The Effects Of TREM2 On Release Of Inflammatory Factors From Microglia And Its Mechanism In Spinal Cord Injury

Background Spinal cord injury(SCI)is a verycommon and frequently occurring disease in the department of rehabilitation medicine.Spinal cord injury(SCI)is common and involves widespread damage to the central nervous system(CNS).SCI often leads to severe neurological symptoms such as varying degree of paralysis,paresthesia,urinary obstruction,and other progressive neurological abnormalities.SCI also involves social loss: data on western countries show that governments spend$40,000–$180,000 for each patient depending on the site of injury.Patients lose their jobs and receive medical treatment,rehabilitation,and maintenance,and each patient costs the country millions of dollars.The pathological process of SCI can be divided into two stages: primary injury and secondary injury.Primary injury occurs immediately after the initial injury,and its pathological processes include demyelination of the spinal cord and necrosis of neurons and axons.Secondary injury occurs throughout the disease,and its pathological processes include demyelination,axonal and neuronal necrosis,nervous tissue ischemia and edema,oxidative stress,inflammatory reaction,and glial scar formation.SCI causes peri-lesional glial cells to robustly alter their phenotypes and activities.Whereas spinal astrocytes typically regulate neurotransmitter availability and blood flow,post-SCI reactive astrocytes form a glial scar that limits both lesion expansion and axon regeneration.Whereas microglia in healthy spinal cord scan for pathogens and control synapse density,post-SCI microglia can promote plasticity,yet also become hyper-activated for prolonged periods and worsen damage.Proinflammatory cytokines IL-6 and TNF-α were increased by microglia activation.And these two cytokines were further reported to be key inflammatory mediators in inflammatory response following the SCI.Nuclear factor κB(NF-κB)is a kind of transcription factor,participated in various processes particularly immune and inflammatory response.Activation of NF-κB pathway is dependent on the secretion of IL-6,TNF-α,and other inflammatory factors induced by pattern recognition receptors(PRRs)such as toll-like receptors(TLRs).Previous studies have found that NF-κB/TLR4 take part in the inflammatory and immune response in microglia under traumatic injury.Triggering receptor expressed on myeloid cells 2(TREM2),a member of TREM family which are immunoreceptors,is correlated to several neurodegenerative diseases.For instance,TREM2 has been reported to be a risk factor for AD.Additionally,TREM2 is usually expressed on myeloid cells like microglia in the central nervous system(CNS).TREM-1 can enhance inflammation,and other members of TREM family and TREM-like receptors were also involved in inflammation and inflammatory bowel disease and so on.What’s more,a variant of TREM2 was discovered to be associated with the high risk of ALS.However,the function of TREM2 in TSCI is little to be known to date.Lipopolysaccharide(LPS),the ligand for TLR4,an endotoxin of bacteria induced several activities such as tissue injury and inflammation,is employed to activate macrophages and to simulate infection after injury.Microglia,a type of macrophage cell,is resident in the brain and spinal cord,accounts for about 10%-20% of all of the glial cells and functions in immune system.There are reports showed that excessive activation and proliferation of microglia is an important manifestation of inflammatory response,and leads to the continuous release of IL-6 and TNF-α,which is similar to the impact after TSCI occurred.Objective To explore the possible mechanism of TREM2 after spinal cord injury and the effects of TREM2 on primary microglia induced by lipopolysaccharide and its mechanism.Materials and Methods1.BV2 cells were cultured in vitro and treated with LPS at different concentrations.Expressions of TREM2 were detected by Western blot at 24 h,and expressions of TREM2,il-6 and TNF-in supernatant at 12 h and 24 h were detected by ELISA.2.The TREM2 gene interfered with LPS(5ug/mL)of lentiviral intervention treated BV2 cells,the expression of TREM2 at 24 h was detected by Western blot,the expression of nf-kb and p-nf-kb at 3h was detected by Western blot,and the expression of il-6 and TNF-in the supernatant of 24 h cells was detected by ELISA.3.The TREM2 gene overexpression lentivirus transfected BV2 cells and treated with NF-kb inhibitor PDTC.The expression of il-6 and TNF-α in 24 h cell supernatant was detected by ELISA.4.To construct TREM2 interference and overexpression lentiviral transfection of BV2 cells,Q-PCR and Western blot were used to detect TREM2 interference and overexpression efficiency.5.Using ELISA to detecte the expression of TREM2,il-6 and TNF-α in peripheral blood of 40 patients with spinal cord injury and 40 healthy people.Results1.Peripheral blood samples of 40 TSCI patients and 40 normal subjects were obtained,and then the concentrations of TREM2,IL-6 and TNF-α were measured by specific ELISA.Through the statistical analysis by t-test,we found that the concentration of TREM2 in peripheral blood of TSCI patients was significant higher than in normal subjects as well as the concentrations of IL-6 and TNF-α were also higher in TSCI patients.It suggested that TREM2,IL-6 and TNF-α may be involved in the process of TSCI.2.Several concentrations of LPS(0,1,5,10 μg/mL)were chose to stimulate cultured BV2,a cell line of microglia,for 24 h.Subsequently,the protein level of TREM2 quantified by western blot showed an obvious increase accompanied with the escalated concentrations of LPS,and reached to the highest level when induced by5μg/mL LPS.Further,the specific ELISA assays were utilized to analyze the concentrations of TREM2,IL-6 and TNF-α.And we discovered that the concentrations of TREM2,IL-6 and TNF-α in the supernatant of cultured medium were also remarkably elevated.What’s more,there was no obvious difference between the cells treated with 10 μg/mL LPS and with 5 μg/mL LPS.So we picked 5 μg/mL LPS to establish the model of TSCI.3.So as to study the function of TREM2 in TSCI,the lentivirus of TREM2 interference and overexpression were employed to infect the cultured BV2 cells,respectively.The mRNA level of TREM2 was determined by RT-PCR while the western blot was used to quantify the protein expression.The mRNA and protein levels of TREM2 were obviously reduced by three siTREM2 lentivirus,and siTREM2-1 had the most significant effect.On the other hand,the mRNA and protein levels of TREM2 were also elevated significantly by infection of oeTREM2 lentivirus.4.Down-regulation of TREM2 had an inhibiting effect on the LPS-induced expression of TREM2 and p-NF-κB.These data revealed that p-NF-κB and the concentrations of IL-6 and TNF-α were positively correlated to the expression of TREM2.5.To further understand the effects of NF-κB on the secretion of IL-6 and TNF-α,PDTC,an inhibitor of NF-κB,was used to treat the BV2 cells infected with oeTREM2.After 24 h of treatment,the specific ELISA assays of IL-6 and TNF-α were performed.The addition of PDTC reduced the high concentrations of IL-6 and TNF-α induced by the overexpression of TREM2.It directly demonstrated that repression of NF-κB showed an inhibitory effect on the release of IL-6 and TNF-α in the BV2 cells.Conclusion The study found that down-regulation of TREM2 can inhibit the release of inflammatory factors from LPS-stimulated microglia by inhibiting the activity of NF-κB signaling pathway.TREM2/NF-κB pathway may provide a new target for SCI treatment.