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.

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.

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.

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.

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.

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.

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.

Experimental Models of Autoimmune Demyelinating Diseases in Nonhuman Primates.

Human idiopathic inflammatory demyelinating diseases (IIDD) are a heterogeneous group of autoimmune inflammatory and demyelinating disorders of the central nervous system (CNS). These include multiple sclerosis (MS), the most common chronic IIDD, but also rarer disorders such as acute disseminated encephalomyelitis (ADEM) and neuromyelitis optica (NMO). Great efforts have been made to understand the pathophysiology of MS, leading to the development of a few effective treatments. Nonetheless, IIDD still require a better understanding of the causes and underlying mechanisms to implement more effective therapies and diagnostic methods. Experimental autoimmune encephalomyelitis (EAE) is a commonly used animal model to study the pathophysiology of IIDD. EAE is principally induced through immunization with myelin antigens combined with immune-activating adjuvants. Nonhuman primates (NHP), the phylogenetically closest relatives of humans, challenged by similar microorganisms as other primates may recapitulate comparable immune responses to that of humans. In this review, the authors describe EAE models in 3 NHP species: rhesus macaques ( Macaca mulatta), cynomolgus macaques ( Macaca fascicularis), and common marmosets ( Callithrix jacchus), evaluating their respective contribution to the understanding of human IIDD. EAE in NHP is a heterogeneous disease, including acute monophasic and chronic polyphasic forms. This diversity makes it a versatile model to use in translational research. This clinical variability also creates an opportunity to explore multiple facets of immune-mediated mechanisms of neuro-inflammation and demyelination as well as intrinsic protective mechanisms. Here, the authors review current insights into the pathogenesis and immunopathological mechanisms implicated in the development of EAE in NHP.

IFN-ß-induced reactive oxygen species and mitochondrial damage contribute to muscle impairment and inflammation maintenance in dermatomyositis.

Dermatomyositis (DM) is an autoimmune disease associated with enhanced type I interferon (IFN) signalling in skeletal muscle, but the mechanisms underlying muscle dysfunction and inflammation perpetuation remain unknown. Transcriptomic analysis of early untreated DM muscles revealed that the main cluster of down-regulated genes was mitochondria-related. Histochemical, electron microscopy, and in situ oxygraphy analysis showed mitochondrial abnormalities, including increased reactive oxygen species (ROS) production and decreased respiration, which was correlated with low exercise capacities and a type I IFN signature. Moreover, IFN-ß induced ROS production in human myotubes was found to contribute to mitochondrial malfunctions. Importantly, the ROS scavenger N-acetyl cysteine (NAC) prevented mitochondrial dysfunctions, type I IFN-stimulated transcript levels, inflammatory cell infiltrate, and muscle weakness in an experimental autoimmune myositis mouse model. Thus, these data highlight a central role of mitochondria and ROS in DM. Mitochondrial dysfunctions, mediated by IFN-ß induced-ROS, contribute to poor exercise capacity. In addition, mitochondrial dysfunctions increase ROS production that drive type I IFN-inducible gene expression and muscle inflammation, and may thus self-sustain the disease. Given that current DM treatments only induce partial recovery and expose to serious adverse events (including muscular toxicity), protecting mitochondria from dysfunctions may open new therapeutic avenues for DM.

Pregabalin enhances myelin repair and attenuates glial activation in lysolecithin-induced demyelination model of rat optic chiasm.

Multiple sclerosis (MS) is an autoimmune disease in which more than 70% of patients experience visual disturbance as the earliest symptoms. Lysolecithin (LPC)-induced focal demyelination model has been developed to evaluate the effects of different therapies on myelin repair improvement. In this study, the effects of pregabalin administration on myelin repair and glial activation were investigated. Local demyelination was induced by administration of LPC (1%, 2µL) into the rat optic chiasm. Rats underwent daily injection of pregabalin (30mg/kg, i.p) or vehicle. Visual-evoked potentials (VEPs) recordings were performed for evaluating the function of optic pathway on days 3, 7, 14 and 28 post lesions. Myelin specific staining and immunostaining against GFAP and Iba1 were also carried out for assessment of myelination and glial activation respectively. Electrophysiological data indicated that pregabalin administration could significantly reduce the P1-N1 latency and increase the amplitude of VEPs waves compared to saline group. Luxol fast blue staining and immunostaining against PLP, as mature myelin marker, showed that myelin repair was improved in animals received pregabalin treatment. In addition, pregabalin effectively reduced the expression of GFAP and Iba1 as activated glial markers in optic chiasm. The present study indicates that pregabalin administration enhances myelin repair and ameliorates glial activation of optic chiasm following local injection of LPC.

Loss of galectin-3 decreases the number of immune cells in the subventricular zone and restores proliferation in a viral model of multiple sclerosis.

Multiple sclerosis (MS) frequently starts near the lateral ventricles, which are lined by subventricular zone (SVZ) progenitor cells that can migrate to lesions and contribute to repair. Because MS-induced inflammation may decrease SVZ proliferation and thus limit repair, we studied the role of galectin-3 (Gal-3), a proinflammatory protein. Gal-3 expression was increased in periventricular regions of human MS in post-mortem brain samples and was also upregulated in periventricular regions in a murine MS model, Theiler's murine encephalomyelitis virus (TMEV) infection. Whereas TMEV increased SVZ chemokine (CCL2, CCL5, CCL, and CXCL10) expression in wild type (WT) mice, this was inhibited in Gal-3(-/-) mice. Though numerous CD45+ immune cells entered the SVZ of WT mice after TMEV infection, their numbers were significantly diminished in Gal-3(-/-) mice. TMEV also reduced neuroblast and proliferative SVZ cell numbers in WT mice but this was restored in Gal-3(-/-) mice and was correlated with increased numbers of doublecortin+ neuroblasts in the corpus callosum. In summary, our data showed that loss of Gal-3 blocked chemokine increases after TMEV, reduced immune cell migration into the SVZ, reestablished SVZ proliferation and increased the number of progenitors in the corpus callosum. These results suggest Gal-3 plays a central role in modulating the SVZ neurogenic niche's response to this model of MS.

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.

Dermatomiosite na infância / Juvenile dermatomyositis

A dermatomiosite juvenil é uma doença autoimune que acomete crianças e adolescentes entre 2 e 17 anos de idade, com média de 7 anos ao diagnóstico. É caracterizada por vasculopatia sistêmica e suas manifestações principais são fraqueza muscular proximal simétrica, elevação de enzimas musculares séricas e lesões cutâneas, sendo que o heliótropo e as pápulas de Gottron são patognomônicas. Sua identificação precoce e instituição rápida de tratamento adequado podem prevenir o aparecimento de calcinose e possibilitam melhor prognóstico e qualidade de vida ao paciente. Embora a base medicamentosa da terapia seja o uso de glicocorticoide, o metotrexato, a ciclosporina, a azatioprina e a ciclofosfamida, dependendo da gravidade, são os imunossupressores mais frequentemente utilizados. Imunoglobulina endovenosa pode ser útil nos casos graves e atualmente o uso de imunobiológicos representa uma nova perspectiva para os casos refratários.

Axonal damage in multiple sclerosis.

Multiple sclerosis is a debilitating disease of the central nervous system that has been characteristically classified as an immune-mediated destruction of myelin, the protective coating on nerve fibers. Although the mechanisms responsible for the immune attack to central nervous system myelin have been the subject of intense investigation, more recent studies have focused on the neurodegenerative component, which is cause of clinical disability in young adults and appears to be only partially controlled by immunomodulatory therapies. Here, we review distinct, but not mutually exclusive, mechanisms of pathogenesis of axonal damage in multiple sclerosis patients that are either consequent to long-term demyelination or independent from it. We propose that the complexity of axonal degeneration and the heterogeneity of the underlying pathogenetic mechanisms should be taken into consideration for the design of targeted therapeutic intervention.

[The expression of the type I interferon system in muscle and lung of autoimmune myositis rat model].

OBJECTIVE: To investigate the expression levels of the type I IFN system in muscle and lung of experimental autoimmune myositis (EAM) model and to evaluate whether the type I IFN system associates with the pathogenesis of the EAM model in rats. METHODS: The EAM model was established to determine creatine kinase (CK) in blood serum. The pathology of muscle and lung tissue was examined by hematoxylin-eosin staining. The concentration of type I IFN system mRNA in muscle and lung tissue was detected by real-time PCR. RESULTS: The concentration of CK in model group [(209.17 ± 91.95) IU/L] was significantly higher than that of two control groups (P < 0.05). The scores of muscle and lung in EAM model were significantly higher than that of control groups (all P < 0.05). The expression levels of the type I IFN system in muscle of EAM model were significantly higher than that of control groups (all P < 0.05). The expression levels of the type I IFN system in muscle with EAM model were positively correlated with CK and the scores of muscle (all P < 0.05). The expression levels of IFNα, IFNß, IFNαR1, signal transducer and activator of transcription 1 (STAT1), myxovirus resistance protein 1 (MX1) in lung of EAM model were significantly higher than those of control groups (P < 0.05), but not seen in INF-induced protein with tetratricopeptide repeats 1 (IFIT1) and IFN-stimulated gene 15 (ISG15). The expression levels of IFNα, IFNß, IFNαR1, STAT1 and MX1 in lung with EAM model were positively correlated with the scores of lung pathology (all P < 0.05). CONCLUSION: The type I IFN system probably played a crucial role in the pathogenesis and the pathology of muscle and lung of EAM model.

Interleukin-2 deficiency-induced T cell autoimmunity in the mouse brain.

Interleukin-2 (IL-2) has been implicated in the pathogenesis of neurodevelopmental and neurodegenerative disorders. Studies from our lab have shown that adult IL-2 knockout (KO) mice exhibit septohippocampal pathology and related behavioral deficits. Compared to IL-2 wild-type (WT) mice, IL-2 KO mice have a marked and selective loss of septal cholinergic neurons that occurs between the third postnatal week and adulthood. Given that the development of septal neurons is completed by embryonic day 17 and that IL-2 KO mice exhibit peripheral autoimmunity that develops progressively post-weaning, our data and others led us to postulate that the loss of septal neurons in adult IL-2 KO mice is due to selective autoimmune neurodegeneration that coincides with increasing levels of peripheral autoimmunity. Thus, the present study tested the hypotheses: (1) that T cells selectively target the septum, and; (2) that T lymphocyte infiltration to the septum would correlate with peripheral autoimmune disease. We quantified CD3(+) T cells in the septum, hippocampus, and cerebellum of IL-2 KO and IL-2 WT mice at ages ranging from 2 to 14 weeks. T cells infiltrated the brains of IL-2 deficient mice, but were not selective for the septum. Brain T lymphocyte levels in IL-2 KO mice correlated positively with the degree of peripheral autoimmunity. We did not detect CD19(+) B lymphocytes, IgG-positive lymphocytes or IgG deposition indicative of autoantibodies in the brains of IL-2 KO mice. Further study is needed to understand how IL-2 deficiency-induced autoimmune T lymphocytes interact with endogenous brain cells to alter function and promote disease.

Role of regulatory T cells in a new mouse model of experimental autoimmune myositis.

Polymyositis is a rare and severe inflammatory muscle disorder. Treatments are partially efficacious but have many side effects. New therapeutic approaches must be first tested in a relevant animal model. Regulatory CD4+CD25+ T cells (Tregs) have been rediscovered as a pivotal cell population in the control of autoimmunity, but the connection between polymyositis and Tregs is currently unknown. To develop a reproducible experimental autoimmune myositis model of polymyositis, mice were immunized once a week for 3 weeks with 1 mg of partially purified myosin emulsified in complete Freund's adjuvant. All mice injected with myosin and complete Freund's adjuvant developed myositis. The infiltrates were composed of CD4(+) and CD8(+) cells, as well as macrophages, but did not contain B lymphocytes. In mice that were depleted of Tregs, the myositis was more severe, as determined by quantitative scoring of muscle inflammation (2.36 +/- 0.9 vs. 1.64 +/- 0.8, P = 0.019). In contrast, injection of in vitro expanded polyclonal Tregs at the time of immunization significantly improved the disease (quantitative score of inflammation 0.87 +/- 1.06 vs. 2.4 +/- 0.67, P = 0.047). Transfer of sensitized or CD4(+)-sorted cells from the lymph nodes of experimental autoimmune myositis mice induced myositis in naïve, irradiated, recipient mice. Thus, experimental autoimmune myositis is a reproducible, transferable disease in mice, both aggravated by Treg depletion and improved by polyclonal Treg injection.

Gene expression profiles of cardiomyocytes in rat autoimmune myocarditis by DNA microarray and increase of regenerating gene family.

Cardiomyocytes with myocarditis compared with the normal state are thought to change the expressions of various genes greatly, some of which may be new biomarkers or new biologic medicinal products. However, until now, little comprehensive analysis has been made of gene-expression changes in cardiomyocytes with myocarditis. In this study, we performed a DNA microarray analysis by using cardiomyocytes from rat experimental autoimmune myocarditis (EAM). On day 0, rats were immunized with porcine cardiac myosin and cardiomyocytes were isolated and purified from EAM hearts and normal hearts by a method that is hardly thought to change gene expressions in cardiomyocytes. RNA from normal cardiomyocytes and cardiomyocytes of EAM on day 18 was analyzed for 7711 gene expressions by DNA microarray. Some gene expressions showed over 10-fold changes. In particular, the regenerated gene (Reg)2/pancreatitis-associated protein (PAP)1 messenger RNA (mRNA) level most markedly increased in the genes, which were clearly expressed in cardiomyocytes rather than in noncardiomyocytes, and it was approximately 2000-fold greater in cardiomyocytes under active myocarditis than normal by real-time reverse transcription polymerase chain reaction analysis. Moreover, we demonstrated that Reg2/PAP1 proteins determined by Western blot analysis and immunohistochemistry and other Reg/PAP family gene expressions were remarkably increased in EAM hearts; in addition, interleukin (IL)-6 expression was significantly related to Reg2/PAP1. It seemed that these data were useful as a reference database of gene-expression changes in cardiomyocytes with myocarditis. The Reg/PAP family, which was found to show dramatically increasing gene expressions by DNA microarray analysis, was suspected to play an important role in myocarditis.