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.

MicroRNA-381 reduces inflammation and infiltration of macrophages in polymyositis via downregulating HMGB1.

The downregulation of microRNA (miR)-381 has been detected in various diseases. The present study aimed to investigate the effects, and underlying mechanisms of miR-381 on inflammation and macrophage infiltration in polymyositis (PM). A mouse model of experimental autoimmune myositis (EAM) was generated in this study. Hematoxylin and eosin staining was conducted to detect the inflammation of muscle tissues. In addition, ELISA and immunohistochemistry were performed to determine the expression levels of associated factors, and reverse transcription-quantitative polymerase chain reaction and western blotting were used to detect the expression levels of related mRNAs and proteins. A luciferase activity assay was used to confirm the binding of miR-381 and high mobility group box 1 (HMGB1) 3' untranslated region. Transwell assays were also performed to assess the migratory ability of macrophages. The results demonstrated that serum creatine kinase (s-CK), HMGB1 and cluster of differentiation (CD)163 expression in patients with PM were increased compared within healthy controls. Conversely, the expression levels of miR-381 were downregulated in patients with PM. Furthermore, high HMGB1 expression was associated with poor survival rate in patients with PM. In the mouse studies, muscle inflammation and CD163 expression were decreased in the anti-IL-17 and anti-HMGB1 groups, compared with in the EAM model group. The expression levels of s-CK, HMGB1, IL-17 and intercellular adhesion molecule (ICAM)-1 were also downregulated in response to anti-IL-17 and anti-HMGB1. These findings indicated that HMGB1 was closely associated with inflammatory responses. In addition, the present study indicated that transfection of macrophages with miR-381 mimics reduced the migration of inflammatory macrophages, and the expression levels of HMGB1, IL-17 and ICAM-1. Conversely, miR-381 inhibition exerted the opposite effects. The effects of miR-381 inhibitors were reversed by HMGB1 small interfering RNA. In conclusion, miR-381 may reduce inflammation and the infiltration of macrophages; these effects were closely associated with the downregulation of HMGB1.

Exacerbation of Murine Experimental Autoimmune Myositis by Toll-Like Receptor 7/8.

OBJECTIVE: Toll-like receptor 7 (TLR-7), TLR-8, and interferon (IFN)-induced genes are expressed in patients with idiopathic inflammatory myositis. This study was undertaken to investigate whether their activation influences the natural history of the disease. METHODS: Experimental autoimmune myositis was induced in mice by injection of the amino-terminal portion of the murine histidyl-transfer RNA synthetase (HisRS). Disease was compared in the presence or the absence of the TLR-7/8 agonist R-848 in wild-type mice and in mice that fail to express the IFNα/ß receptor (IFNα/ßR-null mice). RESULTS: Experimental autoimmune myositis induced by a single intramuscular immunization with HisRS spontaneously abated after 7-8 weeks. In contrast, levels of anti-HisRS autoantibodies, endomysial/perimysial leukocyte infiltration, and myofiber regeneration persisted at the end of the follow-up period (22 weeks after immunization) in mice immunized with HisRS in the presence of R-848. Myofiber major histocompatibility complex (MHC) class I molecules were detectable only in mice immunized with both HisRS and R-848. MHC up-regulation occurred early and in muscles that were not directly injected with HisRS. Muscle MHC expression paralleled with leukocyte infiltration. MHC class I molecules were selectively up-regulated in myotubes challenged with R-848 in vitro. Type I IFN was necessary for the prolonged autoantibody response and for the spreading of the autoimmune response, as demonstrated using IFNα/ßR-null mice. Muscle infiltration was maintained in the injected muscle up to the end of the follow-up period. CONCLUSION: TLR-7/8 activation is necessary to induce and maintain a systemic autoimmune response targeting the skeletal muscle. This experimental autoimmune myositis model reproduces many characteristics of human idiopathic inflammatory myopathies and may represent a tool for preclinical studies.

Roles of Treg/Th17 Cell Imbalance and Neuronal Damage in the Visual Dysfunction Observed in Experimental Autoimmune Optic Neuritis Chronologically.

Optic neuritis associated with multiple sclerosis and its animal model, experimental autoimmune optic neuritis (EAON), is characterized by inflammation, T cell activation, demyelination, and neuronal damage, which might induce permanent vision loss. Elucidating the chronological relationship among the features is critical for treatment of demyelinating optic neuritis. EAON was induced in C57BL/6 mice immunized with myelin oligodendrocyte glycoprotein subcutaneously, and visual function was assessed by flash-visual evoked potential (F-VEP) at days 7, 11, 14, 19, 23, 28 post-immunization. Retinal ganglion cell (RGC) apoptosis was measured by terminal-deoxynucleotidyl transferase-mediated nick-end labeling. Demyelination and axonal damage were verified with myelin basic protein (MBP) and ß-amyloid precursor protein staining, respectively. Real-time polymerase chain reaction quantified IL-17, IL-1ß, TGF-ß, FoxP3, IL-6, and IL-10 mRNA expression in the optic nerve, as well as FoxP3 and IL-17 staining. Systemic changes of Th17 and Treg cells were tested by flow cytometry in spleen. F-VEP latency was prolonged at 11 days and peaked at 23 days commensurate with demyelination. However, F-VEP amplitude was reduced at 11 days, preceding axon damage, and was exacerbated at 23 days when a peak in RGC apoptosis was detected. Th17 cells up-regulated as early as 7 days and peaked at 11 days, while Treg cells down-regulated inversely compared to Th17 cells change as verified by IL-17 and FoxP3 expression; spleen cell samples were slightly different, demonstrating marked changed at 14 days. Treg/Th17 cell imbalance in the optic nerve precedes and may initiate neuronal damage of axons and RGCs. These changes are commensurate with the appearances of visual dysfunction reflected in F-VEP and hence may offer a novel therapeutic avenue for vision preservation.

Development of an improved animal model of experimental autoimmune myositis.

Multiple animal models of experimental autoimmune myositis (EAM) have been developed. However, these models vary greatly in the severity of disease and reproducibility. The goal of this study was to test whether vaccination twice with increased dose of rat myosin and pertussis toxin (PT) could induce EAM with severer disease in mice. BALB/c mice were injected with 1 mg rat myosin in 50% complete Freund's adjuvant (CFA) weekly for four times and one time of PT (EAM) or twice with 1.5 mg myosin in CFA and PT (M-EAM). In comparison with that in the CFA and PT injected controls, vaccination with rat myosin and injection PT significantly reduced the muscle strength and EMG duration, elevated serum creatine kinase levels, promoted inflammatory infiltration in the muscle tissues, leading to pathological changes in the muscle tissues, demonstrating to induce EAM. Interestingly, we found that vaccination twice with the high dose of myosin and PT prevented EAM-related gain in body weights and caused significantly less muscle strength in mice. More importantly, all of the mice receiving high dose of myosin and PT survived while 3 out of 16 mice with four times of low dose of myosin died. Finally, vaccination with high dose of myosin promoted CD4(+) and CD8(+) T cell infiltration in the muscle tissues and up-regulated MHC-I expression in the muscle tissues of mice. Hence, the new model of EAM is a time-saving, efficient and easily replicable tool for studying autoimmune myositis.