Role And Mechanism Of Twist1 In Regulating Macrophage Heterogeneity And Renal Fibrosis

Chronic kidney disease（CKD）,with a high morbidity of 10.8%,poses a tremendous threat to human beings.Renal fibrosis is a common pathological change in many types of chronic kidney diseases（CKD）progressing to end-stage renal disease（ESRD）.At present,patients with ESRD can only receive renal replacement therapy such as hemodialysis,peritoneoclysis and renal transplant,which place a huge financial burden on our society.Therefore,a better understanding of the molecular mechanisms of renal fibrosis and the discovery of potential therapeutic targets will make a sense in the treatment of CKD.Renal fibrosis is characterized by myofibroblast proliferation,inflammatory cell infiltration and excessive deposition of extracellular matrix（ECM）.Although a large number of extracellular matrix produced by fibroblast activation is the direct cause of renal fibrosis,more and more studies have shown that the infiltration and activation of macrophages is an important factor in promoting renal fibrosis.Macrophages can cause renal fibrosis in many ways:1.Macrophages regulate the activation of fibroblasts and the deposition of extracellular matrix by sensing changes in the microenvironment,recruiting to the injured site and undergoing phenotypic changes（M1/M2 mode）to produce cytokines,inflammatory factors and metalloproteinase;2.The synthesis and secretion of extracellular matrix by macrophages directly leads to fibrosis;3.Bone marrow-derived macrophages（mainly M2 macrophages）can be transformed into myofibroblasts.There is no doubt about the role of macrophages in renal interstitial fibrosis.However,the specific molecular mechanisms regulating macrophage migration,activation and MMT in the process of renal fibrosis have not been fully elucidated.The heterogeneity of macrophage activity and function makes its immune regulation more complex in the process of renal injury repair and fibrosis.Therefore,we need to explore the molecular mechanism of macrophage heterogeneity and its role in renal fibrosis,and find the key molecules and signaling pathways.Twist gene belongs to the alkaline helix-loop-helix transcription factor family.It can bind to E-box region of promoter region of many genes and regulate various biological functions.Twist1 can regulate tissue remodeling during embryonic development and endow cells with the ability to migrate.Twist1,as a key regulatory factor in epithelial-mesenchymal transition（EMT）,plays an important role in the invasion and metastasis of tumors.Many studies confirmed that Twist1 gene is involved in the occurrence of fibrosis.Besides,Twist1 participates in the biological processes of metastasis,differentiation and growth of cancer cells by regulating the recruitment of tumor macrophages.Does Twist1 affect the biological function of macrophages?Does specific removal of Twist1 in macrophages alleviate renal pathological damage and delay the progression of renal fibrosis?In this study,we further explored this issue by constructing a macrophage-specific Twist1 knockout mouse model of renal fibrosis.Objective1.To investigate the expression of Twist1 in macrophages and its correlation with renal fibrosis.2.To explore the role of Twist1 in macrophages in renal fibrosis.3.To illuminate the molecular mechanism of Twist1 in regulating macrophage polarization.Methods1.Detection of Twist1 expression in macrophages by RT-PCR,Western blot,immunofluorescence and flow cytometry.2.Construction of macrophage specific knockout Twist1 mice(lyz2-cre/Twist1^Flox/Flox) and control mice(lyz2-cre/Twist1^Flox/-).PCR and agarose gel electrophoresis are used to identify transgenic mice.3.Masson staining,HE staining and immunohistochemical staining are used to detect the degree of renal fibrosis in UUO model mice.4.HE staining,immunofluorescence staining and flow cytometry are used to detect the infiltration of inflammatory cells in the renal of UUO mice.5.HE staining,immunofluorescence staining and flow cytometry are used to detect the degree of renal inflammatory cell infiltration in UUO model mice.6.The UUO model of mice and the transfected siRNA-Twist1 macrophage line are constructed.Immunohistochemistry,immunofluorescence staining,flow cytometry,RT-PCR,Western blot are used to detect the infiltration,activation,chemotaxis,apoptosis and the changes of fibrosis molecules in the macrophage line of UUO model mice.7.RNA-seq detection of gene expression changes in renal macrophages selected by F4/80 magnetic beads.8.Luciferase reporter assay and chromatin immunoprecipitation assay verify the regulatory effect of Twist1 on Lgals3 in macrophage line.9.SPSS19.0 statistical software is used for statistical analysis.All values are represented by mean±standard deviation,kruskal-wallis test is used for comparison of grade data between samples,t test is used for comparison between means,and chi-square test is used for comparison of rates.P<0.05 is considered statistically significant.Results1.Expression of Twist1 in Raw264.7 macrophage line,BMDM derived bone marrow macrophage and renal macrophage by Western blot and RT-PCR.In addition,with the severity of fibrosis increasing,F4/80⁺Twist1⁺cells increase in model mice renal interstitium after UUO surgery 0（n=3）,7（n=3）and 14（n=3）days via immunofluorescence co-localization,these results show that Twist1 expression in macrophages is positively correlated with the severity of renal interstitial fibrosis.2.Identification of transgenic mice by agarose gel electrophoresis;expression of Twist1 is reduced in renal macrophages in knockout mice（n=3）（P<0.05）by Immunofluorescence co-localization staining,RT-PCR and Western blot,indicating that macrophage-specific Twist1 mouse(lyz2-cre⁺Twist1^Flox/Flox)is successfully constructed.3.To observe the role for transcription factor Twist1 in macrophage activation in renal fibrosis,mice with macrophage deletion of Twist1 are generated in Lyz2-Cre⁺,Twist1^fl/fl mice named as Mac–Twist1-KO.The same gender with genotyping Lyz2-Cre^-,Twist1^fl/fll/fl littermates named as Mac–Twist1-WT.Both control littermates and knockouts are subjected to UUO.We observe renal tubular structure destruction including the renal tubular epithelial cells atrophy and fuse in Mac–Twist1-WT mice after UUO 14 days,in the knockout mice renal,tubular injury are obvious attenuated,whereas no difference is observed for renal tubules structure in the sham renal between Mac-Twist-WT and Mac-Twist-KO mice;We then examine renal histologic change in Mac–Twist1-KO and Mac–Twist1-WT mice after UUO.In Mac–Twist1-WT mice,remarkable interstitial extracellular matrix deposition,FN,Col1 and a-SMA induction are detected after UUO,whereas in the knockout mice renal,interstitial fibrosis are markedly attenuated（n=15）（P<0.05）.Thus,these results show that deletion of Twist1 in macrophages alleviates renal tubular injury and diminishes renal fibrosis in the UUO renal.4.To explore the mechanism of Twist1 in macrophages reducing renal fibrosis,a few F4/80-positive macrophages is observed,and no difference is found for macrophage number in the sham renal between Mac-Twist1-WT and Mac-Twist1-KO mice,Macrophage accumulation is largely increased at days 14 after UUO in Mac-Twist-WT kidney,whereas it is less in the knockout kidneys（n=3）（P<0.05）.We also detect the percentage of macrophage in all kidney cells by flow cytometry,it is respectively less around 25.0%,23.1%and 19.3%at days 3,7 and 14 after UUO in Mac-Twist1-KO kidneys than Mac-Twist1-KO kidneys（n=3）（P<0.05）.These data suggest that ablation of Twist1 in macrophages affect macrophage infiltration in the UUO renal.5.Further study for the mechanism of Twist1 reducing macrophage infiltration and delaying renal fibrosis,we detect M2 macropahge（CD11b⁺CD206⁺）percentage respectely is 34.1%、52.6%、71.5%,M1 macropahge（CD11b⁺CD86⁺）percentage respectely is 71.8%、74.3%、78.9%in Mac-Twist1-WT mice after UUO 3,7,14days;CD11b⁺CD206⁺percentage is 25.7%、39.0%、64.6%,CD11b⁺CD86⁺percentage is69.2%、72.9%、76.7%in Mac-Twist1-KO mice after UUO 3,7,14 days,which suggest it is reduced in the knockouts and no difference between Mac-Twist1-WT and Mac-Twist1-KO mice（n=3）（P<0.05）.Western blot analysis shows that the YM1 abundance in macrophages enriched from Mac-Twist1-KO kidneys after UUO 14days is much less compared with that from Mac-Twist1-WT mice（n=3）（P<0.05）.Similarly,Arg-1,MR,chitinase 3–like 3/Ym1,and Fizz1 mRNA expressions are markedly increased in macrophages from Mac-Twist1-WT kidneys,which are much less in macrophages from Mac-Twist1-KO kidneys after UUO（n=3）（P<0.05）.In vitro we assess role of Twist1 in Raw264.7 cells and BMMs stimulated with IL-4（10 ng/ml） and stimulated with INF-γ（20ng/ml）plus LPS（500ng/ml）.Raw264.7 cells are transinfected with siRNA-Twist1 and BMMs isolated from Lyz2-Cre⁺Twist1^fl/fl mice,IL-4 could induce macrophage M2 polarization in Raw264.7 cells and BMMs.siRNA-Twist1 could markedly downregulate IL-4–induced Arg-1,MR,chitinase3–like 3/Ym1,and Fizz1 mRNA expression but INF-γplus LPS-induced iNOS,TNF-α,IL-6 and IL-12 had no significant difference（n=3）（P<0.05）.Western blot analysis shows that the TGF-βand IL-10 abundance in macrophages treated with siRNA-Twist1 is much less compared with normal control group,Col1,α-SMA and FN abundance also decrease in siRNA-Twist1 transinfected macrophages（n=3）（P<0.05）.Western blot analysis shows that the YM1 abundance in macrophages from Mac-Twist1-KO BMMS is much less compared with that from Mac-Twist1-WT mice,（n=3）（P<0.05）.Thus,ablation of Twist1 in Raw264.7 and macropahge prohibit M2 macrophage polarization,which reduce renal fibrosis.6.Further study on the mechanism of Twist1 reducing the number of macrophage infiltration in renal tissue after Twist1 deletion in macrophages,chemokine receptor 2（CCR2）expressions are markedly increase in Mac-Twist1-WT renal tissues,which are much less in Mac-Twist1-KO kidneys after UUO 14 days（n=3）（P<0.05）;CCL2 expressions are markedly increased in macrophages from Mac-Twist1-WT kidneys,which are much less in macrophages from Mac-Twist1-KO kidneys after UUO（n=3）（P<0.05）;Down-regulated Twist1 in Raw264.7 macrophage lines reduced migration ability by Transwell experiment（n=3）（P<0.05）.These results indicate Twist1 may regulate macrophage chemotaxis.7.In order to explore the mechanism of reducing the number of macrophage infiltration in Mac-Twist1-KO kidney tissue,very few F4/80 staining–positive cells with cleaved caspase 3 positive is detected in the sham kidney tissues,at day 14 after UUO,no difference is observed for the macrophages with cleaved caspase 3 positive between in Mac-Twist1-WT and Mac-Twist1-KO kidneys（n=3）（P>0.05）,suggesting Twist1 in macrophage might have no influence on apoptosis.8.To further elucidate the molecular mechanism of Twist1 regulating M2-type macrophages polarization,we seek to identify target genes activation governed by transcription factor Twist1 during the process of renal-infiltrating F4/80⁺macrophages.Temporal gene expression analysis of a microarray data set of renal F4/80⁺ macrophages after UUO reveals that Twist1 knockout down-regulated expression of gene,we focus on Lgals3 because of significant difference.In vitro,we found lgals3, and Arg-1,MR,chitinase 3–like 3/Ym1,Fizz1 mRNA expressions are downregulated in Raw264.7 cells transinfected with siRNA-Twist1 and BMMs isolated from Lyz2-Cre-Twist1fl/fl mice after IL-4 induced（n=3）（P<0.05）.Real time PCR confirmed that upregulation of the lgals3 promote mRNA levels M2 macrophage marker expression（n=3）（P<0.05）.To determine whether lgals3 is a direct target of Twist1,we have predicted five combined sites sharing homology with the Twist1-binding consensus sequence,we performs luciferase reporter assays and Chip experiment,which confirmes Twist1 binds directly to the promoter region of Lgals3 and plays a transcriptional activation role（n=3）（P<0.05）.ConclusionTwist1 is expressed in macrophage lines,bone marrow-derived macrophages and renal macrophages and is closely related to renal fibrosis.Specific removal of Twist1 from macrophages reduce macrophage chemotaxis and infiltration in renal fibrosis model mice,and reduce the polarization of M2 macrophages by inhibiting Lgals3 signaling molecule,ultimately reducing renal fibrosis.Specific removal of Twist1 from macrophages will provide a theoretical basis for exploring intervention targets for CKD.