Immunosubversion du Lymphocyte Natural Killer par Pseudomonas aeruginosa

Pseudomonas aeruginosa (PA) est un pathogène opportuniste responsable d’infections pulmonaires chez le patient immunodéprimé. Parmi ses facteurs de virulence, PA exprime le système de sécrétion de type III (SSTIII) et ses effecteurs (Exoenzymes S, T et Y). Les cellules Natural Killer (NK) jouent un rôle clef dans la défense antibactérienne et particulièrement anti-PA. Leur activation est dépendante du microenvironnement myéloïde. Les NKs présentent 2 fonctions principales : La sécrétion de cytokine et la libération de granules cytotoxiques capables de lyser les cellules anormales. Nous avons étudié l’effet de PA sur ces 2 aspects. Les manipulations in-vitro ont été réalisées sur cellules de volontaires sains (PBMC), NK triées à partir de PBMC et 2 lignées NK humaines (NK92 et NK3.3). Un modèle de pneumonie murine à PA nous a permis de confirmer nos hypothèses in vivo. L’activité cytotoxique a été évaluée par exposition des NKs à des cibles déficientes en HLA de type I (lignée 721.221). Au cours de l’infection, la NK nécessite une stimulation IL-12 pour synthétiser de l’IFN-g. PA augmente la réponse IFN-g comparée à la stimulation IL-12 en condition non infectée. Cette modulation nécessite un contact direct entre PA et la cellule. Parmi les effecteurs du SSTIII, l’ExoT régule la réponse IFN-g via un mécanisme dépendant de ERK. Concernant la fonction cytotoxique, l’activité de la NK diminuait de façon importante après infection à PA. Cette altération de fonction est multifactorielle avec notamment une modification du répertoire activateur (NKG2D) de la cellule NK et une influence du microenvironnement en particulier des lymphocytes T.Pseudomonas aeruginosa (PA) is an opportunistic pathogen that causes lung infections in immunosuppressed patients. Among its virulence factor PA expresses the type III secretion system (T3SS) and effector Exoenzymes (ExoS, T and Y). Natural killer (NK) cell plays a key role in anti-bacterial immunity especially after PA infection. Their activation is highly dependent on their microenvironment especially on myeloid cells. NK cell exhibits two main functions: Cytokines production and cytotoxicity toward stressed or abnormal cells. We studied PA influence on these two main functions. We used peripheral blood mononuclear cells (PBMC), sorted human NK cells and two human NK cell lines (NK92, NK3.3) for in vitro experiments and a PA-pneumonia mouse model to validate our hypothesis in vivo. Degranulation was assessed by cytotoxicity assay, exposing NK cells to 721.221 targets lacking HLA-A, B and C class I antigens. NK cells required IL-12 priming to produce IFN-g in response to the infection and PA increased IFN-g activity as compared to IL-12 stimulation in noninfected conditions. The modulation of IFN-g production after PA infection required bacteria-to-cell contact. Among T3SS and its effector, ExoT is the key regulator of IFN-g activity through a ERK dependant signalisation. Our hypotheses were confirmed in vivo. Alongside with cytokine function, CD107a activity, a surrogate marker of degranulation (Cytotoxic function), dramatically decreased after NK cells infection with PA. Cytotoxicity impairment could be explained by the modification of NK cells receptor expression after infection (notably NKG2D) or accessory cells, especially T cells

Role of IL-12 in overcoming the low responsiveness of NK cells to missing self after traumatic brain injury

International audienceBlood samples from 32 patients with severe Traumatic brain injury (TBI) were studied and compared with 11 cardiac surgery patients, and 29 healthy controls. A dramatic decreased expression of HLA class I molecules on monocytes was associated with increased KIR+ NK cell frequency in TBI patients. Overall, the phenotype of TBI NK cells marked by KIR and CD57 expression and lower level of NKp46 and DNAM-1 reflected a differentiated state. The NK-cell response to missing self was marked by lower degranulation and lower IFN-γ production after stimulation with HLA class I deficient cell line. In contrast, the NK-cell ADCC was not altered. IL-12 was able to restore both IFN-γ production and the cytotoxicity capacities of NK cells. This study provides the first extensive description of the phenotype and functions of NK cells in TBI patients. Further evaluation of IL-12 treatment to overcome immunosuppression-induced nosocomial infections is warranted

Cortisol total/CRP ratio for the prediction of hospital-acquired pneumonia and initiation of corticosteroid therapy in traumatic brain- injured patients

International audienceBACKGROUND:To propose a combination of blood biomarkers for the prediction of hospital-acquired pneumonia (HAP) and for the selection of traumatic brain-injured (TBI) patients eligible for corticosteroid therapy for the prevention of HAP.METHODS:This was a sub-study of the CORTI-TC trial, a multicenter, randomized, double-blind, controlled trial evaluating the risk of HAP at day 28 in 336 TBI patients treated or not with corticosteroid therapy. Patients were between 15 and 65 years with severe traumatic brain injury (Glasgow coma scale score ≤ 8 and trauma-associated lesion on brain CT scan) and were enrolled within 24 h of trauma. The blood levels of CRP and cortisoltotal&free, as a surrogate marker of the pro/anti-inflammatory response balance, were measured in samples collected before the treatment initiation. Endpoint was HAP on day 28.RESULTS:Of the 179 patients with available samples, 89 (49.7%) developed an HAP. Cortisoltotal&free and CRP blood levels upon ICU admission were not significantly different between patients with or without HAP. The cortisoltotal/CRP ratio upon admission was 2.30 [1.25-3.91] in patients without HAP and 3.36 [1.74-5.09] in patients with HAP (p = 0.021). In multivariate analysis, a cortisoltotal/CRP ratio > 3, selected upon the best Youden index on the ROC curve, was independently associated with HAP (OR 2.50, CI95% [1.34-4.64] p = 0.004). The HR for HAP with corticosteroid treatment was 0.59 (CI95% [0.34-1.00], p = 0.005) in patients with a cortisoltotal/CRP ratio > 3, and 0.89 (CI95% [0.49-1.64], p = 0.85) in patients with a ratio  3 upon admission may predict the development of HAP in severe TBI. Among these patients, corticosteroids reduce the occurrence HAP. We suggest that this ratio may select the patients who may benefit from corticosteroid therapy for the prevention of HAP

Tetrafunctional Block Copolymers Promote Lung Gene Transfer in Newborn Piglets

International audienceTetrafunctional block copolymers are molecules capable of complexing DNA. Although ineffective in vitro, studies in mice have shown that the tetrafunctional block copolymer 704 is a more efficient lung gene transfer agent than the cationic liposome GL67A, previously used in a phase II clinical trial in cystic fibrosis patients. In the present study, we compared the gene transfer capacity of the 704-DNA formulation and a cationic liposome-DNA formulation equivalent to GL67A in a larger-animal model, the newborn piglet. Our results indicate an efficacy of the 704-DNA formulation well above one order of magnitude higher than that of the cationic liposome-DNA formulation, with no elevated levels of interleukin-6 (IL-6), taken as a marker of inflammation. Transgene expression was heterogeneous within lung lobes, with expression levels that were below the detection threshold in some samples, while high in other samples. This heterogeneity is likely to be due to the bolus injection procedure as well as to the small volume of injection. The present study highlights the potential of tetrafunctional block copolymers as non-viral vectors for lung gene therapy

Inhaled bacteriophage therapy in a porcine model of pneumonia caused by Pseudomonas aeruginosa during mechanical ventilation

International audienceBackground and purpose: Pseudomonas aeruginosa is a main cause of ventilator-associated pneumonia (VAP) with drug-resistant bacteria. Bacteriophage therapy has experienced resurgence to compensate for the limited development of novel antibiotics. However, phage therapy is limited to a compassionate use so far, resulting from lack of adequate studies in relevant pharmacological models. We used a pig model of pneumonia caused by P. aeruginosa that recapitulates essential features of human disease to study the antimicrobial efficacy of nebulized-phage therapy.Experimental approach: (i) Lysis kinetic assays were performed to evaluate in vitro phage antibacterial efficacy against P. aeruginosa and select relevant combinations of lytic phages. (ii) The efficacy of the phage combinations was investigated in vivo (murine model of P. aeruginosa lung infection). (iii) We determined the optimal conditions to ensure efficient phage delivery by aerosol during mechanical ventilation. (iv) Lung antimicrobial efficacy of inhaled-phage therapy was evaluated in pigs, which were anesthetized, mechanically ventilated and infected with P. aeruginosa.Key results: By selecting an active phage cocktail and optimizing aerosol delivery conditions, we were able to deliver high phage concentrations in the lungs, which resulted in a rapid and marked reduction in P. aeruginosa density (1.5 Log reduction, p<0.001). No infective phage was detected in the sera and urines throughout the experiment.Conclusion and implications: Our findings demonstrated: (i) the feasibility of delivering large amounts of active phages by nebulization during mechanical ventilation, (ii) rapid control of in situ infection by inhaled bacteriophage in an experimental model of P. aeruginosa pneumonia with high translational value