Neutrophil Extracellular Trap Mitochondrial DNA In The Pathogenesis Of Systemic Lupus Erythematosus And A Novel Treatment Approach

Part ⅠNeutrophil extracellular trap mitochondrial DNA and its antibody are are potent inducers of p DC-IFNα-passway activation in systemic lupus erythematosusObjective: To explore the mechanism of Neutrophil extracellular trap(NET) mitochondrial DNA(mt DNA) and anti-mt DNA antibodies(Abs) on type I interferon pathway activation.Methods: We enrolled 102 SLE patients, rheumatoid arthritis patients(n=14) and healthy donors as controls(n=40). NETs were generated from phorbol 12-myristate 13-acetate(PMA)-stimulated peripheral neutrophils. mt DNA levels and the transcriptional levels of five interferon inducible genes(IFIGs)(0AS-1,Mx-1,Ly6 e,IFITl and IFIT4) were measured by quantitative PCR. Interferon scores(IFN scores) were calculated. Anti-mt DNA Abs and IFN-α were detected by enzyme-linked immunosorbent assay. Isolated NETs, mt DNA or double stranded(ds)-DNA, combined with anti-mt DNA or ds DNA Ig G, and other culture conditions were applied to stimulate purified p DCs.Results: Mitochondrial DNA release by netting neutrophils was greatly enhanced in SLE patients compared with healthy controls(P <0.01) and significantly correlated with IFN scores(r =0.4606, P =0.0004) and disease activity index(SLEDAI). Forty-one percent of SLE patients were positive for anti-mt DNA antibodies(Abs), while none of the RA controls displayed a positive serology response to mt DNA. Additionally, the titers of anti-mt DNA Abs were also associated with IFN scores(r=0.2928, P =0.0195) and SLEDAI. The levels of anti-mt DNA Abs significantly correlated with classic anti-ds DNA Abs titers measured by Farr assay(r =0.5429, P <0.0001) and were associated with lupus nephritis(LN)(P =0.012). mt DNA was deposited in NETs in LN renal biopsies. mt DNA/anti-mt DNA were greater inducers of p DC IFN-α production via TLR9 engagement than ds DNA/anti-ds DNA.Conclusion: mt DNA in NETs and anti-mt DNA Abs are involved in SLE type I interferon pathway and serves as a new biomarker for disease activity and active lupus nephritis in SLE patients.Part ⅡMetformin down-regulates NET-p DC-IFNα-passway and its therapeutic effects in lupus patients--- a proof of concept trialObjective: To observe the role of metformin on down-regulating NET-p DC-IFNα passway, and the efficacy and safety of metformin was assessed in lupus patients with a background of Corticosteroids and Conventional Immunosuppressive AgentsMethods: NETs were generated from phorbol 12-myristate 13-acetate(PMA)-stimulated peripheral neutrophils and quantified using Quant-i T? Pico Green R ds DNA kit. mt DNA levels was measured by quantitative PCR IFN-α was detected by enzyme-linked immunosorbent assay. We enrolled 124 SLE patients. Patients were randomly assigned into study group(62 subjects) and control group(62 subjects). Study group received metformin 0.5-2.0g/day for 6 months, and control group received non-intervention follow-up. The primary objective of this design study is to evaluate the effect of metformin comparative with placebo, in terms of reduction inflare(SELENA-SLEDAI Flare Index[SFI]) in SLE patients. Besides, the influence of metformin on corticosteroid sparing effect and reduction in body weight are also assessed during the entire study period.Results: Quantitation of NET showed that metformin inhibited NET formation after stimulation with PMA in a dose dependent manner. 109 patients completed the study(54 patients in study group, 55 patients in control group). Use of prednisone was significantly greater in the control group than in the study group from months 4 to 6(p=0.0806 at month 4, p=0.0307 at month 5, p=0.0482 at month 6). The proportions of patients with at least a 50% reduction in prednisone dose were greater in the study group at month 6(P =0.058).Weight loss and BMI reducing score in study group were significantly higher than those in control group(P<0.001). No significant differences in reduction of disease flares were found between the two groups.Conclusion: Metformin can reduce prednisone use and body weight of SLE patients without increase in disease activity.