The Role And Mechanism Of TIPE2 In Asthma Inflammatory Phenotypes

Background Bronchial asthma(Asthma)is a heterogeneous chronic airway inflammatory disease,and individualized treatment is crucial for disease remission.In 2009,the Global Initiative for Asthma(GINA)first proposed the concept of "phenotype" and suggested that patients with asthma should be classified by phenotype.Professor Peter G Gibson proposed that the "inflammatory phenotype" of asthma should be classified according to the percentages of eosinophils and neutrophils in induced sputum: eosinophilic asthma(EA),neutrophilic asthma(NA),mixed granulocytic asthma(MGA),and paucianulocytic asthma(PGA).There are significant differences in the pathogenesis,clinical characteristics,corticosteroid treatment response,and prognosis of different inflammatory phenotypes of asthma.The classification of asthma inflammatory phenotypes is relatively stable,simple,and easy to popularize in clinical application.Therefore,the 2019 GINA guidelines once again emphasized classifying inflammatory phenotypes for patients with asthma and promoting individualized treatment programs.Further elucidation of the pathogenesis of asthma inflammatory phenotypes,and exploring biomarkers and molecular therapeutic targets of asthma inflammatory phenotypes are necessary to identify effective treatment strategies for patients with asthma.Tumor necrosis factor-α-induced protein 8-like 2(TIPE2)is a newly discovered negative regulator of innate and adaptive immunity,that maintains immune homeostasis and regulates inflammation.TIPE2 gene deletion in mice leads to the spontaneous development of lethal inflammation of multiple organs,including the lung.TIPE2 downregulation in humans leads to various inflammatory diseases.Recent studies have shown that TIPE2 is down-regulated in patients with asthma compared with healthy people,which may be involved in the regulation of asthma airway inflammation,but its role has not been studied in asthma inflammatory phenotypes.It deserves further study whether TIPE2 can serve as a new biomarker and molecular therapeutic target for asthma inflammatory phenotypes.Inflammatory cells and inflammatory mediators constitute the crucial airway inflammatory network in asthma.The imbalance of M1/M2 polarization of macrophages is a critical mechanism for the occurrence and development of asthma inflammatory phenotypes.Excessive M2 polarization of macrophages causes EA dominated by eosinophil infiltration in the airway,in which crucial inflammatory mediators involved are interleukin(IL)-4,IL-5,IL-9,IL-13,etc.Patients with EA are generally sensitive to inhaled corticosteroids(ICS)treatment.The 2019 GINA guideline recommends adding biological therapies(e.g.,anti-IL-4Rα,anti-IL-5/5Rα antibodies)for patients who are insensitive to ICS treatment.Excessive M1 polarization of macrophages causes NA dominated by neutrophil infiltration in the airway,in which crucial inflammatory mediators involved are tumor necrosis factor(TNF)-α and IL-1β,IL-6,IL-8,IL-17,etc.Patients with NA respond poorly to ICS and biological therapies and commonly progress to severe asthma or refractory asthma.Moreover,the pathological mechanism of NA remains unclear and still lacks unified biomarkers and effective drug treatment,which is an urgent clinical problem to be solved.In addition,studies have shown that TIPE2 can promote M2 polarization of macrophages.Therefore,further research on the regulation of TIPE2 on M1 polarization of macrophages is of great significance for elucidating the pathogenesis of NA and exploring new targets for individualized treatment of NA.A variety of cytokines,immune cells,and inflammatory pathways are involved in the polarization of pulmonary macrophages,and its mechanism remains unclear.Recent studies have shown that the nuclear factor E2-related factor 2(Nrf2)pathway is involved in the inhibitory role in the M1 polarization of macrophages.Activating the Nrf2 pathway inhibited M1 polarization of pulmonary macrophages,decreased the expression and secretion of pro-inflammatory cytokines,and reduced the infiltration of neutrophils,macrophages,and lymphocytes in the lungs of mice with asthma,thereby alleviating airway inflammation.Nrf2 activation is a molecular bridge linking macrophage polarization and asthmatic airway inflammation and is a potential treatment strategy for airway inflammation in asthma.It is worth further whether TIPE2 can regulate M1 polarization of macrophages by targeting the Nrf2 pathway and participating in the formation mechanism of NA.Therefore,we detected the expression of TIPE2 in asthma inflammatory phenotypes at the human level,and study the correlation between TIPE2 and different asthma inflammatory phenotypes.Among them,the individualized treatment of NA is a difficult problem to be solved urgently in clinical practice.And excessive M1 polarization of macrophages is a crucial mechanism involved in NA formation.Therefore,we further researched the function and mechanism of TIPE2 on M1 polarization of macrophages,thereby exploring the regulatory role of TIPE2 in the pathogenesis of NA,providing a theoretical basis and new ideas for the discovery of new targets for individualized treatment of NA.Object This study aims to research the expression level and role of TIPE2 in the inflammatory phenotypes of asthma at the human level.And in cell experiments,we further constructed TIPE2-overexpressed and TIPE2-silenced macrophages to explore the mechanism by which TIPE2 regulates the M1 polarization of macrophages by targeting the Nrf2 pathway and thereby participating in the regulation of NA formation.Methods 1.The expression and role of TIPE2 in asthma inflammatory phenotypes Patients with asthma and healthy volunteers were enrolled in strict accordance with the inclusion and exclusion criteria.Subjects underwent pulmonary function tests,fractional exhaled nitric oxide(Fe NO)tests,and whole blood cell count.We evaluated asthma control and quality of life of patients using standardized questionnaires and collected clinical data.We then conducted sputum induction on subjects,collect induced sputum,prepare sputum smears,and classified inflammatory phenotypes of asthma by sputum cell analysis.Meanwhile,we collected induced sputum supernatant and measure the level of inflammatory factors in sputum supernatant using the Multi-Analyte Flow Assay Kit.Quantitative detection of the sputum TIPE2 was performed using the ELISA kit.TIPE2 immunofluorescent staining was performed on sputum cytospins from asthma patients.Finally,we evaluated the expression of TIPE2 in asthma with different inflammatory phenotypes,and studied and analyzed the correlation between TIPE2 and inflammatory mediators,inflammatory cells,and clinical indicators in sputum,thereby studying the role of TIPE2 in inflammatory phenotypes of asthma.2.The possible mechanism of TIPE2 regulating the formation of NA The human peripheral blood mononuclear cell line(THP-1)was induced to M1 polarization by phorbol-12-myristate-13-acetate(PMA)and lipopolysaccharide(LPS).M1 polarization was identified by morphology and flow cytometry.The expression of TIPE2 was detected before and after the M1 polarization of macrophages.To study the function of TIPE2 on M1 polarization of macrophages,we transfected THP-1 using recombinant lentivirus to silence or overexpress the TIPE2 gene in cells.We measure the expression of M1 polarization genes(CD11c,i NOS,IL-6,IL-1β,and TNF-α)in macrophages after LPS stimulation.And M1 cytokines(IL-6,IL-1β,and TNF-α)were detected by ELISA.Similarly,to study the effect of TIPE2 on the Nrf2 pathway during M1 polarization of macrophages,lentivirus transfection was used to silence or overexpress TIPE2 in THP-1.And then the expression of Nrf2 and downstream HO-1 was detected in macrophages after being induced to M1 polarization.Western-blot and immunofluorescence were used to detect the nuclear Nrf2 level in macrophages after LPS stimulation.Finally,the Nrf2 signal in THP-1 and TIPE2 overexpressed THP-1 was blocked by Nrf2 signal blocker ML385,after which we detected the expressions of Nrf2,HO-1,and M1 polarization genes(CD11c,i NOS,IL-6,IL-1β,and TNF-α)in macrophages after stimulated by LPS thus to study the effect of Nrf2 signal block on the inhibition of M1 polarization of macrophages induced by TIPE2 overexpression.Results 1.General characteristics of subjects:(1)Finally,the sputum smears of 102 asthmatic patients and 22 healthy volunteers were qualified(the squamous epithelial cells were <50% and the sputum smear score was ≥8).There was no significant difference in age,sex,and BMI between asthma patients and the healthy group(p>0.05).(2)The lung function of patients with asthma was poor,and the percentages of eosinophils,neutrophils,and lymphocytes in the sputum were significantly higher than those in the healthy group(p<0.05).2.Characteristics of airway inflammation in asthma inflammatory phenotypes:(1)The distribution of asthma inflammatory phenotypes was as follows: PGA: 48(47.1%),EA: 31(30.4%),NA: 12(11.8%),and MGA: 11(10.8%).(2)There was no significant difference in age,sex,and BMI among the four groups(p>0.05).(3)IL-1β,TNF-α,and IL-6 levels in the sputum of patients with NA were significantly higher than those in other groups(p<0.05).(4)The level of IL-13 in the sputum of patients with EA was significantly higher than that of other groups(p<0.05).The level of IL-4 in the sputum of EA patients was significantly higher than that of MGA.The level of IL-5 in MGA was significantly higher than that in PGA and NA(p<0.05).(5)IL-1β,TNF-α and IL-6 in sputum of patients were positively correlated with the percentage of sputum neutrophils(r=0.345,p<0.001;r=0.564,p<0.001;r=0.463,p<0.001).IL-13 in sputum was negatively correlated with the percentage of sputum neutrophils(r=-0.392,p=0.001).(6)IL-13 and IL-5 in sputum were positively correlated with the percentage of sputum eosinophils(r=0.411,p<0.001;r=0.302,p=0.013).IL-1β level in sputum was negatively correlated with the percentage of sputum eosinophils(r=-0.357,p=0.006).3.TIPE2 is lowly expressed in NA and highly expressed in EA:(1)Compared with the healthy group,the level of TIPE2 in the sputum of patients with NA decreased significantly.And the level of TIPE2 in the sputum of the NA group was significantly lower than that in other groups(p<0.05).(2)Compared with the healthy group,the level of TIPE2 in patients with EA increased significantly.And the level of TIPE2 in the sputum of EA was significantly higher than that of PGA and NA(p<0.05).(3)There was no significant difference in TIPE2 levels between MGA,PGA,and the healthy group(p>0.05).(4)In the sputum smears of patients,TIPE2 is lowly expressed in neutrophils and highly expressed in eosinophils.4.TIPE2 was negatively correlated with the neutrophil inflammatory mediator and positively correlated with the eosinophil inflammatory mediator:(1)Level of TIPE2 in induced sputum of patients with asthma was negatively correlated with IL-1β and TNF-α levels(r=-0.245,p=0.038;r=-0.311,p=0.026).(2)TIPE2 in sputum was positively correlated with the levels of IL-4,IL-5 and IL-13(r=0.304,p=0.012;r=0.424,p<0.001;r=0.364,p=0.012).5.Correlation between TIPE2 and clinical characteristics:(1)TIPE2 in sputum was positively correlated with sputum eosinophils and lymphocytes(r=0.308,p=0.005;r=0.288,p=0.012),but negatively correlated with sputum macrophages(r=-0.233,p=0.041).(2)The level of TIPE2 in sputum was positively correlated with blood eosinophil count,blood lymphocyte count and Fe NO value(r=0.228,p=0.045;r=0.226,p=0.050;r=0.337,p=0.016),and was positively correlated with ACQ6 score(r=0.294,p=0.014).6.TIPE2 expression was down-regulated during M1 polarization of macrophages: TIPE2 m RNA and protein expressions were decreased in M1 macrophages compared with M0 macrophages(p<0.05).7.TIPE2 inhibited M1 polarization of macrophages:(1)Silencing TIPE2 promoted M1 polarization of macrophages.Under M1 macrophage–inducing conditions,TIPE2-silenced macrophages exhibited a significant increase in the expression levels of M1 genes(CD11c,i NOS)and the expression and release of M1 cytokines(IL-1β,IL-6,and TNF-α),compared with control macrophages(p<0.05).(2)Overexpressing TIPE2 inhibited M1 polarization of macrophages.Under M1 macrophage–inducing conditions,TIPE2-overexpressed macrophages exhibited a decrease in the expression levels of M1 genes(CD11c,i NOS)and the expression and release of M1 cytokines(IL-1β,IL-6,and TNF-α),compared with control macrophages(p<0.05).8.TIPE2 promoted Nrf2/HO-1 signal pathway activation during M1 polarization of macrophages:(1)Silencing TIPE2 inhibited Nrf2/HO-1 activation.RT-q PCR,Western-blot,and immunofluorescence demonstrated that TIPE2-silenced macrophages showed a significant decrease in the m RNA and protein level of Nrf2 and HO-1 after being stimulated with LPS,compared with the control macrophages(p<0.05).(2)Overexpressing TIPE2 promoted Nrf2/HO-1 activation.RT-q PCR,Western-blot,and immunofluorescence demonstrated that TIPE2-overexpressed macrophages showed a significant increase in the m RNA and protein level of Nrf2 and HO-1 after being stimulated with LPS,compared with the control macrophages(p<0.05).(3)TIPE2 promoted Nrf2 nuclear translocation.Western-blot and cellular immunofluorescence confirmed that the expression of nuclear Nrf2 was decreased in TIPE2-silenced macrophages after stimulated with LPS,compared with the control group.The expression of nuclear Nrf2 was increased in TIPE2-overexpressed macrophages after being stimulated with LPS,compared with the control group(p<0.05).9.TIPE2 inhibited M1 polarization of macrophages by enhancing Nrf2/HO-1 activation:(1)Nrf2 signal block promotes M1 polarization of macrophages.The expression of M1 polarization genes(CD11c,i NOS)and M1 cytokines(IL-1β,IL-6,and TNF-α)were up-regulated in ML385-stimulated macrophages after being stimulated with LPS,compared with the control group(p<0.05).(2)Nrf2 blocker attenuated the inhibition of M1 polarization of macrophages induced by TIPE2 overexpression.The expression of M1 polarization genes(CD11c,i NOS)and M1 cytokines(IL-1β,IL-6,and TNF-α)were up-regulated in TIPE2-overexpressed and ML385-stimulated macrophages after being stimulated with LPS,compared with the TIPE2-overexpressed macrophages(p<0.05).Conclusions 1.TIPE2 is differentially expressed in the inflammatory phenotypes of asthma,TIPE2 is lowly expressed in NA and highly expressed in EA.2.TIPE2 is negatively correlated with airway inflammatory factors of NA and positively correlated with airway inflammatory factors in EA.It is suggested that TIPE2 may be involved in the pathogenesis of asthma inflammatory phenotypes.3.TIPE2 activates the Nrf2/HO-1 pathway by promoting Nrf2 expression and Nrf2 nuclear translocation in macrophages.4.TIPE2 inhibits M1 polarization of macrophages by activating Nrf2/HO-1 pathway,which may be a crucial mechanism for TIPE2 regulating NA formation.