Alternative Splicing Is a Major Factor Shaping Transcriptome Diversity in Mild and Severe Chronic Obstructive Pulmonary Disease.

The role of alternative splicing in chronic obstructive pulmonary disease (COPD) is still largely unknown. We aimed to investigate the differences in alternatively splicing events between patients with mild-to-moderate and severe COPD compared with non-COPD control subjects and to identify splicing factors associated with aberrant alternative splicing in COPD. For this purpose, we performed genome-wide RNA-sequencing analysis of bronchial brushings from 23 patients with mild-to-moderate COPD, 121 with severe COPD, and 23 non-COPD control subjects. We found a significant difference in the frequency of alternative splicing events in patients with mild-to-moderate and severe COPD compared with non-COPD control subjects. There were from two to eight times (depending on event type) more differential alternative splicing events in the severe than in the mild-to-moderate stage. The severe COPD samples showed less intron retention and more exon skipping. It is interesting that the transcript levels of the top 10 differentially expressed splicing factors were significantly correlated with the percentage of many alternatively spliced transcripts in severe COPD. The aberrant alternative splicing in severe COPD was predicted to increase the overall protein-coding capacity of gene products. In conclusion, we observed large and significant differences in alternative splicing between bronchial samples of patients with COPD and control subjects, with more events observed in severe than in mild-to-moderate COPD. The changes in the expression of several splicing factors correlated with prevalence of alternative splicing in severe COPD. Alternative splicing can indirectly impact gene expression by changing the relative abundance of protein-coding isoforms potentially influencing pathophysiological changes. The results provide a better understanding of COPD-related alternative splicing changes.

Astegolimab or Efmarodocokin Alfa in Patients With Severe COVID-19 Pneumonia: A Randomized, Phase 2 Trial.

OBJECTIVES: Severe cases of COVID-19 pneumonia can lead to acute respiratory distress syndrome (ARDS). Release of interleukin (IL)-33, an epithelial-derived alarmin, and IL-33/ST2 pathway activation are linked with ARDS development in other viral infections. IL-22, a cytokine that modulates innate immunity through multiple regenerative and protective mechanisms in lung epithelial cells, is reduced in patients with ARDS. This study aimed to evaluate safety and efficacy of astegolimab, a human immunoglobulin G2 monoclonal antibody that selectively inhibits the IL-33 receptor, ST2, or efmarodocokin alfa, a human IL-22 fusion protein that activates IL-22 signaling, for treatment of severe COVID-19 pneumonia. DESIGN: Phase 2, double-blind, placebo-controlled study (COVID-astegolimab-IL). SETTING: Hospitals. PATIENTS: Hospitalized adults with severe COVID-19 pneumonia. INTERVENTIONS: Patients were randomized to receive IV astegolimab, efmarodocokin alfa, or placebo, plus standard of care. The primary endpoint was time to recovery, defined as time to a score of 1 or 2 on a 7-category ordinal scale by day 28. MEASUREMENTS AND MAIN RESULTS: The study randomized 396 patients. Median time to recovery was 11 days (hazard ratio [HR], 1.01 d; p = 0.93) and 10 days (HR, 1.15 d; p = 0.38) for astegolimab and efmarodocokin alfa, respectively, versus 10 days for placebo. Key secondary endpoints (improved recovery, mortality, or prevention of worsening) showed no treatment benefits. No new safety signals were observed and adverse events were similar across treatment arms. Biomarkers demonstrated that both drugs were pharmacologically active. CONCLUSIONS: Treatment with astegolimab or efmarodocokin alfa did not improve time to recovery in patients with severe COVID-19 pneumonia.

Early plasma angiopoietin-2 is prognostic for ARDS and mortality among critically ill patients with sepsis.

Angiopoietin-2 (Ang-2) is associated with vascular endothelial injury and permeability in the acute respiratory distress syndrome (ARDS) and sepsis. Elevated circulating Ang-2 levels may identify critically ill patients with distinct pathobiology amenable to targeted therapy. We hypothesized that plasma Ang-2 measured shortly after hospitalization among patients with sepsis would be associated with the development of ARDS and poor clinical outcomes. To test this hypothesis, we measured plasma Ang-2 in a cohort of 757 patients with sepsis, including 267 with ARDS, enrolled in the emergency department or early in their ICU course before the COVID-19 pandemic. Multivariable models were used to test the association of Ang-2 with the development of ARDS and 30-day morality. We found that early plasma Ang-2 in sepsis was associated with higher baseline severity of illness, the development of ARDS, and mortality risk. The association between Ang-2 and mortality was strongest among patients with ARDS and sepsis as compared to those with sepsis alone (OR 1.81 vs. 1.52 per log Ang-2 increase). These findings might inform models testing patient risk prediction and strengthen the evidence for Ang-2 as an appealing biomarker for patient selection for novel therapeutic agents to target vascular injury in sepsis and ARDS.

Prognostic and Predictive Biomarkers in Patients With Coronavirus Disease 2019 Treated With Tocilizumab in a Randomized Controlled Trial.

OBJECTIVES: To explore candidate prognostic and predictive biomarkers identified in retrospective observational studies (interleukin-6, C-reactive protein, lactate dehydrogenase, ferritin, lymphocytes, monocytes, neutrophils, d-dimer, and platelets) in patients with coronavirus disease 2019 pneumonia after treatment with tocilizumab, an anti-interleukin-6 receptor antibody, using data from the COVACTA trial in patients hospitalized with severe coronavirus disease 2019 pneumonia. DESIGN: Exploratory analysis from a multicenter, randomized, double-blind, placebo-controlled, phase 3 trial. SETTING: Hospitals in North America and Europe. PATIENTS: Adults hospitalized with severe coronavirus disease 2019 pneumonia receiving standard care. INTERVENTION: Randomly assigned 2:1 to IV tocilizumab 8 mg/kg or placebo. MEASUREMENTS AND MAIN RESULTS: Candidate biomarkers were measured in 295 patients in the tocilizumab arm and 142 patients in the placebo arm. Efficacy outcomes assessed were clinical status on a seven-category ordinal scale (1, discharge; 7, death), mortality, time to hospital discharge, and mechanical ventilation (if not receiving it at randomization) through day 28. Prognostic and predictive biomarkers were evaluated continuously with proportional odds, binomial or Fine-Gray models, and additional sensitivity analyses. Modeling in the placebo arm showed all candidate biomarkers except lactate dehydrogenase and d-dimer were strongly prognostic for day 28 clinical outcomes of mortality, mechanical ventilation, clinical status, and time to hospital discharge. Modeling in the tocilizumab arm showed a predictive value of ferritin for day 28 clinical outcomes of mortality (predictive interaction, p = 0.03), mechanical ventilation (predictive interaction, p = 0.01), and clinical status (predictive interaction, p = 0.02) compared with placebo. CONCLUSIONS: Multiple biomarkers prognostic for clinical outcomes were confirmed in COVACTA. Ferritin was identified as a predictive biomarker for the effects of tocilizumab in the COVACTA patient population; high ferritin levels were associated with better clinical outcomes for tocilizumab compared with placebo at day 28.

Lysophosphatidic acid species are associated with exacerbation in chronic obstructive pulmonary disease.

BACKGROUND: Chronic obstructive pulmonary disease (COPD) exacerbations are heterogenous and profoundly impact the disease trajectory. Bioactive lipid lysophosphatidic acid (LPA) has been implicated in airway inflammation but the significance of LPA in COPD exacerbation is not known. The aim of the study was to investigate the utility of serum LPA species (LPA16:0, 18:0, 18:1, 18:2, 20:4) as biomarkers of COPD exacerbation. PATIENTS AND METHODS: LPA species were measured in the baseline placebo sera of a COPD randomized controlled trial. Tertile levels of each LPA were used to assign patients into biomarker high, medium, and low subgroups. Exacerbation rate and risk were compared among the LPA subgroups. RESULTS: The levels of LPA species were intercorrelated (rho 0.29-0.91). Patients with low and medium levels of LPA (LPA16:0, 20:4) had significantly higher exacerbation rate compared to the respective LPA-high patients [estimated rate per patient per year (95% CI)]: LPA16:0-low = 1.2 (0.8-1.9) (p = 0.019), LPA16:0-medium = 1.3 (0.8-2.0) (p = 0.013), LPA16:0-high = 0.5 (0.2-0.9); LPA20:4-low = 1.4 (0.9-2.1) (p = 0.0033), LPA20:4-medium = 1.2 (0.8-1.8) (p = 0.0089), LPA20:4-high = 0.4 (0.2-0.8). These patients also had earlier time to first exacerbation (hazard ratio (95% CI): LPA16:0-low = 2.6 (1.1-6.0) (p = 0.028), LPA16:0-medium = 2.7 (1.2-6.3) (p = 0.020); LPA20.4-low = 2.8 (1.2-6.6) (p = 0.017), LPA20:4-medium = 2.7 (1.2-6.4) (p = 0.021). Accordingly, these patients had a significant increased exacerbation risk compared to the respective LPA-high subgroups [odd ratio (95% CI)]: LPA16:0-low = 3.1 (1.1-8.8) (p = 0.030), LPA16:0-medium = 3.0 (1.1-8.3) (p = 0.031); LPA20:4-low = 3.8 (1.3-10.9) (p = 0.012), LPA20:4-medium = 3.3 (1.2-9.5) (p = 0.025). For the other LPA species (LPA18:0, 18:1, 18:2), the results were mixed; patients with low and medium levels of LPA18:0 and 18:2 had increased exacerbation rate, but only LPA18:0-low patients had significant increase in exacerbation risk and earlier time to first exacerbation compared to the LPA18:0-high subgroup. CONCLUSIONS: The study provided evidence of association between systemic LPA levels and exacerbation in COPD. Patients with low and medium levels of specific LPA species (LPA16:0, 20:4) had increased exacerbation rate, risk, and earlier time to first exacerbation. These non-invasive biomarkers may aid in identifying high risk patients with dysregulated LPA pathway to inform risk management and drug development.

A Prognostic Model of Persistent Bacteremia and Mortality in Complicated Staphylococcus aureus Bloodstream Infection.

BACKGROUND: Staphylococcus aureus is a leading cause of bacteremia, yet there remains a significant knowledge gap in the identification of relevant biomarkers that predict clinical outcomes. Heterogeneity in the host response to invasive S. aureus infection suggests that specific biomarker signatures could be utilized to differentiate patients prone to severe disease, thereby facilitating earlier implementation of more aggressive therapies. METHODS: To further elucidate the inflammatory correlates of poor clinical outcomes in patients with S. aureus bacteremia, we evaluated the association between a panel of blood proteins at initial presentation of bacteremia and disease severity outcomes using 2 cohorts of patients with S. aureus bacteremia (n = 32 and n = 124). RESULTS: We identified 13 candidate proteins that were correlated with mortality and persistent bacteremia. Prognostic modeling identified interleukin (IL)-8 and CCL2 as the strongest individual predictors of mortality, with the combination of these biomarkers classifying fatal outcome with 89% sensitivity and 77% specificity (P < .0001). Baseline IL-17A levels were elevated in patients with persistent bacteremia (P < .0001), endovascular (P = .026) and metastatic tissue infections (P = .012). CONCLUSIONS: These results demonstrate the potential utility of selected biomarkers to distinguish patients with the highest risk for treatment failure and bacteremia-related complications, providing a valuable tool for clinicians in the management of S. aureus bacteremia. Additionally, these biomarkers could identify patients with the greatest potential to benefit from novel therapies in clinical trials.

miR-144 attenuates the host response to influenza virus by targeting the TRAF6-IRF7 signaling axis.

Antiviral responses must rapidly defend against infection while minimizing inflammatory damage, but the mechanisms that regulate the magnitude of response within an infected cell are not well understood. miRNAs are small non-coding RNAs that suppress protein levels by binding target sequences on their cognate mRNA. Here, we identify miR-144 as a negative regulator of the host antiviral response. Ectopic expression of miR-144 resulted in increased replication of three RNA viruses in primary mouse lung epithelial cells: influenza virus, EMCV, and VSV. We identified the transcriptional network regulated by miR-144 and demonstrate that miR-144 post-transcriptionally suppresses TRAF6 levels. In vivo ablation of miR-144 reduced influenza virus replication in the lung and disease severity. These data suggest that miR-144 reduces the antiviral response by attenuating the TRAF6-IRF7 pathway to alter the cellular antiviral transcriptional landscape.

Evaluation of IFITM3 rs12252 Association With Severe Pediatric Influenza Infection.

Background: Interferon-induced transmembrane protein 3 (IFITM3) restricts endocytic fusion of influenza virus. IFITM3 rs12252_C, a putative alternate splice site, has been associated with influenza severity in adults. IFITM3 has not been evaluated in pediatric influenza. Methods: The Pediatric Influenza (PICFLU) study enrolled children with suspected influenza infection across 38 pediatric intensive care units during November 2008 to April 2016. IFITM3 was sequenced in patients and parents were genotyped for specific variants for family-based association testing. rs12252 was genotyped in 54 African-American pediatric outpatients with influenza (FLU09), included in the population-based comparisons with 1000 genomes. Splice site analysis of rs12252_C was performed using PICFLU and FLU09 patient RNA. Results: In PICFLU, 358 children had influenza infection. We identified 22 rs12252_C homozygotes in 185 white non-Hispanic children. rs12252_C was not associated with influenza infection in population or family-based analyses. We did not identify the Δ21 IFITM3 isoform in RNAseq data. The rs12252 genotype was not associated with IFITM3 expression levels, nor with critical illness severity. No novel rare IFITM3 functional variants were identified. Conclusions: rs12252 was not associated with susceptibility to influenza-related critical illness in children or with critical illness severity. Our data also do not support it being a splice site.

Phase 2 Randomized Trial of the Safety and Efficacy of MHAA4549A, a Broadly Neutralizing Monoclonal Antibody, in a Human Influenza A Virus Challenge Model.

MHAA4549A, a human monoclonal antibody targeting the hemagglutinin stalk region of influenza A virus (IAV), is being developed as a therapeutic for patients hospitalized with severe IAV infection. The safety and efficacy of MHAA4549A were assessed in a randomized, double-blind, placebo-controlled, dose-ranging study in a human IAV challenge model. One hundred healthy volunteers were inoculated with A/Wisconsin/67/2005 (H3N2) IAV and, 24 to 36 h later, administered a single intravenous dose of either placebo, MHAA4549A (400, 1,200, or 3,600 mg), or a standard oral dose of oseltamivir. Subjects were assessed for safety, pharmacokinetics (PK), and immunogenicity. The intent-to-treat-infected (ITTI) population was assessed for changes in viral load, influenza symptoms, and inflammatory biomarkers. MHAA4549A was well tolerated in all IAV challenge subjects. The 3,600-mg dose of MHAA4549A significantly reduced the viral burden relative to that of the placebo as determined by the area under the curve (AUC) of nasopharyngeal virus infection, quantified using quantitative PCR (98%) and 50% tissue culture infective dose (TCID50) (100%) assays. Peak viral load, duration of viral shedding, influenza symptom scores, mucus weight, and inflammatory biomarkers were also reduced. Serum PK was linear with a half-life of ∼23 days. No MHAA4549A-treated subjects developed anti-drug antibodies. In conclusion, MHAA4549A was well tolerated and demonstrated statistically significant and substantial antiviral activity in an IAV challenge model. (This study has been registered at ClinicalTrials.gov under identifier NCT01980966.).

Phase 2 Randomized, Double-Blind, Placebo-Controlled Trial of RG7667, a Combination Monoclonal Antibody, for Prevention of Cytomegalovirus Infection in High-Risk Kidney Transplant Recipients.

Cytomegalovirus (CMV) infection is a significant complication after kidney transplantation. We examined the ability of RG7667, a combination of two monoclonal antibodies, to prevent CMV infection in high-risk kidney transplant recipients in a randomized, double-blind, placebo-controlled trial. CMV-seronegative recipients of a kidney transplant from a CMV-seropositive donor (D+R-) were randomized to receive RG7667 (n = 60) or placebo (n = 60) at the time of transplant and 1, 4, and 8 weeks posttransplant. Patients were monitored for CMV viremia every 1 to 2 weeks posttransplant for 24 weeks. Patients who had seroconverted (D+R+) or withdrawn before dosing were excluded from the analysis (n = 4). CMV viremia occurred in 27 of 59 (45.8%) patients receiving RG7667 and 35 of 57 (61.4%) patients receiving placebo (stratum-adjusted difference, 15.3%; P = 0.100) within 12 weeks posttransplant and in 30 of 59 (50.8%) patients receiving RG7667 and 40 of 57 (70.2%) patients receiving placebo (stratum-adjusted difference, 19.3%; P = 0.040) within 24 weeks posttransplant. Median time to CMV viremia was 139 days in patients receiving RG7667 compared to 46 days in patients receiving placebo (hazard ratio, 0.53; P = 0.009). CMV disease was less common in the RG7667 than placebo group (3.4% versus 15.8%; P = 0.030). Adverse events were generally balanced between treatment groups. In high-risk kidney transplant recipients, RG7667 was well tolerated, numerically reduced the incidence of CMV infection within 12 and 24 weeks posttransplant, delayed time to CMV viremia, and was associated with less CMV disease than the placebo. (This study has been registered at ClinicalTrials.gov under registration no. NCT01753167.).

Generation and Protective Ability of Influenza Virus-Specific Antibody-Dependent Cellular Cytotoxicity in Humans Elicited by Vaccination, Natural Infection, and Experimental Challenge.

BACKGROUND: Nonneutralizing antibodies (Abs) involved in antibody-dependent cellular cytotoxicity (ADCC) may provide some protection from influenza virus infection. The ability of influenza vaccines to induce ADCC-mediating Abs (ADCC-Abs) in adults and children is unclear. METHODS: We quantified ADCC-Abs in serum samples from adults who received a dose of inactivated subunit vaccine (ISV) targeting monovalent 2009 pandemic influenza A(H1N1) virus or live-attenuated influenza vaccine (LAIV) or who had laboratory-confirmed influenza A(H1N1) virus infection. We also measured ADCC-Abs in children who either received a dose of trivalent seasonal ISV followed by trivalent seasonal LAIV or 2 doses of LAIV. Finally, we assessed the ability of low and high ADCC-Ab titers to protect adults from experimental challenge with influenza A/Wisconsin/67/131/2005(H3N2) virus. RESULTS: Adults and children who received a dose of ISV had a robust increase in ADCC-Ab titers to both recombinant hemagglutinin (rHA) protein and homologous virus-infected cells. There was no detectable increase in titers of ADCC-Abs to rHA or virus-infected cells in adults and children who received LAIV. Higher titers (≥320) of preexisting ADCC-Abs were associated with lower virus replication and a significant reduction in total symptom scores in experimentally infected adults. CONCLUSIONS: ADCC-Ab titers increased following experimental influenza virus infection in adults and after ISV administration in both children and adults.

Characterization of innate responses to influenza virus infection in a novel lung type I epithelial cell model.

Type I alveolar epithelial cells are a replicative niche for influenza in vivo, yet their response to infection is not fully understood. To better characterize their cellular responses, we have created an immortalized murine lung epithelial type I cell line (LET1). These cells support spreading influenza virus infection in the absence of exogenous protease and thus permit simultaneous analysis of viral replication dynamics and host cell responses. LET1 cells can be productively infected with human, swine and mouse-adapted strains of influenza virus and exhibit expression of an antiviral transcriptional programme and robust cytokine secretion. We characterized influenza virus replication dynamics and host responses of lung type I epithelial cells and identified the capacity of epithelial cell-derived type I IFN to regulate specific modules of antiviral effectors to establish an effective antiviral state. Together, our results indicate that the type I epithelial cell can play a major role in restricting influenza virus infection without contribution from the haematopoietic compartment.

miR-451 regulates dendritic cell cytokine responses to influenza infection.

MicroRNAs (miRNAs) are important posttranscriptional regulators in immune cells, but how viral infection regulates miRNA expression to shape dendritic cell (DC) responses has not been well characterized. We identified 20 miRNAs that were differentially expressed in primary murine DCs in response to the dsRNA agonist polyinosinic-polycytidylic acid, a subset of which were modestly regulated by influenza infection. miR-451 was unique because it was induced more strongly in primary splenic and lung DCs by live viral infection than by purified agonists of pattern recognition receptors. We determined that miR-451 regulates a subset of proinflammatory cytokine responses. Three types of primary DCs treated with antisense RNA antagomirs directed against miR-451 secreted elevated levels of IL-6, TNF, CCL5/RANTES, and CCL3/MIP1α, and these results were confirmed using miR-451(null) cells. miR-451 negatively regulates YWHAZ/14-3-3ζ protein levels in various cell types, and we measured a similar inhibition of YWHAZ levels in DCs. It is known that YWHAZ can control the activity of two negative regulators of cytokine production: FOXO3, which is an inhibitory transcription factor, and ZFP36/Tristetraprolin, which binds to AU-rich elements within 3'-untranslated regions to destabilize cytokine mRNAs. Inhibition of miR-451 expression correlated with increased YWHAZ protein expression and decreased ZFP36 expression, providing a possible mechanism for the elevated secretion of IL-6, TNF, CCL5/RANTES, and CCL3/MIP1α. miR-451 levels are themselves increased by IL-6 and type I IFN, potentially forming a regulatory loop. These data suggest that viral infection specifically induces a miRNA that directs a negative regulatory cascade to tune DC cytokine production.

Prognostic value of severe acute respiratory syndrome coronavirus-2 viral load and antibodies in patients hospitalized with COVID-19.

Observational studies have identified the potential prognostic value for severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) viral load and anti-SARS-CoV-2 antibodies in coronavirus disease 2019 (COVID-19). However, viral load in nasopharyngeal (NP) swabs produced inconsistent results in prognostic analyses, and the prognostic value of viral load or antibodies has not been confirmed in large clinical trials. COVACTA and REMDACTA were double-blind, randomized, controlled trials with a combined enrollment of 1078 patients hospitalized with COVID-19 treated with tocilizumab or placebo in COVACTA or tocilizumab plus remdesivir or placebo plus remdesivir in REMDACTA. We assessed the potential prognostic value of NP and serum SARS-CoV-2 viral load and serum anti-SARS-CoV-2 antibodies at baseline as biomarkers for clinical outcomes in patients enrolled in these trials. In adjusted Cox proportional hazard models, serum viral load was a more reliable predictor of clinical outcomes than NP viral load; high serum viral load was associated with higher risk for death and mechanical ventilation/death and lower likelihood of hospital discharge (high vs. negative viral load hazard ratios [95% confidence interval {CI}] were 2.87 [1.57-5.25], 3.86 [2.23-6.68], and 0.23 [0.14-0.36], respectively, in COVACTA and 8.11 [2.95-22.26], 10.29 [4.5-23.55], and 0.21 [0.15-0.29], respectively, in REMDACTA) and high serum viral load correlated with levels of inflammatory cytokines and lung damage biomarkers. High anti-SARS-CoV-2 spike protein antibody (ACOV2S) levels were associated with higher likelihood of hospital discharge (high vs. below the limit of quantification hazard ratios [95% CI] were 2.55 [1.59-4.08] for COVACTA and 1.54 [1.13-2.09] for REMDACTA). These results support the role of baseline SARS-CoV-2 serum viral load and ACOV2S antibody titers in predicting clinical outcomes for patients hospitalized with COVID-19.

Transcriptomic and proteomic assessment of tocilizumab response in a randomized controlled trial of patients hospitalized with COVID-19.

High interleukin (IL)-6 levels are associated with greater COVID-19 severity. IL-6 receptor blockade by tocilizumab (anti-IL6R; Actemra) is used globally for the treatment of severe COVID-19, yet a molecular understanding of the therapeutic benefit remains unclear. We characterized the immune profile and identified cellular and molecular pathways modified by tocilizumab in peripheral blood samples from patients enrolled in the COVACTA study, a phase 3, randomized, double-blind, placebo-controlled trial of the efficacy and safety of tocilizumab in hospitalized patients with severe COVID-19. We identified markers of inflammation, lymphopenia, myeloid dysregulation, and organ injury that predict disease severity and clinical outcomes. Proteomic analysis confirmed a pharmacodynamic effect for tocilizumab and identified novel pharmacodynamic biomarkers. Transcriptomic analysis revealed that tocilizumab treatment leads to faster resolution of lymphopenia and myeloid dysregulation associated with severe COVID-19, indicating greater anti-inflammatory activity relative to placebo and potentially leading to faster recovery in patients hospitalized with COVID-19.

A bronchial gene signature specific for severe COPD that is retained in the nose.

Introduction: A subset of COPD patients develops advanced disease with severe airflow obstruction, hyperinflation and extensive emphysema. We propose that the pathogenesis in these patients differs from mild-moderate COPD and is reflected by bronchial gene expression. The aim of the present study was to identify a unique bronchial epithelial gene signature for severe COPD patients. Methods: We obtained RNA sequencing data from bronchial brushes from 123 ex-smokers with severe COPD, 23 with mild-moderate COPD and 23 non-COPD controls. We identified genes specific to severe COPD by comparing severe COPD to non-COPD controls, followed by removing genes that were also differentially expressed between mild-moderate COPD and non-COPD controls. Next, we performed a pathway analysis on these genes and evaluated whether this signature is retained in matched nasal brushings. Results: We identified 219 genes uniquely differentially expressed in severe COPD. Interaction network analysis identified VEGFA and FN1 as the key genes with the most interactions. Genes were involved in extracellular matrix regulation, collagen binding and the immune response. Of interest were 10 genes (VEGFA, DCN, SPARC, COL6A2, MGP, CYR61, ANXA6, LGALS1, C1QA and C1QB) directly connected to fibronectin 1 (FN1). Most of these genes were lower expressed in severe COPD and showed the same effect in nasal brushings. Conclusions: We found a unique severe COPD bronchial gene signature with key roles for VEGFA and FN1, which was retained in the upper airways. This supports the hypothesis that severe COPD, at least partly, comprises a different pathology and supports the potential for biomarker development based on nasal brushes in COPD.

Surfactant protein D is a biomarker of influenza-related pediatric lung injury.

BACKGROUND: Biomarkers that can risk-stratify children with influenza virus lower respiratory infection may identify patients for targeted intervention. Early elevation of alveolar-related proteins in the bloodstream in these patients could indicate more severe lung damage portending worse outcomes. METHODS: We used a mouse model of human influenza infection and evaluated relationships between lung pathophysiology and surfactant protein D (SP-D), SP-A, and Club cell protein 16 (CC16). We then measured SP-A, SP-D, and CC16 levels in plasma samples from 94 children with influenza-associated acute respiratory failure (PICFLU cohort), excluding children with underlying conditions explaining disease severity. We tested for associations between levels of circulating proteins and disease severity including the diagnosis of acute respiratory distress syndrome (ARDS), mechanical ventilator, intensive care unit and hospital days, and hospital mortality. RESULTS: Circulating SP-D showed a greater increase than SP-A and CC16 in mice with increased alveolar-vascular permeability following influenza infection. In the PICFLU cohort, SP-D was associated with moderate-severe ARDS diagnosis (p = 0.01) and with mechanical ventilator (r = 0.45, p = 0.002), ICU (r = 0.44, p = 0.002), and hospital days (r = 0.37, p = 0.001) in influenza-infected children without bacterial coinfection. Levels of SP-D were lower in children with secondary bacterial pneumonia (p = 0.01) and not associated with outcomes. CC16 and SP-A levels did not differ with bacterial coinfection and were not consistently associated with severe outcomes. CONCLUSIONS: SP-D has potential as an early circulating biomarker reflecting a degree of lung damage caused directly by influenza virus infection in children. Secondary bacterial pneumonia alters SP-D biomarker performance.

ATF3 regulates MCMV infection in mice by modulating IFN-gamma expression in natural killer cells.

Activating transcription factor 3 (ATF3) is a negative regulator of proinflammatory cytokine expression in macrophages, and ATF3-deficient mice are more susceptible to endotoxic shock. Here, we demonstrate that ATF3 interacts with a cis-regulatory element of the IFN-gamma gene in natural killer (NK) cells, and that ATF3null NK cells show increased transcription and secretion of IFN-gamma. NK cell-derived IFN-gamma has previously been demonstrated to be protective against murine cytomegalovirus (MCMV) infection, and we show here that ATF3null mice exhibit decreased hepatic viral load and reduced liver histopathology upon challenge with MCMV. Reconstitution of NK-deficient mice with ATF3null NK cells more effectively controlled MCMV infection than mice reconstituted with WT cells, indicating that ATF3 acts within NK cells to regulate antiviral responses.

High serum granulocyte-colony stimulating factor characterises neutrophilic COPD exacerbations associated with dysbiosis.

INTRODUCTION: COPD exacerbations are heterogeneous and can be triggered by bacterial, viral, or noninfectious insults. Exacerbations are also heterogeneous in neutrophilic or eosinophilic inflammatory responses. A noninvasive peripheral biomarker of COPD exacerbations characterised by bacterial/neutrophilic inflammation is lacking. Granulocyte-colony stimulating factor (G-CSF) is a key cytokine elevated during bacterial infection and mediates survival, proliferation, differentiation and function of neutrophils. OBJECTIVE: We hypothesised that high peripheral G-CSF would be indicative of COPD exacerbations with a neutrophilic and bacterial phenotype associated with microbial dysbiosis. METHODS: Serum G-CSF was measured during hospitalised exacerbation (day 0 or D0) and after 30 days of recovery (Day30 or D30) in 37 subjects. In a second cohort, serum and sputum cytokines were measured in 59 COPD patients during stable disease, at exacerbation, and at 2-weeks and 6-weeks following exacerbation. RESULTS: Serum G-CSF was increased during exacerbation in a subset of patients. These exacerbations were enriched for bacterial but not viral or type-2 biologies. The median serum G-CSF level was 1.6-fold higher in bacterial exacerbation compared to nonbacterial exacerbation (22 pg·mL-1 versus 13 pg·mL-1, p=0.0007). Serum G-CSF classified bacterial exacerbations with an area under the curve (AUC) for the receiver operating characteristic (ROC) curve equal to 0.76. Exacerbations with a two-fold or greater increase in serum G-CSF were characterised by neutrophilic inflammation, with increased sputum and blood neutrophils, and high sputum interleukin (IL)-1ß, IL-6 and serum amyloid A1 (SAA1) levels. These exacerbations were preceded by dysbiosis, with decreased microbiome diversity and enrichment of respiratory pathogens such as Haemophilus and Moraxella. Furthermore, serum G-CSF at exacerbation classified neutrophilic-dysbiotic exacerbations (AUC for the ROC curve equal to 0.75). CONCLUSIONS: High serum G-CSF enriches for COPD exacerbations characterised by neutrophilic inflammation with underlying bacterial dysbiosis.