INTRODUCTION:Critically ill patients commonly develop acquired neutrophil dysfunction, which increases susceptibility to intensive care unit-acquired infection (ICU-AI). This study aimed to assess whether interferon gamma (IFN-γ) can restore function in dysfunctional neutrophils from critically ill patients and to uncover potential underlying mechanisms. METHODS:This was an observational cohort study. Neutrophils were isolated from whole blood donated by critically ill patients (n=31) in four separate teaching hospital intensive care units (ICUs). Neutrophils were subsequently treated with recombinant human IFN-γ or vehicle for 1 hour following either Fc gamma receptor (FcγR) blockade, selective inhibition of the gamma isoform of phosphoinositide 3-kinase (PI3K-γ) or vehicle control for 30 min. Neutrophil phagocytosis, bacterial killing, superoxide generation, phagocytic receptor expression and small Rho GTPase activity were assessed. Neutrophil dysfunction was defined as <50% of cells ingesting 2 or more zymosan particles in a phagocytosis assay. RESULTS:IFN-γ significantly improved phagocytosis (control 36.5%, IFN-γ 56.0%), bacterial killing (control 31.6%, IFN-γ 82.1%) and superoxide generation (2.8-fold increase relative to control) in dysfunctional neutrophils. IFN-γ also increased the activity of the small GTPases, Rac and Cdc42 (2.4-fold and 1.5-fold increase relative to control, respectively) in dysfunctional neutrophils. Selective inhibition of PI3K-γ prevented the IFN-γ-mediated improvement of phagocytosis (IFN-γ 62.5%, with inhibitor 27.9%), bacterial killing (IFN-γ 82.1%, with inhibitor 30.5%) and superoxide generation (IFN-γ 2.8-fold change relative to control, 0.7 with inhibitor). The IFN-γ-mediated improvement of bacterial killing in dysfunctional neutrophils was also prevented by FcγR blockade (IFN-γ 82.1%, FcγR inhibition 28.7%). CONCLUSIONS:In critically ill patients with known acquired neutrophil dysfunction, ex vivo application of IFN-γ consistently improved a range of neutrophil effector functions.

BackgroundTracheostomies in children are associated with significant morbidity, poor quality of life, excess healthcare costs and excess mortality. The underlying mechanisms facilitating adverse respiratory outcomes in tracheostomised children are poorly understood. We aimed to characterise airway host defence in tracheostomised children using serial molecular analyses. MethodsTracheal aspirates, tracheal cytology brushings and nasal swabs were prospectively collected from children with a tracheostomy and controls. Transcriptomic, proteomic and metabolomic methods were applied to characterise the impact of tracheostomy on host immune response and the airway microbiome. ResultsChildren followed up serially from the time of tracheostomy up to 3 months postprocedure (n=9) were studied. A further cohort of children with a long-term tracheostomy were also enrolled (n=24). Controls (n=13) comprised children without a tracheostomy undergoing bronchoscopy. Long-term tracheostomy was associated with airway neutrophilic inflammation, superoxide production and evidence of proteolysis when compared with controls. Reduced airway microbial diversity was established pre-tracheostomy and sustained thereafter. ConclusionsLong-term childhood tracheostomy is associated with a inflammatory tracheal phenotype characterised by neutrophilic inflammation and the ongoing presence of potential respiratory pathogens. These findings suggest neutrophil recruitment and activation as potential exploratory targets in seeking to prevent recurrent airway complications in this vulnerable group of patients.

Introduction: Tissue factor expression on monocytes is implicated in the pathophysiology of sepsis-induced coagulopathy. How tissue factor is expressed by monocyte subsets (classical, intermediate and non-classical) is unknown. Methods: Monocytic tissue factor surface expression was investigated during three conditions. Primary human monocytes and microvascular endothelial cell co-cultures were used for in vitro studies. Volunteers received a bolus of lipopolysaccharide (2 ng/kg) to induce endotoxemia. Patients with sepsis, or controls with critical illness unrelated to sepsis, were recruited from four intensive care units. Results: Contact with endothelium and stimulation with lipopolysaccharide reduced the proportion of intermediate monocytes. Lipopolysaccharide increased tissue factor surface expression on classical and non-classical monocytes. Endotoxemia induced profound, transient monocytopenia, along with activation of coagulation pathways. In the remaining circulating monocytes, tissue factor was up-regulated in intermediate monocytes, though approximately 60 % of individuals (responders) up-regulated tissue factor across all monocyte subsets. In critically ill patients, tissue factor expression on intermediate and non-classical monocytes was significantly higher in patients with established sepsis than among non-septic patients. Upon recovery of sepsis, expression of tissue factor increased significantly in classical monocytes. Conclusion: Tissue factor expression in monocyte subsets varies significantly during health, endotoxemia and sepsis.

Chemokine CXCL8 is a key facilitator of the human host immune response, mediating neutrophil migration, and activation at the site of infection and injury. The oxidative burst is an important effector mechanism which leads to the generation of reactive nitrogen species (RNS), including peroxynitrite. The current study was performed to determine the potential for nitration to alter the biological properties of CXCL8 and its detection in human disease. Here, we show peroxynitrite nitrates CXCL8 and thereby regulates neutrophil migration and activation. The nitrated chemokine was unable to induce transendothelial neutrophil migration in vitro and failed to promote leukocyte recruitment in vivo. This reduced activity is due to impairment in both G protein-coupled receptor signaling and glycosaminoglycan binding. Using a novel antibody, nitrated CXCL8 was detected in bronchoalveolar lavage samples from patients with pneumonia. These findings were validated by mass spectrometry. Our results provide the first direct evidence of chemokine nitration in human pathophysiology and suggest a natural mechanism that limits acute inflammation.

BackgroundNeutrophil activation drives lung complications after cardiopulmonary bypass (CPB). Evidence suggests the healthy, ventilated lung may beneficially re-condition pro-inflammatory neutrophils. However, evidence in humans is lacking, due to a paucity of good models. CPB with simultaneous central venous and bilateral pulmonary vein sampling provides an opportunity to model effects of one-lung ventilation. The study's primary objectives were to establish a model of intra-operative, bilateral pulmonary vein sampling and to determine whether neutrophil function differed after passing through inflated or deflated lungs. MethodsSeventeen patients having "on pump" coronary artery bypass grafting (CABG) with one-lung ventilation (in two cohorts with tidal volume 2ml kg(-1) and FiO(2) 0.21, or tidal volume 4 ml kg(-1) and FiO(2) 0.5 respectively) were recruited. Cohort 1 consisted of 9 patients (7 male, median age 62.0 years) and Cohort 2 consisted of 8 male patients (median age 65.5 years). Recruitment was via prospective screening of scheduled elective and non-elective CABG procedures with cardiopulmonary bypass. Each patient had five blood samples taken-central venous blood pre-operatively; central venous blood pre-CPB; central venous blood post-CPB; pulmonary venous blood draining the ventilated lung post-CPB; and pulmonary venous blood draining the deflated lung post-CPB. Neutrophil phagocytosis and priming status were quantified. Plasma cytokines were measured. ResultsPhagocytosis and priming were not significantly different in neutrophils returning from the ventilated lung as compared to the non-ventilated lung. Plasma IL-6, IL-8 and IL-10 were significantly elevated by CPB. ConclusionsThe intra-operative, bilateral pulmonary vein sampling model provides unique opportunities to assess biological effects of interventions to one lung, with the other lung acting as an internal control. Single-lung ventilation during CPB had no significant effects on neutrophil function.

Background: The diagnosis of pneumonia has been hampered by a reliance on bacterial cultures which take several days to return a result, and are frequently negative. In critically ill patients this leads to the use of empiric, broad-spectrum antimicrobials and compromises good antimicrobial stewardship. The objective of this study was to establish the performance of a syndromic molecular diagnostic approach, using a custom TaqMan array card (TAC) covering 52 respiratory pathogens, and assess its impact on antimicrobial prescribing. Methods: The TAC was validated against a retrospective multi-centre cohort of broncho-alveolar lavage samples. The TAC was assessed prospectively in patients undergoing investigation for suspected pneumonia, with a comparator cohort formed of patients investigated when the TAC laboratory team were unavailable. Co-primary outcomes were sensitivity compared to conventional microbiology and, for the prospective study, time to result. Metagenomic sequencing was performed to validate findings in prospective samples. Antibiotic free days (AFD) were compared between the study cohort and comparator group. Results: 128 stored samples were tested, with sensitivity of 97% (95% confidence interval (CI) 88-100%). Prospectively, 95 patients were tested by TAC, with 71 forming the comparator group. TAC returned results 51 hours (interquartile range 41-69 hours) faster than culture and with sensitivity of 92% (95% CI 83-98%) compared to conventional microbiology. 94% of organisms identified by sequencing were detected by TAC. There was a significant difference in the distribution of AFDs with more AFDs in the TAC group (p=0.02). TAC group were more likely to experience antimicrobial de-escalation (odds ratio 2.9 (95%1.5-5.5)). Conclusions: Implementation of a syndromic molecular diagnostic approach to pneumonia led to faster results, with high sensitivity and impact on antibiotic prescribing.

Inflammatory responses are important in cancer, particularly in the context of monocyte-rich aggressive myeloid neoplasm. We developed a label-free cellular phenotypic drug discovery assay to identify anti-inflammatory drugs in human monocytes derived from acute myeloid leukemia (AML), by tracking several features ionizing from only 2500 cells using matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry. A proof-of-concept screen showed that the BCR-ABL inhibitor nilotinib, but not the structurally similar imatinib, blocks inflammatory responses. In order to identify the cellular (off-)targets of nilotinib, we performed thermal proteome profiling (TPP). Unlike imatinib, nilotinib and other later-generation BCR-ABL inhibitors bind to p38α and inhibit the p38α-MK2/3 signaling axis, which suppressed pro-inflammatory cytokine expression, cell adhesion, and innate immunity markers in activated monocytes derived from AML. Thus, our study provides a tool for the discovery of new anti-inflammatory drugs, which could contribute to the treatment of inflammation in myeloid neoplasms and other diseases.

Introduction Sepsis is characterised by a dysregulated immune response to infection, with exaggerated pro-inflammatory and anti-inflammatory responses. A predominant immunosuppressive profile affecting both innate and adaptive immune responses is associated with increased hospital-acquired infection and reduced infection-free survival. While hospital-acquired infection leads to additional antibiotic use, the role of the immunosuppressive phenotype in guiding complex decisions, such as those affecting antibiotic stewardship, is uncertain. This study is a mechanistic substudy embedded within a multicentre clinical and cost-effectiveness trial of biomarker-guided antibiotic stewardship. This mechanistic study aims to determine the effect of sepsis-associated immunosuppression on the trial outcome measures.Methods and analysis RISC-sepsis is a prospective, multicentre, exploratory, observational study embedded within the ADAPT-sepsis trial. A subgroup of 180 participants with antibiotics commenced for suspected sepsis, enrolled in the ADAPT-sepsis trial, will be recruited. Blood samples will be collected on alternate days until day 7. At each time point, blood will be collected for flow cytometric analysis into cell preservation tubes. Immunophenotyping will be performed at a central testing hub by flow cytometry. The primary outcome measures are monocyte human leucocyte antigen-DR; neutrophil CD88; programmed cell death-1 on monocytes, neutrophils and T lymphocytes and the percentage of regulatory T cells. Secondary outcome measures will link to trial outcomes from the ADAPT-sepsis trial including antibiotic days; occurrence of hospital-acquired infection and length of ICU-stay and hospital-stay.Ethics and dissemination Ethical approval has been granted (IRAS 209815) and RISC-sepsis is registered with the ISRCTN (86837685). Study results will be disseminated by peer-reviewed publications, presentations at scientific meetings and via patient and public participation groups and social media.

Acute Respiratory Distress Syndrome (ARDS), an often-fatal complication of pulmonary or systemic inflammation, has no cure. Hypoxemia is a defining feature, yet its impact on inflammation is often neglected. Patients with ARDS are monocytopenic early in the onset of the disease. Endotoxin or Streptococcus pneumoniae acute lung injury (ALI) in the context of hypoxia replicates this finding, through hypoxia-driven suppression of type I interferon signalling. This results in failed lung monocyte-derived interstitial macrophages (IM) niche expansion and unchecked neutrophilic inflammation. Administration of colony stimulating factor 1 (CSF1) rescues the monocytopenia, alters the circulating classical monocyte phenotype in hypoxic endotoxin-driven ALI and enables lung IM population expansion, thus limiting lung injury in endotoxin- and virally-induced hypoxic ALI. Hypoxia directly alters immune dynamics to the detriment of the host and manipulation of this aberrant response offers new therapeutic strategies for ARDS.

Analysis of human blood immune cells provides insights into the coordinated response to viral infections such as severe acute respiratory syndrome coronavirus 2, which causes coronavirus disease 2019 (COVID-19). We performed single-cell transcriptome, surface proteome and T and B lymphocyte antigen receptor analyses of over 780,000 peripheral blood mononuclear cells from a cross-sectional cohort of 130 patients with varying severities of COVID-19. We identified expansion of nonclassical monocytes expressing complement transcripts ( CD16 + C1QA/B/C + ) that sequester platelets and were predicted to replenish the alveolar macrophage pool in COVID-19. Early, uncommitted CD34 + hematopoietic stem/progenitor cells were primed toward megakaryopoiesis, accompanied by expanded megakaryocyte-committed progenitors and increased platelet activation. Clonally expanded CD8 + T cells and an increased ratio of CD8 + effector T cells to effector memory T cells characterized severe disease, while circulating follicular helper T cells accompanied mild disease. We observed a relative loss of IgA2 in symptomatic disease despite an overall expansion of plasmablasts and plasma cells. Our study highlights the coordinated immune response that contributes to COVID-19 pathogenesis and reveals discrete cellular components that can be targeted for therapy.

Significance GDF15, a hormone produced by a wide variety of cells undergoing different types of stress, acts on a receptor in the brain where it transmits signals that are perceived by the organism as aversive. We now report an action of GDF15, whereby it activates the endocrine stress response and increases circulating levels of the principal glucocorticoid (a “stress” steroid). By studying mice genetically deficient in GDF15, we also demonstrate that GDF15 is a key signal through which damage due to toxic chemicals activates the steroid stress response. GDF15 is currently being explored as an antiobesity drug and examination of the degree and duration of the steroid effect will need to be incorporated into any human trials.

Key Points During critical illness, PI3Kδ activation impairs neutrophil phagocytosis. PI3Kδ inhibition consistently restores neutrophil phagocytosis and bacterial killing. PI3Kδ inhibition represents a potential strategy for prevention of infection in ICUs. Visual Abstract Acquired neutrophil dysfunction frequently develops during critical illness, independently increasing the risk for intensive care unit–acquired infection. PI3Kδ is implicated in driving neutrophil dysfunction and can potentially be targeted pharmacologically. The aims of this study were to determine whether PI3Kδ inhibition reverses dysfunction in neutrophils from critically ill patients and to describe potential mechanisms. Neutrophils were isolated from blood taken from critically ill patients requiring intubation and mechanical ventilation, renal support, or blood pressure support. In separate validation experiments, neutrophil dysfunction was induced pharmacologically in neutrophils from healthy volunteers. Phagocytosis and bacterial killing assays were performed, and activity of RhoA and protein kinase A (PKA) was assessed. Inhibitors of PI3Kδ, 3-phosphoinositide-dependent protein kinase-1 (PDK1), and PKA were used to determine mechanisms of neutrophil dysfunction. Sixty-six patients were recruited. In the 27 patients (40.9%) with impaired neutrophil function, PI3Kδ inhibition consistently improved function and significantly increased bacterial killing. These findings were validated in neutrophils from healthy volunteers with salbutamol-induced dysfunction and extended to demonstrate that PI3Kδ inhibition restored killing of clinical isolates of nine pathogens commonly associated with intensive care unit–acquired infection. PI3Kδ activation was associated with PDK1 activation, which in turn phosphorylated PKA, which drove phosphorylation and inhibition of the key regulator of neutrophil phagocytosis, RhoA. These data indicate that, in a significant proportion of critically ill patients, PI3Kδ inhibition can improve neutrophil function through PDK1- and PKA-dependent processes, suggesting that therapeutic use of PI3Kδ inhibitors warrants investigation in this setting.

The nasal epithelium is a plausible entry point for SARS-CoV-2, a site of pathogenesis and transmission, and may initiate the host response to SARS-CoV-2. Antiviral interferon (IFN) responses are critical to outcome of SARS-CoV-2. Yet little is known about the interaction between SARS-CoV-2 and innate immunity in this tissue. Here we apply single-cell RNA sequencing and proteomics to a primary cell model of human nasal epithelium differentiated at air-liquid interface. SARS-CoV-2 demonstrates widespread tropism for nasal epithelial cell types. The host response is dominated by type I and III IFNs and interferon-stimulated gene products. This response is notably delayed in onset relative to viral gene expression and compared to other respiratory viruses. Nevertheless, once established, the paracrine IFN response begins to impact on SARS-CoV-2 replication. When provided prior to infection, recombinant IFNβ or IFNλ1 induces an efficient antiviral state that potently restricts SARS-CoV-2 viral replication, preserving epithelial barrier integrity. These data imply that the IFN-I/III response to SARS-CoV-2 initiates in the nasal airway and suggest nasal delivery of recombinant IFNs to be a potential chemoprophylactic strategy.

ABSTRACTInflammatory responses are important in cancer, particularly in the context of monocyte-rich aggressive myeloid neoplasm. We developed a label-free cellular phenotypic drug discovery assay to identify anti-inflammatory drugs in human monocytes derived from acute myeloid leukaemia (AML), by tracking several biological features ionizing from only 2,500 cells using matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry. A proof-of-concept screen showed that the BCR-ABL inhibitor nilotinib, but not the structurally similar imatinib, blocks inflammatory responses. In order to identify the cellular (off-)targets of nilotinib, we performed thermal proteome profiling (TPP). Unlike imatinib, nilotinib and other later generation BCR-ABL inhibitors inhibit the p38α-MK2/3 signalling axis which suppressed the expression of inflammatory cytokines, cell adhesion and innate immunity markers in activated human monocytes derived from AML. Thus, our study provides a tool for the discovery of new anti-inflammatory drugs, which could contribute to the treatment of inflammation in myeloid neoplasms and other diseases.Key PointsLabel-free cell-based assay identifies new anti-inflammatory drugs using MALDI-TOF MS. Nilotinib reduces inflammation by inhibition of MAPK14-MK2/3 signalling axis in AML.

Citation: Hellyer, T. P., McAuley, D. F., Walsh, T. S., Anderson, N. H., Conway Morris, A., Singh, S., Dark, P., Roy, A. I., Perkins, G. D., McMullan, R., Emerson, L. M. ORCID: 00000002-4250-5758, Blackwood, B., Wright, S. E., Kefala, K., O’Kane, C. M., Baudouin, S. V., Paterson, R. L., Rostron, A. J., Agus, A. M., Bannard-Smith, J., Robin, N. M., Welters, I. D., Bassford, C., Yates, B., Spencer, C., Laha, S. K., Hulme, J., Bonner, S., Linnett, V., Sonksen, J., Van Den Broeck, T., Boschman, G., Keenan, D. W. J., Scott, J., Allen, A. J., Phair, G., Parker, J., Bowett, S. A. and Simpson, A. J. (2020). More research is required to understand factors influencing antibiotic prescribing in complex conditions like suspected ventilatorassociated pneumonia. Annals of Translational Medicine, 8(13), 840.. doi: 10.21037/atm-203701

Background Neutrophils rapidly respond to and clear infection from tissues, but can also induce tissue damage through excessive degranulation, when acute inflammation proceeds unchecked. A number of key neutrophil functions, including adhesion-dependent degranulation, are controlled by src family kinases. Dasatinib is a potent src inhibitor used in treating patients with chronic myeloid leukaemia and treatment-resistant acute lymphoblastic leukaemia. We hypothesized that dasatinib would attenuate acute inflammation by inhibiting neutrophil recruitment, degranulation and endothelial cell injury, without impairing bacterial clearance, in a murine model of bacteria-induced acute lung injury. C57BL/6 mice received intratracheal Escherichia coli , and were treated with intraperitoneal dasatinib or control. Bacterial clearance, lung injury, and markers of neutrophil recruitment and degranulation were measured. Separately, human blood neutrophils were exposed to dasatinib or control, and the effects on a range of neutrophil functions assessed. Results Dasatinib was associated with a dose-dependent significant increase in E. coli in the mouse lung, accompanied by impairment of organ function, reflected in significantly increased protein leak across the alveolar-capillary membrane. However, the number of neutrophils entering the lung was unaffected, suggesting that dasatinib impairs neutrophil function independent of migration. Dasatinib did not cause direct toxicity to human neutrophils, but led to significant reductions in phagocytosis of E. coli , adhesion, chemotaxis, generation of superoxide anion and degranulation of primary and secondary granules. However, no biologically important effect of dasatinib on neutrophil degranulation was observed in mice. Conclusions Contrary to our starting hypothesis, src kinase inhibition with dasatinib had a detrimental effect on bacterial clearance in the mouse lung and therefore does not represent an attractive therapeutic strategy to treat primary infective lung inflammation. Data from human neutrophils suggest that dasatanib has inhibitory effects on a range of neutrophil functions.

Critical illness is accompanied by the release of large amounts of the anaphylotoxin, C5a. C5a suppresses antimicrobial functions of neutrophils which is associated with adverse outcomes. The signaling pathways that mediate C5a-induced neutrophil dysfunction are incompletely understood. Healthy donor neutrophils exposed to purified C5a demonstrated a prolonged defect (7 hours) in phagocytosis of Staphylococcus aureus. Phosphoproteomic profiling of 2712 phosphoproteins identified persistent C5a signaling and selective impairment of phagosomal protein phosphorylation on exposure to S. aureus. Notable proteins included early endosomal marker ZFYVE16 and V-ATPase proton channel component ATPV1G1. An assay of phagosomal acidification demonstrated C5a-induced impairment of phagosomal acidification, which was recapitulated in neutrophils from critically ill patients. Examination of the C5a-impaired protein phosphorylation indicated a role for the PI3K VPS34 in phagosomal maturation. Inhibition of VPS34 impaired neutrophil phagosomal acidification and killing of S. aureus. This study provides a phosphoproteomic assessment of human neutrophil signaling in response to S. aureus and its disruption by C5a, identifying a defect in phagosomal maturation and mechanisms of immune failure in critical illness.

BACKGROUND:Ventilator-associated pneumonia is the most common intensive care unit (ICU)-acquired infection, yet accurate diagnosis remains difficult, leading to overuse of antibiotics. Low concentrations of IL-1β and IL-8 in bronchoalveolar lavage fluid have been validated as effective markers for exclusion of ventilator-associated pneumonia. The VAPrapid2 trial aimed to determine whether measurement of bronchoalveolar lavage fluid IL-1β and IL-8 could effectively and safely improve antibiotic stewardship in patients with clinically suspected ventilator-associated pneumonia.METHODS:VAPrapid2 was a multicentre, randomised controlled trial in patients admitted to 24 ICUs from 17 National Health Service hospital trusts across England, Scotland, and Northern Ireland. Patients were screened for eligibility and included if they were 18 years or older, intubated and mechanically ventilated for at least 48 h, and had suspected ventilator-associated pneumonia. Patients were randomly assigned (1:1) to biomarker-guided recommendation on antibiotics (intervention group) or routine use of antibiotics (control group) using a web-based randomisation service hosted by Newcastle Clinical Trials Unit. Patients were randomised using randomly permuted blocks of size four and six and stratified by site, with allocation concealment. Clinicians were masked to patient assignment for an initial period until biomarker results were reported. Bronchoalveolar lavage was done in all patients, with concentrations of IL-1β and IL-8 rapidly determined in bronchoalveolar lavage fluid from patients randomised to the biomarker-based antibiotic recommendation group. If concentrations were below a previously validated cutoff, clinicians were advised that ventilator-associated pneumonia was unlikely and to consider discontinuing antibiotics. Patients in the routine use of antibiotics group received antibiotics according to usual practice at sites. Microbiology was done on bronchoalveolar lavage fluid from all patients and ventilator-associated pneumonia was confirmed by at least 104 colony forming units per mL of bronchoalveolar lavage fluid. The primary outcome was the distribution of antibiotic-free days in the 7 days following bronchoalveolar lavage. Data were analysed on an intention-to-treat basis, with an additional per-protocol analysis that excluded patients randomly assigned to the intervention group who defaulted to routine use of antibiotics because of failure to return an adequate biomarker result. An embedded process evaluation assessed factors influencing trial adoption, recruitment, and decision making. This study is registered with ISRCTN, ISRCTN65937227, and ClinicalTrials.gov, NCT01972425.FINDINGS:Between Nov 6, 2013, and Sept 13, 2016, 360 patients were screened for inclusion in the study. 146 patients were ineligible, leaving 214 who were recruited to the study. Four patients were excluded before randomisation, meaning that 210 patients were randomly assigned to biomarker-guided recommendation on antibiotics (n=104) or routine use of antibiotics (n=106). One patient in the biomarker-guided recommendation group was withdrawn by the clinical team before bronchoscopy and so was excluded from the intention-to-treat analysis. We found no significant difference in the primary outcome of the distribution of antibiotic-free days in the 7 days following bronchoalveolar lavage in the intention-to-treat analysis (p=0·58). Bronchoalveolar lavage was associated with a small and transient increase in oxygen requirements. Established prescribing practices, reluctance for bronchoalveolar lavage, and dependence on a chain of trial-related procedures emerged as factors that impaired trial processes.INTERPRETATION:Antibiotic use remains high in patients with suspected ventilator-associated pneumonia. Antibiotic stewardship was not improved by a rapid, highly sensitive rule-out test. Prescribing culture, rather than poor test performance, might explain this absence of effect.FUNDING:UK Department of Health and the Wellcome Trust.

Mononuclear phagocytes (MPs) including monocytes, macrophages and dendritic cells (DCs) are critical innate immune effectors and initiators of the adaptive immune response. MPs are present in the alveolar airspace at steady state, however little is known about DC recruitment in acute pulmonary inflammation. Here we use lipopolysaccharide inhalation to induce acute inflammation in healthy volunteers and examine the impact on bronchoalveolar lavage fluid and blood MP repertoire. Classical monocytes and two DC subsets (DC2/3 and DC5) are expanded in bronchoalveolar lavage fluid 8 h after lipopolysaccharide inhalation. Surface phenotyping, gene expression profiling and parallel analysis of blood indicate recruited DCs are blood-derived. Recruited monocytes and DCs rapidly adopt typical airspace-resident MP gene expression profiles. Following lipopolysaccharide inhalation, alveolar macrophages strongly up-regulate cytokines for MP recruitment. Our study defines the characteristics of human DCs and monocytes recruited into bronchoalveolar space immediately following localised acute inflammatory stimulus in vivo.