Studies On Association Of Atopic Dermatitis With Brain-derived Neurotrophic Factor And Filaggrin

IntroductionAtopic dermatitis(AD)is a chronic and relapsing inflammatory skin disorder characterized by intense pruritus and eczematous skin lesions.Genetic,environmental, immunologic and pharmacologic factors are implicated in the pathogenesis of AD. Neural immune factors and skin barrier dysfunction are two major hot spots in the study of AD.Brain-derived neurotrophic factor(BDNF)is the second member of the neurotrophin family.Neurotrophins(NTs)have a wide spectrum of functions outside the nervous system.They appear to be important mediators in the interaction between immune and nerve cells in inflammatory,allergic and autoimmune diseases.BDNF plays an important role in the pathogenesis of AD in that BDNF serum levels provide a useful indicator of disease activity,severity and life quality in AD patients.BDNF inhibits the apoptosis and induces the chemotaxis of eosinophils from AD patients. BDNF can also suppress the production of Th1 type cytokines IFN-γand IL-12 by mononuclear cells,but has no effect on the production of Th2 type cytokines IL-4, IL-10 and IL-13.Consequently,the cytokine balance is skewed toward Th2 type and antigen specific IgE production was enhanced.The BDNF gene locates on human chromosome 11p13,consisting of four short 5'exons with separate promoters and one 3'exon encoding the mature BDNF protein.In the BDNF,several variation and polymorphisms have been reported.The most frequent in research studies include C270T and G196A polymorphisms.Recently,a few studies have been reported in the association of BDNF gene C270T and G196A polymorphisms and atopic diseases in European population.However,until now,none of the above polymorphisms has ever been reported in Chinese AD. Keratinocytes not only constitute a barrier surface of the skin,but also function as important immune cells.Keratinocytes secrete a variety of cytokines and express the corresponding cytokine receptors.Keratinocyte-derived cytokines can affect the migration of inflammatory cells and the proliferation and differentiation of keratinocyte itself.Keratinocytes can also enhance or diminish the secretion of other cytokines and participate in T-cell immune response,even to play an important role in whole-body immunity.Keratinocytes express BDNF receptor TrkB and develop TrkB-typed immunological reaction.BDNF has been confirmed to regulate keratinocytes proliferation and maintain environmental stability of epidermis,but how BDNF regulate the secretion of immunomodulatory cytokines by keratinocytes and involve in allergic inflammatory response of skin subject to further study.Filaggrin is a kind of water-soluble protein,located in epidermal keratinocytes. The natural moisturizing factor(NMF)produced by filaggrin is crucial to maintain the hydration function and barrier function of skin.FLG resides on chromosome 1q21 within the epidermal differentiation complex(EDC),which is a cluster of genes and gene families coding for proteins involved in terminal differentiation of the epidermis. Heritable skin barrier defects caused by mutations in filaggrin gene(FLG)play an important role in the pathogenesis of AD.Reduced or absent filaggrin expression may partially explain the skin barrier defects seen in patients with AD.FLG mutations may have regional or ethnic differences.The strong association of FLG R501X and 2282del4 mutations and AD development have been replicated in several European populations,but they were absent in non-European populations,such as those of Asian or African origin.In Japanese populations,the 3321delA and S2554X mutations were associated with AD,whereas the two mutations were not found in European AD patients.However,until now,none of the FLG mutations has ever been reported in Chinese AD.In this study,we investigated C270T and G196A polymorphisms in the BDNF gene and mutations E2422X,Q2417X,S2554X,S2889X,S3296X,R4307X and 7945delA in FLG in Chinese AD population by polymerase chain reaction-restriction fragment length polymorphisms(PCR-RFLP)and DNA sequencing technique,and tried to discuss AD pathogenesis from the molecular genetics angle.We also investigated the effects of BDNF on the production of cytokines by keratinocyte to study the possible function of BDNF in the cytokine network of AD lesions.Materials and MethodsSubjects(1)AD group:181 AD patients,aged 12.9±13.7 year,including 96 males and 85 females were diagnosed in Department of Dermatology,First Hospital of China Medical University,conforms to the Williams diagnosis standard.They were all active AD patients and had no blood relationships.They had not been treated with systemic corticosteroids or other immuno-suppressants within 6 months.(2)Normal control group:197 examples,aged 31.5±9.4 year,including 103 males and 94 females were random selected as volunteers for health examination.All of them had not AD,allergic asthma,allergic rhinitis,atopic keratoconjunctivitis and other allergic symptoms so far.There were no blood relationships between them.Committee on Genetics of China Medical University approved the study,and written informed consent was obtained from all participants.All patients had not psychiatric disorders and their body mass index were within normal range.(3)HaCaT cellsMaterialsEDTA,chloroform,dimethyl carbinol,alcohol,agarose,2×Taq Plus Master-Mix DNA polymerase,8%negative polyacrylamide gel electrophoresis(negative PAGE), restrictive enzyme HinfⅠ,Eco172Ⅰ,NlaⅢ,BfaⅠ,Hpy188Ⅰ,BamHI,DpnⅡ,DdeⅠ,RPMI1640 cell culture fluid,penicilin,streptomycin,trypsinase,PBS,DEPC, RealMasterMix.Methods(1)The C270T and G196A polymorphisms of BDNF were genotyped by PCR-RFLP.(2)The effects of BDNF on the production of cytokines IL-10,IL-6,IL-12 and IFN-γby keratinocytes were analysed by real-time RT-PCR and ELISA.(3)Mutations E2422X,Q2417X,S2554X,S2889X,S3296X,R4307X and 7945delA were detected by PCR-RFLP and DNA sequencing technique. Statistical AnalysisAll the statistical analysis of data were performed using the statistical software SPSS(version 13.0),including Chi-squared test,ANOVA,a two-tailed P＜0.05 was considered to be of statistical significance.To determine whether genotype was in Hardy-Weinberg equilibrium,a x~2 wax performed..The genotype frequency and the allele frequency of these genes were compared by x~2,and odds ratio(OR)and 95% confidence interval(CI)indicate the relative risk.Results1.Association of C270T and G196A polymorphisms in the BDNF gene with atopic dermatitis(1)BDNF gene C270T and G196A genotype distributions of AD group and normal control group conform to the Hardy-Weinberg equilibrium.(2)Significant difference was observed between AD patients and healthy controls in the T allelic frequencies of C270T polymorphism(OR=3.130,95%CI:1.116～8.778).(3)Significant difference was observed between the male AD patients and male healthy controls in the T allelic frequencies of C270T polymorphism(OR=4.112, 95%CI:1.129～14.973).(4)Significant difference was observed between the IAD patients and EAD patients in the T allelic frequencies of C270T polymorphism(OR=3.554,95%CI: 1.165～10.844).(5)Significant difference was observed between AD patients with atopy in 1 st-degree relatives and AD patients without this condition in the A allelic frequencies of G196A polymorphism(OR=1.579,95%CI:1.021～2.442).2.Effects of BDNF on the production of cytokines by keratinocytes(1)The IL-10 mRNA and IL-10 expressions in 0.1ng/ml BDNF irritated HaCaT cells were the same levels as those in control cells,while the expressions of 1ng/ml and 10ng/ml BDNF irritated cells were significantly higher after 24 hr irration and the effect is dose-dependent.(2)The IL-12 mRNA expression in 0.1ng/ml,1ng/ml,and 10ng/ml BDNF irritated HaCaT cells were all lower than that in control cells after 24 hr irration,and the decrease is dose-dependent.The IL-12 level in 0.1ng/ml BDNF irritated supernatant was the same to that of control supernatant,while the levels of other irritated cells were significantly lower after 24 hr irration and the effect is dose-dependent.3.Association of mutations in FLG with atopic dermatitisMutations of E2422X,Q2417X,S2554X,S2889X,S3296X,R4307X and 7945delA in FLG were not detected either in AD group or normal control.Conclusion1.C270T polymorphism in BDNF gene is associated with AD and carrying T allele increases the risk of AD to male AD patients and IAD patients.2.There are significant differences in genotype distribution and allele frequencies for the G196A polymorphism of BDNF gene in AD patients with atopy in 1st-degree relatives in comparison with AD patients without this condition,and carrying A increases the risk of AD to patients with atopy in 1st-degree relatives.3.The IL-10 mRNA and IL-10 expressions increase after BDNF irritation in a dose-dependent way.The IL-12 mRNA and IL-12 expressions decrease after BDNF irritation in a dose-dependent way.BDNF may affect the production of cytokines by keratinocyte,then change cytokine enviroment and play a role in the Th1/Th2 cytokines imbalance.4.FLG E2422X,Q2417X,S2554X,S2889X,S3296X,R4307X and 7945delA are all wide types in AD and normal controls in North East China and FLG mutations have regional or ethnic differences.