Study On The Mechanism Of Cold Exposure Promoting The Formation Of NETs Through TLR2 And TLR4

Polymorphonuclears（PMNs）are the most abundant white blood cell type in animals,and are the first cells in innate immunity that are recruited to the site of inflammation.They can pass through the unique neutrophil extracellular traps（NETs）in response to external stimuli.Cold stimulation is a common source of stress in Northeast China.It not only causes disturbances in the homeostasis of the body’s environment,but also damages the body’s immune function and leads to inflammation.Studies have shown that cold exposure can activate PMNs,but research on NETs had not been reported yet.So,can cold exposure induce PMNs to form and release NETs?What is the role of the NF-κB signaling pathway mediated by TLR2 and TLR4 in this process?Therefore,while revealing whether cold exposure can induce the formation of NETs,it also preliminarily clarifies how cold exposure affects the formation of NETs through TLR2 and TLR4,laying a theoretical foundation for the study of NETs in the field of cold stress.To explore whether cold exposure can induce the formation of NETs from primary PMNs in the abdominal cavity of mice,8-week-old male mice were randomly divided into a cold exposure group and a normal temperature control group.The mice in the cold exposure group were placed at 4°C at different time points every day.They were kept in a climate room for 3hours for 7 consecutive days.First,by testing the blood biochemical indicators of mice,it was found that the contents of AST,CK,Ca,and AMY all increased to varying degrees,and the contents of TG and GLU decreased significantly（P<0.05,P<0.01,P<0.001）.Then the purity and cell viability of PMNs were detected by immunofluorescence and CCK-8 methods,and it was found that the purity was about 90%,and the viability of PMNs after cold exposure decreased（P<0.001）.Subsequently,immunofluorescence,scanning electron microscopy,cfDNA quantification and western blotting were used to determine whether cold exposure can induce the production of NETs.The results showed that compared with the normal temperature control group,the cold-exposed group had a fibrous network structure,the surface of PMNs had obvious shrinkage,and there was a structure similar to pseudopods protruding;The cfDNA concentration and the protein expression of component proteins citrullinated histone 3（Cit H3）and myeloperoxidase（MPO）were significant increase（P<0.001）.In order to further confirm that cold exposure as an endogenous stimulus can induce the formation of NETs,we selected the exogenous stimulus LPS for comparative analysis.The results showed that compared with the normal temperature control group,other treatment groups formed NETs,and the detection results of nucleic acid backbone cfDNA concentration and component protein expression level were consistent with them（P<0.01,P<0.001）.The results of q-PCR and pathway protein detection showed that the mRNA expression of NETs-related genes PAD4,MMP-9 and inflammatory factors IL-1β,IL-6 and TNF-αin the cold exposure group increased（P<0.01,P<0.001）;The protein expression of TLR2,TLR4 and NF-κB pathway proteins My D88,p65,p-p65,IκB and p-IκB all increased（P<0.05,P<0.01,P<0.001）,indicating that cold exposure is an endogenous source Sexual stimulation can induce the formation of NETs,disrupt the body’s homeostasis,reduce the resistance to exogenous stimuli,activate TLR2 and TLR4,and activate the NF-κB signaling pathway mediated by them.To further explore the role of the NF-κB signaling pathway mediated by TLR2 and TLR4 in the formation of NETs induced by cold exposure,we selected TLR2^-/-and TLR4^-/-knockout（KO）mice for the following research.Firstly,the genotype was identified.After the successful knockout,the blood biochemical indicators of KO mice were tested.The results showed that the contents of ALB,GLOB,TP,ALT,AST,B/C,PHOS,CK,TG,and GLU were different.With the increase of the degree,the content of A/G and Crea decreased to different degrees（P<0.05,P<0.01,P<0.001）.Subsequently,the formation and number of NETs in KO mice and C57BL/6（WT）mice under cold exposure conditions were tested.The results showed that compared with the normal temperature control group,the mice in the cold exposure group formed a bead-like fiber network structure,wrinkled on the cell surface,and protruding pseudopods.The cfDNA test results showed that the concentration of cold exposure group was significantly higher than that of the normal temperature control group（P<0.001）,but the cfDNA concentration of KO mice was lower than that of WT mice（P<0.001）.Next,we tested the effects of endogenous cold exposure and exogenous LPS stimulation on the formation of NETs in KO mice,and found that the formation of fiber network structure,the morphological changes of PMNs,the concentration of cfDNA,and the components of Cit H3 and MPO The protein expression level has the same trend as that of WT mice treated in the same way.q-PCR detection revealed that the m RNA expression of NETs-related genes PAD4,MMP-9 and inflammatory factors IL-1β,IL-6,and TNF-αwere significantly increased in KO mice under cold exposure conditions（P<0.01,P<0.001）.It shows that the knockout of TLR2 and TLR4 genes has a certain impact on the formation of NETs,but there are still a small amount of NETs formed.Conclusion:1.Cold exposure can induce the formation of NETs from primary PMNs in the abdominal cavity of mice.2.The increase in the formation of NETs is through the NF-κB signaling pathway mediated by TLR2 and TLR4.