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Oxysterols provide innate immunity to bacterial infection by mobilizing cell surface accessible cholesterol

Nature Microbiology volume 5, pages 929–942 (2020)Cite this article

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Abstract

Cholesterol 25-hydroxylase (CH25H) is an interferon-stimulated gene that converts cholesterol to the oxysterol 25-hydroxycholesterol (25HC). Circulating 25HC modulates essential immunological processes including antiviral immunity, inflammasome activation and antibody class switching; and dysregulation of CH25H may contribute to chronic inflammatory disease and cancer. Although 25HC is a potent regulator of cholesterol storage, uptake, efflux and biosynthesis, how these metabolic activities reprogram the immunological state of target cells remains poorly understood. Here, we used recently designed toxin-based biosensors that discriminate between distinct pools of plasma membrane cholesterol to elucidate how 25HC prevents Listeria monocytogenes from traversing the plasma membrane of infected host cells. The 25HC-mediated activation of acyl-CoA:cholesterol acyltransferase (ACAT) triggered rapid internalization of a biochemically defined fraction of cholesterol, termed ‘accessible’ cholesterol, from the plasma membrane while having little effect on cholesterol in complexes with sphingomyelin. We show that evolutionarily distinct bacterial species, L. monocytogenes and Shigella flexneri, exploit the accessible pool of cholesterol for infection and that acute mobilization of this pool by oxysterols confers immunity to these pathogens. The significance of this signal-mediated membrane remodelling pathway probably extends beyond host defence systems, as several other biologically active oxysterols also mobilize accessible cholesterol through an ACAT-dependent mechanism.

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Fig. 1: IFN-γ-activated BMDMs secrete an antibacterial factor.
Fig. 2: Functional cDNA screen of γ-ISGs identifies CH25H as an inhibitor of L. monocytogenes infection.
Fig. 3: 25HC inhibits L. monocytogenes infection in local tissue environments.
Fig. 4: 25HC restricts L. monocytogenes cell-to-cell dissemination.
Fig. 5: 25HC reorganizes PM cholesterol.
Fig. 6: Side-chain oxysterols mobilize accessible cholesterol through a concerted mechanism.

Data availability

Data collected during this study is included in the Source Data and Supplementary Information. The data that support the findings of this study are available from the corresponding author on request.

Code availability

No new code was used to analyse the findings in this study.

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Acknowledgements

We thank D. Gammon and E. Rex for assistance with the live-cell imaging, D. Vazquez for assistance with protein purification, and R. Debose-Boyd, D. W. Russell, J. Goldstein and M. Brown for reagents and helpful discussions. We also thank the members of the Alto, Schoggins and Radhakrishnan laboratories for their helpful discussions. This research was supported by grants from the National Institutes of Health (grant nos. AI083359 to N.M.A., AI117922 to J.W.S. and HL20948 to A.R.), Welch Foundation (grant nos. I-1731 to N.M.A. and I-1793 to A.R.), and grants to N.M.A. from the Burroughs Welcome (grant no. 1011019) and the Howard Hughes Medical Institute and Simons Foundation Faculty Scholars Program (grant no. 55108499).

Author information

Author notes

  1. These authors contributed equally: Michael E. Abrams, Kristen A. Johnson.

Authors and Affiliations

  1. Department of Microbiology, University of Texas Southwestern Medical Center, Dallas, TX, USA

    Michael E. Abrams, Sofya S. Perelman, Li-shu Zhang, Katrina B. Mar, John W. Schoggins & Neal M. Alto

  2. Department of Molecular Genetics, University of Texas Southwestern Medical Center, Dallas, TX, USA

    Kristen A. Johnson, Shreya Endapally, Bonne M. Thompson, Jeffrey G. McDonald & Arun Radhakrishnan

  3. Department of Microbiology, New York University School of Medicine, NY, NY, USA

    Sofya S. Perelman

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Contributions

M.E.A., K.A.J., A.R. and N.M.A conceived and designed the study and wrote the manuscript with editorial input from all authors. M.E.A., N.M.A. and J.W.S. designed the γ-ISG screening platform. M.E.A. performed and analysed all of the bacterial infection experiments and most cell-based assays with assistance from S.S.P. K.A.J. and S.E. generated the biochemical reagents, purified recombinant ALOD4 and OlyA, and designed and performed all of the experiments with these toxin sensors. M.E.A. and L.Z. carried out the mouse infections and collected serum samples. K.B.M. and M.E.A. generated the BMDMs. The oxysterol measurements were carried out by B.M.T. and J.G.M.

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Correspondence to Arun Radhakrishnan or Neal M. Alto.

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Extended data

Extended Data Fig. 1 Comparison between flow cytometry and CFUs for L. monocytogenes infection.

a, Schematic of the L. monocytogenes life-cycle (left) and its replication and intercellular dissemination initiated from a low dose of bacterial infection. HEK293A cells were infected with GFP-expressing L. monocytogenes (MOI=1) so that only a small percentage of the host cell monolayer (<1%) are initially infected. Cell-to-cell spread of L. monocytogenes results in robust infection of the monolayer over time. b, Representative flow cytometry plots of L. monocytogenes (GFP) infection of HEK293A cells at the indicated time points. After 90 minutes of infection, the host cell monolayers were washed and incubated with gentamicin to remove and kill extracellular bacteria. These studies were repeated independently four times with similar results. c, Direct comparison between gentamicin protection assays assessed by flow cytometry (as above) or Colony forming Units (CFUs). Samples were harvested for analysis at the indicated time points after infection. Graph showing the percent of infected cells determined by flow cytometry (y-axis, left) were directly compared to CFUs recovered (y-axis, right). Mean values from 4 independent experiments are plotted, and error bars show s.d. We concluded that flow cytometry is an accurate method of enumerating bacterial burden in host cells.

Source data

Extended Data Fig. 2 CH25H inhibits L. monocytogenes through 25HC production.

a, Oxysterol measurements in media collected from SDFs transduced with lentivirus encoding Fluc, CH25H, or a catalytically inactive CH25H mutant with the following mutations: H242H243/Q242Q243. Following 48 hours transduction, the concentration of oxysterols secreted into the media was measured by mass spectrometry. Bars represent mean values. Error bars show s.d. from two independent experiments. See Methods for oxysterol nomenclature. b, To determine if CH25H catalytic activity is necessary for its antibacterial function, HEK293A cells were transduced with lentivirus as in (a), infected with GFP-expressing L. monocytogenes (MOI=10) for 6 hours, and analysed by flow cytometry. Bars represent mean values. Error bars show s.d. from three independent experiments and statistical significance was determined by one-way ANOVA compared to Fluc with Dunnet’s correction. c, Bar graph showing the total L. monocytogenes CFUs recovered from gentamicin protection assays performed on the indicated cell lines treated with vehicle (EtOH) or 25HC (5 μM). The specified cell lines were set up, treated, and infected as in Fig. 3d, and CFU enumerated by lysing cells and plating serial dilutions. Bars represent mean values. Error bars show s.d. from three or four independent experiments as indicated and statistical significance was determined by student’s unpaired t-test (two-tailed).

Source data

Extended Data Fig. 3 25HC does not directly affect bacterial infectivity or host viability.

25HC could inhibit L. monocytogenes infection through different mechanisms. For example, it may (1) directly reduce bacterial viability, (2) inhibit the expression or function of bacterial virulence factors, (3) induce host cell death, or (4) regulate host cellular processes that limit bacterial infection. a, To determine whether 25HC directly reduced bacterial viability, a starting bacterial culture was back-diluted in DMEM (10% FBS) supplemented with vehicle (EtOH) or 25HC (5 μM). Bacterial cultures were incubated at 37 °C while shaking at 200 rpm, and OD600 was measured for each sample at the indicated time points. Mean values from 3 independent experiments are plotted, and error bars show s.d. b, To determine whether 25HC directly modifies bacterial virulence, GFP-expressing L. monocytogenes were cultured overnight in BHI supplemented with vehicle (EtOH) or 25HC (5 μM) and HEK293A cells were then infected with bacteria from either culture (MOI = 20, 6 hours). Infection was analysed by flow cytometry. Bars represent mean values. Error bars show s.d. from three independent experiments and statistical significance was determined by student’s unpaired t-test (two-tailed). c-d, Host cell viability was assessed in cells treated with 25HC (c) or in cells virally transduced with CH25H (d). HEK293A cells were treated with 25HC (5 μM) or vehicle (EtOH) for 6, 16, or 24 hours (c) or transduced with Fluc or CH25H for 72 hours (d). Cell viability was evaluated by measuring ATP production using CellTiter-Glo assays. Data are normalized to vehicle (EtOH) in (c), and Fluc in (d). Bars represent mean values. Error bars show s.d. from three independent experiments and statistical significance was determined before normalization by student’s unpaired t-test (two-tailed).

Source data

Extended Data Fig. 4 25HC has little effect on the early life-cycle stages of L. monocytogenes.

a, 25HC does not inhibit L. monocytogenes adhesion/invasion. The percent adhesion/invasion is shown. Bars represent mean values. Error bars show s.d. from three independent experiments and statistical significance was determined by student’s unpaired t-test (two-tailed). b, 25HC does not inhibit L. monocytogenes vacuole escape. The number of internalized bacteria that escape the phagocytic vacuole was determined by quantifying percent cytosolic L. monocytogenes that polymerize actin (F-actin cages or tails). L. monocytogenes lacking LLO was used as an escape-deficient control. Each data point represents the percent of bacteria that nucleate F-actin per field of view. The total number of individual bacteria assessed for F-actin nucleation is also indicated. Bars represent mean values. Error bars show s.d. and statistical significance was determined by student’s unpaired t-test (two-tailed). c, 25HC does not inhibit L. monocytogenes actin polymerization. Images (left) of GFP-expressing L. monocytogenes (green) and F-actin structures associated with cytosolic bacteria (phalloidin, red). Nuclei were labelled with DAPI (blue). Scale bar, 1 μm. Graph shows the frequency of F-actin structures nucleated by bacteria in host cells treated with vehicle or 25HC (5 μM). These data were collected from the experiments performed in (b). Bars represent mean values, and error bars are s.d. d, 25HC does not inhibit L. monocytogenes replication in host cells. Schematic indicating the time points of sample collection after bacterial infection. The total CFU recovered at each time point is shown (line graph). Bacterial replication was determined by calculating the ratio of CFU recovered at the indicated time points (T2 or T3) relative to the CFUs recovered after 3 hours of infection (T1). Mean values were plotted (left), and bars (right) represent mean values. Error bars show s.d. from three independent experiments and statistical significance was determined using student’s unpaired t-test (two-tailed). e, Left, representative images of L. monocytogenes cell-to-cell dissemination foci in HEK293A monolayers. Scale bar, 1 mm. Right, graph showing mean dissemination foci area formed by L. monocytogenes. Bars represent mean values. Error bars show s.d. of foci area normalized to vehicle-treated cells from three independent experiments. Statistical significance was determined prior to normalization by student’s unpaired t-test (two-tailed).

Source data

Extended Data Fig. 5 25HC has no effect on the total cholesterol content of target cells and mobilizes accessible cholesterol in diverse cell types.

a, Total cellular cholesterol measurements of CHO-K1 cells treated for the indicated times with vehicle (EtOH), 25HC (5 μM), or HPCD (1% w/v), normalized to cellular protein content. Bars represent mean values. Error bars show s.d. from six independent experiments and statistical significance was determined by one-way ANOVA compared to vehicle with Dunnet’s correction. b, Immunoblots showing the effects of SMase treatment on cell surface binding of ALOD4 and OlyA in the indicated cell lines. Cells were treated without or with 25HC (5 μM) for 4 hours, followed by treatment with SMase (100 mU/ml) as indicated. Equal aliquots of cell lysates (10% of total) were subjected to immunoblot analysis. Data are representative of three independent experiments. c, CHO-7 cells were treated with 7α-HC (5μM) or vehicle (EtOH) for 16 hours, infected with GFP-expressing L. monocytogenes (MOI=1, 22 hours), and then subjected to flow cytometry analysis. Bars represent mean values. Error bars show s.d. from four independent experiments and statistical significance was determined by student’s unpaired t-test (two-tailed). d, Representative immunoblots from three independent experiments, measured by ALOD4 binding, quantification of which is shown in Fig. 5c. e, Immunoblots showing the effects of exogenously added epicholesterol or cholesterol on cell surface binding of ALOD4 to the indicated 25HC-treated cell lines. Cells were treated with 5 μM 25HC for 4 hours and then incubated with the indicated concentrations of epicholesterol or cholesterol (complexed to MCD) as described in Methods. Equal aliquots of cell lysates (10% of total; or 20 μg/lane for HEK293A) were subjected to ALOD4 immunoblot analysis. Data are representative of three independent experiments.

Source data

Extended Data Fig. 6 25HC suppresses spread of S. flexneri through mobilization of accessible cholesterol.

a, S. flexneri invades epithelial cells and disseminates from cell-to-cell. However, compared to L. monocytogenes, S. flexneri uses different molecular mechanisms and virulence factors. To test whether 25HC can inhibit S. flexneri by modulating accessible cholesterol, we carried out plaque-forming assays coupled with cholesterol repletion. HEK293A were treated with 25HC (5 μM) or vehicle for 16 hours, then 1 hour prior to S. flexneri infection, cells were treated with Chol/MCD complexes diluted in media (40 μM), or vehicle. Plaques were analysed 72 hours after avicel overlay. Representative images of three independent experiments are shown. Scale bar, 1 mm. b, Plaque area was quantified for assay described in (a), and normalized to vehicle-treated cells. Bars represent mean values. Error bars show s.d. from three independent experiments and statistical significance was determined before normalization by one-way ANOVA compared to vehicle with Dunnet’s correction.

Source data

Extended Data Fig. 7 25HC regulation of cholesterol esterification and ALOD4 binding.

a, b, Examination of lipid droplet formation by microscopy (a; images) or flow cytometry (b; bar graph). CHO-7 cells were treated for 2 hours with SZ58-035 (10 μM) or vehicle (DMSO), and then treated with 25HC (5 μM) along with SZ58-035 (10 μM) or vehicle. For microscopy, CHO-7 cells were plated onto glass culture slides prior to treatments and fixed cells were incubated with DAPI to visualize nuclei (blue) and LipidSpot (green) to detect lipid droplet formation. Representative microscopy images are shown. Scale bar, 20 μm. For flow cytometry measurement of lipid droplets in treated CHO-7 cells, the total fluorescence (LipidSpot, Ex-488 nm) was calculated as the %LipidSpot+ cells multiplied by the geometric mean fluorescence intensity. Bars represent mean values. Error bars show s.d. from four independent experiments and statistical significance was determined by one-way ANOVA compared to vehicle-treated cells, with Dunnet’s correction. c, Immunoblot showing SREBP2 processing in cholesterol-replete (left blot) or cholesterol-depleted (right blot) CHO-K1 cells after treatment without or with 25HC in the presence or absence of 58-035, as described in Methods. P, precursor form of SREBP2; N, nuclear form of SREBP2. Blots are representative of three independent experiments. d, Cholesterol accessibility on PMs of CHO-7 mutant cells (SRD-1) constitutively expressing nuclear SREBP-2 treated with 25HC or 7α-HC was assessed by immunoblot analysis of ALOD4 binding, as described in Methods. A representative immunoblot from three independent experiments quantified in Fig. 6g is shown.

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Extended Data Fig. 8 25HC does not suppress L. monocytogenes infection via LXR stimulation.

a, Strategy to generate LXRα/β-deficient cells via CRISPR/Cas9 editing. Genomic sequencing of LXRα/β-deficient HEK293A demonstrating indel formation in each targeted exon is shown. The 20 bp guides are highlighted in red, while the 3 bp PAM sequence is highlighted in blue. Alignments to WT reference DNA using Needle software is shown for each allele. b, To confirm loss of LXR signalling, Wild-type and LXRα/β-deficient HEK293A were stimulated with LXR agonists (2.5 μM GW3965 or 5 μM 25-HC) or vehicle for 24 hours. Total RNA was extracted and mRNA levels of the LXR target gene ABCA1 was determined by qPCR. Expression levels were normalized to non-treated samples, for LXR WT and LXRα/β-deficient cells, respectively. Bars represent mean values. Error bars show s.d. from three independent experiments and statistical significance was determined by one-way ANOVA compared to vehicle-treated cells, with Dunnet’s correction. c, Wild-type and LXRα/β-deficient HEK293A cells were transduced with lentivirus co-expressing Tag-RFP and Fluc or CH25H. After 48 hour transduction, cells were infected with GFP-L. monocytogenes (MOI =10) for 6 hours and analysed by flow cytometry. Bars represent mean values. Error bars show s.d. from three independent experiments and statistical significance was determined by student’s unpaired t-test (two-tailed).

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Extended Data Fig. 9 Model of plasma membrane cholesterol remodelling by circulating oxysterols.

Model of 25HC-mediated regulation of accessible cholesterol. (1) 25HC secreted from IFN-γ activated macrophage enters the target cell and stimulates the enzymatic activity of ACAT (Step 1). ACAT activation results in production of cholesteryl esters that are incorporated into lipid droplets (Step 2). Cholesterol esterification lowers the free cholesterol levels in the ER, triggering internalization of accessible cholesterol from the plasma membrane (Step 3). Long-term suppression of accessible cholesterol is achieved through 25HC-mediated inhibition of the SREBP2 pathway which leads to lower cholesterol synthesis and uptake (Step 4).

Supplementary information

Reporting Summary

Supplementary Table 1

γ-ISG lentiviral library and screening results.

Supplementary Video 1

Live-cell imaging of L. monocytogenes membrane protrusions.

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Abrams, M.E., Johnson, K.A., Perelman, S.S. et al. Oxysterols provide innate immunity to bacterial infection by mobilizing cell surface accessible cholesterol. Nat Microbiol 5, 929–942 (2020). https://doi.org/10.1038/s41564-020-0701-5

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