Expression Of Toll-like Receptor 2 In Psoriatic Lesions And It's Effect On The Proliferation Of Human Keratinocytes

PartⅠThe expressions of Toll-like receptor 2 and its related factors in psoriatic lesionsObjective To investigate the expressions of Toll-like receptor 2(TLR2), nuclear-factor-κB(NF-κB) p65 and transforming growth factor-alpha(TGF-alpha) in the epidemis of psoriasis and their correlation with disease severity in psoriatic patients.Methods Forty samples of lesional psoriatic skin,eleven samples of nonlesional psoriatic skin and fifteen samples of normal skin were analyzed by EliVision immunohistochemical technique for the expressions of TLR2,NF-κBp65 and TGF-alpha protein.Correlation analysis was performed among the expressions of three factors as well as between the expressions and disease severity.Results Compared with normal epidermis and nonlesional psoriatic epidermis,TLR2,NF-κB p65 and TGF-alpha were more highly expressed in lesional epidermis from patients with psoriasis(all P＜0.05).In nonlesional psoriatic epidermis,TLR2 expression was also upregulated compared with normal epidermis(P＜0.05).The expressions of TLR2 and NF-κBp65 in lesional epidermis positively correlated individually with the psoriasis area and severity index(PASI) scores in patients(both P＜0.05).Positive correlation was also found between the expressions of any two factors(TLR2,NF-κB p65 or TGF-alpha) in lesional epidermis(all P＜0.05).Conclusion The upregulation of TLR2,NF-κBp65 and TGF-alpha expressions in psoriatic lesions may be involved in the pathogenesis of psoriasis.PartⅡEffect of Toll-like receptor 2 on the proliferation of human keratinocytesObjective To investigate the effect of TLR2 on the proliferation of human keratinocytes.Methods Keratinocytes were isolated from the foreskin of children, and subjected to primary culture.After 3-5 passages,the keratinocytes were incubated with peptidoglycan(PGN),a TLR2 agonist.Cell proliferation was detected by MTT colorimetric assay and the suitable concentrations of PGN were determined.The mRNA and protein expressions of TLR2 and Ki67 were detected by real-time quantitative PCR and Western blot.Antibody blocking test was utilized to evaluate the effect on TLR2 and Ki67 expressions of keratinocytes by blocking TLR2 with specific anti-TLR2 neutralizing monoclonal antibody before incubation with PGN.Results The keratinocytes proliferation was promoted by the incubation with PGN of 1.25,2.5 and 5ug/mL for 24 hours(all P＜0.05).1.25ug/mL PGN up-regulated TLR2 mRNA expression at 12h and 24h after the incubation,and Ki67 mRNA expression at 24h and 48h.In the keratinocytes incubated with PGN of 1.25,2.5 and 5ug/mL for 24 h,TLR2 mRNA and protein,Ki67 protein were all more highly expressed in different groups,and Ki67 mRNA expression was upregulated at the concentrations of 1.25 and 2.5ug/mL(all P＜0.05).The mRNA and protein expressions of TLR2 and Ki67 were all inhibited by blocking TLR2 when incubation with PGN(all P＜0.05).Conclusion Activation of TLR2 by PGN could promote the in vitro proliferation of cultured human keratinocytes,which provide supporting evidence for the role of TLR2 in psoriasis.PartⅢStudy on the mechanism of Toll-like receptor 2 in promoting human keratinocytes proliferationObjective To evaluate the effect of TLR2 on NF-κBp65 activation and TGF-alpha expression in human keratinocytes.Methods Cultured keratinocytes were incubated with peptidoglycan(PGN).Real-time quantitative PCR and Western blot were used to detect the mRNA and protein expressions ofNF-κBp65 and TGF-alpha,respectively. Antibody blocking test was utilized to evaluate the effect on NF-κBp65 activation and TGF-alpha expression of keratinocytes by blocking TLR2 with specific anti-TLR2 neutralizing monoclonal antibodie before incubation with PGN.Results 1.25ug/mL PGN up-regulated NF-κBp65 mRNA expression in keratinocytes at 24h after the incubation,and TGF-αmRNA expression at 6h and 24h.In the keratinocytes incubated with PGN of 1.25,2.5 and 5ug/mL for 24 h,nuclear NF-κB p65 protein was more highly expressed in different groups,and TGF-αmRNA and protein expressions were upregulated at the concentrations of 1.25 and 5ug/mL(all P＜0.05). NF-κB p65 activation and TGF-alpha expression were both inhibited by blocking TLR2 when incubation with PGN(all P＜0.05).Conclusion Activation of TLR2 by PGN could promote NF-kappaB activation and TGF-αexpression,which may be one of the mechanisms of human keratinocytes proliferation.