Lysosomal BK channels facilitate silica-induced inflammation in macrophages.

BACKGROUND: Lysosomal ion channels are proposed therapeutic targets for a number of diseases, including those driven by NLRP3 inflammasome-mediated inflammation. Here, the specific role of the lysosomal big conductance Ca2+-activated K+ (BK) channel was evaluated in a silica model of inflammation in murine macrophages. A specific-inhibitor of BK channel function, paxilline (PAX), and activators NS11021 and NS1619 were utilized to evaluate the role of lysosomal BK channel activity in silica-induced lysosomal membrane permeabilization (LMP) and NLRP3 inflammasome activation resulting in IL-1ß release. METHODS: Murine macrophages were exposed in vitro to crystalline silica following pretreatment with BK channel inhibitors or activators and LMP, cell death, and IL-1ß release were assessed. In addition, the effect of PAX treatment on silica-induced cytosolic K+ decrease was measured. Finally, the effects of BK channel modifiers on lysosomal pH, proteolytic activity, and cholesterol transport were also evaluated. RESULTS: PAX pretreatment significantly attenuated silica-induced cell death and IL-1ß release. PAX caused an increase in lysosomal pH and decrease in lysosomal proteolytic activity. PAX also caused a significant accumulation of lysosomal cholesterol. BK channel activators NS11021 and NS1619 increased silica-induced cell death and IL-1ß release. BK channel activation also caused a decrease in lysosomal pH and increase in lysosomal proteolytic function as well as a decrease in cholesterol accumulation. CONCLUSION: Taken together, these results demonstrate that inhibiting lysosomal BK channel activity with PAX effectively reduced silica-induced cell death and IL-1ß release. Blocking cytosolic K+ entry into the lysosome prevented LMP through the decrease of lysosomal acidification and proteolytic function and increase in lysosomal cholesterol.

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

Lung diseases characterized by type 2 inflammation are reported to occur with a female bias in prevalence/severity in both humans and mice. This includes previous work examining multi-walled carbon nanotube (MWCNT)-induced eosinophilic inflammation, in which a more exaggerated M2a phenotype was observed in female alveolar macrophages (AMs) compared to males. The mechanisms responsible for this sex difference in AM phenotype are still unclear, but estrogen receptor (ER) signaling is a likely contributor. Accordingly, male AMs downregulated ERα expression after MWCNT exposure while female AMs did not. Thus, ER antagonist Fulvestrant was administered prior to MWCNT instillation. In females, Fulvestrant significantly attenuated MWCNT-induced M2a gene expression and eosinophilia without affecting IL-33. In males, Fulvestrant did not affect eosinophil recruitment but reduced IL-33 and M2a genes compared to controls. Regulation of cholesterol efflux and oxysterol synthesis is a potential mechanism through which estrogen promotes the M2a phenotype. Levels of oxysterols 25-OHC and 7α,25-OHC were higher in the airways of MWCNT-exposed males compared to MWCNT-females, which corresponds with the lower IL-1ß production and greater macrophage recruitment previously observed in males. Sex-based changes in cholesterol efflux transporters Abca1 and Abcg1 were also observed after MWCNT exposure with or without Fulvestrant. In vitro culture with estrogen decreased cellular cholesterol and increased the M2a response in female AMs, but did not affect cholesterol content in male AMs and reduced M2a polarization. These results reveal the modulation of (oxy)sterols as a potential mechanism through which estrogen signaling may regulate AM phenotype resulting in sex differences in downstream respiratory inflammation.

Cholesterol content regulates silica-induced lysosomal membrane permeability.

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-1ß release. Conversely, increasing lysosomal and cellular cholesterol with U18666A reduced IL-1ß 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.

Cholesterol-dependent molecular mechanisms contribute to cationic amphiphilic drugs' prevention of silica-induced inflammation.

Silicosis is considered an irreversible chronic inflammatory disease caused by the inhalation of crystalline silica (cSiO2). The cycle of inflammation that drives silicosis and other particle-caused respiratory diseases is mediated by NLRP3 inflammasome activity in macrophages resulting in the release of IL-1ß. Lysosomal membrane permeability (LMP) initiated by inhaled particles is the key regulatory step in leading to NLRP3 activity. In addition to its role in LMP, the lysosome is crucial to cellular cholesterol trafficking. Lysosomal cholesterol has been demonstrated to regulate LMP while cationic amphiphilic drugs (CADs) reduce cholesterol trafficking from lysosomes and promote endolysosomal cholesterol accumulation as seen in Niemann Pick disease. Using a bone marrow derived macrophage (BMdM) model, four CADs were examined for their potential to reduce cSiO2-induced inflammation. Here we found that FDA-approved CAD drugs imipramine, hydroxychloroquine, fluvoxamine, and fluoxetine contributed to reduced LMP and IL-1ß release in cSiO2 treated BMdM. These drugs inhibited lysosomal enzymatic activity of acid sphingomyelinase, decreased lysosomal proteolytic function, and increased lysosomal pH. CADs also demonstrated a significant increase in lysosomal-associated free cholesterol. Increased lysosomal cholesterol was associated with a significant reduction in cSiO2 induced LMP and IL-1ß release. In contrast, reduced lysosomal cholesterol significantly exacerbated cSiO2-induced IL-1ß release and reduced the protective effect of CADs on IL-1ß release following cSiO2 exposure. Taken together, these results suggest that CAD modification of lysosomal cholesterol may be used to reduce LMP and cSiO2-induced inflammation and could prove an effective therapeutic for silicosis and other particle-caused respiratory diseases.

Self-replicating murine ex vivo cultured alveolar macrophages as a model for toxicological studies of particle-induced inflammation.

Alveolar macrophages (AM) are integral to maintaining homeostasis within the lungs following exposure to inhaled particles. However, due to the high animal number requirements for in vitro research with primary AM, there remains a need for validated cell models that replicate alveolar macrophages in form and function to better understand the mechanisms that contribute to particle-induced inflammation and disease. A novel, easily adaptable, culture model that facilitates the continued expansion of murine alveolar macrophages for several months, termed murine ex vivo cultured AM (mexAM) has been recently described. Therefore, the present work evaluated the use of mexAMs as a suitable model for primary AM interactions with nano- and micro-sized particles. mexAM displayed a comparable profile of functional phenotype gene expression as primary AM and similar particle uptake capabilities. The NLRP3 inflammasome-driven IL-1ß inflammatory response to crystalline silica and various nanoparticles was also assessed, as well as the effects of cationic amphiphilic drugs to block particle-induced inflammation. For all endpoints, mexAM showed a comparable response to primary AM. Altogether, the present work supports the use of mexAM as a validated replacement for primary AM cultures thereby reducing animal numbers and serving as an effective model for mechanistic investigation of inflammatory pathways in particle-induced respiratory disease.

Molecular recognition between membrane epitopes and nearly free surface silanols explains silica membranolytic activity.

Inhaled crystalline silica causes inflammatory lung diseases, but the mechanism for its unique activity compared to other oxides remains unclear, preventing the development of potential therapeutics. Here, the molecular recognition mechanism between membrane epitopes and "nearly free silanols" (NFS), a specific subgroup of surface silanols, is identified and proposed as a novel broad explanation for particle toxicity in general. Silica samples having different bulk and surface properties, specifically different amounts of NFS, are tested with a set of membrane systems of decreasing molecular complexity and different charge. The results demonstrate that NFS content is the primary determinant of membrane disruption causing red blood cell lysis and changes in lipid order in zwitterionic, but not in negatively charged liposomes. NFS-rich silica strongly and irreversibly adsorbs zwitterionic self-assembled phospholipid structures. This selective interaction is corroborated by density functional theory and supports the hypothesis that NFS recognize membrane epitopes that exhibit a positive quaternary amino and negative phosphate group. These new findings define a new paradigm for deciphering particle-biomembrane interactions that will support safer design of materials and what types of treatments might interrupt particle-biomembrane interactions.

The role of lysosomal ion channels in lysosome dysfunction.

Lysosomes offer a unique arrangement of degradative, exocytic, and signaling capabilities that make their continued function critical to cellular homeostasis. Lysosomes owe their function to the activity of lysosomal ion channels and transporters, which maintain concentration gradients of H+, K+, Ca2+, Na+, and Cl- across the lysosomal membrane. This review examines the contributions of lysosomal ion channels to lysosome function, showing how ion channel function is integral to degradation and autophagy, maintaining lysosomal membrane potential, controlling Ca2+ signaling, and facilitating exocytosis. Evidence of lysosome dysfunction in a variety of disease pathologies creates a need to understand how lysosomal ion channels contribute to lysosome dysfunction. For example, the loss of function of the TRPML1 Ca2+ lysosome channel in multiple lysosome storage diseases leads to lysosome dysfunction and disease pathogenesis while neurodegenerative diseases are marked by lysosome dysfunction caused by changes in ion channel activity through the TRPML1, TPC, and TMEM175 ion channels. Autoimmune disease is marked by dysregulated autophagy, which is dependent on the function of multiple lysosomal ion channels. Understanding the role of lysosomal ion channel activity in lysosome membrane permeability and NLRP3 inflammasome activation could provide valuable mechanistic insight into NLRP3 inflammasome-mediated diseases. Finally, this review seeks to show that understanding the role of lysosomal ion channels in lysosome dysfunction could give mechanistic insight into the efficacy of certain drug classes, specifically those that target the lysosome, such as cationic amphiphilic drugs.