| Macrophages are innate immune cells that are widely distributed in the human body,which have the ability to recognize and eliminate dead cells and pathogens,and participate in inflammation and wound healing after injury.It plays an important role in tissue homeostasis,immune defense,and pathological processes.Plasticity and heterogeneity are crucial characteristics of macrophages,thus different cytokines and microenvironments are able to induce macrophages into different phenotypes.According to the pathway of activation,polarized macrophages are broadly classified into two distinct subsets: classically activated macrophages(M1)for pro-inflammatory responses;and alternatively activated macrophages(M2)for anti-inflammatory action.Moreover,M2 macrophages can be further classified into four subtypes termed M2 a,M2b,M2 c,and M2 d.Different phenotypes of macrophages perform different biological functions in inflammatory responses,immune regulation,and related diseases.At present,the proteins and glycoproteins of M1 and M2 macrophages have been characterized,but studies on the subtypes of M2 macrophages are lacking,which is one of the obstacles to explore the function of macrophages in immune mechanisms and to identify macrophage phenotypes in pathological tissues.Therefore,systematic proteomic and glycoproteomic analysis of M2 macrophage subtypes is essential for their biological functions analysis and potential biomarker discovery.In this study,a human-derived THP-1 cell line was used as a model to generate M0,M1,M2 a,M2b,M2 c and M2 d type macrophages by a standard polarization process.Proteomics results showed that a total of 4910 proteins were quantified in differentially polarized macrophages.By analyzing differentially expressed proteins,we found that all four M2 macrophages subtypes have the ability to response and kill extracellular pathogens.M2 a and M2 c play critical roles in tissue repair and fibrosis,while M2 b and M2 d are mainly involved in humoral immunity,angiogenesis and hematopoiesis.In addition,we validated common or unique up-regulated proteins from four M2 macrophages subtypes,which may serve as potential biomarkers for identifying subtypes of M2 macrophages.Glycoproteomics results showed that a total of 2,303 intact N-glycopeptides were quantified in different polarized macrophages.We analyzed the differential N-glycopeptides in the four M2 macrophage subtypes and found a regular pattern in the type and modification of glycosylation: more glycopeptides were modified by high-mannan glycan in M2 a and M2 c macrophages,and more glycopeptides were modified by complex glycan in M2 b and M2 d macrophages.Disease enrichment analysis of the differentially expressed proteins and glycoproteins showed that all the four M2 macrophage subtypes were associated with similar diseases,including systemic lupus erythematosus,multiple sclerosis,dilated cardiomyopathy,etc.It suggested that the four M2 macrophage subtypes may influence pathological changes of the same disease through specific protein or glycosylation.In summary,this study systematically analyzed four M2 macrophage subtypes by proteomics and glycoproteomics,thereby elucidating their biological functions and contributing to explore the functions of macrophages in immune regulatory mechanisms.It also illustrated a series of candidate biomarkers for the identification of different M2 macrophage subtypes,and provided an indirect reference for the identification of macrophage phenotypes in normal or pathological tissues.In addition,this project investigated the intact N-glycopeptides of the four subtypes of M2 macrophages for the first time,which will lay the foundation for further glycosylation-related research of macrophage.Finally,this study will be important for studying the plasticity of macrophages and the diagnosis and treatment of macrophagerelated diseases. |
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