Alexis Broquet

Hypoxia modulates infection of epithelial cells by Pseudomonas aeruginosa.

Pseudomonas aeruginosa (P. aeruginosa) is an opportunistic pathogen commonly associated with lung and wound infections. Hypoxia is a frequent feature of the microenvironment of infected tissues which induces the expression of genes associated with innate immunity and inflammation in host cells primarily through the activation of the hypoxia-inducible factor (HIF) and Nuclear factor kappaB (NF-κB) pathways which are regulated by oxygen-dependent prolyl-hydroxylases. Hypoxia also affects virulence and antibiotic resistance in bacterial pathogens. However, less is known about the impact of hypoxia on host-pathogen interactions such as bacterial adhesion and infection. In the current study, we demonstrate that hypoxia decreases the internalization of P. aeruginosa into cultured epithelial cells resulting in decreased host cell death. This response can also be elicited by the hydroxylase inhibitor Dimethyloxallyl Glycine (DMOG). Reducing HIF-2α expression or Rho kinase activity diminished the effects of hypoxia on P. aeruginosa infection. Furthermore, in an in vivo pneumonia infection model, application of DMOG 48 h before infection with P. aeruginosa significantly reduced mortality. Thus, hypoxia reduces P. aeruginosa internalization into epithelial cells and pharmacologic manipulation of the host pathways involved may represent new therapeutic targets in the treatment of P. aeruginosa infection.

Efficacy of ceftolozane in a murine model of Pseudomonas aeruginosa acute pneumonia: in vivo antimicrobial activity and impact on host inflammatory response

OBJECTIVES To assess the activity of ceftolozane, a novel oxyimino-cephalosporin, in comparison with ceftazidime and piperacillin/tazobactam against a multidrug-resistant Pseudomonas aeruginosa strain using a murine model of pneumonia. METHODS Quantitative bacteriology, survival, histological examination, myeloperoxidase activity, proinflammatory cytokine levels in lungs and endothelial permeability were evaluated to determine the effects of ceftolozane and comparators on P. aeruginosa-induced pneumonia. RESULTS After 48 h of treatment, ceftolozane reduced the bacterial load by 3-4 log(10) cfu/g of lung. Systemic dissemination of the pulmonary infection and development of lung damage were inhibited in all β-lactam-treated animals. P. aeruginosa-induced pneumonia led to elevated concentrations of tumour necrosis factor-α, interleukin (IL)-1β and macrophage inflammatory protein (MIP)-2 in the lungs. While the levels of proinflammatory cytokines decreased following ceftazidime and piperacillin/tazobactam therapy, ceftolozane exhibited increased concentrations of IL-1β and MIP-2 after 24 h of infection, resulted in significantly increased levels of recruited neutrophils within the infected lung without increasing lung endothelial permeability. CONCLUSIONS These data strongly support ceftolozane as an effective option for the treatment of severe P. aeruginosa respiratory infections by improving the early pulmonary inflammatory response without impairing 48 h post-infection homeostasis.

Depletion of natural killer cells increases mice susceptibility in a Pseudomonas aeruginosa pneumonia model.

Objectives:Pseudomonas aeruginosa infection is a clinically relevant infection involved in pneumonia in ICUs. Understanding the type of immune response initiated by the host during pneumonia would help defining new strategies to interfere with the bacteria pathogenicity. In this setting, the role of natural killer cells remains controversial. We assessed the role of systemic natural killer cells in a Pseudomonas aeruginosa mouse pneumonia model. Design:Experimental study. Setting:Research laboratory from a university hospital. Subjects:RjOrl:SWISS and BALB/cJ mice (weight, 20–24 g). Interventions:Lung injuries were assessed by bacterial load, myeloperoxidase activity, endothelial permeability (pulmonary edema), immune cell infiltrate (histological analysis), proinflammatory cytokine release, and Ly6-G immunohistochemistry. Bacterial loads were assessed in the lungs and spleen. Natural killer cell number and status were assessed in spleen (flow cytometry and quantitative polymerase chain reaction). Depletion of natural killer cells was achieved through an IV anti-asialo-GM1 antibody injection. Measurements and Main Results:Pseudomonas aeruginosa tracheal instillation led to an acute pneumonia with a rapid decrease of bacterial load in lungs and with an increase of endothelial permeability, proinflammatory cytokines (tumor necrosis factor-&agr; and interleukin-1&bgr;), and myeloperoxidase activity followed by Ly6-G positive cell infiltrate in lungs. Pseudomonas aeruginosa was detected in the spleen. Membrane markers of activation and maturation (CD69 and KLRG1 molecules) were increased in splenic natural killer cells during Pseudomonas aeruginosa infection. Splenic natural killer cells activated upon Pseudomonas aeruginosa infection produced interferon-&ggr; but not interleukin-10. Ultimately, mice depleted of natural killer cells displayed an increased neutrophil numbers in the lungs and an increased mortality rate without bacterial load modifications in the lungs, indicating that mice depleted of natural killer cells were much more susceptible to infection compared with control animals. Conclusions:We report for the first time that natural killer cells play a major role in the mice susceptibility toward a Pseudomonas aeruginosa–induced acute pneumonia model.

Hydrocortisone prevents immunosuppression by interleukin-10+ natural killer cells after trauma-hemorrhage.

Objective:Trauma induces a state of immunosuppression, which is responsible for the development of nosocomial infections. Hydrocortisone reduces the rate of pneumonia in patients with trauma. Because alterations of dendritic cells and natural killer cells play a central role in trauma-induced immunosuppression, we investigated whether hydrocortisone modulates the dendritic cell/natural killer cell cross talk in the context of posttraumatic pneumonia. Design:Experimental study. Settings:Research laboratory from an university hospital. Subjects:Bagg Albino/cJ mice (weight, 20–24 g). Interventions:First, in an a priori substudy of a multicenter, randomized, double-blind, placebo-controlled trial of hydrocortisone (200 mg/d for 7 d) in patients with severe trauma, we have measured the blood levels of five cytokines (tumor necrosis factor-&agr;, interleukin-6, interleukin-10, interleukin-12, interleukin-17) at day 1 and day 8. In a second step, the effects of hydrocortisone on dendritic cell/natural killer cell cross talk were studied in a mouse model of posttraumatic pneumonia. Hydrocortisone (0.6 mg/mice i.p.) was administered immediately after hemorrhage. Twenty-four hours later, the mice were challenged with Staphylococcus aureus (7 × 105 colony-forming units). Measurements and Main Results:Using sera collected during a multicenter study in patients with trauma, we found that hydrocortisone decreased the blood level of interleukin-10, a cytokine centrally involved in the regulation of dendritic cell/natural killer cell cluster. In a mouse model of trauma-hemorrhage–induced immunosuppression, splenic natural killer cells induced an interleukin-10–dependent elimination of splenic dendritic cell. Hydrocortisone treatment reduced this suppressive function of natural killer cells and increased survival of mice with posthemorrhage pneumonia. The reduction of the interleukin-10 level in natural killer cells by hydrocortisone was partially dependent on the up-regulation of glucocorticoid-induced tumor necrosis factor receptor-ligand (TNFsf18) on dendritic cell. Conclusions:These data demonstrate that trauma-induced immunosuppression is characterized by an interleukin-10–dependent elimination of dendritic cell by natural killer cells and that hydrocortisone improves outcome by limiting this immunosuppressive feedback loop.

Toll-like receptor-4 agonist in post-haemorrhage pneumonia: role of dendritic and natural killer cells

Haemorrhage-induced immunosuppression has been linked to nosocomial infections. We assessed the impact of monophosphoryl lipid A, a Toll/interleukin-1 receptor-domain-containing adaptor protein inducing interferon-biased Toll-like receptor-4 agonist currently used as a vaccine adjuvant in humans, on post-haemorrhage susceptibility to infection. We used a mouse model of post-haemorrhage pneumonia induced by methicillin-susceptible Staphylococcus aureus. Monophosphoryl lipid A was administered intravenously after haemorrhage and before pneumonia onset. Haemorrhage altered survival rate, increased lung damage (neutrophil accumulation, oedema and cytokine release) and altered the functions of dendritic and natural killer cells. Here, we show that monophosphoryl lipid A decreased systemic dissemination of S. aureus and dampened inflammatory lung lesions. Monophosphoryl lipid A partially restored the capacity for antigen presentation and the transcriptional activity in dendritic cells. Monophosphoryl lipid A did not restore the interferon-&ggr; mRNA but prevented interleukin-10 mRNA overexpression in natural killer cells compared with untreated mice. Ex vivo monophosphoryl lipid A-stimulated dendritic cells or natural killer cells harvested from haemorrhaged animals were adoptively transferred into mice undergoing post-haemorrhage pneumonia. Stimulated dendritic cells (but not stimulated natural killer cells) improved the survival rate compared with mice left untreated. In vivo depletion of natural killer cells decreased survival rate of monophosphoryl lipid A-treated mice. Dendritic and natural killer cells are critically involved in the beneficial effects of monophosphoryl lipid A within post-haemorrhage pneumonia. Dendritic cells and NK cells are critically involved in the beneficial effects of MPLA within post-haemorrhage pneumonia http://ow.ly/oc4Zp

Linezolid Dampens Neutrophil-Mediated Inflammation in Methicillin-Resistant Staphylococcus aureus–Induced Pneumonia and Protects the Lung of Associated Damages

BACKGROUND Linezolid is considered as a therapeutic alternative to the use of glycopeptides for the treatment of pneumonia caused by methicillin-resistant Staphylococcus aureus (MRSA). Clinical studies reported a potent survival advantage conferred by the oxazolidinone and called into question the use of glycopeptides as first-line therapy. METHODS In a mouse model of MRSA-induced pneumonia, quantitative bacteriology, proinflammatory cytokine concentrations in lung, myeloperoxidase activity, Ly6G immunohistochemistry, and endothelial permeability were assessed to compare therapeutic efficacy and immunomodulative properties of linezolid and vancomycin administered subcutaneously every 12 hours. RESULTS Significant antibacterial activity was achieved after 48 hours of treatment for linezolid and vancomycin. Levels of interleukin 1β, a major proinflammatory cytokine, and macrophage inflammatory protein 2, a chemokine involved in the recruitment of neutrophils, were decreased by both antimicrobials. Only linezolid was able to dramatically reduce the production of tumor necrosis factor α. Analysis of myeloperoxidase activity and Ly6G immunostaining showed a dramatic decrease of neutrophil infiltration in infected lung tissues for linezolid-treated animals. A time-dependent increase of endothelial permeability was observed for the control and vancomycin regimens. Of interest, in the linezolid group, decreased endothelial permeability was detected 48 hours after infection. CONCLUSIONS Our results indicate that linezolid could be superior to vancomycin for the management of MRSA pneumonia by attenuating an excessive inflammatory reaction and protecting the lung from pathogen-associated damages.

Pathogenic potential of Escherichia coli clinical strains from orthopedic implant infections towards human osteoblastic cells

Escherichia coli is one of the first causes of Gram-negative orthopedic implant infections (OII), but little is known about the pathogenicity of this species in such infections that are increasing due to the ageing of the population. We report how this pathogen interacts with human osteoblastic MG-63 cells in vitro, by comparing 20 OII E. coli strains to two Staphylococcus aureus and two Pseudomonas aeruginosa strains. LDH release assay revealed that 6/20 (30%) OII E. coli induced MG-63 cell lysis whereas none of the four control strains was cytotoxic after 4 h of coculture. This high cytotoxicity was associated with hemolytic properties and linked to hlyA gene expression. We further showed by gentamicin protection assay and confocal microscopy that the non-cytotoxic E. coli were not able to invade MG-63 cells unlike S. aureus strains (internalization rate <0.01% for the non-cytotoxic E. coli versus 8.88 ± 2.31% and 4.60 ± 0.42% for both S. aureus). The non-cytotoxic E. coli also demonstrated low adherence rates (<7%), the most adherent E. coli eliciting higher IL-6 and TNF-α mRNA expression in the osteoblastic cells. Either highly cytotoxic or slightly invasive OII E. coli do not show the same infection strategies as S. aureus towards osteoblasts.

Hypoxia reduces the pathogenicity of Pseudomonas aeruginosa by decreasing the expression of multiple virulence factors

Our understanding of how the course of opportunistic bacterial infection is influenced by the microenvironment is limited. We demonstrate that the pathogenicity of Pseudomonas aeruginosa strains derived from acute clinical infections is higher than that of strains derived from chronic infections, where tissues are hypoxic. Exposure to hypoxia attenuated the pathogenicity of strains from acute (but not chronic) infections, implicating a role for hypoxia in regulating bacterial virulence. Mass spectrometric analysis of the secretome of P. aeruginosa derived from an acute infection revealed hypoxia-induced repression of multiple virulence factors independent of altered bacterial growth. Pseudomonas aeruginosa lacking the Pseudomonas prolyl-hydroxylase domain-containing protein, which has been implicated in bacterial oxygen sensing, displays reduced virulence factor expression. Furthermore, pharmacological hydroxylase inhibition reduces virulence factor expression and pathogenicity in a murine model of pneumonia. We hypothesize that hypoxia reduces P. aeruginosa virulence at least in part through the regulation of bacterial hydroxylases.

Interleukin-22 level is negatively correlated with neutrophil recruitment in the lungs in a Pseudomonas aeruginosa pneumonia model

Pseudomonas aeruginosa is a major threat for immune-compromised patients. Bacterial pneumonia can induce uncontrolled and massive neutrophil recruitment ultimately leading to acute respiratory distress syndrome and epithelium damage. Interleukin-22 plays a central role in the protection of the epithelium. In this study, we aimed to evaluate the role of interleukin-22 and its soluble receptor IL-22BP in an acute Pseudomonas aeruginosa pneumonia model in mice. In this model, we noted a transient increase of IL-22 during Pseudomonas aeruginosa challenge. Using an antibody-based approach, we demonstrated that IL-22 neutralisation led to increased susceptibility to infection and to lung damage correlated with an increase in neutrophil accumulation in the lungs. On the contrary, rIL-22 administration or IL-22BP neutralisation led to a decrease in mouse susceptibility and lung damage associated with a decrease in neutrophil accumulation. This study demonstrated that the IL-22/IL-22BP system plays a major role during Pseudomonas aeruginosa pneumonia by moderating neutrophil accumulation in the lungs that ultimately leads to epithelium protection.

Apoptosis induced by Pseudomonas aeruginosa: a lonely killer?

Apoptosis is a fundamental biological process allowing tissue homeostasis through the regulation of cell populations by eliminating unnecessary elements. During infection, pathogens have evolved to take advantage of this process for their own and are able to induce the apoptosis of cells, i.e. immune cells by the host itself. Pseudomonas aeruginosa is one of the most studied opportunistic bacteria due to its significant involvement worldwide in pneumonia, corneal infections and wound burns. Several research groups have pointed out the ability of these bacteria to interfere and/or evade host immune system by inducing apoptosis of the targeted cells. In May 2014, looking up ‘Pseudomonas aeruginosa’ and ‘apoptosis’ keywords in PubMed search engine retrieve more than 300 hits. Pseudomonas aeruginosa seems to induce apoptosis through direct interaction with the host cells (the most studied system being the type-III secretion system: T3SS) or through secreting factors such as pyocyanin. T3SS, the most well-studied virulence apparatus of P. aeruginosa is composed of a needle complex through which exoenzymes are injected into the host cells (Galle et al., 2012,2012). Recently, Beyaerts laboratory described an exotoxin-independent function of the T3SS in the killing of macrophages in an acute lung infection model (Galle et al., 2012,2012). Although, T3SS is a major virulence system, it is not fully required for the bacteria to display virulence as T3SS negative strains are shown to exihibit signficant virulence (example of Elsens paper). In short, a wide variety of P. aeruginosa virulence factors are involved in inducing apoptosis by several distinct mechanisms, from the activation of the mitochondrial pathway, the generation of reactive oxygen species to the activation of the caspase pathways (Table 1). Table 1 Virulence factors inducing apoptosis in targeted host cells (non-exhaustive list) The ability of P. aeruginosa to induce apoptosis in various in vitro model of infection (macrophages, neutrophils, epithelial cells …) or in vivo models such as lungs, cornea and burn wounds infections is not mediated by a single bacterial cell but rather by a multicellular population of P. aeruginosa. Members of such population interact with each other through a number of chemical signals known globally on for quorum sensing (QS). Quoting Rutherford and Bassler (2012), ‘Quorum sensing is a bacterial cell-cell communication process that involves the production, detection, and response to extracellular signaling molecules called autoinducers’. QS molecules were shown to regulate virulence factors such as toxins, exotoxin A, pyocyanin, … and so in fine apoptosis (Rutherford and Bassler, 2012). The best described QS signalling systems in P. aeruginosa are the N-acyl homoserine lactones systems Las and Rhl. The Las system produces and responds to N-oxododecanoyl homoserine lactone and the Rhl system to N-butanoyl homoserine lactone respectively. Las system is known to control the production of various virulence factors involved in host cell damages such as exotoxin A (Jones et al., 1993). On the other hand, Rhl system was described to repress the expression of genes responsible for the assembly and function of the T3SS (Bleves et al., 2005). Last but not least, QS molecule such as 3-oxododecanoyl-L-homoserine lactone (3-oxo-C12-HSL) itself has been shown to induce apoptosis. Several studies have demonstrated that incubation of different cell lines with 3-oxo-C12-HSL molecule resulted in the induction of apoptosis involving calcium signalling, the mitochondrial pathway and caspase activations (Table 2). Interestingly, N-butanoyl-L-homoserine lactone (known as C4-HSL, the second major QS molecule in P. aeruginosa) harbouring a shorter fatty acid chain has not been shown to induce apoptosis compared with 3-oxo-C12-HSL (Tateda et al., 2003; Holban et al., 2014). Table 2 Apoptosis pathways activated by 3-oxo-C12-HSL (non-exhaustive list) Knowing that P. aeruginosa is able to induce apoptosis through its QS systems molecule, studies focusing on apoptosis induction should be considered with the context of QS signalling. Particularly, QS considerations should be taken into account when comparing studies using different multiplicity of infection or bacteria preparation protocols, processes that influence QS molecules concentration and/or bacteria population numbers.