| Proinflammation Cytokines enhanced the AntibacterialActivity of Human Urinary Bladder Epithelial Cellsagainst Klebsiella PneumoniaeThe epithelium provides the first barrier protecting the host frommicrobe infections. Besides their function as a mechanical barrier, epithelialcells produce cytokines, chemokines and antimicrobial peptides which arecritical components of the innate and acquired immunity.Klebsiella pneumoniae is a common opportunistic pathogen thatfrequently caused nosocomial infections, especially in patients withimmunodeficiency. Klebsiella pneumoniae infections range from mildurinary infections to severe bacteremia and pneumonia with a high rate ofmortality and morbidity.The pathogenetic process of infections caused by Klebsiella pneumoniaeinvolves receptor-ligand recognition between bacteria and host cells, bacterialadherence to and entry into epithelial cells. It has been proved that theadherence of Klebsiella pneumoniae to the epithelial cells activates cellularsignal transduction, which leads to internatization of bacteria into epithelialcells.The factors involved in Klebsiella pneumoniae pathogenesis includecapsular antigen, adhesin such as type-1 pilus, LPS, anti-serum reactionsystem and iron carrier. The natural host defense to bacteria recognizesconserve microbial components known as pathogen-associated molecularpattems (PAMP) with pattern recognition receptors (PRR) and induces theproduction of inflammation and protective immunity. Toll-like receptors(TLR) and Nods receptors are two classes of PRR that are located on the cellular membrane and in cytosol respectively.The mechanisms of inflammatory and immunity in epithelial cells arecomplicated, involving inflammatory signal transduction and the expressionof a wide variety of genes. During the long-term of co-evolution betweenpathogens and their hosts, complex interrelationship of infection andanti-infection has taken shape at the epithelial surface. With the developmentof bacterial virulent factors such as adhesin and invasin, pathogens maycolonize and grow at the mucosa surface, subjecting the host to infectiousdiseases. Therefore, it is necessary for the host to evolve potent defensemechanisms against microbes. Thus, epithelial cells functioning as thefrontline barrier preventing infection not only produce antibacterial peptideswith directly microbicidal effect, but also they secrete chemokines andcytokines modulating inflammatory and acquired immune responses.The purpose of this study was to investigate the interaction betweenintracellular Klebsiella pneumoniae and human urinary epithelial cells. Twoclinical isolates of Klebsiella pneumoniae, K5 and 03183, were incubatedwith T24 cells for 2 hours. The extracellular bacteria were killed by theaddition of gentamicin. At different time points of incubation, cells werelysed with TritonX-100. Cell lysates were appropriately diluted and plated onLB agar plates to quantify viable intracellular bacteria. The results showedthat thought in the early phase of bacterial entry into T24 cells (within 24hours) intracellular Klebsiella pneumoniae K5 and 03183 multiplied slightly,viable intracellular bacteria decreased after 24 hours. These findings suggestthat urinary bladder epithelial cells are able to contain the growth ofintracellular bacteria and moreover, to clear intracellular Klebsiellapneumoniae.TNF-α, INF-γand IL-1βare proinflammatory cytokines that playimportant roles in host defense. To observe the effects of these cytokines onthe clearance of intracellular Klebsiella pneumoniae by urinary bladder epithelial cells, confluent T24 cell monolayers were incubated with Klebsiellapneumoniae strain K5 for 2 hours and treated for another 2 hours with freshmedium containing gentamicin (100ug/ml) to kill extracellular bacteria. Thecells were then exposed to difference concentrations (5ng/ml, 10ng/ml,20g/ml) of TNF-α, INF-γand IL-1βrespectively and were lysed withTritonX-100 at 24hr and 48 hr after the initial incubation to quantify viableintracellular bacteria. The results showed that in the presence of TNF-α,INF-γand IL-1β, the viable intracellular Klebsiella pneumoniae decreasedsignificantly compared. to untreated control cells. The cytokine-enhancedclearance of intracellular Klebsiella pneumoniae by T24 cells wasdose-dependant and INF-γwas more potent than other two cytokines.To ascertain if there is synergetic effect on the enhancement ofantibacterial activity between these cytokines, T24 cells were treated withTNF-αplus INF-γand TNF-αplus IL-1βrespectively. The results revealedthat combined application of TNF-αplus IL-1βare slightly more effectivethan separate cytokine application. However, combined application of TNF-αplus TNF-αstrongly enhanced the capacity of T24 cells to clear intracellularbacteria and suggested that these two cytokines induced synergisticallycellular antibacterial activityRespiratory burst creating a variety of reactive oxygen species (ROS) inneutrophils and macrophages is one of the major mechanisms by which theykill phagocytized bacteria. To determine whether ROS is relevant tocytokine-induced antibacterial activity in urinary bladder epithelial cells, weperformed experiments in the presence of catalase (CATA) andNG-monomethyl-L-arginine (L-NAME). The results shown that the additionof CATA significantly decreased the antibacterial activity of cytokine-treatedT24 cells, while L-NAME had slight effect, indicating that the production ofH2O2 by cytokine-treated T24 cells is involved in clearance of intracellularbacteria. In short, results of this study proved that urinary bladder epithelial cellspossessed innate capability to control the growth of and clear intracellularbacteria that may contribute to maintenance of urinary bladder in sterilesituation. Proinflammation cytokines such as TNF-α,INF-γand IL-1βcouldenhance antibacterial activity of epithelial cell, suggesting that during urinarybacterial infections cytokines producing by in situ inflammatory cells couldmodify the defense function of epithelial cells.
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