Mechanisms Of Tim-3 In Idiopathic Pulmonary Fibrosis By Regulating Macrophage Function

Backgroud:Idiopathic pulmonary fibrosis(IPF)is an interstitial lung disease(ILD)with unknown etiology and characterized by pulmonary interstitial fibrosis.IPF has no obvious symptom in the primary stage nor uniform diagnostic criteria.The median survival time of patients after diagnosis is often only 3 to 5 years,and the mortality rate is very high.Due to the unclear etiology and lack of targeted treatment strategies,the current treatment plan is mainly for symptomatic treatment and improving the quality of patients' life.There are only two drugs currently approved for the treatment of IPF,Nintedanib and Pirfenidone.Nidanib is a tyrosine kinase inhibitor that inhibits neovascularization and fibroblast differentiation.Pirfenidone is a broadspectrum antifibrotic drug that inhibits fibroblast proliferation and collagen production.Different ways,such as myofibroblast differentiation,are involved in the process of fibrosis.However,these two drugs have serious side effects,such as nausea and vomiting,skin photoreaction,dizziness,liver function damage,etc.,and patient compliance is poor.Most importantly,clinical studies have found that nidanib and pirfenidone do not fundamentally improve survival in patients with IPF or prevent progression of fibrosis.Therefore,re-examining the pathological mechanisms of IPF and identifying key targets will provide an important complement to existing treatment strategies.At present,the basic pathological features of IPF are: unknown reasons of oxidative stress damages type I alveolar epithelial cells,and the alveolar macrophage amplifies the damage signal,further stimulates the abnormal proliferation of type II alveolar epithelial cells,recruited fibroblasts and differentiated into myofibroblasts to repair the damage,and finally into an uncontrolled repair process to form scar tissue.Exocrine collagen gradually deposits in the pulmonary interstitial,severely blocking the gas exchange between the alveoli and the capillaries.Patients eventually develop severe respiratory failure and even death.It can be said that there are many kinds of cell interactions in the development of IPF,and they participate in the microenvironment of alveolar inflammation.In other words,the key to control IPF is to block the above-mentioned fibrosis cascade and correct the disorder of the alveolar inflammation microenvironment.Alveolar macrophages are the main cell type colonized in alveoli.Similar to colonized macrophages in other parts of the body,alveolar macrophages participate in the construction of barriers and sentinels that communicate with the outside world,and maintain the normal state of alveolar inflammation microenvironment through the secretion of various cytokines.At the same time,in some lung diseases,alveolar macrophages can also adjust the activation state,which aggravates the imbalance of the alveolar inflammation microenvironment.In IPF,alveolar macrophages mainly play a role in profibrosis,by identifying alveolar epithelial damage and amplifying the inflammatory response in the alveoli,secreting growth factor beta 1(TGF-?1),interleukin-10(IL-10)and other chemotaxis,then recruiting fibroblasts,promoting its activation and proliferation,and finally accelerating the fibrotic process in lung.From a certain perspective,alveolar macrophages act as bridges and amplifiers in IPF.Therefore,in-depth study of the mechanism of alveolar macrophages and targeted intervention to restore the normal immune regulation of alveolar inflammation microenvironment will effectively delay the progression of IPF.However,the molecular mechanism of alveolar macrophages in IPF is not well understood,and related targets need to be further discovered and clarified.T cell immunoglobulin and mucin-domain containing-3(Tim-3)was first reported in 2002.Tim-3 is expressed on the surface of Th1(T helper cell 1),Tc1(T cytotoxic cell 1),macrophages,etc.,and belongs to the Tim family.As a research hotspot in the past decade,Tim-3 can combined with ligand galectin-9(Gal-9),high-mobility group box protein 1,HMGB1,to regulate the functional status of T cells and shows great potential in the field of tumor therapy.It is a new generation of immune checkpoint protein after programmed cell death protein 1(PD-1),cytotoxic T-lymphocyte-associated protein 4(CTLA-4).A large number of studies have confirmed that Tim-3 plays an important role in chronic and infectious diseases such as chronic hepatitis C and rheumatoid arthritis.Studies by our research group and others have shown that Tim-3 is also an important regulator of macrophages,which can enhance the repair function of macrophage in certain diseases,thereby affecting the disease outcome.More articles have reported that Tim-3 can inhibit the production of reactive oxygen species(ROS)and the activation of NOD-like receptors 3(NLRP3)in macrophages.However,in pulmonary fibrosis,there is currently no systematic study on Tim-3,and its molecular mechanism in alveolar macrophages needs to be elucidated.In addition,oxidative stress injury is an important initiating factor of pulmonary fibrosis,which is involved in the regulation of alveolar macrophages and interferes with the intracellular metabolic response of alveolar macrophage.The last key rate-limiting enzyme in the intracellular glycolytic pathway,pyruvate kinase(PK),is an important metabolic node switch.PK has four isozymes,and pyruvate kinase isozyme M2(PKM2)is closely related to the production of ROS.Other studies have shown that PKM2 can also be used as an important co-transcription factor to regulate the activation of macrophages,such as promoting secretion of IL-1?.This indicate that PKM2 may be associated with Tim-3 in regulating macrophage function.However,it is unclear how Tim-3 and PKM2 regulate macrophage function,which in turn affects IPF.In summary,we put forward the following questions: Does Tim-3 participate in the pathological process of IPF? And is this process achieved by regulating PK and macrophages? Objective:(1)Identify the changes of Tim-3 in the development of IPF(alveolar macrophages).(2)Clarify the role and mechanism of Tim-3 in the development of IPF.(3)Evaluate the feasibility of Tim-3 as a target for the diagnosis and treatment of pulmonary fibrosis.Interpretation of the changes and internal mechanisms of Tim-3 in idiopathic pulmonary fibrosis will provide important clues and supplements for the diagnosis and treatment of IPF,and has great theoretical significance and application value.Methods:Our study intends to use bleomycin(BLM)-induced mouse pulmonary fibrosis model as the main research strategy,focusing on the alveolar inflammation microenvironment in IPF,combined with in vivo and in vitro experimental studies,systematically explore the changes and mechanisms of Tim-3 in IPF.First,the changes of Tim-3 level in idiopathic pulmonary fibrosis(1)Online mining and analysis of Gene Expression Omnibus(GEO)database data to obtain the expression level of Tim-3 in IPF patient samples.(2)Establishing of BLM-induced mouse pulmonary fibrosis model,using qRT-PCR,ELISA and other methods to detect the expression levels of Tim-3 and fibrosis-related molecules,combined with H&E staining,Masson trichrome staining and other pathological analysis to clarify the trends of Tim-3 in IPF and to explore the correlation between Tim-3 and IPF.(3)In the BLM-induced mouse pulmonary fibrosis model,the correlation between Tim-3 and macrophages in IPF was detected by immunohistochemical staining.Second,the effect of Tim-3 intervention on pulmonary fibrosis in mice(1)A BLM-induced mouse pulmonary fibrosis model was constructed using wild type(WT)and Tim-3 transgenic(Tim-3-TG)mice.The effects of Tim-3 transgene on pulmonary fibrosis were clarified by qRT-PCR,ELISA,H&E staining,and Masson's trichrome staining.(2)On the basis of BLM-induced pulmonary fibrosis model in mice,WT or Tim-3-TG mouse-derived macrophages were adoptively transferred into WT mouse alveolar.Then,qRT-PCR,ELISA,H&E staining,Masson's trichrome staining and other methods were used to determine the effect of Tim-3 on macrophage function on IPF.Third,molecular mechanism of Tim-3 regulating pulmonary fibrosis(1)BLM was used to stimulate the mouse macrophage cell line RAW264.7,and the reactivity of Tim-3 to bleomycin was determined by qRT-PCR and FCM.(2)Tim-3 was knocked out in RAW264.7 using CRISPR/Cas9 gene editing tool,and then the effects of Tim-3 knockdown on the expression of TGF-?1 and other molecules were detected by qRT-PCR and ELISA.(3)Introducing PKM2 inhibitor(compound 3K)to intervene in RAW264.7 cells,and further detect TGF-?1 and other molecules by qRT-PCR,ELISA based on BLM stimulation and Tim-3 knockout.(4)In the BLM-induced WT and Tim-3-TG mouse pulmonary fibrosis models,by using the Compound 3K intervention,subsequent detection methods such as qRT-PCR,ELISA,H&E staining,and Masson trichrome staining were performed.The effect of PKM2 on the regulation of IPF by Tim-3 was clarified in in vivo experiments.Result:(1)According to the results of the database and our mouse model of pulmonary fibrosis,it was found that when fibrosis occurs,the level of Tim-3 in the lung tissue is significantly increased.(2)Tim-3 is mainly expressed on the surface of alveolar macrophages and is increased in the development of pulmonary fibrosis.(3)The lung fibrosis of Tim-3 transgenic mice was aggravated compared to wild type mice.(4)Wild-type mice that accepted the Tim-3 transgenic mouse macrophage induced more severe pathological changes in lung tissue and more expression of the profibrotic factor TGF-?1.(5)In vitro,the Tim-3 knockout RAW264.7 cell line secreted less profibrotic factor TGF-?1;however,this effect disappeared after intervention with the specific inhibitor of PKM2,Compound 3K.(6)PKM2 specific activator,DASA-58,can reverse the profibrotic effect of Tim-3 and alleviate the pathological changes of lung tissue in both wild-type and Tim-3 transgenic mice,while the specificity inhibitor of PKM2,compound 3K has the opposite effect.Conclusion:This study systematically studied the role and mechanism of Tim-3 in IPF,and clarified the molecular mechanism by which Tim-3 affects IPF progeny by regulating alveolar macrophages,and elucidated of PKM2 as a downstream key molecule of Tim-3 mediates the process by which Tim-3 regulates macrophage function.This study provides an important laboratory basis for Tim-3 targeted therapy of IPF,and proposes a new theoretical basis and ideas for intervention of IPF from the perspective of immune regulation.