Toll-like Receptors Expression Profile And Function Of Toll-like Receptor 2 In Human Epidermal Keratinocytes

Toll-like receptors(TLRs) are a recently discovered family of pattern recognition receptors which play a critical role in innate immune and adaptive immune. Epidermal keratinocytes are the first barier between external environment and internal organs.Keratinocytes are invaded by diverse pathogens.TLRs are important in the defense of keratinocytes,and associated with many infected and inflammatory skin diseases.The TLRs profile of keratinocytes is not very clear yet.There are few researches about regulation and function of TLRs in keratinocytes In this research,the TLRs profile of keratinocytes is investigated,the effect of peptidoglycan from Staphylococcus aureus on keratinocytes TLRs expression and chemokines secretion is discussed.This study consisted of the following three parts.PartⅠ:Toll-like receptors expression in human epidermal keratinocytesObjective To investigate TLRs profile of human epidermal keratinocytes. Methods Immortalized human keratinocyte cell line HaCaT cells and normal human epidermal keratinocytes(NHEK) were cultured,epidermis were seprated with dispase from foreskins.Reverse transcription polymerase chain reaction(RT-PCR) was used to detect TLR 1-10 mRNA expression.Flow cytometry was used to assess TLR 2 and 4 protein expression on surface of HaCaT cells and NHEK.Results HaCaT cells,NHEK and seprated epidermis all expressed TLR 1-10 mRNA with different intensity.TLR 4 protein could be detected on the surface of HaCaT cells and NHEK,TLR 2 protein expression was few.Conclusions Human epidermal keratinocytes constitutively express all TLR 1-10 mRNA which may enable human keratinocytes to respond to a wide range of pathogenic micro-organisms. PartⅡThe effect of peptidoglycan from Staphylococcus aureus on TLR2 and TLR4 expression in HaCaT cellsObjective To study TLRs modulation in HaCaT cells by peptidoglycan(PGN). Methods Different concentration of PGN was co-cultured with HaCaT cells in different time,RT-PCR was used to detect TLR2 and TLR4 mRNA expression,Flow cytometry was used to assess TLR 2 and 4 protein expression on surface of HaCaT cells.Results HaCaT cells stimulated with 30μg/ml PGN in 6 hours showed significant increased expression of TLR2 and TLR4 mRNA,TLR 2 and 4 protein expressions were the highest with 100μg/ml PGN co-culture in 24 hours.Conclusions PGN up-regulates TLR2 and TLR4 expression in HaCaT cells.PartⅢChemokines modulation of PGN from Staphylococcus aureus in normal human epidermal keratinocytes and the effect of TLR2Objective To investigate the effect of PGN from Staphylococcus aureus on the release of several chemokines in NHEK.Methods After different concentration of PGN was co-cultured with NHEK in different time,enzyme linked immunosorbent assay(ELISA) was used to detect interleukin-8(IL-8),regulated upon activation, normal T-cell expressed and secreted(RANTES) and macrophage-derived chemokine (MDC) in culture supernatants.Pretreated with functional grade purified anti-human TLR2 monoclonal antibody,NHEK were stimulated with 100μg/ml PGN in 12 hours, chemokines in culture supernatants were measured.Results NHEK spontaneously released IL-8 and RANTES.With the concentration of PGN increased,the co-culture time prolongation,IL-8 was gradually induced more and RANTES decreased more. Anti-human TLR2 monoclonal antibody abrogated the up-regulatory effects of PGN on IL-8 production and had no effect on RANTES release.MDC could not be detected with or without PGN stimulation.Conclusions PGN induces the IL-8 production in NHEK via TLR2.PGN inhibits RANTES production in NHEK.