Neutrophils Generate Higher Levels of Reactive Oxygen Species after Stimulation by Inactive vs. Active Lupus Nephritis Serum

Background: There is increasing evidence for the role of neutrophils in lupus nephritis (LN) pathogenesis. Release of reactive oxygen species (ROS) by neutrophils may be a source of glomerular damage in LN. Our previous work investigating LN neutrophil ROS generation suggested the possibility of endogenous neutrophil activators in LN serum. To further this investigation, we aimed to identify differences in healthy donor neutrophil activation in response to different LN serum treatments based on active versus inactive disease. Methods: Neutrophils were isolated from whole blood of 6 healthy donors. Cell treatment groups included untreated, 10% pooled active LN serum, 10% pooled inactive LN serum, or 10% pooled healthy donor platelet poor plasma (PPP). Active disease was defined as urine-protein-to-creatinine ratio greater than 500. Active and inactive LN serum samples were paired from 3 LN patients on different dates. ROS generation was measured using superoxide assay. Results: Results were calculated using Dunn’s multiple comparisons test. Statistics were performed with GraphPad Prism 10.2.1. Neutrophil stimulation with healthy donor PPP, active LN serum, and inactive LN serum resulted in more ROS generation than in the untreated condition (p=0.009, p<0.0001, p<0.0001). Healthy donor PPP resulted in less ROS generation than either active or inactive LN serum (p=0.0009, p<0.0001). Treatment with inactive LN serum caused the highest level of ROS generation, more than the active LN serum treatment (p<0.0001). Conclusion: Serum from inactive LN stimulated neutrophils to produce the most ROS despite lower clinical disease activity than active LN. These results may be attributable to differences in serum contents related to disease status. These data warrant further investigation to characterize serum contents for active and inactive LN. Greater sample size and a bigger patient pool may further our understanding of these differences. Improving understanding of serum characteristics by disease status may have implications for experimental designs in LN research, for elucidating patterns of remission and relapse in LN, and for future clinical LN management. Funding: NIDDK Support