Mitochondria And Peroxisome-mediated Fatty Acid Oxidation Regulate Macrophage Phenotype And Function

BackgroundThe incidence of malignant tumors in China is high and showing an increasing trend year by year.Recent studies have found that the occurrence of tumors is often accompanied by abnormalities in the immune microenvironment.The composition of tumor immune microenvironment is complex.Some metabolites associated with tumor cells can reshape the phenotype and function of immune cells,and inhibit their anti-tumor ability,and even play a pro-tumor role,leading to tumor immune escape.Therefore,in recent years,immunotherapy has gradually become one of the important options for clinical tumor comprehensive treatment.Hepatocellular carcinoma(HCC)is the most common malignant tumor of the liver,accounting for the sixth incidence of malignant tumors in the world,and the third cause of cancer mortality in the world.It seriously endangers human health and has poor response to immunotherapy.The liver is rich in macrophages,and tumor-associated macrophages(TAMs)are an important part of the immune microenvironment of liver cancer.With high plasticity and heterogeneity,TAMs play an important role in determining the occurrence and development of liver cancer and the response to clinical immunotherapy.Therefore,further revealing the regulatory mechanism of TAMs will provide experimental basis for elucidating the pathogenesis of HCC and discovering new targets for tumor intervention.Macrophages have a high degree of phenotypic plasticity and functional pluripotency.There are classically activated macrophages of M1 type and alternatively activated macrophages of M2 type and numerous transitional states in between.Both M1 and M2 TAMs exist in tumor microenvironment.The infiltration and distribution of M2 TAMs in tumors are closely related to the occurrence and development of tumors and poor prognosis of patients.Numerous mechanisms are involved in the regulation of macrophage phenotypic differentiation and functional remodeling,and lipid metabolic reprogramming plays an important role in this process.Previous studies have shown that M1 macrophages rely on aerobic glycolysis to consume glucose and glutamine to produce ATP,while M2 macrophages tend to use oxidative metabolism,especially fatty acid oxidation(FAO),as a source of ATP.Mitochondria,as the main site of aerobic respiration of cells,participates in the regulation of lipid metabolism of TAMs and affect their function,affecting the occurrence and development of tumors.However,more and more evidences indicate that another organelle,peroxisome,is required for the oxidation of long-chain fatty acids in cells.After the initial oxidative cleavage of long-chain fatty acids by peroxisomes,the short-chain fatty acids are transferred to mitochondria in the form of acyl-coa for further β-cycle oxidation.However,little is known about the role of peroxisomes in the regulation of macrophage phenotype and function,as well as the relationship between mitochondria and peroxisomes in the regulation of macrophage lipid oxidation.In this study,we found that IL-4 induced up-regulation of lipid enrichment in M2 macrophages,and this abnormal lipid enrichment was closely related to the FAO of mitochondria and peroxisomes.Because the distribution and abnormal lipid metabolism of TAMs lead to poor prognosis of patients,we futher studied TAMs.After in vitro experiments and analysis of samples from HCC patients,we found that there is abnormal lipid enrichment in TAMs,and mitochondrial and peroxisomal FAO metabolism were related to the maintenance of M2 phenotype of TAMs.In summary,we investigated the role and interaction of mitochondrial and peroxisome mediated fatty acid oxidation in regulating lipid metabolism,phenotype and function of M2 macrophages and TAMs,which provided new evidences for elucidating the metabolic remodeling mechanism of macrophages and potential targets for interfering with the tumor-promoting function of TAMs.Methods and results1.Lipid enrichment level was upregulated in M2 macrophagesLipid metabolism plays an important role in the regulation of macrophage phenotype and function.We first stimulated mouse macrophage cell line RAW264.7 with mIL-4(20ng/ml)or human monocyte cell line THP1 with hIL-4 and hIL-13(20ng/ml).RT-qPCR was used to verify the up-regulation of RAW264.7 cell M2 polarization related marker molecules Argl and Tgf-β and CD206,TGF-β and IL-10 of THP1 to confirm the successful induction of M2-like macrophages.Fluorescence microscopy and flow cytometry were used to detect intracellular lipid accumulation after BODIPY staining,and the results showed that lipid accumulation levels were significantly up-regulated in M2 macrophages compared with control cells.At the same time,bone marrow-derived macrophages(BMDMs)and peritoneal macrophages(PEMs)were induced with IL-4,and the M2 phenotype was verified by RT-qPCR.Flow cytometry was used to detect lipid enrichment levels.These results suggested that lipid enrichment level was significantly upregulated in M2 macrophages.2.The expression of FAO-related genes in mitochondria and peroxisome was up-regulated in M2 macrophagesSince mitochondria and peroxisomes are the main organelles involved in cellular FAO metabolism,we further examined the expression of genes involved in the mitochondrial and peroxisomal FAO pathway in M2 macrophages.The results of RT-qPCR showed that the expressions of mitochondrial FAO metabolism-related enzymes Cpt1a,Crat and Acads and peroxisomal FAO metabolism-related enzymes Acot4,Acot8,Ehhadh and Acox1 were up-regulated in BMDMs and PEMs after IL-4 stimulation.These results suggested that the FAO pathway of mitochondria and peroxisomes may be activated in M2 macrophages.3.Blocking the mitochondrial and peroxisomal FAO pathway further upregulated lipid enrichment in M2 macrophagesTo further test the role of mitochondrial and peroxisomal FAO metabolic pathways in the regulation of lipid metabolism in M2 macrophages,we used Etomoxir,an inhibitor of mitochondrial FAO rate-limiting enzyme CPT1A,and TDYA,an inhibitor of peroxisomal FAO rate-limiting enzyme ACOX1.Lipid accumulation in BMDMs and PEMs stimulated with IL-4 was detected by BODIPY staining and flow cytometry.The results showed that both Etomoxir and TDYA treatment significantly upregulated lipid enrichment levels in BMDMs and PEMS,suggesting that both mitochondria and peroxisomes were involved in the lipid catabolic pathway in M2 macrophages.4.Disruption of the mitochondrial and peroxisomal FAO pathways hinderd the M2 macrophage phenotype and functionLipid metabolism plays an important role in the regulation of macrophage phenotype.To further clarify whether the FAO pathway of mitochondria and peroxisome is involved in the regulation of M2 macrophage polarization phenotype,we examined the effects of Etomoxir and TDYA treatment on macrophage phenotypes.RT-qPCR results showed that Etomoxir and TDYA treatment significantly down-regulated the expression levels of M2 macrophage markers Arg1 and Ym1 in IL-4-stimulated BMDMs and PEMs.Flow cytometry data showed that Etomoxir and TDYA treatment downregulated CD206,TGF-β and IL-10 expression levels in BMDMs and PEMs.These results suggested that both mitochondrial and peroxisomal FAO pathways were involved in regulating the phenotype and function of M2 macrophages.For tumor cells,macrophages can specifically show different anti-tumor and pro-tumor effects.We then investigated whether Etomoxir and TDYA treatment,which hinderd the M2-type macrophage phenotype,affected the proliferation of mouse hepatoma cell line Hepa1-6.The supernatant of M2 macrophages treated with inhibitors Etomoxir and TDYA were collected,and the mouse hepatoma cell line Hepa1-6 cells were cultured.CCK8 cell proliferation assay showed that the proliferation of Hepa1-6 cells were inhibited to a certain extent after 48h treatment with the supernatant,and the addition of TDYA produced a more significant inhibitory effect than Etomoxir.5.Lipid enrichment level of TAMs was up-regulatedTumor microenvironment can reshape the phenotype and function of TAMs to mediate tumor immune escape and promote disease progression.TAMs are known to be predominantly polarized to a tumor-promoting M2 phenotype,so this study further validated the lipid enrichment in TAMs.A co-culture system of tumor cells and macrophages was established in vitro.BODIPY staining,fluorescence microscopy and flow cytometry showed that lipid accumulation was significantly up-regulated in THP1 and RAW264.7 cells co-cultured with Huh7 or Hepa1-6 cells,respectively.RT-qPCR and flow cytometry were used to verify the up-regulation of M2 phenotyping markers Arg1 and Ym1 in mouse primary macrophages(BMDMs and PEMs)co-cultured with Hepa1-6,and the lipid enrichment level was detected by flow cytometry.The results showed that the lipid enrichment levels in BMDMs and PEMs co-cultured with Hepa1-6 cells were significantly higher than those comtrol cells.At the same time,the lipid levels of TAMs in HCC tissues and adjacent tissues were quantitatively analyzed by Raman spectroscopy combined with immunofluorescence staining.It was found that lipid enrichment levels were significantly increased in TAMs from HCC tissues compared with macrophages from paracancerous tissues.These results suggested that tumor microenvironment up-regulated the level of lipid enrichment in macrophages.6.Mitochondria and peroxisome were involved in regulating the FAO metabolic pathway of hepatocellular carcinoma related macrophagesBodipy-labeled neutral lipids are mainly localized in lipid droplets,and the interaction between lipid droplets and mitochondria and peroxisome may promote the FAO pathway.Therefore,we detected the association of lipid droplets with mitochondria and peroxisome by fluorescence staining in THP1 cells co-cultured with Huh7 cells.Laser scanning confocal microscopy showed that BODIPY fluorescence was co-localized with MitoTracker Deep Red and PMP70.These results suggested that mitochondria and peroxisomes may be involved in the process of lipid metabolism in TAMs.To clarify the changes of mitochondrial and peroxisomal FAO pathway in TAMs,we further examined the expression of FAO-related genes in BMDMs and PEMs co-cultured with Hepa1-6 cells.RT-qPCR results showed that the expression of mitochondrial FAO metabolism-related enzymes Cpt1a,Crat and Acads,as well as peroxisomal FAO metabolism-related enzymes Acot4,Acot8,Ehhadh and Acox1 in BMDMs and PEMs were significantly up-regulated.The protein levels showed that the FAO rate-limiting enzyme CPT1A in mitochondria and ACOX1 in peroxisomes were up-regulated in PEMs co-cultured with Hepa1-6 cells.The above results suggested that the FAO pathways were activated in mitochondria and peroxisomes in TAMs.7.Blockade of mitochondrial and peroxisomal FAO pathways further promoted lipid enrichment in Hepatoma-associated macrophagesTo further verify the role of mitochondrial and peroxisomal FAO pathways of lipid metabolism in TAMs,BMDMs and PEMs co-cultured with Hepa1-6 cells were treated with Etomoxir and TDYA,and lipid accumulation was detected by BODIPY staining and flow cytometry.The results showed that Etomoxir and TDYA treatment significantly upregulated lipid enrichment levels in BMDMs and PEMs co-cultured with Hepa1-6 cells,suggesting that both mitochondrial and peroxisomal FAO pathways were involved in lipid metabolism in TAMs.8.Inhibition of mitochondrial and peroxisome FAO pathway regulated TAMs phenotypeTo test the role of mitochondrial and peroxisomal FAO pathways in the phenotypic remodeling of TAMs,we examined the effects of Etomoxir and TDYA treatments on the phenotypes of TAMs.The results showed that the expression of Argl,Ym1 and CD206 of M2 phenotype markers in BMDMs and Argl,Ym1,CD206,TGF-β and IL-10 in PEMs were significantly down-regulated after co-cultured with Etomoxir and TDYA inhibitor.These results suggested that both mitochondrial and peroxisomal FAO pathways were involved in the M2-like phenotype remodeling of TAMs cells.Conclusion and significanceLipid metabolism plays an important regulatory role in the phenotypic and functional remodeling of macrophages,and FAO pathway is one of the main energy sources of M2-like macrophages.In this study,IL-4 activated M2 macrophages and TAMs were used to detect lipid enrichment and changes in mitochondrial and peroxisomal FAO pathways in the above macrophages,and to preliminarily explore the role of mitochondrial and peroxisomal FAO pathways in the regulation of macrophage phenotype and function.Increased lipid enrichment was found in M2 macrophages and TAMs,accompanied by up-regulation of mitochondrial and peroxisome FAO pathway related genes.Blockade of the mitochondrial and peroxisome FAO pathway further up-regulated lipid enrichment in macrophages and hinderd the M2-like macrophage phenotype.Our study provides a new experimental basis for elucidating the mechanism of macrophage metabolic remodeling in the tumor microenvironment,and this provides a new idea for finding the intervention targets of tumor-associated macrophage immunotherapy.