Study On The Mechanism Of 3OC12HSL’s Participation In The Immune Modulation Between HCE And Pseudomonas Aeruginosa

Background:The keratitis induced by Pseudomonas aeruginosa is a potential blinding bacterial infection keratitis,normally the healthy cornea has a strong resistance to Pseudomonas aeruginosa,only when the corneal epithelial barrier were damaged can cause the corneal infection.A variety of factors such as trauma,ocular surface disease,immunodeficiency and long-term use of contact lenses are associated with the development of Pseudomonas aeruginosa keratitis.The persistent unhealed Pseudomonas aeruginosa keratitis can cause the damage of normal corneal physiological structure,even corneal scars.Pseudomonas aeruginosa keratitis is one of the most serious corneal acute suppurative inflammation,such as failure to take timely and effective measures can cause serious consequences.Pseudomonas aeruginosa affects host immune and evades immune surveillance through a population sensing mechanism(Quorum sensing,QS system)and modulate the gene expression.The detection of homoserine lactone(AHL)secretion can reflect the density of Pseudomonas aeruginosa.Pseudomonas aeruginosa mainly produces two kinds of AHL,3-oxododecanoyl-homoserine lactone(30C12HSL)and butyralyl serine lactone(C4-HSL,also known as PAI-2 or BHL).Although these two AHLs can be spread inside and outside the bacteria,but 30C12HSL can also be from the bacteria to the extracellular environment by MexAB OprM efflux system.Such as the density of Pseudomonas aeruginosa increases in the environment,the AHLs content will increase.When the threshold concentration is reached,AHLs bind and activate the transcription factor and then modulate the expression of the important gene to increase the expression level,which also lead to increased bacterial toxicity.Recently,it has been initially confirmed that 3OC12HSL is not only important to regulate bacterial virulence,but also critical in interacting with eukaryotic cells and stimulating the immune response.30C12HSL can stimulate human corneal epithelial cells to induce the production of chemokine IL-6.This induction of IL-6 is regulated by the activation of the mitogen-activated protein kinase pathway,which in turn leads to activation of the transcription factor NF-κB.30C12HSL also activates classic cellular immunity.For example,it has recently been shown that 30C12HSL can directly stimulate T cells and lead to an increase in IFN expression.These data indicate that 30C12HSL secreted by Pseudomonas aeruginosa is a potent activator of many different eukaryotic cells and may greatly affect the pathogenicity of Pseudomonas aeruginosa.30C12HSL is a molecular basis not only for microbial communication but also between microbes and the cells,and is an indispensable molecule of Pseudomonas aeruginosa virulence.When the cornea is infected with Pseudomonas aeruginosa,Pseudomonas aeruginosa will release 3 OC12HSL molecules,when the body’s immune system detects the signal molecules,the macrophages and epithelial cells will react to the signal molecules,and then increase the production of various inflammatory factors,which is an important reason for the delayed healing of Pseudomonas aeruginosa keratitis.Therefore,the study on the impact of 30C12HSL in the immune regulation has a positive significance.In this paper,we mainly study the mechanism of 3OC12HSL’ participation in the immune modulation between HCE and Pseudomonas aeruginosa.Purpose:As known that human corneal epithelial cells(HCE)contribute to the inflammatory process in the ocular surface by releasing inflammatory cytokines.N-acyl-homoserine lactone(AHL)quorum-sensing molecules produced by gram-negative bacteria can influence the innate immune of corneal epithelium.In this study,we investigated the effects of N-3-oxododecanoyl-homoserine lactone(30C12-HSL)on cell viability and inflammatory cytokines secretion in human corneal epithelial cells(HCE).Methods:In this study,immortalized human corneal epithelial cells were cultured in vitro,and different concentrations of 30C12HSL were added.The cell viability were measured by Crystal violet staining and CCK-8.The expression of Toll-like receptors(TLR2、4、5、6)in corneal epithelial cells under different stimulation conditions was detected by Real-time PCR.Western Blotting was used to detect the expression of Toll-like receptor and signal pathway molecule NF-κB.The expression of NF-κB was observed under immunofluorescence after immunofluorescence staining.The secretion of cytokines IL-6,IL-8,IL-10 and TNF-α was detected by Real-time PCR.The secretion of IL-8 in the supernatant was detected by ELISA and analyzed statistically.Effects of different concentrations of dexamethasone on Pseudomonas aeruginosa biofilm were measured by quantitative crystal violet staining.The statistical analysis was performed by the software GraphPad Prism 7.0.Results:We investigated the effect of 30C12HSL on the downstream immune response of NF-kB.We found that 30C12HSL can present the expression of Toll-like receptors mRNA and protein in THCEs in a concentration-dependent manner by RT-PCR and Western-blot,and the low concentration promotes the expression of TLR,and the high concentration inhibits the expression of TLR,especiall-y TLR2 and TLR4 Has a positive correlation with the processing time.The mRNA of IL-6,IL-8,IL-10,TNF-a were found increased after treated with 30C12HSL.In order to explore the relationship between IL-8 expression and TOLL-like receptors,the specific monoclonal antibodies of TLR2,4,5,6 were used to block TLR2,4,5,6 and the specific agonists of TLR2,4,5,6 were used to activate TLR2,4,5,6.The expression of IL-8 was significantly decreased after treated with the specific monoclonal antibodies of TLR2 and the other agonists and antibodies had no obvious effect.The pretreatment of dexamethasone can inhibit the formation of Pseudomonas aeruginosa biofilm,while the pretreatment of 30C12HSL did not have a significant effect on Pseudomonas aeruginosa biofilm.Conclusions:30C12HSL can regulate the immune response of human corneal epithelial cells,and can regulate the expression of immune-related inflammatory factors.30C12HSL has specific TLRs activation mechanism on the regulation of the innate immunity of corneal epithelial cells,especially the TLR2 and TLR4;The specific TLRs activation mechanism is related to the activation of nuclear transcription factor NF-κB,the transcription factor NF-κB may be one of the important pathways in the early stage of immune regulation.