A Novel Role for Cholesterol: Regulating Inflammatory Signalling in Macrophages

By Kelly Macdonald-Ramsahai 

Acute inflammation is a physiological local and systemic response of the innate immune system to injury and infection, in which myeloid cells of the hematopoietic system – including monocytes, macrophages, neutrophils, and dendritic cells (DCs) – are activated in infected tissue upon contact with pro-inflammatory stimuli, inducing the secretion of pro-inflammatory cytokines and chemokines (Rogero and Calder, 2018).

Multi-domain inflammasome complexes, which are molecular platforms for pro-caspase-1 auto-cleavage and activation, are responsible for inflammatory responses. Active hetero-tetrameric caspase-1 is a critical molecule during the inflammatory response, through processing hydrolytic cleavage and maturation of pro-inflammatory cytokines interleukin (IL)-1β and IL-18 in macrophages. These are subsequently secreted from macrophages to induce inflammation and immune cell recruitment towards the local site of infection or injury. A further substrate of caspase-1 is Gasdermin-D (GSDMD). The 31kDa N-terminal fragment (GSDMD-N) cleavage product assembles into a membrane-associated pore inducing cell lysis and rupture to facilitate pyroptosis – a microbial and inflammatory-associated form of Programmed Cell Death (PCD) (He, Hara and Núñez, 2016). 

Whilst acute inflammation is essential for survival, chronic (sustained) inflammation causes deregulated immune responses and local tissue destruction, contributing to the development of disease. Resultantly, sustained inflammation is a hallmark of multiple pathological subtypes, including metabolic, auto-immune, cardiovascular diseases (CVDs) and tumours.

To avoid chronic inflammation, activation of the NLRP3 inflammasome is regulated at a transcription and maturation level (Two-Step Checkpoint Hypothesis). During NLRP3 ‘priming’, Toll-Like Receptor (TLR) 4, which is a host membrane-bound Pathogen Recognition Receptor (PRR), is activated upon contact with its Pathogen Associated Molecular Pattern (PAMP) agonist, Lipopolysaccharide (LPS). This induces MyD88-dependent MAPK/NF-κB pathway activation, resulting in up-regulated pro-IL-1β and Nlrp3 transcription. During an ‘activation’ step, host-derived Damage Associated Molecular Patterns (DAMPs) agonists, which arise during cellular stress, injury or infection, (such as extracellular ATP (eATP)), activate the Purinergic 2X7 Receptor (P2X7R). In turn, P2X7R agonist stimulation induces inotropic changes which stimulate the assembly of the multi-protein NLRP3 inflammasome complex (He, Hara and Núñez, 2016).

Various activators of the NLRP3 inflammasome have been characterized, including inotropic changes, lysosomal rupture, and an increase in reactive oxygen species (Yang et al, 2018). More recently, there is a growing appreciation for the direct and indirect influence of lipid species and metabolic pathways on immune responses, including how cells respond to infectious and ‘sterile’ stimuli (Yan et al, 2019). Cholesterol is an essential lipid sterol, which is diet-derived and biosynthesized from animal cells. The sterol regulates a plethora of cellular functions, including serving as a precursor of hormones (Vitamin D), bile salts, and steroid hormones. However, its primary function involves regulating the dynamics, fluidity and permeability, and cellular events at biological membranes such as the plasma membrane (PM) and of sub-cellular organelles including the endoplasmic reticulum (ER) and lysosomes. Loss-of-function (LOF) mutations in the cholesterol-related molecules manifest into metabolic disorders, such as LOF mutations in the cholesterol efflux transporter ATP-Binding Cassette (ABC) A1 (ABCA1) associates with Tangiers Disease, whilst Niemann-Pick Intracellular Cholesterol Transporter 1 (NPC1) deficiency results in Niemann-Pick Disease (NPD) due to aberrant intracellular cholesterol trafficking between the lysosome and ER (Tarling, Vallim, Edwards, 2013; Platt, Boland, & Van Der Spoel, 2012). In concordance with this, lipid metabolism disorders often display chronic inflammation, suggesting metabolic regulation of the inflammasome is likely. Additionally, perturbation of cholesterol pathways are a common manifestation of other subsets of inflammatory-related diseases including auto-immune and cardiovascular, as well as tumours. 

Studies have demonstrated the expression of cholesterol biosynthesis-related genes in macrophages are modulated upon exposure to pro-inflammatory stimuli, such as LPS. For example, Sterol Regulatory Element Binding Proteins 2 (SREBP2) and 3-Hydroxy-3-methylglutaryl-CoA reductase (HMGCR) expression are significantly up-regulated in LPS-stimulated human HCC, HepG2 and Huh7 cells compared to control cells. Conversely, Proprotein onvertase subtilisin/kexin 9 (PCSK9), which has a major regulatory role in Low-Density Lipoprotein Receptor (LDL-R) expression, is reduced in LPS-primed HepG2 and Huh7 cells. Additionally, LPS-primed THP-1 macrophages express reduced gene and protein SREBP-Cleavage Activating Protein (SCAP) expression. SCAP binds SREBPs to facilitate their export to the Golgi apparatus from the ER in cholesterol-depleted cells to initiate cholesterol biosynthesis, reflecting its essential role in cholesterol homeostasis (He et al, 2017). Cholesterol influx, efflux, and intracellular transport programs rely on the function of transporters. The expression of LDLR, which facilitates cholesterol influx, is significantly up-regulated in human HCC cells, HepG2 and Huh7 exposed to LPS compared to control cells (Li et al, 2013). Additionally, ABCA1 and ABCG1, which facilitate reverse cholesterol export, are down-regulated in macrophages upon LPS exposure (Lai et al, 2016). These observations are indicative of a relationship between cholesterol metabolism and immune signalling, however, whether these molecules regulate inflammasome-mediated caspase-1 activation and IL-1b secretion remain to be explored.

Cholesterol localized to the PM is essential for NLRP3 activation, specifically at the ‘priming’ stage. Lipid rafts are small, dynamic micro-domains (10-200 nm) present in the extracellular and cytoplasmic leaflet of the PM, dominantly enriched with cholesterol and sphingolipids. Lipid rafts function as molecular platforms within the PM, facilitating its organization and functionalities including receptor-mediated signal transduction, protein recruitment, membrane trafficking events, and host-pathogen interactions. Cholesterol-rich lipid rafts harbor the glycophosphatidylinositol (GPI)-anchored protein Cluster of Differentiation 14 (CD14), which is a co-receptor for TLR4. During LPS stimulation, the lipid A moiety of LPS complexes with NH2 terminal of LPS-binding protein (LBP), a 60kDa glycoprotein in lipid rafts. The COOH-terminal of LBP binds hydrophobic regions within its N-terminal cleft of GPI-anchored CD14. Overall, LPS stimulation in macrophages induces colocalization of a multi-receptor PRR complex containing TLR4, CD14, and MD-2 specifically within lipid rafts to induce downstream NLRP3 priming and pro-inflammatory signalling. Additionally, through harboring CD14, lipid rafts enable the recruitment of downstream adaptor proteins to facilitate NF-kB signalling (Â Katagiri, Kiyokawa, Fujimoto, 2001). The requirement of cholesterol-rich lipid micro-domains for facilitating LPS-TLR4 interactions during inflammasome priming and downstream inflammatory signalling have been demonstrated in cholesterol depletion studies: Methyl-β-cyclodextrin and nystatin, which sequester cholesterol, blunt NF-kB signalling in LPS-induced macrophages due to defective lipid-raft formation (Varshney, Yadav and Saini, 2016).

Additionally, the administration of cholesterol crystals to murine immortalized bone marrow-derived macrophages (iBMDMs) increases pro-inflammatory cytokine secretion, suggesting cholesterol positively regulates NLRP3 inflammasome activation. Cholesterol crystals, similar to other large particulate activators such as silica and album, cause aggravated phagocytosis and lysosomal rupture which release cathepsin-B into the cytosol. Cathepsin-B is known to directly interact with NLRP3 protein to stimulate inflammasome assembly. These observations are significant, as cholesterol crystals are observed during early Atherosclerosis (AS) as a result of an increase in dietary cholesterol – suggesting aggravated phagocytosis of cholesterol crystals may be a primary inflammatory stimulus during AS onset (Anand, 2020)

Besides the roles of cholesterol in PM-localised lipid rafts and cholesterol crystals in regulating immune signalling pathways, homeostatic cholesterol metabolism and trafficking pathways appear to have a more complex role. A previous study demonstrated pharmacological inhibition and CRISPR/Cas9 mediated knock-out (KO) of NCP1, which facilitates lysosome-to-ER cholesterol transport, in iBMDMs reduced nlrp3, tnf-a and il-18 transcription compared to wild-type (WT) cells. At the protein level, reduced caspase-1 activation, secretion of pro-inflammatory cytokines, and GSDMD-N were observed indicating a decrease in inflammation and pyroptosis as a result of defective inflammasome activation. Overall, the findings indicate sterol tracking into the ER from lysosomes, via the intracellular cholesterol transporter NCP1, is essential for NLRP3 inflammasome activation. Sequestration of ER cholesterol blunted caspase-1 activation, indicating the ER-cholesterol pool is essential for NLRP3 inflammasome activation (De La Roche et al, 2018). 

In conclusion, cholesterol is an essential component of viable animal cells. In addition to its diverse cellular roles, mounting evidence suggests cholesterol influences immune signalling and NLRP3 inflammasome activation. Whilst the roles of cholesterol crystals and PM-localised lipid rafts in regulating NLRP3 inflammasome activation are well defined, the influences of many homeostatic intracellular cholesterol trafficking events remain to be resolved. Their elucidation is important to provide novel insights in inflammatory-related pathologies where aberrant lipid metabolism is observed. Additionally, recognition of these processes will provide novel avenues for their intervention for therapeutic benefit.

References:

Anand, PK., 2020. Lipids, inflammasomes, metabolism, and disease. Immunological Reviews, 297, pp. 108–122.

 Katagiri Y, Kiyokawa N, Fujimoto J.,  2001. A Role for Lipid Rafts in Immune Cell Signaling. Microbiology and Immunology, 45(1), pp.1-8

De La Roche M, Hamilton C, Mortensen R, Jeyaprakash A, Ghosh S, Anand P.,  2018. Trafficking of cholesterol to the ER is required for NLRP3 inflammasome activation. Journal of Cell Biology, 217(10) pp. 3560-3576

He M, Zhang W, Dong Y, Wang L, Fang T, Tang W et al., 2017. Pro-inflammation NF-κB signaling triggers a positive feedback via enhancing cholesterol accumulation in liver cancer cells. Journal of Experimental & Clinical Cancer Research, 36(1).

He, Y., Hara, H. and Núñez, G., 2016. Mechanism and Regulation of NLRP3 Inflammasome Activation. Trends in Biochemical Sciences, 41(12), pp.1012-1021.

Lai L, Azzam K, Lin W, Rai P, Lowe J, Gabor K et al., 2016. MicroRNA-33 Regulates the Innate Immune Response via ATP Binding Cassette Transporter-mediated Remodeling of Membrane Microdomains. Journal of Biological Chemistry.291(37), pp.19651-19660.

Li L, Varghese Z, Moorhead J, Lee C, Chen J, Ruan X., 2013. Cross-talk between TLR4-MyD88-NF-κB and SCAP-SREBP2 pathways mediates macrophage foam cell formation. American Journal of Physiology-Heart and Circulatory Physiology.;304(6), pp. 874-884.

Platt, F. M., Boland, B. & Van Der Spoel, A. C., 2012 Lysosomal storage disorders: The cellular impact of lysosomal dysfunction. J. Cell Biol, 199 (5), pp.723–734.

Rogero M, Calder P., 2018. Obesity, Inflammation, Toll-Like Receptor 4 and Fatty Acids. Nutrients,10(4) pp. 432.

Tarling E, Vallim T, Edwards P., 2013. Role of ABC transporters in lipid transport and human disease. Trends in Endocrinology & Metabolism, 24(7), pp. 342-350.

Varshney P, Yadav V, Saini N., 2016. Lipid rafts in immune signalling: current progress and future perspective. Immunology, 149(1), pp. 13-24.

Yang Q, Liu R, Yu Q, Bi Y, Liu G.,2019. Metabolic regulation of inflammasomes in inflammation. Immunology,157(2), pp. 95-109.

Yang Y, Wang H, Kouadir M, Song H, Shi F., 2019. Recent advances in the mechanisms of NLRP3 inflammasome activation and its inhibitors. Cell Death & Disease,10(2).

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