Functional Characterization And Global Analysis Of Essential Molecular Clusters Targeting Phosphorylation In Phenotypically-Polarized Macrophages

Maintenance Macrophages, acting as immune effector cells, are essential components of innate immunity, making them the first line of defense against pathogens. Pathogens that invade bodies are recognized eliminated by macrophages and induce macrophage activation, which is characterized by releasing a variety of pro-inflammatory factors, oxygen and nitrogen radicals to kill pathogens, making them as potent mediators of inflammation and important components of host defense.It is worth noting that macrophage activation induces obvious morphological change, including cytoskeletal rearrangement, resulting in the formation of lamellipodia, filopodia and membrane ruffles. This change is referred to as macrophage polarization, which is a marker of macrophage activation.In this work, we introduced an impedance based method to characterize macrophage polarization in vitro as specific time-and dose-dependent cell response profiles (TCRPs) measured by a real-time cell analyzer. Applying the microelectronic sensor technology to record the impedance, the method provides outputs that are well correlated with macrophage morphological changes, in a way of label-free, real-time and high-throughput. Our data showed that TCRPs of IFN-y and LPS induced RAW264.7cell polarization had a distinct profile. Using traditional methods, we demonstrated that macrophage polarization associated TCRPs is in line with the inherent cytoskeletal rearrangements, and is induced by macrophage activation. Based on it, the demonstrated function of Transforming growth factor-β (TGF-β) in inhibiting macrophage activation is verified in our model. Moreover, we found that only cytochalasin, but not Cytochalasin D, contributed to the macrophage polarization associated TCRPs. Finally, an inhibitor library including more than160inhibitors for either kinases or phosphatases was screened and analyzed. Among the inhibitors, Lck inhibitor (damnacanthal) was found to inhibit macrophage polarization, which was further confirmed by expression of inflammatory cytokines and cytoskeletal rearrangements. Interestingly, this was reported for the first time that PHPS1, the inhibitor of phosphatase shp2, inhibit macrophage polarization associated TCRPs. PHPS1could also relieve mouse model of acute lung injury in vivo, so as macrophage conditional knock out shp2mouse. Considering the vital role of macrophage activation in inflammation, the results implicate the potential application of targeting shp2in treatment of inflammation.In summary, our work demonstrated that recording macrophage polarization featured TCRPs is a promising and powerful tool for monitoring, screening and identifying macrophage activation.