Protease-Anti-Protease Compartmentalization in SARS-CoV-2 ARDS: Therapeutic Implications

Rationale Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is characterised by an IL-6 driven cytokinemia, associated with a rapidly developing acute respiratory distress syndrome (ARDS). A blunted AAT response to IL-6 in SARS-CoV-2 has been associated with increased morbidity and mortality. One of the main functions of IL-6 is regulation of acute-phase proteins such as alpha-1 antitrypsin (AAT), a key lung anti-protease. We investigated the proteaseanti- protease balance in the circulation and pulmonary compartments in SARS-CoV-2 acute respiratory distress syndrome (ARDS). In addition, we investigated the effect of anti-IL-6 therapy on anti-protease defence. Methods Levels and activity of AAT and neutrophil elastase (NE) were measured in plasma (n=20), airway tissue (n=8) and tracheal secretions (n=13) of people with severe SARS-CoV-2 infection. AAT and IL-6 levels were also evaluated over time in people with moderate SARS-CoV-2 infection who received standard of care +/- tocilizumab (n=30). Results AAT plasma levels doubled in severe SARS-CoV-2 ARDS patients (329g/L +/- 08 g/L as compared to baseline levels 174g/L +/- 011 g/L, P<0001). In lung parenchyma AAT levels were increased. Despite no increase in neutrophils, an increased percentage of neutrophils involved in NET formation were observed in the alveoli. A protease-anti-protease imbalance was detected in tracheal aspirates (TA). NE was active and AAT inactivated, reflecting cleavage and complexation with NE. The major airway anti-protease, secretory leukoprotease inhibitor (SLPI) was decreased in SARS-CoV-2-infected lungs and cleaved in TAs. Induction of AAT in SARS-CoV-2 infection occurred mainly through IL-6 signalling. Tocilizumab (IL-6 receptor antagonist) down-regulated AAT during infection (13g/L+/-0225 from 2469 g/L+/-0197, P<00001) while IL-6 remained elevated (NS=0.0998) as reflected by the IL-6/AAT ratio (P=0046). Conclusion This study shows that the AAT response to SARS-CoV-2 infection is compartmentalized with an appropriate increase in plasma and alveoli but an inadequate response in airways. This underlines a significant, but potentially treatable, protease-antiprotease imbalance in SARS-CoV-2 ARDS as well as highlighting IL-6's importance in SARS-CoV-2 pathology not only as a pro-inflammatory cytokine but as an anti-inflammatory regulator. In conclusion there is unopposed NE activity in the airways of people with SARS-CoV-2 ARDS which could be amenable to AAT therapy. Our data suggest caution in the use of IL-6 blocking therapies in SARS-CoV-2-infected individuals.

The clinical impact of maternal COVID-19 on mothers, their infants, and placentas with an analysis of vertical transfer of maternal SARS-CoV-2-specific IgG antibodies.

INTRODUCTION: The effect of SARS-CoV-2 severity or the trimester of infection in pregnant mothers, placentas, and infants is not fully understood. METHODS: A retrospective, observational cohort study in Chapel Hill, NC of 115 mothers with SARS-CoV-2 and singleton pregnancies from December 1, 2019 to May 31, 2021 via chart review to document the infants' weight, length, head circumference, survival, congenital abnormalities, hearing loss, maternal complications, and placental pathology classified by the Amsterdam criteria. RESULTS: Of the 115 mothers, 85.2% were asymptomatic (n = 37) or had mild (n = 61) symptoms, 13.0% had moderate (n = 9) or severe (n = 6) COVID-19, and 1.74% (n = 2) did not have symptoms recorded. Moderate and severe maternal infections were associated with increased C-section, premature delivery, infant NICU admission, and were more likely to occur in Type 1 (p = 0.0055) and Type 2 (p = 0.0285) diabetic mothers. Only one infant (0.870%) became infected with SARS-CoV-2, which was not via the placenta. Most placentas (n = 63, 54.8%) did not show specific histologic findings; however, a subset showed mild maternal vascular malperfusion (n = 26, 22.6%) and/or mild microscopic ascending intrauterine infection (n = 28, 24.3%). The infants had no identifiable congenital abnormalities, and all infants and mothers survived. DISCUSSION: Most mothers and their infants had a routine clinical course; however, moderate and severe COVID-19 maternal infections were associated with pregnancy complications and premature delivery. Mothers with pre-existing, non-gestational diabetes were at greatest risk of developing moderate or severe COVID-19. The placental injury patterns of maternal vascular malperfusion and/or microscopic ascending intrauterine infection were not associated with maternal COVID-19 severity.

Proteome Characterictics of Endotracheal Tube Washes from COVID-19 ICU Patients: Assessing the Host Response and Understanding the Pathways Involved in Generation of Severe Disease

RATIONALE There is a great need to understand the pathogenesis of COVID-19, to predict the host responses to COVID-19 infection, and to efficiently assess clinical trial data for effectiveness. Data describing the sequence and predictability of progression of COVID-19 lung disease are not available, nor are biomarkers available to estimate lung damage. Using a proteomic approach, we measured the composition of tracheal aspirate of intubated COVID-19 positive subject and compared to COVID-19 negative ARDS subjects. METHODS EndoTracheal Tube (ETT) washes from critically ill 20 COVID-19+ patients and from 13 COVID19 negative ARDS patients were obtained from UNC COVID-19 Biospecimens Core. This BSL2+ facility obtained ICU samples and treated them with 8M urea to sterilize pathogens and solubilize the samples. Samples were normalized by total biomolecular concentration by SEC-MALLS dRI and prepared with filter aided sample preparation for mass spectrometry based label free quantiative proteome analyses. Proteins were quantified using Scaffold software (normalized total precursor intensity). Unpaired, non-parametric, Mann-Whitney test was performed in GraphPad Prism 9.0.0. RESULTS More than 1,200 proteins were identified. Of these, 209 were unique to COVID-19 samples. Of 823 proteins common to disease controls, the level of 32 proteins were decreased, and 10 proteins incresased in the in COVID-19 samples comparing to non-COVID controls (p value <0.05). Proteins significantly reduced in COVID-19 were mostly in the innate immune category including SFTPA1, BPIB1, PIGRS, and ezrin. Acute-phase proteins (alpha 1 acid glycoprotein 1, prothrombin), proteins related to iron homeostasis (serotransferrin, hemopexin), and complement pathway proteins (Complement C9 and vitronectin) were significantly increased in the COVID-19 lung. Pathways analysis indicated that acute phase signalling and LXR/RXR pathway were activated, while the glycolysis I pathway was inhibited in the COVID-19 group CONCLUSIONS These data reveal several important aspects of COVID-19-induced lung injury: 1) TNF-alpha and/or IL-6 pathways are involved;2) the pulmonary epithelia are severely injured as acute phase serum components including albumin and serotransferrin, are excessively leaking into the lung from serum;3) Activation of LXR/RXR pathways in the macrophages and the inhibition of glycolysis pathway could be an important pathological outcome of the severe form of COVID-19. These data help elucidate cell-type-specific host responses and identify these pathways and their roles in generation of disease sequelae useful in the development of new therapies to reduce virus-relate injury and/or improve resolution of injury.