A concerted mechanism involving ACAT and SREBPs by which oxysterols deplete accessible cholesterol to restrict microbial infection
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eLife assessment
This work provides valuable new insights into the mechanisms by which 25-hydroxycholesterol (which is known to be rapidly produced in macrophages and other cells during acute infections) acts to protect cells and animals from microbial infection. The authors provide compelling evidence that the cholesterol-esterifying enzyme acylCoA:cholesterol acyltransferase (ACAT) that is induced by 25-hydroxycholesterol promotes the depletion of an accessible pool of plasma membrane cholesterol, producing anti-microbial effects.
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Abstract
Most of the cholesterol in the plasma membranes (PMs) of animal cells is sequestered through interactions with phospholipids and transmembrane domains of proteins. However, as cholesterol concentration rises above the PM’s sequestration capacity, a new pool of cholesterol, called accessible cholesterol, emerges. The transport of accessible cholesterol between the PM and the endoplasmic reticulum (ER) is critical to maintain cholesterol homeostasis. This pathway has also been implicated in the suppression of both bacterial and viral pathogens by immunomodulatory oxysterols. Here, we describe a mechanism of depletion of accessible cholesterol from PMs by the oxysterol 25-hydroxycholesterol (25HC). We show that 25HC-mediated activation of acyl coenzyme A: cholesterol acyltransferase (ACAT) in the ER creates an imbalance in the equilibrium distribution of accessible cholesterol between the ER and PM. This imbalance triggers the rapid internalization of accessible cholesterol from the PM, and this depletion is sustained for long periods of time through 25HC-mediated suppression of SREBPs and continued activation of ACAT. In support of a physiological role for this mechanism, 25HC failed to suppress Zika virus and human coronavirus infection in ACAT-deficient cells, and Listeria monocytogenes infection in ACAT-deficient cells and mice. We propose that selective depletion of accessible PM cholesterol triggered by ACAT activation and sustained through SREBP suppression underpins the immunological activities of 25HC and a functionally related class of oxysterols.
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Author Response
Reviewer #1 (Public Review):
The data presented throughout are solid, however, some of the structures drawn of the oxysterols in Figure 1 are not chemically correct. 24(S)HC is drawn as 24(R)HC and visa versa, also the oxysterol sulfate should have a bond between C-3 and the O of OSO3H. It would also help the reader if the vehicle for oxysterol additions was clarified.
We thank the reviewer for pointing out these embarrassing errors! All structures have been corrected. The vehicle for oxysterol (ethanol) is indicated in the Methods.
The data presented in Figures 2 and 3 show that inhibition of SREBP processing by 25HC is important for the long-term maintenance of depletion of plasma membrane accessible cholesterol, but I wonder if activation of LXR may also be important here. I appreciate that the data in Figure 2 …
Author Response
Reviewer #1 (Public Review):
The data presented throughout are solid, however, some of the structures drawn of the oxysterols in Figure 1 are not chemically correct. 24(S)HC is drawn as 24(R)HC and visa versa, also the oxysterol sulfate should have a bond between C-3 and the O of OSO3H. It would also help the reader if the vehicle for oxysterol additions was clarified.
We thank the reviewer for pointing out these embarrassing errors! All structures have been corrected. The vehicle for oxysterol (ethanol) is indicated in the Methods.
The data presented in Figures 2 and 3 show that inhibition of SREBP processing by 25HC is important for the long-term maintenance of depletion of plasma membrane accessible cholesterol, but I wonder if activation of LXR may also be important here. I appreciate that the data in Figure 2 points against LXR being involved in the rapid depletion of accessible cholesterol in HEK293 cells, but perhaps it is important for the long-term depletion of accessible cholesterol. Could there be some cell type specificity here?
We agree with the reviewer that 25HC’s effects on multiple signaling pathways complicates mechanistic interpretations. Our studies suggest that ACAT activity is absolutely required for the rapid depletion of accessible PM cholesterol and LXRs play a minor role at this stage. The long-term contributions could very well arise from any of the other 25HC targets, including LXRs, and the relative contributions of ACAT, SREBPs, and LXRs could vary between cell types.
Something that always concerns me when the antimicrobial activity of 25HC is discussed is the fact that 25HC is usually a minor side-chain oxysterol compared to 24(S)HC and 27HC (and 22(R)HC in steroidogenic tissue), except for a short time after infection. Perhaps any long-term antimicrobial activity, and diminishment of accessible cholesterol, results from these other side-chain oxysterols. This may be worthy of some additional discussion.
We agree with the reviewer that we cannot rule out the contribution of other oxysterols to long-term antimicrobial activity. While we have kept our focus on 25HC in this study, we point out in the Discussion that other ACAT-activating oxysterols such as 20(R)HC, 24(R)HC, 24(S)HC, and 27HC, all of which diminish accessible cholesterol, could also have long-term immunological effects.
Reviewer #2 (Public Review):
The paper describes a fairly complete set of experiments describing a mechanism by which 4-hour treatment with 25HC can provide reductions in plasma membrane cholesterol for up to 22 hours. The basic finding is that 25HC depletes the ER of cholesterol by stimulating esterification and that SREBP activation is also inhibited. This effect is associated with the slow loss of 25HC from the cells.
The paper describes detailed studies of the long-lasting effects of a 4-hour exposure to 25HC on the loss of plasma membrane cholesterol. The paper characterizes the effects on SREBP processing to account for this. The possible long-lasting effects of ACAT stimulation were not investigated but may play an equal role.
The paper presents data that the effects on plasma membrane cholesterol can account for the inhibitory effects on some bacterial toxins and viruses.
We thank the reviewer for their positive comments.
Reviewer #3 (Public Review):
The paper uses multiple approaches in cultured cells to show that the rapid depletion of accessible plasma membrane cholesterol by 25-hydroxycholesterol is mediated by the activation of the cholesterol-esterifying enzyme acylCoA:cholesterol acyltransferase (ACAT). They carefully consider and exclude other potential mechanisms that could explain the effects of 25-OH cholesterol on the plasma membrane cholesterol pool, such as decreased cholesterol biosynthesis or activation of LXR transcription factors. Cell lines with mutations in ACAT and in cholesterol homeostatic factors are used in an ingenious fashion to support the role of ACAT and exclude these other mechanisms. The in vivo relevance of accessible membrane cholesterol and ACAT is then demonstrated for toxic cytolysin binding to cells, Listeria infection in vivo, and Zika and Coronavirus infections of cultured liver cells. Overall, the evidence is exceptional that ACAT modulates the plasma membrane accessible cholesterol pool as a strategy of the host to protect against various infectious agents. The discussion of the paper could be broadened to include other mechanisms that are known concerning the role of 25-OH cholesterol in infectious processes and the body's responses.
We thank the reviewer for their positive assessment.
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eLife assessment
This work provides valuable new insights into the mechanisms by which 25-hydroxycholesterol (which is known to be rapidly produced in macrophages and other cells during acute infections) acts to protect cells and animals from microbial infection. The authors provide compelling evidence that the cholesterol-esterifying enzyme acylCoA:cholesterol acyltransferase (ACAT) that is induced by 25-hydroxycholesterol promotes the depletion of an accessible pool of plasma membrane cholesterol, producing anti-microbial effects.
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Reviewer #1 (Public Review):
In this manuscript, the authors propose a model to explain how oxysterols provide protection against bacteria and viruses by modulating a cell's "accessible cholesterol". The paper concentrates on one particular oxysterol, 25-hydroxycholesterol (25HC) which provides an example of a wider group of side-chain oxysterols. Previous studies have shown that 25HC can protect cells against microbial infection and have suggested that this is achieved by depleting "un-sequestered" or "accessible" cholesterol from the plasma membrane. Here, Heisler et al provide convincing evidence that this is achieved initially by activation of the enzyme acyl coenzyme A: cholesterol acyltransferase (ACAT also known as Sterol O-acyltransferase, SOAT, not to be confused with acetyl-coenzyme A acetyltransferase) to rapidly reduce …
Reviewer #1 (Public Review):
In this manuscript, the authors propose a model to explain how oxysterols provide protection against bacteria and viruses by modulating a cell's "accessible cholesterol". The paper concentrates on one particular oxysterol, 25-hydroxycholesterol (25HC) which provides an example of a wider group of side-chain oxysterols. Previous studies have shown that 25HC can protect cells against microbial infection and have suggested that this is achieved by depleting "un-sequestered" or "accessible" cholesterol from the plasma membrane. Here, Heisler et al provide convincing evidence that this is achieved initially by activation of the enzyme acyl coenzyme A: cholesterol acyltransferase (ACAT also known as Sterol O-acyltransferase, SOAT, not to be confused with acetyl-coenzyme A acetyltransferase) to rapidly reduce plasma membrane accessible cholesterol by conversion of cholesterol to its cholesteryl ester. This is followed by 25HC-induced inhibition of the processing of SREBPs, the master transcription factors for genes of the cholesterol biosynthesis pathway and also the LDL receptor, to maintain cholesterol depletion.
The data presented throughout are solid, however, some of the structures drawn of the oxysterols in Figure 1 are not chemically correct. 24(S)HC is drawn as 24(R)HC and visa versa, also the oxysterol sulfate should have a bond between C-3 and the O of OSO3H. It would also help the reader if the vehicle for oxysterol additions was clarified.
The data presented in Figures 2 and 3 show that inhibition of SREBP processing by 25HC is important for the long-term maintenance of depletion of plasma membrane accessible cholesterol, but I wonder if activation of LXR may also be important here. I appreciate that the data in Figure 2 points against LXR being involved in the rapid depletion of accessible cholesterol in HEK293 cells, but perhaps it is important for the long-term depletion of accessible cholesterol. Could there be some cell type specificity here?
Something that always concerns me when the antimicrobial activity of 25HC is discussed is the fact that 25HC is usually a minor side-chain oxysterol compared to 24(S)HC and 27HC (and 22(R)HC in steroidogenic tissue), except for a short time after infection. Perhaps any long-term antimicrobial activity, and diminishment of accessible cholesterol, results from these other side-chain oxysterols. This may be worthy of some additional discussion.In summary, the authors present a convincing model for the depletion of accessible cholesterol by oxysterols and their involvement in antimicrobial activities.
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Reviewer #2 (Public Review):
The paper describes a fairly complete set of experiments describing a mechanism by which 4-hour treatment with 25HC can provide reductions in plasma membrane cholesterol for up to 22 hours. The basic finding is that 25HC depletes the ER of cholesterol by stimulating esterification and that SREBP activation is also inhibited. This effect is associated with the slow loss of 25HC from the cells.
The paper describes detailed studies of the long-lasting effects of a 4-hour exposure to 25HC on the loss of plasma membrane cholesterol. The paper characterizes the effects on SREBP processing to account for this. The possible long-lasting effects of ACAT stimulation were not investigated but may play an equal role.
The paper presents data that the effects on plasma membrane cholesterol can account for the inhibitory …
Reviewer #2 (Public Review):
The paper describes a fairly complete set of experiments describing a mechanism by which 4-hour treatment with 25HC can provide reductions in plasma membrane cholesterol for up to 22 hours. The basic finding is that 25HC depletes the ER of cholesterol by stimulating esterification and that SREBP activation is also inhibited. This effect is associated with the slow loss of 25HC from the cells.
The paper describes detailed studies of the long-lasting effects of a 4-hour exposure to 25HC on the loss of plasma membrane cholesterol. The paper characterizes the effects on SREBP processing to account for this. The possible long-lasting effects of ACAT stimulation were not investigated but may play an equal role.
The paper presents data that the effects on plasma membrane cholesterol can account for the inhibitory effects on some bacterial toxins and viruses.
-
Reviewer #3 (Public Review):
The paper uses multiple approaches in cultured cells to show that the rapid depletion of accessible plasma membrane cholesterol by 25-hydroxycholesterol is mediated by the activation of the cholesterol-esterifying enzyme acylCoA:cholesterol acyltransferase (ACAT). They carefully consider and exclude other potential mechanisms that could explain the effects of 25-OH cholesterol on the plasma membrane cholesterol pool, such as decreased cholesterol biosynthesis or activation of LXR transcription factors. Cell lines with mutations in ACAT and in cholesterol homeostatic factors are used in an ingenious fashion to support the role of ACAT and exclude these other mechanisms. The in vivo relevance of accessible membrane cholesterol and ACAT is then demonstrated for toxic cytolysin binding to cells, Listeria …
Reviewer #3 (Public Review):
The paper uses multiple approaches in cultured cells to show that the rapid depletion of accessible plasma membrane cholesterol by 25-hydroxycholesterol is mediated by the activation of the cholesterol-esterifying enzyme acylCoA:cholesterol acyltransferase (ACAT). They carefully consider and exclude other potential mechanisms that could explain the effects of 25-OH cholesterol on the plasma membrane cholesterol pool, such as decreased cholesterol biosynthesis or activation of LXR transcription factors. Cell lines with mutations in ACAT and in cholesterol homeostatic factors are used in an ingenious fashion to support the role of ACAT and exclude these other mechanisms. The in vivo relevance of accessible membrane cholesterol and ACAT is then demonstrated for toxic cytolysin binding to cells, Listeria infection in vivo, and Zika and Coronavirus infections of cultured liver cells. Overall, the evidence is exceptional that ACAT modulates the plasma membrane accessible cholesterol pool as a strategy of the host to protect against various infectious agents. The discussion of the paper could be broadened to include other mechanisms that are known concerning the role of 25-OH cholesterol in infectious processes and the body's responses.
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