ZHX2 Participates In Sepsis And Tumor Process By Reprogramming Macrophages

Macrophages are one of the most plastic cells,which exhibit different phenotypes under external stimuli with corresponding functions,therefore,play critical roles in various diseases,such as tumor and acute inflammation.Macrophages phenotypes mainly include pro-inflammatory M1 macrophages,anti-inflammatory M2 macrophages,and intermediate continuous functions State of macrophages.During acute inflammation,macrophages display a pro-inflammatory phenotype.Sustained excessive inflammation lead to shock and multiple organ dysfunction.During tumorigenesis,the tumor microenvironment recruits monocytes and educate into tumor-associated macrophages(TAMs).Most TAMs exhibit M2-like phenotype and promote the tumor progression.Clinical studies have shown the beneficial effects of reprogramming TAMs into M1 phenotypes in tumor therapy.Therefore,elucidating the regulatory mechanism of macrophage reprogramming is of great significance for the treatment of various diseases.Complicated mechanisms are involved in regulating macrophage phenotype,including signal pathway activation,metabolism,and transcription factors.Anaerobic glycolysis is the mainly metabolism pathway in M1-like pro-inflammatory macrophages,and M2-like anti-inflammatory macrophages show increased levels of oxidative phosphorylation.Enhanced glycolysis reinforces the synthesis and release of pro-inflammatory factors from macrophages.Transcription factors also play a key role in the establishment and maintenance of macrophage gene expression profiles.Blocking the activation of related signaling pathways and inhibiting the expression of transcription factors can reprogram the phenotype of macrophages.Indentification of the key transcription factors in macrophages polarization of and reveal its regulatory mechanisms are crucial for regulating the phenotype and function of macrophages and may provide a promising strategy for treatment of diseases.ZHX2(Zinc fingers and homeoboxes 2)belongs to the zinc finger and homeoboxes(ZHX)protein family and is involved in many pathophysiological processes such as tumor progression,development and metabolic regulation.Our previous studies identified ZHX2 as tumor suppressor in HCC by regulating cyclins,MDR1 and lipid lipase.Recent research demonstrated the critical role of ZHX2 in transcriptional regulatory network in monocytes.Animal studies have found that ZHX2 participates in the process of atherosclerosis by affecting the apoptosis of macrophages.All above data suggest that ZHX2 is involved in macrophages regulation.However,whether ZHX2 takes roles in macrophage reprogramming has not been reported so far.Our study here tried to explore the role of ZHX2 in macrophage reprogramming and related disease,such as sepsis and liver cancer.Part 1 ZHX2 reprograms macrophages through promoting glycolysisIn order to clarify the role of ZHX2 in macrophage polarization,we examined the expression of ZHX2 in macrophages stimulated with different stimuli.Subsequently,we bred myeloid cell-specific Zhx2 knockout(MKO)mice,and BMDMs(Bone marrow-derived macrophages)from WT and MKO mice were used for RNA-seq and in vitro and in vivo experiments.?.ZHX2 is one of the important regulatory transcription factors in macrophage polarizationIn order to explore transcriptional factors important for macrophages polarization,RNA sequencing was performed with BMDM from C57BL/6 wild-type(WT)mice sitimulated with/without LPS.GSEA(Gene Set Enrichment Analysis)analysis identified ZHX2 as one of the important regulatory transcription factors in LPS stimulated macrophage.?.The expression of ZHX2 altered in macrophages with different polarizationTo clarify the correlation between ZHX2and macrophage polarization,we stimulated macrophages from different sources with LPS,IL-4,and liver cancer cell conditioned medium(HCM)which mimics the tumor microenvironment.Results of RT-qPCR and Western Blot(WB)showed that LPS significantly increased,while IL-4 greatly decreased the expression of ZHX2 in RAW264.7 cells,in time-dependent and dose dependent manner.In addition,HCM derived from both mouse and human liver cancer cell lines greatly inhibited the expression of ZHX2 in macrophages in time and dose dependent manner.?.Zhx2 knockout in macrophages significantly inhibits its pro-inflammatory phenotype and function1.RNA-seqencing release that Zhx2 knockout significantly inhibits the expression of proinflammatory genes in macrophagesRNA of BMDMs from WT and MKO mice was extracted for sequencing.Analysis of GSEA showed that compared with the MKO group,M1-like macrophages phenotype and inflammatory response related pathways were significantly enriched in BMDMs from wild-type mice,Argl and CD206 were significantly increased in MKO BMDMs and the expressions of iNOS,TNF-?,IL-6 and IL-1? were significantly reduced.2.ZHX2 participates in macrophage polarization under multiple stimuliTo clarify the role of ZHX2 in macrophage reprogramming,BMDMs from WT and MKO mice were stimulated with LPS or IL-4.Results of RT-qPCR,WB and ELISA showed that ZHX2 significantly enhanced the expression of proinflammatory factors,iNOS,TNF-?,IL-6 and IL-1? in LPS-induced macrophages and inhibited the expression of anti-inflammatory factors Argl,CD206,TGF-? and IL-10 in IL-4 induced macrophages.Rescue experiments further confirmed the results.In order to verify the effect of ZHX2 in macrophages on liver cancer progression,IL-4 stimulated BMDMs from WT and MKO mice were co-cultured with HCC cell line Hepa1-6 cells for 24 h,then transwell assay was used to detect the migration of Hepa1-6 cells,or the supernatant from IL-4 induced WT and MKO BMDMs was collected to stimulate Hepa1-6 cells for CCK-8 assay.The results showed that compared with WT BMDMs,the MKO BMDMs significantly promoted the migration and proliferation of Hepa1-6 cells under the induction of IL-4,further confirming that ZHX2 inhibited the function of IL-4 induced M2 polorization.?.ZHX2 reprograms macrophages through switching metabolism to glycolysis in PFKFB3 dependent mannerGSEA analysis of RNA-seq data showed that,compared with MKO macrophages,the glycolytic pathway in WT-derived macrophages was significantly enriched,accompanied by the increased expression of multiple glycolytic-related enzymes.RT-qPCR and WB confirmed the reduced expression of key glycolytic enzymes such as PFKFB3 in the MKO BMDMs.Accordingly,data of seahorse analysis showed that,under the stimulation of LPS,the glycolytic ability of BMDMs in the MKO group was significantly reduced accompanied with decreased secretion of lactate.While,on the orther hand,the level of oxidative phosphorylation was significantly increased in IL-4 induced MKO BMDMs.Furthermore,pretreatment with 2-DG,the glycolytic inhibitor,almost completely revised the decreased pro-inflammatory function of Zhx2 in LPS-induced macrophages,suggesting that ZHX2 promotes the pro-inflammatory phenotype of macrophages by enhancing glycolysis.RNA-seq analysis indicated PFKFB3,the key glycolytic enzyme as one the most differentially expressed genes in BMDMs from MKO and WT.WB and dual fluorescence report experiments confirmed that ZHX2 promoted the transcription of Pfkfb3.Further ChIP experiments demonstrated the accumulation of ZHX2 on Pfkfb3 promoter region.More importantly,overexpression of PFKFB3 significantly reversed the reduced glycolytic ability of MKO BMDMs.Collectively,ZHX2 participated in the regulation of macrophage reprogramming via promoting PFKFB3 initiated glycolysis.Macrophage polarization plays an important role in many diseases,such as tumors,chronic infections,acute infections,and inflammation.In order to further clarify the pathophysiological function of ZHX2 in macrophage reprogramming,sepsis and liver cancer were selected as representatives of acute and chronic diseases to explore the role of ZHX2 in macrophage reprogramming in disease developmentPart 2 ZHX2 in Macrophage exacerbates sepsis through switching metabolism to glycolysis in PFKFB3 dependent mannerSepsis is a systemic inflammatory syndrome caused by infections,with a high incidence,danger,and high mortality.Results in part one showed that knockout of Zhx2 inhibited the inflammatory response of macrophages.In this part,the role of ZHX2 in macrophages reprogramming during sepsis was studied with two sepsis mouse models.?.Deletion of Zhx2 in macrophages alleviates sepsis significantlyWT and MKO mice were induced by LPS injection and cecal ligation puncture(CLP),respectively.Then,mice survival,the expression of inflammatory factors and lactate in serum were detected and the lung injury was assayed by HE staining.The results showed that the deletion of Zhx2 in macrophages significantly reduced the mortality of sepsis,reduced the concentration of serum inflammatory factors TNF-,IL-6 and IL-1?,and improved the lung injury in both LPS and CLP treated mice.?.ZHX2 promotes the secretion of proinflammatory factors in macrophages during sepsisFlow cytometry results showed that the deletion of Zhx2 did not affect the percentage and number of immune cells including T cell,B cell and NK cell,but the percentage and fluorescence intensity of TNF-?,IL-6 and IL-1? in macrophages were significantly reduced in MKO septic mice.Similarly,dereased level of proinflammatory factors were detected in macrophages purified from spleen and peritoneal resident macrophage from MKO septic mice.Above data suggested that ZHX2 promotes the secretion of inflammatory factors in macrophages in septic mice.?.ZHX2 in macrophages exacerbates sepsis through glycolysis regulation in PFKFB3 dependent mannerIn order to verify the role of glycolysis in ZHX2 enhanced sepsis,glycolytic capacity and lactate secretion were detected in peritoneal resident macrophages collected from septic mice(WT and MKO).The results showed that the glycolytic capacity was significantly reduced and lactate secretion was reduced in macrophages of MKO sepsis mice.Furthermore,pretreatment of 2-DG significantly eliminated the effect of ZHX2 in macrophage during sepsis.This result was further verified BMDMs transfer assay.Overexpression of PFKFB3 partially rescued the protective effect of MKO mice on sepsis,accompanied by increased levels of serum inflammatory factors and lactate secretion.All above suggested that ZHX2 in macrophages exacerbated sepsis through promoting glycolysis in PFKFB3 dependent manner.Part 3 ZHX2 inhibits the development of liver cancer through repression of tumor-associated macrophage reprogrammingMacrophages are the most important immune cells in tumor microenvironment,and the infiltration of tumor associated macrophages(TAMs)is significantly correlated with the poor prognosis of liver cancer.In this part,the effect and mechanism of ZHX2 on TAMs reprogramming in liver cancer are explored.?.Decreased expression of ZHX2 in tumor-associated macrophagesThe first part of the results showed that the expression of ZHX2 in macrophages was significantly inhibited after the stimulation of tumor microenvironment with HCM.Subsequently,we purified tumor associated macrophages from subcutaneous H22 homografts in BALB/c mice,H22 homografts orthotopic transplanted liver tumor,and clinic liver cancer tissues to detect ZHX2 expression.RT-qPCR results showed that the expression of Zhx2 in TAMs was significantly reduced and the expression of Zhx2 in TAMs gradually decreased with the development of tumor.Consistently,results of RT-qPCR and WB confirmed that ZHX2 expression in human monocyte cell line THP1 cels was decreased after the stimulation with homogenate of liver tumor tissue compared with that stimulated with homogenate of para-tumor tissue.Similarly,the results of double immunofluorescence staining of the liver cancer tissue showed that the expression of ZHX2 in TAMs in the HCC tissues was significantly reduced,and the decreased ZHX2 in TAMs was positively correlated with the survival of the patients.?.Lactate in tumor microenvironment reduces the expression of ZHX2 in TAMsIn order to identify factors in tumor microenvironment(TEM)leading to reduced ZHX2 in TAMs,HCM was either separated with a 3kD boundary,or heat treated,and then stimulated BMDMs from WT and MKO mice.Results of RT-qPCR and WB showed that the heat stable components with molecular weight less than 3 kD in the tumor microenvironment caused the decreased expression of ZHX2 in macrophages.Warburg effect exists in tumor cells,so a large amount of lactate is accumulated in tumor microenvironment.As expected,RT-qPCR and WB results showed that lactate significantly inhibited the expression of ZHX2 in macrophages in a time-dependent and dose-dependent manner.Moreover,lactate inhibitors almost completely eliminated the inhibitory effect of HCM on the expression of ZHX2 in macrophages.Accordingly,transfection and dual-luciferase assay showed that lactate significantly inhibited the activity of Zhx2 promoter.These results demonstrated that lactate is a key factor in tumor microenvrionment that inhibits the expression of ZHX2 in macrophages.?.Deletion of Zhx2 in macrophages promote the development of liver cancerIn order to clarify the effect of decreased ZHX2 in TAMs in liver cancer,we stimulated WT and MKO BMDMs with HCM,then co-cultured with liver cancer cells Hepa1-6 or collected the supernatant to stimulate Hepa1-6.Transwell and CCK-8 were performed to detect the migration and proliferation ability of Hepa1-6.The results showed that BMDMs with Zhx2 deletion significantly promoted the migration and proliferation of Hepa1-6 under HCM stimulation.To further verify the above results in vivo,three mouse models of liver cancer were established:H22 tumor-bearing mice,STZ-HFD induced liver cancer and the cholangiocarcinoma model in mice.As expected,compared with mice injected with WT BMDMs,injection of MKO BMDMs promoted tumor growth.Similarly,H22 exografts grew more faster in MKO mice than that in WT mice.WB results showed increased PCNA and Ki67 expression in tumor cells from MKO mice.In accordance,in the model of STZ-HFD induced liver cancer,the survival of MKO mice was significantly reduced and the liver tumors were larger.Moreover,same results were got with mice bearing cholangiocarcinoma induced by sleeping beauty transposon and AKT-cMYC-luc plasmid.Compared with WT mice,MKO mice showed significantly treater tumor burdon.In summary,all three models prove that the deletion of Zhx2 in macrophages significantly promotes the development of tumors.?.ZHX2 reprograms TAMs and improves tumor microenvironmentTo identify the mechanism of ZHX2 in macrophages during the development of liver cancer,flow cytometry was used to detect expression of key molecules in macrophages from WT and MKO tumor-bearing mice.The results showed that TAMs of MKO mice had a significantly tumor-promoting phenotype,displaying as reduced expression of MCH-? and TNF-?,and increased expression of CD206,Argl,and TGF-?.Further analysis showed that the immunosuppressive environment of the tumors in the MKO group was aggravated,accompanied with the increased Treg and the enhanced expression of PD-1 and Tim-3 in CD8+T cells.V.ZHX2 attenuates the pro-tumor function of TAMs by inhibiting the secretion of TGF-?In the first part,we found that ZHX2 inhibited TGF-? expression in macrophages.To verify the effect of TGF-? secreted by MKO macrophages in process of liver cancer cell proliferation and migration,TGF-?-myeloid specific knockout(TGF-? MKO)mice and TGF-?/Zhx2-myeloid double specific knockout(TZ MKO)mice were constructed.BMDMs from TGF-? MKO/TZ MKO mice were stimulated with HCM and co-cultured with liver cancer cells.Results of transwell and CCK-8 showed that compared with the MKO group,coculture with macrophages from TZ MKO significantly reduced the migration and proliferation ability of Hepa1-6 cells,and the migration and proliferation ability was enhanced again after the addition of exogenous TGF-?.Conclusion:Our data for the first time identified ZHX2 as the critical regulator for macrophages polarization.ZHX2 switches the macrophage to pro-inflammatory phenotype by promoting the glycolysis ability in PFKFB3 dependent manner.We further demonstrate the important role of ZHX2 in macrophage related diseases,such as sepsis and liver cancer.In sepsis,ZHX2 enhances the secretion of proinflammatory factors in macrophages,aggravating the inflammatory response,tissue damage,lactate accumulation,and mortality.In liver cancer,ZHX2 switches tumor-related macrophages into proinflammatory and anti-tumor phenotypes,inhibiting the secretion of TGF-? and the development of liver cancer.In summary,ZHX2 plays an important role in macrophages reprogramming and related diseases including tumor and sepesis.