| BackgroudHuman idiopathic inflammatory myopathies (IIM) are a heterogeneous andacquired group of skeletal muscle inflammatory diseases, which characterizedby the presence of symmetry of proximal muscles weakness and infiltration ofnon-necrotic muscle fibers by lymphocytes, and polymyositis is one of thesubtypes. Due to the exact etiology and pathogenesis are unclear, there is noeffective treatment. In the clinical, the drug used for PM is efficacious to acertain extent but has the long duration of treatment, lots of side effects and limitefficacy. Therefore, we should systematically and deeply study the specificetiology and pathogenesis of polymyositis.In recent years, little is known regarding its etiology and pathogenesis, atleast because a good animal model for PM is not available at present. Uponimmunization with increasing dose of skeleton myosin from different species ofgenus along with pertussis toxin (PT) injection, experimental autoimmune myositis (EAM) animal models have been successfully induced. This is atime-saving and reproducible animal model, which serves as a goodexperimental tool to study and explore new treatment for idiopathicinflammatory myopathies.Present studies revealed that polymyositis are a group of autoimmunedisease, which is mediated predominantly by T lymphocyte. In2005, a thirdsubset of effector Th cells that produce IL-17(Th17) was discovered and itexhibit effector functions distinct from Th1and Th2cells. The primary functionof Th17cells appears to be the clearance of pathogens that are not adequatelyhandled by Th1or Th2cells. However, Th17cells are potent inducers of tissueinflammation and have been associated with the pathogenesis of manyexperimental autoimmune diseases and human inflammatory condition. RORγt,as the main but not specific regulatory factor, is believed to be essential for thedifferentiation of Th17cells.Is Th17cells involved in the pathogenesis of PM or EAM? Currently thereare no relevant reports.Rapamycin, a novel immunosuppressant drug, can inhibit IL-6、IFN-γinduced lymphocyte proliferation, including inhibition of proliferation of Th17cells. In recent years, rapamycin can be used to treat transplant rejectionreaction、MS and many other autoimmune disease, but has not been reported inthe IIM.ObjectivesExplore whether Th17cells participate in the pathogenesis of humanpolymyositis and mouse EAM model. And then evaluate the treatment ofrapamycin and observe the influences of Th17cells in experimental autoimmune myositis. We preliminary investigate the etiology and pathogenesis of PM andmay be provide new insight into the treatment of autoimmune inflammation.MethodsPart one: the infiltration of th17Cells in muscle tissues of polymyositis.Collect muscle specimen of suspected patients with polymyositis, thesepatients is adimitted to our department during the past two years. To make adefinite diagnosis, immunofluorescent studies of the muscle taken by biopsyrevealed that MHC class I expressed in muscle fiber memberances(stronglyexpressed) and CD8+T lymphocyte infiltrated non-necrotic muscle fibers(16cases). Select patients with non-idiopathic myositis performance and histologyin patients with normal muscle biopsy spacimens as control. After accessing tomuscle frozen section, and then observe: pathological changes in muscle tissue、immunofluorescent stained IL-17.Part two: the investigation of Th17cells in experimental autoimmunemyositis model.First, observation of animal model: BALB/c mice were randomly divided intotwo groups: normal group and model group. Normal group: without anyintervention. The model group was immunized two times using1.5mg musclemyosin emulsified with equal volume of CFA. Immunization, once a week fortwo weeks. At the same time, intraperitoneal injection PT two times. And thenwe can observe and treat the model group at10days after the secondimmunization.By testing muscle strength and histological score to determine the animalmodel was successfully established, and then continue to observe theintervention of rapamycin. The RAPA group, after the animal model was successfully established（10days after the second immunization）, the rapamycinwere injected intraperitoneally for continuous two weeks；adjuvant controlgroup（DMSO and NS）：the method and time, as above. The last, the day afterthe end of the intervention, we start to observe the following indicators: musclestrength、pathological changes in muscle tissue、immunohistochemistry stainedIL-17and RORγt mRNA expression in muscle and spleen.ResultsPart one: The muscle tissues of patients with non-inflammatory disease arearranged regularly and basically the same size, there is only very small amountof inflammatory cell infiltration; the polymyositis patients’ muscle fibers are in awild range of sizes and infiltrated a large number of inflammatory cells. A smallportion of muscle fiber has different degrees of atrophy、degeneration andnecrosis. The muscle fiber membranes of patients with non-inflammatorydisease do not express MHC class I, small vessel can be expressed. However,the sarcolemma of PM patients expressed strongly; CD8+T lymphocytes aremainly distributed in endomysium, some cells surrounded or infiltratednon-necrotic muscle fibers. IL-17is positively expressed in patient withpolymyositis which is mainly in cells from inflammatory infiltrates. The numberof IL-17positive cells is positively correlated with the severity of inflammatorymuscle tissue. However, in muscle tissue of non-inflammatory myopathy, IL-17expression is negative.Part two:Observation of animal model the muscle strength of model group issignificantly worse than normal group（0.91±0.11、60±5，P<0.05）；Thehistological score show that normal group, no inflammation, and there are obvious inflammatory cells infiltration in the model group（0、2.47±0.27，P<0.05）. Immunohistochemical studies have shown that IL-17is positivelyexpressed in model group which is mainly in cells from inflammatory infiltrates.The number of IL-17positive cells is positively correlated with the severity ofinflammatory muscle tissue, while the normal group expresses a small amount.RT-PCR： The muscle tissue RORγt mRNA level of the model group issignificantly higher than the normal group, while in the spleen, the result isopposite (interblock P<0.05, there were significant differences).Intervention effectiveness of rapamycin the muscle strength of RAPA groupis significantly improved than adjuvant control group (5.13±0.53、1.56±0.18，P<0.05). The histological score: RAPA group, only a few inflammatory cellsinfiltration, while there is still a large number of diffuse inflammation cellinfiltration (1.16±0.13、2.06±0.16, P<0.05). Immunohistochemical studies haveshown that there is a small amount of IL-17positive expression in RAPA group,while IL-17is positively expressed in adjuvant control group which is mainly incells from inflammatory infiltrates. RT-PCR：The muscle tissue RORγt mRNAlevel of the RAPA group is significantly lower than the adjuvant control group,while in the spleen, the result is opposite (interblock P<0.05, there weresignificant differences).ConclusionThe immunohistochemistry results of patients with polymyositis suggest thatIL-17is positively expressed in patient with polymyositis which is mainly incells from inflammatory infiltrates. This result suggests that Th17cells may beinvolved in the inflammatory pathogenesis of PM.In EAM, we can observe that the increase of Th17cells in muscle tissues of EAM and the upregulation of RORγt mRNA expression,these results alsosuggest that Th17cells may be involved in the inflammatory pathogenesis ofEAM.After EAM is intervented two weeks, the general condition of RAPA mice issignificantly improved, which suggest that rapamycin has promising prospects forthe treatment of EAM. Rapamycin may play a role through regulating Th17cells. |
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