The phospholipids 1,2-dioleoyl-sn-glycero-3-phospho-(1′-rac-glycerol) (18:1 Δ9-cis) (DOPG) (Item No. 840475) and 1,2-dioleoyl-sn-glycero-3-phosphocholine 18:1 (Δ9-Cis) (DOPC) (Item No. 850375) were purchased in chloroform from Avanti Polar Lipids, inc. (Alabaster, AL). Cholesterol powder (Item No. 700000) was also bought from Avanti Polar Lipids, inc. (Alabaster, AL). The fluorescence probe used for anisotropy measurements was Di-4-ANEPPDHQ, which was purchased from ThermoFisher (Waltham, MA). U18666a (Item No. 10009085) was obtained from Cayman Chemical (Ann Arbor, MI). The fluorescent substrate for n-acetyl-β-glucosaminidase was 4-Methylumbelliferyl-2-acetamido-2-deoxy-β-D-Glucopyranoside (Item No. 26953), and it was purchased from Cayman Chemical (Ann Arbor, MI). Filipin complex (Item No. F9765), digitonin (Item No. 300410), and Pefabloc SC (Item No. 11429868001) were from Sigma Aldrich (St. Louis, MO). For staining lysosomes, LysoView 633 (Item No. 70058) was purchased from Biotium, Inc. (Fremont, CA). The Cellview cell culture dishes (Item No. 627871) were from Greiner Bio-One North America, Inc. (Monroe, NC). Lactate dehydrogenase (LDH) in the cellular supernatant was measured with a CytoTox 96 Non-radioactive Cytotoxicity Assay (Item No. G1780) Promega (Madison, WI). Crystalline silica (Min-u-sil-5) was obtained from the Pennsylvania Glass Sand Corporation (Pittsburgh, PA). Min-u-sil-5 has a circle equivalent average diameter of 1.3 µm and has been further characterized in our previous work (Pavan et al., 2022).

C57BL/6J mice (Jackson Laboratories) were housed in the University of Montana’s specific-pathogen-free Laboratory Animal Resources facility with controlled environmental conditions and a 12 h light/dark cycle. Mice were euthanized by overdose of sodium pentobarbital (Euthasol™) prior to collection of legs for bone marrow. The animal procedures used in this study were approved by the Institutional Animal Care and Use Committee (IACUC) at the University of Montana.

C57Bl/6 mice were sacrificed, and the hind legs removed for bone marrow isolation. Bone marrow cells were collected by flushing the tibia and femur bones with complete media (RPMI, 10% FBS, 1% Penicillin/Streptomycin, 1% Sodium Pyruvate, 0.1% β-mercaptoethanol) with a 1/2” long 27-gauge needle. Cells were incubated overnight (37°C; 3.0 × 10⁷ cells/T75 flask) to eliminate stromal cells by adherence. The following day, non-adherent cells were transferred to new T75 flasks (1.5 × 10⁷ seeding density) with macrophage colony stimulating growth factor (M-CSF) (20 ng/mL, R&D Systems). Cells were maintained with additional M-CSF (10 ng/mL) every 3–4 days. BMdM were collected by trypsin and gentle scraping and used in all described experiments after day 9.

BMdM were primed with 20 ng/mL lipopolysaccharide (Sigma) for NLRP3 inflammasome activation and treated with 100-nm DOPG liposomes (50 μM) for 1 h before silica (50 μg/mL) application. BMdM were incubated (37°C; 5% CO₂) for 24 h in 96 well plate (100,000 cells/well) in complete RPMI 1640 media (10% fetal bovine serum, 1% Penicillin/Streptomycin). Cell death was measured in supernatants by Promega CytoTox 96 Non-Radioactive LDH assay. IL-1β release was quantified in supernatants by R&D Systems IL-1β ELISA kit.

The measurement of lysosomal membrane permeability was adapted from methods that were previously described (Aits et al., 2015; Jessop et al., 2017). BMdM were plated at an amount of 1 × 10⁵ cells per well and were treated with 50 μg/mL silica and/or 50 µM DOPG liposomes. After 24 h, the supernatant was removed for use in an LDH assay. The cells were then washed twice with PBS before 100 µL of cytosolic extraction buffer was added. The composition of the cytosol extraction buffer was 250 mM sucrose, 20 mM HEPES, 10 mM KCl, 1.5 mM MgCl₂, 1 mM EDTA, 1 mM EGTA, 0.5 mM pefabloc SC, and 15 μg/mL digitonin. A digitonin titration was performed in advance to select the optimal working concentration (15 μg/mL) of digitonin needed to fully permeabilize the plasma membrane but have minimal effect on lysosomes. The plate was then placed on ice with rocking for 15 min. After 15 min, the extracted cytosol was removed from the cells and placed in a new 96 well plate on ice. The activity of n-acetyl-β-glucosaminidase (NAG) was measured by adding 30 µL of extracted cytosol to 100 µL of NAG reaction buffer (0.2 M sodium citrate pH 4.5, 300 μg/mL 4-Methylumbelliferyl-2-acetamido-2-deoxy-β-D-Glucopyranoside). Cleavage of the fluorescent NAG substrate (4-Methylumbelliferyl-2-acetamido-2-deoxy-β-D-Glucopyranoside) was measured for 20 min with 45 s intervals at 30°C in a Molecular Devices (San Jose, CA) SpectraMax M4 microplate reader. The cleaved substrate was excited at 356-nm, and the fluorescence emissions were detected at 444-nm. Measured NAG activity was normalized by dividing NAG activity from the working concentration of digitonin (15 μg/mL) by the activity from the total lysis concentration of digitonin 200 μg/mL. This was done for each condition.

BMdM were added to a 96-well plate (100,000 cells per well) with TopFluor cholesterol (5 μg/mL) in serum-free media for 24 h (37°C), washed, and incubated with U18666A for 24 h or DOPG for 1 h in complete media (RPMI 1640, 10% FBS), in a similar fashion to that previously described (Liu et al., 2014). Supernatants were transferred to separate wells in black 96 well plate and TopFluor fluorescence measured by a Molecular Devices (San Jose, CA) SpectraMax M4 microplate reader. Efflux of treated BMdM was compared to control efflux. TopFluor was excited at 488-nm and detected at 525-nm.

All fluorescence images were collected on a Zeiss 880 LSM (Carl Zeiss Microscopy, LLC., White Plaines, NY). The objective used to collect the images was a ×63 Plan Apo 1.4 NA oil immersion objective. A confocal aperture of 1 AU was used for all image acquisition. For imaging of cholesterol and lysosomes, a procedure similar to that of (Kummer et al., 2022) was used. BMdM were seeded the prior day at 1.5 × 10⁵ cells per well in a 4-well Greiner Cellview cell culture dish. The next day, BMdM were stained with LysoView 633 for 1 h at a concentration of 1x in complete media per the manufacturer’s instructions. The cells were then washed twice with PBS before being fixed for 10 min at room temperature in 4% paraformaldehyde. Again, the cells were washed twice with PBS before addition of filipin staining solution in complete media. Filipin staining solution was made by diluting a 2.5 mg/mL stock of Filipin in DMSO 1:2 with 2% BSA/PBS. BMdM were then incubated at 37°C in the dark for 2 hours. The staining solution was then removed and replaced with phenol-red free complete media. Filipin was excited with a 405-nm laser, and LysoView 633 was excited with a 633-nm laser. The laser power and detector gain were held contestant across all conditions. At least six images of each condition were acquired per experiment. Experiments were conducted over three separate days. The laser power and detector gain were held constant across all conditions. Fluorescence overlays were generated with Zeiss Zen 2.3 software.

All lipids were dissolved in chloroform and allowed to dry under a stream of nitrogen gas for approximately 1 h. The dried lipids were rehydrated with Dulbecco’s PBS or tris buffered saline (TBS) (150 mM NaCl, 50 mM tris). The lipids were then sonicated in a water bath for 10 min prior to extrusion through an Avanti mini extruder set with a 100-nm pore size filter. DOPG liposomes in PBS were used to treat cells, while DOPC liposomes in TBS were incubated with silica particles at 37°C with tumbling for 2 h. Lipid order in 100-nm DOPC liposomes was assessed by time-resolved fluorescence anisotropy measurements of the lipophilic probe, Di-4-ANEPPDHQ. The fluorescence probe, Di-4-ANEPPDHQ has been previously demonstrated to be sensitive to changes in the lipid environment surrounding the probe (Owen et al., 2011; Steele et al., 2019; Sydor et al., 2020; Sydor et al., 2021; Pavan et al., 2022). Di-4-ANEPPDHQ was dissolved in spectral grade dimethyl sulfoxide (DMSO) and used at a working concentration of 400 nM with the final DMSO concentration being <1%. Di-4-ANEPPDHQ was added 15 min before data collection and was allowed to incubate with the liposome sample at room temperature, as previously described (Steele et al., 2019). An in-house built fluorimeter with parts from Quantum Northwest (Liberty Lake, WA) was used to acquire time-resolved anisotropy measurements as previously reported (Minazzo et al., 2009). A PicoQuant 470-nm pulsed diode laser (LDH-P-C-470) was used to excite the Di-4-ANEPPDHQ. Fluorescence emissions were collected with a Chorma 500-nm longpass filter and an Edinburgh Instruments photomultiplier tube (H10720-01). All data was collected from a sample chamber at 20°C ± 0.02°C. The anisotropy decay curves were fit using the PicoQuant FluoFit software (V4.6.6). The range of motion of Di-4-ANEPPDHQ in the lipid membrane was presented as a wobble-in-a-cone-angle. This was calculated from equation #1 and has been previously used to describe lipid membrane order (Steele et al., 2019; Sydor et al., 2020; Pavan et al., 2022).

Statistical significance was defined as p < 0.05. An unpaired-student’s t-test was used to compare the statistical difference of two means. For the comparison of more than two means, one or two-way ANOVA were used. A Dunnett post-test was used to compare multiple means back to the control. For comparisons between all groups, a Tukey HSD or Sidak-Holmes post-test was used. A minimum of three experimental replicates were performed for each data set. All statistical tests and graphs were generated with Prism 9.0 by GraphPad Software (San Diego, CA).

It has previously been demonstrated that crystalline silica promotes increased IL-1β release and cell death in BMdM (Hornung et al., 2008). Because NLRP3 inflammasome activation requires a priming as well as an activating signal, BMdM were primed with LPS (20 ng/mL) immediately prior to incubation with silica (50 μg/mL) for 24 h. Silica caused a significant increase in cell death measured by LDH release (Figure 1A) compared to untreated BMdM. This increase in cytotoxicity was accompanied by a significant increase in IL-1β release following silica exposure (Figure 1B).

The protective effect of cholesterol in lysosomal membranes was confirmed by addition of U18666A (10 μg/mL) to BMdM for 1 h prior to silica for 24 h. U18666A interacts with the NPC1 cholesterol transporter by binding the sterol-sensing domain and causes an increase in lysosomal cholesterol (Liscum and Faust, 1989; Lu et al., 2015; Chung et al., 2018). U18666A significantly reduced silica-induced cell death (Figure 1A) and IL-1β release compared to silica treated control BMdM (Figure 1B).

In contrast to U18666A, DOPG is reported to increase cholesterol trafficking out of the lysosome, thereby reducing lysosomal cholesterol (Ilnytska et al., 2021b). Here 100-nm liposomes comprised of DOPG (50 μM) were applied to BMdM for 1 h before 24 h silica treatment. While DOPG alone did not impact cell death compared to control BMdM, DOPG significantly increased cell death compared to silica-only treated BMdM (Figure 1A). Additionally, DOPG treatment significantly increased IL-1β release compared to silica-only BMdM (Figure 1B). These results demonstrate that U18666A treatment reduces silica-induced cell death and IL-1β release while DOPG treatment increases silica-induced cell death and IL-1β release in BMdM.

DOPG and its derivative, lysobisphosphatidic acid (LBPA) are reported to increase cholesterol trafficking from lysosomes (McCauliff et al., 2019; Ilnytska et al., 2021a; Ilnytska et al., 2021b). Lysosomal cholesterol trafficking patterns impact cellular cholesterol efflux from macrophages, therefore the impact of U18666A and DOPG on cholesterol efflux was confirmed. BMdM were loaded with fluorescently labeled TopFluor cholesterol in serum-free conditions for 24 h. After washing, cells were treated with U18666A (10 μg/mL) for 24 h or DOPG (50 μM) for 1 h in complete RPMI 1640 media (10% FBS). Fluorescence intensity was measured in supernatants after 24 h and compared to untreated BMdM to assess cholesterol efflux. DOPG treatment significantly increased cholesterol efflux compared to untreated control BMdM (Figure 2). In contrast, U18666A treated BMdM demonstrated a significant decrease in cholesterol efflux (Figure 2). These results demonstrate that DOPG promotes cholesterol efflux from BMdM while U18666A prevents cholesterol efflux.

To demonstrate that the increased DOPG-induced cholesterol efflux corresponded to decreased lysosomal cholesterol upon DOPG treatment, free cholesterol within BMdM was measured by filipin staining. Filipin is a fluorescent antibiotic that binds unesterified (free) cholesterol in fixed cells (Maxfield and Wustner, 2012). Previously, it has been demonstrated that U18666A treatment causes an accumulation of filipin-stained free cholesterol within lysosomes (Reiners et al., 2011; Meyer et al., 2021). Here, BMdM were incubated with U18666A to demonstrate free cholesterol accumulation or DOPG + U18666A for 24 h to assess whether DOPG could reverse free cholesterol accumulation induced by U18666A. Treated BMdM were stained with Lysoview 633 prior to fixation and filipin staining. U18666A caused an observable increase in distinct puncta with Lysoview 633 and filipin staining compared to control BMdM indicating an accumulation of cholesterol within the lysosomes (Figure 3A). DOPG + U18666A-treated BMdM showed a similar colocalization of Lysoview 633 and Filipin as cholesterol is sequestered within the lysosomes (Figure 3A). Quantification of filipin stained puncta with ImageJ showed that U18666A significantly increased filipin puncta compared to untreated BMdM (Figure 3B). DOPG + U18666A treated cells also showed a significant increase in filipin puncta compared to untreated BMdM; however, they were significantly reduced in number compared to U18666A only treated cells (Figure 3B). DOPG alone produced no demonstratable change in filipin stained puncta, likely due to the considerably low number of observable puncta in control cells (data not shown). An analysis of the Manders Overlap Coefficient (using ImageJ JACoP) showed a significant increase in the amount of Lysoview 633 staining overlapping with filipin staining (Figure 3C) upon U18666A treatment, which was then significantly reduced by DOPG treatment.

DOPG is proposed to reduce lysosomal cholesterol by promoting increased trafficking of cholesterol through the NPC2 protein (McCauliff et al., 2019; Ilnytska et al., 2021a; Ilnytska et al., 2021b). In order to assess that lysosomal cholesterol was lowered, digitonin treatment was used. Digitonin is a cell membrane permeabilizer that specifically interacts with membrane cholesterol (Nishikawa et al., 1984). Therefore, since a change in membrane cholesterol would affect the efficacy of digitonin to permeabilize membranes, we assessed the impact of DOPG treatment on a digitonin titration curve compared to untreated BMdM. Increasing cholesterol content in the membrane corresponds with increased susceptibility to digitonin-induced lysis (Sudji et al., 2015). Detection of lysosomal enzymes in the extracted cytosol can then be used to assess lysosomal membrane integrity (Aits et al., 2015; Jessop et al., 2017).

Digitonin was applied (0–200 μg/mL) for 15 min on ice, then cytosolic extracts were measured for the cytosolic enzyme lactate dehydrogenase (LDH) and the lysosomal enzyme n-acetyl-β-glucosaminidase (NAG). The highest dose of digitonin (200 μg/mL) was selected because it was much higher than the rest of the range and will permeabilize both the plasma membrane and lysosomal membranes in all conditions. DOPG treatment did not change LDH measurements compared to untreated BMdM with the exception of digitonin at 10 μg/mL (Figure 4A). In contrast, NAG values were significantly decreased compared to control BMdM at multiple digitonin concentrations (15–35 μg/mL) (Figure 4B).

The lack of demonstratable difference between LDH values for BMdM with and without DOPG treatment (Figure 4A) suggests that the digitonin extraction is extracting the cytosol to a comparable degree between the two groups due to their similar plasma membrane cholesterol levels. The significant decrease in measured NAG fluorescent intensity between untreated and DOPG treated BMdM points to a significant difference in lysosomal cholesterol content between the two groups, as the lower lysosomal cholesterol of the DOPG treated BMdM would make it less susceptible to digitonin extraction. Thus, these results demonstrate that DOPG treatment had no significant impact on plasma membrane cholesterol but significantly lowered the lysosomal cholesterol content.

The indicated change in susceptibility to digitonin titration upon treatment of DOPG supported the hypothesis that DOPG decreased lysosomal cholesterol consistent with previous reports (Ilnytska et al., 2021a; Ilnytska et al., 2021b). Increased lysosomal cholesterol has been demonstrated to be protective of LMP from a variety of causes (Appelqvist et al., 2011; Reiners et al., 2011; Biswas et al., 2014; Fucho et al., 2014). In this work, DOPG treatment of BMdM promoted cholesterol efflux, which in turn increased silica-induced cell death and IL-1β release. Therefore, we assessed the impact of decreased lysosomal cholesterol with DOPG treatment on LMP following silica exposure. BMdM were pre-treated with and without DOPG 1 h prior to silica application for 24 h. Cytosolic NAG activity was assessed using digitonin in a similar manner as described above. The digitonin dose was optimized by the previous titration to determine a working concentration (15 μg/mL) that selectively permeabilized the plasma membrane while leaving intracellular membranes intact and was compared to a complete lysis with 200 μg/mL for each condition. NAG activity was then expressed as NAG₁₅/NAG₂₀₀. This was done to account for cell death caused by silica, which could cause lower NAG levels in the extracts of silica treated BMdM. Silica exposure significantly increased LMP compared to untreated BMdM (Figure 5). DOPG alone did not impact NAG activity; however, DOPG pre-treatment before silica exposure resulted in a significant increase in NAG activity compared to silica-only BMdM (Figure 5). These results demonstrate that DOPG significantly increased silica-caused LMP.

We have previously demonstrated that crystalline silica can interact with and disrupt lipid membranes, which resulted in changes to lipid order in the membrane. This is likely through preferential interactions with phosphatidylcholine (DOPC) phospholipid headgroups (Pavan et al., 2020; Sydor et al., 2021; Pavan et al., 2022). Because of this our current work utilized 100-nm liposomes composed of DOPC with or without cholesterol that were incubated with crystalline silica for the purpose of detecting silica-induced changes to membrane order. Time-resolved fluorescence anisotropy was used to measure the movement of fluorescent membrane probe Di-4-ANEPPDHQ. The wobble-in-a-cone angle of Di-4-A-NEPPDHQ is reduced when the lipid order of the membrane is increased and prevents the movement of the probe within the membrane. Treatment with silica (200 μg/mL) caused a significant reduction of the cone angle compared to control DOPC liposomes (Figure 6A). In order to assess the effects of cholesterol on mitigating silica induced membrane disruption, DOPC liposomes were prepared with a 4:1 DOPC to cholesterol molar ratio. Addition of cholesterol to the liposomes prevented the decrease in cone angle with 200 μg/mL silica (Figure 6B). Addition of 400 μg/mL silica to the DOPC 4:1 Chol liposome also failed to significantly decrease the cone angle of Di-4-ANEPPDHQ (Figure 6B).

These results demonstrate that silica causes an increase in membrane lipid order as indicated by the reduced Di-4-ANEPPDHQ cone angle (Figure 6A) that is prevented by the addition of cholesterol to the DOPC liposome (Figure 6B).