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Acid ceramidase of macrophages traps herpes simplex virus in multivesicular bodies and protects from severe disease

ABSTRACT: Macrophages have important protective functions during infection with herpes simplex virus type 1 (HSV-1). However, molecular mechanisms that restrict viral propagation and protect from severe disease are unclear. Here we found that macrophages take up HSV-1 via endocytosis and transport the virions into multivesicular bodies (MVBs). In MVBs, acid ceramidase (aCDase) converts ceramide into sphingosine and increases the formation of sphingosine-rich intraluminal vesicles (ILVs). Once HSV-1 particles pass MVBs, sphingosine-rich ILVs bind to HSV-1 particles, which restricts fusion with the limiting endosomal membrane and prevents cellular infection. Lack of aCDase in macrophage cultures or in Asah1–/– mice results in replication of HSV-1 and Asah1–/– mice died soon after systemic or intravaginal inoculation. The treatment of macrophages with sphingosine enhancing compounds blocks HSV-1 propagation, suggesting a therapeutic potential of this pathway. In conclusion, we demonstrate that aCDase loads ILVs with sphingosine, which prevents HSV-1 capsids from penetrating into the cytosol.

ORGANISM(S): Mus musculus

PROVIDER: GSE142175 | GEO | 2020/01/14

REPOSITORIES: GEO

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Acid ceramidase regulates innate immune memory

Project description:Innate immune memory, also called "trained immunity," is a metabolically and epigenetically regulated functional state of myeloid cells. This phenomenon is important for host defense, but also plays a role in various immune-mediated conditions. We found that exogenously administered sphingolipids and inhibition of enzymes involved in sphingolipid metabolism modulate trained immunity. In particular, we found that acid ceramidase, an enzyme that converts ceramide to sphingosine, is a potent regulator of trained immunity. We discovered that acid ceramidase regulates the expression of genes encoding histone-modifying enzymes, resulting in profound changes in the epigenetic landscape. We confirmed our findings by identifying single-nucleotide polymorphisms in the region of ASAH1, the gene encoding acid ceramidase, that are associated with the trained immunity cytokine response. Our findings reveal a novel immunomodulatory effect of sphingolipids, provide new insight into the metabolic regulation of trained immunity, and identify acid ceramidase as a therapeutic target to modulate it.

2023-11-27 | GSE188641 | GEO

Intermittent bulk release of human cytomegalovirus

Project description:Human Cytomegalovirus (HCMV) significantly remodels host cell processes and membranes to enable effective production of virus progeny. Nascent capsids exit the nucleus and undergo a final morphogenesis process in the cytoplasm where they associate with tegument and membranes. During this process called secondary envelopment, fully assembled virions are formed which are wrapped inside of host vesicles. Both the origin of the receiving membrane as well as the dynamic organization of the envelopment and exocytosis process are not well understood. Enveloped virus particles can be both found individually wrapped in small vesicles but also as accumulations in multivesicular bodies (MVBs). While individually wrapped particles are believed to exit the cell by exocytosis, it is unclear if particles in MVBs ever leave the cell or are actually targeted for lysosomal degradation. Using a novel correlative light and electron microscopy workflow that allows three-dimensional imaging of whole cells, we present evidence that HCMV capsids can bud into MVBs, generating large intracellular accumulations of enveloped virions. Immunofluorescence and fusion assays suggest that these MVBs are positive for markers of the exosomal pathway. Time-lapse as well as functional live-cell imaging showed that MVBs fuse with the plasma membrane and release patch-like accumulations of virus particles. Our data suggests a so far undescribed envelopment and exocytosis pathway of HCMV, possibly involving the late-endosome/exosome pathway, leading to the intermittent bulk release of virus particles.

2022-06-23 | PXD023444 | Pride

Mass spectrometric characterization of HSV-1 L-particles from human dendritic cells and BHK21 cells and analysis of their functional role

Project description:Herpes simplex virus type 1 (HSV-1) is a very common human pathogenic virus among the world’s 12 population. The lytic replication cycle of HSV-1 is, amongst others, characterized by a tripartite viral 13 gene expression cascade, the assembly of nucleocapsids involving their subsequent nuclear egress, 14 tegumentation, re-envelopment and the final release of progeny viral particles. During productive 15 infection of a multitude of different cell types, HSV-1 generates not only infectious heavy (H-) 16 particles, but also non-infectious light (L-) particles, lacking the capsid. In monocyte-derived mature 17 dendritic cells (mDCs), HSV-1 causes a non-productive infection with the predominant release of L-18 particles. Until now, the generation and function of L-particles is not well understood, however, they 19 are described as factors transferring viral components to the cellular microenvironment. To obtain 20 deeper insights into the L-particle composition, we performed a mass-spectrometry-based analysis of 21 L-particles derived from HSV-1-infected mDCs or BHK21 cells and H-particles from the latter one. 22 In total, we detected 63 viral proteins in both H- and L-particle preparations derived from HSV-1-23 infected BHK21 cells. In L-particles from HSV-1-infected mDCs we identified 41 viral proteins 24 which are differentially distributed compared to L-particles from BHK21 cells. In this study, we 25 present data suggesting that L-particles modify mDCs and suppress their T cell stimulatory capacity. 26 Due to the plethora of specific viral proteins incorporated into and transmitted by L-particles, it is 27 tempting to speculate that L-particles manipulate non-infected bystander cells for the benefit of the 28 virus.

2021-09-09 | PXD020845 | Pride

Mass spectrometric characterization of HSV-1 L-particles from human dendritic cells and BHK21 cells and analysis of their functional role Part B

2021-09-09 | PXD020846 | Pride

Pseudomonas-derived Ceramidase Induces Production of Inflammatory Mediators from Human Keratinocytes via Sphingosine-1-Phosphate

2014-12-25 | GSE53670 | GEO

Presence of ceramidase activity in electronegative LDL

Project description:Electronegative LDL (LDL(-)) is a minor modified fraction of human plasma LDL with several atherogenic properties. Among them, LDL(-) has increased content of bioactive lipid mediators, such as lysophosphatidylcholine (LPC), non-esterified fatty acids (NEFA), ceramide (Cer), and sphingosine (Sph), which are related to the presence in LDL(-) of some phospholipolytic activities, including platelet-activating factor acetylhydrolase (PAF-AH), phospholipase C (PLC) and sphingomyelinase (SMase). However, the activity of these enzymes does not explain the increased content of Sph, which should derive from Cer degradation. In the present study we analyzed the putative presence of a ceramidase (CDase) activity that could explain the increased content of Sph. Thin layer chromatography (TLC) and lipidomic analysis showed that Cer, Sph and NEFA spon-taneously increased in LDL(-) incubated alone at 37ºC, in contrast with native LDL(+). An inhibitor of neutral CDase prevented the formation of Sph and, in contrast, increased the content of Cer in LDL(-). In addition, a fluorescently-labelled Cer (NBD-Cer) was efficiently degraded by LDL(-) to form Sph and NEFA. These observations point to the existence of a CDase activity associated to LDL(-). However, neither proteomic analysis nor western blot detected the presence of a known CDase enzyme. Further studies are warranted to define the origin of the CDase activity detected in LDL(-).

2023-01-09 | PXD036168 | Pride

Caveolae-mediated endocytosis of VLDL-sized emulsion particles in macrophages requires NPC1 and STARD3 for further lysosomal processing

Project description:Triglyceride-rich lipoproteins and their remnants contribute to atherosclerosis, possibly by carrying remnant cholesterol and/or by exerting a pro-inflammatory effect on macrophages. Nevertheless, little is known about how macrophages process triglyceride-rich lipoproteins. We show that uptake by macrophages of VLDL-sized emulsion particles is dependent on the enzyme lipoprotein lipase via its C-terminal domain. Subsequent internalization of VLDL-triglycerides by macrophages is carried out by caveolae-mediated endocytosis, followed by hydrolysis by lysosomal acid lipase. STARD3 is required for the transfer of lysosomal fatty acids to the ER for lipid storage, while NPC1 likely is involved in promoting the extracellular efflux of fatty acids. Our data provide novel insights into how macrophages process VLDL-derived triglycerides and suggest that macrophages have the remarkable capacity to excrete internalized triglycerides as fatty acids.

2022-05-21 | GSE203250 | GEO

Sequencing of HSV-1 genomic DNA to identify genomic variation resulting from selective pressure imposed by mutation of the PP1 binding site in the pUL21 gene

Project description:The pUL21 gene is essential for efficient replication and dissemination in herpes simplex virus 1 (HSV-1) but the molecular basis for its function is not well understood. Experimental work revealed that pUL21 is a virus-encoded adaptor of protein phosphatase 1 (PP1). Mutation of the specific motif in pUL21 responsible for PPI recruitment, followed by propagation in cell culture, was performed in order to identify the impact of preventing PP1 recruitment on the viral genome. This allowed identification of compensatory mutations disrupting the viral kinase gene pUS3, which restored viral growth and spread, revealing that pUL21 directly antagonises pUS3. Three mutant strains of HSV-1 were generated, mutating the conserved residues F242 and V243 in the PP1 binding sequence, individually and in combination (pUL21F242E, pUL21V243D, pUL21FV242AA), along with a knockout strain lacking pUL21 (ΔpUL21). These four virus stocks, plus wild-type HSV-1, were propagated in Vero cells for three passages. Genomic DNA was extracted from the P3 viruses and sequenced with Illumina. Reads were trimmed and mapped to an edited version of the HSV-1 genome (based on JQ673480.1) and read depth and variants were quantified.

2021-07-31 | E-MTAB-10788 | biostudies-arrayexpress

Pseudomonas-derived Ceramidase Induces Production of Inflammatory Mediators from Human Keratinocytes via Sphingosine-1-Phosphate

Project description:A Pseudomonas aeruginosa-derived neutral ceramidase (PaCDase) isolated from a patient with atopic dermatitis was shown to effectively degrade ceramide in the presence of Staphylococcus aureus-derived lipids or neutral detergents. To understand the effect of ceramide metabolites on the functions of differentiating keratinocytes, we have conducted genes expressions analysis from PaCDase-, sphingosine-1-phosphate (S1P)-treated or untreated three-dimensionally cultured human primary keratinocytes, which form a stratum corneum, in the presence of Triton X-100 using high resolution DNA microarray. To evaluate the effects of PaCDase and S1P on keratinocyte functions, we applied an epicutaneous 24-h patch test on the EPI-Model in keratinocyte serum-free medium (keratinocyte-SFM) (Invitrogen; Carlsbad, CA, USA) by the method of Spiekstra et al. [25] with some modification. The Triton X-100 treated samples were labeled using Cy5, and the PaCDase or S1P samples were labeled with Cy3.

2014-12-25 | E-GEOD-53670 | biostudies-arrayexpress

RNA-seq analysis of Sphingosine 18:1 treated mice bone marrow-derived macrophages

Project description:Purpose: Study of the influence of Sphingosine 18:1 in macrophages Method: 2μM Sphingosine 18:1 was added to BMDMs for 6 hours, solvent was added as control. mRNA profiles were generated by deep sequencing. Results: Several signaling pathways were changed after Sphingosine administration, including hypoxia-related signal pathway. RT-quantitive PCR was performed to validate the expression of Epas1 and its downstream target genes, results showed that Sphingosine 18:1 significantly inhibits Epas1 expression.

2024-03-21 | GSE262135 | GEO

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