Data analysis was performed using FlowJo version 9.3 by gating on Batefenterol live cells based on forward scatter versus side scatter profiles, then on singlets using forward scatter area versus height, followed Batefenterol by cell subset?specific gating. Methods Healthy individuals (n?=?790) were screened for autoantibodies specific for 11 antigens associated with lupus, systemic sclerosis, and Sj?gren’s syndrome. From this screening, 31 European American ANA\positive healthy individuals were selected and demographically matched to ANA\negative controls and SLE patients. Serum cytokine profiles, leukocyte subset frequency, and reactivity were analyzed by multiplex assays, immunophenotyping, and phosphospecific flow cytometry. Results Of 790 individuals screened, 57 (7%) were ANA\positive. The majority of proinflammatory cytokines, including interferon\ (IFN), tumor necrosis factor, interleukin\17 (IL\17), and granulocyte colony\stimulating factor, exhibited a stepwise increase in serum levels from ANA\negative controls to ANA\positive healthy individuals to SLE patients (0.0001). IFN, IFN, IL\12p40, and stem cell factor/c\Kit ligand were increased in SLE patients only (0.05). B lymphocyte stimulator (BlyS) was elevated in SLE patients but decreased in ANA\positive individuals (0.001). Further, IL\1 receptor antagonist (IL\1Ra) was down\regulated in SLE patients only (0.0001). ANA\positive individuals had increased frequencies of monocytes, memory B cells, SERPINF1 and plasmablasts and increased levels of pSTAT\1 and pSTAT\3 following IFN stimulation compared with ANA\negative controls (0.05). Conclusion ANA\positive healthy individuals exhibit dysregulation in multiple immune pathways yet differ from SLE patients by the absence of elevated IFNs, BLyS, IL\12p40, and stem cell factor/c\Kit ligand. Further, severely decreased levels of IL\1Ra in SLE patients compared with ANA\positive individuals may contribute to disease development. These results highlight the importance of IFN\related pathways and regulatory elements in SLE pathogenesis. Antinuclear antibodies (ANAs) are detected in almost one\fifth of the general population, yet few individuals are diagnosed as having an autoimmune disease 1, 2. Thus, a substantial percentage of the population carries detectable levels of circulating autoantibodies without developing Batefenterol clinical symptoms. Autoantibodies are also present in the sera of patients Batefenterol with systemic autoimmune diseases, such as systemic lupus erythematosus (SLE), many years before clinical disease onset 3, 4. Although some ANA\positive healthy individuals eventually develop clinical autoimmunity, many do not. The transition to clinical SLE has been correlated with autoantibody profiles combined with female sex, but other risk factors, including age, ANA titer, number of autoantibody specificities, and type I interferon (IFN) signature, do not definitively identify ANA\positive healthy individuals in whom autoimmune disease develops eventually 5, 6, 7, 8. Comparing ANA\positive healthy individuals with SLE patients and ANA\negative healthy controls provides a unique perspective in the dissection of pathogenic mechanisms in autoimmunity and may reveal the means to provide regulatory control. A delicate balance of inflammatory and regulatory immune cells is required to control infection without promoting autoreactivity. Evolving work has suggested that interactions between both the adaptive and innate immune systems are critical for autoimmune disease pathogenesis 9, 10, 11, 12. For example, IFN\associated gene signatures are elevated in peripheral blood of SLE patients and correlate with increased disease activity 13, 14, 15, 16. Plasmacytoid dendritic cells (DCs) are a primary source of type I IFN in SLE 17, 18 and produce type I IFN upon Toll\like receptor (TLR) stimulation, particularly TLR\7 and TLR\9. These receptors may be activated by immune complexes containing double\stranded DNA (dsDNA) and/or RNA\associated binding proteins in SLE 19, 20, 21, 22, 23. The IFN signature in SLE is detected in leukocytes involved in both innate and adaptive immunity, such as B cells, T cells, neutrophils, and myeloid cells. However, whether ANA\positive healthy individuals have a heightened IFN response remains unknown 15, 16. The cellular response Batefenterol to IFN and many cytokines is mediated by signaling through cytokine\specific tyrosine kinase receptors to activate JAK/STAT. SLE patients have increased basal levels of pSTAT\3 in T cells and monocytes and pSTAT\5 in T cells and B cells, suggesting that abnormal STAT signaling plays a role in SLE pathophysiology 24, 25. Studies in SLE patients and in the MRL\Fas/mouse lupus model have shown ablation of STAT phosphorylation in response to type I IFNs and other inflammatory cytokines, suggesting a disturbance of negative and positive feedback mechanisms in autoimmunity 25, 26. In this.

Data analysis was performed using FlowJo version 9