Marie-Hélène Ruchaud-Sparagano

Proteasome-independent Degradation of Canonical NFκB Complex Components by the NleC Protein of Pathogenic Escherichia coli

The NFκB transcription factor is a key component of immune and inflammatory signaling as its activation induces the expression of antimicrobial reagents, chemokines, cytokines, and anti-apoptotic factors. Many pathogens encode effector proteins that target factors regulating NFκB activity and can provide novel insights on regulatory mechanisms. Given the link of NFκB dysfunction with inflammatory diseases and some cancers, these effectors have therapeutic potential. Here, screening enteropathogenic Escherichia coli proteins for those implicated in suppressing NFκB function revealed that eGFP-NleC, unlike eGFP, strongly inhibited basal and TNFα-induced NFκB reporter activity to prevent secretion of the chemokine, IL-8. Work involving NleC variants, chemical inhibitors, and immunoprecipitation studies support NleC being a zinc metalloprotease that degrades NFκB-IκBα complexes. The findings are consistent with features between residues 33–65 recruiting NFκB for proteasomal-independent degradation by a mechanism inhibited by metalloprotease inhibitors or disruption of a consensus zinc metalloprotease motif spanning NleC residues 183–187. This raises the prospect that mammalian cells, or other pathogens, employ a similar mechanism to modulate NFκB activity. Moreover, NleC represents a novel tool for validating NFκB as a therapeutic target and, indeed, as a possible therapeutic reagent.

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 EPECs NF-κB inhibitory capacity linked to Tirs 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 EPECs 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 >104 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.

Effect of granulocyte-macrophage colony-stimulating factor on neutrophil function in idiopathic bronchiectasis.

Neutrophils are consistently found in inflamed and infected airways in idiopathic bronchiectasis, but relatively little is known about the function of blood neutrophils in this condition. We hypothesized that peripheral blood neutrophil (PBN) phagocytosis and superoxide generation are impaired in bronchiectasis, and that granulocyte‐macrophage colony‐stimulating factor (GM‐CSF) is capable of improving neutrophil function.

MPLA inhibits release of cytotoxic mediators from human neutrophils while preserving efficient bacterial killing

Monophosphoryl lipid A (MPLA) is a lipopolysaccharides (LPS) derivative associated with neutrophil‐dependent anti‐inflammatory outcomes in animal models of sepsis. Little is known about the effect of MPLA on neutrophil function. This study sought to test the hypothesis that MPLA would reduce release of cytotoxic mediators from neutrophils without impairing bacterial clearance. Neutrophils were isolated from whole blood of healthy volunteers. The effects of MPLA and LPS on autologous serum‐opsonised Pseudomonas aeruginosa killing by neutrophils and phagocytosis of autologous serum‐opsonised zymosan were examined. Neutrophil oxidative burst, chemotaxis, enzyme and cytokine release as well as Toll‐like receptor 4 (TLR4) expression were assessed following exposure to LPS or MPLA. LPS, but not MPLA, induced significant release of superoxide and myeloperoxidase from neutrophils. However, MPLA did not impair neutrophil capacity to ingest microbial particles and kill P. aeruginosa efficiently. MPLA was directly chemotactic for neutrophils, involving TLR4, p38 mitogen‐activated protein kinase and tyrosine and alkaline phosphatases. LPS, but not MPLA, impaired N‐formyl‐methionyl‐leucyl phenylalanine‐directed migration of neutrophils, increased surface expression of TLR4, increased interleukin‐8 release and strongly activated the myeloid differentiation primary response 88 pathway. Phosphoinositide 3‐kinase inhibition significantly augmented IL‐8 release from MPLA‐treated neutrophils. The addition of MPLA to LPS‐preincubated neutrophils led to a significant reduction in LPS‐mediated superoxide release and TLR4 surface expression. Collectively, these findings suggest that MPLA directs efficient chemotaxis and bacterial killing in human neutrophils without inducing extracellular release of cytotoxic mediators and suggest that MPLA warrants further attention as a potential therapeutic in human sepsis.

Randomised controlled trial of GM-CSF in critically ill patients with impaired neutrophil phagocytosis

Background Critically ill patients with impaired neutrophil phagocytosis have significantly increased risk of nosocomial infection. Granulocyte-macrophage colony-stimulating factor (GM-CSF) improves phagocytosis by neutrophils ex vivo. This study tested the hypothesis that GM-CSF improves neutrophil phagocytosis in critically ill patients in whom phagocytosis is known to be impaired. Methods This was a multicentre, phase IIa randomised, placebo-controlled clinical trial. Using a personalised medicine approach, only critically ill patients with impaired neutrophil phagocytosis were included. Patients were randomised 1:1 to subcutaneous GM-CSF (3 μg/kg/day) or placebo, once daily for 4 days. The primary outcome measure was neutrophil phagocytosis 2 days after initiation of GM-CSF. Secondary outcomes included neutrophil phagocytosis over time, neutrophil functions other than phagocytosis, monocyte HLA-DR expression and safety. Results Thirty-eight patients were recruited from five intensive care units (17 randomised to GM-CSF). Mean neutrophil phagocytosis at day 2 was 57.2% (SD 13.2%) in the GM-CSF group and 49.8% (13.4%) in the placebo group, p=0.73. The proportion of patients with neutrophil phagocytosis≥50% at day 2, and monocyte HLA-DR, appeared significantly higher in the GM-CSF group. Neutrophil functions other than phagocytosis did not appear significantly different between the groups. The most common adverse event associated with GM-CSF was fever. Conclusions GM-CSF did not improve mean neutrophil phagocytosis at day 2, but was safe and appeared to increase the proportion of patients with adequate phagocytosis. The study suggests proof of principle for a pharmacological effect on neutrophil function in a subset of critically ill patients.

Excess Mucin Impairs Subglottic Epithelial Host Defense in Mechanically Ventilated Patients

&NA; 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 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 min); isolation and culture of primary subglottic epithelial cells from control patients; 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 the ICU was characterized by neutrophilic inflammation, significantly increased proinflammatory 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 impairment was reversible on 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 the ICU is characterized by substantial mucin secretion and neutrophilic inflammation. Mucin impairs neutrophil function 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.

Mitochondrial DNA depletion induces innate immune dysfunction rescued by IFN-γ

County Council (ALF), the ChAMP Consortium, the Swedish Strategical Research Foundation (SSF), the V ardal Foundation, and Karolinska Institutet. Disclosure of potential conflict of interest: S. E. Dalhen has received grants from the Swedish Science Foundation, the Swedish Research Council, and the Swedish Heart-Lung Foundation; has a board membership with RSPR Pharma; and has consultant arrangements with AstraZeneca, GlaxoSmithKline, and Regeneron. B. Dahlen has received grants from the Swedish Medical Research Council and the Swedish Heart-Lung Foundation. M. van Hage has consultant arrangements with Biomay AG and Hycor Biomedical LLC and has received payment for lectures from Thermo Fisher Scientific. S. Gabrielsson has received grants from the Swedish Research Council, the Swedish Heart-Lung Foundation, the Hesselman’s Foundation, the Cancer and Allergy Research Foundation, the Stockholm County Council (ALF), the ChAMP Consortium, and Karolinska Institutet. The rest of the authors declare that they have no relevant conflicts of interest.

P142 Src kinase inhibition attenuates neutrophil degranulation without impairing bacterial killing: a possible therapeutic strategy for acute lung injury?

Background A key mechanism in the pathogenesis of acute lung injury (ALI) is excessive neutrophil degranulation in response to an overwhelming inflammatory or infective insult. To date, no pharmacological therapy for ALI has proven beneficial. Aim To investigate our hypothesis that extracellular neutrophil degranulation can be inhibited without necessarily impairing phagocytosis or live bacterial killing. Methods Whole blood or purified neutrophils from healthy volunteers were pre-treated with a src kinase inhibitor (PP1) or vehicle control, before stimulation with either Phorbol 12-myristate 13-acetate (PMA), cytochalasin B + N-formylmethionyl-leucyl-phenylalanine (fMLP), lipopolysaccharide (LPS), live serum-opsonized Staphylococcus aureus (SA) or Pseudomonas aeruginosa (PA), to induce degranulation. Degranulation was measured in whole blood using CD63/CD66b expression and in purified neutrophils by extracellular release of myeloperoxidase (MPO) and lactoferrin (LTF). Neutrophil phagocytosis of fluorescent killed bacteria, cell viability, apoptosis and bacterial killing (by serial dilution and colony counting) were also measured. All experiments carried out using n = 4–6 healthy volunteers. Results PP1 pre-treatment using concentrations above 10 μM significantly attenuated primary and secondary granule exocytosis from healthy neutrophils in response to LPS, cytochalasin B/fMLP, SA and PA but not to PMA. The same effect was observed in whole blood assays and in purified neutrophils, both in free suspension and when adhered to tissue culture plastic. PP1 treatment did not increase neutrophil death in response to the stimuli, nor did it significantly alter baseline apoptosis rates. Importantly, PP1 did not impair neutrophil phagocytosis or live bacterial killing of SA and PA. Conclusions Our study supports our hypothesis and proposes src kinases as an attractive target for anti-inflammatory therapy in conditions such as ALI.