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Pseudomonas-derived ceramidase induces production of inflammatory mediators from human keratinocytes via sphingosine-1-phosphate.

ABSTRACT: Ceramide is important for water retention and permeability barrier functions in the stratum corneum, and plays a key role in the pathogenesis of atopic dermatitis (AD). A Pseudomonas aeruginosa-derived neutral ceramidase (PaCDase) isolated from a patient with AD was shown to effectively degrade ceramide in the presence of Staphylococcus aureus-derived lipids or neutral detergents. However, the effect of ceramide metabolites on the functions of differentiating keratinocytes is poorly understood. We found that the ceramide metabolite sphingosine-1-phosphate (S1P) stimulated the production of inflammatory mediators such as TNF-? and IL-8 from three-dimensionally cultured human primary keratinocytes (termed "3D keratinocytes"), which form a stratum corneum. PaCDase alone did not affect TNF-? gene expression in 3D keratinocytes. In the presence of the detergent Triton X-100, which damages stratum corneum structure, PaCDase, but not heat-inactivated PaCDase or PaCDase-inactive mutant, induced the production of TNF-?, endothelin-1, and IL-8, indicating that this production was dependent on ceramidase activity. Among various ceramide metabolites, sphingosine and S1P enhanced the gene expression of TNF-?, endothelin-1, and IL-8. The PaCDase-enhanced expression of these genes was inhibited by a sphingosine kinase inhibitor and by an S1P receptor antagonist VPC 23019. The TNF-?-binding antibody infliximab suppressed the PaCDase-induced upregulation of IL-8, but not TNF-?, mRNA. PaCDase induced NF-?B p65 phosphorylation. The NF-?B inhibitor curcumin significantly inhibited PaCDase-induced expression of IL-8 and endothelin-1. VPC 23019 and infliximab inhibited PaCDase-induced NF-?B p65 phosphorylation and reduction in the protein level of the NF-?B inhibitor I?B?. Collectively, these findings suggest that (i) 3D keratinocytes produce S1P from sphingosine, which is produced through the hydrolysis of ceramide by PaCDase, (ii) S1P induces the production of TNF-? via S1P receptors, and (iii) released TNF-? stimulates the production of inflammatory mediators such as IL-8.

SUBMITTER: Oizumi A

PROVIDER: S-EPMC3934885 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature

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Pseudomonas-derived ceramidase induces production of inflammatory mediators from human keratinocytes via sphingosine-1-phosphate.

Oizumi Ami A Nakayama Hitoshi H Okino Nozomu N Iwahara Chihiro C Kina Katsunari K Matsumoto Ryo R Ogawa Hideoki H Takamori Kenji K Ito Makoto M Suga Yasushi Y Iwabuchi Kazuhisa K

PloS one 20140225 2

Ceramide is important for water retention and permeability barrier functions in the stratum corneum, and plays a key role in the pathogenesis of atopic dermatitis (AD). A Pseudomonas aeruginosa-derived neutral ceramidase (PaCDase) isolated from a patient with AD was shown to effectively degrade ceramide in the presence of Staphylococcus aureus-derived lipids or neutral detergents. However, the effect of ceramide metabolites on the functions of differentiating keratinocytes is poorly understood. ...[more]

PMID: 24586752

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Project description:Background: The pathophysiology of non-alcoholic steatohepatitis involves hepatocyte lipotoxicity due to excess saturated free fatty acids and concomitant proinflammatory macrophage effector responses. These include the infiltration of macrophages into hepatic cords in response to incompletely understood stimuli. Stressed hepatocytes release an increased number of extracellular vesicles (EVs), which are known to participate in intercellular signaling and coordination of the behavior of immune cell populations via their cargo. We hypothesized that hepatocyte-derived lipotoxic EVs that are enriched in sphingosine 1-phosphate (S1P) are effectors of macrophage infiltration in the hepatic microenvironment. Methods: Lipotoxic EVs were isolated from palmitate treated immortalized mouse hepatocytes and characterized by nanoparticle tracking analysis. Lipotoxic EV sphingolipids were quantified using tandem mass spectrometry. Wildtype and S1P1 receptor knockout bone marrow-derived macrophages were exposed to lipotoxic EV gradients in a microfluidic gradient generator. Macrophage migration toward EV gradients was captured by time-lapse microscopy and analyzed to determine directional migration. Fluorescence-activated cell sorting along with quantitative PCR and immunohistochemistry were utilized to characterize the cell surface expression of S1P1 receptor on intrahepatic leukocytes and hepatic expression of S1P1 receptor, respectively. Results: Palmitate treatment induced the release of EVs. These EVs were enriched in S1P. Palmitate-induced S1P enriched EVs were chemoattractive to macrophages. EV S1P enrichment depended on the activity of sphingosine kinases 1 and 2, such that, pharmacological inhibition of sphingosine kinases 1 and 2 resulted in a significant reduction in EV S1P cargo without affecting the number of EVs released. When exposed to EVs derived from cells treated with palmitate in the presence of a pharmacologic inhibitor of sphingosine kinases 1 and 2, macrophages displayed diminished chemotactic behavior. To determine receptor-ligand specificity, we tested the migration responses of macrophages genetically deleted in the S1P1 receptor toward lipotoxic EVs. S1P1 receptor knockout macrophages displayed a marked reduction in their chemotactic responses toward lipotoxic palmitate-induced EVs. Conclusions:Palmitate-induced lipotoxic EVs are enriched in S1P through sphingosine kinases 1 and 2. S1P-enriched EVs activate persistent and directional macrophage chemotaxis mediated by the S1P1 receptor, a potential signaling axis for macrophage infiltration during hepatic lipotoxicity, and a potential therapeutic target for non-alcoholic steatohepatitis.

Dataset Information

Pseudomonas-derived ceramidase induces production of inflammatory mediators from human keratinocytes via sphingosine-1-phosphate.

Publications

Pseudomonas-derived ceramidase induces production of inflammatory mediators from human keratinocytes via sphingosine-1-phosphate.

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