TIPE2 Attenuates Lipopolysaccharide-Induced Acute Lung Injury By Regulating Macrophage Polarization

BackgroundAcute lung injury is the acute and progressive respiratory failure caused by various direct and indirect pathogenic factors.The clinical manifestations were high respiratory rate,respiratory distress and intractable hypoxemia.Study found that acute lung injury is not an uncommon complication(60%-89.5%)after sepsis,which might be associated with poor prognosis due to consequent acute respiratory failure and death.Indeed,it has already become one of the most important prognostic factors in patients with sepsis.There are no specific drugs for the sepsis-induced acute lung injury yet,so it’s important to study how to prevent or reduce lung injury.Macrophages serve as different roles in homeostasis,innate immunity against microbes and tissue repair.Macrophages are differentiated from monocytes,and may polarize into the inflammatory Ml(classically activated)lineage or the immunomodulatory M2(alternatively activated)lineage when escaping from the blood vessels,depending on different tissue microenvironment,specific pathogens or cytokine stimulation.Ml polarization is induced by lipopolysaccharide(LPS)and/or interferon-γ(IFN-y)during acute infections to release pro-inflammatory cytokines and chemokines that mount an immune response against various intracellular pathogens,whereas M2 polarization is primarily activated by interleukin-4(IL-4)to produce anti-inflammatory cytokines(e.g.,IL-10),promote tissue remodeling and enhance adaptive T helper 2 cell immunity.Tumor necrosis factor alpha induced protein 8 like molecular 2(TIPE2)was expressed mainly in the inflammatory tissues and lymphoid tissues.TIPE2 can negatively regulate the activation of T receptors and TLRs,thereby preventing the development of severe inflammatory diseases.TIPE2 can inhibit the activation of JNK amino terminal kinase and p38MAPK,and enhance the activation of transcription factor activator protein-1,thus negatively regulating the signal transduction pathway of p38 and MAPK C-JUN.At the same time,TIPE2 can regulate the signal transduction of NF-kappa B and inhibit the transcription and translation of many cytokines,thus inhibiting the activation of inflammatory cells.The present study assessed the role of TIPE2 in the process of macrophage activation and polarization.M2 polarization was hallmarked by increased TIPE2 expression during M2-like differentiation.Exogenous overexpression of TIPE2 in M2 macrophages drove global expression of M2-specific cytokines.Conversely,in Ml macrophages with ectopic TIPE2 expression,the induction of an M1-specific phenotype was impaired.Studies have suggested that TIPE2 is a negative regulator of the innate and adaptive immune response.It can inhibit the immune response,so as to maintain the immune homeostasis.Macrophages also serve as different roles in homeostasis,innate immunity against microbes and tissue repair.TIPE2 attenuates lipopolysaccharide-induced acute lung injury by regulating macrophage polarization is not reported.Therefore,this research explores the involvement of macrophage polarization and reveals the role of TIPE2 on macrophage polarization at the cellular level by TIPE2 interference and overexpression on the LPS-induced acute inflammation reaction.The present study provided a basis for understanding how TIPE2 control macrophage activation and polarization.It is also to provide new ideas and methods for the treatment of acute lung injury and provide basis and potential targets to find and develop new drugs to cure the LPS-induced acute lung injury caused by immune responses.Chapter I Study of the relationship between TIPE2 and LPS-induced acute lung injury in miceObjective:The histological changes of lung in mice were detected in this study.The expression level of TIPE2 in mice lung tissue and peripheral blood mononuclear cells after LPS treatment was detected by Western blot method.The expression levels of TGF-β、IL-1p、IL-6、TNF-a in mice BALF were detected by real-time PCR method.Method:The lungs were harvested,fixed in 10%formalin,embedded in paraffin After staining with hematoxylin and eosin(H&E),the sections were observed under a light microscope.The mice lungs and peripheral blood mononuclear cells were collected and homogenized after LPS treatment.Then total proteins from the lungs and mononuclear cells were extracted by tissue protein extraction solution.Equal amounts of proteins were separated and transferred onto nitrocellulose membranes.The blots were developed using Western blotting reagents and densitometric analysis was performed.The mice BALF were collected and the levels of inflammatory cytokines TGF-β、IL-1β、IL-6、TNF-α in the BALF were measured by ELISA after LPS treatment.The relationship of TIPE2 expression level and the development of LPS-induced acute lung injury in mice were analyzed.Results:The lung histological sections of LPS-treated mice showed alveolar wall thickening,edema,and infiltration of inflammatory cells.The expression level of TIPE2 in mice lungs and peripheral blood mononuclear cells after LPS treatment was significantly lower than that of control group.The effects of LPS treatment in inflammatory cytokines production were detected by ELISA.The production of TGF-β、IL-1β、IL-6、TNF-α in BALF significantly increased in LPS-treated mice in comparison with the control group.Conclusion:The expression level of TIPE2 in LPS-induced acute lung injury was decreased.The expression level of TIPE2 and the severity of lung injury were negative correlated.We found the correlation between TIPE2 and the expression of TGF-β、IL-1β、IL-6、TNF-α in LPS-induced acute lung injury.The TIPE2 and inflammatory cytokines productions were negative correlated.Chapter ⅡTIPE2 Attenuates LPS-Induced Acute Lung Injury by Regulating Macrophage PolarizationObjective:TIPE2 mRNA and protein expressions were detected following LPS/IFNy stimulation of MO macrophages,and they were also detected in IL-4-challenged MO macrophages.Ml cytokines(TNFa,IL-6 and IL-12b)and M1 genes were detected in BMDMs treated with TIPE2 siRNA and LPS/IFNy,and these were also detected in TIPE2 overexpressed BMDMs treated with LPS/IFNy.The expression levels of IL-10 and M2 genes were detected in BMDMs treated with TIPE2 siRNA and IL-4;and these were also detected in TIPE2 overexpressed BMDMs treated with IL-4.Method:TIPE2 mRNA and protein expressions were detected following LPS/IFNy stimulation of MO macrophages to induce polarization into M1 macrophages by RT-qPCR analysis and immunoblotting,and they were also detected in IL-4-challenged MO macrophages polarized into M2 macrophages.Ml cytokines(TNFa,IL-6 and IL-12b)were detected in BMDMs treated with TIPE2 siRNA and LPS/IFNy by ELISA analysis,and M1 genes were detected by RT-qPCR analysis.The expression level of IL-10 was detected in BMDMs treated with TIPE2 siRNA and IL-4 by ELISA analysis,and M2 genes were detected by RT-qPCR analysis.Ml cytokines were also detected in TIPE2 overexpressed BMDMs treated with LPS/IFNy by ELISA analysis,and Ml genes were detected by RT-qPCR analysis.IL-10 was detected in TIPE2 overexpressed BMDMs treated with IL-4 by ELISA analysis;and M2 genes were also detected by RT-qPCR analysis.Results:T-IPE2 expression was selectively inhibited during the M1 polarization of macrophages by LPS/IFNγ,although it was elevated in M2 macrophages.M1 cytokines(TNFa,IL-6 and IL-12b)and M1 genes were significantly elevated in BMDMs treated with TIPE2 siRNA and LPS/IFNy,whereas TIPE2 deletion negatively regulating the expression levels of IL-10 and M2 genes in BMDMs stimulated by IL-4.TIPE2 overexpression in BMDMs impeded LPS/IFBNγ-induced M1 inflammation,whereas these cells exhibited enhanced M2 responses to IL-4 stimulation.Conclusion:The expression of TIPE2 was associated with Ml and M2 polarization of macro-phages.TIPE2 serves as an important role in orchestrating the different polarization status of macrophages.TIPE2 attenuates LPS/IFNy-induced M1 polarization,whereas it enhances IL-4 induced M2 polarization.Chapter ⅢStudy on the signal pathway of TIPE2 regulation of macrophage polarizationObjective:This part of the study aims to investigate the underlying mechanisms of TIPE2 on regulation of macrophage polarization.Method:The expression levels of IKKβ,phospho-IKKP,IRF5,p65,phospho-p65 were detected in TIPE2 overexpressed macrophages by immunoblotting,and IKKP,IRF5,p65,phospho-p65 were detected in the macrophages with mt-IKKβ.TNFa,IL-6 were detected in the macrophages with mt-IKKβ and during exogenous co-expression of mt-IKKβ and TIPE2 in macrophages by ELISA analysis;and M1 genes were detected by RT-qPCR analysis.The expression levels of IKKβ,p65,phospho-p65,S6K1 and phospho-S6K1 were detected in BMDMs with TSC1 deletion by immunoblotting.S6K1 and phospho-S6K1 were detected in TIPE2 overexpressed BMDMs treated with IL-4 by RT-qPCR analysis.After IL-4 treatment,IL-10 was detected in BMDMs with TSC1 deletion and TSC1 deletion BMDMs with TIPE2 overexpression by ELISA analysis,and M2 genes were detected by RT-qPCR analysis.Results:1.Prior to or during LPS/IFNy stimulation,IKKβ,IRF5 and phospho-p65 in the macrophages with mt-IKKβ were significantly higher than that of control group.After LPS/IFNγ stimulation,the expression levels of phospho-IKKβ,IRF5,phospho-p65 in macrophages with TIPE2 overexpression were significantly lower than that of control group,and the signal pathway of IKKβ-NFκB was inhibited,M1 cytokines(TNFa,IL-6)and Ml genes in macrophages with exogenous co-expression of mt-IKKβ and TIPE2 were significantly lower than that of macrophages with mt-IKKβ.2.The expression levels of IKKβ,phospho-p65 and phospho-S6Kl in BMDMs with TSC1 deletion were significantly higher than that of control group.After IL-4 stimulation,phospho-S6Kl in TIPE2 overexpressed BMDMs was significantly lower than that of control group,and the activation of mTORC1 pathway was weakened,IL-10 and M2 genes in TSC1 deletion BMDMs with TIPE2 overexpression was significantly higher than that in BMDMs with TSC1 deletion.Conclusion:TIPE2 impeded M1 polarization by interfering with IKKβ-NFκB pathway activation.TIPE2 overexpression accelerated IL4 induced M2 polarization by dampening mTORC1 activation.