P152 Synergistic activation of proteinase-activated receptor 2 and toll-like receptor 4 induces production of thymic stromal lymphopoietin production by human keratinocytes

Abstract

Thymic stromal lymphopoietin (TSLP) plays an important role in allergic inflammation by instructing myeloid dendritic cells (mDCs) to induce inflammatory T helper 2 (Th2) cells. TSLP is highly expressed by keratinocytes in skin lesions from patients with atopic dermatitis (AD), and act as a master switch of AD pathogenesis. However factors inducing TSLP production from human keratinocytes are uncertain. Protease-activated receptors (PARs) and Toll-like receptors (TLRs) play a role in cytokine secretion from kertinocytes. Recent studies described synergistic PAR2- and TLR4-mediated cytokine production; but little information is available on TSLP production. In this study, we investigated the secretion of TSLP from human keratinocytes after combined stimulation with PAR2- and TLR4-agonists. Normal human epidermal keratinocytes (NHEK) were stimulated with PAR2-activating peptide (AP) and/or Kdo2-Lipid A in vitro . TSLP secretion from NHEK cells was measured by enzyme-linked immunosorbent assay (ELISA). The expression of TSLP mRNA was determined by reverse transcription-polymerase chain reaction (RT-PCR). Combined stimulation of NHEK cells with PAR2-AP and Kdo2-Lipid A resulted in a marked secretion of TSLP. The stimulation with Kdo2-Lipid A alone showed a moderate and lower increase in TSLP production compared to the synergistic stimulation. The stimulation with PAR2-AP alone failed to induce TSLP production by human keratinocytes. RT-PCR analysis confirmed these results in TSLP gene transcriptional levels. It appears that PAR2 and TLR4 synergistically up-regulate TSLP production by human keratinocytes. Recent studies have revealed aberrant expression and activation of serine proteases and PAR-2 in the lesional skin of AD patients. This implies that PAR2 activation and TLR4 triggering may amplify Th2 inflammation via the induction of TSLP in AD. The knowledge about the molecular basis of AD will allow the development of novel, rational-based therapeutic strategies.