Sphingosine kinase 1 activation enhances epidermal innate immunity through sphingosine-1-phosphate stimulation of cathelicidin production

Abstract

Background: The ceramide metabolite, sphingosine-1-phosphate (S1P), regulates multiple cellular functions in keratinocytes (KC). We recently discovered that production of a key innate immune element, cathelicidin antimicrobial peptide (CAMP), is stimulated via a NF-κB-dependent mechanism that is activated by S1P when S1P is generated by sphingosine kinase (SPHK) 1.

Objective: We investigated whether pharmacological modulation of SPHK1 activity, using a novel synthetic SPHK1 activator, (S)-methyl 2-(hexanamide)-3-(4-hydroxyphenyl) propanoate (MHP), stimulates CAMP expression.

Methods: MHP-mediated changes in both S1P and CAMP downstream mediators were analyzed in normal cultured human KC by qRT-PCR, Western immunoblot, ELISA, confocal microscopy for immunohistochemistry, HPLC and ESI-LC/MS/MS, and microbial pathogen invasion/colonization in a human epidermal organotypic model.

Results: Treatment with MHP directly activated SPHK1 and increased cellular S1P content in normal cultured human KC. Because MHP did not inhibit S1P lyase activity, which hydrolyses S1P, augumented S1P levels could be attributed to increased synthesis rather than blockade of S1P degradation. Next, we found that exogenous MHP significantly stimulated CAMP mRNA and protein production in KC, increases that were significantly suppressed by siRNA directed against SPHK1, but not by a scrambled control siRNA. NF-κB activation, assessed by nuclear translocation of NF-κB, occurred in cells following incubation with MHP. Conversely, pretreatment with a specific inhibitor of SPHK1 decreased MHP-induced nuclear translocation of NF-κB, and significantly attenuated the MHP-mediated increase in CAMP production. Finally, topical MHP significantly suppressed invasion of the virulent Staphylococcus aureus into murine skin explants.

Conclusion: MHP activation of SPHK1, a target enzyme of CAMP production, can stimulate innate immunity.

Keywords: Antimicrobial defense; Cathelicidin antimicrobial peptide; Small molecule; Sphingosine kinase 1.

Fig. 1. MHP increases CAMP mRNA and protein expression

The chemical structure of MHP (A). Primary cultured keratinocytes (KC) were incubated with the indicated concentrations of MHP for 24 h. Cell toxicity was assessed by MTT assay (B). CAMP mRNA (C) and protein expression (D) was determined by qRT-PCR and ELISA, respectively. Similar results were obtained when the experiment was repeated (more than twice) using different cell preparations. Values are means ± SD (n=3). Means with different letters differ at p < 0.01 by one-way ANOVA coupled with Duncan’s multiple comparison test (C).

Fig. 2. SPHK1 activity is required for MHP-induced increase in CAMP expression

KC pretreated with or without siRNA against SPHK1 were treated with MHP for 24 h. CAMP protein levels were assessed by Western immunoblot analysis (A). Intensity of individual bands was quantified using a LAS-3000 chemiluminescent image analyzer (Fujifilm). The ratio of CAMP band to that of β-actin in the scrambled siRNA (siControl)-transfected, vehicle-treated cells was set to 100%, to which the corresponding ratios in the other samples were normalized (B). Similar results were obtained when the experiment was repeated (more than twice) using different cell preparations.

Fig. 3. NF-κB activation is required for MHP-induced upregulation of CAMP production

KC were pretreated with or without either SPHK1 inhibitor, SKI (1 μM, 30 min) or NF-κB inhibitor, BAY11-7082 (1 μM, 30 min), were incubated with MHP for 30 min or 24 h. MHP-induced nuclear translocation of NF-κB was determined by immunofluorescence (A). CAMP protein expression was determined by western immunoblot analysis (B). The ratio of CAMP band to that of β-actin in the vehicle-treated cells was set to 100%, to which the corresponding ratios in the other samples were normalized (C). Similar results were obtained when the experiment was repeated (in triplicate) using different cell preparations.

Fig. 4. Inhibition of S. aureus invasion into murine skin

S. aureus was applied epicutaneouly to full-thickness pieces of murine skin (n=3) treated with or without MHP, followed by incubation for 6 h at 37°C. Hematoxylin and eosin (H&E) staining (A, top panel). MHP-induced mCAMP expression was determined by immunofluorescence (A, middle panel). Bacterial invasion in stratum corneum was assessed by Gram staining (A, bottom panel). The number of invasive bacteria was counted in stratum corneum (B). Data are means ± SD (n=3). Arrows indicate invaded bacteria.