The Effect Of TLR9 On NETs Formation In LPS-induced Acute Lung Injury

[Background]Acute lung injury(Acute Lung Injury,ALI)/Acute Respiratory Distress Syndrome(ARDS)is a common clinical critical illness.The study found that in ALI/ARDS,neutrophils accumulate in large amounts,secrete inflammatory mediators and cytokines,and continuous activation causes inflammatory cascade reactions.At the same time,neutrophil extracellular trapping nets(NETs)are also produced.NETs is a new type of neutrophil antibacterial method,but abnormal NETs can cause damage to its own tissues and organs.Studies have shown that,after acute lung injury,the interaction between the production of NETs and inflammatory mediators may directly aggravate the degree of acute lung injury.Toll-like receptor 9(TLR9)is a key pattern recognition receptor of the natural immune system and can be expressed in a variety of immune cells.It plays an important role in the body's immune response,inflammation and tissue repair.Acute lung injury is the result of systemic uncontrolled inflammatory response,therefore TLR9 may be a potential target for the treatment of acute lung injury.Recent studies have shown that the formation of NETs depends on the release of DAMPs from stress or injured cells,and DAMPs may produce networks through TLR9-dependent pathway,aggravating the degree of injury.However,the role of TLR9 in acute lung injury is not completely clear,and the role of TLR9 in the formation of NETs in LPS-induced acute lung injury is unclear.[Objective]To investigate the role of TLR9 in the process of acute lung injury,and to investigate the role of TLR9 in the formation of NETs induced by LPS in acute lung injury.[Methods]1.Construction of TLR9 gene knockout mice and preliminary identification of their phenotypesFirst,the CRISPR/Cas9 technology was used to establish the TLR9 gene knockout mouse model.Genotype identification was performed using capillary gel electrophoresis technology and PCR product sequencing analysis.Using qRT-PCR,Western blot and immunohistochemical techniques to identify the effect of TLR9 knockout.The phenotypes of TLR9 gene knockout mice were preliminarily analyzed by detecting the genetic traits,body weight and blood parameters of TLR9 gene knockout mice and hematoxylin-eosin(H-E)staining to observe the pathological morphological changes of mouse tissues.2.Effect of TLR9 knockout on neutrophil immune functionTLR9+/+and TLR9-/-mouse bone marrow-derived neutrophils were isolated and identified.TLR9 agonist CpGODN stimulated neutrophils.qRT-PCR was used to detect the expression of neutrophil inflammatory factors and chemokines.qRT-PCR and Western blot were used to detect the release of degranulation in neutrophils and cell supernatant MMP8,MMP9.Western blot was used to detect the effect of TLR9 knockout on MAPKs signaling pathway in neutrophils.3.Establish mouse model of LPS-induced acute lung injury and explore the effect of TLR9 knockout on the formation of NETs in LPS-induced acute lung injury.TLR9+/+ and TLR9-/-mice were injected intraperitoneally with LPS to construct mouse model of acute lung injury,and the survival time of the mice was monitored.Lung tissue H&E staining,pathological scoring and dry and wet specific gravity measurement were used to detect the effects of TLR9 on the pathological morphology and dry and wet specific gravity of lung tissue in mice with acute lung injury.qRT-PCR was used to detect the effect of TLR9 knockout on the expression of proinflammatory cytokines and chemokines in the lung tissue of mice with acute lung injury.To investigate the effect of TLR9 knockout on the generation of NETs in the lung tissue of mice with acute lung injury,qRT-PCR and Western blot and were used to detect the expression of PAD4 in lung tissue,Western blot and immunofluorescence staining were used to detect Cit-H3 in lung tissue.TLR9+/+ and TLR9-/-bone marrow-derived neutrophils were isolated,and PMA,A23187,LPS were used as NETs inducers to stimulate neutrophils.Immunofluorescence staining and Western blot were used to detect the generation of Cit-H3,qRT-PCR and Western blot were used to detect the experession of PAD4 and the effect of TLR9 knockout on the generation of neutrophil NETs was explored in vitro.[Results]1.Successfully construct TLR9 knockout mice.The expression of TLR9 mRNA and protein in spleen and liver tissues of TLR9 gene knockout mice was significantly lower than that of wild type mice;TLR9 gene knockout mice can grow and reproduce normally,and their body weight and peripheral blood routine indicators are normal.And histomorphological characteristics were not significantly different from wild type mice.2.PMN was purified and identified from the bone marrow of TLR9+/+and TLR9-/-mice.Detection revealed that the PMN derived from mouse bone marrow was successfully extracted and purified.TLR9 lacked mRNA and protein expression in TLR9-/-PMN cells.Further detection of PMN-related immune function showed that TLR9 knockdown can reduce the neutrophil proinflammatory cytokines TNF-?,IL-6,IL-1?,chemokine IL-8 and degranulation activity MMP8,MMP9 expression.And TLR9 knockout inhibits the activation of p38 MAPK and ERK1/2 in the MAPK signaling pathway.Detection revealed that TLR9 expression was lost at the mRNA and protein levels in TLR9-/-PMN cells.Further examination of PMN-related functions showed that TLR9 knockdown can reduce the expression of neutrophil proinflammatory cytokines TNF-?,IL-6,IL-1?,IL-8 and degranulation activity MMP8 and MMP9.And TLR9 knockout inhibits the activation of MAPK signaling pathway p38 MAPK and ERK1/2.3.LPS was used to construct ALI/ARDS mouse model.Compared with TLR9-/-mice,TLR9+/+ mice showed earlier onset and higher mortality;And TLR9 knockout can significantly reduce the lung tissue pathological changes,inflammatory cell infiltration,pulmonary tissue bleeding after LPS-induced acute lung injury,reduce the wet-dry weight ratio of lung tissue(P<0.05),and reduce the expression of inflammatory cytokines TNF-?,IL-6,IL-10 and chemokine IL-8,MCP-1 in lung tissue of acute lung injury.To further investigate the effects of TLR9 knockout on the generation of NETs in acute lung injury,qRT-PCR,Western blot and immunohistochemical results showed that compared with WT mice,the expression levels of PAD4 and Cit-H3 is low in lung tissues of TLR9-/-mice.Immunofluorescence results showed that the LPS group compared with the PBS control group,the production of Cit-H3 in the lung tissue was significantly increased,and after LPS treatment,compared with TLR9+/+mouse,the amount of Cit-H3 produced by TLR9-/-mouse was significantly reduced.After the NETs inducer stimulated neutrophils,the results of immunofluorescence,qRT-PCR and Western blot showed that the knockout of TLR9 could inhibit the generation of neutrophils NETs.[Conclusion]1.TLR9 gene knockout mouse model was successfully established.2.TLR9 knockout can inhibit neutrophil immune function.3.TLR9 is involved in the pathogenesis of acute lung injury.TLR9 knockout can reduce the production of neutrophil NETs and regulate lipopolysaccharide-induced acute lung injury,which may be a potential new target for prevention and treatment of acute lung injury.