Abstract
Plasma membranes of animal cells are enriched for cholesterol. Cholesterol-dependent cytolysins (CDCs) are pore-forming toxins secreted by bacteria that target membrane cholesterol for their effector function. Phagocytes are essential for clearance of CDC-producing bacteria; however, the mechanisms by which these cells evade the deleterious effects of CDCs are largely unknown. Here, we report that interferon (IFN) signals convey resistance to CDC-induced pores on macrophages and neutrophils. We traced IFN-mediated resistance to CDCs to the rapid modulation of a specific pool of cholesterol in the plasma membrane of macrophages without changes to total cholesterol levels. Resistance to CDC-induced pore formation requires the production of the oxysterol 25-hydroxycholesterol (25HC), inhibition of cholesterol synthesis and redistribution of cholesterol to an esterified cholesterol pool. Accordingly, blocking the ability of IFN to reprogram cholesterol metabolism abrogates cellular protection and renders mice more susceptible to CDC-induced tissue damage. These studies illuminate targeted regulation of membrane cholesterol content as a host defense strategy.
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Acknowledgements
This research was supported by NIH grants AI093768 (to S.J.B.), HL146358 (to S.J.B. and P.T.), AR073940 (to P.O.S), and HL136543 (to E.J.T.). M.S.L. is supported by Ruth L. Kirschstein National Research Service Award AI007323. The research described was also supported by an NIH/National Center for Advancing Translational Science (NCATS) UCLA CTSI grant (UL1TR001881). We thank S. Young, T. Weston and R.S. Jung for help with protein purification. We thank T. Weston for NanoSIMS sample preparation and SEM imaging. We thank A. Divakaruni for guidance with PFO permeabilization assays. We thank A. Radhakrishnan for ALO-D4 and full-length ALO plasmids. We thank S. Young, A. Hoffmann, Y. Du, R. Sun, T.-T. Wu, J. F. Miller and M. Li for thoughtful discussions.
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Contributions
S.J.B. conceived the study. Q.D.Z. led the design and execution of experiments. X.C. codesigned and performed all flow cytometry experiments and data analysis. V.L.B., W.Y.H. and J.J.M. contributed to flow cytometry experiments. J.J.M. and M.S.L. contributed to protein purification and staining experiments. Q.D.Z., M.S.L. and R.D. developed and performed live-cell imaging assays. C.H. performed NanoSIMS analysis and contributed to protein purification. J.J.M. and E.B.K. contributed to RNA analysis. W.Y.H. and A.G.Y. performed GC–MS analysis with help from Q.D.Z. and E.B.K. K.J.W. conducted lipidomic studies. X.X., A.F., P.T. and E.J.T. contributed to Abcg1, Abca1 and SCAP KO studies. A.E.D. analyzed gene expression data. A.-C.F., P.O.S., M.S.L. and S.T.S. conceptualized and developed the in vivo SLO challenge assay. W.Y.H. contributed to data visualization. S.J.B., Q.D.Z., W.Y.H., X.C. and P.T. contributed to construction of the manuscript.
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Extended data
Extended Data Fig. 1 Interferon signaling mediates resistance to cholesterol-dependent cytolysins.
a, Percentage of PI-positive BMDMs treated with TLR1/2 agonist (Pam3CSK4; 50 ng/mL), TLR3 agonist (Poly(I:C); 1 μg/mL), TLR4 agonist (LPS; 50 ng/mL), TLR7 agonist (CL307; 100 nM), TLR9 (ODN1668; 100 nM) agonist, or unstimulated (NT) for 24 h and then challenged with PFO for up to 60 min in the presence of PI. Cells were imaged every 10 min to assess changes in PI incorporation. b, Percentage of PI-positive BMDMs treated with the various concentrations of TLR4 agonist for 24 h and then challenged with PFO for up to 60 min in the presence of PI. Cells were imaged every 7 min to assess changes in PI incorporation. c, Percentage of PI-positive BMDMs treated with IFN-β or IFN-γ (20 ng/mL) for 24 h and then challenged with PFO for 60 min in the presence of PI. d, Percentage of PI-positive BMDMs treated with NOD2 agonist (N-Glycolyl-MDP; 20 μg/ml), STING agonist (2’,3’cGAMP and c-di-GMP; both 2 μg/mL) for 24 h and then challenged with PFO for 60 min in the presence of PI. e, Percentage of PI-positive BMDMs treated with STING ligand for 24 h and then challenged with Streptolysin O (SLO) for 4 h in the presence of PI. f, Percentage of PI-positive BMDMs treated with IFN-β or IFN-γ (20 ng/mL) for 24 h and then challenged with SLO for 4 h in the presence of PI. g, Percentage of PI-positive BMDMs treated with IFN-β or IFN-γ (20 ng/mL) for 24 h and then challenged with ALO for 60 min in the presence of PI. h, Percentage of PI-positive BMDMs treated with IFN-β (100 ng/mL) or TLR1/2 agonist, or IFN-β (100 ng/mL) together with TLR1/2 agonist for 24 h and then challenged with PFO for 60 min in the presence of PI. i, Percentage of PI-positive BMDMs treated with IFN-β (100 ng/mL) or TLR1/2 agonist, or IFN-β (100 ng/mL) together with TLR1/2 agonist for 24 h and then challenged with SLO for 4 h in the presence of PI. Data are representatives of three independent experiments, and are shown as mean ± s.e.m. (n = 3). Statistical significance was determined using an RM one-way ANOVA with Dunnett’s correction (a-g) or a two-way ANOVA with Dunnett’s correction (h, i). ***P<0.001.
Extended Data Fig. 2 IFN signals decrease plasma membrane binding to ALO-D4 protein.
a, Confocal images of neutrophils stimulated with IFN-β or IFN-γ (20 ng/mL) for 6 h, and then stained with fluorescent ALO-D4 and DAPI. b, Violin plots of cellular fluorescent intensity quantified from a (n = 20334, 18546, 16290). c, Confocal images of WT or type I interferon receptor–deficient (IFNAR KO) BMDMs stimulated with TLR3 agonist (1 μg/mL) or IFN-β (20 ng/mL) for 24 h, and then stained with fluorescent ALO-D4 and DAPI. d, Violin plots of cellular fluorescent intensity quantified from c (n = 5543, 6682, 4673, 8231, 5201, 7906). Data are representatives of three independent experiments. Violin plots are shown with median (solid lines in b, d) and 25% and 75% percentiles (dashed lines in b), and statistical significance was determined using a Kruskal-Wallis test with Dunn’s correction. ***P<0.001. Scale bars in a, c represent 50 μm.
Extended Data Fig. 3 IFN signals reprogram cholesterol metabolism to decrease the pool of cholesterol targeted by CDCs.
a, Total cholesterol (nmol/107 cells) from C57BL/6 bone marrow–derived macrophages (BMDMs) stimulated with cGAMP (2 μg/mL), TLR3 agonist (Poly(I:C); 1 μg/mL), or unstimulated (NT) for 48 h. Total cholesterol was determined by GC-MS (n = 4). b, Total plasma membrane cholesterol (normalized to total FAMEs) from C57BL/6 bone marrow–derived macrophages (BMDMs) stimulated with IFN-γ (40 ng/mL) or unstimulated (NT) for 24 h (n = 4). c, Relative Fillipin III fluorescence intensity of plasma membranes of untreated macrophages or macrophages stimulated with IFN-β (20 ng/mL) or IFN-γ (20 ng/mL) for 24 h (n = 32, 38, 34, 42). MβCD-Cholesterol loaded macrophages indicate dynamic range of Fillipin III fluorescence and are included as a positive control. d, Confocal images of BMDM stimulated with IFN-β (20 ng/mL) for 24 h, and then stained with fluorescent ALO-D4 or OlyA and DAPI. Scale bar, 50 μm. e, Violin plots of cellular fluorescent intensity quantified from d (n = 2225, 2021, 2225, 2021). f, Cholera Toxin B staining of BMDM stimulated with IFN-β or IFN-γ (20 ng/mL) for 24 h. Median fluorescence intensity (MFI) are indicated on the left. Data are representative of three (a, d, e, f) independent experiments, three independent samples (c) or from 4 biological replicates (b). Data in a-c are shown as mean ± s.e.m., violin plots in e are shown with median (solid lines). Statistical significance was determined using an unpaired two-tailed Student’s t-test (a, b), a one-way ANOVA with Dunnett’s correction (c), or a two-tailed Mann–Whitney test (e) ***P<0.001.
Extended Data Fig. 4 Cholesterol synthesis is linked to CDC susceptibility.
a, Net synthesized cholesterol (nmol/107 cells) from C57BL/6 bone marrow–derived macrophages (BMDMs) stimulated with cGAMP (2 μg/mL), or unstimulated (NT) for 48 h. Synthesized cholesterol was determined by GC-MS and isotopomer spectral analysis modeling (n = 4). b, Net synthesized cholesterol (nmol/107 cells) from C57BL/6 bone marrow–derived macrophages (BMDMs) stimulated with TLR1/2 agonist (Pam3CSK4; 50 ng/mL), TLR3 agonist (Poly(I:C); 1 μg/mL), TLR4 agonist (LPS; 50 ng/mL), TLR7 agonist (CL307; 100 nM), TLR9 (ODN1668; 100 nM) agonist, or unstimulated (NT) for 48 h. Synthesized cholesterol was determined by GC-MS and isotopomer spectral analysis modeling (n = 4). c, Percentage of PI–positive WT BMDMs treated with Simvastatin (1 μM) for 4 h and then challenged with PFO for 60 min in the presence of PI (n = 3). Data are representative of three independent experiments and are shown as mean + s.e.m. Statistical significance was determined using an unpaired two-tailed Student’s t-test (a), a one-way ANOVA with Dunnett’s correction (b), or a paired two-tailed Student’s t-test (c). ***P<0.001.
Extended Data Fig. 5 Production of 25-hydroxycholesterol is required to maintain changes in plasma membrane cholesterol and mediates resistance to CDCs.
a, Percentage of PI–positive control or CH25H KO BMDMs stimulated with IFNs (20 ng/mL) for 24 h and then challenged with PFO for 60 minutes in the presence of PI. b, Percentage of PI–positive control or CH25H KO BMDMs stimulated with IFNs (20 ng/mL) for 24 h and then challenged with SLO for 2 h in the presence of PI. Data are representative of three independent experiments and are shown as mean + s.e.m. (n = 3) and statistical significance was determined using a two-way ANOVA with Dunnett’s correction. ***P<0.001.
Extended Data Fig. 6 Cholesterol esterification contributes to CDC resistance of macrophages.
a, Quantification (nmol/107 cells) of cholesterol ester species (16:0. 18:1, 20:4) in BMDMs stimulated with TLR3 agonist (1 μg/mL) in FBS or LPDS for 48 h. CE species pool sizes were determined by direct infusion MS. b, Percentage of PI–positive WT BMDMs treated with IFN-β, or IFN-γ (20 ng/mL), or in combination with ACATi 58-035 (4.3 μM) for 24 h and then challenged with PFO for 60 min in the presence of PI. c, Quantification (nmol/107 cells) of total cholesterol ester (CE) in control or CH25H KO BMDMs stimulated with IFN-β (20 ng/mL) or IFN-γ (20 ng/mL) for 48 h. CE pool sizes were determined by direct infusion mass spectrometry. d, Percentage of PI-positive CH25H KO BMDMs treated with ACATi 58-035 (4.3 μM) for 24 h and then challenged PFO for 60 min in the presence of PI. e, Violin plots of cellular fluorescent intensity quantified from control or ABCA1 KO or ABCG1 KO BMDMs stimulated with IFNs (20 ng/mL) for 24 h and then stained with fluorescent ALO-D4 and DAPI (n = 5943, 4126, 5727, 6914, 5740, 7898; n=7201, 7532, 7563, 7417). f, Percentage of PI-positive control or ABCA1 KO BMDMs treated with IFNs (20 ng/mL) for 24 h and then challenged PFO for 60 min in the presence of PI. g, Percentage of PI-positive control or CH25H KO BMDMs treated with LXR agonist GW3965 (1 μM) for 24 h and then challenged PFO for 60 min in the presence of PI. Data are representatives of two (a, c) or three (b, d, e, f, g) independent experiments. Data in a, b, c, d, f and g are shown as mean + s.e.m. (n = 3 in a, b, d, g; n = 4 in c). Violin plots in e are shown with median (solid lines) and 25% and 75% percentiles (dashed lines). Statistical significance was determined using an unpaired two-tailed Student’s t-test (a), a two-way ANOVA with Tukey’s correction (b, c, f, g), a paired two-tailed Student’s t-test (d), or a Kruskal–Wallis test with Dunn’s correction (e). ***P<0.001.
Extended Data Fig. 7 25HC mediates protection to CDC induced tissue damage.
a, Lesion images of control or CH25H KO mice challenged intradermally with SLO (8 kU/mouse) for 48 h. b, Lesion images of vehicle or 25HC pretreated mice challenged intradermally with ALO (20 nM) for 48 h.
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Zhou, Q.D., Chi, X., Lee, M.S. et al. Interferon-mediated reprogramming of membrane cholesterol to evade bacterial toxins. Nat Immunol 21, 746–755 (2020). https://doi.org/10.1038/s41590-020-0695-4
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DOI: https://doi.org/10.1038/s41590-020-0695-4
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