Discovering a Dihydrofluorescein Analogue as a Promising Fluorescence Substrate to HRP

Horseradish peroxidase (HRP) combined with its fluorescence substrates is attracting increasing attention for biochemical analysis. Amplex red is the most widely used fluorescence substrate to HRP;however, it suffers from some drawbacks, such as nonspecific responsiveness toward carboxylesterases. Discovering a new small molecular fluorescence substrate with improved sensitivity and selectivity for HRP is thus desired. Herein, three dihydrofluorescein derivatives (DCFHs) are presented to serve as HRP substrates through fluorescence turn-on methods. The most promising one, 2,7-dichloro-9-(2-(hydroxymethyl)phenyl)-9H-xanthene-3,6-diol (DCFH-1), exhibited excellent sensitivity in the detection of HRP. Moreover, DCFH-1 does not respond to carboxylesterase, thus holding advantages over Amplex red. In the further study, the detection reagent in the commercial ELISA kits was replaced with DCFH-1 to establish a new fluorescence ELISA, which works very well in the quantification of inflammatory cytokine biomarkers from in vitro models.

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.

Exposures to Heat-Inactivated SARS-CoV-2 Causes Reactive Oxygen Species Generation And Inflammatory Response in RAW 264.7 Cells

Introduction and Objectives Novel SARS-CoV-2 virus has been implicated in prompting a bold immune response that leads to severe Coronavirus disease 2019 (COVID-19). Recent studies have shown that SARSCoV-2-infected monocytes and macrophages are stimulated to produce an overabundance of pro-inflammatory cytokines and chemokines to generate a cytokine storm. Cytokines in excess can contribute to local tissue inflammation and the pathogenesis of COVID-19. However, the mechanism by which SARS-CoV-2 signal macrophage-derived inflammatory response remains unclear. In the present study, we used RAW 264.7 cells, a wellcharacterized macrophage model, to study the in vitro effects of SARS-CoV-2 on reactive oxygen species (ROS) production and its potential role in the signal transduction of cytokine production. Methods The effect of SARS-CoV-2 on ROS and cytokine generation in macrophages was assessed by treating RAW 264.7 cells with SARS-CoV-2 heat inactivated virus (0-20 million viral particles) or recombinant proteins for 24 hours. 2',7'-Dichlorodihydrofluorescein (2',7'-DCF) fluorescence analysis was utilized to quantify ROS generation within the RAW 264.7 macrophage cell line. Cell culture medium was sampled to quantify the levels of tumor necrosis factor (TNF) using enzyme-linked immunosorbent assay (ELISA). To assess the effects of SARS-CoV-2 on mitochondrial function, cells were treated with SARS-CoV-2 heat inactivated virus (0-20 million viral particles) for 24 hrs. Mitochondria-derived superoxide was measured using the MitoSOX™ red mitochondrial superoxide indicator. Results Treatment of RAW 264.7 cells with inactivated SARS-CoV-2 viral particles or recombinant proteins stimulated ROS production. Mitochondria-derived superoxide and hydrogen peroxide production were increased in response to inactivated SARS-CoV-2 viral particles and recombinant protein exposure. The increased ROS generation is linked to macrophage activation induced by SARS-CoV-2 exposures. Along with the ROS generation, increased TNF production was observed. Conclusions The results of this study suggest that both SARS-CoV-2 viral proteins and heat-inactivated viral particle exposures cause significant generation of ROS and cytokines by RAW 264.7 cells. ROS generation and the subsequent cytokine release apparently play a significant role in the pathogenesis associated with the SARS-CoV-2 viral infection. The imbalanced cellular defense system against oxidative stress commonly associated with aging could explain the increased occurrence of more severe SARS-CoV-2 illness in seniors and in patients with underlying health conditions. Based on the results from this study, we propose that antioxidants such as N-acetyl-L-cysteine, resveratrol, or Vitamin E in combination with antiinflammatory drug could be used to control excess ROS and cytokines in patients with severe COVID-19.

Red Blood Cells from COVID-19 Patients Show Evidence of Increased Oxidative Stress and Increased Lactate Influx

Red Blood Cells from COVID-19 Patients Show Evidence of Increased Oxidative Stress and Increased Lactate Influx Corona Disease 19 (COVID-19) is caused by SARS-CoV-2, a novel, highly infectious, single stranded RNA virus. In severe cases, excess oxidative stress produced by a ‘cytokine storm’ may generate excess reactive oxygen species (ROS) and lead to tissue damage in the lungs and elsewhere. As the potential role of RBCs in the pathophysiology of COVID-19 remains controversial (1), we investigated for evidence of increased oxidative stress and increased thrombotic tendency in RBCs from patients with COVID-19 infection. Following ethical approval and written informed consent, we used flow cytometry (BD FACSCanto II) to measure baseline RBC ROS following incubation with 2‘-7‘-dichlorofluorescein diacetate (DCF). RBC ROS were also measured following pre-incubation with hydrogen peroxide (H2O2) (2mM) +/- antioxidant N-acetyl cysteine (NAC) (0.6mM). We also measured RBC surface expression of adhesion molecules CD44, CD47 and CD242, as well as CD147. Results were expressed as mean +/- standard deviation (SD). RBC ROS were measured in 22 COVID-19 positive patients and in 10 age matched healthy controls. One patient died from respiratory failure, whilst only 3 others required ITU admission for continuous positive airway pressure (CPAP) or intubation. There was no statistical difference in mean basal RBC DCF mean fluorescence intensity (MFI) levels between COVID-19 positive patients and controls. However, mean increase in RBC DCF MFI following H2O2 incubation was significantly higher in the COVID-19 positive group (1105.7+/-336.3) compared to the control group (843.4+/-256.7)( p= 0.042). The increase in RBC DCF MFI in the COVID-19 positive group correlated with CRP (p=0.014) but not with D-dimer, serum ferritin or any complete blood count (CBC) parameters. Incubation of RBC with 0.6 mM NAC for 30 minutes prior to H2O2 exposure caused a mean reduction in DCF MFI of 26.7% in the COVID-19 positive group. RBC expression of CD44, CD47, CD242 and CD147 were measured In a separate cohort of COVID-19 positive patients (n=32), and in 22 age matched controls. There were no statistically significant differences in mean expression levels of CD44, CD47 and CD242 between the 2 groups. However, mean RBC CD147 MFI expression was higher in the COVID-19 group (1319.64+/-374.76) compared to controls (1061.59+/-253.33) (p=0.018). There was no significant correlation between RBC CD147 MFI and D-dimer, CRP, serum ferritin or any CBC parameters in the COVID-19 positive group. However, 21 of the 32 COVID-19 positive patients had blood lactate levels measured and there was a positive correlation between CD147 MFI expression and blood lactate (R=0.56, p=0.0077). Induction of oxidative stress by H2O2 resulted in a greater increase in ROS in RBCs from COVID-19 patients compared to controls and with correlation to CRP, despite the fact that there were very few patients with severe disease in the study. This suggests a role for oxidative stress in disease pathogenesis. Pre-incubation with NAC attenuated this increase in ROS, suggesting a possible role for antioxidants in therapy. Increased RBC cell surface expression of adhesion molecules CD44, CD47 and CD242 can facilitate RBC interaction with platelets and/or endothelial cells, potentially contributing to thrombosis. We found no increase in their expression in COVID-19 patients compared to controls although RBCs may contribute to thrombosis in COVID-19 infection by other means (1). CD147 is tightly associated with and enables proper expression of monocarboxylate transporter 1, the lactate transporter for RBCs. We found increased surface expression of CD147 on RBCs of COVID-19 patients, whilst CD147 expression showed a moderate correlation with serum lactate levels, suggesting that RBCs in COVID-19 infection may be acting as a lactate sink to protect against lactic acidosis. In summary, our study suggests that COVID-19 infection causes increased oxidative stress and increased lactate influx i RBCs. Further studies are warranted into the role of RBCs in COVID-19 infection. Reference: (1) Murphy P, Glavey S, Quinn J. Anemia and red blood cell abnormalities in COVID-19. Leuk Lymphoma 2021;62:1539 Disclosures: Quinn: Takeda: Honoraria. Glavey: Abbvie: Research Funding;Celgene and BMS company: Research Funding;Janssen: Honoraria, Research Funding;Amgen: Honoraria, Research Funding.