Investigatiaon On Effects Of TNF-α, IL-1β And IL-6 On In Vitro Bacterial Growth

Background and objectivesEvery cell, no matter in what forms it is existing, individually or organizationally, will responses to its environmental signals, and with which establishes intercellular interactions. In maintenance of tissue hemostasis and host defense against infection or in host response to non - infectious damage, cells can modulate their growth and multiplication by way of autocrine or paracrine with the aid of series of low molecular - weight polypeptides so - called cytokines.Infection is an outcome from mutual interactions between pathogen and host. Researchers have found that pathogen and/or its products are possibly only a triggering or initiating factors in Systemic Inflammatory Response Syndrome (SIRS) and organ dysfunction caused by infection, whereas the host response to inflammatory insults seems to be much more important. Scientists find that an important way of host response is to release pro - inflammatory cytokines in responding to infectious insults leading to either positive or negative influences in controlling infection. The research on correlation between cytokines ( eg. TNF -α, IL-1β and IL - 6 ) and infectious diseases found that certain degree of cytokines release can be helpful in controlling infections, and cytokines quantity was to some degree correlated with disease severity and prognosis in some studies; Patients with continuous elevated cytokines release are more likely to have hospital - acquired infections.But in recent years, some preliminary studies found that pathogens may possibly evades or even utilize cytokines. Some scientists found that some cytokines can contribute to in vitro growth of some bacteria, but the mechanism is still not clear and many questions are waiting to be investigated.Three cytokines, TNF - α, IL-1β and IL - 6, which are the most important in host response, are selected in this study to investigate their possible effects on in vitro bacterial growth. Pseudomonas aeruginosa, Staphylococcus aureus and Acinetobacter baumanni are 3 bacteria common in hospital infections , and so are used in this study. The present study can possibly be contributing to understanding of interactions between bacteria and host cytokines, and of the mechanisms of bacterial multiplication in persisting inflammation, and also provide further evidence in evaluating therapeutic approaches that is directed in preventing cytokines imbalance.Materials and methods1. Materials(1) Bacteria: Pseudomonas aeruginosa, Staphylococcus aureus and Acinetobacter baumanni are fresh isolates by No. 1 Hospital of China Medical University.(2) Reagents: Frozen cytokines (TNF -α, IL - 1β and IL -6) are from JingMei Biotech; Anti - human TNF - α, IL-1β and IL - 6 monoclonal antibodies from WuHan Boster Biological Technology Co. Ltd.(3) Instruments and others; nutrient broth, nutrient agar plate, incubator, centrifuge, transmissometer, Vortex, inoculating loop, sterile tubes, micropi-pette, PBS, RPMI1640 culture solution and 10% calf serum.2. Experimental procedures(1) cytokines Preparation of different concentrations and bacteria suspension(2) Bacterium quota raise; add 10μl (106/ml, viz, one CFU) to tubes prefilled with 1ml RPMI1640 culture solution containing 0, 10pg, 50pg, 100μg, 500μg, 1ng, and 10ng cytokines respectively, Incubated at 35℃ and took out after 2h, 4h, 6h, 8h, 16h ~ 18h. Then diluted in 10 ratio and inoculated on agar plates with 10 times dilution, and after 16 - 18h incubation at 35℃, CFUs are counted and the mean were calculated.(3) Neutralizing efects of monoclonal antibody: Mix the monoclonal anti-body with according cytokines and make bacterium quota raise.(4) Statistics analysis-. Analyze the data with SPSS11.5 Software System. Use Spearman rank correlation to determine correlation between cytokines concentrations and bacteria CFU. Changes before and after monoclonal antibodies are analyzed with t test.Results1. Effects of the 3 cytokines at different concentrations on growth of 3 bacteria selectedAt 6 hour time point after incubation, Pseudomonas aeruginosa growth and IL - 6 concentration ( rsO. 856), Staphylococcus aureus and IL -1β ( rs0. 856) , Acinetobacter baumanni and IL - 1β (rsO. 937) are correlated significantly , and the growth enhancing effects of cytokines are dose - dependent.2. ffects of cytokines monoclonal antibodiesAfter adding neutralizing antibody, the concentration - dependent growth -enhancing effects of cytokines are remarkably inhibited.IscussionThe studies on how bacteria can evade or utilize host cytokines are seldom carried out, and the mechanism is till not clarified. It was reported by Denis that IL - 6 in vitro can directly promote mycobacterium avium multiplication. Hogan 's research on γ - interferon on E coli as well as Porat s on IL-1β on pathogenic E coli found that the bacterial growth - enhancing effects were concentration - dependent. But the mechanisms were not clear. It is regarded that cytokines do not change the quantity of bacteria in GO phase but only exert influence on how faster can bacteria progress to GO. phaseThe present study shows that IL - 6, IL-1β and IL-1β do have a growth - enhancing effects on newly isolated Pseudomonas aeruginosa, Staphylococcus aureus and Acineitobacter baumanni in a concentration - dependent manner. The defects can be neutralized by specific cytokines monoclonal antibodies.