Poster AA20 - Small lipid droplets in macrophages suppress engulfment and cause confinement-induced rupture but phagocytosis is rescued by LDs’ abundance via actomyosin activation

Introduction: Lipid droplets (LDs) are cytoplasmic condensates in which a lipid ester core is surrounded by phospholipid monolayer. They are found in various cell types and can be a source of damage [1]. LDs’ mechanical interaction with other organelles is determined by their interfacial tension  [mN/m] and we found a high  of mechanically isolated LD from fat tissue ~ tens of mN/m [2]. We have recently shown that small LDs are rigid enough to deform the cell nucleus and actomyosin network. LDs can indent the nuclear lamina, dilute Lamin B1 at high curvature indentation sites and cause rupture with mis-localization of DNA repair factors and subsequently increased DNA damage [2]. Macrophages often have to engulf and digest dead cells which can be lipid loaded, for example - dead adipocytes in obesity or in the case of formation of hyperlipidemic lung lining in response to acute injury. Studies to date on how LDs’ presence in macrophages affects phagocytosis are conflicting and their biophysical interaction with the other organelles are understudied.

Materials and

Methods: THP-1 cells were cultured in RPMI 1640 medium with 10% FBS and 1% Penn-Strep. To obtain macrophages, THP-1 monocytes were differentiated using 100 nM phorbol 12-myristate-13-acetate for 48 hours. Mcherry-cGAS expressing THP-1 cells were developed via lentiviral transduction as described in [3]. LDs formation is performed as described in [2]. In vitro phagocytosis assays were done using 6 µm, 3 µm and 100 nm streptavidin coated polystyrene beads prior to opsonization with anti-streptavidin. The phagocytosis assay was done for 45 minutes at ratio of 10:1 while cells were kept at 37°C with 5% CO2 . Beads that remained in suspension were removed by rinsing with PBS before fixation and immunostaining. Phagocytic uptake was quantified by presence or absence of secondary antibody stain on beads. Confinement experiments were done using confinement micropillar system, previously described in [4] in collaboration with dr. Nader at Penn. Briefly, PDMS micropillars, are attached to a modified 6 well plate lid, which is then lowered onto the 6 well plate to push the micropillars on top of the cells. Microscopy and Live Imaging: Fixed sample imaging was performed on both epifluorescence with (Olympus IX71 microscope with a 40x/0.6-NA objective, EMCCD camera) and on confocal with Leica TCS SP8 system or a Zeiss LSM 980 system with a 63x/1.4-NA oil immersion objective. Live imaging during confinement experiments were performed at 37°C with 5% CO2 using a Zeiss LSM 900 with Airyscan and a 40x/1.2-NA water immersion objective for 0.5- 2 hours. Images were processed with Fiji software.

Results, Conclusions, and Discussions: We found that oleic acid induced formation of small lipid droplets in the cytosol of macrophages impairs phagocytosis and does not alter macrophages’ polarization, measured here by levels of proinflammatory (CD80) and anti-inflammatory (CD206) surface markers. LDs loaded macrophages show suppressed engulfment of nano and micron sized beads and opsonized cancer cells including CD47-SIRP “don’t eat me” knockout cells. While moderate inhibition of actomyosin typically suppresses uptake, engulfment by LD-loaded macrophages remains unaffected after drug treatment suggesting an independent disruption mechanism. Visually actomyosin filaments are excluded and displaced by LDs, which remain spherical consistent with a high interfacial tension for LDs. Although F-actin levels are unaffected by LDs, cortical F-actin at the apical surface that drives engulfment is depleted in +LD cells and instead accumulates at the cell-substrate interface. To investigate how LDs impact phagocytosis under physiologically relevant 3D conditions, macrophages were confined to a 4 µm height using PDMS micropillars and permitted to phagocytose 3 µm diameter opsonized beads for 3 hours. We found that confined macrophages have increased phagocytosis by ~ 50%, which is in part due to enhanced actomyosin contractility caused by confinement.
Surprisingly, in vitro assays showed that similar mechanically-induced myosin activation measured here using fluorescent intensity of phosphomyosin light chain (p-MLC2) also enables macrophages densely filled with either LDs or beads to engulf targets more efficiently than sparsely loaded cells. However, the presence of LDs during macrophage confinement increases the frequency of nuclear rupture events 10 fold compared to macrophages in 2D despite their relatively high Lamin A:B ratio. F-actin dependent infiltration of macrophages through pores is also perturbed by LDs, with droplets acting as additional barriers confining the nucleus and reducing migration speed. Lipid droplets thus exert physical effects on uptake and motility processes by modulating organization and activation of actomyosin.

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