Anh Tuan Vu

Cytokine milieu modulates release of thymic stromal lymphopoietin from human keratinocytes stimulated with double-stranded RNA

BACKGROUND Thymic stromal lymphopoietin (TSLP) plays a key role in allergic diseases, such as atopic dermatitis (AD) and asthma. TSLP is highly expressed by keratinocytes in skin lesions of patients with AD, but environmental triggers for its release from keratinocytes with endogenous factors are not well understood. Patients with AD, in whom allergic sensitization is already established, are susceptible to viral dissemination. OBJECTIVES We investigated TSLPs release from primary human keratinocytes stimulated with a Toll-like receptor (TLR) 3 ligand, polyinosinic-polycytidylic acid, which mimics viral double-stranded RNA (dsRNA), and its modulation by cytokines. METHODS Primary human keratinocytes were stimulated with TLR ligands, cytokines, or both. TSLP released into culture supernatants was measured by means of ELISA. RESULTS Stimulation of keratinocytes with dsRNA induced release of TSLP and upregulated gene expression of TSLP and other cytokines and chemokines. The release of TSLP was enhanced by the addition of IL-4, IL-13, and/or TNF-alpha. With or without the T(H)2/TNF cytokines, the dsRNA-induced release of TSLP was upregulated by IFN-alpha and IFN-beta and suppressed by IFN-gamma, TGF-beta, or IL-17. CONCLUSIONS The effect of the TLR3 ligand on keratinocytes suggests contribution of viral dsRNA to skin inflammations under the influence of a cytokine milieu. The results imply that viral dsRNA and a T(H)2 cytokine milieu might promote T(H)2-type inflammation through an induction of TSLP expression, suggesting that a vicious cycle exists between AD with T(H)2-type inflammation and viral infections and a possible blockade of this cycle by other cytokine milieus provided by cells, such as T(H)1, regulatory T, and T(H)17 cells.

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

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 TSLPs 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 T(H)2/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 T(H)2-type inflammation through TSLP production in keratinocytes, providing an overall picture of the vicious cycles between colonization by S. aureus and AD in the T(H)2-skewed sensitization process, exacerbation of the disease, or both.

Flagellin induces the expression of thymic stromal lymphopoietin in human keratinocytes via toll-like receptor 5.

Background: Thymic stromal lymphopoietin (TSLP), highly expressed by keratinocytes in skin lesions of atopic dermatitis patients and bronchial epithelial cells in asthma, plays a key role in allergic diseases. Information on triggers for the release of TSLP in keratinocytes is still limited. Keratinocytes express Toll-like receptor (TLR) 5, the ligand for which is flagellin, the major structural protein of the flagella of Gram-negative bacteria. IL-4, IL-13 and TNF-α (Th2/TNF) are associated with allergic diseases. TGF-α, one of the ligands for the epidermal growth factor receptor, is overexpressed in keratinocytes in atopic dermatitis. We investigated the induction of TSLP expression in keratinocytes stimulated with flagellin and its modulation by the Th2/TNF cytokines and TGF-α. Methods: Primary human keratinocytes were stimulated with flagellin with or without cytokines. The TSLP released was measured by ELISA. Gene expression was analyzed by quantitative real-time PCR. Results: Stimulation of keratinocytes with flagellin induced the release of TSLP protein and upregulation of the gene expression of TSLP and other pro-inflammatory molecules. The flagellin-induced release of TSLP was enhanced by the Th2/TNF cytokines or TGF-α. Small interfering RNA-mediated knockdown of TLR5 expression suppressed the flagellin-induced TSLP gene expression. Conclusions: Flagellin induces TSLP expression in keratinocytes via TLR5 and the expression can be upregulated by a cytokine milieu with Th2/TNF or TGF-α, suggesting that exposure of barrier-defective skin to Gram-negative bacteria or environmental flagellin contributes to the initiation and/or amplification of Th2-type skin inflammation including atopic dermatitis through the induction of TSLP expression in keratinocytes.

Extracellular Double-Stranded RNA Induces TSLP via an Endosomal Acidification- and NF-κB-Dependent Pathway in Human Keratinocytes

Double-stranded RNA (dsRNA) causes keratinocytes to release thymic stromal lymphopoietin (TSLP), which plays a key role in allergic diseases. Endosomal Toll-like receptor 3 (TLR3) and cytosolic RIG-like receptors (RLRs) and PKR have been reported to recognize dsRNA. Here, we demonstrate that dsRNA induces TSLP in keratinocytes via an endosomal acidification-dependent and NF-κB-mediated pathway. After treatment with pharmacologic inhibitors or transfection with small interfering RNAs (siRNAs), primary human keratinocytes were stimulated. Bafilomycin A1, which inhibits endosomal acidification to block the TLR3 pathway, blocked the dsRNA-induced expression of TSLP, IL-8, IFN-β, and other molecules including the dsRNA sensors, whereas it did not inhibit diacyllipopeptide-induced expression of TSLP and IL-8. The dsRNA-induced gene expression of TSLP depended on RelA, a component of NF-κB, but not IRF3, similar to IL-8 but different from IFN-β, which depended on both IRF3 and RelA. The results indicate that endosomal acidification and the subsequent activation of NF-κB are necessary to sense extracellular dsRNA, suggesting the importance of the TLR3-NF-κB axis to trigger production of TSLP against the self dsRNA released from damaged cells or viral dsRNA, in the epidermis, relating to skin inflammation including atopic dermatitis (AD).

TSLP expression induced via Toll-like receptor pathways in human keratinocytes.

The skin epidermis and mucosal epithelia (airway, ocular tissues, gut, and so on) are located at the interface between the body and environment and have critical roles in the response to various stimuli. Thymic stromal lymphopoietin (TSLP), a cytokine expressed mainly by epidermal keratinocytes (KCs) and mucosal epithelial cells, is a critical factor linking the innate response at barrier surfaces to Th2-skewed acquired immune response. TSLP is highly expressed in skin lesions of atopic dermatitis patients. Here, we describe on Toll-like receptor (TLR)-mediated induction of TSLP expression in primary cultured human KCs, placing emphasis on experimental methods used in our studies. Double-stranded RNA (TLR3 ligand), flagellin (TLR5 ligand), and diacylated lipopeptide (TLR2-TLR6 ligand) stimulated human KCs to express TSLP and Staphylococcus aureus membranes did so via the TLR2-TLR6 pathway. Atopic cytokine milieu upregulated the TLR-mediated induction of TSLP. Culturing in the absence of glucocorticoid before stimulation enhanced the TSLP expression. Extracellular double-stranded RNA induced TSLP via endosomal acidification- and NF-κB-dependent pathway. Specific measurement of the long TSLP transcript, which contributes to the production of the TSLP protein, rather than total or the short transcript is useful for accurate detection of functional human TSLP gene expression. The results suggest that environment-, infection-, and/or self-derived TLR ligands contribute to the initiation and/or amplification of Th2-type skin inflammation including atopic dermatitis and atopic march through the induction of TSLP expression in KCs and include information helpful for understanding the role of the gene-environment interaction relevant in allergic diseases.

Glucocorticoids Inhibit Double-Stranded RNA-Induced Thymic Stromal Lymphopoietin Release from Keratinocytes in an Atopic Cytokine Milieu More Effectively than Tacrolimus

Background: Thymic stromal lymphopoietin (TSLP), highly expressed by keratinocytes in skin lesions in atopic dermatitis and bronchial epithelial cells in asthma, plays a key role in allergic diseases. Double-stranded RNA (dsRNA) stimulates keratinocytes to release TSLP in vitro. Objective: To examine the potential of glucocorticoids and calcineurin inhibitors to suppress dsRNA-induced release of TSLP from keratinocytes. Methods: Primary human kerarinocytes were stimulated with dsRNA in the presence of IL-4, IL-13 and TNF-α. TSLP release was measured by ELISA. The effects of glucocorticoids and 2 calcineurin inhibitors, cyclosporin A and FK506/tacrolimus, were analyzed. Results: The glucocorticoids inhibited dsRNA-induced release of TSLP. The inhibitory effect became saturated (50–70% reduction) at concentrations higher than 10–10M. Cyclosporin A inhibited the release of TSLP by 50–60% at 10–5 and 10–4M. FK506 had no effect at 10–5M or less, but almost completely inhibited the release of TSLP at 10–4M. No synergistic effect was obtained with a glucocorticoid plus either of the calcineurin inhibitors. An additive inhibitory effect was obtained with a glucocorticoid plus 10–5M cyclosporin A. Glucocorticoid inhibited dsRNA-induced TSLP transcription in the absence of Th2/TNF cytokines. Conclusions: Glucocorticoids inhibited the dsRNA-induced release of TSLP in the atopic cytokine milieu at much lower concentrations than calcineurin inhibitors, suggesting that they could be effective in the treatment of atopic dermatitis when exogenous or endogenous dsRNA is involved in the pathogenesis. In addition, the in vitro system established in this study would be useful for screening of therapeutic reagents which target TSLP expression.