Study On The Relationship Between Latent Cytomegalovirus Infection And IPF By Activating TGF(?)β1

BackgroundIdiopathic pulmonary fibrosis（Idiopathic pulmonary,fibrosis,IPF）is a chronic pulmonary interstitial disease which mainly occurs in the elderly over the age of50,most sporadically while rare in familial aggregation.It is one of the most common form of interstitial lung disease and characterized with common pathological type of interstitial pneumonia pathologically.The etiology and pathogenesis of IPF is still not fully understood.Studies found that many IPF patients had a previous viral infection history.SARS coronavirus,H5N1influenza virus,and highly pathogenic avian influenza virus are all high fatality viruses which have caused severe respiratory symptoms and threatened the public health safety nationally and worldwide since the year of 2000.Pulmonary fibrosis was found in the survival patients after the viral infection was controlled.In contrast to SARS and influenza viruses which often cause severe acute respiratory infections,some weak virulence virus infection were also found related to IPF,which is particularly concerned about the herpes virus,especially cytomegalovirus（CMV）,EB virus,human herpes virus-7（HHV-7）and human herpes virus-8（HHV-8）.Moreover,studies have also found that chronic hepatitis C virus is associated with pulmonary fibrosis.As IPF is a chronic disease rather than an acute disease,it is more likely that a latent viral infection which targets at alveoli and/or interstitium in the lung and causes alveolitis and interstitial lung disease.Human cytomegalovirus（CMV）,one of the largest virus of human herpes virus group in the herpes simplex virus family,can cause human cytomegalovirus infection.CMV can cause latent infection without clinical symptoms in people with normal immune-state.The virus often aggregates in the white blood cells,kidney,uterus neck,salivary gland,testis,and breast.The continuous or intermittent release of viruses from these their latent organs will stimulate the body to produce some cytokines,especially the TGF-β1 factor.CMV infection increases the quantity and activity of TGF-β1,which then actives the TGF-β1/SMAD2 signal pathway which further causes pulmonary epithelial-mesenchymal transition（EMT）and pulmonary fibrosis.Objective:This research studied 1)the relationship between latent CMV infection and IPF by comparing the measurements of detection copy numbers of CMV DNA and PP65 gene and quantality of CMV IgG in IPF patients and control patients without IPF;2)the role of TGF-β1/SMAD2 signal pathway in the development of EMT and pulmonary fibrosis in mouse models with latent MCMV infection and bleomycin induced pulmonary fibrosis.Methods Thirty-eight IPF patients and twenty age and sex matched lung cancer patients were recruited in this study after consent.CMV IgG antibody,CMVpp65 antigen,and CMV DNA copy number in peripheral blood and BALF were detected by Indirect Chemiluminescence immunoassay,immunofluorescence quantitative PCR,and indirect immunofluorescence,respectively.The results were further analyzed with standard analytic methods.The salivary gland?passed MCMV was prepared by homogenizing the salivary glands of the BALB/c mice,which had been infected with 1×10⁴ plaque forming units（PFU）for 3 weeks.Aliquots of the homogenized supernatants of the salivary glands were stored in liquid nitrogen with RPMI 1640 with 5%fetal bovine serum,and titrated onto NIH?3T3 cells.The salivary gland-passaged MCMV was suspended in 200μl of Hanks’balanced salt solution with 3%fetal bovine serum.The experimental group mice were infected via intra-peritoneal injection with 1×10⁵ PFU and the control group mice were injected with Hanks’balanced salt solution/3%fetal bovine serum.The viral loads in mouse salivary gland,spleen,liver,and lung were analyzed using PCR at 3,14 and 28 days following infection.After 4 weeks,the MCMV-infected and uninfected mice were anesthetized with sevoflurane inhalation and placed in dorsal recumbency.Transtracheal insertion of a 24G animal feeding needle was used to instill bleomycin（0.75 U/ml）or vehicle（PBS）,in a volume of 80μl.The mice were sacrificed 7 and 14 days after instillation,and the lungs were removed for further analysis.The lung tissue of mice was divided into 4 parts,of which 1 were fixed with formaldehyde after paraffin sections were prepared for histopathological examination,a total protein extraction by Western blot quantitative detection of EMT signaling pathway related protein expression in a tissue homogenate was prepared to detect TGF-1 beta activity,after extracting RNA by detecting a TGF-Real-time PCR beta 1 and EMT signaling pathway related gene mRNA expression.Results Through analysis of peripheral blood and bronchoalveolar fluid content CMV IgG antibody detection CMV IgM antibody screening,CMV detection rate and copy number of DNA and pp65 antigen,and bronchoalveolar cells in BAL fluid in 38 cases of IPF group and 20 patients in control group,CMV IgG and CMV DNA were detected in the bronchoalveolar lavage fluid of the IPF patients group,suggesting that IPF was associated with the infection of CMV.The CMV IgG antibody in the bronchoalveolar lavage fluid,CMV DNA copy number were significantly higher in the IPF experimental group than those in the control group.However,in the group of patients with IPF,CMV pp65 antigen was not detected in the in peripheral blood and alveolar,which indicates that CMV latent infection is dominant in the newly diagnosed stable IPF patients.Mouse lung epithelial cells were prepared and the mouse CMV（MCMV）was then inoculated into the cells.The expression of viral proteins and TGF-β1 was detected by indirectimmunofluorescenceandWesternblot,respectively.Thesalivary gland-passaged MCMV was successfully prepared,the titer of the MCMV was5×10⁶PFU/ml.The results of the RT-qPCR analyses showed that MCMV invaded the lung tissues.The viral DNA loads in the salivary glands,spleen,liver and lungs were highest for 3 days following inoculation.Then,the numbers gradually reduced and maintained at a low level,which is similar the latent infection in humans.Changes in the body weights of the mice suggested that the weights of the mice decreased sharply following infection for 3 days,and then the weights recovered rapidly in the infected group.No significant differences were observed between the control group and infected group at day 15 post infection.Compared with the activity of TGF-β1 in the beomycin group and control group following treatment for 7 days,the activity of TGF-β1 was higher in the MCMV+beomycin group and the activity reached to the highest level at day 14 following treatment.The results of the pathological detection showed that the degree of pulmonary fibrosis was higher in the MCMV+beomycin group comparing with the beomycin group following treatment for 7 and 14 days.The results of the western blot analysis showed that the protein expression levels of Vimentin and phospho-SMAD2 increased following treatment with beomycin and MCMV+beomycin,whereas the protein levels of E?cadherin decreased following treatment with beomycin and MCMV+beomycin.Conclusion This study revealed that（1）CMV infection is associated with IPF and latent infection is a mechanism for IPF development in patient with CMV infection;（2）MCMV infection may activate pulmonary epithelial cell EMT and aggravate pulmonary fibrosis through the TGF-β1/SMAD signal pathways which increase Vimentin expression and decrease E-Cadherin expression.