We biochemically characterized the PTMs accompanying in vitro NETosis and broadly especially pro filed the in vivo humoral immune responses of patients with SLE and mice immunized with NETS by applying multiple proteomic approaches, including autoantigen microarrays, PTM modified histone peptide arrays and a high throughput immunoblotting assay. Consis tent with recent findings, we found that sera from patients with SLE reacted to acetyl H2B histone pro teins. Inhibitors,Modulators,Libraries In broadly profiling the PTMs of NETs from human and mouse sources, we observed their enrich ment for distinctive PTMs characteristic of transcrip tional silencing. However, these marks only partly overlapped with autoantibody profiles in histone reactive sera of patients with SLE and with those in sera from mice prone to spontaneous autoimmunity.
Nonetheless, we found that NETs could serve as weak autoantigens in vivo, capable of eliciting mouse IgG and IgM responses. Materials and methods Human subjects, Inhibitors,Modulators,Libraries specimens and controls In accordance Inhibitors,Modulators,Libraries with approved Institutional Review Board protocols, serum samples Inhibitors,Modulators,Libraries from patients with SLE were obtained with informed consent from the Autoimmune Biomarkers Collaborative Network, a multi disciplinary, multi institutional effort to identify clini cally useful biomarkers for the management of autoim mune diseases. Normal sera and neutrophils were similarly obtained from healthy donors as part of the Stanford Chronic Immunologic Disease Registry and Repository and IRB protocol 17036, respectively. Human neutrophils were isolated from peripheral blood as previously described, using Percoll density gradient separation.
A mixture of commer cially available autoimmune sera with defined reactivities was used as Inhibitors,Modulators,Libraries a positive control, and secondary antibody alone was used as a negative control. Mice and NET immunization protocol All animal experiments were approved by, and per formed in compliance with, a protocol approved by the Institutional Animal Care and Use Committee of Stan ford University. Female Balb c mice 9 to 12 weeks of age were obtained from the Jackson Laboratory. All mice used in this study were main tained under standard conditions at the Stanford Uni versity Research Animal Facility. NETs from the EPRO cell line were prepared as described below. NETs were prepared and stored at a concentration of 10 ug ml of which 200 ul was subcutaneously injected at each immunization.
Each treatment group included 3 mice, immunized weekly over 4 weeks with NETs alone, or NETs com bined with murine cathelicidin related antimicrobial peptide at a 5,1 w w ratio to NETs. Pro teinuria was assessed by dipstick analysis using Albustix. Mouse serum samples were obtained by saphenous vein inhibitor expert bleeding immediately before the first injection and once every 4 weeks after immuni zation for up to 12 weeks.