Fig 1: aCDase in multivesicular bodies traps HSV-1.a, b Immunofluorescence (a) and tissue culture infection dose 50 (TCID50) from supernatants (b) of fibroblasts or bone marrow derived macrophages (BMDMs) that were infected with herpes simplex virus type 1 (HSV-1) at a multiplicity of infection (MOI) of 10 and stained after 6 h (a, n = 4; scale bar 50 µm) or MOI 0.01 or MOI 1 and supernatant was collected after 24 h (b, n = 8; one-way ANOVA [Tukey’s multiple comparison]). c Gene set enrichment analysis (GSEA) of microarrays showing gene sets enriched in macrophages (red) vs. gene sets enriched in fibroblasts (blue). See Supplementary Table 1a, b for gene sets, ranking and grouping. NES: normalized enrichment score. ER: endoplasmic reticulum. d, e Representative electron microscopic images (d) and quantification (e) of wild-type (WT) BMDMs and/or fibroblasts infected with HSV-1 (MOI 250) analyzed after 30 min (BMDMs: n = 77 images from three independent experiments, scale bar 5 µm; Fibroblasts: n = 106 images from three independent experiments, scale bar 5 µm, one-way ANOVA [Tukey’s multiple comparison]). Overview of one macrophage and one fibroblast is shown. Details show specific compartments containing HSV-1. MVB: multivesicular bodies. f: Expression of membrane-modulating proteins analyzed from microarrays of fibroblasts and macrophages with or without 50 U/ml interferon-a4 (IFN-a4) treatment. g Real-time polymerase chain reaction (RT-PCR) for Asah1 mRNA expression of fibroblasts and macrophages from WT mice (n = 6, two-tailed student’s t-test). h Western Blot for aCDase protein expression of fibroblasts and macrophages from WT mice (1 of 2 is shown). All data are shown as mean ± SEM. *p = 0.05, **p = 0.01, #p = 0.001, ##p = 0.0001.
Fig 2: Deficiency of progranulin leads to ganglioside accumulation in mouse and human brain tissues.a Ganglioside degradation pathway in the lysosome. The names of glycosyl hydrolases (green), activator proteins (purple), and associated metabolic diseases (red) are indicated in the scheme. b Quantification of mono-sialyated and di-sialyated ganglioside species isolated from Grn+/+ (gray) (n = 6), Grn+/R493X (blue) (n = 4), and GrnR493X/R493X (purple) (n = 4) mouse brains. c Quantification of mono-sialyated and di-sialyated ganglioside species isolated from the frontal lobes of control (pink), FTD-TDP43-A (sporadic-non-GRN) (green), and FTD-TDP43-A (GRN) (blue) human brains. Box plots display mean ± the minimum and maximum number in the data set of control (n = 3), FTD-TDP43-A (sporadic-non-GRN) (n = 6) or FTD-TDP43-A (GRN) (n = 12). Box plots display mean ± the minimum and maximum number. One-way ANOVA, followed by multigroup comparison (Dunn’s) test, was performed. *p < 0.05, **p < 0.01. G is for ganglioside; M/D/T are for monosialic, disialic, or trisialic; and the number refers to the order of discovery. GM2-AP GM2 ganglioside activator protein, SAP-B/C/D saposin-B/C/D, GLB1 galactosidase beta 1, HEXA beta-hexosaminidase subunit alpha, NEU3/4 neuraminidase 3/4, GALC galactosylceramidase, GCase glucosylceramidase beta, ASAH1 N-acylsphingosine amidohydrolase 1, SAP-C/D saposin-C/D.
Fig 3: aCDase limits HSV-1 infection and prevents disease.a, b Representative images (a) and quantification (b) of immunofluorescence from wild-type (WT) and Asah1-/- bone marrow derived macrophages (BMDMs) infected with HSV-1 for 6 h (multiplicity of infection [MOI] 10, n = 5, scale bar 50 µm, two-tailed student’s t-test). N.D.: not done. c Immunofluorescence of livers from WT and Asah1-/- mice that were infected with 7 × 107 plaque forming units (PFU) HSV-1 and analyzed after 12 h (n = 6–9, scale bar 100 µm). d, e Real-time polymerase chain reaction (RT-PCR) of lymph nodes (LN), spleens and livers (d, n = 7–10, 2-way ANOVA [Sidak’s multiple comparison]) and tissue culture infection dose 50 (TCID50, e, n = 4–6, two-tailed student’s t-test) of spleens and livers from WT mice and Asah1-/- mice that were infected with 2 × 106 TCID50 HSV-1 and analyzed on day 3. f TCID50 of spleens and livers from WT (n = 9), Samhd1-/- (n = 4), Ifnar-/- (n = 4), and MyD88-/- x Trif-/- x Cardif-/- (n = 3, 2-way ANOVA [Tukey’s multiple comparison]) mice that were infected with 2 × 106 TCID50 HSV-1 and analyzed on day 3. g RT-PCR of spleens and livers from bone marrow chimera mice receiving Cre- Asah1fl/fl or Cre+ Asah1fl/fl bone marrow that were tamoxifen-treated, infected with 6 × 106 TCID50 HSV-1 and analyzed on day 3 (n = 4–5, two-tailed student’s t-test). h Survival of tamoxifen-treated Cre- Asah1fl/fl and Cre+ Asah1fl/fl mice which were infected intravaginally with 2 × 107 TCID50 HSV-1 (n = 11–12, p = 0.0004, Log-rank [Mantel-Cox] test). i Survival of tamoxifen-treated Cre– Asah1fl/fl and Cre+ Asah1fl/fl mice which were infected intravenously with 5 × 105 TCID50 HSV-1 (n = 11–13, p = 0.0004, Log-rank [Mantel-Cox] test) or left uninfected (Cre+ Asah1fl/fl, naïve; n = 5). All data are shown as mean ± SEM. *p = 0.05, **p = 0.01, #p = 0.001, ##p = 0.0001.
Fig 4: Sphingosine protects against HSV-1 infection.a Immunofluorescence of wild-type (WT) macrophages, which were infected with HSV-1 (multiplicity of infection [MOI] 10) and stained for acid ceramidase (aCDase) and HSV-1 (n = 5, blue represents Hoechst staining, scale bar 10 µm) after 30 min. b Schematic representation showing the metabolism of sphingomyelin (SM) to sphingosine-1-phosphate (S1P). c, d Representative image (c) and quantification (d) of immunofluorescence from bone marrow derived macrophages (BMDMs) that have been incubated for 30 min with ß-D-galactosyl ceramide (Cer; 100 µM), D-erythro-sphingosine (Sph; 100 µM), sphingosine kinase inhibitor (SKI-II; 100 µM), ceramidase (CDase; 250 U/L) or sphingomyelinase (SMase; 6.5 U/ml) and subsequently infected with HSV-1 at a multiplicity of infection (MOI) of 100 (n = 5, scale bar 50 µm, one-way ANOVA [Dunnett’s multiple comparison]), fixed and stained after 6 h. N.D.: not done. e Tissue culture infection dose 50 (TCID50) of BMDMs that have been incubated for 30 min with D-erythro-sphingosine (Sph; 40 µM), sphingosine kinase inhibitor (SKI-II; 200 µM), or sphingomyelinase (SMase; 6.5 U/ml), subsequently infected with HSV-1 at a MOI of 1 and analyzed after 24 h (n = 3–4, one-tailed student’s t-test). f TCID50 of THP-1 cells that have been incubated for 30 min with D-erythro-sphingosine (Sph; 400 µM) and subsequently infected with HSV-1 at a MOI of 0.01, analyzed after 6 h (n = 3, two-tailed student’s t-test). g TCID50 of HeLa cells that have been incubated for 30 min with D-erythro-sphingosine (Sph; 100 µM), and sphingosine kinase inhibitor (SKI-II; 100 µM), and subsequently infected with HSV-1 at a MOI of 0.001 analyzed after 24 h (n = 3, one-way ANOVA [Dunnett’s multiple comparison]). h TCID50 of Vero cells that have been incubated for 30 min with D-erythro-sphingosine (Sph; 100 µM), and sphingosine kinase inhibitor (SKI-II; 100 µM), and subsequently infected with HSV-1 at a MOI of 0.01 analyzed after 24 h (n = 3, two-tailed student’s t-test). All data are shown as mean ± SEM. *p = 0.05, **p = 0.01, #p = 0.001, ##p = 0.0001.
Fig 5: Sphingosine-rich intraluminal vesicles trap HSV-1.a, b Representative images (a) and quantification (b) of immunofluorescence from wild-type (WT) and Asah1-/- bone marrow derived macrophages (BMDMs) incubated with HSV-1-VP16-GFP (multiplicity of infection [MOI] 30) for 30 min. Intact viral particles are visible through GFP-tagged tegument protein VP16 (green). Exposed capsids were stained with anti-VP5 (red, n = 17 images, one of two experiments is shown, scale bar 10 µm). c, d Representative electron microscopy images (c) and quantification (d) of WT and Asah1-/- BMDMs infected with HSV-1 (MOI 250) analyzed after 30 min (n = 68 WT images and n = 63 Asah1-/- images from three independent experiments; upper scale bar 400 nm, lower scale bar 200 nm, Mann-Whitney test). e Immunofluorescence of WT BMDMs, which were loaded with clickable ?-azido-sphingosine for 3 h, infected with HSV-1 (MOI 10) for 30 min and then stained for sphingosine, the intraluminal vesicle (ILV) marker CD9, HSV-1 anti-capsid and Hoechst (blue, n = 5, scale bar 20 µm). f, g Representative dot plots (f) and quantification (g) of control liposomes, ceramide-loaded liposomes and sphingosine-loaded liposomes that were incubated with fluorescently labeled HSV-1 for 10 min and then analyzed for HSV-1 binding in flow cytometry (n = 6, 2-way ANOVA [Tukey’s multiple comparison]). All data are shown as mean ± SEM. *p = 0.05, **p = 0.01, #p = 0.001, ##p = 0.0001.
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