Impact Of Quorum Sensing Systems On Regulatory T Cells In Pseudomonas Aeruginosa Biofilm Infections In Vitro

Objective: Quorum sensing(QS)systems in Pseudomonas aeruginosa has been widely believed to participate in modulating biofilm formation as well as the host immune system.In the meantime,the newly found subset of T cells,called regulatory T cells(Tregs),were found in chronic infections.Therefore,our research aims to investigate the impact of quorum sensing systems on Tregs in Pseudomonas aeruginosa biofilm infections in vitro.Methods: Biofilm-conditioned medium(BCM)of PAO1,?lasI?rhlI and ?lasR?rhlR strains were collected respectively,and then used to co-culture with human PBMCs for 16 h.CCK-8 were used to measure the cell cytotoxicity.Cell suspension were collected and centrifuged.The cell pellets were used to extract for total RNA,and mRNA levels of Foxp3 were measured by RT-PCR.The supernatant was used to measure TGF-?1,IL-10 and IL-6 protein levels by ELISA.Flow cytometry analys is was used to identify Tregs.Biofilm infection rat models were conducted and the levels of Foxp3 and TGF-?1 were measured.Results: We found that all three experimental groups showed significant cytotoxicity after the PBMCs were co-cultured with BCM obtained from different strains.Additionally,the Foxp3 mRNA level was significantly higher in PAO1-BCM treatment group compared with those in ?lasI?rhlI-BCM and ?lasR?rhlR-BCM treatment groups(1.15±0.16 & 0.97±0.11,0.93±0.12,P<0.05).The protein level of TGF-?1 was also higher in PAO1-BCM group(844.49±40.97 pg/ml,P<0.05),however,no significant differences were observed between ?lasI?rhlI-BCM and ?lasR?rhlR-BCM treatment groups(725.69±129.16 pg/ml & 740.29±35.86 pg/ml).Interestingly,IL-10 protein level was much lower in PAO1-BCM treatment group(2.46±1.22 pg/ml)compared with those in ?lasI?rhlI-BCM and ?lasR?rhlR-BCM treatment groups(33.29±6.11 pg/ml & 35.11±7.72 pg/ml,P<0.05),and still,no significant difference was observed between the two QS-mutant groups.Whereas the levels of IL-6(799.27±75.66 pg/ml &1305.42±198.48 pg/ml)were higher in ?lasI?rhlI-BCM and ?lasR?rhlR-BCM treatment groups when compared with that in PAO1-BCM treatment group(12.63±8.31 pg/ml,P<0.05).The results of Foxp3 and TGF-?1 in animal experiments were consistent with the results of those in vitro experiments.Conclusion: Our present study has demonstrated that QS systems in Pseudomonas aeruginosa can up-regulate the number of Tregs,and the function of Tregs is also activated.This mechanism might partially explain the phenomenon that Pseudomonas aeruginosa biofilm always result in chronic infections.