The Enhancing Role And Its Mechanisms Of TIPE2 On M2 Macrophage Polarization In Asthma Via Negative Regulation Of MTORC1

Background:Bronchial asthma(asthma)is the most common chronic respiratory disease in children.Repeated attacks seriously affect the health of children and the quality of life.In the past 30 years,the prevalence of asthma among children in cities of China has increased significantly.Asthma is a chronic inflammatory disease characterized by airway hyperresponsiveness,infiltration of inflammatory cells,excessive mucus production and airway remodeling.It can be induced by respiratory infection,allergen exposure,severe exercise,climate change and so on.Inhaling glucocorticoid is the classic drug for the treatment of asthma,and some children are not well treated.Some asthmatic patients(5%to 10%of asthmatic patients)receive repeated attacks which can be life-threatening,even if they are treated with high doses of inhaled corticosteroids and sometimes with systemic glucocorticoids.It not only affects the quality of life,but also has a significant impact on medical expenditure.Therefore,elucidating the immunoregulatory mechanism of asthma is of great significance and may provide new target candidates for the new therapies of asthma.The pathogenesis of asthma is very complicated.The researchs on the pathogenesis of asthma have made progress over the past few years.It is considered that the Th1/Th2 imbalance is one of the pathogenesis of asthma.Th2 cytokines interleukin(IL)4 and IL-13 play an important role in the pathogenesis of asthma.In recent years,it has been found that macrophage polarization is closely related to asthma.Macrophage polarization is a process whereby macrophages phenotypically mount a specific phenotype and a functional response to the microenvironmental stimuli and signals that encounter in each specific tissue.The local cytokine milieu can orientate macrophage polarization.Two major macrophage subpopulations with different functions include classically activated or inflammatory(M1)and alternatively activated or anti-inflammatory(M2)macrophages have been recognized.M1 macrophages are typically induced by Th1 cytokines,such as IFN-? or by bacterial lipopolysaccharide(LPS)recognition and produce and secrete pro-inflammatory cytokines,such as IL-1?,IL-6,IL-12,IL-23 and TNF-?.M2 macrophages have anti-inflammatory and immunomodulatory effects.Depending on the activating stimulus which received,M2 macrophages can be further divided into four different subsets consisting of M2a,M2b,M2c,and M2d.The M2a subset of macrophages could be induced by IL-4 and IL-13 and produces anti-inflammatory cytokines IL-10 and transforming growth factor beta(TGF-beta).The polarization state of M1/M2 can be induced and completely reversed or repolarized by complex endogenous signaling pathways and its regulatory factors.IL-4 can induce M2 polarization of macrophages through the JAK/STAT and PI3K/Akt signaling pathways and participate in the regulation of asthma.Therefore,the study on the regulatory mechanisms of macrophage polarization induced by IL-4 may provide a theoretical basis for the new treatment of asthma.Tumor necrosis factor-alpha-induced protein 8-like 2(TIPE2)is one of the family members of tumor necrosis factor-alpha-induced protein 8(TNFAIP8)and was first found in mice models of experimental autoimmune encephalomyelitis.TIPE2 is mainly expressed in the thymus,lymph nodes,spleen and small intestinal mucosa and is highly expressed in macrophages,T lymphocytes and B lymphocytes.TIPE2 plays an important negative regulatory role in innate immune response and adaptive immune response.TIPE2 is involved in the regulation of multiple signaling pathways,such as JNK and NF-kappa B.Previous studies have shown that TIPE2 expression changes in systemic lupus erythematosus,chronic hepatitis B virus infection and asthma.TIPE2 is down-regulated in patients with systemic lupus erythematosus.One study showed that TIPE2 could alleviate systemic lupus erythematosus by inducing M2 type macrophage polarization.TIPE2 is down-regulated in peripheral blood mononuclear cells of asthmatic children,however,it is not clear whether TIPE2 participates in the regulation of asthma.Because the M2 polarization of macrophages is closely related to asthma,we will study the effect and mechanisms of TIPE2 on the M2 polarization of macrophages.This study may provide a theoretical basis for further study of the role of TIPE2 in asthma.In this study,we identified the changes of macrophage polarization and IL-4 in the lung of asthmatic mice induced by ovalbumn.We also identified the regulatory effect of TIPE2 on macrophage polarization and explored the molecular mechanism of TIPE2 regulating M2 polarization induced by IL-4 in vitro.This study may provide a theoretical basis for exploring the effect and mechanisms of TIPE2 on asthma.Chapter ? Study on macrophage polarization in lung of asthmatic mice induced by ovalbuminObjective:To identify the changes of macrophage polarization and IL-4 in the lung of asthmatic mice induced by ovalbumin(OVA).Method:The model of asthmatic mice was induced by OVA and the expression of IL-4,M1 polarization markers(iNOS),M1 polarizing gene(CD38)and M2 polarizing gene(Arg-1)in the lung tissues of asthmatic mice and control mice were detected by RT-qPCR.Results:IL-4 was significantly higher in the lung of asthmatic mice induced by OVA compared with control mice.The expression of M2 polarization gene(Arg-1)in the lung of asthmatic mice was significantly higher than that in the control group,but there was no significant difference in the expression of M1 polarization markers(iNOS)and Ml polarizing gene(CD38).Conclusion:IL-4 and M2 polarization gene expression increased in the lung of asthmatic mice induced by OVA.Chapter ? Effect of TIPE2 on the regulation of macrophage polarizationObjective:To detect the changes of TIPE2 expression during M1 and M2 polarization in bone marrow-derived macrophages(BMDMs),and study the regulatory effect of TIPE2 on the polarization of macrophages through TIPE2 silenced BMDMs and TIPE2 overexpressed BMDMs.Method:BMDMs were used as the models of undifferentiated(MO)macrophages in our study.TIPE2 expression levels were determined by RT-qPCR and Western-blot during M1 polarization induced by LPS/IFN-y and M2 polarization induced by IL-4.To make clear the regulating role of TIPE2 on macrophage polarization,Ml cytokines(TNF-?,IL-6)and M1 genes(CD38?FPR2?GPR18)of BMDMs with TIPE2 deletion or overexpression were determined during M1 polarization,and M2 cytokine IL-10 and M2 genes(EGR2?MGL1?MGL2?FIZZ1?YM1?MRC1?PGC1?)of BMDMs with TIPE2 deletion or overexpression were determined during M2 polarization.Cytokines were detected by ELISA,and M1 genes and M2 genes were detected by RT-qPCR.Results:TIPE2 was down-regulated during M1 polarization of macrophages induced by LPS/IFN-y and up-regulated during M2 polarization induced by IL-4.Ml cytokines(TNF-?,IL-6)and M1 genes were up-regulated in TIPE2 silenced BMDMs during Ml polarization induced by LPS/IFN-y,and M2 cytokine IL-10 and M2 genes were down-regulated in TIPE2 silenced BMDMs during M2 polarization induced by IL-4.Ml cytokines(TNF-?,IL-6)and M1 genes were down-regulated in TIPE2 overexpressed BMDMs during M1 polarization induced by LPS/IFN-?,and M2 cytokine IL-10 and M2 genes were up-regulated in TIPE2 overexpressed BMDMs during M2 polarization induced by IL-4.Conclusion:TIPE2 serves as an important role in orchestrating the different polarization status of macrophages.TIPE2 attenuates M1 polarization induced by LPS/IFN-?,whereas it enhances M2 polarization induced by IL-4.Chapter ? TIPE2 enhances M2 polarization of macrophage via negative regulation of mTORC1Objective:To investigate the underlying mechanisms of TIPE2 on enhancing M2 polarization of macrophage.Method:The key proteins(STAT6,Akt,S6K1,4E-BP1 and phosphorylations of these proteins)in the signaling pathways of M2 polarization induced by IL-4 were detected by Westem-blot in TIPE2 overexpressed BMDMs and control BMDMs to explore the effect of TIPE2 on the M2 polarization signaling pathways.Mammalian target of rapamycin complex 1(mTORCl)was constitutively activated by silencing tuberous sclerosis complex 1(TSC1).M2 cytokine IL-10 and M2 genes were detected in BMDMs with and without TIPE2 overexpression during M2 polarization to explore the effect of TIPE2 on the M2 polarization of BMDMs with mTORCl constitutive activation.IL-10 was detected by ELISA,and M2 genes were detected by RT-qPCR.The key proteins(IRS2,Akt and Akt phosphorylation)of the Akt signaling pathway in BMDMs with and without mTORC1 constitutive activation were detected by Western-blot before and after being induced by IL-4.Results:Akt phosphorylation in BMDMs with TIPE2 overexpression was more marked and phosphorylations of the mTORCl downstream molecules ribosomal protein s6 kinase p-1(S6K1)and eukaryotic translation initiation factor 4E-binding protein 1(4E-BP1)were weaker comppared with wild-type BMDMs during M2 macrophage polarization induced by IL-4,while the activation of STAT6 was unaltered between BMDMs with TIPE2 overexpression and wild-type BMDMs.M2 cytokine IL-10 and M2 genes were up-regulated in BMDMs with TIPE2 overexpression and constitutive mTORC1 activation compared with BMDMs with mTORC1 activation during M2 macrophage polarization induced by IL-4.TIPE2 overexpression enhanced the M2 polarization which was weakened by constitutive mTORC1 activation.Akt phosphorylation and IRS2 were attenuated in BMDMs with constitutive mTORC1 activation compared with wild-type BMDMs during M2 macrophage polarization induced by IL-4.Conclusion:TIPE2 enhanced M2 polarization of macrophage induced by IL-4 via the negative regulation of mTORC1.