C5a impairs phagosomal maturation in the neutrophil through phosphoproteomic remodeling.

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.AJTW was a Gates Cambridge Scholar supported by the Gates Cambridge Trust from 2015-2018. ACM is supported by a Clinical Research Career Development Fellowship from the Wellcome Trust (WT 2055214/Z/16/Z). Grants to ACM from the Academy of Medical Sciences and European Society for Intensive Care Medicine supported this work. The study was also supported by the National Institute for Health Research (NIHR) Newcastle Biomedical Research Centre (IS-BRC-1215-20001) and the Medical Research Council SHIELD antimicrobial resistance consortium (MR/ N02995X/1

Excess Mucin Impairs Subglottic Epithelial Host Defense in Mechanically Ventilated Patients

Rationale: Aspiration of infective subglottic secretions causes ventilator-associated pneumonia (VAP) in mechanically ventilated patients. Mechanisms underlying subglottic colonization in critical illness have not been defined, limiting strategies for targeted prevention of VAP. Objectives: To characterize subglottic host defense dysfunction in mechanically ventilated patients in the intensive care unit (ICU). To determine whether subglottic mucin contributes to neutrophil phagocytic impairment and bacterial growth. Methods: Prospective subglottic sampling in mechanically ventilated patients (intubated for four or more days), and newly intubated control patients (intubated for less than 30 minutes). Isolation and culture of primary subglottic epithelial cells from controls. Laboratory analysis of host innate immune defenses. Measurements and Main Results: Twenty-four patients in the ICU and 27 newly intubated control patients were studied. Subglottic ICU samples had significantly reduced microbiological diversity and contained potential respiratory pathogens. The subglottic microenvironment in ICU was characterized by neutrophilic inflammation, significantly increased pro-inflammatory cytokines and neutrophil proteases, and altered physical properties of subglottic secretions, including accumulation of mucins. Subglottic mucin from ICU patients impaired the capacity of neutrophils to phagocytose and kill bacteria. Phagocytic function was reversible upon treatment with a mucolytic agent. Subglottic mucus promoted growth and invasion of bacterial pathogens in a novel air-liquid interface model of primary human subglottic epithelium. Conclusions: Mechanical ventilation in ICU is characterized by substantial mucin secretion and neutrophilic inflammation. Mucin impairs neutrophil dysfunction and promotes bacterial growth. Mucolytic agents reverse mucin-mediated neutrophil dysfunction. Enhanced mucus disruption and removal has potential to augment preventive benefits of subglottic drainage

The Enteropathogenic E. coli (EPEC) Tir Effector Inhibits NF-κB Activity by Targeting TNFα Receptor-Associated Factors

Enteropathogenic Escherichia coli (EPEC) disease depends on the transfer of effector proteins into epithelia lining the human small intestine. EPEC E2348/69 has at least 20 effector genes of which six are located with the effector-delivery system genes on the Locus of Enterocyte Effacement (LEE) Pathogenicity Island. Our previous work implied that non-LEE-encoded (Nle) effectors possess functions that inhibit epithelial anti-microbial and inflammation-inducing responses by blocking NF-κB transcription factor activity. Indeed, screens by us and others have identified novel inhibitory mechanisms for NleC and NleH, with key co-operative functions for NleB1 and NleE1. Here, we demonstrate that the LEE-encoded Translocated-intimin receptor (Tir) effector has a potent and specific ability to inhibit NF-κB activation. Indeed, biochemical, imaging and immunoprecipitation studies reveal a novel inhibitory mechanism whereby Tir interaction with cytoplasm-located TNFα receptor-associated factor (TRAF) adaptor proteins induces their proteasomal-independent degradation. Infection studies support this Tir-TRAF relationship but reveal that Tir, like NleC and NleH, has a non-essential contribution in EPEC's NF-κB inhibitory capacity linked to Tir's activity being suppressed by undefined EPEC factors. Infections in a disease-relevant intestinal model confirm key NF-κB inhibitory roles for the NleB1/NleE1 effectors, with other studies providing insights on host targets. The work not only reveals a second Intimin-independent property for Tir and a novel EPEC effector-mediated NF-κB inhibitory mechanism but also lends itself to speculations on the evolution of EPEC's capacity to inhibit NF-κB function

Diagnostic accuracy of pulmonary host inflammatory mediators in the exclusion of ventilator-acquired pneumonia.

BACKGROUND: Excessive use of empirical antibiotics is common in critically ill patients. Rapid biomarker-based exclusion of infection may improve antibiotic stewardship in ventilator-acquired pneumonia (VAP). However, successful validation of the usefulness of potential markers in this setting is exceptionally rare. OBJECTIVES: We sought to validate the capacity for specific host inflammatory mediators to exclude pneumonia in patients with suspected VAP. METHODS: A prospective, multicentre, validation study of patients with suspected VAP was conducted in 12 intensive care units. VAP was confirmed following bronchoscopy by culture of a potential pathogen in bronchoalveolar lavage fluid (BALF) at >10(4) colony forming units per millilitre (cfu/mL). Interleukin-1 beta (IL-1β), IL-8, matrix metalloproteinase-8 (MMP-8), MMP-9 and human neutrophil elastase (HNE) were quantified in BALF. Diagnostic utility was determined for biomarkers individually and in combination. RESULTS: Paired BALF culture and biomarker results were available for 150 patients. 53 patients (35%) had VAP and 97 (65%) patients formed the non-VAP group. All biomarkers were significantly higher in the VAP group (p<0.001). The area under the receiver operator characteristic curve for IL-1β was 0.81; IL-8, 0.74; MMP-8, 0.76; MMP-9, 0.79 and HNE, 0.78. A combination of IL-1β and IL-8, at the optimal cut-point, excluded VAP with a sensitivity of 100%, a specificity of 44.3% and a post-test probability of 0% (95% CI 0% to 9.2%). CONCLUSIONS: Low BALF IL-1β in combination with IL-8 confidently excludes VAP and could form a rapid biomarker-based rule-out test, with the potential to improve antibiotic stewardship

A human model of bilateral pulmonary vein sampling to assess the effects of one-lung ventilation on neutrophil function

Background: Neutrophil 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. Methods: Seventeen patients having “on pump” coronary artery bypass grafting (CABG) with one-lung ventilation (in two cohorts with tidal volume 2ml kg-1 and FiO2 0.21, or tidal volume 4 ml kg-1 and FiO2 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. Results: Phagocytosis 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. Conclusions: The 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

Exchange protein directly activated by cyclic AMP (EPAC) activation reverses neutrophil dysfunction induced by β₂-agonists, corticosteroids, and critical illness

BACKGROUND: Neutrophils play a role in the pathogenesis of asthma, chronic obstructive pulmonary disease, and pulmonary infection. Impaired neutrophil phagocytosis predicts hospital-acquired infection. Despite this, remarkably few neutrophil-specific treatments exist. OBJECTIVES: We sought to identify novel pathways for the restoration of effective neutrophil phagocytosis and to activate such pathways effectively in neutrophils from patients with impaired neutrophil phagocytosis. METHODS: Blood neutrophils were isolated from healthy volunteers and patients with impaired neutrophil function. In healthy neutrophils phagocytic impairment was induced experimentally by using β2-agonists. Inhibitors and activators of cyclic AMP (cAMP)-dependent pathways were used to assess the influence on neutrophil phagocytosis in vitro. RESULTS: β2-Agonists and corticosteroids inhibited neutrophil phagocytosis. Impairment of neutrophil phagocytosis by β2-agonists was associated with significantly reduced RhoA activity. Inhibition of protein kinase A (PKA) restored phagocytosis and RhoA activity, suggesting that cAMP signals through PKA to drive phagocytic impairment. However, cAMP can signal through effectors other than PKA, such as exchange protein directly activated by cyclic AMP (EPAC). An EPAC-activating analog of cAMP (8CPT-2Me-cAMP) reversed neutrophil dysfunction induced by β2-agonists or corticosteroids but did not increase RhoA activity. 8CPT-2Me-cAMP reversed phagocytic impairment induced by Rho kinase inhibition but was ineffective in the presence of Rap-1 GTPase inhibitors. 8CPT-2Me-cAMP restored function to neutrophils from patients with known acquired impairment of neutrophil phagocytosis. CONCLUSIONS: EPAC activation consistently reverses clinical and experimental impairment of neutrophil phagocytosis. EPAC signals through Rap-1 and bypasses RhoA. EPAC activation represents a novel potential means by which to reverse impaired neutrophil phagocytosis