Effects Of Oral Bacteria On The In-vitro Infection Of Pseudomonas Aeruginosa To Respiratory Epithelial Cells

Effects of oral bacteria on the in-vitro infection of Pseudomonas aeruginosa to respiratory epithelial cellsObjectivePeriodontal disease is the most common chronic infectious disease.In recent years,many investigations have indicated that periodontal infection may be the real risk factor for some systemic diseases(e.g.cardiovascular disease,diabetes,respiratory disease,gastrointestinal disease,osteoporosis,low birth weight premature infants, herpes virus infection).The relationship between periodontal disease and pulmonary infection has received more and more attention.Bartlett et al.have found that 35%of nosocomial infections and pneumonia-are associated with pathogenic anaerobes.Later on,the periodontal anaerobic pathogens have been isolated from the lungs infected in many experimental studies,which confirmed their findings.In 1990,Slots isolated Pseudomonas aeruginosa from the oral cavity in patients with refractory periodontitis. He Let al.confirmed that acquired pneumonia pathogens are highly homogenous with oropharyngeal bacteria through molecular biological techniques.All the findings above indicate that periodontal bacteria and lung infection are closely related.Pseudomonas aeruginosa,an important opportunistic bacterial pathogen,causes infections of the respiratory and other organs in susceptible hosts.Pseudomonas aeruginosa infection is initiated by adhesion to and invasion of mucosal epithelial cells.The failure of host defenses to eliminate Pseudomonas aeruginosa from mucosal surfaces results in Pseudomonas aeruginosa proliferation,sometimes followed by overt infection and tissue destruction.Recent studies have associated poor oral health with respiratory infection.An in vitro model system for bacterial invasion of respiratory epithelial cells was used to investigate the role of oral bacteria in Pseudomonas aeruginosa invasion. Oral pathogens,including Porphyromonas gingivalis,Fusobacterium nucleatum and Aggregatibacter actinomycetemcomitans,increased invasion of Pseudomonas aeruginosa into HEp-2 cells from one to three fold.In contrast,non-pathogenic oral bacteria,such as Actinomyces naeslundii and Streptococcus gordonii,showed no significant influence on Pseudomonas aeruginosa invasion.Pseudomonas aeruginosa together with oral bacteria stimulated greater cytokine production from HEp-2 cells than did PAO1 strain alone.In oral bacteria,P.gingivalis bacteria is the major pathogen of chronic periodontal disease.In this study,P.gingivalis together with Pseudomonas aeruginosa stimulated lung epithelial cells to produce inflammatory cytokines IL-6 and IL-8.IL-6 may activate the JAK-STAT3 signal transduction pathway.Targets of STAT3 include anti-apoptotic Bcl-2 family members and Survivin protein from IAP family.In terms of mRNA alteratations of pro-apoptotic protein(Bad, Bak) and anti-apoptotic proteins(Bcl-2,Survivin) at different time points when P. gingivalis bacteria concentrations reach a certain level,it was suggested that P. gingivalis may adhere to,be internalized and proliferate in lung epithelial cells in short-term to further promote the apoptosis of lung epithelial cells,which led to more severe destruction of lung tissue infected.The purpose of this study is to investigate the changes in the number of host cells invaded by bacteria and the degree of host cell apoptosis when the lung epithelial cells were exposed to P.gingivalis together with Pseudomonas aeruginosa,to clarify the apoptosis pathway and dynamic pattern of cytokine released in response to bacteria and to explore the synergistic role of oral pathogens in lung infection and their mechanisms of action.Methods1.SubjectsRespiratory bacteria:Pseudomonas aeruginosa(TAO1 strain)Oral bactria:P.gingivalis;F.nucleatum;A.actinomycetemcomitans; A.naeslundii;S.gordoniiCell:HEp-2 epithelial cells and A549 epithelial cells2.MethodsBacterial adhesion and invasion experiments,including the culture of bacteria and cells,selection of bacteria concentration and culture time,the number of bacteria in HEp-2 cells infected by PAO1 alone or in the presence of oral bacteria.Detection of cell survival and apoptosis:(1) Measurement of the number of viable cells cultured with bacteria for different periods of time using MTT assay;(2) Fluorescent spectrophotometric analysis of apoptosis.Evaluation of the effects of oral bacteria on IL-6 and IL-8 production by HEp-2 cells cultured with Pseudomonas aeruginosa using ELISA.Observation of pulmonary cell apoptosis pathway induced by P.gingivalis and PA01 strain as well the cytokines involved in above process using RT-PCR and Realtime PCR.3.Statistical AnalysisData were statistically analyzed using SPSS 13.0.Independent student t test or one-way ANOVA was used to compare differences in normally-distributed data between groups,which are shown as mean±SD.Otherwise,rank sum test orχ~2 test was used for comparison between groups.Logistic regression model was used for multi-factor analysis.Differences were considered statistically significant if p＜0.05.Results1.The regulation of PAO1 invasion to HEp-2 cells by Oral bacteriaThe adhesion and invasion of Pseudomonas aeruginosa to HEp-2 cells without or with other bacteria.When Pseudomonas aeruginosa were incubated with epithelial cells in the presence of oral bacteria,Pseudomonas aeruginosa adhered to HEp-2 cells at different levels depending on the species of oral bacteria used.Co-incubation with F. nucleatum,S.gordonii,or A.naeslundii;did not significantly alter the interation of PAO1 to HEp-2 cells.In contrast,both P.gingivalis and A.actinomycetemcomitans significantly decreased the interaction of PAO1 with the epithelial cells.P.gingivalis,F. nucleatum and A.actinomycetemcomitans significantly increased the percentage of Pseudomonas aeruginosa that invaded,up to three fold when compared to Pseudomonas aeruginosa alone,whereas A.naeslundii and S.gordonii showed no significant influence on Pseudomonas aeruginosa invasion activity.2.The effects of oral bacteria on the response of Hep-2 cells to Pseudomonas aeruginosa invasion(1) Epithelial cell survival and proliferationWhen HEp-2 cells were incubated with Pseudomonas aeruginosa alone or co-cultured with oral bacteria(MOI 100:1),the number of viable epithelial cells decreased slightly at 1h of infection,as indicated by the MTT assay.When Pseudomonas aeruginosa was incubated together with oral bacteria,more epithelial cells were killed than were killed by Pseudomonas aeruginosa alone at all time points. Most of the epithelial cells remained viable between 0 and 6 h of exposure to all of thebacterial strains tested.However,by 12 h,increased epithelial cell death was seen in most cases.Fewer epithelial cells survived when Pseudomonas aeruginosa was co-incubated with A.actinomycetemcomitans(32%average epithelial cell survival;p＜0.001),followed by F.nucleatum(39%;p＜0.01) and S.gordonii(41%;p＜0.01), compared to Pseudomonas aeruginosa challenge alone(55%).P.gingivalis(average epithelial cell survival 50%) in combination with Pseudomonas aeruginosa also induced more cell damage than Pseudomonas aeruginosa alone,although the difference was not statistically significant.Control experiments were also performed to examine the effect of each oral strain alone on HEp-2 cell viability.Results showed that oral bacteria alone damaged HEp-2 cells,usually beginning at 12 h,with an average epithelial cell survival of 48%,52%,and 65%for S.gordonii, A.actinomycetemcomitans and A.naeslundii,respectively.In contrast,P.gingivalis and F.nucleatum demonstrated no obvious damage to epithelial cells at 12h.(2) Epithelial cell apoptosisCaspase-3 activity is a marker for eukaryotic cell apoptosis.HEp-2 cell apoptosis was triggered after both Pseudomonas aeruginosa contact with host cells and Pseudomonas aeruginosa co-cultured with oral bacteria.Pseudomonas aeruginosa induced epithelial cell apoptosis increased with time,and coincubation with oral bacteria enh]anced apoptotic activity.After 1 h,apoptosis of HEp-2 cells was significantly increased only by the incubation of Pseudomonas aeruginosa with P. gingivalis as compared to incubation with Pseudomonas aeruginosa alone(p＜0.001). However,by 12 h,co-incubation of Pseudomonas aeruginosa with each of the other oral species significantly triggered apoptosis in HEp-2 cells to a greater extent than when Pseudomonas aeruginosa was incubated with the epithelial cells alone for the same length of time(p＜0.001).Pseudomonas aeruginosa co-incubation with F. nucleatum induced the highest level of caspase-3 activity,F.nucleatum alone showed lower caspase-3 activity to HEp-2 cells.(3) Effects of oral bacteria on production of IL-6 and IL-8 from HEp-2 cells upon infection by PAO1 with oral bacteriaAll of the oral bacterial strains tested,with the exception of P.gingivalis,induced a higher level of cytokine release than did Pseudomonas aeruginosa alone.P. gingivalis appeared to reduce the detectable IL-6 and IL-8 released into the supernatant by HEp-2 cells by 4 and 12 h,respectively.Co-incubation of Pseudomonas aeruginosa with the oral bacteria slightly reduced cytokine release as compared to incubation with the oral bacteria alone.F.nucleatum was the most potent stimulator of IL-6 and IL-8 release from HEp-2 cells after 6 and 12 h of incubation.In general,IL-6 was released earlier than IL-8 from epithelial cells and IL-8 release reached a peak concentration later than did IL-6.3,The mechanism for epithelial cell apoptosis incubated with Pseudomonas aeruginosa in the presence of P.gingivalisRT-PCR and realtime PCR were used to qualitatively and qualitively analyze the pathway for pulmonary cell apoptosis induced by P.gingivalis co-cultured with Pseudomonas aeruginosa STAT3 was activated in respiratory epithelial cells after 2-hour incubation with P.gingivalis alone or in the presence of Pseudomonas aeruginosa,which was stronger in the latter.Expression of pro-apoptotic(Bad) and anti-apoptotic(Bc1-2) members of Bc1-2 family in respiratory epithelial cells incubated with P.gingivalis and Pseudomonas aeruginosa.The expression of bad were increased after 2-hour incubation of bacteria, inhibited after 4h,and then reached the peak levels after 8h.The latter indicates that apoptosis was accelerated at that time.After 12-hour of incubation,most of the cells had undergone apoptosis or death.The anti-apoptotic protein Bc1-2 and also showed corresponding alterations at all time points tested above.Survivin can inhibit apoptosis and promote cell proliferation,which is located downstream of STAT3.It was increased after 2-hour incubation,which indicates the self-repair in stress responses at the early period of damage.Survivin expression was markedly increased after 4 hours.However,it then was gradually decreased after 8 hours,suggesting that the damaged cells could not recover at that time.Conclusion1.P.gingivalis,F.nucleatum and A.actinomycetemcomitans significantly increased the percentage of Pseudomonas aeruginosa that invaded,up to three fold when compared to Pseudomonas aeruginosa alone,whereas A.naeslundii and S.gordonii showed no significant influence on Pseudomonas aeruginosa invasion activity.It further indicates that poor oral health is associated with respiratory infection.2.Pseudomonas aeruginosa together with oral bacteria stimulated greater cytokine production from HEp-2 cells than did Pseudomonas aeruginosa alone. Pseudomonas aeruginosa in combination with periodontal pathogens also increased apoptosis of HEp-2 cells.Extrapolating these findings to the in vivo situation,invasion of the mucosal surface and subsequent induction of cell apoptosis and the release of cytokine by oral bacteria may alter the local microenvironment,and facilitate the onset and/or progression of respiratory disease in susceptible individuals.3.When pulmonary epithelial cells are exposed to Pseudomonas aeruginosa together with P.gingivalis at certain levels,JAK-STAT3 signal pathway may be activated to regulate the expressions of the downstream proteins-Bc1-2 and Survivin, which may result in a transient inhibition of apoptosis,cause more bacteria to adhere and invade into the cells,and bring about more severe respiratory inflammation.The findings above suggest an important role of periodontal pathogens in respiratory infection.