Hydrolysis of sphingomyelin to ceramide induced by heat-killed Lactoplantibacillus plantarum APsulloc 331261 via neutral sphingomyelinase 2 activation protects against Staphylococcal α-toxin–induced cytotoxicity in keratinocytes
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Presented by: Minjung Chae
Introduction
Atopic dermatitis (AD) is a chronic inflammatory skin disease associated with skin barrier dysfunction as the initial step of the development of AD. The function of skin barrier is based on lipid matrix, which are composed of ceramides, cholesterol and free fatty acids in the outermost layer of the skin, the stratum corneum (SC). In particularly, reduction of ceramides in the SC is involved in barrier impairment in AD. In addition, the colonization of Staphylococcus aureus (S. aureus) in the skin have an important role in the pathogenesis of AD. The most prominent and destructive virulence factor produced by S.aureus is α-toxin, inducing cell death by forming heptameric pores that penetrate though the lipid bilayer. Recently, oral or topical application of probiotics has shown to provide incredible health benefits to AD treatment. The effect of a selected probiotic extract in increasing ceramide levels on the SC in AD patients is reported, there was no direct evidence how probiotic extract can modulate ceramide production and S. aureus α-toxin sensitivity in human keratinocytes.
Aim of the paper
In the present study, we investigated the effect of Lactoplantibacillus plantarum APsulloc 331261 (APsulloc 331261) extract on the mechanism of ceramide synthesis and the S. aureus α-toxin-induced toxicity.
Methods
APsulloc 331261 adjusted to 109 cfu/ml was heat killed at 80°C for 10 min in a water bath. Normal human epidermal keratinocytes (NHEKs) were incubated with heat-killed APsulloc 331261 at various concentrations (0, 107, and 108 cfu/ml) under either low calcium (50 μM) or high calcium (1.2 mM). RNA expression responsible for ceramide synthesis was determined using quantitative real time PCR and protein expression of neutral sphingomyelinase 2 (SMPD3) was determined by western blotting. Lipids of NHEKs were extracted by Bligh Dyer method and analyzed ceramide species (NDS, NS, NP, AP, AS) by liquid chromatography-mass spectrometry. α-toxin-induced cytotoxicity was measured by lactate dehydrogenase release.
Results
We found that, among ten genes responsible for ceramide synthesis, SMPD3 which catalyzes the hydrolysis of sphingomyelin to form ceramide and phosphocholine, was the only gene that was upregulated by heat-killed APsulloc 331261 under both low calcium and high calcium in NHEKs. Western blot analysis also showed that heat-killed APsulloc 331261 increased the expression of SMPD3. mRNA and protein expression of SMPD3 was drastically upregulated during NHEK differentiation and the upregulation of SMPD3 expression induced by APsulloc 331261 was blunted upon peroxisome proliferator-activated receptor (PPAR) δ antagonist (GSK3787, 10 μM), suggesting PPAR dependent mechanism of action of heat-killed APsulloc 331261. Lipid analysis showed that total ceramides level was upregulated, which was diminished by SMPD3 inhibitor (GW4869, 5 μM). Finally, we determined that heat-killed APsulloc 331261 protected α-toxin cytotoxicity in a dose dependent manner and the protection was abrogated by SMPD3 inhibitor.
Conclusion and Discussion
The present study demonstrated that heat-killed APsulloc 331261 stimulated SMPD3 expression, resulting in the increase of ceramide at the expense of sphingomyelin. SMPD3 expression was upregulated during NEHK differentiation and the induction of SMPD3 expression dependent on heat-killed APsulloc 331261 was mediated by PPAR activation. Heat-killed APsulloc 331261 could confer protection against α-toxin–induced cell death. The action mechanism underlying the beneficial effects of heat-killed APsulloc 331261 on the α-toxin–induced cytotoxicity may be attributed to possibly the reduction of the number of α-toxin binding sites on the cell surface due to enzymatic activity of SMPD3-mediated cleavage of sphingomyelin because sphingomyelin is one of the known α-toxin receptors. The effect of heat-killed APsulloc 331261 on sphingomyelin level is undergoing in our laboratory. These results suggest that heat-killed APsulloc 331261 could contribute to the improvement of lipid barrier and a more effective resistance against S. aureus infection. Thus, heat-killed APsulloc 331261 are potential bioactive supplement to provide health benefits to skin health.
Atopic dermatitis (AD) is a chronic inflammatory skin disease associated with skin barrier dysfunction as the initial step of the development of AD. The function of skin barrier is based on lipid matrix, which are composed of ceramides, cholesterol and free fatty acids in the outermost layer of the skin, the stratum corneum (SC). In particularly, reduction of ceramides in the SC is involved in barrier impairment in AD. In addition, the colonization of Staphylococcus aureus (S. aureus) in the skin have an important role in the pathogenesis of AD. The most prominent and destructive virulence factor produced by S.aureus is α-toxin, inducing cell death by forming heptameric pores that penetrate though the lipid bilayer. Recently, oral or topical application of probiotics has shown to provide incredible health benefits to AD treatment. The effect of a selected probiotic extract in increasing ceramide levels on the SC in AD patients is reported, there was no direct evidence how probiotic extract can modulate ceramide production and S. aureus α-toxin sensitivity in human keratinocytes.
Aim of the paper
In the present study, we investigated the effect of Lactoplantibacillus plantarum APsulloc 331261 (APsulloc 331261) extract on the mechanism of ceramide synthesis and the S. aureus α-toxin-induced toxicity.
Methods
APsulloc 331261 adjusted to 109 cfu/ml was heat killed at 80°C for 10 min in a water bath. Normal human epidermal keratinocytes (NHEKs) were incubated with heat-killed APsulloc 331261 at various concentrations (0, 107, and 108 cfu/ml) under either low calcium (50 μM) or high calcium (1.2 mM). RNA expression responsible for ceramide synthesis was determined using quantitative real time PCR and protein expression of neutral sphingomyelinase 2 (SMPD3) was determined by western blotting. Lipids of NHEKs were extracted by Bligh Dyer method and analyzed ceramide species (NDS, NS, NP, AP, AS) by liquid chromatography-mass spectrometry. α-toxin-induced cytotoxicity was measured by lactate dehydrogenase release.
Results
We found that, among ten genes responsible for ceramide synthesis, SMPD3 which catalyzes the hydrolysis of sphingomyelin to form ceramide and phosphocholine, was the only gene that was upregulated by heat-killed APsulloc 331261 under both low calcium and high calcium in NHEKs. Western blot analysis also showed that heat-killed APsulloc 331261 increased the expression of SMPD3. mRNA and protein expression of SMPD3 was drastically upregulated during NHEK differentiation and the upregulation of SMPD3 expression induced by APsulloc 331261 was blunted upon peroxisome proliferator-activated receptor (PPAR) δ antagonist (GSK3787, 10 μM), suggesting PPAR dependent mechanism of action of heat-killed APsulloc 331261. Lipid analysis showed that total ceramides level was upregulated, which was diminished by SMPD3 inhibitor (GW4869, 5 μM). Finally, we determined that heat-killed APsulloc 331261 protected α-toxin cytotoxicity in a dose dependent manner and the protection was abrogated by SMPD3 inhibitor.
Conclusion and Discussion
The present study demonstrated that heat-killed APsulloc 331261 stimulated SMPD3 expression, resulting in the increase of ceramide at the expense of sphingomyelin. SMPD3 expression was upregulated during NEHK differentiation and the induction of SMPD3 expression dependent on heat-killed APsulloc 331261 was mediated by PPAR activation. Heat-killed APsulloc 331261 could confer protection against α-toxin–induced cell death. The action mechanism underlying the beneficial effects of heat-killed APsulloc 331261 on the α-toxin–induced cytotoxicity may be attributed to possibly the reduction of the number of α-toxin binding sites on the cell surface due to enzymatic activity of SMPD3-mediated cleavage of sphingomyelin because sphingomyelin is one of the known α-toxin receptors. The effect of heat-killed APsulloc 331261 on sphingomyelin level is undergoing in our laboratory. These results suggest that heat-killed APsulloc 331261 could contribute to the improvement of lipid barrier and a more effective resistance against S. aureus infection. Thus, heat-killed APsulloc 331261 are potential bioactive supplement to provide health benefits to skin health.