Investigation Of SAA Facilitates NETs Formation Through Toll-like Receptor 2 Signaling Pathway In Rheumatoid Arthritis

ObjectiveRheumatoid arthritis(RA)is a common systemic autoimmune disease characterized by chronic inflammation of the joints,which induce the formation of vasculature and eventually lead to joint destruction.The pathogenesis is very complicated and has not yet been fully elucidated.Previous studies have confirmed that both innate immunity and adaptive immunity are involved in the pathology of RA.As we all known,neutrophils are the main participants of host innate immunity.Studies have shown that they play a crucial role in RA inflammation,such as neutrophil extracellular traps(NETs).NETs are a fibrous network structure composed of DNA,histones and neutrophil proteins(such as myeloperoxidase,etc.),which can trap pathogens and exert anti-infection effects.However,if too many NETs are formed or the clearance is hindered,it may induce autoimmune diseases such as RA.At present,studies have reported that in the inflammatory environment of RA,NETs are easily formed in neutrophils in peripheral blood and local joints,and NETs are an important source of autoantibodies against RA citrullinated protein antigens.Our team has previously studied that in RA,an acute phase response protein(SAA)can induce the formation of NETs and promote angiogenesis.Subsequently,the main of this present study is to explore the molecular mechanism of SAA inducing the formation of NETs by TLR2 in RA patients,and provide a basis for the diagnosis and therapeutic approach of RA.Methods 1.Peripheral blood neutrophils from healthy subjects and RA patients were isolated by density gradient centrifugation.the neutrophil purity and cell morphology were observed by Wright staining and DAPI staining.2.DAPI staining and immunofluorescence microscopy were used to evaluate the formation of NETs in each group(SAA stimulation;LPS stimulation(positive control group);negative control(with the addition of tris-HCL buffer))of RA and HC groups,respectively.The number of NETs formed per field/total number of cells,that is,the percentage of NETs formed,was used as a measure stantard;the enzyme-linked immunosorbent assay(ELISA)was used to detect the content of histone H3 in the supernatant of each group.3.A fluorescence microplate reader was used to detect the ROS content during SAA-induced neutrophils,and NADPH oxidase inhibitor was added to analyze the results.4.Western Blot was used to observe the expression of TLR2 in neutrophils stimulated by SAA.5.Isolated neutrophils as cell models,evaluate the effect of TLR2 neutralizing antibody on SAA-induced NETs formation by immunofluorescence,and ELISA was used to detect histone H3 content in the supernatant of cell culture in each group.6.Analysis of the role of SAA-TLR2 in neutrophils through Western Blot,and add TLR2 neutralizing antibody and PI3K/Akt inhibitor respectively to analyze its effect on the activation of PI3K/Akt signal pathway.7.Use immunofluorescence and ELISA to explore the effect of PI3K/Akt signal pathway activation on SAA-TLR2 induced NETs formation.Results 1.The results of Wright’s staining and DAPI staining show that the purity and morphology of neutrophils meet the experimental requirements.2.Immunofluorescence microscopy results showed that compared with HC,RA patients had more NETs;SAA stimulation group had a higher percentage of NETs formation than negative control group,but lower than LPS stimulation group;detection The H3 content in the cell culture supernatant of each group was found to be consistent with the percentage change of NETs formed.3.Fluorescence microplate reader test results showed that compared with the negative control,the ROS content of the SAA group was significantly increased,slightly lower than the PMA group(positive control group);after adding NADPH inhibitor,a small amount of ROS was detected in SAA group and PMA group Almost no ROS can be detected.4.Western Blot Results showed that the expression of Toll-like Receptor 2 increased in SAA-stimulated neutrophils.5.DAPI staining and immunofluorescence microscopy observation results showed that: the percentage of NETs formed by the treatment with TLR2 neutralizing antibody,that is,the SAA+anti-TLR2 antibody group was much lower than that of the SAA group;The change of H3 content in the cell culture supernatant of each group was consistent with the level of NETs formed.6.Western Blot results showed that the expression of TLR2 and p-Akt was down-regulated after neutrophils were stimulated by SAA and added TLR2 neutralizing antibody;after adding PI3K/Akt inhibitor,the expression of p-Akt was significantly down-regulated,while the expression of TLR2 No significant changes.7.The results of immunofluorescence microscopy showed that the formation percentage of NETs in SAA group was significantly higher than that in the other groups,while the formation percentage of NETs in the SAA+anti-TLR2 antibody group and SAA+PI3K/Akt inhibitor group were reduced;cell culture supernatant was detected by ELISA The content of H3 is consistent with the percentage change of NETs.Conclusion 1.Neutrophils in RA patients are prone to form NETs after SAA stimulation,and this process is induced by SAA through TLR2 receptor.In addition,SAA induced ROS production in the formation of NETs,and may be involved in the NADPH oxidase-dependent pathway.2.During the formation of SAA-induced NETs,they are involved through the SAA-TLR2-PI3 K / Akt signaling pathway,which may be directly involved in the occurrence and development of RA arthritis.