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. 2022 Oct 20:13:993720.
doi: 10.3389/fimmu.2022.993720. eCollection 2022.

Abrogation of neutrophil inflammatory pathways and potential reduction of neutrophil-related factors in COVID-19 by intravenous immunoglobulin

Affiliations

Abrogation of neutrophil inflammatory pathways and potential reduction of neutrophil-related factors in COVID-19 by intravenous immunoglobulin

Jorge Adrian Masso-Silva et al. Front Immunol. .

Abstract

Pathogenesis of lung injury in COVID-19 is not completely understood, leaving gaps in understanding how current treatments modulate the course of COVID-19. Neutrophil numbers and activation state in circulation have been found to correlate with COVID-19 severity, and neutrophil extracellular traps (NETs) have been found in the lung parenchyma of patients with acute respiratory distress syndrome (ARDS) in COVID-19. Targeting the pro-inflammatory functions of neutrophils may diminish lung injury in COVID-19 and ARDS. Neutrophils were isolated from peripheral blood of healthy donors, treated ex vivo with dexamethasone, tocilizumab and intravenous immunoglobulin (IVIG) and NET formation, oxidative burst, and phagocytosis were assessed. Plasma from critically ill COVID-19 patients before and after clinical treatment with IVIG and from healthy donors was assessed for neutrophil activation-related proteins. While dexamethasone and tocilizumab did not affect PMA- and nigericin-induced NET production ex vivo, IVIG induced a dose-dependent abrogation of NET production in both activation models. IVIG also reduced PMA-elicited reactive oxygen species production, but did not alter phagocytosis. COVID-19 patients were found to have elevated levels of cell-free DNA, neutrophil elastase and IL-8 as compared to healthy controls. Levels of both cell-free DNA and neutrophil elastase were lower 5 days after 4 days of daily treatment with IVIG. The lack of impact of dexamethasone or tocilizumab on these neutrophil functions suggests that these therapeutic agents may not act through suppression of neutrophil functions, indicating that the door might still be open for the addition of a neutrophil modulator to the COVID-19 therapeutic repertoire.

Keywords: COVID-19; NETosis; corticosteroids; dexamethasone; intravenous immunoglobulin (IVIG); neutrophils; oxidative burst; tocilizumab.

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Conflict of interest statement

Author GS has received consulting and research fees from Octapharma USA (Hoboken, NJ). The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1
IVIG has a dose-dependent suppressive effect on NETosis, which suggests that it may be a complimentary treatment in acute lung injury. Ex vivo functional assays of healthy neutrophils stimulated with either PMA or nigericin shows that abrogation of NETosis is induced in a dose-dependent manner by: IVIG (A, D), but not tocilizumab (B, E) or dexamethasone (C, F). An alternative approach of assessing NETosis using fluorescence microscopy of neutrophils stained for myeloperoxidase (green; a key component of NETs) shows clear inhibition of NETosis by IVIG (G). Data in panels A–F was analyzed with one-way ANOVA with multiple comparisons. Data are presented as individual data points ± SD with n = 6–7 individuals per group. Error bars represent 95% confidence interval. *P < .05, **P < .01, ***P < .001, ****P < .0001.
Figure 2
Figure 2
Treatment of primary human neutrophils with IVIG inhibits oxidative burst in a dose-dependent manner, while preserving phagocytosis. Circulating neutrophils from healthy controls were used to assess antimicrobial functions including reactive oxygen species production (oxidative burst) and phagocytosis. (A) Neutrophil oxidative burst, while being an important antimicrobial function can cause significant collateral damage to tissues, was inhibited in a dose-dependent manner by IVIG (solid circle markers) but not by dexamethasone (hollow purple markers). (B) Neutrophil phagocytosis was retained (unaffected) by treatment with IVIG or dexamethasone. Data were analyzed with a 2-way ANOVA with Geisser-Greenhouse correction and Dunnett’s multiple comparisons test. Data are presented as individual data points ± SEM with n = 9 and n = 5 individuals per group in oxidative burst and phagocytosis assays, respectively. Error bars represent 95% confidence interval. *P < .05, **P < .01, ***P < .001, ****P < .0001.
Figure 3
Figure 3
Treatment of severe COVID-19 with IVIG may lead to decreased NET-related factors and IL-8 in the circulation of patients. We assessed NET related components in the circulation of IVIG-treated COVID-19 patients, hours before the first IVIG dose and up to 5 days after treatment with IVIG. Moreover, we did a paired comparison of pre-IVIG plasma versus 5 days post-IVIG. We assessed NET components such as cell-free DNA (A, B), neutrophil elastase (C, D), and myeloperoxidase (MPO)-DNA complexes (E, F). Cell-free DNA and neutrophil elastase were significantly elevated in COVID-19 patients relative to controls (A, C), and decreased after IVIG treatment (B, D). MPO-DNA was not elevated in the plasma of patients with severe COVID-19 and did not change after IVIG treatment (E, F). The neutrophil chemokine and activating cytokine IL-8 was elevated in the plasma of COVID-19 patients (G) but did not significantly decrease after IVIG treatment (H). Data for panels A, C, E, and G was analyzed via one-way ANOVA with Tukey’s multiple comparisons test, although data points from same patients are connected to observe data trends. Data for panels B, D, F and H was analyzed with paired t-tests. Data are presented as individual data points ± SD with healthy controls (n = 10-14) and COVID-19 (n = 12). Error bars represent 95% confidence interval. *P < .05, **P < .01, ***P < .001, ****P < .0001.

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