Effect And Molecular Mechanism Of Mammalian Cathelicidins(LL37 And CRAMP)-Mediated Promotion Of Coxsackievirus B3 Replication In Intestinal Epithelial Cells

Background The antimicrobial peptide cathelicidin is a kind of cationic peptide with broad-spectrum antimicrobial activity.There is only one cathelicidin found in human?LL-37?and in mice?CRAMP?.AMP cathelicidins are mainly expressed in tissue cells?epithelial cells,keratinocytes?and immune cells?neutrophils,macrophages?.The antimicrobial mechanism involves direct electrostatic interactions between the cationic AMPs and anionic LPS or lipoteichoic acids of pathogens,leading to membrane disruption.Owing to this membrane-disrupting mechanism,cathelicidin has been demonstrated to disrupt enveloped viruses?vaccinia virus,respiratory syncytial virus,influenza Virus?.However,the antiviral effect and mechanism of cathelicidin on non-enveloped viruses are rarely reported.Coxsackievirus B3?CVB3?,a positive-stranded RNA virus belonging to non-enveloped enterovirus,is the most common cause of viral Myocarditis?VMC?.Our preliminary data have demonstrated that cathelicidin significantly inhibits CVB3 replication in cardiomyocytes in vitro and in vivo,leading to ameliorated acute myocarditis in mice.To explore the antiviral molecular mechanism of cathelicidin against non-enveloped CVB3,first we verified that exogenous cathelicidin?LL37/CRAMP?administration profoundly inhibited CVB3 replication in the primary cardiomyocytes in vitro in dose-dependent manner.However in another 2 kinds of cells,intestinal epithelial HCT116?the initial infection sites for CVB3?and cervical carcinoma epithelial Hela?the most widely used propagating cell for CVB3?cells,exogenous cathelicidin was found to strikingly promote CVB3 replication in dose-dependent manner.CVB3 belongs to enterovirus family and intestinal epithelial cells are the primary entry site for CVB3.It is believed that CVB3 disseminates into other permissive organs like heart and pancreas after vivid propagation in the intestinal epithelium and MLNs.Therefore intestinal replication event is critical for the subsequent organ dissemination and the disease development.Meanwhile,epithelial cells are one of the major sources of cathelicidin.We hypothesize that the phenomenon that cathelicidin AMPs promote CVB3 replication in intestinal epithelial cells is of great significance and needs further exploration.In this study,focusing on the phenomenon that cathelicidin AMPs promote CVB3 replication in intestinal epithelial cells,we plan to first verify this result by administration of cathelicidin onto a variety of epithelial cells and by knock-down of cathelicidin expression in HCT116 cells.Then,the antiviral mechanism of cathelicidin would be explored according to various steps limiting CVB3 replication on cells including virus adhesion,virus entry as well as cell autophagy and cell apoptosis.This study is the first study focusing on the viral-enhancing effect of cathelicidin AMPs.Our data would provide new insights into the effect and mechanism involving role of cathelicidin AMPs on non-enveloped viruses.Purpose Explore the effect and mechanism underlying cathelicidin AMPs-mediated promotion of CVB3 replication in intestinal epithelial cells.Method1.Preparation of primary cardiac myocytes:Hearts of suckling mice born within 24 hours were minced and subjected to 2 rounds of digestion with collagenase II.Pooed cell suspensions were collected and myocardial cells were isolated by differential pasting method then cultured in DMEM-10%FBS at 37?.2.Cell culture of intestinal epithelial cell:Intestinal epithelial cell line HCT116 was cultured in DMEM-10%FBS at 37?.3.CVB3 infection of cells:0.1 ml of CVB3?MOI=10,PBS?was added on to cells and incubated at 37?for 1 hr before moving.4.Western blotting detection of CVB3 capsid protein VP1.Cell proteins were separated by SDS-page gels and transferred on to PVDF membranes.The membranes were blocked with 5%BSA and probed with anti-VP1 or anti-actin overnight at 4? and subsequently incubated with a HRP-anti-rabbit IgG.Chemiluminescence detection was performed using ECL.5.Quanticative RT-PCR detection of CVB3 RNA:Fluorescence quantitative PCR was used to detect the RNA level of positive and negative strand of CVB3.Total RNA was extracted by RNAiso and reversely transcribed into cDNA,and SYBR Green was used for Q-RT-PCR assay.The 2-??CT method was used to calculate the relative expression of RNA of the virus.6.TCID₅₀assay of CVB3 viral titers:CVB3 was titrated by TCID₅₀assay on Hela cell monolayers.30?l CVB3 was added onto Hela cells and the cytopathic effect was observed every day till 5 days p.i..Viral titer=lg?diluted more than 50%lesion positive rate?+?percentage of greater than 50%positive rate of lesions-50?/?percentage of more than 50%positive rate of lesions-less than 50%of positive rate of lesions?.The PFU of CVB3?TCID₅₀/ml?0.7.7.Knock-down of endogenous cathelicidin by CRISPR/CAS9 technology:Cas9 gene was transfected into HCT116 by LV-cas9,stable cas9 expression was acquired by puromycin screening.Then designed LV-sgRNAs targeting cathelicidin were transfected into the HCT116-cas9 and the knock-down cell line was screened by fluorescence evaluation.8.Effect of cathelicidin on viral adhesion and entry into cells:1)Viral adhesion assay:cells were infected with CVB3?MOI=20?,in presence with LL37 at4? for 1h?support viral adhesion but not entry?.After three times of washing,cells were placed in DMEM-10%FBS at 37? for 18 hrs.2)Viral entry assay:cells were infected with CVB3?MOI=20?at 4? for 1 hr to finish adhesion.Afterwashing cells were cultured in DMEM-10%FBS in presence with LL37 for 1 hr at 37?.Then cells were washed and cultured in DMEM-10%FBS at 37? for 18 hrs.9.Detection of autophagy:1)Western Blot detection of autophagy-related protein LC3:Cell proteins were collected at 0,2,4,6 and 8 h after infection,samples were probed for LC3-I and LC3-II via WB assay.2)Immunofluorescent detection of LC3:Cells were infected by CVB3 for 6 h and fixed with 4%paraformaldehyde for 15 min.Then 0.25%TRITON X-100 was added to disrupt membrane.Samples were blocked with 10%BSA before incubation with anti-LC3-I/II Ab.After incubation with FITC-Secondary Ab,nuclei was stained with DAPI before observation under laser confocal microscope.10.Flow cytometry of apoptotic cells:Single cell suspensions of CVB3 infected HCT116 were stained with Anexin V-APC and 7-AAD to stain the apoptitic cells.The data were collected using FACS Canto II flow cytometer and analyzed using Flowjo 7.0software.11.Western blot analysis of ERK and AKT phosphorylation:Proteins were separated by SDS-page gels and transferred on to PVDF membranes.The membranes were blocked with 5%BSA and probed with anti-ERK,anti-p-ERK,anti-AKT,anti-p-AKT,overnight and subsequently incubated with a HRP-anti-rabbit IgG.Chemiluminescence detection was performed using ECL.12.Statistical methods:Data were expressed as mean±standard deviation;One way ANOVA was used for comparison between groups;statistical analysis was performed with GraphPad Prism 5 and SPSS 11 software,and P<0.05 was considered to be significantly different.Results1.5 to 20?g/m L of cathelicidin?LL37 and CRAMP?was safe for cardiomyocytes and epithelial cells.2.LL37 and CRAMP inhibit CVB3 replication in primary cardiomyocytes in dose-dependent manner.3.Cathelicidin promoted replication of CVB3 in intestinal epithelial cell HCT116 in a dose-dependent manner.HCT116 were infected with GFP-CVB3 or common CVB3?MOI=10?for 1hr in absence or presence of 5 to 20?g/m L CRAMP and LL37,using sLL37 and sCARMP as controls.Then the protein,RNA levels as well as viral titers were detected.We found that:1)Flow cytometry detection of GFP-positive cells confirmed that both CRAMP and LL37 increased CVB3 replication in HCT116;2)Western blot assay demonstrated that LL37 and CRAMP significantly increased the protein levels of VP1 in a dose-dependent manner.3)Q-RT-PCR assay indicated that LL37 and CRAMP significantly promoted the RNA expression of CVB3;4)TCID₅₀assay demonstrated that 20?g/mL of LL37 and CRAMP profoundly enhanced CVB3 particle production with viral titer increasing from 10⁵ pfu to 10⁷ pfu/0.1ml.4.Cathelicidin also promoted the replication of CVB3 in human cervical cancer epithelial HeLa,human lung cancer epithelial cell A549 and human renal epithelial cell HepG-2,suggesting that cathelicidin AMPs promote CVB3 replication in epithelial cells.5.CVB3 infection up-regulated the expression of endogenous cathelicidin as early as 4 hrs p.i..6.Konck-down of cathelicidin significantly blocked the CVB3 replication in HCT116 cells.CRISPR-Cas9 gene editing technology was used to knock down intracellular CRAMP expression in HCT116 cells.In this stable knockdown HCT116-Cas9 cell line,viral replication at 18 hes p.i.was significantly reduced after CVB3 infection including capsid protein level of VP1,RNA levels and viral titer.7.Cathelicidin promoted adhesion and entry of CVB3 into intestinal epithelial cells.By 4? viral adhesion assay and by 37? viral entry assay,our data revealed that cathelicidins promoted the adhesion and entry of CVB3 into HCT116 cells.8.Cathelicidin administration did not affect CVB3-induced LC3I/LC3 II upregulation?early event of autophagy?at 0⁸ hrs after CVB3 infection.9.Flow cytometry found that cathelicidin AMPs promoted Annexin V+7-AAD-early apoptotic cells after CVB3 infection,which may increase viral replication.10.Cathelicidin administration affected p-ERK phosphorylation in HCT116 cells.ERK signaling inhibitor U0126 significantly inhibited CVB3 replication,confirming that antimicrobial peptides affected ERK pathway activation.11.CVB3 infection activated p-AKT and VP1 protein expression only after 6 hrs p.i..LL37 administration significantly enhanced AKT phosphorylation and VP1 expression at as early as 4hr p.i..It indicates that cathelicidin AMPs activate intracellular AKT signaling pathway in advance therefore promote the transcription and replication of CVB3 RNA.Conclusion1.The cathelicidin AMPs effectively inhibit the replication of CVB3 in cardiomyocytes in vitro and in vivo,but significantly promote CVB3 replication in intestinal epithelial cells?HCT116?and other epithelial cells in vitro.2.Knocking down of endogenous cathelicidin significantly blocks replication of CVB3 in intestinal epithelial cells.3.The molecular mechanism underlying the viral replication-promoting effect by cathelicidin in intestinal epithelial cells may lie in that,cathelicidin promotes viral adhesion and entry into as well as the early apoptosis of intestinal epithelial cells;and cathelicidin may enhance the transcription of viral RNA by activating intracellular Akt phosphorylation in advance at early phase of CVB3 infection.