Impact of the NRF2 activator, stabilised synthetic sulforaphane, on systemic inflammation in COVID19: results from the STAR-COVID19 trial

Introduction: Dysregulated immune responses are implicated in the pathogenesis of severe COVID19 and may be modulated by the transcription factor Nrf2. Hypothesis: Treatment with stabilised, synthetic sulforaphane (S-SFN)-an Nrf2 inducer-improves clinical status in hospitalised patients with suspected COVID19 pneumonia by curbing the inflammatory response. Method(s): Double-blind RCT of S-SFN (300mg, once daily, 14 days;EudraCT 2020-003486-19) in patients hospitalised with confirmed or suspected COVID19, in Dundee, UK. The primary outcome was the 7 point WHO Clinical Status scale at day 15. Blood samples were taken on days 1, 8 and 15 for measurement of 45 serum cytokines using the Olink Target48 panel. Key neutrophil functions were assessed including migration, phagocytosis and bacterial killing. Result(s): 133 participants were randomized (placebo n=68, S-SFN n=65) from Nov 2020 to May 2021. S-SFN treatment did not improve clinical status at day 15 (adjusted OR 0.87 95%CI 0.41-1.83). In serum, Nrf2 target TGFalpha was significantly increased at day 15 in those receiving S-SFN treatment compared with placebo (p=0.004;linear mixed effects model). Other targets implicated in cytokine storm, including IL6, IL1beta and TNFalpha, were unchanged. Patients receiving Tocilizumab (n=20) were excluded from exploratory analyses due to a strong impact upon IL6 levels, leading to significant increases at day 8 across the study population (p=0.015). S-SFN treatment did not significantly affect neutrophil function. Conclusion(s): S-SFN treatment modulated select Nrf2 targets but did not modulate key cytokines. Further analyses to delineate drug activity are ongoing.

Dipeptidyl Peptidase-1 Inhibition in Patients Hospitalized with COVID-19: a Multicentre Randomized Double-Blind Placebo Controlled Trial

Background: Neutrophil serine proteases (NSPs) are involved in the pathogenesis of COVID19 and are increased in severe and fatal infection. We investigated whether treatment with Brensocatib, an inhibitor of dipeptidyl peptidase-1, an enzyme responsible for the activation of NSPs, would improve outcomes in hospitalized patients with COVID19. Method(s): In a randomized, double-blind, placebo-controlled trial, 406 hospitalized patients with COVID19 with at least one risk factor for severe disease were randomized 1:1 to once-daily Brensocatib 25mg (n=192) or placebo (n=214) for 28 days. Primary outcome was the 7-point World Health Organisation Clinical Status scale at day 29. Secondary outcomes included time to clinical improvement, national early warning score, new oxygen and ventilation use, neutrophil elastase activity in blood and mortality. Finding(s): Brensocatib treatment was associated with worse clinical status at day 29 (adjusted odds ratio 0 72, 95%CI 0 57-0 92) compared to placebo. The adjusted hazard ratio (aHR) for time to clinical improvement was 0 87 (95%CI 0 76-1 00) and time to hospital discharge was 0 98 (95%CI 0 84-1 13). During the 28-day follow-up period, 23 (11%) and 29 (15%) patients died in the placebo and Brensocatib treated groups respectively). Oxygen and new ventilation use were greater in the Brensocatib treated patients. Neutrophil elastase activity in blood was significantly reduced in the Brensocatib group from baseline to day 29. Prespecified subgroup analyses of the primary outcome supported the primary results.

Investigating neutrophil AMPK activity in COVID-19 and pneumonia

Introduction: COVID19 can cause neutrophilic inflammation and reaction oxygen species (ROS) production, leading to acute lung injury and mortality. AMPK is a key regulator of cellular energy with profound effects on neutrophil function. This study aims to investigate the role of AMPK activity in neutrophils during COVID-19 and pneumonia caused by pathogens other than SARS-CoV-2. Method(s): Patients hospitalised due to pneumonia or COVID-19 were recruited from Ninewells Hospital (Dundee, UK). Blood was sampled at day 1, 8, and 15. ROS production, phospho-AMPK (pAMPK), and NQO1 were stained in neutrophils, and then analysed by flow cytometry. The endogenous AMPK inhibitor, resistin, was quantified by ELISA, in serum (day 1, 8, 15). WHO clinical scale and CURB65 score were utilised to define severity. Result(s): 133 patients were enrolled (mean age 63.6 years). Resistin was not different between pneumonia and COVID-19 on day1. However, day 1 resistin was higher in severe disease defined by CURB65 Score (p=0.0220) and WHO score (p=0.0184). Resistin reduced over time at day 1 (mean 63.1pg/mL;n=121) to day 15 (mean 59.5pg/mL;n=66)(p=0.0002). Zymosan stimulation significantly increases neutrophil ROS production (p<0.0001), and significantly decreases NQO1 (p<0.0001), but caused no changes with pAMPK. There were no changes in these markers over time. pAMPK significantly correlated with NQO1 in unstimulated neutrophils (p=0.0388), but not when stimulated with zymosan. There were no associations between resistin and pAMPK, and no difference in these markers between pneumonia and COVID-19 groups. Conclusion(s): Our study suggests resistin as a marker of severity and disease course in COVID-19, independent of neutrophil AMPK signalling.

Stabilised, synthetic sulforaphane in hospitalized patients with suspected COVID-19 pneumonia: a double-blind, placebo-controlled RCT

Introduction: The transcription factor Nrf2 downregulates key inflammatory cytokines in COVID-19 (IL-6, IL-1b, COX-2 and TNF-a). We investigated the efficacy of S-SFN (stabilised sulforaphane, activator of Nrf2) to improve clinical outcomes in patients hospitalized with suspected COVID-19. Method(s): Randomized, double-blind, placebo-controlled trial, patients hospitalised with suspected or confirmed COVID-19, radiological pneumonia and a CURB65 score of >= 1 were randomized 1:1 to once-daily S-SFN 300mg or placebo for 14 days. The primary outcome was the 7-point WHO Clinical Status (CS) scale at day 15. Key secondary outcomes included time to clinical improvement, national early warning score (NEWS), oxygen and ventilation use, and mortality. Result(s): The trial was terminated due to futility after 133 patients had been enrolled (S-SFN, n=65 and placebo, n=68). 103 had PCR confirmed COVID-19 infection. S-SFN treatment was not associated with improved CS at day 15 (OR 0.87 95%CI (0.41-1.83, p=0.712). There was no difference in time to clinical improvement (HR 1.02 (0.70- 1.49)). S-SFN was not associated with a reduced length of hospital stay (6.2days vs 7.4days (S-SFN)). There were 26 deaths during the 29-day follow-up, 11 (16.2%) and 15 (23.1%) patients died in the placebo and S-SFN treated groups respectively (HR 1.45 (0.67-3.16)). There were no differences between treatment groups with respect to oxygen or ventilation free days. Adverse events were reported in 44.1% of placebo treated and 64.6% of S-SFN treated patients. Conclusion(s): S-SFN treatment did not improve day 15 clinical status in hospitalized patients with suspected or confirmed COVID-19 infection.

Longitudinal profiling of the peripheral blood transcriptome identifies risk of Major Cardiac Events after Hospitalization for COVID-19

Introduction: SARS-CoV-2 infection has profound effects on endothelial and immune cell function and coagulation, and better understanding of these events in COVID-19 would allow for targeted cardiovascular treatment and followup. Method(s): Longitudinal observational study of patients with PCR-confirmed SARS-CoV-2 infection admitted to hospital at two UK sites. Patients were enrolled within 96 hours of admission, with sampling up to day 29. RNAstabilised whole blood was processed for mRNA sequencing. Gene expression levels were compared between patients who did and did not suffer a major cardiac event (MACE) from admission to 1-year post-hospitalization. Result(s): At day 1, in acute COVID-19, no differences in gene expression were observed between those with (n=23) and without (n=140) a MACE. However, 93 significant differentially expressed genes (DEGs;adjusted pvalue<0.05;Wald test with Benjamini-Hochberg correction) were identified at day 29 between patients who suffered a MACE (n=16) or not (n=85) post-hospitalization. Neutrophil elastase (ELANE), tissue factor pathways inhibitor (TFPI) and integrin subunit alpha-2 (ITGA2B) were significantly elevated in patients who suffered a MACE. Significantly enriched pathways associated with cardiovascular events included type I interferon signalling and neutrophil chemotaxis. Conclusion(s): COVID-19 patients who experienced a MACE demonstrated significant changes in peripheral blood transcriptome 29 days after hospital admission. Significant DEGs were related to neutrophil activity, coagulation and interferon signalling, suggesting a relationship between these pathways and increased cardiovascular risk.

Altered neutrophil proteomes in COVID19 patients 29-days post hospital admission are associated with delayed recovery: results from the PREDICT-COVID19 study

Introduction and ObjectivesNeutrophils are increasingly recognised for a role in acute COVID19, contributing to hyperinflammatory responses, immunothrombosis and tissue damage. However, less is known about the cellular changes occurring within neutrophils in acute disease, as well as neutrophil function in patients recovering from COVID19. Mass spectrometry-based proteomics of neutrophils from hospitalised COVID19 patients sampled longitudinally was utilised to characterise these cells in both acute and long COVID19 (i.e. symptoms for ≥4 weeks).MethodsProspective observational study of hospitalised patients with PCR-confirmed SARS-CoV-2 infection (May 2020–December 2020). Patients were enrolled within 96 hours of admission, with longitudinal sampling up to day 29. Control groups comprised hospitalised patients with non-COVID19 acute respiratory infection and age-matched non-infected controls. Neutrophils isolated from peripheral blood were processed for mass spectrometry. COVID19 severity was defined using the WHO 7-point ordinal scale.Results84 COVID19 patients were included (mean age±SD 65.5±14.6 years;52.4% male), 91 non-COVID19 respiratory infection patients (age 65.7±16.7 years;49.5% male) and 42 non-infected controls (age 58.5±17.9;40% male). 1,748 proteins were significantly different (q-value≤0.05) in COVID19 neutrophils compared to those of non-infected controls. Major differences included a robust interferon response at baseline, with markers of neutrophil immaturity (CD10, CD71), increased neutrophil activation (CD64), and changes in metabolism which associated with COVID19 disease severity. Delayed recovery (WHO score 2–3) at day 29 was associated with significant changes in 1,107 proteins compared to the control population. Features of non-recovery included significantly reduced abundance of migratory receptors (e.g. C3AR1, LTB4R), integrins (CD11b, CD18), inhibitory molecules (e.g. SHP-1, SHIP-1) and indications of increased activation (CD64). Overall, ficolin and specific granule content was decreased in COVID19 patient neutrophils at day 29 compared with controls, however, comparing those who had recovered and those who had not, granule content was found to be significantly lower in the non-recovery group.ConclusionNeutrophils undergo significant changes in acute COVID19 associated with disease severity. Neutrophil proteomics revealed that these cells may have an ongoing role in non-recovered patients, including profiles associated with increased potential for neutrophil activation and reduced migratory capacity, highlighting neutrophils as potential therapeutic targets in long COVID19.

Results from the STAR-COVID19 trial, a double-blind RCT of stabilised, synthetic sulforaphane in hospitalised patients with suspected COVID19

Introduction and ObjectivesThe transcription factor, Nrf2, can directly promote beneficial anti-oxidant and anti-inflammatory responses. In the STAR-COVID19 trial, hospitalised patients with confirmed or suspected COVID19 were treated with stabilised, synthetic sulforaphane (S-SFN)—an Nrf2 inducer—to evaluate impact on clinical status and systemic inflammation.MethodsDouble-blind, randomised, placebo-controlled trial of S-SFN (300 mg S-SFN or placebo once daily for 14 days;allocation ratio 1:1;EudraCT 2020–003486-19) in Dundee, UK. Inclusion criteria were age ≥18 years, suspected or confirmed COVID19 or pneumonia and CURB65 score ≥1. The primary outcome was the 7-point WHO Clinical Status scale at day 15. Secondary outcomes included time to clinical improvement, length of hospital stay, and mortality. Blood samples were taken on days 1, 8 and 15 for exploratory analyses. To assess Nrf2 activity and inflammation, 45 serum cytokines were measured using the Olink Target48 panel and mRNA sequencing of peripheral blood leukocytes performed. Further, as key immune cells in COVID19 responses, select neutrophil functions such as migration, phagocytosis and extracellular trap formation were evaluated.Results133 participants (77.4% PCR-confirmed SARS-CoV-2 infection) were randomized from Nov 2020 to May 2021. 68 received placebo (61.8% male;age 63.6±13.8) and 65 received S-SFN (53.8% male;age 61.6±12.7).S-SFN treatment did not improve clinical status at day 15 (Intention-to-treat population;adjusted OR 0.87, 95%CI 0.41–1.83, p=0.712) and the trial was terminated due to futility. Time to clinical improvement (adjusted HR 1.02(0.70–1.49)), length of hospital stay (aHR 0.84(0.56–1.26)), or 29-day mortality (aHR 1.45(0.67–3.16)) were not improved with S-SFN treatment.230 samples in total were utilised for serum cytokine measurement;Nrf2 targets implicated in cytokine storm, including IL6, IL1β and TNFα, were not significantly changed by S-„SFN treatment. Interestingly, serum TGFα was significantly increased at day 15 in those receiving S-SFN compared with placebo (p=0.004;linear mixed effects model). S-SFN treatment did not significantly affect neutrophil functions investigated.ConclusionS-SFN treatment did not improve clinical status at day 15 or modulate key inflammatory cytokines—however, changes in other factors were indicated. Further analyses, including transcriptomics, to delineate drug activity are currently ongoing.

Inflammatory biomarkers predict clinical outcomes in patients with COVID-19 infection: results from the PREDICT-COVID19 study

IntroductionCOVID-19 is reported to cause profound systemic inflammation. Anti-inflammatory treatments such as corticosteroids and anti-IL-6 receptor monoclonal antibodies reduce mortality. Identifying inflammatory biomarkers associated with increased morbidity and mortality may allow both prediction of outcomes and identification of further therapeutic targets.MethodsA prospective observational study of patients with PCR-confirmed SARS-CoV-2 admitted to a single centre in Dundee, UK. Patients were enrolled within 96 hours of hospital admission. 45 inflammatory biomarkers were measured in serum using the Olink Target48 proteomic-based biomarker panel. Additional markers were measured by ELISA/immunoassay and enzyme activity assays. Severe disease was defined as the requirement for non-invasive or mechanical ventilation or death within 28 days of admission. Discrimination between groups was evaluated using the area under the receiver operator characteristic curve (AUC).Results176 patients were included (mean age 64.9 years, SD 13.6), 101 were male (57.4%). 56 patients developed severe disease (31.8%), mortality was 16.5%. Using ROC analysis, the strongest predictors of severity (p<0.0001) were CCL7/MCP3 (AUC 0.78 95%CI 0.70–0.85), IL6 (0.73 95%CI 0.66–0.81), IL15 (0.73 95%CI 0.65–0.81), CXCL10/IP10 (0.73 95%CI 0.65–0.81). Further significant predictors of severity included CXCL11, IL10, CCL2/MCP1 and CSF2/GM-CSF. Predictors of mortality were CXCL10 (0.78 95%CI 0.69–0.86), IL6 (0.76 95%CI 0.67–0.85), IL15 (0.75 95%CI 0.66–0.84), IL10 (0.73 95%CI 0.64–0.82). Further significant predictors of mortality were CXCL9 and CCL7.ConclusionMultiple circulating biomarkers were identified which predicted disease severity and mortality in COVID19, indicating clinical value in measurement upon hospital admission to highlight high-risk patients. Associated biological processes for these proteins included anti-viral and interferon responses and immune cell chemotaxis. In particular, CCL7 and CXCL10, the strongest predictors of severity and mortality in this dataset, are key players in the cytokine storm and immune cell recruitment linked with COVID19. These chemokines are not currently therapeutic targets, highlighting key avenues for further clinical research.

Comparison of inflammatory profiles between COVID-19 and other acute lower respiratory tract infections: Results from the PREDICT-COVID19 study

IntroductionCOVID-19 has been reported to induce a ‘cytokine storm’ distinct from other acute respiratory tract infections (LRTIs). Understanding the similarities and differences in inflammatory profiles between SARS-CoV-2 infection and other respiratory infections may aid diagnosis, as well as the potential to repurpose therapies such as steroids and anti-IL-6 receptor antagonists for other respiratory infections.MethodsA prospective observational study of patients in 3 groups 1) PCR confirmed SARS-CoV-2 infection, 2) community-acquired pneumonia (CAP) without SARS-CoV-2, and 3) controls hospitalized for reasons other than infection. Patients were enrolled from a single centre in Dundee, UK. Patients were enrolled within 96 hours of hospital admission. 45 inflammatory biomarkers were measured in blood using the Olink target proteomic based biomarker panel. Additional markers were measured by ELISA/immunoassay and enzyme activity assay as appropriate. Discrimination between groups was evaluated using the area under the receiver operator characteristic curve (AUC).Results294 patients were included (COVID-19 n=176, CAP n=76, controls n=42), mean age 64 (SD±15.2) and 150 subjects were male (51.0%). Using ROC analysis the most discriminating biomarkers for COVID-19 compared to CAP were CXCL-10 (AUC 0.84 95%CI 0.78–0.90 p<0.001), CCL-8 (0.87 95%CI 0.82–0.92, p<0.001), CCL-7 (0.84 95%CI 0.78–0.89, p<0.001), CXCL-11 (0.80 95%CI 0.73–0.88, p<0.001). Further biomarkers included IL-18, IL-7, IL-10 and IL-33. The most discriminating biomarkers for COVID-19 compared to controls were CXCL-10 (0.89 95%CI 0.85–0.93, p<0.001, CCL-7 (0.88 95%CI 0.83–0.92, p<0.001), CCL-8 (0.87 95%CI 0.82–0.92, p<0.001). Further biomarkers included IL-10, CXCL-11 and IL-18. IL-4 was significantly lower in COVID-19 patients compared to controls (0.27 95%CI 0.16–0.38, p<0.001). No significant difference in IL-6 was seen between COVID-19 and CAP (median 21.9pg/ml vs 19.8pg/ml,p=0.59).ConclusionDifferential markers of inflammation were identified between COVID-19, CAP and control samples, indicating distinct immunological pathways. The identification of a similar IL-6 signature between COVID-19 and CAP indicates that IL-6 targeting therapies currently being used to treat COIVD-19 may also be beneficial in the treatment of CAP.

Clinical characteristics, management, and outcomes in COVID-19-positive dialysis patients from three London Renal Centres, United Kingdom

Background: Dialysis patients, with frequent co-morbidities, advanced age and frailty, visiting treatment facilities frequently are perhaps more prone to SARS-Cov-2 infection and related death - the risk-factors and dynamics of which are unknown. The aim of this study was to investigate the hospital outcomes in SARS-CoV-2 infected dialysis patients. Methods: This prospective, observational, multi-centre study collected data on SARS-CoV-2 infected HD patients between 29/02/2020 and 15/05/2020. Data was collected on demographics, comorbidities, WHO performance status, clinical symptoms, laboratory parameters, hospital management and outcomes. Treatment was predominantly supportive, unless patients were part of an approved clinical trial. The study was approved by NHS Research Ethics Committee 20/SW/0077 and Heath Research Authority IRAS 283130. Results: Of 1737 HD patients at the 3 renal centres, 224 (13%) were COVID-19 positive over the study period. The characteristics of the COVID-19 HD patients were: mean age 65.8;59% male;38% Caucasian;81% hypertension;54% diabetes;25% chronic lung disease;29% ischaemic heart disease and 22% cerebrovascular disease. The most common symptoms at presentation were fever (62%) and cough (53%). About 143 (64%) patients were managed as an inpatient and 81 (36%) as an outpatient. Of 9 patients that required mechanical ventilation: 6 died, 1 patient was discharged and 2 are still under clinical care. Overall 51 patients died (23%), 154 (69%) were discharged alive and 19 (8%) were still under clinical care as of 15/05/2020. Preliminary analyses suggested that those that died were significantly older (p=0.0028), more likely to have ischaemic heart disease (p=0.003), cerebrovascular disease (p=0.019), smoking history (p=0.006), WHO performance status 3-4 (p=0.004), higher neutrophil: lymphocyte ratio at presentation (p=0.0001) and higher CRP at presentation (p=0.0021). Conclusions: This large cohort of COVID-19 positive haemodialysis demonstrates a high case fatality ratio, which increased significantly with age, cardiovascular disease, smoking history, frailty and markers of inflammation.