Immune Mechanism Of NKp46+NK Cells Promoting Inflammation And Bone Destruction In Rheumatoid Arthritis And Intervention Of Astragalus Polysaccharides

IntroductionRheumatoid arthritis(RA)is a chronic,systemic inflammatory autoimmune disease.The etiology of RA is still unclear.It is generally believed that immune cells infiltration caused by immune disorders,such as myeloid cells,helper T cells,and natural killer cells(NK),plays a vital role in the occurrence and development of RA.Collagen-induced arthritis(CIA)is a classic animal model of RA.Our team’s previous research identified a new subset of cells that exacerbate inflammation and bone destruction in RA and CIA:myeloid-derived suppressor cells(MDSC).In addition,our team found that NK cells greatly expanded in peripheral blood and synovial fluid of RA patients and positively correlated with disease activity.NKp46 is a specific receptor of mouse and human NK cells.The role of NKp46~+NK cells in the pathological process of RA and whether NKp46~+NK cells regulate MDSC remains to be elucidated.Astragalus is widely used in treating arthralgia,and astragalus polysaccharides(APS)is one of the most abundant main active components in astragalus.The potential therapeutic value of APS in RA and the immune mechanism of its action remains to be further elucidated.Objectives1.To clarify the role and mechanism of NKp46~+NK cells in the pathological process of CIA.2.To reveal the therapeutic effect of APS on CIA inflammation and bone destruction and related immune mechanisms.Methods1.CIA model was constructed in DBA/1 mice.The arthritis index score,joint pathology score,and micro-CT analysis of bone tissue were used to evaluate the severity of the disease at different stages.Flow cytometry was used to analyze the proportions of NK cells and their subsets in the peripheral blood,spleen,lymph nodes,and paws.The changes in cell proportions and their correlation with the arthritis index were analyzed.Immunofluorescence was utilized to detect the infiltration of NKp46~+NK cells in the joints at different time points,and its correlation with the severity of arthritis was evaluated.NKp46~+NK cells were adoptively transferred to observe the effect of NKp46~+NK cells on the pathological process of CIA.2.The CIA model in NKp46 knockout mice(Ncr1gfp/gfp)was constructed to observe the effect of NKp46 on CIA inflammation and bone destruction.The proportion of MDSC in CIA was detected,and its correlation with NKp46~+NK cells in the paws was analyzed.The NKp46~+NK cells were isolated from the paws of CIA mice,and their capacity to secret TNF-α,IFN-γ,and GM-CSF,and their chemotactic effects on MDSC were detected.3.The CIA model of DBA/1 mice was treated with different doses of APS.The effect of APS intervention was evaluated by arthritis index score,joint histopathological analysis,and microCT.At the same time,the effects of APS on NKp46~+NK cells and MDSC in the peripheral blood and paws were evaluated.Results1.NKp46~+NK cells in the paws increased with the progression of CIA and were positively correlated with the arthritis index.Immunofluorescence staining showed that the number of NKp46~+NK cells in the joints increased with disease progression and was positively correlated with disease severity.Adoptive transfer of different numbers of NKp46~+NK cells aggravated the progression of CIA to various degrees.2.Knockout of the receptor NKp46 did not affect the incidence and severity of CIA arthritis.MDSC in the peripheral blood and paws of CIA mice were greatly expanded,and the proportion of MDSC and NKp46~+NK cells in the paws was highly positively correlated.CIA-derived paw NKp46~+NK cells secreted more TNF-α,IFN-γ,and GM-CSF,and their chemotactic ability of MDSC was enhanced.3.High dose(4g/kg body weight)of APS significantly reduces the arthritis index,the infiltration of joint inflammatory cells,the extent of synovial hyperplasia and cartilage destruction,and bone destruction and bone loss in CIA mice.High-dose APS can significantly increase the proportion of NKp46~+NK cells in the peripheral blood of CIA mice and,at the same time,reduce the infiltration of NKp46~+NK cells and MDSC in the paws.Medium and low doses of APS had no significant effect on the progression of CIA or the proportion of NKp46~+NK cells and MDSC.Conclusions1.NKp46~+NK cells infiltrate the inflamed joints,exacerbate CIA inflammation and bone destruction by secreting pro-inflammatory cytokines and recruiting MDSC.2.APS can significantly improve CIA inflammation and bone destruction,and its mechanism may be through reducing the infiltration of NKp46~+NK cells and MDSC in inflammatory joints.