Background Anti-nuclear antibody (ANA) positivity is a principal feature of individuals with an autoimmune disease, yet up to one in five healthy individuals are ANA-positive (ANA+) and will never develop overt disease. Understanding differences in immune cell physiology between ANA+healthy individuals and individuals with clinical SLE remains a critical goal in the understanding of SLE pathogenesis across ethnicities.
Methods Blood specimens and information on disease activity were collected from European (EA) and African American (AA) individuals classified and matched in groups as ANA- healthy controls (n=24), ANA +healthy (n=24) or SLE patients (n=24). Single-cell analysis of cell surface markers was completed by mass cytometry on PBMCs and cellular heterogeneity was visualized using tSNE (figure 1A–B) and manual gating. Further, phospho-specific flow cytometry was used to measure basal levels of pERK, pPLCg2 and p38 and expression following CD3/CD28 (TCR) and anti-IgG and IgM (BCR) stimulation. Whole genome RNA-sequencing was performed on flow cytometry sorted T cells, B cells and monocytes from 35 matched ANA-, ANA +and SLE patients followed by weighted correlation network analysis (WGCNA) and pathway enrichment analyses.
Results Both European and African American SLE patients were distinguished from healthy individuals by T cell proliferation (p=0.002) (figure 1C), plasmacytoid dendritic cell activation (p=0.021) and elevated stem cell factor (p=0.0003). EA ANA+healthy individuals exhibited greater immune regulation with reduced T cell numbers (p=0.002) (figure 1C), decreased activation of dendritic cells (p=0.039) and transitional B cells (0.033), and elevated expression of the inhibitory receptor CD85j (p=0.042) on specific immune cell subsets compared to ANA- healthy subjects. Further, a module associated with hematopoiesis, T cell activation and intrinsic apoptosis signaling pathways is expressed at a higher level in T cells of EA ANA+individuals. In contrast, AA ANA+healthy individuals had elevated plasma levels of IL-6 (p=0.018) and reduced inhibitory receptor expression (p=0.0089) compared to ANA- healthy controls. Gene expression modules associated with viral responses and type I IFN pathway activation were identified in AA SLE patient B cells, while similar expression modules were only found in the monocytes of European American SLE patients.
Conclusions These results highlight the importance of stem cell factor and T cell expansion in SLE pathogenesis, and suggest that mechanisms of SLE pathogenesis differ by ethnicity. ANA +European Americans may have more effective regulatory mechanisms in place to prevent transition to classified autoimmune disease.
Funding Source(s): This work was supported by the NIH under award numbers U54GM104938,
U01AI101934, U19AI082714, and P30AR053483.
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