The RNA binding proteins ZFP36L1 and ZFP36L2 are dysregulated in airway epithelium in human and a murine model of asthma.

Introduction: Asthma is the most common chronic inflammatory disease of the airways. The airway epithelium is a key driver of the disease, and numerous studies have established genome-wide differences in mRNA expression between health and asthma. However, the underlying molecular mechanisms for such differences remain poorly understood. The human TTP family is comprised of ZFP36, ZFP36L1 and ZFP36L2, and has essential roles in immune regulation by determining the stability and translation of myriad mRNAs encoding for inflammatory mediators. We investigated the expression and possible role of the tristetraprolin (TTP) family of RNA binding proteins (RBPs), poorly understood in asthma. Methods: We analysed the levels of ZFP36, ZFP36L1 and ZFP36L2 mRNA in several publicly available asthma datasets, including single cell RNA-sequencing. We also interrogated the expression of known targets of these RBPs in asthma. We assessed the lung mRNA expression and cellular localization of Zfp36l1 and Zfp36l2 in precision cut lung slices in murine asthma models. Finally, we determined the expression in airway epithelium of ZFP36L1 and ZFP36L2 in human bronchial biopsies and performed rescue experiments in primary bronchial epithelium from patients with severe asthma. Results: We found ZFP36L1 and ZFP36L2 mRNA levels significantly downregulated in the airway epithelium of patients with very severe asthma in different cohorts (5 healthy vs. 8 severe asthma; 36 moderate asthma vs. 37 severe asthma on inhaled steroids vs. 26 severe asthma on oral corticoids). Integrating several datasets allowed us to infer that mRNAs potentially targeted by these RBPs are increased in severe asthma. Zfp36l1 was downregulated in the lung of a mouse model of asthma, and immunostaining of ex vivo lung slices with a dual antibody demonstrated that Zfp36l1/l2 nuclear localization was increased in the airway epithelium of an acute asthma mouse model, which was further enhanced in a chronic model. Immunostaining of human bronchial biopsies showed that airway epithelial cell staining of ZFP36L1 was decreased in severe asthma as compared with mild, while ZFP36L2 was upregulated. Restoring the levels of ZFP36L1 and ZFP36L2 in primary bronchial epithelial cells from patients with severe asthma decreased the mRNA expression of IL6, IL8 and CSF2. Discussion: We propose that the dysregulation of ZFP36L1/L2 levels as well as their subcellular mislocalization contributes to changes in mRNA expression and cytoplasmic fate in asthma.

Repair of Acute Respiratory Distress Syndrome in COVID-19 by Stromal Cells (REALIST-COVID Trial): A Multicenter, Randomized, Controlled Clinical Trial.

Rationale: Mesenchymal stromal cells (MSCs) may modulate inflammation, promoting repair in coronavirus disease (COVID-19)-related acute respiratory distress syndrome (ARDS). Objectives: We investigated the safety and efficacy of ORBCEL-C (CD362 [cluster of differentiation 362]-enriched, umbilical cord-derived MSCs) in COVID-19-related ARDS. Methods: In this multicenter, randomized, double-blind, allocation-concealed, placebo-controlled trial (NCT03042143), patients with moderate to severe COVID-19-related ARDS were randomized to receive ORBCEL-C (400 million cells) or placebo (Plasma-Lyte 148). The primary safety and efficacy outcomes were the incidence of serious adverse events and oxygenation index at Day 7, respectively. Secondary outcomes included respiratory compliance, driving pressure, PaO2:FiO2 ratio, and Sequential Organ Failure Assessment score. Clinical outcomes relating to duration of ventilation, lengths of ICU and hospital stays, and mortality were collected. Long-term follow-up included diagnosis of interstitial lung disease at 1 year and significant medical events and mortality at 2 years. Transcriptomic analysis was performed on whole blood at Days 0, 4, and 7. Measurements and Main Results: Sixty participants were recruited (final analysis: n = 30 received ORBCEL-C, n = 29 received placebo; 1 participant in the placebo group withdrew consent). Six serious adverse events occurred in the ORBCEL-C group and three in the placebo group (risk ratio, 2.9 [95% confidence interval, 0.6-13.2]; P = 0.25). Day 7 mean (SD) oxygenation index did not differ (ORBCEL-C, 98.3 [57.2] cm H2O/kPa; placebo, 96.6 [67.3] cm H2O/kPa). There were no differences in secondary surrogate outcomes or in mortality at Day 28, Day 90, 1 year, or 2 years. There was no difference in the prevalence of interstitial lung disease at 1 year or significant medical events up to 2 years. ORBCEL-C modulated the peripheral blood transcriptome. Conclusion: ORBCEL-C MSCs were safe in subjects with moderate to severe COVID-19-related ARDS but did not improve surrogates of pulmonary organ dysfunction.

Comparison of systemic inflammatory profiles in COVID-19 and community-acquired pneumonia patients: a prospective cohort study.

BACKGROUND: Inflammatory responses contribute to tissue damage in COVID-19 and community-acquired pneumonia (CAP). Although predictive values of different inflammatory biomarkers have been reported in both, similarities and differences of inflammatory profiles between these conditions remain uncertain. Therefore, we aimed to determine the similarities and differences of the inflammatory profiles between COVID-19 and CAP, and their association with clinical outcomes. METHODS: We report a prospective observational cohort study; conducted in a reference hospital in Latin America. Patients with confirmed COVID-19 pneumonia and CAP were included. Multiplex (Luminex) cytokine assays were used to measure the plasma concentration of 14 cytokines at hospital admission. After comparing similarities and differences in the inflammatory profile between COVID-19 and CAP patients, an unsupervised classification method (i.e., hierarchical clustering) was used to identify subpopulations within COVID-19 and CAP patients. RESULTS: A total of 160 patients were included, 62.5% were diagnosed with COVID-19 (100/160), and 37.5% with CAP (60/160). Using the hierarchical clustering, COVID-19 and CAP patients were divided based on its inflammatory profile: pauci, moderate, and hyper-inflammatory immune response. COVID-19 hyper-inflammatory subpopulation had the highest mortality. COVID-19 hyper-inflammatory subpopulation, compared to pauci-inflammatory, had higher levels of IL-10 (median [IQR] 61.4 [42.0-109.4] vs 13.0 [5.0-24.9], P: < 0.001), IL-6 (48.1 [22.3-82.6] vs 9.1 [0.1-30.4], P: < 0.001), among others. Hyper-inflammatory vs pauci-inflammatory CAP patients were characterized by elevation of IFN2 (48.8 [29.7-110.5] vs 3.0 [1.7-10.3], P: < 0.001), TNFα (36.3 [24.8-53.4] vs 13.1 [11.3-16.9], P: < 0.001), among others. Hyper-inflammatory subpopulations in COVID-19 and CAP compared to the corresponding pauci-inflammatory subpopulations had higher MCP-1. CONCLUSIONS: Our data highlights three distinct subpopulations in COVID-19 and CAP, with differences in inflammatory marker profiles and risks of adverse clinical outcomes. TRIAL REGISTRATION: This is a prospective study, therefore no health care intervention were applied to participants and trial registration is not applicable.

Highly Sensitive Lineage Discrimination of SARS-CoV-2 Variants through Allele-Specific Probe PCR.

Tools to detect SARS-CoV-2 variants of concern and track the ongoing evolution of the virus are necessary to support public health efforts and the design and evaluation of novel COVID-19 therapeutics and vaccines. Although next-generation sequencing (NGS) has been adopted as the gold standard method for discriminating SARS-CoV-2 lineages, alternative methods may be required when processing samples with low viral loads or low RNA quality. To this aim, an allele-specific probe PCR (ASP-PCR) targeting lineage-specific single nucleotide polymorphisms (SNPs) was developed and used to screen 1,082 samples from two clinical trials in the United Kingdom and Brazil. Probit regression models were developed to compare ASP-PCR performance against 1,771 NGS results for the same cohorts. Individual SNPs were shown to readily identify specific variants of concern. ASP-PCR was shown to discriminate SARS-CoV-2 lineages with a higher likelihood than NGS over a wide range of viral loads. The comparative advantage for ASP-PCR over NGS was most pronounced in samples with cycle threshold (CT) values between 26 and 30 and in samples that showed evidence of degradation. Results for samples screened by ASP-PCR and NGS showed 99% concordant results. ASP-PCR is well suited to augment but not replace NGS. The method can differentiate SARS-CoV-2 lineages with high accuracy and would be best deployed to screen samples with lower viral loads or that may suffer from degradation. Future work should investigate further destabilization from primer-target base mismatch through altered oligonucleotide chemistry or chemical additives.

Virological Characterization of Critically Ill Patients With COVID-19 in the United Kingdom: Interactions of Viral Load, Antibody Status, and B.1.1.7 Infection.

BACKGROUND: Convalescent plasma containing neutralizing antibody to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is under investigation for coronavirus disease 2019 (COVID-19) treatment. We report diverse virological characteristics of UK intensive care patients enrolled in the Immunoglobulin Domain of the REMAP-CAP randomized controlled trial that potentially influence treatment outcomes. METHODS: SARS-CoV-2 RNA in nasopharyngeal swabs collected pretreatment was quantified by PCR. Antibody status was determined by spike-protein ELISA. B.1.1.7 was differentiated from other SARS-CoV-2 strains using allele-specific probes or restriction site polymorphism (SfcI) targeting D1118H. RESULTS: Of 1274 subjects, 90% were PCR positive with viral loads 118-1.7 × 1011IU/mL. Median viral loads were 40-fold higher in those IgG seronegative (n = 354; 28%) compared to seropositives (n = 939; 72%). Frequencies of B.1.1.7 increased from <1% in November 2020 to 82% of subjects in January 2021. Seronegative individuals with wild-type SARS-CoV-2 had significantly higher viral loads than seropositives (medians 5.8 × 106 and 2.0 × 105 IU/mL, respectively; P = 2 × 10-15). CONCLUSIONS: High viral loads in seropositive B.1.1.7-infected subjects and resistance to seroconversion indicate less effective clearance by innate and adaptive immune responses. SARS-CoV-2 strain, viral loads, and antibody status define subgroups for analysis of treatment efficacy.