Effect of Intravenous Immunoglobulin on Neutrophil Antimicrobial and Inflammatory Functions - Implications in Neutrophil-Mediated Immunopathology

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Rationale. Neutrophils are the most abundant circulating leukocytes and the first line of defense against invading pathogens. Known for their function as phagocytic cells, neutrophils also capture and kill pathogens via elaboration of Neutrophil Extracellular Traps (NETosis), and production of reactive oxygen species (ROS), antimicrobial peptides and proteases. Conversely, neutrophils also have been associated with immunopathology. For example, numerous studies have indicated that neutrophils play a role in acute respiratory distress syndrome (ARDS), which is a major cause of death in COVID-19. We found that COVID-19 patients with ARDS have circulating neutrophils with a hyper-activated phenotype: elevated NETosis, ROS production and phagocytosis. While a small prospective clinical trial showed benefit of intravenous immunoglobulin (IVIG) in mitigating respiratory comorbidity in COVID-19, and prior literature showed benefit of IVIG in viral ARDS, the exact mechanisms are not known. Current clinical therapeutic trials have targeted NET clearance following their release, but not aimed at preventing their formation. In concordance with previous findings, we hypothesized that inhibition of systemic activation of neutrophils with IVIG may reduce collateral tissue damage by blocking NETosis. Methods. Neutrophils from healthy controls were treated ex vivo with IVIG and NETosis, ROS production and phagocytosis quantified. Stimulants phorbol myristate acetate (PMA), for the canonical pathway, and nigericin, for the non-canonical pathway, were used. NETosis was quantified with Quant-iT™ PicoGreen™ dsDNA Assay (Invitrogen) and by NET visualization using myeloperoxidase staining;ROS production was assessed by OxyBURST™ Green H2DCFDA (Invitrogen). Phagocytosis of pHrodo™ Red S. aureus Bioparticles™ (Invitrogen) was also quantified.Results. Ex vivo treatment of healthy neutrophils with IVIG was associated with a significant decrease in NETosis at concentrations of 5 and 10 mg/ml of IVIG (p<0.0001;Figure 1A). IVIG suppression of NETosis was associated with a dose effect. ROS production was also diminished in all concentrations of IVIG tested, including as low as 0.2 mg/ml (p<0.0001;Figure 1B). Phagocytosis was not only preserved but was boosted in the setting of IVIG treatment at 10 mg/ml (p<0.05;Figure 1C).Conclusion. These ex vivo data utilizing fresh human neutrophils demonstrate the potential beneficial effect of IVIG, an FDAapproved treatment, in the context of neutrophil-mediated immunopathology. IVIG may be a potential treatment to prevent progression of vascular and lung inflammation and damage in COVID-19, consistent with emerging evidence of its therapeutic benefit in this disorder. Results from an FDA Phase 3 prospective randomized international study that evaluates IVIG in COVID-19 are highly anticipated.