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.

[The expression and significance of TLR4, MyD88 and NF-κB mRNA in mouse lymph node of experimental autoimmune myositis].

AIM: To investigate TLR4, MyD88 and NF-κB mRNA levels in mouse lymph node with experimental autoimmune myositis(EAM)and determine the role of TLR4 in autoimmune myositis. METHODS: Thirty femal BALB/c mice were randomly divided into five groups (n=6 animals per group): group 1 was the control, while animals in other four groups were killed at different time point: group 2 in the first week, group 3 in the second week, group 4 in the third week and group 5 in the fourth week since they had been given myosin for preparing EAM. The expressions of TLR4, MyD88 and NF-κB mRNA were measured with real-time fluorescent quantitative polymerase chain reaction. RESULTS: (1)The expressions of TLR4, MyD88 and NF-κB mRNA in each EAM group were significantly high compared with those in the normal control group, which was significantly highest in group 3 of all(P<0.01) and significantly higher in group 4 than in group 5(P<0.01).(2)The expression level of TLR4 mRNA had significant positive correlations with the expressions of MyD88 mRNA and NF-κB mRNA(r=0.906, r=0.967, P<0.01), and the latter two also had significant positive correlations(r=0.919, P<0.01). CONCLUSION: TLR4 played an important role in the development of autoimmune myositis and run its function mainly by MyD88-dependent pathway that could activate NF-κB for promoting the release of inflammatory factors.

Genomics studies of immune-mediated diseases using the BN-LEW rat model.

LEW and BN rats, that behave in opposite ways for their susceptibility to various immune-mediated diseases, provide a powerful model to investigate the molecular and genetic bases of immune system physiology and dysregulation. Using this model, we addressed the question of the genetic control of central nervous system autoimmunity, of xenobiotic-induced allergic diseases, and of T cell subsets that differ by their cytokine profiles. By linkage analysis and genetic dissection, using a panel of congenic rats, we identified a 120 Kb region on chromosome 9 that controls all these phenotypes, indicating that this region contains a gene or set of genes that plays an important role in the immune system homeostasis and susceptibility to immune mediated diseases. In this review, we will describe these rat genomics studies and will discuss the cellular and genetic factors that may be involved in the differences between these rat strains.

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.

Molecular mimicry between neurons and an intracerebral pathogen induces a CD8 T cell-mediated autoimmune disease.

To identify basic mechanisms of how infections may induce a neuron-specific autoimmune response, we generated mice expressing OVA as neuronal autoantigen under control of the neuron-specific enolase promoter (NSE-OVA mice). Intracerebral, but not systemic, infection with attenuated Listeria monocytogenes-secreting OVA induced an atactic-paretic neurological syndrome in NSE-OVA mice after bacterial clearance from the brain, whereas wild-type mice remained healthy. Immunization with attenuated Listeria monocytogenes-secreting OVA before intracerebral infection strongly increased the number of intracerebral OVA-specific CD8 T cells aggravating neurological disease. T cell depletion and adoptive transfer experiments identified CD8 T cells as decisive mediators of the autoimmune disease. Importantly, NSE-OVA mice having received OVA-specific TCR transgenic CD8 T cells developed an accelerated, more severe, and extended neurological disease. Adoptively transferred pathogenic CD8 T cells specifically homed to OVA-expressing MHC class I(+) neurons and, corresponding to the clinical symptoms, approximately 30% of neurons in the anterior horn of the spinal cord became apoptotic. Thus, molecular mimicry between a pathogen and neurons can induce a CD8 T cell-mediated neurological disease, with its severity being influenced by the frequency of specific CD8 T cells, and its induction, but not its symptomatic phase, requiring the intracerebral presence of the pathogen.

Inhibition of CX3CL1 (fractalkine) improves experimental autoimmune myositis in SJL/J mice.

Idiopathic inflammatory myopathy is a chronic inflammatory muscle disease characterized by mononuclear cell infiltration in the skeletal muscle. The infiltrated inflammatory cells express various cytokines and cytotoxic molecules. Chemokines are thought to contribute to the inflammatory cell migration into the muscle. We induced experimental autoimmune myositis (EAM) in SJL/J mice by immunization with rabbit myosin and CFA. In the affected muscles of EAM mice, CX3CL1 (fractalkine) was expressed on the infiltrated mononuclear cells and endothelial cells, and its corresponding receptor, CX3CR1, was expressed on the infiltrated CD4 and CD8 T cells and macrophages. Treatment of EAM mice with anti-CX3CL1 mAb significantly reduced the histopathological myositis score, the number of necrotic muscle fibers, and infiltration of CD4 and CD8 T cells and macrophages. Furthermore, treatment with anti-CX3CL1 mAb down-regulated the mRNA expression of TNF-alpha, IFN-gamma, and perforin in the muscles. Our results suggest that CX3CL1-CX3CR1 interaction plays an important role in inflammatory cell migration into the muscle tissue of EAM mice. The results also point to the potential therapeutic usefulness of CX3CL1 inhibition and/or blockade of CX3CL1-CX3CR1 interaction in idiopathic inflammatory myopathy.

Biozzi mice: of mice and human neurological diseases.

In 1972 Guido Biozzi selectively bred mice to study the immunopathological mechanisms underlying polygenic diseases. One line, the Biozzi antibody high (AB/H) mouse (now designated the ABH strain) was later found to be highly susceptible to many experimentally induced diseases such as autoimmune encephalomyelitis, autoimmune neuritis, autoimmune uveitis, as well as virus-induced demyelination and has thus been a key mouse strain to study human inflammatory neurological diseases. In this paper we discuss the background of the Biozzi ABH mouse and review how studies with these mice have shed light on the pathogenic mechanisms operating in chronic neurological disease.

Adult gonadal hormones selectively regulate sexually dimorphic quantitative traits observed in experimental allergic encephalomyelitis.

Experimental allergic encephalomyelitis (EAE) and multiple sclerosis (MS) are characterized by strong sexual dimorphisms, many of which may be due to genetically controlled sex hormone effects on the immune system, the central nervous system (CNS), or both. In the present study we used 487 gonadectomized and 376 intact age-matched F(2) mice generated through crosses of B10.S/SgMcdJ and SJL/J mice to assess the role of adult gonadal hormones in regulating clinical and histopathological quantitative traits (QT) associated with EAE in the context of genetic heterogeneity. We found that gonadectomy resulted in different effects, depending on the QT and the sex of the mouse. Ovariectomized mice on average had lower cumulative clinical disease scores, shorter duration of clinical signs, and increased peak disease scores. This trend was accompanied by a significant increase in the incidence of acute, progressive EAE which is more frequently seen in intact and orchiectomized males. Although spinal cord (SC) inflammation was the better predictor of clinical signs of EAE in both sexes, ovariectomized females had considerable reductions in nearly all histopathological QT in both the brain and SC. Orchiectomy resulted in modestly significant increases in disease severity and peak score and earlier onset of clinical signs. With the exception of SC demyelination and lesion scores, orchiectomy had no effect on histopathological QT. Importantly, gonadectomy reduced but did not completely abolish any of the sexually dimorphic clinical QT seen in intact mice. It did however, lead to a significant sexual dimorphism in incidence and severity not seen in intact mice. For histopathological QT, no sexual dimorphism was detected for brain lesions in either intact or gonadectomized mice. In contrast, SC histopathological QT exhibited significant sexual dimorphisms, which were impacted by gonadectomy. The results from this study indicate that within the context of genetic heterogeneity, circulating gonadal hormones influence both clinical and histopathological QT in this model of MS, but they do not solely account for the sexual dimorphisms seen in these traits. Thus, additional mechanisms must play a role in regulating gender differences in autoimmune disease of the CNS.

Paralytic autoimmune myositis develops in nonobese diabetic mice made Th1 cytokine-deficient by expression of an IFN-gamma receptor beta-chain transgene.

Nonobese diabetic (NOD) mice and some human type 1 diabetes (T1D) patients manifest low to high levels of other autoimmune pathologies. Skewing their cytokine production from a Th1 (primarily IFN-gamma) to a Th2 (primarily IL-4 and IL-10) pattern is a widely proposed approach to dampen the pathogenicity of autoreactive diabetogenic T cells. However, it is important that altered cytokine balances not enhance any other autoimmune proclivities to dangerous levels. Murine CD4 T cells are characterized by a reciprocal relationship between the production of IFN-gamma and expression of the beta-chain component of its receptor (IFN-gamma RB). Thus, NOD mice constitutively expressing a CD2 promoter-driven IFN-gamma RB transgene in all T cells are Th1-deficient. Unexpectedly, NOD.IFN-gamma RB Tg mice were found to develop a lethal early paralytic syndrome induced by a CD8 T cell-dependent autoimmune-mediated myositis. Furthermore, pancreatic insulitis levels were not diminished in 9-wk-old NOD.IFN-gamma RB Tg females, and overt T1D developed in the few that survived to an older age. Autoimmune-mediated myositis is only occasionally detected in standard NOD mice. Hence, some manipulations diminishing Th1 responses can bring to the forefront what are normally secondary autoimmune pathologies in NOD mice, while also failing to dependably abrogate pancreatic beta cell destruction. This should raise a cautionary note when considering the use of protocols that induce alterations in cytokine balances as a means of blocking progression to overt T1D in at-risk humans.

Paclitaxel (Taxol) attenuates clinical disease in a spontaneously demyelinating transgenic mouse and induces remyelination.

Treatment with paclitaxel by four intraperitoneal injections (20 mg/kg) 1 week apart attenuated clinical signs in a spontaneously demyelinating model, if given with onset of clinical signs. If given at 2 months of age (1 month prior to clinical signs), disease was almost completely prevented The astrogliosis, prominent in our model, was reversed by paditaxel as determined by astrocyte counts and quantitation of GFAP. Electron microscopic examination of affected regions at 2.5 months demonstrated that the myelin was generally normal. By 4 months of age, demyelination was common in the superior cerebellar peduncle, maximal at 6 months, but continued to 8 months. In addition to myelin vacuolation and nude axons, the presence of many thin myelin sheaths suggested remyelination or partial demyelination. Although no evidence of oligodendrocyte loss was seen, nuclear changes were observed. To substantiate that remyelination was occurring, we measured MBP (18.5 kDa), MBP-exon II, Golli-MBP, TP8, Golli-MBP-J37, platelet-derived growth factor alpha (PDGFR alpha) and sonic hedgehog (SHH). Of these TP8, PDGFR alpha and SHH were up-regulated in the untreated transgenic. After paditaxel treatment, MBP-Exon II, TP8, PDGFR alpha and SHH were further up-regulated. We concluded that some of the effects of paditaxel were to stimulate proteins involved in early myelinating events possibly via a signal transduction mechanism.

Development of spontaneous autoimmune peripheral polyneuropathy in B7-2-deficient NOD mice.

An increasing number of studies have documented the central role of T cell costimulation in autoimmunity. Here we show that the autoimmune diabetes-prone nonobese diabetic (NOD) mouse strain, deficient in B7-2 costimulation, is protected from diabetes but develops a spontaneous autoimmune peripheral polyneuropathy. All the female and one third of the male mice exhibited limb paralysis with histologic and electrophysiologic evidence of severe demyelination in the peripheral nerves beginning at 20 wk of age. No central nervous system lesions were apparent. The peripheral nerve tissue was infiltrated with dendritic cells, CD4(+), and CD8(+) T cells. Finally, CD4(+) T cells isolated from affected animals induced the disease in NOD.SCID mice. Thus, the B7-2-deficient NOD mouse constitutes the first model of a spontaneous autoimmune disease of the peripheral nervous system, which has many similarities to the human disease, chronic inflammatory demyelinating polyneuropathy (CIDP). This model demonstrates that NOD mice have "cryptic" autoimmune defects that can polarize toward the nervous tissue after the selective disruption of CD28/B7-2 costimulatory pathway.