Intravenous Immunoglobulin but Not Dexamethasone Inhibits Neutrophil Pro-Inflammatory Activity While Maintaining Key Antimicrobial Functions

Abstract

Rationale. Acute respiratory distress syndrome (ARDS) is a heterogeneous clinical disease. ARDS immunopathology due to lung infection involves an array of immune cells and the importance of granulocytes, and in particular neutrophils and neutrophil extracellular trap production (NETosis), has recently come to light. Despite over 20 well run, randomized, controlled trials, no specific therapies for ARDS are available and mortality remains high. Current treatments for ARDS are primarily limited to supportive therapies, including lung protective ventilation, and in certain situations, systemic steroid administration. Recently, clinical studies adding intravenous immunoglobulin (IVIG), an FDA approved drug, to standard ARDS therapy have shown faster recovery with less severe symptoms, suggesting a complementary beneficial effect, but the mechanism(s) remain unknown. Interestingly, previous in vitro studies found that IVIG can impair some inflammatory pathways in neutrophils. Our study assessed effects of IVIG with and without dexamethasone (a key glucocorticoid used in COVID-19 ARDS) in neutrophils ex vivo and in vivo in COVID-19 patients. Methods. Ex vivo treatment of neutrophils with IVIG or dexamethasone was conducted, followed by assessment of NETosis, oxidative burst and phagocytosis. Additionally, cell-free DNA was quantified in the blood of COVID-19 patients before and after treatment with IVIG. Ex vivo NETosis and plasma cell-free DNA was quantified using the QuantiT ™ PicoGreen™ dsDNA Assay Kit (Invitrogen). Oxidative burst was assessed by OxyBURST™ Green H2DCFDA, SE (Invitrogen) and phagocytosis of pHrodo™ Red S. aureus Bioparticles™ (Invitrogen) was quantified. Results. IVIG inhibits crucial neutrophil inflammatory pathways such as NETosis and oxidative burst while concomitantly enhancing phagocytic activity (Figure panels A-C). Notably, dexamethasone does not impact any of these critical pathways. Moreover, COVID-19 patients undergoing standard treatment plus IVIG had decreased cell-free DNA in the circulation 5 days after initiation of a 4 day treatment course, suggesting decreased NETs in circulation (Figure panel D) which possibly reverted at a later timepoint. Conclusion. Our data demonstrate potential targeted beneficial effects of IVIG in the context of neutrophil-mediated immunopathology. We demonstrate an ex vivo inhibitory effect of IVIG on pro-inflammatory pathways in neutrophils, which may lead to diminished immunopathology in disease states worsened by neutrophil-driven destruction. Based on the compelling evidence of the contribution of neutrophils to development and severity in ARDS, our evidence of IVIG impairing key pro-inflammatory functions in neutrophils (where dexamethasone does not) suggests a theoretical potential complementary beneficial effect of adding IVIG to standard treatment for infection induced ARDS although further research is needed.