Reprogramming cholesterol metabolism in macrophages and its role in host defense against cholesterol-dependent cytolysins

Cholesterol is a critical lipid for all mammalian cells, ensuring proper membrane integrity, fluidity, and biochemical function. Accumulating evidence indicates that macrophages rapidly and profoundly reprogram their cholesterol metabolism in response to activation signals to support host defense processes. However, our understanding of the molecular details underlying how and why cholesterol homeostasis is specifically reshaped during immune responses remains less well understood. This review discusses our current knowledge of cellular cholesterol homeostatic machinery and introduces emerging concepts regarding how plasma membrane cholesterol is partitioned into distinct pools. We then discuss how proinflammatory signals can markedly reshape the cholesterol metabolism of macrophages, with a focus on the differences between MyD88-dependent pattern recognition receptors and the interferon signaling pathway. We also discuss recent work investigating the capacity of these proinflammatory signals to selectively reshape plasma membrane cholesterol homeostasis. We examine how these changes in plasma membrane cholesterol metabolism influence sensitivity to a set of microbial pore-forming toxins known as cholesterol-dependent cytolysins that specifically target cholesterol for their effector functions. We also discuss whether lipid metabolic reprogramming can be leveraged for therapy to mitigate tissue damage mediated by cholesterol-dependent cytolysins in necrotizing fasciitis and other related infections. We expect that advancing our understanding of the crosstalk between metabolism and innate immunity will help explain how inflammation underlies metabolic diseases and highlight pathways that could be targeted to normalize metabolic homeostasis in disease states.

Sterols, Oxysterols, and Accessible Cholesterol: Signalling for Homeostasis, in Immunity and During Development

In this article we discuss the concept of accessible plasma membrane cholesterol and its involvement as a signalling molecule. Changes in plasma membrane accessible cholesterol, although only being minor in the context of total cholesterol plasma membrane cholesterol and total cell cholesterol, are a key regulator of overall cellular cholesterol homeostasis by the SREBP pathway. Accessible cholesterol also provides the second messenger between patched 1 and smoothened in the hedgehog signalling pathway important during development, and its depletion may provide a mechanism of resistance to microbial pathogens including SARS-CoV-2. We revise the hypothesis that oxysterols are a signalling form of cholesterol, in this instance as a rapidly acting and paracrine version of accessible cholesterol.

Computational drug repurposing against SARS-CoV-2 reveals plasma membrane cholesterol depletion as key factor of antiviral drug activity

Comparing SARS-CoV-2 infection-induced gene expression signatures to drug treatment-induced gene expression signatures is a promising bioinformatic tool to repurpose existing drugs against SARS-CoV-2. The general hypothesis of signature based drug repurposing is that drugs with inverse similarity to a disease signature can reverse disease phenotype and thus be effective against it. However, in the case of viral infection diseases, like SARS-CoV-2, infected cells also activate adaptive, antiviral pathways, so that the relationship between effective drug and disease signature can be more ambiguous.To address this question, we analysed gene expression data from in vitro SARS-CoV-2 infected cell lines, and gene expression signatures of drugs showing anti-SARS-CoV-2 activity. Our extensive functional genomic analysis showed that both infection and treatment with in vitro effective drugs leads to activation of antiviral pathways like NFkB and JAK-STAT. Based on the similarity - and not inverse similarity - between drug and infection-induced gene expression signatures, we were able to predict the in vitro antiviral activity of drugs. We also identified SREBF1/2, key regulators of lipid metabolising enzymes, as the most activated transcription factors by several in vitro effective antiviral drugs. Using a fluorescently labeled cholesterol sensor, we showed that these drugs decrease the cholesterol levels of plasma-membrane. Supplementing drug-treated cells with cholesterol reversed the in vitro antiviral effect, suggesting the depleting plasma-membrane cholesterol plays a key role in virus inhibitory mechanism.Our results can help to more effectively repurpose approved drugs against SARS-CoV-2, and also highlights key mechanisms behind their antiviral effect.Abstract Figure

High-Fat and Resveratrol Supplemented Diets Modulate Adenosine Receptors in the Cerebral Cortex of C57BL/6J and SAMP8 Mice

Neurodegenerative disorders are devastating diseases in which aging is a major risk factor. High-fat diet (HFD) seems to contribute to cognition deterioration, but the underlying mechanisms are poorly understood. Moreover, resveratrol (RSV) has been reported to counteract the loss of cognition associated with age. Our study aimed to investigate whether the adeno-synergic system and plasma membrane cholesterol are modulated by HFD and RSV in the cerebral cortex of C57BL/6J and SAMP8 mice. Results show that HFD induced increased A1R and A2AR densities in C57BL/6J, whereas this remained unchanged in SAMP8. Higher activity of 5′-Nucleotidase was found as a common effect induced by HFD in both mice strains. Furthermore, the effect of HFD and RSV on A2BR density was different depending on the mouse strain. RSV did not clearly counteract the HFD-induced effects on the adeno-synergic system. Besides, no changes in free-cholesterol levels were detected in the plasma membrane of cerebral cortex in both strains. Taken together, our data suggest a different modulation of adenosine receptors depending on the mouse strain, not related to changes in plasma membrane cholesterol content.

Alteration of Membrane Cholesterol Content Plays a Key Role in Regulation of Cystic Fibrosis Transmembrane Conductance Regulator Channel Activity

Altered cholesterol homeostasis in cystic fibrosis patients has been reported, although controversy remains. As a major membrane lipid component, cholesterol modulates the function of multiple ion channels by complicated mechanisms. However, whether cholesterol directly modulates cystic fibrosis transmembrane conductance regulator (CFTR) channel function remains unknown. To answer this question, we determined the effects of changing plasma membrane cholesterol levels on CFTR channel function utilizing polarized fischer rat thyroid (FRT) cells and primary human bronchial epithelial (HBE) cells. Treatment with methyl-β-cyclodextrin (MβCD) significantly reduced total cholesterol content in FRT cells, which significantly decreased forskolin (FSK)-mediated activation of both wildtype (WT-) and P67L-CFTR. This effect was also seen in HBE cells expressing WT-CFTR. Cholesterol modification by cholesterol oxidase and cholesterol esterase also distinctly affected activation of CFTR by FSK. In addition, alteration of cholesterol increased the potency of VX-770, a clinically used potentiator of CFTR, when both WT- and P67L-CFTR channels were activated at low FSK concentrations; this likely reflects the apparent shift in the sensitivity of WT-CFTR to FSK after alteration of membrane cholesterol. These results demonstrate that changes in the plasma membrane cholesterol level significantly modulate CFTR channel function and consequently may affect sensitivity to clinical therapeutics in CF patients.

Effects of plasma membrane cholesterol content on ultrasound and microbubble mediated sonoporation

Ultrasonically-stimulated microbubbles can increase cell membrane permeability and allow otherwise impermeable molecules to enter the intracellular space of cells; a phenomenon known as

Effects of plasma membrane cholesterol content on ultrasound and microbubble mediated sonoporation

Ultrasonically-stimulated microbubbles can increase cell membrane permeability and allow otherwise impermeable molecules to enter the intracellular space of cells; a phenomenon known as

DHCR24 Knock-Down Induced Tau Hyperphosphorylation at Thr181, Ser199, Thr231, Ser262, Ser396 Epitopes and Inhibition of Autophagy by Overactivation of GSK3β/mTOR Signaling

Accumulating evidences supported that knock-down of DHCR24 is linked to the pathological risk factors of AD, suggesting a potential role of DHCR24 in AD pathogenesis. However, the molecular mechanism link between DHCR24 and tauopathy remains unknown. Here, in order to elucidate the relationship between DHCR24 and tauopathy, we will focus on the effect of DHCR24 on the tau hyperphosphorylation at some toxic sites. In present study, we found that DHCR24 knock-down significantly lead to the hyperphosphorylation of tau sites at Thr181, Ser199, Thr231, Ser262, Ser396. Moreover, DHCR24 knock-down also increase the accumulation of p62 protein, simultaneously decreased the ratio of LC3-II/LC3-I and the number of autophagosome compared to the control groups, suggesting the inhibition of autophagy activity. In contrast, DHCR24 knock-in obviously abolished the effect of DHCR24 knock-down on tau hyperphosphrylation and autophagy. In addition, to elucidate the association between DHCR24 and tauopathy, we further showed that the level of plasma membrane cholesterol, lipid raft-anchored protein caveolin-1, and concomitantly total I class PI3-K (p110α), phospho-Akt (Thr308 and Ser473) were significantly decreased, resulting in the disruption of lipid raft/caveola and inhibition of PI3-K/Akt signaling in silencing DHCR24 SH-SY5Y cells compared to control groups. At the same time, DHCR24 knock-down simultaneously decreased the level of phosphorylated GSK3β at Ser9 (inactive form) and increased the level of phosphorylated mTOR at Ser2448 (active form), leading to overactivation of GSK3β and mTOR signaling. On the contrary, DHCR24 knock-in largely increased the level of membrane cholesterol and caveolin-1, suggesting the enhancement of lipid raft/caveola. And synchronously DHCR24 knock-in also abolished the effect of DHCR24 knock-down on the inhibition of PI3-K/Akt signaling as well as the overactivation of GSK3β and mTOR signaling. Collectively, our data strongly supported DHCR24 knock-down lead to tau hyperphosphorylation and the inhibition of autophagy by a lipid raft-dependent PI3-K/Akt-mediated GSK3β and mTOR signaling. Taking together, our results firstly demonstrated that the decrease of plasma membrane cholesterol mediated by DHCR24 deficiency might contribute to the tauopathy in AD and other tauopathies.

P2X7 Receptors and TMEM16 Channels Form a Hub with Implications for Macropore Formation and Current Facilitation

P2X7 receptors (P2X7) are cationic channels involved in many diseases. They exhibit unique behaviors, such as “macropore” formation, which corresponds to enhanced large molecule cell membrane permeability, and current facilitation, which is caused by prolonged activation. These two phenomena have often been confounded, but thus far no clear mechanisms have been resolved. Here we provide evidence that current facilitation and macropore formation involve functional complexes comprised of P2X7 and TMEM16, a family of Ca2+-activated ion channel/scramblases. We found that current facilitation results in an increase of complex-embedded P2X7 open channel probability, a result mimicked by plasma membrane cholesterol depletion. We further show that macropore formation entails two distinct large molecule permeation components, one of which requires protein complexes featuring TMEM16F subtype, the other likely being direct permeation through the P2X7 pore itself. Such protein complexes can be considered to represent a regulatory hub intimately involved in distinct P2X7 functionalities.