Elucidating mammalian cellular responses to the uptake of nanoparticles (NPs), pathogens, and lipoproteins: similarities and differences

Soft poly-ethylene-glycol (PEG)-based soft nanoparticles (NPs) including cylindrical (CNPs) micelles, spherical (SNPs) micelles, and lipid bilayer vesicles (LNPs) are thought to be treated as foreign objects by mammalian phagocytes. If this hypothesis is true, NPs should trigger a proinflammatory, autophagic phenotype that is similar to the one seen when macrophages phagocytose pathogens or when macrophage surface expressed proteins bind pathogen surface factors such as lipopolysaccharide (LPS). Here, we show that macrophage responses to the above NPs are almost completely unique from those triggered by group A streptococcus (GAS) pathogens (JRS4 cells) and LPS. Instead, macrophages treat these soft NPs more like high-density lipoprotein (HDL) and low-density lipoprotein (LDL). RNA sequencing of macrophage transcripts after hHDL, hLDL, JRS4 cell, LPS, and LNP incubation showed three diverse response clusters triggered by JRS4 cells (1), LPS (2), and hHDL-hLDL-LNP-PBS (3). Of these reagents, LNPs triggered the fewest post-incubation transcript changes from macrophages to which PBS was added (control). LNPs did not increased the transcription of factors associated with foreign object identification including Fc and complement receptors. LNPs did not increase transcripts whose translated proteins are involved in phagocytosis, autophagy, lysosome biogenesis, or inflammation. LNPs did not increase Tfeb transcripts, which is a master regulator of lysosome biogenesis and a necessary component of autophagy. To further determine the effects of these NPs on cells, we performed fluorescence microscopy and flow cytometry experiments. CNPs, SNPs, and LNPs lowered macrophage autophagosome levels that were raised by the canonical challenges: starvation, rapamycin, and LPS. CNPs, SNPs, and LNPs lowered reaction oxygen species (ROS) levels, did not increase lysosome acidification, and the reduced the secretion of proinflammatory cytokines compared to basal conditions and LPS addition to macrophage cultures. CNPs and SNPs triggered low lysosome acidification and LNPs did not increase Tfeb levels, the master regulator of lysosome biogenesis and a necessary component of autophagy. Thus, the terminology that macrophages "clear" NPs, which has been used over many decades, is most likely misleading. Our findings challenge the hypothesis that the main uptake mechanism of soft PEG-NPs by M1-polarized murine macrophages in vitro is phagocytosis.