Atopic dermatitis and skin disease
Staphylococcus aureus membrane and diacylated lipopeptide induce thymic stromal lymphopoietin in keratinocytes through the Toll-like receptor 2–Toll-like receptor 6 pathway

https://doi.org/10.1016/j.jaci.2010.09.002Get rights and content

Background

Staphylococcus aureus heavily colonizes the lesions of patients with atopic dermatitis (AD) and is known to trigger a worsening of AD. However, the exact mechanism by which S aureus promotes AD is unknown. Thymic stromal lymphopoietin (TSLP), which is highly expressed by keratinocytes in skin lesions of patients with AD and bronchial epithelial cells in asthmatic patients, represents a critical factor linking responses at interfaces between the body and the environment to allergic type 2 immune responses.

Objectives

We sought to examine the ability of synthetic lipopeptides and S aureus to induce TSLP expression in human keratinocytes and identify the pathway of induction.

Methods

We stimulated primary human keratinocytes with lipopeptides and S aureus–derived materials. The release and gene expression of TSLP were measured by means of ELISA and quantitative PCR, respectively.

Results

Diacylated lipopeptide upregulated the expression of TSLP and other proinflammatory molecules. Heat-killed S aureus and the subcellular fractions of S aureus induced TSLP's release, with the membranous fraction having the greatest activity. Small interfering RNA–mediated knockdown of either Toll-like receptor (TLR) 2 or TLR6 inhibited the diacylated lipopeptide– and S aureus membrane–induced TSLP gene expression. S aureus membrane– and diacylated lipopeptide–induced release of TSLP was enhanced by TH2/TNF-α cytokines and partially suppressed by IFN-γ and TGF-β.

Conclusions

The results suggest that ligands for the TLR2-TLR6 heterodimer in S aureus membranes, including diacylated lipoproteins, could promote TH2-type inflammation through TSLP production in keratinocytes, providing an overall picture of the vicious cycles between colonization by S aureus and AD in the TH2-skewed sensitization process, exacerbation of the disease, or both.

Section snippets

Reagents

The recombinant human cytokines used to stimulate keratinocytes were TNF-α (20 ng/mL), IL-4 (100 ng/mL), IL-13 (100 ng/mL), IFN-γ (100 ng/mL), TGF-β (10 ng/mL), and IL-17A (IL-17; 100 ng/mL; R&D Systems, Minneapolis, Minn). The TLR ligands used to stimulate keratinocytes were (S,R)-(2,3-bispalmitoyloxypropyl)-Cys-Gly-Asp-Pro-Lys-His-Pro-Lys-Ser-Phe (10-1000 ng/mL; (FSL-1; InvivoGen, San Diego, Calif); (S)-[2,3-Bis(palmitoyloxy)-(2-RS)-propyl]-N-palmitoyl-(R)-Cys-(S)-Ser-(S)-Lys4-OH, 3HCl (5

Diacylated lipopeptide induced release of TSLP in keratinocytes

We examined whether PGN from S aureus, the synthetic triacylated lipopeptide Pam3CSK4, and the synthetic diacylated lipopeptide FSL-1 induce the release of TSLP. Keratinocytes treated with FSL-1 released detectable amounts of TSLP and IL-8 (Fig 1). PGN and Pam3CSK4 did not induce the release of TSLP at the concentrations tested. Pam3CSK4 induced the release of IL-8 but not TSLP.

The stimulation of keratinocytes with FSL-1 induced the gene (Fig 2, A) or protein (Figs 1 and 2, B) expression of not

Discussion

The human innate immune system recognizes bacterial lipoproteins through TLR2. Some recent reports revealed that, among bacterial products reported as TLR2 agonists, only lipoproteins/lipopeptides are sensed at physiological concentrations by TLR2 and suggested that the TLR2 agonistic activity of the fractions prepared from bacteria, such as PGN and lipoteichoic acid (LTA), is likely due to contaminating, highly active natural lipoproteins/lipopeptides.26, 27, 29, 30 TLR2 forms a heterodimer

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    Supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology, Japan (to T. T.).

    Disclosure of potential conflict of interest: The authors have declared that they have no conflict of interest.

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