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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.

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., Competing Interests: Conflict of interest: W. Timens declares consultancy payments to their institution from Merck Sharp & Dohme and Bristol Myers Squibb, in the 36 months prior to manuscript submission; as well as board membership of the Dutch Society of Pathology and membership of the Council for Research and Innovation of the Federation of Medical Specialists. Conflict of interest: D-J. Slebos declares grants or contracts to their institution from the following US companies in the 36 months prior to manuscript submission: PulmonX Corp., PneumRx/BTG/Boston Scientific, FreeFlowMedical, Nuvaira, PulmAir, GALA, CSA Medical and Apreo; consulting fees paid to their institution from PulmonX Corp., PneumRx/BTG/Boston Scientific, Nuvaira and Apreo; payment or honoraria for lectures, presentations, speakers’ bureaus, manuscript writing or educational events from PulmonX Corp, PneumRx/BTG/Boston Scientific and Nuvaira; support for attending meeting and/or travel from PulmonX Corp., PneumRx/BTG/Boston Scientific and Nuvaira; and receipt by their institution of study material/devices from PulmonX Corp., PneumRx/BTG/Boston Scientific, FreeFlowMedical, Nuvaira, PulmAir, GALA and CSA Medical, all in the 36 months prior to manuscript submission. Conflict of interest: D.F. Choy is an employee of Genentech, Inc., a member of the Roche Group, and is a co-inventor, as part of that employment, for pending patents related to the diagnosis and treatment of chronic respiratory diseases. They also hold stocks and/or options in Roche. Conflict of interest: A. Chakrabarti is an employee of Genentech, Inc., a member of the Roche Group. Conflict of interest: M. Grimbaldeston is an employee of Genentech, Inc., a member of the Roche Group, and has received stock options. Conflict of interest: C.M. Rosenberger is an employee of Genentech, Inc., a member of the Roche Group, and has received support for attending conferences from their employer. They hold stocks and stock options in Roche. Conflict of interest: C-A. Brandsma declares a research grant from Genentech, related to the current manuscript. Conflict of interest: M. van den Berge declares a research grant from Genentech, related to the current manuscript. Conflict of interest: All other authors declare no competing interests., (Copyright ©The authors 2023.)

Altered myeloid inflammation and lymphopenia are hallmarks of severe infections. We identified the upregulated EN-RAGE gene program in airway and blood myeloid cells from patients with acute lung injury from SARS-CoV-2 or other causes across 7 cohorts. This program was associated with greater clinical severity and predicted future mechanical ventilation and death. EN-RAGE hi myeloid cells express features consistent with suppressor cell functionality, including low HLA-DR and high PD-L1. Sustained EN-RAGE program expression in airway and blood myeloid cells correlated with clinical severity and increasing expression of T cell dysfunction markers. IL-6 upregulated many EN-RAGE program genes in monocytes in vitro. IL-6 signaling blockade by tocilizumab in a placebo-controlled clinical trial led to rapid normalization of EN-RAGE and T cell gene expression. This identifies IL-6 as a key driver of myeloid dysregulation associated with worse clinical outcomes in COVID-19 patients and provides insights into shared pathophysiological mechanisms in non-COVID-19 ARDS., Competing Interests: J.A.H., H.S., J.V.H., C.O., L.O., X.G., N.W., A.Q., D.C., A.C, D.F.C., T.R., J.M.M., F.C., A.T., M.B., L.T., A.R., S.B.K., R.N.B., and C.M.R. were employees of Genentech, Inc. at the time of this study and own equity in Roche. The COMET study was supported in part by Genentech funding. C.J.Y. is a scientific advisory board member for and holds equity in Related Sciences and ImmunAI, a consultant for and holds equity in Maze Therapeutics, and a consultant for TRex Bio. C.J.Y. has received research support from Chan Zuckerberg Initiative, Chan Zuckerberg Biohub, and Genentech. C.S.C. has received research funding from Roche-Genentech for an unrelated project as well as from NIH, DOD, and Quantum Leap Healthcare Collaborative. C.S.C. is a consultant for Vasomune, Quark, and GEn1E Lifesciences. C.H. is a consultant for Spring Discovery but does not have any financial interest in the company nor is the work related to what is covered in this manuscript. A.R. is a co-founder and equity holder of Celsius Therapeutics, an equity holder in Immunitas Therapeutics and, until 31 July 2020, was a scientific advisory board member of Thermo Fisher Scientific, Syros Pharmaceuticals, Asimov, and Neogene Therapeutics. A.R. is a named inventor on multiple patents related to single-cell and spatial genomics filed by or issued to the Broad Institute., (© 2023 The Authors.)

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., Competing Interests: H.S., J.A.H., C.M.R., A.Q., O.O., J.M., F.C., M.B., L.T., A.R., and R.N.B. are employees of Roche/Genentech and hold stock and/or stock options in Roche/Genentech. A.T. is an employee of Roche/Genentech. F.C. has a patent pending to Genentech for biomarkers for predicting a response to an interleukin (IL)-6 antagonist (P36367-US). L.T. is an author of a patent Method for Treating Pneumonia, including COVID-19 Pneumonia with an IL-6 Antagonist pending, owned by Genentech/Roche. A.R. is a co-founder and equity holder of Celsius Therapeutics, an equity holder in Immunitas Therapeutics and, until July 31, 2020, was a scientific advisory board member of ThermoFisher Scientific, Syros Pharmaceuticals, Asimov, and Neogene Therapeutics. A.R. is a named inventor on multiple patents related to single cell and spatial genomics filed by or issued to the Broad Institute. I.O.R. has nothing to declare., (© 2023 The Authors.)

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., (© 2023. The Author(s).)

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., (© 2023 F. Hoffmann-La Roche Ltd and The Authors. Clinical and Translational Science published by Wiley Periodicals LLC on behalf of American Society for Clinical Pharmacology and Therapeutics.)

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., Competing Interests: Dr. Waters has received funding from Genentech paid to his institution for the conduct of this trial. Dr. McKinnell has received funding from Genentech paid to his institution for the conduct of this trial; he reports receiving research funding paid to his institution from Gilead, Eli Lilly and Company, and Duke University; and he received funding from Pfizer and ThermoFisher. Dr. Martin has received compensation for service as a monitor of the COVID-astegolimab-interleukin trial. Dr. Buchman serves as a Senior Advisor to Biomedical Advanced Research and Development Authority (BARDA), U.S. Department of Health and Human Services under an agreement between his employer, Emory University, and that agency; he also serves as Editor-in-Chief of Critical Care Medicine, and Emory University similarly receives payment for that service from the Society of Critical Care Medicine. Drs. McKinnell, Martin, Buchman, Theess, Lekkerkerker, Staton, Rosenberger, Pappu, Wang, Zhang, Brooks, Cheung, Galanter, Chen, Mohan, and Peck disclosed the off-label product use of Astegolimab and Efmarodocokin Alfa. Dr. Theess is currently an employee of F. Hoffmann-La Roche and owns Roche stock. Drs. Staton and Wang are former employees of Genentech and own Roche stock. Dr. Chen is a former employee of Genentech. Drs. Martin, Theess, Yang, Lekkerkerker, Staton, Rosenberger, Pappu, Wang, Zhang, Cheung, Galanter, Chen, Mohan, and Peck received funding from Genentech. Drs. Theess’s, Lekkerkerker’s, Staton’s, Rosenberger’s, Wang’s, Zhang’s, Cheung’s, Chen’s, Mohan’s, and Peck’s institutions received funding from the Department of Health and Human Services, the Office of the Assistant Secretary for Preparedness and Response, and the BARDA (OT number: HHSO100201800036C). Drs. Theess, Yang, Lekkerkerker, Staton, Rosenberger, Pappu, Wang, Zhang, Brooks, Cheung, Galanter, Chen, Mohan, and Peck are employees of Genentech, a member of the Roche group, and own Roche stock. Drs. Theess and Brooks disclosed they are employed by F. Hoffman-LaRoche. Drs. Theess, Lekkerkerker, Staton, Rosenberger, Pappu, Wang, Zhang, Brooks, Cheung, Galanter, Chen, Mohan, and Peck disclosed work for hire. Dr. Brooks disclosed that he is a Principal Computational Researcher at Genentech. Dr. Kalil has disclosed that he does not have any potential conflicts of interest., (Copyright © 2022 The Author(s). Published by Wolters Kluwer Health, Inc. on behalf of the Society of Critical Care Medicine and Wolters Kluwer Health, Inc.)

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., Competing Interests: Drs. Tom, Bao, Tsai, Carrasco-Triguero, and Cai received funding from the Biomedical Advanced Research and Development Authority (BARDA) under OT number HHSO100201800036C. Drs. Tom, Bao, Tsai, Qamra, Summers, Carrasco-Triguero, McBride, Rosenberger, Lin, Stubbings, and Cai disclosed they are employees of Genetech/Roche. Drs. Tom and Cai disclosed that they have a patent pending to Genetech for biomarkers for predicting response to an interleukin (IL)–6 antagonist (P36367-US). Drs. Bao, Tsai, and van der Leest received funding from Roche/Genetech. Dr. Bao received support for article research from BARDA. Drs. Bao and Tsai disclosed a patent pending for a method for treating pneumonia, including coronavirus disease 2019 pneumonia with an IL-6 antagonist (EFS ID 38946141). Drs. Bao and Blyth disclosed government work. Drs. Bao, Tsai, Qamra, Summers, Carrasco-Triguero, McBride, Stubbings, Haslem, and Cai disclosed the off-label product use of tocilizumab (Actemra). Dr. Qamra received funding from Hoffman-La Roche Canada. Dr. Summers received funding from Roche Products Ltd. Drs. McBride, Rosenberger, and Lin disclosed they own stock/stock options of Genetech. Drs. McBride, Rosenberger, and Cai disclosed work for hire. Dr. Stubbings is an employee of F Hoffmann-La Roche AG. Dr. Blyth received funding from Rocket Medical UK Ltd. Dr. Carratalà’s institution received funding from Gilead and Roche. Dr. François received funding from Aridis, AM-Pharma, Asahi Kasei, Inotrem, GlaxoSmithKline, Enlivex, Polyphor, Takeda, Transgene, and Biomérieux. Dr. Benfield received funding from Novo Nordisk, Simonsen, GlaxoSmithKline, Pfizer, Gilead, Lundbeck, Kai Hansen Foundation and personal fees from GlaxoSmithKline, Pfizer, Boehringer Ingelheim, Gilead, and Merck Sharp & Dohme outside the submitted work. Dr. Bonfanti received funding from Viiv, Gilead, and Janssen. Dr. van der Leest received funding from Bristol Myers Squibb, Merck Sharpe & Dohme, AbbVie, Boehringer Ingelheim, and AstraZeneca. Dr. Luyt’s institution received funding from Roche; he received funding from Correvio, Bayer Healthcare, Aerogen, ThermoFisher Brahms, Merck Sharpe & Dohme, Carmat, and Biomérieux. Dr. Luyt reports a grant from Roche to the Institute of Cardiometabolism and Nutrition, Sorbonne Université, Hôpital de la Pitié Salpêtrière, Assistance Publique - Hôpitaux de Paris for the COVACTA trial and a grant from Correvio and personal fees from Bayer Healthcare, Aerogen, ThermoFisher Brahms, Merck Sharp & Dohme, and Biomérieux outside the submitted work. Dr. Kheradmand received support for article research from the National Institutes of Health. Dr. Rosas received funding from Boehringer Ingelheim, Bristol Myers Squibb, and Immunomet. He reports a grant from Roche for the COVACTA trial and a grant and personal fees from Genentech/Roche outside the submitted work. Dr. Cai’s institution received funding from F. Hoffman-La Roche Ltd and the U.S. Department of Health and Human Services, the Office of the Assistant Secretary for Preparedness and Response. The remaining authors have disclosed that they do not have any potential conflicts of interest., (Copyright © 2021 The Author(s). Published by Wolters Kluwer Health, Inc. on behalf of the Society of Critical Care Medicine and Wolters Kluwer Health, Inc.)

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., (© 2021 Wiley Periodicals LLC.)

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., (© 2021. The Author(s).)

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)-1β, 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., Competing Interests: Conflict of interest: A. Chakrabarti reports personal fees from and stock in Genentech, Inc., during the conduct of the study and outside the submitted work. Conflict of interest: J.S. Mar reports personal fees from and equity in Genentech, Inc., outside the submitted work. Conflict of interest: D.F. Choy is an employee of Genentech, Inc. Conflict of interest: Y. Cao reports personal fees from and stock in Genentech, Inc., during the conduct of the study and outside the submitted work. Conflict of interest: N. Rathore reports personal fees from and stock in Genentech, Inc., during the conduct of the study and outside the submitted work. Conflict of interest: X. Yang reports personal fees from Genentech, Inc., during the conduct of the study and outside the submitted work. Conflict of interest: G.W. Tew reports personal fees from Genentech, Inc., during the conduct of the study and outside the submitted work. Conflict of interest: O. Li is a salaried employee of Genentech, Inc. and received nonfinancial support from Genentech, Inc., during the conduct of the study. Conflict of interest: P.G. Woodruff reports consulting fees from Regeneron, Sanofi, Glenmark Pharma, Theravance and NGM Pharma, and visiting professor honoraria from Amgen and Genentech, outside the submitted work. Conflict of interest: C.E. Brightling is an employee of the University of Leicester. Conflict of interest: M. Grimbaldeston is an employee of Genentech, Inc., and reports personal fees and nonfinancial support during the conduct of the study. Conflict of interest: S.A. Christenson reports consulting fees from AstraZeneca, GlaxoSmithKline, Amgen and Glenmark; personal fees for invited lectures from Sunovion and Genentech; and personal fees for writing for UpToDate, all outside the submitted work. Conflict of interest: M. Bafadhel reports grants from AstraZeneca; honoraria for consulting and advisory boards, as well as travel to conferences, from AstraZeneca, Chiesi and GSK. She is a scientific advisor to and minor shareholder in AlbusHealth, all outside the submitted work. Conflict of interest: C.M. Rosenberger reports personal fees from and stock in Genentech, Inc., during the conduct of the study and outside the submitted work., (Copyright ©The authors 2021.)

Background: Biomarkers are needed for early identification of patients at risk of severe complications from influenza infection, including prolonged respiratory failure and death. Eicosanoids are bioactive lipid mediators with pro- and anti-inflammatory properties produced in response to infection. This study assessed the relationships between the host bioactive lipid response, influenza viral load, and clinical outcomes., Methods: Influenza-positive, intubated children ≤18 years old were enrolled across 26 US pediatric intensive care units (PICUs). Mass spectrometry was used to measure >100 lipid metabolites in endotracheal and nasopharyngeal samples. Influenza viral load was measured by quantitative polymerase chain reaction., Results: Age and bacterial co-infection were associated with multiple bioactive lipids ( P  < .05). Influenza viral load was lower in patients with bacterial co-infection compared with those without, and pro-inflammatory bioactive lipids positively correlated with viral load in bacterially co-infected children ( P  < .05). Lipids associated with disease resolution correlated with viral load in patients without bacterial co-infection ( P  < .01). After adjusting for age and bacterial co-infection status, elevated pro- and anti-inflammatory lipids measured early in the intensive care unit course were associated with higher mortality, whereas influenza viral load and endotracheal cytokine levels were not associated with clinical outcomes. Prostaglandin E 2 , arachidonic acid, docosahexaenoic acid, and 12-hydroxyeicosatetraenoic acid measured within 72 hours of PICU admission predicted death or prolonged (≥28 days) mechanical ventilator support (area under the curve, 0.72-0.79; P  < .02) not explained by admission illness severity., Conclusions: Children with influenza-related complications have early bioactive lipid responses that may reflect lung disease severity. Respiratory bioactive lipids are candidate prognostic biomarkers to identify children with the most severe clinical outcomes., (© The Author(s) 2020. Published by Oxford University Press on behalf of Infectious Diseases Society of America.)

Objectives: To identify risk stratification biomarkers to enrich for the subset of Staphylococcus aureus bacteraemia patients who develop deep-seated tissue infections with high morbidity and mortality to guide clinical trial enrolment and clinical management., Methods: We evaluated the prognostic value of eight biomarkers for persistent bacteraemia, mortality and endovascular infection foci in a validation cohort of 160 patients with S. aureus bacteraemia enrolled consecutively over 3 years., Results: High levels of IL-17A, IL-10 or soluble E-selectin at bacteraemia diagnosis correlated with the duration of positive blood cultures. When thresholds defined in an independent cohort were applied, these biomarkers were robust predictors of persistent bacteraemia or endovascular infection. High serum levels of IL-17A and IL-10 often preceded the radiographic diagnosis of infective endocarditis, suggesting potential utility for prioritising diagnostic radiographic imaging. High IL-8 was prognostic for all-cause mortality, while IL-17A and IL-10 were superior to clinical metrics in discriminating between attributable mortality and non-attributable mortality. High IL-17A and IL-10 identified more patients who developed microbiological failure or mortality than were identified by infective endocarditis diagnosis., Conclusion: These biomarkers offer potential utility to identify patients at risk of persistent bacteraemia to guide diagnostic imaging and clinical management. Low biomarker levels could be used to rule out the need for more invasive TEE imaging in patients at lower risk of infective endocarditis. These biomarkers could enable clinical trials by enriching for patients with the greatest need for novel therapies., Competing Interests: YC, AOG, MCP, OM, KH, MC‐T and CMR were employees of Genentech, Inc. during the execution of this study. VGF served as Chair of V710 Scientific Advisory Committee (Merck); has received grant support from Cerexa/Actavis/Allergan, Pfizer, Advanced Liquid Logics, NIH, MedImmune, Basilea, Karius, ContraFect, Regeneron and Genentech; has NIH STTR/SBIR grants pending with Affinergy, Locus and Medical Surface, Inc; has been a paid consultant for Achaogen, Astellas, Arsanis, Affinergy, Basilea, Bayer, Cerexa, ContraFect, Cubist, Debiopharm, Durata, Grifols, Genentech, MedImmune, Merck, Medicines Co., Pfizer, Novartis, NovaDigm, Theravance and xBiotech; has received honoraria from Theravance and Green Cross; and has a patent pending in sepsis diagnostics. Other authors have no competing interests., (© 2020 The Authors. Clinical & Translational Immunology published by John Wiley & Sons Australia, Ltd on behalf of Australian and New Zealand Society for Immunology Inc.)

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., (© The Author(s) 2018. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.)

Background: Staphylococcus aureus is a leading global cause of bacteremia that can cause invasive tissue infections with high morbidity and mortality despite appropriate antibiotic therapy. Clinicians lack sufficient tools to rapidly identify patients with a poor prognosis to guide diagnostic workup and treatment decisions. Host cell-free DNA provides prognostic value across a spectrum of critical illnesses, including S. aureus bacteremia and sepsis. Metrics of high bacterial load are associated with disease severity in S. aureus bacteremia, and the objective of this study was to evaluate whether incorporating quantitation of cell-free bacterial DNA would provide additive prognostic value when combined with biomarkers of the inflammatory response., Methods: S. aureus cell-free DNA was measured by quantitative polymerase chain reaction (PCR) in baseline serum samples from an observational cohort of 111 patients with complicated S. aureus bacteremia and correlated with host inflammatory markers and clinical outcomes., Results: High levels of S. aureus cell-free DNA at the time of positive index blood culture were prognostic for all-cause and attributable mortality and persistent bacteremia and were associated with infective endocarditis. However, they did not provide additive value to biomarkers of the host response to infection in multivariate analysis., Conclusions: Measurements of bacterial load by PCR are a clinically feasible candidate biomarker for stratifying patients at higher risk for complications and poor outcomes. Their diagnostic and prognostic value for identifying foci of infection and influencing treatment remain to be evaluated in additional cohorts.

Background: Staphylococcus aureus (SA) bacteremia often requires a long treatment duration with antibiotics to prevent relapse due to the ability of SA to establish reservoirs of infection in sites such as heart and bone. These metastatic sites of infection cannot be serially sampled to monitor the clearance of SA infection. This study aimed to establish a link between persistence of circulating SA deoxyribonucleic acid (SA-DNA) and tissue reservoirs in patients with SA bacteremia., Methods: A highly sensitive quantitative polymerase chain reaction was used to measure whole blood SA-DNA and plasma-derived SA cell-free DNA (SA-cfDNA) in a set of longitudinal samples from 73 patients with confirmed SA bacteremia and correlated with clinical features., Results: Blood SA-DNA was detected for longer than the duration of positive blood cultures. Longer duration of circulating bacterial DNA was observed in complicated SA bacteremia infections, such as endocarditis and osteoarticular infections, compared with uncomplicated bloodstream infections. In contrast, traditional blood cultures demonstrated similar time to clearance regardless of foci of infection. Plasma-derived SA-cfDNA showed concordance with blood SA-DNA levels. Baseline levels of SA-DNA were higher in patients presenting with greater clinical severity and complicated bacteremia., Conclusions: Prolonged levels of circulating SA-DNA in patients with complicated tissue reservoirs after clearance of blood cultures observed in this single-center study should be validated in additional cohorts to assess the potential utility for monitoring clearance of infection in patients with SA bacteremia.

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 (TCID 50 ) (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.)., (Copyright © 2017 American Society for Microbiology.)

Previous studies have reported associations of IFITM3 SNP rs12252 with severe influenza, but evidence of association and the mechanism by which risk is conferred remain controversial. We prioritized SNPs in IFITM3 on the basis of putative biological function and identified rs34481144 in the 5' UTR. We found evidence of a new association of rs34481144 with severe influenza in three influenza-infected cohorts characterized by different levels of influenza illness severity. We determined a role for rs34481144 as an expression quantitative trait locus (eQTL) for IFITM3, with the risk allele associated with lower mRNA expression. The risk allele was found to have decreased IRF3 binding and increased CTCF binding in promoter-binding assays, and risk allele carriage diminished transcriptional correlations among IFITM3-neighboring genes, indicative of CTCF boundary activity. Furthermore, the risk allele disrupts a CpG site that undergoes differential methylation in CD8 + T cell subsets. Carriers of the risk allele had reduced numbers of CD8 + T cells in their airways during natural influenza infection, consistent with IFITM3 promoting accumulation of CD8 + T cells in airways and indicating that a critical function for IFITM3 may be to promote immune cell persistence at mucosal sites.Our study identifies a new regulator of IFITM3 expression that associates with CD8 + T cell levels in the airways and a spectrum of clinical outcomes.

Background: Interferon-induced transmembrane protein 3 (IFITM3) restricts endocytic fusion of influenza virus. IFITM3 rs12252&#95;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&#95;C was performed using PICFLU and FLU09 patient RNA., Results: In PICFLU, 358 children had influenza infection. We identified 22 rs12252&#95;C homozygotes in 185 white non-Hispanic children. rs12252&#95;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., (© The Author 2017. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail journals.permissions@oup.com.)

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.

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.)., (Copyright © 2017 American Society for Microbiology.)

DSTA4637A, a novel THIOMAB™ antibody antibiotic conjugate (TAC) against Staphylococcus aureus (S. aureus), is currently being investigated as a potential therapy against S. aureus infections. Structurally, TAC is composed of an anti-S. aureus antibody linked to a potent antibiotic, dmDNA31. The goal of the current study was to characterize the pharmacokinetics (PK) of TAC in mice, assess the effect of S. aureus infection on its PK, and evaluate its pharmacodynamics (PD) by measuring the bacterial load in various organs at different timepoints following TAC treatment. Plasma concentrations of 3 analytes, total antibody (TAb), antibody-conjugated dmDNA31 (ac-dmDNA31), and unconjugated dmDNA31, were measured in these studies. In non-infected mice (target antigen absent), following intravenous (IV) administration of a single dose of TAC, systemic concentration-time profiles of both TAb and ac-dmDNA31 were bi-exponential and characterized by a short distribution phase and a long elimination phase as expected for a monoclonal antibody-based therapeutic. Systemic exposures of both TAb and ac-dmDNA31 were dose proportional over the dose range tested (5 to 50 mg/kg). In a mouse model of systemic S. aureus infection (target antigen present), a single IV dose of TAC demonstrated PK behavior similar to that in the non-infected mice, and substantially reduced bacterial load in the heart, kidney, and bones on 7 and 14 d post dosing. These findings have increased our understanding of the PK and PK/PD of this novel molecule, and have shown that at efficacious dose levels the presence of S. aureus infection had minimal effect on TAC PK.

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., (Published by Oxford University Press for the Infectious Diseases Society of America 2016. This work is written by (a) US Government employee(s) and is in the public domain in the US.)

The Systems Biology for Infectious Diseases Research program was established by the U.S. National Institute of Allergy and Infectious Diseases to investigate host-pathogen interactions at a systems level. This program generated 47 transcriptomic and proteomic datasets from 30 studies that investigate in vivo and in vitro host responses to viral infections. Human pathogens in the Orthomyxoviridae and Coronaviridae families, especially pandemic H1N1 and avian H5N1 influenza A viruses and severe acute respiratory syndrome coronavirus (SARS-CoV), were investigated. Study validation was demonstrated via experimental quality control measures and meta-analysis of independent experiments performed under similar conditions. Primary assay results are archived at the GEO and PeptideAtlas public repositories, while processed statistical results together with standardized metadata are publically available at the Influenza Research Database (www.fludb.org) and the Virus Pathogen Resource (www.viprbrc.org). By comparing data from mutant versus wild-type virus and host strains, RNA versus protein differential expression, and infection with genetically similar strains, these data can be used to further investigate genetic and physiological determinants of host responses to viral infection.

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.

Influenza viruses exhibit large, strain-dependent differences in pathogenicity in mammalian hosts. Although the characteristics of severe disease, including uncontrolled viral replication, infection of the lower airway, and highly inflammatory cytokine responses have been extensively documented, the specific virulence mechanisms that distinguish highly pathogenic strains remain elusive. In this study, we focused on the early events in influenza infection, measuring the growth rate of three strains of varying pathogenicity in the mouse airway epithelium and simultaneously examining the global host transcriptional response over the first 24 hours. Although all strains replicated equally rapidly over the first viral life-cycle, their growth rates in both lung and tracheal tissue strongly diverged at later times, resulting in nearly 10-fold differences in viral load by 24 hours following infection. We identified separate networks of genes in both the lung and tracheal tissues whose rapid up-regulation at early time points by specific strains correlated with a reduced viral replication rate of those strains. The set of early-induced genes in the lung that led to viral growth restriction is enriched for both NF-κB binding site motifs and members of the TREM1 and IL-17 signaling pathways, suggesting that rapid, NF-κB -mediated activation of these pathways may contribute to control of viral replication. Because influenza infection extending into the lung generally results in severe disease, early activation of these pathways may be one factor distinguishing high- and low-pathogenicity strains.

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.

Host resistance against Salmonella enterica serovar Typhimurium (S. Typhimurium) is mediated by natural resistance-associated macrophage protein 1 (Nramp1/Slc11a1). Nramp1 is critical to host defence, as mice lacking Nramp1 fail to control bacterial replication and succumb to low doses of S. Typhimurium. Despite this crucial role, the mechanisms underlying Nramp1's protective effects are unclear. Dendritic cells (DCs) that sample the intestinal lumen are among the first cells encountered by S. Typhimurium following oral infection and act as a conduit for S. Typhimurium to cross the intestinal epithelial barrier. We report that DCs, including intestinal, splenic and bone marrow-derived DCs (BMDCs), express Nramp1 protein. In the small intestine, Nramp1 expression is greater in a subset of DCs (CD11c(+)CD103(-)) characterized by the elevated expression of pro-inflammatory cytokines in response to bacterial products. While Nramp1 expression did not affect S. Typhimurium replication in BMDCs, infected Nramp1+/+ BMDCs and intestinal CD11c(+)CD103(-) DCs secreted more inflammatory cytokines (IL-6, IL-12 and TNF-alpha) than Nramp1-/-, suggesting that Nramp1 expression may promote a more rapid inflammatory response following infection. Collectively, these findings reveal a new role for DCs and Nramp1 in modulating the host inflammatory response to S. Typhimurium.

Enteropathogenic Escherichia coli (EPEC) and the murine pathogen Citrobacter rodentium belong to the attaching and effacing (A/E) family of bacterial pathogens. These noninvasive bacteria infect intestinal enterocytes using a type 3 secretion system (T3SS), leading to diarrheal disease and intestinal inflammation. While flagellin, the secreted product of the EPEC fliC gene, causes the release of interleukin 8 (IL-8) from epithelial cells, it is unclear whether A/E bacteria also trigger epithelial inflammatory responses that are FliC independent. The aims of this study were to characterize the FliC dependence or independence of epithelial inflammatory responses to direct infection by EPEC or C. rodentium. Following infection of Caco-2 intestinal epithelial cells by wild-type and DeltafliC EPEC, a rapid activation of several proinflammatory genes, including those encoding IL-8, monocyte chemoattractant protein 1, macrophage inflammatory protein 3alpha (MIP3alpha), and beta-defensin 2, occurred in a FliC-dependent manner. These responses were accompanied by mitogen-activated protein kinase activation, as well as the Toll-like receptor 5 (TLR5)-dependent activation of NF-kappaB. At later infection time points, a subset of these proinflammatory genes (IL-8 and MIP3alpha) was also induced in cells infected with DeltafliC EPEC. The nonmotile A/E pathogen C. rodentium also triggered similar innate responses through a TLR5-independent but partially NF-kappaB-dependent mechanism. Moreover, the EPEC FliC-independent responses were increased in the absence of the locus of enterocyte effacement-encoded T3SS, suggesting that translocated bacterial effectors suppress rather than cause the FliC-independent inflammatory response. Thus, we demonstrate that infection of intestinal epithelial cells by A/E pathogens can trigger an array of proinflammatory responses from epithelial cells through both FliC-dependent and -independent pathways, expanding our understanding of the innate epithelial response to infection by these pathogens.

Macrophages are versatile immune cells that can detect a variety of pathogen-associated molecular patterns through their Toll-like receptors (TLRs). In response to microbial challenge, the TLR-stimulated macrophage undergoes an activation program controlled by a dynamically inducible transcriptional regulatory network. Mapping a complex mammalian transcriptional network poses significant challenges and requires the integration of multiple experimental data types. In this work, we inferred a transcriptional network underlying TLR-stimulated murine macrophage activation. Microarray-based expression profiling and transcription factor binding site motif scanning were used to infer a network of associations between transcription factor genes and clusters of co-expressed target genes. The time-lagged correlation was used to analyze temporal expression data in order to identify potential causal influences in the network. A novel statistical test was developed to assess the significance of the time-lagged correlation. Several associations in the resulting inferred network were validated using targeted ChIP-on-chip experiments. The network incorporates known regulators and gives insight into the transcriptional control of macrophage activation. Our analysis identified a novel regulator (TGIF1) that may have a role in macrophage activation.

Background: As part of a National Institute of Allergy and Infectious Diseases funded collaborative project, we have performed over 150 microarray experiments measuring the response of C57/BL6 mouse bone marrow macrophages to toll-like receptor stimuli. These microarray expression profiles are available freely from our project web site http://www.innateImmunity-systemsbiology.org. Here, we report the development of a database of computationally predicted transcription factor binding sites and related genomic features for a set of over 2000 murine immune genes of interest. Our database, which includes microarray co-expression clusters and a host of web-based query, analysis and visualization facilities, is available freely via the internet. It provides a broad resource to the research community, and a stepping stone towards the delineation of the network of transcriptional regulatory interactions underlying the integrated response of macrophages to pathogens., Description: We constructed a database indexed on genes and annotations of the immediate surrounding genomic regions. To facilitate both gene-specific and systems biology oriented research, our database provides the means to analyze individual genes or an entire genomic locus. Although our focus to-date has been on mammalian toll-like receptor signaling pathways, our database structure is not limited to this subject, and is intended to be broadly applicable to immunology. By focusing on selected immune-active genes, we were able to perform computationally intensive expression and sequence analyses that would currently be prohibitive if applied to the entire genome. Using six complementary computational algorithms and methodologies, we identified transcription factor binding sites based on the Position Weight Matrices available in TRANSFAC. For one example transcription factor (ATF3) for which experimental data is available, over 50% of our predicted binding sites coincide with genome-wide chromatin immnuopreciptation (ChIP-chip) results. Our database can be interrogated via a web interface. Genomic annotations and binding site predictions can be automatically viewed with a customized version of the Argo genome browser., Conclusion: We present the Innate Immune Database (IIDB) as a community resource for immunologists interested in gene regulatory systems underlying innate responses to pathogens. The database website can be freely accessed at http://db.systemsbiology.net/IIDB.

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.

Inflammatory bowel diseases and infectious gastroenteritis likely occur when the integrity of intestinal barriers is disrupted allowing luminal bacterial products to cross into the intestinal mucosa, stimulating immune cells and triggering inflammation. While specific Toll-like receptors (TLR) are involved in the generation of inflammatory responses against enteric bacteria, their contributions to the maintenance of intestinal mucosal integrity are less clear. These studies investigated the role of TLR2 in a model of murine colitis induced by the bacterial pathogen Citrobacter rodentium. C. rodentium supernatants specifically activated TLR2 in vitro while infected TLR2-/- mice suffered a lethal colitis coincident with colonic mucosal ulcerations, bleeding and increased cell death but not increased pathogen burden. TLR2-/- mice suffered impaired epithelial barrier function mediated via zonula occludens (ZO)-1 in naïve mice and claudin-3 in infected mice, suggesting this could underlie their susceptibility. TLR2 deficiency was also associated with impaired production of IL-6 by bone marrow-derived macrophages and infected colons cultured ex vivo. As IL-6 has antiapoptotic and epithelial repair capabilities, its reduced expression could contribute to the impaired mucosal integrity. These studies report for the first time that TLR2 plays a critical role in maintaining intestinal mucosal integrity during infection by a bacterial pathogen.

Innate immunodeficiency has recently been reported as resulting from the Q293X IRAK-4 mutation with consequent defective TLR/IL-1R signaling. In this study we report a method for the rapid allele-specific detection of this mutation and demonstrate both cell type specificity and ligand specificity in defective IL-1R-associated kinase (IRAK)-4-deficient cellular responses, indicating differential roles for this protein in human PBMCs and primary dermal fibroblasts and in LPS, IL-1beta, and TNF-alpha signaling. We demonstrate transcriptional and post-transcriptional defects despite NF-kappaB signaling and intact MyD88-independent signaling and propose that dysfunctional complex 1 (IRAK1/TRAF6/TAK1) signaling, as a consequence of IRAK-4 deficiency, generates specific defects in MAPK activation that could underpin this patient's innate immunodeficiency. These studies demonstrate the importance of studying primary human cells bearing a clinically relevant mutation; they underscore the complexity of innate immune signaling and illuminate novel roles for IRAK-4 and the fundamental importance of accessory proinflammatory signaling to normal human innate immune responses and immunodeficiencies.

Enteropathogenic Escherichia coli (EPEC) and enterohemorrhagic E. coli (EHEC) are noninvasive bacterial pathogens that infect their hosts' intestinal epithelium, causing severe diarrheal disease. These infections also cause intestinal inflammation, although the mechanisms underlying the inflammatory response, as well as its potential role in host defense, are unclear. Since these bacteria are gram-negative, Toll-like receptor 4 (TLR4), the innate receptor for bacterial lipopolysaccharide may contribute to the host response; however, the role of TLR4 in the gastrointestinal tract is poorly understood, and its impact has yet to be tested against this family of enteric bacterial pathogens. Since EPEC and EHEC are human specific, we infected mice with Citrobacter rodentium, a mouse-adapted attaching and effacing (A/E) bacterium that infects colonic epithelial cells, causing colitis and epithelial hyperplasia, using a similar array of virulence proteins as EPEC and EHEC. We demonstrated that C. rodentium activates TLR4 and rapidly induced NF-kappaB nuclear translocation in host cells in a partially TLR4-dependent manner. Infection of TLR4-deficient mice revealed that TLR4-dependent responses mediate much of the inflammation and tissue pathology seen during infection, including the induction of the chemokines MIP-2 and MCP-1, as well as the recruitment of macrophages and neutrophils into the infected intestine. Surprisingly, spread of C. rodentium through the colon was delayed in TLR4-deficient mice, whereas the duration of the infection was unaffected, indicating that TLR4-mediated responses against this A/E pathogen are not host protective and are ultimately maladaptive to the host, contributing to both the morbidity and the pathology seen during infection.

Pseudomonas aeruginosa is a pulmonary pathogen in individuals with impaired mucociliary clearance such as cystic fibrosis or mechanical ventilation. Non-opsonic phagocytosis of P. aeruginosa can be mediated by either CR3 or CD14 and different strains appear to have a bias towards one or the other receptor. Strain Fc808 is ingested through CD14 whereas P1 (Fc194) uses CR3. In an in vitro culture system, the inflammatory response of macrophages to these two different strains of P. aeruginosa was divergent at the protein level, with higher IL-6 and tumour necrosis factor (TNF)-alpha production generated in response to strain P1 and higher IL-1 beta production in response to strain Fc808. Interaction of macrophages with these two bacterial strains induced distinct gene expression patterns as detected by gene array analysis, with prominence of genes encoding pro-inflammatory cytokines, surface receptors, transcription factors and proteins involved in phagocytosis. However, comparison of gene expression data and cytokine response data with the two bacterial strains indicated that production of IL-1 beta, IL-6 and TNF-alpha was under differential post-transcriptional control. Interestingly, this effect did not correlate with receptor bias but instead was related to the different LPSs of the two strains. The use of specific mitogen-activated protein kinase (MAPK) inhibitors suggested a role for extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK) in the differential cytokine production by strains P1 and Fc808. These results indicate that strains of the same species of bacteria may induce differential macrophage phagocytic and inflammatory responses with likely consequence for bacterial clearance and host injury.

Antimicrobial peptides have established an important role in the defense against extracellular infections, but the expression of cationic peptides within macrophages as an antibacterial effector mechanism against intracellular pathogens has not been demonstrated. Macrophage expression of the murine cathelicidin-related antimicrobial peptide (CRAMP) was increased after infection by the intracellular pathogen Salmonella typhimurium, and this increase required reactive oxygen intermediates. By using CRAMP-deficient mice or synthetic CRAMP peptide, we found that CRAMP impaired Salmonella cell division in vivo and in vitro, resulting in long filamentous bacteria. This impaired bacterial cell division also depended on intracellular elastase-like serine protease activity, which can proteolytically activate cathelicidins. Macrophage serine protease activity induced filamentation and enhanced the activity of CRAMP in vitro. A peptide-sensitive Salmonella mutant showed enhanced survival within macrophages derived from CRAMP-deficient mice, indicating that Salmonella can sense and respond to cationic peptides in the intracellular environment. Although cationic peptides have been hypothesized to have activity against pathogens within macrophages, this work provides experimental evidence that the antimicrobial arsenal of macrophages includes cathelicidins. These results show that intracellular reactive oxygen intermediates and proteases regulate macrophage CRAMP expression and activity to impair the replication of an intracellular bacterial pathogen, and they highlight the cooperativity between macrophage antibacterial effectors.

Macrophages function at the front line of immune defences against incoming pathogens. But the ability of macrophages to internalize bacteria, migrate, recruit other immune cells to the site of infection and influence the nature of the immune response can provide unintended benefits for bacterial pathogens that are able to subvert or co-opt these normally effective defences. This review highlights recent advances in our understanding of the many interference strategies that are used by bacterial pathogens to undermine macrophage signalling.

Host responses during the later stages of Salmonella-macrophage interactions are critical to controlling infection but have not been well characterized. After 24 h of infection, nearly half of interferon-gamma-primed murine RAW 264.7 macrophage-like cells infected by Salmonella enterica serovar Typhimurium contained filamentous bacteria. Bacterial filamentation indicates a defect in completing replication and has been previously observed in bacteria responding to a variety of stresses. To understand whether macrophage gene expression was responsible for this effect on Salmonella Typhimurium replication, we used gene arrays to profile interferon-gamma-primed RAW 264.7 cell gene expression following infection. We observed an increase in MEK1 kinase mRNA at 8 h, an increase in MEK protein at 24 h, and measured phosphorylation of MEK's downstream target kinase, ERK1/2, throughout the 24-h infection period. Treatment of cells with MEK kinase inhibitors significantly reduced numbers of filamentous bacteria observed within macrophages after 24 h and increased the number of intracellular colony-forming units. Phagocyte NADPH oxidase inhibitors and antioxidants also significantly reduced bacterial filamentation. Either MEK kinase or phagocyte oxidase inhibitors could be added 4-8 h after infection and still significantly decrease bacterial filamentation. Oxidase activity appears to mediate bacterial filamentation in parallel to MEK kinase signaling, while inducible nitric-oxide synthase inhibitors had no significant effect on bacterial morphology. In summary, Salmonella Typhimurium infection of interferon-gamma-primed macrophages triggers a MEK kinase cascade at later infection times, and both MEK kinase and phagocyte NADPH oxidase activity impair bacterial replication. These two signaling pathways mediate a host bacteriostatic pathway and may play an important role in innate host defense against intracellular pathogens.

Gene expression array technology is a powerful new tool that has already been used to expand our understanding of host-pathogen interactions. There has been a rapid increase in published reports describing use of this approach to profile host responses to pathogenic bacteria and viruses. The large number of array studies currently in progress coupled with increasing accessibility of this new technology promises a plethora of gene expression data on host response to infection in the near future. Recent insights into macrophage and epithelial cell responses to Salmonella infection garnered from array studies are outlined and used as a basis to discuss various future research directions using gene arrays that will advance the field of cellular microbiology. There is an exciting potential for the gene expression data generated in such studies to provide insights into host physiology, the pathophysiology of disease and novel therapeutics.

Enteropathogenic Escherichia coli (EPEC) is an extracellular bacterial pathogen that infects the human intestinal epithelium and is a major cause of infantile diarrhea in developing countries. EPEC belongs to the group of attaching and effacing (A/E) pathogens. It uses a type III secretion system to deliver proteins into the host cell that mediate signal transduction events in host cells. We used gene array technology to study epithelial cell responses to EPEC infection at the level of gene expression. We found that EPEC induces the expression of several genes in infected HeLa cells by a lipopolysaccharide (LPS)-independent mechanism, including cytokines and early growth response factor 1 (Egr-1). The transcription factor Egr-1 is an immediate-early-induced gene that is activated in most cell types in response to stress. EPEC-induced upregulation of egr-1 is mediated by the activation of the MEK/extracellular signal-regulated kinase signal transduction pathway and is dependent on the type III secretion system. egr-1 is also induced during infection of mice by the A/E pathogen Citrobacter rodentium, suggesting that both Egr-1 and the activation of this mitogen-activated protein kinase signal transduction pathway may play a role in disease.

SifA was originally identified as a virulence factor required for formation of Salmonella-induced filaments (Sifs), elongated tubules rich in lysosomal glycoproteins that extend from the Salmonella-containing vacuole in infected epithelial cells. Here, we demonstrate that deletion mutants of ssaR, a component of the SPI-2 type III secretion system, do not form Sifs in HeLa epithelial cells. This suggests that SifA is a translocated effector of this system, acting within host cells to form Sifs. In support of this hypothesis, transfection of HeLa cells with a vector encoding SifA fused to the green fluorescent protein caused extensive vacuolation of LAMP-1-positive compartments. Filamentous tubules that closely resembled Sifs were also observed in transfected cells, demonstrating that SifA is sufficient to initiate alteration of host cell endosomal structures. deltasifA mutants were impaired in their ability to survive/replicate in RAW 264.7 murine macrophages, a phenotype similar to ssaR mutants. Our findings suggest that SifA is an effector of the SPI-2 type III secretion system and allows colonization of murine macrophages, the host niche exploited during systemic phases of disease in these animals. A family of SifA-related proteins and their importance to Salmonella pathogenesis is also discussed.

The route of initial entry influences how host cells respond to intracellular pathogens. Recent studies have demonstrated that a wide variety of pathogens target lipid microdomains in host cell membranes, known as lipid rafts, to enter host cells as an infectious strategy.

Certain cationic antimicrobial peptides block the binding of LPS to LPS-binding protein and reduce the ability of LPS to induce the production of inflammatory mediators by macrophages. To gain a more complete understanding of how LPS activates macrophages and how cationic peptides influence this process, we have used gene array technology to profile gene expression patterns in macrophages treated with LPS in the presence or the absence of the insect-derived cationic antimicrobial peptide CEMA (cecropin-melittin hybrid). We found that CEMA selectively blocked LPS-induced gene expression in the RAW 264.7 macrophage cell line. The ability of LPS to induce the expression of >40 genes was strongly inhibited by CEMA, while LPS-induced expression of another 16 genes was relatively unaffected. In addition, CEMA itself induced the expression of a distinct set of 35 genes, including genes involved in cell adhesion and apoptosis. Thus, CEMA, a synthetic alpha-helical peptide, selectively modulates the transcriptional response of macrophages to LPS and can alter gene expression in macrophages.

Changes in macrophage phenotype induced during infection result from the recognition of bacterial products as well as the action of bacterial virulence factors. We used the unprecedented opportunity provided by gene arrays to simultaneously study the expression of hundreds of genes during Salmonella typhimurium infection of macrophages and to assess the contribution of the bacterial virulence factor, LPS, in initiating the host responses to Salmonella. We found that S. typhimurium infection caused significant changes in the expression of numerous genes encoding chemokines, cell surface receptors, signaling molecules, and transcriptional activators at 4 h postinfection of the RAW 264.7 murine macrophage cell line. Our results revealed changes in the expression of several genes that had not been previously implicated in the host responses to S. typhimurium infection, as well as changes in the expression of several genes previously shown to be regulated by S. typhimurium infection. An overlapping spectrum of genes was expressed in response to virulent S. typhimurium and purified S. typhimurium LPS, reinforcing the major role of this surface molecule in stimulating the early response of macrophages to bacterial infection. The macrophage gene expression profile was further altered by activation with IFN-gamma, indicating that host cell responses depend on the activation state of the cell.