Cholesterol content regulates silica-induced lysosomal membrane permeability

Abstract

Inhalation of crystalline silica has been well documented to cause pulmonary inflammation and lung disease such as silicosis. Respirable silica particles deposit in the lungs and are phagocytosed by alveolar macrophages. Subsequently, phagocytosed silica remains undegraded within lysosomes causing lysosomal damage known as phagolysosomal membrane permeability (LMP). LMP can trigger the assembly of the NLRP3 inflammasome resulting in release of inflammatory cytokines that contribute to disease. In order to better understand the mechanisms of LMP this study used murine bone marrow derived macrophages (BMdM) as a cellular model to investigate the mechanism of silica-induced LMP. Reduction of lysosomal cholesterol in bone marrow derived macrophages with 18:1 phosphatidylglycerol (DOPG) liposome treatment increased silica-induced LMP and IL-1β release. Conversely, increasing lysosomal and cellular cholesterol with U18666A reduced IL-1β release. Co-treatment of bone marrow derived macrophages with 18:1 phosphatidylglycerol and U18666A resulted in a significant reduction of the effects of U18666A on lysosomal cholesterol. Phosphatidylcholine 100-nm liposome model systems were used to examine the effects of silica particles on lipid membrane order. Time-resolved fluorescence anisotropy of the membrane probe, Di-4-ANEPPDHQ, was used to determine changes to membrane order. Silica increased lipid order that was attenuated by inclusion of cholesterol in the phosphatidylcholine liposomes. These results demonstrate that increased cholesterol can attenuate silica-induced membrane changes in liposomes and cell models, while decreasing cholesterol exacerbates silica-induced membrane changes. Selective manipulation of lysosomal cholesterol may be a way of attenuating lysosomal disruption and preventing silica-induced chronic inflammatory disease progression.

Estrogen contributes to sex differences in M2a macrophages during multi‐walled carbon nanotube‐induced respiratory inflammation

“…Therefore, the increased 25‐OHC in male airways (Figure 4B) and resulting inhibition of NLRP3/IL‐1β would be protective against macrophage‐mediated MWCNT lung inflammation. Likewise, increased cholesterol content in lysosomal membranes is proposed to be protective against LMP 51,52 . Therefore, lower levels of cholesterol in the lysosomes of female AMs, as mediated by E 2 (Figure 6A), would increase susceptibility to LMP and downstream inflammation.…”

“…Likewise, increased cholesterol content in lysosomal membranes is proposed to be protective against LMP. 51,52 Therefore, lower levels of cholesterol in the lysosomes of female AMs, as mediated by E 2 (Figure 6A), would increase susceptibility to LMP and downstream inflammation. Both of these ideas are supported by the greater IL-1β production previously observed in AMs from MWCNT-females.…”

“…Bone marrow-derived macrophages (BMDM) were made from male and female WT mice, and age-matched male SR-BI mice, as previously described by our laboratory. 51,52 On BMDM culture day 1, BM cells were collected by flushing the tibia and femur bones with complete media (RPMI, 10% FBS, 1% Penicillin/Streptomycin, 1% Sodium Pyruvate, 0.1% βmercaptoethanol) using a 1/2″ long 27-gauge needle on a 5-10 mL syringe. All BM cells were incubated overnight (37°C; 3.0 × 10 7 cells/20 mL complete media in a T75 flask) to eliminate stromal cells by adherence.…”

Imipramine Treatment Alters Sphingomyelin, Cholesterol, and Glycerophospholipid Metabolism in Isolated Macrophage Lysosomes

“…It was concluded that the imipramine-induced increase in lysosomal cholesterol stabilized the lysosome membrane, preventing LMP and subsequent inflammation. This conclusion was supported by multiple studies, demonstrating that increases in lysosomal membrane cholesterol induced biophysical membrane changes imparting resistance to LMP [17][18][19]. Further support came from an in vivo study in which imipramine pre-and post-treatment blocked crystalline silicainduced inflammation and lung pathology in C57BL/6 mice [20].…”

“…LysoTracker Red DND-99 (LTR) (cat # L7528, Thermo Fisher Scientific) and the Nacetyl-β-d-glucosaminidase (NAG) enzyme activity were used to validate the lysosome integrity and functionality. The enzyme NAG has been used previously in multiple studies as a marker for lysosome membrane permeabilization [16,18,24]. In this study, the NAG activity in the control lysosomes relative to the fully lysed lysosomes was used as an indicator of the lysosome integrity.…”