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

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.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

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.

References

  1. 1.

    Schroder, K., Hertzog, P. J., Ravasi, T. & Hume, D. A. Interferon-γ: an overview of signals, mechanisms and functions. J. Leukoc. Biol. 75, 163–189 (2004).

    CAS  PubMed  Article  Google Scholar 

  2. 2.

    Jouanguy, E. et al. A human IFNGR1 small deletion hotspot associated with dominant susceptibility to mycobacterial infection. Nat. Genet. 21, 370–378 (1999).

    CAS  PubMed  Article  Google Scholar 

  3. 3.

    Jouanguy, E. et al. IL-12 and IFN-γ in host defense against mycobacteria and salmonella in mice and men. Curr. Opin. Immunol. 11, 346–351 (1999).

    CAS  PubMed  Article  Google Scholar 

  4. 4.

    Radoshevich, L. & Cossart, P. Listeria monocytogenes: towards a complete picture of its physiology and pathogenesis. Nat. Rev. Microbiol. 16, 32–46 (2018).

    CAS  PubMed  Article  Google Scholar 

  5. 5.

    Portnoy, D. A., Auerbuch, V. & Glomski, I. J. The cell biology of Listeria monocytogenes infection: the intersection of bacterial pathogenesis and cell-mediated immunity. J. Cell Biol. 158, 409–414 (2002).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  6. 6.

    Bauman, D. R. et al. 25-Hydroxycholesterol secreted by macrophages in response to Toll-like receptor activation suppresses immunoglobulin A production. Proc. Natl Acad. Sci. USA 106, 16764–16769 (2009).

    CAS  PubMed  Article  Google Scholar 

  7. 7.

    Park, K. & Scott, A. L. Cholesterol 25-hydroxylase production by dendritic cells and macrophages is regulated by type I interferons. J. Leukoc. Biol. 88, 1081–1087 (2010).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  8. 8.

    Blanc, M. et al. The transcription factor STAT-1 couples macrophage synthesis of 25-hydroxycholesterol to the interferon antiviral response. Immunity 38, 106–118 (2013).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  9. 9.

    Lund, E. G., Kerr, T. A., Sakai, J., Li, W. P. & Russell, D. W. cDNA cloning of mouse and human cholesterol 25-hydroxylases, polytopic membrane proteins that synthesize a potent oxysterol regulator of lipid metabolism. J. Biol. Chem. 273, 34316–34327 (1998).

    CAS  PubMed  Article  Google Scholar 

  10. 10.

    Dang, E. V., McDonald, J. G., Russell, D. W. & Cyster, J. G. Oxysterol restraint of cholesterol synthesis prevents AIM2 inflammasome activation. Cell 171, 1057–1071 (2017).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  11. 11.

    Reboldi, A. et al. Inflammation. 25-Hydroxycholesterol suppresses interleukin-1-driven inflammation downstream of type I interferon. Science 345, 679–684 (2014).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  12. 12.

    Perelman, S. S. et al. Cell-based screen identifies human interferon-stimulated regulators of Listeria monocytogenes infection. PLoS Pathog. 12, e1006102 (2016).

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  13. 13.

    Kandutsch, A. A. & Chen, H. W. Inhibition of sterol synthesis in cultured mouse cells by 7α-hydroxycholesterol, 7β-hydroxycholesterol, and 7-ketocholesterol. J. Biol. Chem. 248, 8408–8417 (1973).

    CAS  PubMed  Google Scholar 

  14. 14.

    Brown, M. S. & Goldstein, J. L. Suppression of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity and inhibition of growth of human fibroblasts by 7-ketocholesterol. J. Biol. Chem. 249, 7306–7314 (1974).

    CAS  PubMed  Google Scholar 

  15. 15.

    Radhakrishnan, A., Ikeda, Y., Kwon, H. J., Brown, M. S. & Goldstein, J. L. Sterol-regulated transport of SREBPs from endoplasmic reticulum to Golgi: oxysterols block transport by binding to Insig. Proc. Natl Acad. Sci. USA 104, 6511–6518 (2007).

    CAS  PubMed  Article  Google Scholar 

  16. 16.

    Brown, M. S., Dana, S. E. & Goldstein, J. L. Cholesterol ester formation in cultured human fibroblasts. Stimulation by oxygenated sterols. J. Biol. Chem. 250, 4025–4027 (1975).

    CAS  PubMed  Google Scholar 

  17. 17.

    Chang, T. Y., Chang, C. C., Ohgami, N. & Yamauchi, Y. Cholesterol sensing, trafficking, and esterification. Annu. Rev. Cell Dev. Biol. 22, 129–157 (2006).

    CAS  PubMed  Article  Google Scholar 

  18. 18.

    Repa, J. J. & Mangelsdorf, D. J. The liver X receptor gene team: potential new players in atherosclerosis. Nat. Med. 8, 1243–1248 (2002).

    CAS  PubMed  Article  Google Scholar 

  19. 19.

    Das, A., Brown, M. S., Anderson, D. D., Goldstein, J. L. & Radhakrishnan, A. Three pools of plasma membrane cholesterol and their relation to cholesterol homeostasis. eLife 3, e02882 (2014).

  20. 20.

    Infante, R. E. & Radhakrishnan, A. Continuous transport of a small fraction of plasma membrane cholesterol to endoplasmic reticulum regulates total cellular cholesterol. eLife 6, e25466 (2017).

  21. 21.

    Endapally, S. et al. Molecular Discrimination between two conformations of sphingomyelin in plasma membranes. Cell 176, 1040–1053 (2019).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  22. 22.

    Gay, A., Rye, D. & Radhakrishnan, A. Switch-like responses of two cholesterol sensors do not require protein oligomerization in membranes. Biophys. J. 108, 1459–1469 (2015).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  23. 23.

    Johnson, K. A., Endapally, S., Vazquez, D. C., Infante, R. E. & Radhakrishnan, A. Ostreolysin A and anthrolysin O use different mechanisms to control movement of cholesterol from the plasma membrane to the endoplasmic reticulum. J. Biol. Chem. 294, 17289–17300 (2019).

    CAS  PubMed  Article  Google Scholar 

  24. 24.

    Ray, K., Marteyn, B., Sansonetti, P. J. & Tang, C. M. Life on the inside: the intracellular lifestyle of cytosolic bacteria. Nat. Rev. Microbiol. 7, 333–340 (2009).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  25. 25.

    Brown, M. S., Radhakrishnan, A. & Goldstein, J. L. Retrospective on cholesterol homeostasis: the central role of scap. Annu. Rev. Biochem. 87, 783–807 (2018).

    CAS  PubMed  Article  Google Scholar 

  26. 26.

    Janowski, B. A., Willy, P. J., Devi, T. R., Falck, J. R. & Mangelsdorf, D. J. An oxysterol signalling pathway mediated by the nuclear receptor LXRα. Nature 383, 728–731 (1996).

    CAS  PubMed  Article  Google Scholar 

  27. 27.

    Chang, T. Y., Chang, C. C. & Cheng, D. Acyl-coenzyme A:cholesterol acyltransferase. Annu. Rev. Biochem. 66, 613–638 (1997).

    CAS  PubMed  Article  Google Scholar 

  28. 28.

    Cheng, D., Chang, C. C., Qu, X. & Chang, T. Y. Activation of acyl-coenzyme A:cholesterol acyltransferase by cholesterol or by oxysterol in a cell-free system. J. Biol. Chem. 270, 685–695 (1995).

    CAS  PubMed  Article  Google Scholar 

  29. 29.

    Ross, A. C., Go, K. J., Heider, J. G. & Rothblat, G. H. Selective inhibition of acyl coenzyme A:cholesterol acyltransferase by compound 58-035. J. Biol. Chem. 259, 815–819 (1984).

    CAS  PubMed  Google Scholar 

  30. 30.

    Yang, J., Sato, R., Goldstein, J. L. & Brown, M. S. Sterol-resistant transcription in CHO cells caused by gene rearrangement that truncates SREBP-2. Genes Dev. 8, 1910–1919 (1994).

    CAS  PubMed  Article  Google Scholar 

  31. 31.

    Hannedouche, S. et al. Oxysterols direct immune cell migration via EBI2. Nature 475, 524–527 (2011).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  32. 32.

    Nelson, E. R. et al. 27-Hydroxycholesterol links hypercholesterolemia and breast cancer pathophysiology. Science 342, 1094–1098 (2013).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  33. 33.

    Corcoran, R. B. & Scott, M. P. Oxysterols stimulate Sonic hedgehog signal transduction and proliferation of medulloblastoma cells. Proc. Natl Acad. Sci. USA 103, 8408–8413 (2006).

    CAS  PubMed  Article  Google Scholar 

  34. 34.

    Dwyer, J. R. et al. Oxysterols are novel activators of the hedgehog signaling pathway in pluripotent mesenchymal cells. J. Biol. Chem. 282, 8959–8968 (2007).

    CAS  PubMed  Article  Google Scholar 

  35. 35.

    Kinnebrew, M. et al. Cholesterol accessibility at the ciliary membrane controls hedgehog signaling. eLife 8, e50051 (2019).

  36. 36.

    Liu, S. Y. et al. Interferon-inducible cholesterol-25-hydroxylase broadly inhibits viral entry by production of 25-hydroxycholesterol. Immunity 38, 92–105 (2013).

    PubMed  Article  CAS  Google Scholar 

  37. 37.

    Choi, W. S. et al. The CH25H–CYP7B1–RORα axis of cholesterol metabolism regulates osteoarthritis. Nature 566, 254–258 (2019).

    CAS  PubMed  Article  Google Scholar 

  38. 38.

    Ortiz, A. et al. An interferon-driven oxysterol-based defense against tumor-derived extracellular vesicles. Cancer Cell 35, 33–45 e36 (2019).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  39. 39.

    Flanagan, J. J., Tweten, R. K., Johnson, A. E. & Heuck, A. P. Cholesterol exposure at the membrane surface is necessary and sufficient to trigger perfringolysin O binding. Biochemistry 48, 3977–3987 (2009).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  40. 40.

    Metherall, J. E., Goldstein, J. L., Luskey, K. L. & Brown, M. S. Loss of transcriptional repression of three sterol-regulated genes in mutant hamster cells. J. Biol. Chem. 264, 15634–15641 (1989).

    CAS  PubMed  Google Scholar 

  41. 41.

    Brown, A. J., Sun, L., Feramisco, J. D., Brown, M. S. & Goldstein, J. L. Cholesterol addition to ER membranes alters conformation of SCAP, the SREBP escort protein that regulates cholesterol metabolism. Mol. Cell 10, 237–245 (2002).

    CAS  PubMed  Article  Google Scholar 

  42. 42.

    Sanda, C. et al. Differential gene induction by type I and type II interferons and their combination. J. Interferon Cytokine Res. 26, 462–472 (2006).

    CAS  PubMed  Article  Google Scholar 

  43. 43.

    Sa, S. M. et al. The effects of IL-20 subfamily cytokines on reconstituted human epidermis suggest potential roles in cutaneous innate defense and pathogenic adaptive immunity in psoriasis. J. Immunol. 178, 2229–2240 (2007).

    CAS  PubMed  Article  Google Scholar 

  44. 44.

    Xue, J. et al. Transcriptome-based network analysis reveals a spectrum model of human macrophage activation. Immunity 40, 274–288 (2014).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  45. 45.

    Wang, S. H. et al. Microarray analysis of cytokine activation of apoptosis pathways in the thyroid. Endocrinology 148, 4844–4852 (2007).

    CAS  PubMed  Article  Google Scholar 

  46. 46.

    Waddell, S. J. et al. Dissecting interferon-induced transcriptional programs in human peripheral blood cells. PLoS ONE 5, e9753 (2010).

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  47. 47.

    Pawliczak, R. et al. Influence of IFN-γ on gene expression in normal human bronchial epithelial cells: modulation of IFN-γ effects by dexamethasone. Physiol. Genomics 23, 28–45 (2005).

    CAS  PubMed  Article  Google Scholar 

  48. 48.

    Rock, R. B. et al. Transcriptional response of human microglial cells to interferon-γ. Genes Immun. 6, 712–719 (2005).

    CAS  PubMed  Article  Google Scholar 

  49. 49.

    Johnson-Huang, L. M. et al. A single intradermal injection of IFN-γ induces an inflammatory state in both non-lesional psoriatic and healthy skin. J. Invest. Dermatol. 132, 1177–1187 (2012).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  50. 50.

    Indraccolo, S. et al. Identification of genes selectively regulated by IFNs in endothelial cells. J. Immunol. 178, 1122–1135 (2007).

    CAS  PubMed  Article  Google Scholar 

  51. 51.

    Hu, X., Park-Min, K. H., Ho, H. H. & Ivashkiv, L. B. IFN-γ-primed macrophages exhibit increased CCR2-dependent migration and altered IFN-γ responses mediated by Stat1. J. Immunol. 175, 3637–3647 (2005).

    CAS  PubMed  Article  Google Scholar 

  52. 52.

    He, X. S. et al. Differential transcriptional responses to interferon-α and interferon-γ in primary human hepatocytes. J. Interferon Cytokine Res. 30, 311–320 (2010).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  53. 53.

    Fairfax, B. P. et al. Innate immune activity conditions the effect of regulatory variants upon monocyte gene expression. Science 343, 1246949 (2014).

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  54. 54.

    Cheon, H. et al. IFNβ-dependent increases in STAT1, STAT2, and IRF9 mediate resistance to viruses and DNA damage. EMBO J. 32, 2751–2763 (2013).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  55. 55.

    Schoggins, J. W. et al. A diverse range of gene products are effectors of the type I interferon antiviral response. Nature 472, 481–485 (2011).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  56. 56.

    Seiler, C. Y. et al. DNASU plasmid and PSI:Biology-Materials repositories: resources to accelerate biological research. Nucleic Acids Res. 42, D1253–D1260 (2014).

    CAS  PubMed  Article  Google Scholar 

  57. 57.

    Campeau, E. et al. A versatile viral system for expression and depletion of proteins in mammalian cells. PLoS ONE 4, e6529 (2009).

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  58. 58.

    McDonald, J. G., Smith, D. D., Stiles, A. R. & Russell, D. W. A comprehensive method for extraction and quantitative analysis of sterols and secosteroids from human plasma. J. Lipid Res. 53, 1399–1409 (2012).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  59. 59.

    Endapally, S., Infante, R. E. & Radhakrishnan, A. Monitoring and modulating intracellular cholesterol trafficking using ALOD4, a cholesterol-binding protein. Methods Mol. Biol. 1949, 153–163 (2019).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  60. 60.

    Schindelin, J. et al. Fiji: an open-source platform for biological-image analysis. Nat. Methods 9, 676–682 (2012).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  61. 61.

    Bourdeau, R. W. et al. Cellular functions and X-ray structure of anthrolysin O, a cholesterol-dependent cytolysin secreted by Bacillus anthracis. J. Biol. Chem. 284, 14645–14656 (2009).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

Download references

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

Affiliations

Authors

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.

Corresponding authors

Correspondence to Arun Radhakrishnan or Neal M. Alto.

Ethics declarations

Competing interests

The authors declare no competing interests.

Additional information

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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. Source data

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). Source data

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

41564_2020_701_MOESM3_ESM.avi

Live-cell imaging of L. monocytogenes membrane protrusions.

Reporting Summary

Supplementary Table 1

γ-ISG lentiviral library and screening results.

Supplementary Video 1

Live-cell imaging of L. monocytogenes membrane protrusions.

Source data

Source Data Fig. 1

Statistical Source Data.

Source Data Fig. 2

Statistical Source Data.

Source Data Fig. 3

Statistical Source Data.

Source Data Fig. 4

Statistical Source Data.

Source Data Fig. 5

Statistical Source Data.

Source Data Fig. 5

Full-length unprocessed western blot.

Source Data Fig. 6

Statistical Source Data.

Source Data Fig. 6.

Full-length unprocessed western blot.

Source Data Extended Data Fig. 1

Statistical Source Data.

Source Data Extended Data Fig. 2

Statistical Source Data.

Source Data Extended Data Fig. 3

Statistical Source Data.

Source Data Extended Data Fig. 4

Statistical Source Data.

Source Data Extended Data Fig. 5

Statistical Source Data.

Source Data Extended Data Fig. 5

Full-length unprocessed western blot.

Source Data Extended Data Fig. 6

Statistical Source Data.

Source Data Extended Data Fig. 7

Statistical Source Data.

Source Data Extended Data Fig. 7

Full-length unprocessed western blot.

Source Data Extended Data Fig. 8

Statistical Source Data.

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

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

Download citation

Further reading