Interferon-mediated reprogramming of membrane cholesterol to evade bacterial toxins

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.

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.

Fig. 1 |. Interferon signaling mediates resistance to cholesterol-dependent cytolysins

a. Percentage of propidium iodide (PI)–positive BMDMs treated with the 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 perfringolysin O (PFO) for 60 min in the presence of PI. b. Percentage of PI–positive BMDMs treated with IFN-β (20 ng/mL) and IFN-γ (20 ng/mL) for 24 h and then challenged with PFO for 60 min in the presence of PI. c. 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. d. Percentage of PI–positive BMDMs treated with the indicated IFNs (20 ng/mL) for 24 h and then challenged with Streptolysin-O (SLO) for 2 h in the presence of PI. e. Percentage of PI–positive BMDMs treated with the indicated IFNs (20 ng/mL) for 24 h and then challenged with anthrolysin-O (ALO) for 2 h in the presence of PI. f. Percentage of PI–positive BMDMs treated with TLR1/2 (50 ng/mL) together with IFNs (100 ng/mL) for 24 h and then challenged with PFO for 60 min in the presence of PI. g. Percentage of PI–positive BMDMs treated with TLR1/2 (50 ng/mL) together with IFNs (100 ng/mL) for 24 h and then challenged with SLO for 2 h in the presence of PI. h. Percentage of PI–positive neutrophils treated with IFN-β (20 ng/mL) and IFN-γ (20 ng/mL) for 4 h and then challenged with PFO for 60 min in the presence of PI (n = 3). i. Flow cytometry plots of S. aureus phagocytosed by control or IFNs-stimulated BMDMs. Macrophage cultures were stimulated with IFNs (100 ng/mL) for 24 h. BMDMs were then washed and then incubated with PFO for 15 min. PFO containing media was then replaced with fresh media containing pHrodo-red-labeled S. aureus. Median fluorescence intensity (MFI) are indicated on the right. Data are representatives of three independent experiments. Data in a-h are shown as mean ± s.e.m.(n=3). Statistical significance was determined using a one-way ANOVA with Dunnett’s correction (a-e), a two-way ANOVA with Tukey’s correction (f, g), or an RM one-way ANOVA with Dunnett’s correction (h). ***P<0.001.

Fig. 2 |. IFN signals decrease plasma membrane binding to ALO-D4 protein

a. Super resolution confocal images of macrophage cultures stimulated with the indicated IFNs (20 ng/mL) for 24 h and then stained with fluorescent ALO-D4 and DAPI. b. Confocal images of macrophage cultures stimulated with the indicated IFNs (20 ng/mL) for 24 h and then stained with fluorescent ALO-D4 and DAPI. c. Violin plots of cellular fluorescent intensity quantified from b (from left to right: n = 6539, 4561, 5367). d. Confocal images of macrophage cultures stimulated with the indicated PRR ligands for 24 h and then stained with fluorescent ALO-D4 and DAPI. e. Violin plots of cellular fluorescent intensity quantified from d (n = 5931, 4766, 5660). f. Confocal images of macrophage cultures stimulated with the indicated TLR agonists for 24 h and then stained with fluorescent ALO-D4 and DAPI. g. Violin plots of cellular fluorescent intensity quantified from f (n = 4358, 3270). h. Confocal images of human peripheral blood monocyte (hPBMC)–derived macrophages stimulated with human IFN-α or IFN-β (10 ng/mL) for 24 h and then stained with fluorescent ALO-D4 and DAPI. i. Violin plots of cellular fluorescent intensity quantified from h (n = 2084, 1818, 1728). j. Confocal images of macrophage cultures stimulated with IFN-β (20 ng/mL) for 2 h and then stained with fluorescent ALO-D4 and DAPI. k. Violin plots of cellular fluorescent intensity quantified from j (n = 6550, 7012). l. Confocal images of WT or type I interferon receptor–deficient (IFNAR KO) BMDMs stimulated with IFN-γ (20 ng/mL) for 24 h and then stained with fluorescent ALO-D4 and DAPI. m. Violin plots of cellular fluorescent intensity quantified from l (n = 4358, 2853, 7089, 5458). Data in a are representatives of three independent samples. Data in b-m are representatives of three (b-k) or two (l, m) independent experiments. Violin plots in c, e, g, i, k, and m are shown with median (solid line) and 25% and 75% percentiles (dashed lines), and statistical significance was determined using a Kruskal-Wallis test with Dunn’s correction (c, e, i) or a two-tailed Mann-Whitney test (g, k, m). ***P<0.001. Scale bars represent 10 μm (a) or 50 μm (b, d, f, h, j, l).

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 IFN-β (20 ng/mL), IFN-γ (20 ng/mL), or unstimulated (NT) for 48 h. Synthesized cholesterol was determined by GC-MS and isotopomer spectral analysis modeling (n = 4). b. Total plasma membrane cholesterol (nmol/10⁷ cells) from C57BL/6 bone marrow–derived macrophages (BMDMs) stimulated with IFN-γ (40 ng/mL) or unstimulated (NT) for 24 h (n = 4). c. Quantification of [¹⁵N]ALO-D4 or [¹⁵N]OlyA binding on untreated or IFN-β (100 ng/mL, 16 h)-stimulated BMDMs determined by NanoSIMS. Quantification based on average ¹⁵N/¹⁴N ratio by cell. Data are mean ± s.e.m (n = 18, 14 for ALO and n = 8, 10 for OlyA). d. Confocal images of WT BMDM cultures stimulated with IFNs (20 ng/mL) for 24 h, treated with Sphingomyelinase (SMase; 200 mU/mL) for 30 min at 37 ˚C, and then stained with fluorescent ALO-D4 and DAPI. Scale bars represent 50 μm. e. Violin plots of cellular fluorescent intensity quantified from d (n = 6868, 6811, 3404, 3838, 2930, 2642). Data are representative of three (a, d, e) independent experiments, three independent samples (c) or from 4 biological replicates (b). Data in a-c are shown as mean ± s.e.m., violin plot in e is shown with median (solid lines) and 25% and 75% percentiles (dashed lines). Statistical significance was determined using a one-way ANOVA with Dunn’s correction (a), an unpaired two-tailed Student’s t test (b), or a two-tailed Mann-Whitney test (c,e). ***P<0.001.

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 IFN-β (20 ng/mL), IFN-γ (20 ng/mL), or unstimulated (NT) for 48 h. Synthesized cholesterol was determined by GC-MS and isotopomer spectral analysis modeling (n = 4). b. Confocal images of WT BMDM treated with Simvastatin (1 μM-5 μM) for 24 h and then stained with fluorescent ALO-D4 and DAPI. c. Violin plots of cellular fluorescent intensity quantified from b (n = 5713, 2252, 2808, 5556). d. Violin plots of cellular fluorescent intensity quantified from control or SCAP KO BMDMs stained with fluorescent ALO-D4 and DAPI (n = 3306, 2927). e. Percentage of PI–positive control or SCAP KO BMDMs challenged with PFO for 60 min in the presence of PI (n = 3). f. Confocal images of WT BMDMs cultures incubated with 25HC (3 μM) for 4 h, and then stained with fluorescent ALO-D4 and DAPI. g. Violin plots of cellular fluorescent intensity quantified from f (n = 5861, 5769). Data are representatives of three independent experiments. Data in a, and e are shown as mean ± s.e.m. Violin plots in c, d, and g are shown with median (solid lines) and 25% and 75% percentiles (dashed lines). Statistical significance was determined using a Kruskal-Wallis test with Dunn’s correction (c), a two-tailed Mann-Whitney test (d, g), or an RM one-way ANOVA (e). ***P<0.001. Scale bars in b and f represent 50 μm.

Fig. 5 |. Production of 25-hydroxycholesterol is required to maintain changes in plasma membrane cholesterol and mediates resistance to CDCs

a. Confocal images of control or cholesterol 25-hydroxylase-deficient (CH25H KO) BMDMs cultures stimulated with IFN-β (20 ng/mL), or IFN-γ (20 ng/mL) for 24 h, and then stained with fluorescent ALO-D4 and DAPI. b. Violin plots of cellular fluorescent intensity quantified from a (from left to right: n = 5294, 5505, 4564, 4066, 5370, 4962). c. 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. d. 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. e. Flow cytometry plots of S. aureus phagocytosed by control or CH25H KO BMDMs. Macrophage cultures were treated with IFNs (100 ng/mL) or 25HC (3 μM). After 24 h, BMDMs were washed and then incubated with PFO for 15 min. PFO containing media was then replaced with fresh media containing pHrodo-red-labeled S. aureus. Median fluorescence intensity (MFI) are indicated on the right. f. Percentage of PI-positive control or CH25H KO BMDMs incubated with 25HC (1 μM) overnight and then challenged with PFO for 60 minutes in the presence of PI. g. Net synthesized and total cholesterol (nmol/10⁷ cells) from CH25H-deficient or control bone marrow–derived macrophages (BMDMs) stimulated with IFN-β (20 ng/mL) or unstimulated (NT) for 48 h. Synthesized cholesterol was determined by GC-MS and isotopomer spectral analysis modeling. h. Confocal images of CH25H KO BMDMs treated with Simvastatin (5 μM) for 24 h and then stained with fluorescent ALO-D4 and DAPI. i. Violin plots of cellular fluorescent intensity quantified from h (n = 6666, 2867). Data are representative of three independent experiments. Data in c, d, f, and g are shown as mean ± s.e.m. (n = 3 in c, d, f and n = 4 in g). Violin plots are show with median (solid lines in b, i) and 25% and 75% percentiles (dashed lines in i). Statistical significance was determined using a Kruskal-Wallis test with Dunn’s correction (b), a two-way ANOVA with Tukey’s correction (c, d, f, g), or a two-tailed Mann-Whitney test (i). ***P<0.001. Scale bars in a and h represent 50 μm.

Fig. 6 |. Cholesterol esterification contributes to CDC resistance of macrophages

a. qPCR analysis of genes that regulate cholesterol homeostasis in WT BMDM treated with IFNs (20 ng/mL) for 24 h. b. Quantification (nmol/10⁷ cells) of total cholesterol ester (CE) in WT 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 (left panel: n = 3, right panel: n = 4). c. Percentage of PI–positive BMDMs treated with IFN-β (20 ng/mL) and IFN-γ (20 ng/mL) for 24 h in the presence of the ACAT inhibitor 58–035 (4.3 μM) and then challenged with PFO for 60 min in the presence of PI. d. Violin plots of cellular fluorescent intensity quantified from control or CH25H KO BMDMs treated with ACAT inhibitor 58–035 (4.3 μM) for 24 h and then stained with fluorescent ALO-D4 and DAPI (n = 6666, 9997). e. Percentage of PI–positive control or ABCA1 KO BMDMs stimulated with IFNs (20 ng/mL) for 24 h and then challenged with PFO for 60 minutes in the presence of PI. f. Violin plots of cellular fluorescent intensity quantified from control or CH25H KO BMDMs treated with LXR agonist GW3965 (1 μM) for 24 h and then stained with fluorescent ALO-D4 and DAPI (n= 2304, 3954, 2712, 2322). g. Percentage of PI–positive control or CH25H KO BMDMs treated with LXR agonist GW3965 (1 μM) for 24 h and then challenged with PFO for 60 min in the presence of PI. Data are representative of three independent experiments. Data in a, b, c, e, and g are shown as mean ± s.e.m. (n = 3) unless otherwise specified. Violin plots in d, and f are shown with median (solid lines) and 25% and 75% percentiles (dashed lines). Statistical significance was determined using a one-way ANOVA with Dunnett’s correction (a), an unpaired two-tailed Student’s t test (b), a two-way ANOVA with Tukey’s correction (c, e, g), a two-tailed Mann-Whitney test (d), or a Kruskal-Wallis test with Dunn’s correction (f). ***P<0.001, n.s., not significant.

Fig. 7 |. 25HC mediates protection to CDC induced tissue damage

a. Quantification of lesion size from WT (n = 13) and CH25H KO mice (n = 9) challenged with SLO for 48 h. b. Representative histology from mice demonstrates serum crust overlying partial thickness erosion with a small full-thickness ulceration (green dotted line) of the epidermis, and a robust inflammatory response in the dermis and adipose tissue in control (B6) mice (left panel). Wound areas in CH25H-deficient mice demonstrate a large ulceration (green dotted line) with necrosis/destruction of the papillary and much of the reticular dermis with a less pronounced inflammatory response (right panel). c. Quantification of lesion size from WT mice (n = 10 each) pre-treated with vehicle or 25HC for 6 h and challenged with ALO for 48 h. d. Representative histology of wounds demonstrates full thickness ulceration (green dotted line) with overlying neutrophilic/serum crust by ALO in wounds pretreated with ethanol vehicle (left panel), but no ulceration and only edema and a mixed mononuclear and neutrophilic inflammatory host response in the dermis and adipose tissue of mice pretreated with 25HC (right panel). Data in a, c are shown as mean ± s.e.m. and statistical significance was determined using a two-tailed Mann-Whitney test. Scale bars in b, d represent 500 μm.