Exploring New Inflammatory Biomarkers and Pathways during LPS-Induced M1 Polarization

Figure 8

Schematic representation of inflammatory players implicated in lipopolysaccharide- (LPS-) induced M1 polarization of N9 microglial cells and of exosome upregulated microRNAs. (A) We first studied functions and pathways commonly described for microglia classical activation. We observed that LPS causes a moderate degree of apoptosis in the N9 cells and a switch from the ramified to an amoeboid cell shape. LPS-stimulated cells lose the ability to migrate towards ATP but show increased phagocytic ability, as revealed by the elevated number of latex beads ingested. LPS also triggers microglia proliferation as indicated by the increased number of Cd11b positive cells and Ki-67 stained nuclei. Inflammatory events are mediated through the signaling pathway involving the toll-like receptor 4 (TLR4)/TLR2 and nuclear factor kappa B (NF-κB) in LPS-treated N9 microglia and implicate the upregulation of microRNA-155 (miR-155) and miR-146a as well as the release of nitric oxide (NO) and matrix metalloproteinase-9 (MMP-9) to the extracellular milieu. (B) Our study provided new evidence that N9 cells treated with LPS have increased expression of inflammasome complex comprehending the upregulation of interleukin- (IL-) 1beta, IL-18, and NOD-like receptor family pyrin domain containing 3 (Nlrp3), together with enhanced caspase-1 activation. Increased expression of Nos2 and major histocompatibility complex class II (Mhc-II) (markers of M1 polarization or classically activated microglia), in conjunction with decreased expression of arginase 1 (Arg1), found in inflammatory zone 1 (Fizz1), miR-124 (markers of M2 polarization or alternatively activated microglia), and reduced CX3C chemokine receptor 1 (CX3CR1) expression corroborate the acquisition of a prevalent M1 phenotype in microglial cells exposed to LPS. (C) Finally, we described for the first time that increased MFG-E8 expression and release are induced in M1 polarized microglia and that miRs expression profile is recapitulated in cell-derived exosomes, further supporting M1 polarization in LPS-treated N9 cells and dissemination of inflammatory mediators by extracellular vesicles.