Borrelia Burgdorferi And Its Membrane Protein BmpA Initiate Cytokine Production In Rhesus Brain Tissue And Human Microglial Cell Line

Objectives:1.This study aims to explore the expression of cytokines in an ex vivo model of interaction of Borrelia burgdorferi(B.burgdorferi,B.b)and its membrane protein BmpA with rhesus brain frontal cortex tissue explants.2.To study B.burgdorferi and its membrane protein BmpA initiate IL-6 production in human microglial cell line.Research content and Methods:1.Exploration of the cytokines expression in an ex vivo model of interaction of live B.burgdorferi and its membrane protein BmpA with rhesus brain frontal cortex tissue explants.(1)Experimental groups:Control group,rBmpA experimental group and B.burgdorferi experimental group.(2)The concentration of cytokines in the culture medium was assessed by Quantibody(?)Non-Human Primates Cytokine Array 1(Quantitative measurement of 10 Non-Human Primate(NHP)cytokines).(3)The concentration of IL-6 and TNF-α was later confirmed using ELISA technique with the RayBio Rhesus Macaque IL-6 and TNF-α ELISA kits.Freshly harvested frontal cortex brains tissues were collected immediately at necropsy from rhesus macaque and washed two times with PBS.The frontal cortex tissues were then sliced as thin as possible.Each section weighed 0.5 g and was placed in a separate T-25 cell culture flask containing 4 mL of 10%FBS-RPMI 1640 medium(Control group)or 4 mL of 1×107/mL B.burgdorferi bacterial suspension(experimental group)or 4 mL of 40 μg/mL rBmpA solution(experimental group);and incubated in a humidified incubator,conditions:5%CO2 at 37℃ for 6 h,12 h,and 24 h,respectively.At the end of the incubation times,the culture mediums were collected and first subjected to Quantibody(?)Non-Human Primates Cytokine Array 1 for cytokines detection.After the results of the cytokine array,ELISA technique was used to confirmed the concentration of highly expressed cytokines IL-6 and TNF-α.2.To study B.burgdorferi and its membrane protein BmpA initiate IL-6 production in human microglial cells.(1)Experimental groups:PBS(control)group,rBmpA experimental group and B.burgdorferi experimental group.(2)The concentration of IL-6 in cells culture medium was assessed using ELISA technique.(3)The mRNA level of IL-6 in cells was assessed using Real-time PCR technique.The human microglial HMC3 cell were cultured in 6-well microplates at 37℃,5%CO2 incubator.The suitable preparation of PBS,B.burgdorferi and rBmpA were added into corresponding stimulation groups and cultured for 6 h,12 h,24 h and 48 h.At the end of the incubation times,the culture mediums were collected and Trizolwas used to completely lyse the cells,and the cells were collected.The concentration of IL-6 in the cells culture medium was assessed using ELISA technique and the mRNA level of IL-6 in cells was assessed using Real-time PCR.Results:1.The results of the cytokine array showed that,compared to the control group,three cytokines(IL-6,IL-16 and TNF-α)increased in the experimental groups.-IL-6:6 h and 12 h following stimulation,the protein rBmpA induce the highest IL-6 production compared to the control.-IL-16:6 h following stimulation,B.burgdorferi induced the highest IL-16 production while 12 h following stimulation,the protein rBmpA induced the highest IL-16 production in comparison with the control.-TNF-α:6 h and 12 h following stimulation,the protein rBmpA induced the highest production of TNF-α compared to the control.2.The results of RayBio Rhesus Macaque IL-6 ELISA showed that the protein rBmpA significantly increased the production of IL-6 compared to the control 6 h and12 h after stimulation.RayBio Rhesus Macaque TNF-α ELISA results showed that B burgdorferi initiate TNF-α production compared to the control 6h after stimulation,while at 12h following stimulation,rBmpA initiate TNF-α production compared to the control.3.6 h,12 h,24 h and 48 h after HMC3 cell co-culture with rBmpA and B burgdorferi the IL-6 concentration significantly increased compared to the control4.After 6h,12h,24h and 48h of HMC3 cell co-culture with rBmpA and B burgdorferi the relative mRNA expression of IL-6 significantly increased compared to the control.Conclusions:1.B.burgdorferi and rBmpA can initiate brain tissue and microglial cells to produce inflammatory mediators such as IL-6 and TNF-α.1.B.burgdorferi and rBmpA might stimulate microglial cells to produce inflammatory mediators which results to brain injury and Lyme neuroborreliosis.