The Mechanism Of Haemophilusparasuis Serotype 4 Across Tracheal Epithelium Barrier By Breaking Tight Junctions

Haemophilus parasuis(HPS)is an important swine pathogen.It can invade the body and cause the swine Glasser’s disease with the main clinical symptoms are meningitis,multiple serositis and arthritis as an opportunistic pathogen.HPS includes at least 15 serotypes and about 25%of isolates were still clinically indeterminate.The main epidemic strains in China are serotype 4 and serotype 5,which have highly virulence so far.Further identification of the serotypes of HPS isolates from 17 provinces in China found that the isolation rate of serotype 4 was slightly higher than serotype 5.HPS leads to mixed infection with other pig pathogens such as porcine circovirus type 2,porcine reproductive and respiratory syndrome virus,Streptococcus suis and so on,which causes huge economic losses to the pig industry in the world every year.Therefore,the research on its pathogenic mechanism as well as effective prevention and control has been paid more and more attention.HPS can colonize the respiratory tract of healthy pigs and cause pig disease under stress or co infection.As a respiratory pathogen,respiratory epithelium is the first line to defense against HPS.Recently researchs found that HPS can invade endothelial cells or epithelial cells in vitro,but the mechanism of its invasion into the body through the respiratory epithelial barrier is not clear.In this study,HPS colonized porcine respiratory epithelial cells(STEC)were used as the cell model of HPS infection and the respiratory epithelial cell barrier model was constructed in vitro to study the mechanism of HPS infection from the upper respiratory tract of pigs and provide reference basis for the effective control the disease caused by HPS.1.Establishment of STEC model of porcine tracheal epithelial cells infected with Haemophilus parasuis serotype 4 HPS4-YCIn this study,the porcine tracheal epithelial cell(STEC)colonized by HPS,was used as the cell model of HPS infection to study the mechanism of HPS infection through upper respiratory tract.Giemsa staining showed that HPS4-YC could adhere to STEC.According to the number of HPS bacteria adhered to each STEC cell,it was found that the adhesion of HPS to STEC was dose-dependent of bacteria.The multiplicity of infection(MOI)and time of HPS infection were studied by using adhesion rate and invasion rate as the criterion.These results showed that the optimal condition of STEC infected with HPS4-YC was 2h,MOI=100.Also,the LDH release assay indicated that there was no cytotoxic effect on STEC during the adhesion and invasion of HPS.In conclusion,this study preliminarily established the infection model of STEC infected with HPS,which was a foundation for the follow-up study.2.The mechanism of HPS4 breaking through the epithelial barrier of STEC.HPS infects the body through the respiratory tract.The highly virulent strain can cause acute systemic inflammation in pigs,and it is easy to cause mixed infection or even sudden death with other infectious diseases of pigs.In this study,Transwell’s epithelial barrier model was constructed by STEC which can be colonized by HPS.The effect of HPS4-YC strain on the respiratory epithelial barrier of STEC was observed with MOI= 100 bacteria infection.The results showed that no bacteria penetrated the barrier within 0-7 h of infection;at 8 h,bacteria began to cross the barrier.The CFU was about 100 and penetration rate was less than 0.001‰.While after 10 h,the penetration rate of bacteria accelerated dramatically,and the penetration rate increased 65 times to 0.02 ‰ at 12h,with 2.6×10~4CFU,which indicated that the cell barrer was obviously damaged and bacteria penetrated the barrier in large quantities.The trans-epithelium electrical resistant(TEER)is widely accepted as a quantitative technique for measuring the integrity of tight junction dynamics in endothelial and epithelial monolayer cell culture models.The TEER value of the barrier infected with HPS were recorded in real time,and results showed that the resistance value decreased significantly at 6-8h.To further detect the effect of HPS4-YC on barrier integrity,fitc-dextran(fd-4)was used to detect paracellular permeability.It was found that,fd-4 permeability of the group infected with HPS4-YC increased after the 6th hour,compared with the control group.LDH release assay was conducted after STEC infected with HPS.It was found that there was no cytotoxicity on STEC after HPS infection 24h.The above results indicated that HPS can destroy the integrity of the respiratory epithelial barrier constructed by STEC and cross the barrier,meanwhile,the penetration mechanism has nothing to do with cytotoxicity.3.The effect of STEC infection by HPS4 on tight junction proteinThe swine tracheal epithelial cell(STEC)is a host cell which can be colonized by HPS and previous studies have confirmed that HPS can penetrate the epithelial barrier model in vitro,increasing paracellular permeability and damaging barrier integrity.The epithelial barrier is maintained by tight and adhesive junctions that form apical junction complexes in the epithelium of the respiratory tract.In this study,STEC monolayer cells were incubated with HPS HPS4-YC and the level of TJ mRNA were detected at different time.Results showed that HPS4-YC infection down regulated the transcription levels of ZO-1,occludin and claudin-1 and the difference was significant,but the transcription levels of claudin-4 increased.In addition,Western blotting of ZO-1,occludin and claudin-1 showed that the protein expression decreased.The indirect immunofluorescence results also suggested that the integrity of the tightly connected distribution was compromised.These results showed that HPS could disrupt the tight junction structure and break through epithelial barrier model by down-regulating the transcription and expression level of ZO-1,occludin and claudin-1.