Proinflammatory Role Of IL-36α In The Autoimmune Myocarditis In Mice

Background:Myocarditis is a myocardial inflammatory disease, which manifest as the myocardial cell necrosis and interstitial inflammatory cell infiltration.It is usually induced by infection, autoimmunity, poisoning,etc.It often implicates the myocardial cells, the mesenchymal cell, interstitial, heart valve and pericardium, eventually leads to the entire heart structure damage.Because of its complex pathogenesis, myocarditisis characterized by different degree of dyspnea, heart failure, stroke and other symptoms.The morbidity of young people is on the increase, including asymptomatic patients, which accounts for 12 percent of the sudden death under the age of 40.Although most patients can be restored to health, part of patients who has the genetic susceptibility can gradually progress to chronic myocarditis and dilated cardiomyopathy (DCM) associated with congestive heart failure.The definition of myocarditis seems to be simple, but the diagnosis and treatment of myocarditisis still a major clinical problem.Viral myocarditis (VMC) is a kind of clinical common diseases of the cardiovascular system. It is a disease caused by viralinfection, and the myocardial cell degeneration, necrosis, interstitial inflammatory cell infiltration and fiber leakage is the main pathological changes of this diseases.Fiedler KL reported 3 cases of pathological concept of VMC, and there has been more and more reports of VMC in Europe and the United States since then.In the 1970s, China, Japan and other Asian countries also have reported the cases related VMC.In recent years, VMC has become one of the main reasons for teenagers’unexplained sudden death, with the increasing morbidity of VMC.Patients with acute VMC have no self-conscious symptom or have mild symptomsonly.The body can clear the virus itself, and then to be recovery.10-20% of the patients can develop into chronic VMC, congestive heart failure, and even dilated cardiomyopathy. Moreover, chronic VMC often cause dilated heart disease. An investigation from the United States shows that the annual increase of the dilated heart disease patients has been up to 100000, and it has become one of the major causes for the increasing of the heart transplantation. As is known to all, VMC is endangering the public health. So far, due to the pathogenesis of VMC is still not be entirely cleared, there is no efficient treatment of VMC.The pathogenesis involves the virus directly damage, immune mechanism, biochemical mechanism, myocardial fibrosis, apoptosis, etc.Most research at present point that the virus triggered myocardial autoimmune reaction as the autoimmune myocarditis (AMC)is the major cause of the progressive myocardial injury by VMC. The way to reduce the immuneinjury becomes the hot issues to be resolved. Therefore, to clarify the pathogenesis of VMC has important theoretical and practical significance.The traditionally treatment of viral myocarditis in a clinical context is the intravenous gamma globulin to adjust the immune status reducing the myocardial damage, and improving myocardial energy metabolism without a clear and effective way.At home and abroad in recent years, many experts has also made various attempts, developing a large number of experimental studies of drug treatment to this disease, such as the gene targeting therapy of costimulatory molecule,inhibition of inflammatory factors, the drug adjusting the redox reaction, the drug adjusting the secretion of Th1/Th2 inflammation factors, the drug inhibiting of cardiac remodeling, etc. Since the research on cytokines has been much more intensive, most experts are focusing attention on the cytokines to treat the myocarditis, and this method has also obtained some certain results. Some cytokines, such as rIL-2 and IFN-γ has been used in clinical currently.IL-36 was recently named in the IL-lfamily, consisting of 3molecule with similar biology function, IL-36α, IL-36β and IL-36γ.IL-36 members have co-receptor as IL-36R, which combines with IL-1 receptor assistant protein (IL-1RAcP) to be a dimer. Extracellular IL-36 is not a transmembrane protein IL-36R and IL-1RAcP extracellular form complexes upon binding by intracellular domain of the adapter protein myeloid differentiation protein 88 (myeloid differentiation primary response 88, MyD88) combined activation of MAPKs, NF-κB signaling cascade pathway plays a pro-inflammatory activation of the immune reaction. Studies have shown, IL-36 members of the support and immune and immune activation effect than the conventional IL-1 family members stronger, dendritic cells and T cells is a potent modulator involved in activation of dendritic cells and Th cells, and antigen presentation stimulate pro-inflammatory cytokine production, has great potential in the immune response. Mainly:①role in immune cells:in vitro, bone marrow-derived dendritic cells (BMDC) mice and CD4+T cells to IL-36 can produce significant specific reaction, IL-36 activation of CD4+T cells the Thl cell response; in vivo, IL-36 can promote the maturation of dendritic cells, dendritic cells upregulate the expression of surface MHC-Ⅱ and costimulatory molecules CD80/CD86 enhance antigen-presenting function of dendritic cells and stimulate cell ThO to Thl cells. Nicklin and other studies have shown that dendritic cell response to IL-36 to IL-1 than the more strongly in promoting antigen presentation. Th1 and Th2 cells express IL-36R mRNA levels than IL-1 high.②the role of cytokines:mouse bone marrow-derived dendritic cells (BMDCs) and CD4+T cells in persistent expression IL-36R, IL-36 stimulation when subjected, BMDCs produce pro-inflammatory cytokines such as IL-12, IL-18, IL-6, TNF-y, IL-23, etc., CD4+T cells produce INF-y, IL-4, IL-17, etc. These responses can be antagonized by IL-36Ra.Part 1:In the process of AMC, IL-36 a is positively associated with the degree of myocardial injuryObjective:Build experimental autoimmune myocarditis (AMC) animal model with BALB/c mice to prove the relationship between the upregulation of IL-36 a in AMC and the level of myocardial immune injury.Method:1.Wild type male BALB/c mice (SPF level) of 6 weeks old and 14-18g body weight were bought from Laboratory animal center, southern medical university. All experimental animals’operational processes have received the approval of the animal protection authority, and followed guidelines foranimal experiments Southern Medical University.2.Build AMC animal model:24 BALB/c mice are divided into 4 groups as the normal control group (group C), low dose short peptide group (group L), middle dose short peptide group (group M), high dose short peptide group (group H). The specific short peptide is the weight chain of the mice myocardial α-myosin (MyHc-a614-629[Ac-S LKLM ATLFSTYAS AD-OH]), which is used to build experimental autoimmune myocarditis animal model in last three groups. Each mouse is injected on the back with this shout peptide of emulsifier at the first and seventh day. Observe the rats’change of activity, diet, skin color and weight. Cardiac ultrasound, detection of IL-36a expression by Western-blot, HE staining and histopathological grading of myocardialsection have been taken at 7th and 21st day after first injection.Result:Myocarditis is raised by the immune since the shout peptide from weight chain of the mice myocardial α-myosin in complete freund’s adjuvant. IL-36 a has low expression in the normal control group, though it has high expression in AMC model. The myocarditis damage degree is aggravating along with the expression of IL-36 a increasing.Conclusion:1.AMC model can be successfully built by the immune since the shout peptide from weight chain of the mice myocardial a-myosin in BALB/c mice.2.There is a positive correlation between IL-36 a in AMC and the level of myocardial immune injury.Part 2:The IL-36 a promotes the deterioration of cardiac function in the AMC miceObjective:Discuss the inflammatory response of the IL-36a in the AMC miceMethod:1.40 BALB/c mice are divided into 4 groups as the Group Ctrl, Group AMC, Group AMC+IL-36a, and Group AMC+IL-36Ra.AMC animal model is built by 200μg the shout peptide from weight chain. The last two groups are intraperitoneal injected with the IL-36 a or the IL-36Ra (0.1 ug/rat/day)for 7 days at the 15th day after modeling, when the Group Ctrl and the Group AMC are intraperitoneal injected of with the same measure of physiological saline. Check the indicators at the 21st day of the experiment.2. Transthoracic echocardiography:The mice were first anesthetized finished measuring body weight of each mouse, do transthoracic echocardiography were measured left ventricular end-systolic and end-diastolic diameter, is calculated by the instrument automatically get left ventricular ejection fraction and left ventricular shortening fraction short sleeves.3. Hematoxylin-eosin (HE) staining cardiac pathology:All mice were euthanized, the hearts were immersed in formalin for 24 hours, after paraff in processing, slicing, hematoxylin and eosin staining.4. Determination of ELISA serum IL-23, IL-17 concentrations:ELISA test kit to detect serum levels of interleukin-23 (IL-23) and interleukin-17 (IL-17) level.Result:After the successful establishment of AMC model mice, compared with the Ctrl group, the experimental group of mice with varying degrees of heart failure, serum IL-23, IL-17 levels and myocardial IL-36a protein expression levels were significantly increased myocardial biopsy visible inflammatory cell infiltration, which went AMC+IL-36a mice increased inflammatory cytokines, and increased expression of inflammatory cell infiltration and increased cardiac dysfunction most obvious, while the more AMC Group, AMC+IL-36Ra mice each indicators was significantly inhibited.Conclusion:1.Exogenous IL-36a injected before the process of AMC enhances the immune response, and aggravates the myocardial injury.2.Blocking the IL-36a by the IL-36Ra injected during the process of AMC inhibits he immune response, and reduces the myocardial injury.Full Summary:1. By subcutaneousa-cardiac myosin heavy chain peptide immunized BALB/c mice successfully induced AMC.2. Upregulation IL-36a AMC may aggravate myocardial inflammation injury in mice.3.IL-36a as proinflammatory cytokines play an important role in AMC’s own immune response, it is by promoting proliferation and cytokine secretion of T cells, directly involved in the course of myocarditis, inhibit the expression of IL-36a reduce cardiac immune response, suppression AMC’s progress.