Mechanical ventilation affects respiratory microbiome of COVID-19 patients and its interactions with the host (preprint)

Understanding the pathology of COVID-19 is a global research priority. Early evidence suggests that the microbiome may be playing a role in disease progression, yet current studies report contradictory results. Here, we examine potential confounders in COVID-19 microbiome studies by analyzing the upper (n=58) and lower (n=35) respiratory tract microbiome in well-phenotyped COVID-19 patients and controls combining microbiome sequencing, viral load determination, and immunoprofiling. We found that time in the intensive care unit and the type of oxygen support explained the most variation within the upper respiratory tract microbiome, dwarfing (non-significant) effects from viral load, disease severity, and immune status. Specifically, mechanical ventilation was linked to altered community structure, lower species- and higher strain-level diversity, and significant shifts in oral taxa previously associated with COVID-19. Single-cell transcriptomic analysis of the lower respiratory tract of ventilated COVID-19 patients identified increased oral microbiota compared to controls. These oral microbiota were found physically associated with proinflammatory immune cells, which showed higher levels of inflammatory markers. Overall, our findings suggest confounders are driving contradictory results in current COVID-19 microbiome studies and careful attention needs to be paid to ICU stay and type of oxygen support, as bacteria favored in these conditions may contribute to the inflammatory phenotypes observed in severe COVID-19 patients.

The metabolic fingerprint of COVID-19 severity (preprint)

Corona virus disease 2019 (COVID-19) has been associated with a wide range of divergent pathologies, and risk of severe disease is reported to be increased by a similarly broad range of co-morbidities. The present study investigated blood metabolites in order to elucidate how infection with severe acute respiratory syndrome coronavirus 2 can lead to such a variety of pathologies and what common ground they share. COVID-19 patient blood samples were taken at hospital admission in two Belgian patient cohorts, and a third cohort that included longitudinal samples was used for additional validation (total n=581). A total of 251 blood metabolite measures and ratios were assessed using nuclear magnetic resonance spectroscopy and tested for association to disease severity. In line with the varied effects of severe COVID-19, the range of severity-associated biomarkers was equally broad and included increased inflammatory markers (glycoprotein acetylation), amino acid concentrations (increased leucine and phenylalanine), increased lipoprotein particle concentrations (except those of very low density lipoprotein, VLDL), decreased cholesterol levels (except in large HDL and VLDL), increased triglyceride levels (only in IDL and LDL), fatty acid levels (decreased poly-unsaturated fatty acid, increased mono-unsaturated fatty acid) and decreased choline concentration, with association sizes comparable to those of routine clinical chemistry metrics of acute inflammation. Our results point to systemic metabolic biomarkers for COVID-19 severity that make strong targets for further fundamental research into its pathology (e.g. phenylalanine and omega-6 fatty acids).

A randomized, open-label, adaptive, proof-of-concept clinical trial of modulation of host thromboinflammatory response in patients with COVID-19: the DAWn-Antico study (preprint)

Background: The peak of the global COVID-19 pandemic has not yet been reached and many countries face the prospect of a second wave of infections before effective vaccinations will be available. After an initial phase of viral replication, some patients develop a second illness phase in which the host thrombotic and inflammatory responses seems to drive complications. Severe COVID-19 disease is linked to high mortality, hyperinflammation, and a remarkably high incidence of thrombotic events. We hypothesize a crucial pathophysiological role for the contact pathway of coagulation and the kallikrein-bradykinin pathway. Therefore, drugs that modulate this excessive thromboinflammatory response should be investigated in severe COVID-19. Methods: In this adaptive, open-label multicenter randomized clinical trial we compare low molecular weight heparins at 50 IU anti-Xa/kg twice daily - or 75 IU anti-Xa twice daily for intensive care (ICU) patients - in combination with aprotinin to standard thromboprophylaxis in hospitalized COVID-19 patients. In the case of hyperinflammation, the interleukin-1-receptor antagonist anakinra will be added on top of the drugs in the interventional arm. In a pilot phase, the effect of the intervention on thrombotic markers (D-dimer) will be assessed. In the full trial, the primary outcome is defined as the effect of the interventional drugs on clinical status as defined by the WHO ordinal scale for clinical improvement. Discussion: In this trial we target the thromboinflammatory response at multiple levels. We intensify the dose of low molecular weight heparins to reduce thrombotic complications. Aprotinin is a potent kallikrein pathway inhibitor that reduces fibrinolysis, activation of the contact pathway of coagulation, and local inflammatory response. Additionally, aprotinin has shown in vitro inhibitory effects on SARS-CoV-2 cellular entry. Because the excessive thromboinflammatory response is one of the most adverse prognostic factors in COVID-19, we will add anakinra, a recombinant interleukin-1 receptor antagonist, to the regimen in case of severely increased inflammatory parameters. This way, we hope to modulate the systemic response to SARS-CoV-2 and avoid disease progressions with a potentially fatal outcome. Trial registration This trial is registered in the EU Clinical Trials Register. Registration number: 2020-001739-28. Registered on 2020-04-10.

Monocyte-driven atypical cytokine storm and aberrant neutrophil activation as key mediators of COVID-19 disease severity (preprint)

Epidemiological and clinical reports have indicated that the host immune response to SARS-CoV-2, more so than viral factors, determines COVID-19 disease severity. To elucidate the immunopathology underlying COVID-19 severity, cytokine and multiplex immune profiling was performed in mild-moderate and critically-ill COVID-19 patients. Hypercytokinemia in COVID-19 differed from the IFN-γ-driven cytokine storm in macrophage activation syndrome, and was more pronounced in critical versus mild-moderate COVID-19. Systems modelling of cytokine levels followed by deep-immune profiling showed that classical monocytes drive this hyper-inflammatory phenotype and that a reduction in T-lymphocytes correlates with disease severity, with CD8+ cells being disproportionately affected. Expression of antigen presenting machinery was reduced in critical disease, while also neutrophils contributed to disease severity and local tissue damage by amplifying hypercytokinemia and neutrophil extracellular trap formation. We suggest a myeloid-driven immunopathology, in which hyperactivated neutrophils and an ineffective adaptive immune system act as mediators of COVID-19 disease severity.

Discriminating Mild from Critical COVID-19 by Innate and Adaptive Immune Single-cell Profiling of Bronchoalveolar Lavages (preprint)

How innate and adaptive lung immune responses to SARS-CoV-2 synchronize during COVID-19 pneumonitis and regulate disease severity is poorly established. To address this, we applied single-cell profiling to bronchoalveolar lavages from 44 patients with mild or critical COVID-19 versus non-COVID-19 pneumonia as control. Viral RNA-tracking delineated the infection phenotype to epithelial cells, but positioned mainly neutrophils at the forefront of viral clearance activity during COVID-19. In mild disease, neutrophils could execute their antiviral function in an immunologically controlled fashion, regulated by fully-differentiated T-helper-17 (TH17)-cells, as well as T-helper-1 (TH1)-cells, CD8+ resident-memory (TRM) and partially-exhausted (TEX) T-cells with good effector functions. This was paralleled by orderly phagocytic disposal of dead/stressed cells by fully-differentiated macrophages, otherwise characterized by anti-inflammatory and antigen-presenting characteristics, hence facilitating lung tissue repair. In critical disease, CD4+ TH1- and CD8+ TEX-cells were characterized by inflammation-associated stress and metabolic exhaustion, while CD4+ TH17- and CD8+ TRM-cells failed to differentiate. Consequently, T-cell effector function was largely impaired thereby possibly facilitating excessive neutrophil-based inflammation. This was accompanied by impaired monocyte-to-macrophage differentiation, with monocytes exhibiting an ATP-purinergic signalling-inflammasome footprint, thereby enabling COVID-19 associated fibrosis and worsening disease severity. Our work represents a major resource for understanding the lung-localised immunity and inflammation landscape during COVID-19.