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

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.

Circulating neutrophils from COVID-19 patients have elevated rates of phagocytosis

Rationale: Neutrophils are an important component of the immune system and are crucial for the inflammatory response during infection from a foreign pathogen. Phagocytosis is a vital cellular mechanism in which pathogens are engulfed in a phagosome to be eliminated. Once taken inside the cell, the pathogen is subject to a cytotoxic environment inside the phagolysosome. While neutrophils are critical in suppressing the spread of infection, too much activity has been linked to tissue damage. Inflammatory lung conditions such as acute respiratory distress syndrome (ARDS) can occur, often leading to death. Our aim is to determine whether the rate of phagocytosis is associated with disease severity among patients with COVID-19. Methods: Neutrophils were isolated from the peripheral blood of 20 COVID-19 human subjects. Neutrophils were isolated with Polymorphoprep and mixed in a 1:1 ratio with pHrodo TM Red Staphylococcus aureus Bioparticles TM (Invitrogen) in a 96-well plate and centrifuged at 1200 rpm for 6 min. Fluorescence was quantified via plate reader (Infinite M200, TECAN) every 15min for 2 hours to define the rate of phagocytosis, with excitation at 560nm and emission at 585nm. Cells were incubated at 37°C with 5% CO2 in-between reads. Statistical analysis was performed via simple linear regression with detection of significant differences in slopes. Paired two-tailed non-parametric t-tests (Wilcoxon) were conducted for each time point. Results: Rates of phagocytosis were significantly higher in neutrophils from critically ill COVID-19 patients relative to neutrophils from healthy controls (487.5 versus 374.5 at 120min;p<0.05 at 75, 90, 105 and 120 min). Conclusion: Despite the functional role of neutrophils in suppression of foreign pathogens, increased activity of neutrophils can lead to collateral damage and thus exacerbated lung injury. We found that neutrophils from COVID-19 patients have higher rates of phagocytosis than those from healthy subjects. These findings suggest that neutrophils circulating in COVID-19 patients have an elevated activation state and may contribute to increased severity of disease. However, the elevation in phagocytosis may also suggest that neutrophils in critically ill COVID patients are well able to phagocytose and kill pathogens which cause secondary infections in this at-risk cohort. Figure: .

Neutrophils from the circulation of critically ill COVID-19 patients may promote inflammation via increased production of reactive oxygen species

Background: Neutrophils are an important part of the innate immune system and play a vital role in host defense. Neutrophils target invading pathogens through both intracellular and extracellular mechanisms. One of their primary antimicrobial mechanisms is the production of reactive oxygen species (ROS) within phagolysosomes during oxidative burst. ROS production is a marker of the inflammatory/activity state of neutrophils. Although neutrophils are crucial in the defense against microorganisms, recruitment and hyperactivation of neutrophils can also cause collateral damage to the host. Recent studies have shown that neutrophils may be playing a role in acute respiratory distress syndrome (ARDS) in COVID-19. We hypothesized that ROS production would be increased secondary to elevated activation of circulating neutrophils in COVID-19 patients and may be playing a role in immunopathology.Methods: Circulating neutrophils were isolated from the blood of 20 human subjects with COVID-19 and 12 healthy controls. Neutrophils at 2x106 cells/ml were incubated with 10μM 2',7'-dichlorodihydrofluorescein diacetate (H2DCFDA) for 20min at 37°C. Neutrophils were centrifuged at 500g, resuspended in HBSS-/-, plated at 2x105 in 96-well plates and exposed to 0, 2.5, 25 and 250nM phorbol myristate acetate (PMA;in triplicate). Fluorescence was measured (Infinite M200, TECAN) at 495nm:520nm every 15min for 2hr. ROS production was analyzed using mixed-effects models with Geisser- Greenhouse correction and Sidak's multiple comparisons test.Results: ROS production was increased in neutrophils isolated from the circulation of critically ill COVID-19 patients as compared to healthy controls (2581nm versus 3790nm, respectively, at 120min, p<0.05). Upon stimulation with PMA, ROS production was also increased in COVID-19 patients relative to controls (2.5 PMA: 16597 versus 10549, respectively, <0.001;25 PMA: 20633 versus 11312, respectively, p<0.0001). At the final timepoint, 1.57, 1.82 and 1.85-fold difference in ROS production was noted at 2.5, 25 and 250nM respectively between patient populations. Conclusion: Increased ROS production at baseline and upon stimulation demonstrates that neutrophils in the circulation of critically ill COVID-19 patients with ARDS have an elevated activation state. These patients are known to have recruitment of neutrophils to the lung parenchyma, where the production and release of ROS may cause collateral damage. Further, neutrophils have been implicated in the vascular and systemic immunopathology of COVID-19, which may be driven by release of these damaging and toxic molecules. Neutrophil-associated pathways may serve as therapeutic targets and quantitative markers of disease in COVID-19 patients.

Neutrophil extracellular trap formation (NETOSIS) increases with severity of disease in COVID-19 patients

Background: Neutrophils are key players in the immune and aid in the defense against microorganisms. Neutrophil extracellular traps (NETs) are extracellular DNA complexes, which are released during NETosis, a programmed form of cell death. Although NETs are crucial in the fight against infectious agents, an overabundance of neutrophils has been implicated in many inflammatory lung conditions. Our aim is to determine whether an overabundance of NETosis is associated with clinical deterioration of patients with COVID-19. Methods: Circulating polymorphonuclear cells (neutrophils) were isolated from human peripheral blood of 20 human subjects with COVID-19. Neutrophils were seeded in 96-well plates and treated with 0, 2.5 nM, 25 nM, and 250 nM of phorbol 12-myrisate 13-acetate (PMA) or 12 uM nigericin for 2 hours to stimulate NET production via canonical and noncanonical pathways, respectively. Following incubation, wells were treated with micrococcal nuclease, supernatants were collected from each well, and extracellular DNA content to quantify NETosis was detected by fluorescent plate reader. We calculated acute physiology and chronic health evaluation (APACHE-II) scores for every human subject. These were calculated at the same time point at which the neutrophils were collected. They were then compared to the degree of NETosis and absolute neutrophil count (ANC). These were analyzed using a simple linear regression model. We also categorized participants based on APACHE-II scores (APACHE-II <15, APACHE-II>15) and compared them to rates of NETosis using a bar graph. Results: APACHE II is a widely used ICU mortality prediction score that is used to risk-stratify patients. We found that participants with higher APACHE-II scores had higher rates of NETosis, both at 0 nM PMA and when stimulated with nigericin (figure 1a-b). This suggests that higher rates of NETosis correlate with increased disease severity. Additionally, we found a positive correlation between ANC and NETosis (Figure 1c-1d), suggesting that ANC itself is a reliable marker of NETosis and disease severity. Conclusion: NETosis is an important player in immune system defense but has also been implicated in various inflammatory lung conditions. We found that in patients with COVID-19, there was a positive correlation between worsening disease state, measure by APACHE II scores, and increased NETosis. This suggests that over-activation of neutrophils may play a role in disease progression. We also found a positive correlation between NETosis and ANC, indicating that the degree of circulating neutrophils is a reliable marker of the functional state of neutrophils, as well as disease severity.

Effect of intravenous immunoglobulin on neutrophil antimicrobial and inflammatory functions-implications in neutrophil-mediated immunopathology

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.

Circulating neutrophils from critically ill covid-19 patients have increased netosis via canonical and non-canonical pathways

Introduction. The pathophysiology of infection with SARS-CoV-2 involves the lower airways and host-launched aggressive inflammatory responses leading to exacerbated lung damage in these vital tissues. Early clinical studies found that COVID-19 patients have higher levels of neutrophils in the circulation. Neutrophils are the most abundant leukocyte in circulation and are known to be highly proinflammatory due to production of neutrophil extracellular traps (NETosis). NETs are web-like chromatin structures coated with histones and proteases that both capture and kill invading pathogens. However, while being an effective countermeasure towards foreign microbes, this process also causes undesirable damage in host tissues. Therefore, we sought to characterize NETosis in circulating neutrophils from COVID-19 patients to determine whether this immunological response might be exacerbating or driving the disease state in COVID-19, rather than mitigating the virus. Methods.Blood was drawn daily from critically ill COVID-19 patients (n=16) after consent was obtained. Healthy controls (n=13) were screened for COVID-19 and gave blood once a week. Blood was drawn into lithium heparin tubes (BD Vacutainer). Neutrophils were isolated using PolymorphprepTM(PROGEN) per manufacturer's instructions. Cells were resuspended at 2x106 cells/ml for functional assays. Neutrophils were stimulated with increasing concentrations of PMA (Phorbol 12-myristate 13-acetate) of 2.5nM, 25nM and 250nM to stimulate NETosis via the canonical pathway, and nigericin at 15uM for the non-canonical pathway. NETosis was quantified using the Quant-iT™ PicoGreen™ dsDNA Assay Kit (Invitrogen) and by NET visualization via myeloperoxidase and nuclear staining (using Polyclonal Rabbit Anti-Human Myeloperoxidase by Dako and Hoescht stain by Invitrogen). Results.Functional NETosis assays of circulating neutrophils from COVID-19 patients demonstrate overall increased NETosis determined by increased release of dsDNA. This enhanced NETosis occurred at baseline and after stimulation with PMA when compared to healthy controls (Figure 1A, p <0.0001). Fluorescent microscopy also demonstrated increased NETosis in neutrophils from COVID-19 patients (Figure 1B;MPO-green and nucleus-blue). NETosis via the non-canonical pathway (induction with nigericin) was also increased in COVID-19 patients versus controls (p=0.02). Conclusions.Circulating neutrophils from critically ill COVID-19 patients are more prone to produce NETs than circulating neutrophils from healthy individuals. This is likely to lead to NETmediated tissue injury once neutrophils enter inflamed tissue, where they can potentially drive acute lung injury and acute respiratory distress syndrome, common causes of mortality in COVID-19. The finding of increased production of NETs by both canonical and non-canonical pathways is consistent with an overall hyper-activated state in COVID-19.