhibit the mTOR pathway, and the inflammatory factors IL-17A and IFN-γ were downregulated in EAM mouse muscle and serum. Consistently, muscle inflammatory injury was significantly reduced, and muscle strength was significantly improved. Our results suggest that TLR4 may regulate inflammatory muscle injury in EAM by activating the mTOR and NF-κB pathways, which provides both an experimental complement for the pathological mechanism of IIM and an encouraging target for the selection of effective treatments.

CD40L protects against mouse hepatitis virus-induced neuroinflammatory demyelination.

Neurotropic mouse hepatitis virus (MHV-A59/RSA59) infection in mice induces acute neuroinflammation due to direct neural cell dystrophy, which proceeds with demyelination with or without axonal loss, the pathological hallmarks of human neurological disease, Multiple sclerosis (MS). Recent studies in the RSA59-induced neuroinflammation model of MS showed a protective role of CNS-infiltrating CD4+ T cells compared to their pathogenic role in the autoimmune model. The current study further investigated the molecular nexus between CD4+ T cell-expressed CD40Ligand and microglia/macrophage-expressed CD40 using CD40L-/- mice. Results demonstrate CD40L expression in the CNS is modulated upon RSA59 infection. We show evidence that CD40L-/- mice are more susceptible to RSA59 induced disease due to reduced microglia/macrophage activation and significantly dampened effector CD4+ T recruitment to the CNS on day 10 p.i. Additionally, CD40L-/- mice exhibited severe demyelination mediated by phagocytic microglia/macrophages, axonal loss, and persistent poliomyelitis during chronic infection, indicating CD40-CD40L as host-protective against RSA59-induced demyelination. This suggests a novel target in designing prophylaxis for virus-induced demyelination and axonal degeneration, in contrast to immunosuppression which holds only for autoimmune mechanisms of inflammatory demyelination.

Anti-MAG neuropathy: From biology to clinical management.

The acquired chronic demyelinating neuropathies include a growing number of disease entities that have characteristic, often overlapping, clinical presentations, mediated by distinct immune mechanisms, and responding to different therapies. After the discovery in the early 1980s, that the myelin associated glycoprotein (MAG) is a target antigen in an autoimmune demyelinating neuropathy, assays to measure the presence of anti-MAG antibodies were used as the basis to diagnose the anti-MAG neuropathy. The route was open for describing the clinical characteristics of this new entity as a chronic distal large fiber sensorimotor neuropathy, for studying its pathogenesis and devising specific treatment strategies. The initial use of chemotherapeutic agents was replaced by the introduction in the late 1990s of rituximab, a monoclonal antibody against CD20+ B-cells. Since then, other anti-B cells agents have been introduced. Recently a novel antigen-specific immunotherapy neutralizing the anti-MAG antibodies with a carbohydrate-based ligand mimicking the natural HNK-1 glycoepitope has been described.

Inducible knockout of Clec16a in mice results in sensory neurodegeneration.

CLEC16A has been shown to play a role in autophagy/mitophagy processes. Additionally, genetic variants in CLEC16A have been implicated in multiple autoimmune diseases. We generated an inducible whole-body knockout, Clec16aΔUBC mice, to investigate the loss of function of CLEC16A. The mice exhibited a neuronal phenotype including tremors and impaired gait that rapidly progressed to dystonic postures. Nerve conduction studies and pathological analysis revealed loss of sensory axons that are associated with this phenotype. Activated microglia and astrocytes were found in regions of the CNS. Several mitochondrial-related proteins were up- or down-regulated. Upregulation of interferon stimulated gene 15 (IGS15) were observed in neuronal tissues. CLEC16A expression inversely related to IGS15 expression. ISG15 may be the link between CLEC16A and downstream autoimmune, inflammatory processes. Our results demonstrate that a whole-body, inducible knockout of Clec16a in mice results in an inflammatory neurodegenerative phenotype resembling spinocerebellar ataxia.

Immune response to dermatomyositis-specific autoantigen, transcriptional intermediary factor 1γ can result in experimental myositis.

OBJECTIVES: To investigate whether autoimmunity to transcriptional intermediary factor 1 (TIF1)γ, a ubiquitous nuclear autoantigen for myositis-specific autoantibodies detected in patients with dermatomyositis (DM) is pathogenetic for inflammatory myopathy. METHODS: Wild-type, ß2-microglobulin-null, perforin-null, Igµ-null and interferon α/ß receptor (IFNAR)-null mice were immunised with recombinant human TIF1γ whole protein. A thymidine incorporation assay was performed using lymph node T cells from TIF1γ-immunised mice. Plasma was analysed using immunoprecipitation followed by western blot analysis and enzyme-linked immunosorbent assays. Femoral muscles were histologically and immunohistochemically evaluated. CD8+ or CD4+ T cells isolated from lymph node T cells or IgG purified from plasma were adoptively transferred to naïve mice. TIF1γ-immunised mice were treated with anti-CD8 depleting antibody and a Janus kinase inhibitor, tofacitinib. RESULTS: Immunisation with TIF1γ-induced experimental myositis presenting with necrosis/atrophy of muscle fibres accompanied by CD8+ T cell infiltration successfully in wild-type mice, in which TIF1γ-specific T cells and antihuman and murine TIF1γ IgG antibodies were detected. The incidence and severity of myositis were significantly lower in ß2-microglobulin-null, perforin-null, CD8-depleted or IFNAR-null mice, while Igµ-null mice developed myositis normally. Adoptive transfer of CD8+ T cells induced myositis in recipients, while transfer of CD4+ T cells or IgG did not. Treatment with tofacitinib inhibited TIF1γ-induced myositis. CONCLUSIONS: Here we show that TIF1γ is immunogenic enough to cause experimental myositis, in which CD8+ T cells and type I interferons, but not CD4+ T cells, B cells or antibodies, are required. This murine model would be a tool for understanding the pathologies of DM.

Eccentric Resistance Training Ameliorates Muscle Weakness in a Mouse Model of Idiopathic Inflammatory Myopathies.

OBJECTIVE: High-force eccentric contractions (ECCs) have traditionally been excluded from rehabilitation programs that include patients with idiopathic inflammatory myopathies (IIMs) due to unverified fear of causing muscle damage and inflammation. In an IIM animal model that used mice with experimental autoimmune myositis (EAM), we undertook this study to investigate whether ECC training can safely and effectively be used to counteract muscle weakness in IIM. METHODS: EAM was induced in BALB/c mice by immunization with 3 injections of myosin emulsified in Freund's complete adjuvant. Controls (n = 12) and mice with EAM (n = 12) were exposed to either an acute bout of 100 ECCs or 4 weeks of ECC training (20 ECCs every other day). To induce ECCs, plantar flexor muscles were electrically stimulated while the ankle was forcibly dorsiflexed. RESULTS: Less cell damage, as assessed by Evans blue dye uptake, was observed in the muscles of mice with EAM, compared to controls, after an acute bout of 100 ECCs (P < 0.05). Maximum Ca2+ -activated force was decreased in skinned gastrocnemius muscle fibers from mice with EAM, and this was accompanied by increased expression of endoplasmic reticulum (ER) stress proteins, including Gsp78 and Gsp94 (P < 0.05). ECC training prevented the decrease in force and the increase in ER stress proteins and also enhanced the expression and myofibrillar binding of small heat-shock proteins (HSPs) (P < 0.05), which can stabilize myofibrillar structure and function. CONCLUSION: ECC training protected against the reduction in myofibrillar force-generating capacity in an IIM mouse model, and this occurred via inhibition of ER stress responses and small HSP-mediated myofibrillar stabilization.

Upregulated expression of NMDA receptor in the paraventricular nucleus shortens ejaculation latency in rats with experimental autoimmune prostatitis.

BACKGROUND: Estimated 30%-40% of patients with chronic prostatitis/chronic pelvic pain syndrome (CP/CPPS) suffer from premature ejaculation (PE), which is difficult to cure, but the mechanism is still unknown. Based on the results of our previous clinical studies and animal experiments, we propose that the glutamatergic system dysfunction in the paraventricular nucleus (PVN) may be involved. METHODS: To test this hypothesis, we used experimental autoimmune prostatitis (EAP) rats to investigate the effects of CP/CPPS on ejaculation behavior through integrating copulatory behavior testing, neuroelectrophysiologic experiments, and molecular biology technologies. RESULTS: Histological examination of prostate tissue in EAP rats exhibited consistent pathological findings with that in CP/CPPS patients. Behavior testing showed that ejaculation latency (EL) of EAP rats significantly shortened compared with the controls (5.1 ± 1.8 vs 9.1 ± 2.4 min, P < .001). Sympathetic nervous system (SNS) activity testing revealed that EAP rats displayed significantly higher plasma norepinephrine (NE) level (1780 ± 493 vs 1421 ± 453 pg/mL, P = .043) and SNS sensitivity (67.8 ± 9.6 vs 44.6 ± 8.7%, P < .001). Immunohistochemical detection and Western blot analysis both displayed that NR1 subunit expression of N-methyl-D-aspartic acid (NMDA) receptors in the PVN of EAP rats was significantly upregulated (P = .007 and P < .001). Furthermore, the expression of NMDA NR1 subunit positively correlated both with SNS sensitivity (r = .917, P < .001) and prostatic inflammation scores (r = .964, P < .001). CONCLUSION: This study shows that EAP rats suffer from the same PE symptom as CP/CPPS patients. CP/CPPS-induced inflammatory-immune response can significantly upregulate the expression of NMDA receptors in the PVN, which shortening the EL by enhancing SNS sensitivity. However, the exact mechanism of chronic inflammation in the prostate causing the upregulated expression of NMDA receptors needs to be further studied.

Haploinsufficient Rock1+/- and Rock2+/- Mice Are Not Protected from Cardiac Inflammation and Postinflammatory Fibrosis in Experimental Autoimmune Myocarditis.

Progressive cardiac fibrosis is a common cause of heart failure. Rho-associated, coiled-coil-containing protein kinases (ROCKs) have been shown to enhance fibrotic processes in the heart and in other organs. In this study, using wild-type, Rock1+/- and Rock2+/- haploinsufficient mice and mouse model of experimental autoimmune myocarditis (EAM) we addressed the role of ROCK1 and ROCK2 in development of myocarditis and postinflammatory fibrosis. We found that myocarditis severity was comparable in wild-type, Rock1+/- and Rock2+/- mice at day 21 of EAM. During the acute stage of the disease, hearts of Rock1+/- mice showed unaffected numbers of CD11b+CD36+ macrophages, CD11b+CD36-Ly6GhiLy6chi neutrophils, CD11b+CD36-Ly6G-Ly6chi inflammatory monocytes, CD11b+CD36-Ly6G-Ly6c- monocytes, CD11b+SiglecF+ eosinophils, CD11b+CD11c+ inflammatory dendritic cells and type I collagen-producing fibroblasts. Isolated Rock1+/- cardiac fibroblasts treated with transforming growth factor-beta (TGF-ß) showed attenuated Smad2 and extracellular signal-regulated kinase (Erk) phosphorylations that were associated with impaired upregulation of smooth muscle actin alpha (αSMA) protein. In contrast to cardiac fibroblasts, expanded Rock1+/- heart inflammatory myeloid cells showed unaffected Smad2 activation but enhanced Erk phosphorylation following TGF-ß treatment. Rock1+/- inflammatory cells responded to TGF-ß by a reduced transcriptional profibrotic response and failed to upregulate αSMA and fibronectin at the protein levels. Unexpectedly, in the EAM model wild-type, Rock1+/- and Rock2+/- mice developed a similar extent of cardiac fibrosis at day 40. In addition, hearts of the wild-type and Rock1+/- mice showed comparable levels of cardiac vimentin, periostin and αSMA. In conclusion, despite the fact that ROCK1 regulates TGF-ß-dependent profibrotic response, neither ROCK1 nor ROCK2 is critically involved in the development of postinflammatory fibrosis in the EAM model.

Reduced miR-146a Promotes REG3A Expression and Macrophage Migration in Polymyositis and Dermatomyositis.

Background: Growing evidence from studies elsewhere have illustrated that microRNAs (miRNAs) play important roles in polymyositis and dermatomyositis (PM/DM). However, little has been reported on their relationship with regenerating islet-derived protein 3-alpha (REG3A) as well as their associative roles in macrophage migration. Therefore, this study sought to establish the association between miR-146a and REG3A as well as investigate their functional roles in macrophage migration and PM/DM pathogenesis. Methods: Peripheral blood mononuclear cells (PBMCs) were isolated from PM/DM patients and healthy controls through density centrifugation. Macrophages were obtained from monocytes purified from PBMCs via differentiation before their transfection with miRNA or plasmids to investigate cell migration with transwell assay. An experimental autoimmune myositis murine model was used to investigate PM/DM. Real-time PCR and Western blot analysis were conducted to determine the expression levels of miR-146a, interferon gamma (IFN-γ), interleukin (IL)-17A, and REG3A. Results: The messenger RNA (mRNA) expression level of miR-146a markedly decreased, while the mRNA level of REG3A, IFN-γ, and IL-17A expression increased substantially in PBMCs from PM/DM patients compared with the healthy controls. The levels of IFN-γ and IL-17A in serum from PM/DM patients was much higher than the healthy controls. Immunohistochemistry analysis showed that REG3A expression increased in muscle tissues from patients. Consistent with clinical data, the mRNA expression level of miR-146a also decreased, whereas the mRNA and protein level of REG3A, IFN-γ, and IL-17A significantly increased in the muscle tissues of experimental autoimmune myositis mice. Moreover, miR-146a inhibited monocyte-derived macrophage migration, and REG3A promoted macrophage migration. In addition, IL-17A induced REG3A expression, while miR146a inhibited expression of REG3A in monocyte-derived macrophages from the PBMCs of the healthy donors. Notably, inhibition of macrophage migration by miR-146a was via the reduction in REG3A expression. Conclusions: Reduced miR-146a expression in PM/DM leads to increased REG3A expression that increases inflammatory macrophage migration, which may be a possible underlying mechanism of DM/PM pathogenesis.

Regulated Tristetraprolin Overexpression Dampens the Development and Pathogenesis of Experimental Autoimmune Uveitis.

Non-infectious uveitis, a common cause of blindness in man, is often mediated by autoimmunity, a process in which cytokines play major roles. The biosynthesis and secretion of pro-inflammatory cytokines are regulated in part by tristetraprolin (TTP), an endogenous anti-inflammatory protein that acts by binding directly to specific sequence motifs in the 3'-untranslated regions of target mRNAs, promoting their turnover, and inhibiting synthesis of their encoded proteins. We recently developed a TTP-overexpressing mouse (TTPΔARE) by deleting an AU-rich element (ARE) instability motif from the TTP mRNA, resulting in increased accumulation of TTP mRNA and protein throughout the animal. Here, we show that homozygous TTPΔARE mice are resistant to the induction of experimental autoimmune uveitis (EAU) induced by interphotoreceptor retinoid-binding protein (IRBP), an established model for human autoimmune (noninfectious) uveitis. Lymphocytes from TTPΔARE mice produced lower levels of the pro-inflammatory cytokines IFN-γ, IL-17, IL-6, and TNFα than wild type (WT) mice. TTPΔARE mice also produced lower titers of antibodies against the uveitogenic protein. In contrast, TTPΔARE mice produced higher levels of the anti-inflammatory cytokine IL-10, and had higher frequencies of regulatory T-cells, which, moreover, displayed a moderately higher per-cell regulatory ability. Heterozygous mice developed EAU and associated immunological responses at levels intermediate between homozygous TTPΔARE mice and WT controls. TTPΔARE mice were able, however, to develop EAU following adoptive transfer of activated WT T-cells specific to IRBP peptide 651-670, and naïve T-cells from TTPΔARE mice could be activated by antibodies to CD3/CD28. Importantly, TTPΔARE antigen presenting cells were significantly less efficient compared to WT in priming naïve T cells, suggesting that this feature plays a major role in the dampened immune responses of the TTPΔARE mice. Our observations demonstrate that elevated systemic levels of TTP can inhibit the pathogenic processes involved in EAU, and suggest the possible use of TTP-based treatments in humans with uveitis and other autoimmune conditions.

Beneficial role of simvastatin in experimental autoimmune myositis.

OBJECTIVE: Statins have immunomodulatory potential in autoimmune diseases but had not been studied as a disease-modifying agent in inflammatory myopathies. The objective of this study is to assess the effect of simvastatin in an experimental model of autoimmune myositis in mice on muscle strength and histopathology. METHODS: Four groups of mice (n = 5 per group) were selected for experimentally induced myositis. Mice were immunized with 1.5 mg myosin in complete Freund's adjuvant weekly for two times and injected with 500 ng pertussis toxin twice immediately after each immunization. From day 1 before immunization to 10 days after the last immunization, mice were treated with oral simvastatin (10 or 20 or 40 mg/kg) diluted in DMSO. The control group mice were injected with complete Freund's adjuvant weekly for two times and did not receive treatment. Non-immunized mice (n = 5 per group) were treated either with simvastatin (5 mg/kg or 20 mg/kg or 40 mg/kg of simvastatin diluted in DMSO) or with DMSO. RESULTS: Inflammation was observed in myositis groups with positive myositis-specific antibodies. Muscle strength dropped significantly after immunization. Immunized simvastatin 20 mg/kg treated group had significantly higher muscle strength versus non-treated myositis mice and versus other simvastatin doses. Besides, a trend toward higher serum Th17 percentage population was found in immunized non-treated mice, versus immunized simvastatin- treated mice, without significant difference. CONCLUSION: Simvastatin at 20 mg/kg decreases the severity of myositis in experimental autoimmune myositis and is a candidate of being a disease-modifying agent in inflammatory myopathies.

Blocking LFA-1 Aggravates Cardiac Inflammation in Experimental Autoimmune Myocarditis.

The lymphocyte function-associated antigen 1 (LFA-1) is a member of the beta2-integrin family and plays a pivotal role for T cell activation and leukocyte trafficking under inflammatory conditions. Blocking LFA-1 has reduced or aggravated inflammation depending on the inflammation model. To investigate the effect of LFA-1 in myocarditis, mice with experimental autoimmune myocarditis (EAM) were treated with a function blocking anti-LFA-1 antibody from day 1 of disease until day 21, the peak of inflammation. Cardiac inflammation was evaluated by measuring infiltration of leukocytes into the inflamed cardiac tissue using histology and flow cytometry and was assessed by analysis of the heart weight/body weight ratio. LFA-1 antibody treatment severely enhanced leukocyte infiltration, in particular infiltration of CD11b+ monocytes, F4/80+ macrophages, CD4+ T cells, Ly6G+ neutrophils, and CD133+ progenitor cells at peak of inflammation which was accompanied by an increased heart weight/body weight ratio. Thus, blocking LFA-1 starting at the time of immunization severely aggravated acute cardiac inflammation in the EAM model.

Enteric neuroplasticity and dysmotility in inflammatory disease: key players and possible therapeutic targets.

Intestinal functions, including motility and secretion, are locally controlled by enteric neural networks housed within the wall of the gut. The fidelity of these functions depends on the precision of intercellular signaling among cellular elements, including enteric neurons, epithelial cells, immune cells, and glia, all of which are vulnerable to disruptive influences during inflammatory events. This review article describes current knowledge regarding inflammation-induced neuroplasticity along key elements of enteric neural circuits, what is known about the causes of these changes, and possible therapeutic targets for protecting and/or repairing the integrity of intrinsic enteric neurotransmission. Changes that have been detected in response to inflammation include increased epithelial serotonin availability, hyperexcitability of intrinsic primary afferent neurons, facilitation of synaptic activity among enteric neurons, and attenuated purinergic neuromuscular transmission. Dysfunctional propulsive motility has been detected in models of colitis, where causes include the changes described above, and in models of multiple sclerosis and other autoimmune conditions, where autoantibodies are thought to mediate dysmotility. Other cells implicated in inflammation-induced neuroplasticity include muscularis macrophages and enteric glia. Targeted treatments that are discussed include 5-hydroxytryptamine receptor 4 agonists, cyclooxygenase inhibitors, antioxidants, B cell depletion therapy, and activation of anti-inflammatory pathways.

Low-Dose Subcutaneous Anti-CD20 Treatment Depletes Disease Relevant B Cell Subsets and Attenuates Neuroinflammation.

To explore the B cell depleting capacity of a low-dose (20 µg) subcutaneous mouse anti-CD20 antibody treatment on disease-relevant B cell populations within lymph nodes and the spleen. B cell depleting capacity was explored in healthy female C57BL/6 and BALB/c mice; following immune activation in two different mouse models: trinitrophenylated lipopolysaccharide model (thymus-independent response) and dinitrophenyl-keyhole limpet hemocyanin model (thymus-dependent response); and in a chronic neuroinflammation experimental autoimmune encephalomyelitis model. CD20 protein expression on B cell subpopulations was also studied. The subcutaneous anti-CD20 regimen resulted in rapid depletion of B cells in blood, lymph nodes and spleen. Low-dose subcutaneous treatment did not reduce antigen-specific immunoglobulin M and immunoglobulin G titers in all subgroups, and relatively spared splenic marginal zone (MZ) B cells in both T cell dependent and T cell independent B cell immunization models. Analysis of immune compartments during anti-CD20-modulated autoimmune neuroinflammation showed that the maximal B cell depletion was achieved within 2 days of treatment and was highest in the lymph node. Regardless of the tissues analyzed, low-dose subcutaneous treatment was characterized by rapid B cell repletion following treatment cessation. CD20 protein expression was consistent on all B cell subsets in blood, and was more pronounced in germinal center B cells of lymph nodes and MZ B-cells of the spleen. Low-dose subcutaneous anti-CD20 therapy effectively depleted B cells within lymphatic tissues and reduced the severity of neuroinflammation. These data suggest that subcutaneous anti-CD20 therapies can effectively target disease-relevant B cell populations, have shorter repletion kinetics and maintain vaccination responses, thereby achieving autoimmune amelioration without severely impacting immune surveillance functions. Graphical Abstract *p < 0.05; **p < 0.01. CD, cluster of differentiation; DNP-KLH, dinitrophenyl-keyhole limpet hemocyanin; EC50, concentration of a drug that gives half-maximal response; Ig, immunoglobulin; MZ, marginal zone; s.c., subcutaneous; SEM, standard error of mean; TNP-LPS, trinitrophenylatedlipopolysaccharide.

Plakophilin-2 Haploinsufficiency Causes Calcium Handling Deficits and Modulates the Cardiac Response Towards Stress.

Human variants in plakophilin-2 (PKP2) associate with most cases of familial arrhythmogenic cardiomyopathy (ACM). Recent studies show that PKP2 not only maintains intercellular coupling, but also regulates transcription of genes involved in Ca2+ cycling and cardiac rhythm. ACM penetrance is low and it remains uncertain, which genetic and environmental modifiers are crucial for developing the cardiomyopathy. In this study, heterozygous PKP2 knock-out mice (PKP2-Hz) were used to investigate the influence of exercise, pressure overload, and inflammation on a PKP2-related disease progression. In PKP2-Hz mice, protein levels of Ca2+-handling proteins were reduced compared to wildtype (WT). PKP2-Hz hearts exposed to voluntary exercise training showed right ventricular lateral connexin43 expression, right ventricular conduction slowing, and a higher susceptibility towards arrhythmias. Pressure overload increased levels of fibrosis in PKP2-Hz hearts, without affecting the susceptibility towards arrhythmias. Experimental autoimmune myocarditis caused more severe subepicardial fibrosis, cell death, and inflammatory infiltrates in PKP2-Hz hearts than in WT. To conclude, PKP2 haploinsufficiency in the murine heart modulates the cardiac response to environmental modifiers via different mechanisms. Exercise upon PKP2 deficiency induces a pro-arrhythmic cardiac remodeling, likely based on impaired Ca2+ cycling and electrical conduction, versus structural remodeling. Pathophysiological stimuli mainly exaggerate the fibrotic and inflammatory response.

The autoimmune basis of hypopituitarism in traumatic brain injury: fiction or reality?

Post-traumatic hypopituitarism has remained as an obscured cause of worsening morbidity and mortality in head injury patients. Researchers have for decades been puzzled by the mechanism of pituitary dysfunction in these cases. Amongst other causes like direct injury, vascular injury etc, an immunological basis of hypopituitarism has been suggested in some animal studies as well as human research. In this article, we have reviewed the latest articles and compiled the evidence which suggests for or against the role of autoimmunity in post-traumatic hypopituitarism or which defines the strength to which autoimmunity has been established as a cause of head-injury induced pituitary dysfunction.

Human Stem Cell-Derived Models: Lessons for Autoimmune Diseases of the Nervous System.

Autoimmunity of the peripheral and central nervous system is an important cause of disease and long-term neurological disability. Autoantibodies can target both intracellular and extracellular neuronal epitopes. Autoantibodies that target cell-surface epitopes infer pathogenicity through several distinct mechanisms, while patients often respond to immunotherapy. However, the underlying pathogenesis of these autoantibodies is yet to be fully understood. Human stem cell-based disease modeling, and the rise of induced pluripotent stem cell technology in particular, has revolutionized the fields of disease modeling and therapeutic screening for neurological disorders. These human disease models offer a unique platform in which to study autoimmunity of the nervous system. Here, we take an in-depth look at the possibilities that these models provide to study neuronal autoantibodies and their underlying pathogenesis.

Nrf2/ARE pathway inhibits inflammatory infiltration by macrophage in rats with autoimmune myositis.

BACKGROUND: Idiopathic inflammatory myopathies (IIM) are a group of autoimmune diseases characterized by muscle disorders. We conducted this study to detect whether NF-E2-related factor 2 (Nrf2) pathway inhibit inflammatory infiltration by macrophage in experimental autoimmune myositis (EAM) rat model. METHODS: CD163 levels were examined by immunohistochemistry (IHC), while serum creatine kinase (CK), reactive oxygen species (ROS), and serum monocyte chemoattractant protein-1 (MCP-1), tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) levels were determined by enzyme linked immunosorbnent assay (ELISA), both in IIM patients and EAM rat. We also detected MCP-1, TNF-α, IL-6, and Nrf2 levels by Realtime quantitative PCR (RT-PCR) in patients' muscles, and MCP-1, TNF-α, IL-6, and Nrf2, HO-1, NQO-1 levels by RT-PCR and Western blot in EAM rats' muscles. EAM macrophages were separated, and Nrf2 over-expression macrophages were constructed. ROS level and cell migration were detected by flow cytometer and transwell assay respectively. Then, levels of MCP-1, TNF-α, IL-6, Nrf2, Heme oxygenase-1 (HO-1) and NAD(P)H: quinine oxidoreductase 1 (NQO-1) were detected by RT-PCR and Western blot. RESULTS: Results showed that EAM rats were histopathologically inflammatory cell infiltration. Levels of CD163, serum CK and ROS, serum/muscle MCP-1, TNF-α and IL-6 increased and muscle Nrf2 level decreased in IIM patients and EAM rats. Cell migration ability and levels of ROS, MCP-1, TNF-α, IL-6, and plasma Nrf2 were down-regulated, and total/nucleus Nrf2, HO-1, NQO-1 were up-regulated notably when Nrf2 over-expressed. CONCLUSION: Nrf2 inhibited EAM macrophage infiltration by activating Nrf2/ARE pathway which could induce ROS degradation and inhibit pro-inflammatory factors expression.