Background/Purpose: The production of anti-nuclear auto-antibodies (Ab) and the formation of immune complexes (IC) are hallmarks of Systemic Lupus Erythematosus (SLE). Enhanced neutrophil (PMN) activation and formation of neutrophil extracellular traps (NETs), through engulfment of nucleic acid-containing ICs, have been implicated in disease pathogenesis, particularly in the acceleration of tissue inflammation, end-organ and vascular damage. Upon activation, PMNs may contribute to B cell activation and auto-Ab production by three main mechanisms: indirectly by NETs-stimulated interferon production, or directly, by releasing B cell activating factor (BAFF) and extruding autoantigens, such as mitochondria, during NETosis. Still, little is known about the link between the PMN activation and B cell immune responses in human SLE. This study was undertaken to test whether IC-driven PMN activation and NET formation contribute to BAFF increase, auto-Ab production and B cell activation in SLE patients.

Methods: BAFF levels were analyzed in serum samples from 60 SLE patients and 20 healthy controls (HC) by ELISA. Markers of neutrophil activation and NETosis (S100A8/A9, MPO-DNA complexes, cell-free oxidized (8-OHdG) DNA and mitochondrial (mt) DNA levels) were analyzed by ELISA, fluorimetric assays, and qPCR. FcγRIIA internalization, a bioassay for IC quantification, was analyzed by flow cytometry. Anti-DNA IgG titers, anti-cardiolipin (CP) IgM and IgG, C3 and C4 complement and CRP levels were obtained from clinical records. PMNs from healthy donors were stimulated with RNP-containing ICs and PMN activation, BAFF production, and B cell activation were studied in vitro.

Results: Levels of BAFF were markedly elevated in SLE as compared to HC serum samples (p< 0.0001). Consistent with our hypothesis of neutrophil-driven BAFF release in SLE, levels of BAFF correlated with markers of PMN activation (S100A8/A9) and NET-derived components, including mtDNA and cell-free 8-OHdG DNA (r=0.34-0.38, p<0.05 for both analyses). Increased BAFF levels associated significantly with decreased serum C3 and C4 levels, suggesting an association with ongoing IC-driven disease. Furthermore, levels of serum ICs correlated with an increase in BAFF levels (r=0.38, p=0.02) as determined by a flow cytometry-based bioassay. Auto-Ab analysis showed positive correlations between anti-DNA and anti-CL Ab titers, BAFF levels and markers of NETosis, including 8-OHdG DNA and S100A8/A9 levels. Stimulation of PMNs with ICs in vitro induced PMN activation, NET formation, and a significant increase in BAFF release three-to-six hours post-stimulation. PMN-B cell co-culture experiments showed increased B cell activation in response to IC-induced PMN activation.

Conclusion: Our results support the hypothesis that PMNs, through IC-mediated activation, release BAFF and auto-Ags, including DNA and mitochondrial components, which may contribute to B cell activation and production of pathogenic auto-Abs. These findings bring new insight into the mechanisms of PMN-B cell interactions involved in the pathogenesis of SLE.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/immune-complex-driven-neutrophil-activation-and-baff-production-promote-b-cell-activation-and-autoantibody-production-in-human-sle/