Comparison of rapamycin and methylprednisolone for treating inflammatory muscle disease in a murine model of experimental autoimmune myositis.
ABSTRACT: Idiopathic inflammatory myopathies (IIMs) are a group of autoimmune inflammatory muscle diseases. Rapamycin has been shown to ameliorate inflammation and improve muscle function in a mouse model of experimental autoimmune myositis (EAM). In the present study, the therapeutic effect of rapamycin was compared with methylprednisolone (MP) on EAM. Mice were injected with myosin for 10 days to induce EAM and were subsequently treated with rapamycin (1.5 mg/kg), MP (40 mg/kg) or placebo (DMSO) for 14 days. The rapamycin-treated group exhibited significantly decreased severe inflammation and improved muscle strength compared with the MP-treated group. The plasma transforming growth factor-? (TGF-?) concentration in the rapamycin-treated group was significantly higher compared with the placebo group. However, both treatment groups exhibited significantly lower plasma interleukin-10 levels compared with the placebo group. Moreover, splenic regulatory T cell frequency in both the rapamycin- and MP-treated animals was significantly lower than that in the animals of the placebo group. Rapamycin showed better immune suppressive effects than MP in this model of EAM, and these effects were likely to be mediated by the TGF-? signaling pathway.
Project description:OBJECTIVE:Toll-like receptor 4 (TLR4) is one of the key players in the development of many autoimmune diseases. To determine the possible role of TLR4 in polymyositis (PM) development, we collected muscle samples from PM patients and mice subjected to an experimental autoimmune myositis (EAM) model. METHODS:We measured TLR4-MyD88 pathway-related factors, interferon-? (IFN-?), and interleukin-17A (IL-17A) in EAM mice and PM patients. Then, we observed the changes of above factors and the inflammatory development of EAM mice with TLR4 antagonist TAK-242, IFN-?, or IL-17A antibody treatment. RESULTS:The expression of TLR4, MyD88, and NF-?B was significantly upregulated in the muscle tissues both in 22 patients with PM and in the EAM model. As expected, increased levels of various cytokines, such as IL-1?, IL-6, IL-10, IL-12, tumor necrosis factor-?, TGF-?, IFN-?, and IL-17A, were evident in the serum of EAM mice. Moreover, mRNA expression levels of IFN-? and IL-17A were significantly increased in both PM patients and EAM mice. Consistently, the levels of these factors were positively correlated with the degree of muscle inflammation in EAM mice. However, when EAM mice were treated with TLR4 antagonist TAK-242, the expression of IFN-? and IL-17A was decreased. When the cytokines were neutralized by anti-IFN-? or anti-IL-17A antibody, the inflammatory development of EAM exacerbated or mitigated. CONCLUSION:The present study provided the important evidence that the TLR4-MyD88 pathway may be involved in the immune mechanisms of PM by mediating IFN-? and IL-17A.
Project description:We developed an experimental autoimmune myositis (EAM) mouse model of polymyositis where we outlined the role of regulatory T (Treg) cells. Rapamycin, this immunosuppressant drug used to prevent rejection in organ transplantation, is known to spare Treg. Our aim was to test the efficacy of rapamycin in vivo in this EAM model and to investigate the effects of the drug on different immune cell sub-populations.EAM is induced by 3 injections of myosin emulsified in CFA. Mice received rapamycin during 25 days starting one day before myosin immunization (preventive treatment), or during 10 days following the last myosin immunization (curative treatment).Under preventive or curative treatment, an increase of muscle strength was observed with a parallel decrease of muscle inflammation, both being well correlated (R(2) = -0.645, p<0.0001). Rapamycin induced a general decrease in muscle of CD4 and CD8 T cells in lymphoid tissues, but spared B cells. Among T cells, the frequency of Treg was increased in rapamycin treated mice in draining lymph nodes (16.9 ± 2.2% vs. 9.3 ± 1.4%, p<0.001), which were mostly activated regulatory T cells (CD62L(low)CD44(high): 58.1 ± 5.78% vs. 33.1 ± 7%, treated vs. untreated, p<0.001). In rapamycin treated mice, inhibition of proliferation (Ki-67(+)) is more important in effector T cells compared to Tregs cells (p<0.05). Furthermore, during preventive treatment, rapamycin increased the levels of KLF2 transcript in CD44(low) CD62L(high) naive T cell and in CD62L(low) CD44(high) activated T cell.Rapamycin showed efficacy both as curative and preventive treatment in our murine model of experimental myositis, in which it induced an increase of muscle strength with a parallel decrease in muscle inflammation. Rapamycin administration was also associated with a decrease in the frequency of effector T cells, an increase in Tregs, and, when administered as preventive treatment, an upregulation of KFL2 in naive and activated T cells.
Project description:BACKGROUND Recent data have demonstrated the potential immunosuppressive roles of interleukin-37 (IL-37) in several diseases, but whether it is involved in the pathogenesis of inflammatory myopathy has not been elucidated. MATERIAL AND METHODS An experimental autoimmune myositis (EAM) model was built by subcutaneous injections of pertussis toxin (PTX) and purified rabbit myosin (10mg/kg) emulsified with an equal volume of conventional complete Freund's adjuvant (CFA) in a Lewis model. Autoimmune myositis Lewis model rats were divided into 3 groups: group A rats (control group) were injected with CFA in saline weekly; group B (IL-37 group) rats were injected with saline with IL-37 and CFA in saline weekly; and group C (IL-37 + SIS3 group) rats were injected with IL-37, CFA, and SIS3. ELISA was also used to assess the expressions of TNF-?, IL-6, IL-1?, TGF-?1, and CK. HE staining was performed to assess pathological changes in lung and muscle tissues. RESULTS The expressions of TNF-?, IL-6, IL-1?, TGF-?1, and CK significantly increased in autoimmune myositis Lewis model rats. After IL-37 treatment, the expression of TNF-?, IL-6, IL-1?, TGF-?1, and CK was significantly reduced, as were the inflammatory responses of lung and muscle. However, SIS3 reduced the effects of IL-37 on the autoimmune myositis Lewis model rats. CONCLUSIONS These findings indicate that IL-37 protects against inflammatory response via regulating Smad3 in autoimmune myositis Lewis model rats.
Project description: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-?) showed attenuated Smad2 and extracellular signal-regulated kinase (Erk) phosphorylations that were associated with impaired upregulation of smooth muscle actin alpha (?SMA) protein. In contrast to cardiac fibroblasts, expanded Rock1+/- heart inflammatory myeloid cells showed unaffected Smad2 activation but enhanced Erk phosphorylation following TGF-? treatment. Rock1+/- inflammatory cells responded to TGF-? by a reduced transcriptional profibrotic response and failed to upregulate ?SMA 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 ?SMA. In conclusion, despite the fact that ROCK1 regulates TGF-?-dependent profibrotic response, neither ROCK1 nor ROCK2 is critically involved in the development of postinflammatory fibrosis in the EAM model.
Project description:Idiopathic Inflammatory Myopathies (IIMs) are a heterogeneous group of autoimmune diseases affecting skeletal muscle tissue homeostasis. They are characterized by muscle weakness and inflammatory infiltration with tissue damage. Amongst the cells in the muscle inflammatory infiltration, dendritic cells (DCs) are potent antigen-presenting and key components in autoimmunity exhibiting an increased activation in inflamed tissues. Since, the IIMs are characterized by the focal necrosis/regeneration and muscle atrophy, we hypothesized that DCs may play a role in these processes. Due to the absence of a reliable in vivo model for IIMs, we first performed co-culture experiments with immature DCs (iDC) or LPS-activated DCs (actDC) and proliferating myoblasts or differentiating myotubes. We demonstrated that both iDC or actDCs tightly interact with myoblasts and myotubes, increased myoblast proliferation and migration, but inhibited myotube differentiation. We also observed that actDCs increased HLA-ABC, HLA-DR, VLA-5, and VLA-6 expression and induced cytokine secretion on myoblasts. In an in vivo regeneration model, the co-injection of human myoblasts and DCs enhanced human myoblast migration, whereas the absolute number of human myofibres was unchanged. In conclusion, we suggest that in the early stages of myositis, DCs may play a crucial role in inducing muscle-damage through cell-cell contact and inflammatory cytokine secretion, leading to muscle regeneration impairment.
Project description:Myocarditis is a critical inflammatory disorder which causes life-threatening conditions. No specific or effective treatment has been established. DPP-4 inhibitors have salutary effects not only on type 2 diabetes but also on certain cardiovascular diseases. However, the role of a DPP-4 inhibitor on myocarditis has not been investigated. To clarify the effects of a DPP-4 inhibitor on myocarditis, we used an experimental autoimmune myocarditis (EAM) model in Balb/c mice. EAM mice were assigned to the following groups: EAM mice group treated with a DPP-4 inhibitor (linagliptin) (n = 19) and those untreated (n = 22). Pathological analysis revealed that the myocardial fibrosis area ratio in the treated group was significantly lower than in the untreated group. RT-PCR analysis demonstrated that the levels of mRNA expression of IL-2, TNF-?, IL-1? and IL-6 were significantly lower in the treated group than in the untreated group. Lymphocyte proliferation assay showed that treatment with the DPP-4 inhibitor had no effect on antigen-induced spleen cell proliferation. Administration of the DPP-4 inhibitor remarkably suppressed cardiac fibrosis and reduced inflammatory cytokine gene expression in EAM mice. Thus, the agents present in DPP-4 inhibitors may be useful to treat and/or prevent clinical myocarditis.
Project description:BACKGROUND: The idiopathic inflammatory myopathies represent a group of autoimmune diseases that are characterized by lymphocyte infiltration of muscle and muscle weakness. Insulin-like 6 (Insl6) is a poorly characterized member of the insulin-like/relaxin family of secreted proteins, whose expression is upregulated upon acute muscle injury. METHODS: In this study, we employed Insl6 gain or loss of function mice to investigate the role of Insl6 in a T cell-mediated model of experimental autoimmune myositis (EAM). EAM models in rodents have involved immunization with human myosin-binding protein C with complete Freund's adjuvant (CFA) emulsions and pertussis toxin. RESULTS: Insl6-deficiency in mice led to a worsened myositis phenotype including increased infiltration of CD4 and CD8 T cells and the elevated expression of inflammatory cytokines. Insl6-deficient mice show significant motor function impairment when tested with treadmill or Rotarod devices. Conversely, muscle-specific overexpression of Insl6 protected against the development of myositis as indicated by reduced lymphocyte infiltration in muscle, diminished inflammatory cytokine expression and improved motor function. The improvement in myositis by Insl6 could also be demonstrated by acute hydrodynamic delivery of a plasmid encoding murine Insl6. In cultured cells, Insl6 inhibits Jurkat cell proliferation and activation in response to phytohemagglutinin/phorbol 12-myristate 13-acetate stimulation. Insl6 transcript expression in muscle was reduced in a cohort of dermatomyositis and polymyositis patients. CONCLUSIONS: These data suggest that Insl6 may have utility for the treatment of myositis, a condition for which few treatment options exist.
Project description:Autoimmune hepatitis (AIH) is characterized by massive immune cell-mediated hepatocyte destruction. Glucocorticoids, particularly methylprednisolone (MP), are the most effective treatment for AIH; however, the mechanism underlying the effects of glucocorticoid treatment has not been fully elucidated. The present study explored the effects of MP on damaged hepatocytes in mice with concanavalin A (ConA)-induced experimental autoimmune hepatitis (EAH). C57BL/6 mice were divided into three groups: a normal control group (injected with normal saline), a ConA (20 mg/kg) group, and a ConA + MP (3.12 mg/kg) group. The serum levels of liver enzymes, cytokines, activated T cells, and apoptosis- and autophagy-associated marker proteins were determined 12 h after ConA injection. Human hepatocyte cell line LO2 was used to verify the effects of ConA and MP in vitro. MP treatment significantly decreased inflammatory reactions in the serum and liver tissues and activated the Akt/mTOR signaling pathway to inhibit apoptosis and autophagy in hepatocytes in vivo. Transmission electron microscopy (TEM) revealed fewer autophagosomes in the MP-treated group than in the ConA-treated group. MP treatment obviously suppressed apoptosis and mitochondrial membrane potential (??m) loss in hepatocytes in vitro. Furthermore, ConA treatment increased the levels of LC3-II, p62/SQSTM1, and Beclin-1, while bafilomycin A1 did not augment the levels of LC3-II. MP treatment decreased the levels of LC3-II, p62/SQSTM1, and Beclin-1 and upregulated the levels of phosphorylated (p)-Akt and p-mTOR. In conclusion, MP ameliorated mitochondria-mediated apoptosis and autophagy dysfunction in ConA-induced hepatocyte injury in vivo and in vitro via the Akt/mTOR signaling pathway.
Project description:We have shown previously that rapamycin, the canonical inhibitor of the mechanistic target of rapamycin (mTOR) complex 1, markedly inhibits the growth of focal lesions in the resistant hepatocyte (Solt-Farber) model of hepatocellular carcinoma (HCC) in the rat. In the present study, we characterized the proteome of persistent, pre-neoplastic focal lesions in this model. One group was administered rapamycin by subcutaneous pellet for 3 weeks following partial hepatectomy and euthanized 4 weeks after the cessation of rapamycin. A second group received placebo pellets. Results were compared to unmanipulated control animals and to animals that underwent an incomplete Solt-Farber protocol to activate hepatic progenitor cells. Regions of formalin-fixed, paraffin-embedded tissue were obtained by laser capture microdissection (LCM). Proteomic analysis yielded 11,070 unique peptides representing 2,227 proteins. Quantitation of the peptides showed increased abundance of known HCC markers (e.g., glutathione S-transferase-P, epoxide hydrolase, 6 others) and potential markers (e.g., aflatoxin aldehyde reductase, glucose 6-phosphate dehydrogenase, 10 others) in foci from placebo-treated and rapamycin-treated rats. Peptides derived from cytochrome P450 enzymes were generally reduced. Comparisons of the rapamycin samples to normal liver and to the progenitor cell model indicated that rapamycin attenuated a loss of differentiation relative to placebo. We conclude that early administration of rapamycin in the Solt-Farber model not only inhibits the growth of pre-neoplastic foci but also attenuates the loss of differentiated function. In addition, we have demonstrated that the combination of LCM and mass spectrometry-based proteomics is an effective approach to characterize focal liver lesions.
Project description:We developed a novel poly(lactic-co-glycolic acid)-based, microparticle (MP) system providing concurrent delivery of multiple encapsulated immuno-suppressive factors and antigen, for in vivo conditioning of dendritic cells (DCs) toward a tolerance promoting pathway. Subcutaneous administration prevents onset of type 1 diabetes (T1D) in NOD mice. Two MP sizes were made: phagocytosable MPs were fabricated encapsulating vitamin D3 or insulin B(9-23) peptide, while unphagocytosable MPs were fabricated encapsulating TGF-?1 or GM-CSF. The combination of Vit D3/TGF-?1 MPs confers an immature and LPS activation-resistant phenotype to DCs, and MP-delivered antigen is efficiently and functionally presented. Notably, two subcutaneous injections into 4week old NOD mice using the combination of MPs encapsulating Vit D3, Ins B, TGF-?1 and GM-CSF protected 40% of mice from T1D development, significant in comparison to the control. This work represents one of the first applications of a biomaterial-based, MP vaccine system to successfully prevent autoimmune diabetes.