| Macrophages are a set of specialized phagocytic cells that play important roles in innate and adaptive immune responses. They contributed to the recognition, uptake and killing of non-self agents, the setting off and progression of the adaptive immune response, and also mediated the physiological and pathological progression. Macrophages are heterogeneous populations and respond to environmental stimulations (e.g., microbial products, damaged cells, activated lymphocytes) with the acquisition of distinct functional phenotypes. In response to various signals, macrophages may undergo classical Ml activation (typically stimulated by TLR ligands and IFN-y) or alternative M2 activation (typically stimulated by IL-4/IL-13 etc.). Compared to the molecular mechanisms that have been illustrated in the process of macrophage classical activation, the molecular mechanisms involved macrophage alternative activation are still elusive. Here, we have made use of a sensitive LC-MS/MS based system biology approach to study the changes in protein composition taking place during macrophage alternative activation for the first time. By using SILAC, we profiled the plasma membrane protein and whole cellular protein abundance of alternatively activated macrophages. Based on the proteomic data, we identified two new regulatorsPax4 and capase-6, which play critical roles in alternative activation of macrophages and tumor-associated macrophage biology.We first labeled mouse RAW264.7 cell by using SILAC. IL-4 stimulated light isotope-labeled RAW264.7 cells, which belonged to alternative M2 activation. Heavy isotope-labeled RAW264.7 cells were served as control sample. Plasma membrane proteins of two groups were extracted by biotin-streptavidin affinity enrichment and mixed at a ratio of protein amounts at 1:1 for a total of 200μg. Using FASP, the samples were prepaired and digested into peptides, which were separated on OFFGEL. Peptides were eluted with 50% methanol in 5% formic acid directly into a nanospray needle, and subjected to MS and MS/MS analysis. The data were treated by Maxqaunt software and 2238 proteins were quantified, from which the expression levels of 108 proteins were significantly changed. Meanwhile, we also undertook a comparative MS analysis of proteins from unstimulated and alternatively activated macrophages in a time-dependent pattern, leading to the quantification of 3442 proteins, from which the expression levels of 342 proteins were significantly changed. Bioinformatics analysis of differentially expressed poteins provided evidence indicating that phagocytosis functional properties were. greatly impaired by down-regulation of nearly 20 proteins of phagocytosis pathway, including integrins, toll-like receptors, Arp2/3, rab5, dynein, lampl and sec22b, key proteins regulating every biological steps of phagocytosis. Functional study confirmed that the phagocytic ability was impaired during macrophage alternative activation.Bioinformatics analysis showed that transcription factor Pax4 may participate in the regulation of macrophage alternative activation. Therefore, we measured the expression levels and transcriptional activity of Pax4 in the macrophage alternative activation. The results indicated that Pax4 expression and transcriptional activity up-regulated significantly. The overexpression and knock-down of Pax4 further showed that Pax4 can affect the macrophage alternative activation. Meanwhile, our analysis also revealed that Pax4 was also responsible for the attenuated phagocytic ability of M2 macrophages through the transactivation of Bcl-xL and alternation of cellular calcium level. Furthermore, we showed that the impairment of phagocytosis pathway and phagocytic abilitywas a general biological function alternation not only in M2 macrophages but also in TAMs, and so as to the underlying molecular mechanisms.Quantitative proteomic data also revealed that the expression level of caspase-6 was significantly up-regulated. We confirmed that the expression level and activity were both upregulated in M2 macrophages and provided evidence for the involvement of caspase-6 in the process of macrophage alternative activation by regulating M2 marker genes. We also reported that caspase-6 was also upregulated in TAMs, where it controlled the invasion of tumor cells by regulating the expression of MMP9.In summary, the present study describes a comprehensive quantitative plasma membrane proteomics and dynamic proteomics of M2 macrophages playing pivotal roles in immune system. The application of a comprehensive proteomics enabling the profiling of protein abundance provided unprecedented molecular details on the dynamic changes in protein composition of alternatively activated macrophages.Based on the proteomic data, we identified two new regulators playing critical roles in alternative activation of macrophages and tumor-associated macrophage biology, contributing to the elucidation ofcomplicated molecular mechanisms and functional alternations of macrophage alternative activation. |
