Study On The Expression Of Toll-like Receptor3Etc In Condyloma Acuminatum With Low-risk HPV

Background and Objective:Condyloma acuminata (CA) refers to an epidermal lesions with genital skin or mucous membrane except for subclinical infection caused by human papillomavirus (HPV) infectionclinically. The characteristics of CA are infectious, proliferative and likely to relapse. At present situation, CAincidence rate is in the second place of the sexually transmitted diseases (STD) in China now.The major clinical manifestations of Condyloma acuminatum (CA) is associated with benign epithelial proliferations of skin and internal squamous mucosae mainlycaused by low-risk HPV. Malignant transformation probability of CA is low. The main infection sources are from the CA patients,persons with subclinicalHPV infection and latent infection. The incidence of CA in China is higher and higher year by year, so the increasing attention has been paid.Human papillomavirus is a tiny DNA virus with epidermotropic and tissue-specific. The only host of HPV is the complex layer squamous epithelium of human skin and mucous membrane. There are at least180HPV genotypes now. According to capability of the virus in malignant transformation, HPV divided into high-risk type and low-risk type. More than40HPV genotypes can infect in the genital area. CA is caused by low-risk HPV type mainly. Host does not establish an effective immune response lead to HPV difficult to removal. Therefore, human normal immune play an important role in the developing of disease caused by HPV.The immune evasion mechanism of HPV may associate with HPV inhibits and delays the host local immune response.Toll-like receptors (TLRs) is widely distributed in the surface of immune cells, especially in non-immune cells and some somatic cells, as a kind of pattern recognition receptors (PRR). They can play a role inantimicrobial immunity through recognition and combiningmicroorganism Pathogen associated molecular patterns (PAMPs) induce cytokines released and costimulatory moleculesup-regulated, which can induction the innate immune response as well as provide the necessary activation signal for the adaptive immune. TLRs as bridge link the innate immunity to adaptive immunity.TLRs play an important role in activating the host antiviral immunity, especially the TLR3/TLR7/TLR9.TLR3start TRIF-dependent pathways, resulting in activation of NF-κB.Both TLR7-and TLR9-mediated MyD88-dependent pathways induce the production of inflammatory cytokines and type I IFNs. IRF3/IRF7is phosphorylated and then translocated to the nucleus, where it induces the IFNa and IFNβ promoters. IRF7regulate the production of IFNa as well as IRF3is key transcription factors in the production of IFNPThepersistence infection of HPV associate with HPV inhibits and delays the host local immune response,largely affecting the process of antigen presenting. Langerhans cell (LC) as profesionalantigen presented cell (APC) process and present antigen in the epithelium. HPV may inhibits and delays host local immune response through influencing some factors during the present antigen process.Chemotactic factor CXCL12combine with its receptor CXCR4particularly, which inducedendritic cell (DC) to intake and present antigen, produce cytokines including lipopolysaccharide (LPS), tumor necrosis factor (TNF) and interleukin-1(IL-1) etc, and promote mature dendritic cell migration into draining lymph nodes. There was reported that the different density of CXCL12has contrary affection for T cells, which suggested that the expression of CXCL12regulate the function of host antigen presenting. CXCL12has the potential to modulate efficacious antitumor responses in view of its properties as a powerful chemoattractant for T and pre-B lymphocytes and dendritic cells to the tumor site. It suggested that abnormal expression of CXCL12can cause the body’s immune imbalance.Epithelial cell adhesion molecule E-cadherin plays a crucial role in maintaining the integrity of the organizational structure and cell polarity. However, recent findings have uncovered an immunological role for this adhesion molecule, linked to its expression in dendritic cells (DCs) and alternatively activated macrophages and its impact on intracellular signaling pathways.E-cadherin is known to mediate interactions with keratinocytes, thereby immobilizing immature LCs in the epidermis and preventing their maturation. HPV16E6/E7inhibit the expression of E-cadherin directly has be reported recently.Cyclooxygnasis-2(COX-2) and Membrane type combined with prostaglandin E2synthetase1(mPGEs-1) are important enzymes during the synthesis of PGE2and suppress the immune function. They are positioned on the microsome membrane and With stimulation of inflammation factors, COX-2and mPGEs-1induce increasing the production of PGE2markedly which could inhibit immune by inducing suppressor T cells and suppressor macrophage production and prevent immature DC towards to mature DC.However, recent findings have uncovered COX2、mPGEs-lalso influence host antigen presentation.The TNF superfamily molecule receptor activator of NF-κB (RANK) and its ligand RANKL, play a key role in the regulation of several DC functions including enhancement of their capacity to activate T cells. RANKL has been shown to sustain viability of blood dendritic cells in addition to its role in promoting proliferation and differentiation of several cell types, notably osteoclasts.Human KC expressed RANKL and epidermal LC expressed cell surface RANK. Barbaroux JB’s study shows for the first time that the TNF superfamily molecule RANKL is a key regulator of epidermal LC proliferation and survival.The research screen CA lesions infection with low-risk HPV (HPV6/HPV11) and target genes related with TLRs and its signaling pathways include TLR3, TLR7, TLR9, IRF3, IRF7, TRIF and6target genes associate with antigen presentation include CXCL12, E-cadherin, COX2, mPGEs-1, RANK, RANKL. We used real-time fluorescent quantitative PCR detected the expression of target genes in local lesion with low-risk HPV infection and study the possible mechanism of HPV effects on local immunity.MethodsFor screening low risk human papilloma virus (HPV6,11) from43clinical specimens collection of CA patients,HPV DNA-typing used for21HPV types by DNA-PCRamplification and diversion hybridization technology, While18cases of normal adult foreskin are as normal control group. Total RNA of all CA specimens being extracted by Trizol reverse transcription to cDNA, and real-time quantitative fluorescence PCR with SYBR Green dyedetection method are appllied. We based on P-actin as the reference gene and detect the expression of TLR3, TLR7, TLR9, IRF3, IRF7, TRIF, CXCL12, E-cadherin, mPGEs-1, COX-2, RANK, RANKL in the two groups. Statistical processing use2-△△Ct and Mann-Whitney U Test, and α=0.05.ResultsIn the43cases of condyloma acuminatum specimens, HPV6infections only found in1cases (2.32%,1/43cases), HPV11has8cases (18.60%,8/43cases), HPV6and11were13cases (41.86%,13/43cases), HPV6+other (except HPV11) was1cases (2.32%,1/43), HPV11+other (except HPV6) were3cases (6.98%,3/43),HPV6/11+other were12cases (27.90%,12/43).27cases were infected by low risk human papilloma virus (HPV6/11) and infection rates was62.79%. Between the two groups, the expression of β-actin relatively constant.2-△△Ct method is used to calculate the each sample2-△△Ct result. With the Mann-Whitney U Test found that IRF7have been up-regulation expression in CA groups (fold change=10.083) with statistical significance (P<0.05), while the differences of the two groups of other target genes related TLRs signaling pathways including TLR3, TLR7, TLR9, IRF3, TRIF were no statistical significant (P>0.05). Inantigen-presentation target genes, expressions of CXCL12and mPGEs-1had the statistical significance (P<0.05) in two groups. CXCL12has lower expression (fold change=20.231) in CA groups, while the expression of mPGEs-1was down-regulation (fold change=3.233).Between the two groups, the differences of genes expressions of E-cadherin, COX2, RANK, RANKL were no statistical significant (P>0.05).Conclusions43specimens of condyloma acuminatum cases have low-risk HPV infection (100%,43/43). There were27single or mixed infection cases of HPV6/11specimens,62.79%in the CA groups. This data suggested that low-risk type HPV6/11are the main infection types of condyloma acuminatum.According to the real-time quantitative fluorescence PCR shows that the expression of TLR3, TLR7, TLR9had trend of down-regulation, we speculated that HPV may inhibit the expression of TLRs.TRIF, IRF3, IRF7are the key factors in downstream of TLRs signaling pathway. All of them have trend of up-regulation expression in CA group, while IRF7expression raised obviously. It suggested that host activated the NF-κB pathway when HPV infected, inducing production of type I and type II interferon to activate natural immunity and providing a necessary signal to acquired immunity.Low-risk HPVs (6and11) have close relationship with TLRs and its downstream pathway. TLRs signaling pathway in HPV infections is relatively complex, so we need to further study in the future.6genes of CXCL12, E-cadherin, mPGEs-1, COX-2, RANKL, and RANK which associated with antigen presentation were detected. There was lower expression of CXCL12and mPGEs-lin lesions of CA caused by low-risk HP V.It suggested that HPV may inhibit CXCL12, imbalancing in antigen presenting, suppressing maturity and migration of LC, and reducing production of PGE2through suppressing mPGEs-l,and influencing local immune response finally. According to these gene expressions in CA and their functions, our data suggested that low-risk HPV (HPV6/11) can affect the TLRs pathways,inhibit antigen presenting process and delay host local immune response, so HPV infections are consistently.