Publications by authors named "Mustapha Si-Tahar"

70 Publications

Lung compartmentalization of inflammatory biomarkers in COVID-19-related ARDS.

Crit Care 2021 03 24;25(1):120. Epub 2021 Mar 24.

INSERM, Centre d'Etude des Pathologies Respiratoires (CEPR), UMR 1100, Tours, France.

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http://dx.doi.org/10.1186/s13054-021-03513-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7988241PMC
March 2021

Outcome of SARS-CoV-2 infection is linked to MAIT cell activation and cytotoxicity.

Nat Immunol 2021 03 2;22(3):322-335. Epub 2021 Feb 2.

Université de Paris, Institut Cochin, Inserm U1016, Centre National de la Recherche Scientifique UMR 8104, Inflamex Laboratory, Paris, France.

Immune system dysfunction is paramount in coronavirus disease 2019 (COVID-19) severity and fatality rate. Mucosal-associated invariant T (MAIT) cells are innate-like T cells involved in mucosal immunity and protection against viral infections. Here, we studied the immune cell landscape, with emphasis on MAIT cells, in cohorts totaling 208 patients with various stages of disease. MAIT cell frequency is strongly reduced in blood. They display a strong activated and cytotoxic phenotype that is more pronounced in lungs. Blood MAIT cell alterations positively correlate with the activation of other innate cells, proinflammatory cytokines, notably interleukin (IL)-18, and with the severity and mortality of severe acute respiratory syndrome coronavirus 2 infection. We also identified a monocyte/macrophage interferon (IFN)-α-IL-18 cytokine shift and the ability of infected macrophages to induce the cytotoxicity of MAIT cells in an MR1-dependent manner. Together, our results suggest that altered MAIT cell functions due to IFN-α-IL-18 imbalance contribute to disease severity, and their therapeutic manipulation may prevent deleterious inflammation in COVID-19 aggravation.
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http://dx.doi.org/10.1038/s41590-021-00870-zDOI Listing
March 2021

Controlled Heat and Humidity-Based Treatment for the Reuse of Personal Protective Equipment: A Pragmatic Proof-of-Concept to Address the Mass Shortage of Surgical Masks and N95/FFP2 Respirators and to Prevent the SARS-CoV2 Transmission.

Front Med (Lausanne) 2020 20;7:584036. Epub 2020 Oct 20.

Université de Tours, Tours, France.

The coronavirus infectious disease-2019 (COVID-19) pandemic has led to an unprecedented shortage of healthcare resources, primarily personal protective equipment like surgical masks, and N95/filtering face piece type 2 (FFP2) respirators. Reuse of surgical masks and N95/FFP2 respirators may circumvent the supply chain constraints and thus overcome mass shortage. Methods, design, setting, and measurement: Herein, we tested the effects of dry- and moist-air controlled heating treatment on structure and chemical integrity, decontamination yield, and filtration performance of surgical masks and FFP2 respirators. We found that treatment in a climate chamber at 70°C during 1 h with 75% humidity rate was adequate for enabling substantial decontamination of both respiratory viruses, oropharyngeal bacteria, and model animal coronaviuses, while maintaining a satisfying filtering capacity. Further studies are now required to confirm the feasibility of the whole process during routine practice. Our findings provide compelling evidence for the recycling of pre-used surgical masks and N95/FFP2 respirators in case of imminent mass shortfall.
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http://dx.doi.org/10.3389/fmed.2020.584036DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7607499PMC
October 2020

Evidence of early increased sialylation of airway mucins and defective mucociliary clearance in CFTR-deficient piglets.

J Cyst Fibros 2021 Jan 23;20(1):173-182. Epub 2020 Sep 23.

Univ.lille, CNRS, UMR8576-UGSF-Unité de Glycobiologie Structurale et Fonctionnelle, Lille F59000, France. Electronic address:

Background: Bacterial colonization in cystic fibrosis (CF) lungs has been directly associated to the loss of CFTR function, and/or secondarily linked to repetitive cycles of chronic inflammation/infection. We hypothesized that altered molecular properties of mucins could contribute to this process.

Methods: Newborn CFTR and CFTR were sacrificed before and 6 h after inoculation with luminescent Pseudomonas aeruginosa into the tracheal carina. Tracheal mucosa and the bronchoalveolar lavage (BAL) fluid were collected to determine the level of mucin O-glycosylation, bacteria binding to mucins and the airways transcriptome. Disturbances in mucociliary transport were determined by ex-vivo imaging of luminescent Pseudomonas aeruginosa.

Results: We provide evidence of an increased sialylation of CF airway mucins and impaired mucociliary transport that occur before the onset of inflammation. Hypersialylation of mucins was reproduced on tracheal explants from non CF animals treated with GlyH101, an inhibitor of CFTR channel activity, indicating a causal relationship between the absence of CFTR expression and the sialylation of mucins. This increased sialylation was correlated to an increased adherence of P. aeruginosa to mucins. In vivo infection of newborn CF piglets by live luminescent P. aeruginosa demonstrated an impairment of mucociliary transport of this bacterium, with no evidence of pre-existing inflammation.

Conclusions: Our results document for the first time in a well-defined CF animal model modifications that affect the O-glycan chains of mucins. These alterations precede infection and inflammation of airway tissues, and provide a favorable context for microbial development in CF lung that hallmarks this disease.
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http://dx.doi.org/10.1016/j.jcf.2020.09.009DOI Listing
January 2021

High Dimensional Single-Cell Analysis Reveals iNKT Cell Developmental Trajectories and Effector Fate Decision.

Cell Rep 2020 09;32(10):108116

INSERM, Centre d'Etude des Pathologies Respiratoires (CEPR), UMR 1100, Tours, France; Université de Tours, Faculté de Médecine de Tours, Tours, France. Electronic address:

CD1d-restricted invariant Natural Killer T (iNKT) cells represent a unique class of T lymphocytes endowed with potent regulatory and effector immune functions. Although these functions are acquired during thymic ontogeny, the sequence of events that gives rise to discrete effector subsets remains unclear. Using an unbiased single-cell transcriptomic analysis combined with functional assays, we reveal an unappreciated diversity among thymic iNKT cells, especially among iNKT1 cells. Mathematical modeling and biological methods unravel a developmental map whereby iNKT2 cells constitute a transient branching point toward the generation of iNKT1 and iNKT17 cells, which reconciles the two previously proposed models. In addition, we identify the transcription co-factor Four-and-a-half LIM domains protein 2 (FHL2) as a critical cell-intrinsic regulator of iNKT1 specification. Thus, these data illustrate the changing transcriptional network that guides iNKT cell effector fate.
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http://dx.doi.org/10.1016/j.celrep.2020.108116DOI Listing
September 2020

Phenotypical and functional alteration of unconventional T cells in severe COVID-19 patients.

J Exp Med 2020 12;217(12)

Institut national de la santé et de la recherche médicale, Centre d'Etude des Pathologies Respiratoires, UMR 1100, Tours, France.

COVID-19 includes lung infection ranging from mild pneumonia to life-threatening acute respiratory distress syndrome (ARDS). Dysregulated host immune response in the lung is a key feature in ARDS pathophysiology. However, cellular actors involved in COVID-19-driven ARDS are poorly understood. Here, in blood and airways of severe COVID-19 patients, we serially analyzed unconventional T cells, a heterogeneous class of T lymphocytes (MAIT, γδT, and iNKT cells) with potent antimicrobial and regulatory functions. Circulating unconventional T cells of COVID-19 patients presented with a profound and persistent phenotypic alteration. In the airways, highly activated unconventional T cells were detected, suggesting a potential contribution in the regulation of local inflammation. Finally, expression of the CD69 activation marker on blood iNKT and MAIT cells of COVID-19 patients on admission was predictive of clinical course and disease severity. Thus, COVID-19 patients present with an altered unconventional T cell biology, and further investigations will be required to precisely assess their functions during SARS-CoV-2-driven ARDS.
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http://dx.doi.org/10.1084/jem.20200872DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7472174PMC
December 2020

Intrinsic alterations in peripheral neutrophils from cystic fibrosis newborn piglets.

J Cyst Fibros 2020 09 9;19(5):830-836. Epub 2020 Mar 9.

ISP, INRA, Université de Tours, UMR 1282, 37380, Nouzilly, France. Electronic address:

Background: The hallmark of the cystic fibrosis (CF) lung disease is a neutrophil dominated lung environment that is associated to chronic lung tissue destruction and ultimately the patient's death. It is unclear whether the exacerbated neutrophil response is primary related to a defective CFTR or rather secondary to chronic bacterial colonization and inflammation. Here, we hypothesized that CF peripheral blood neutrophils present intrinsic alteration at birth before the start of an inflammatory process.

Methods: Peripheral blood neutrophils were isolated from newborn CFTR and CFTR piglets. Neutrophils immunophenotype was evaluated by flow cytometry. Lipidomic and proteomic profile were characterized by liquid chromatography/tandem mass spectrometry (LC-MS/MS), intact cell matrix-assisted laser desorption/ionization mass spectrometry (ICM-MS) followed by top-down high-resolution mass spectrometry (HRMS), respectively. The ability of CF neutrophils to kill pseudomonas aeruginosa was also evaluated.

Results: Polyunsaturated fatty acid metabolites analysis did not show any difference between CFTR and CFTR neutrophils. On the other hand, a predictive mathematical model based on the ICM-MS proteomic profile was able to discriminate between both genotypes. Top-down proteomic analysis identified 19 m/z differentially abundant masses that corresponded mainly to proteins related to the antimicrobial response and the generation of reactive oxygen species (ROS). However, no alteration in the ability of CFTR neutrophils to kill pseudomonas aeruginosa in vitro was observed.

Conclusions: ICM-MS demonstrated that CFTR neutrophils present intrinsic alterations already at birth, before the presence of any infection or inflammation.
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http://dx.doi.org/10.1016/j.jcf.2020.02.016DOI Listing
September 2020

CFTR-deficient pigs display alterations of bone microarchitecture and composition at birth.

J Cyst Fibros 2020 05 29;19(3):466-475. Epub 2019 Nov 29.

Université de Reims Champagne Ardenne, BIOS EA 4691, Biomatériaux et Inflammation en site osseux, SFR CAP-Santé (FED 4231), 1, Avenue du Maréchal Juin, 51097 Reims, France. Electronic address:

Background: The lack of cystic fibrosis transmembrane conductance regulator (CFTR) function causes cystic fibrosis (CF), predisposing to severe lung disease, reduced growth and osteopenia. Both reduced bone content and strength are increasingly recognized in infants with CF before the onset of significant lung disease, suggesting a developmental origin and a possible role in bone disease pathogenesis. The role of CFTR in bone metabolism is unclear and studies on humans are not feasible. Deletion of CFTR in pigs (CFTR pigs) displays at birth severe malformations similar to humans in the intestine, respiratory tract, pancreas, liver, and male reproductive tract.

Methods: We compared bone parameters of CFTR male and female pigs with those of their wild-type (WT) littermates at birth. Morphological and microstructural properties of femoral cortical and trabecular bone were evaluated using micro-computed tomography (μCT), and their chemical compositions were examined using Raman microspectroscopy.

Results: The integrity of the CFTR bone was altered due to changes in its microstructure and chemical composition in both sexes. Low cortical thickness and high cortical porosity were found in CFTR pigs compared to sex-matched WT littermates. Moreover, an increased chemical composition heterogeneity associated with higher carbonate/phosphate ratio and higher mineral crystallinity was found in CFTR trabecular bone, but not in CFTR cortical bone.

Conclusions: The loss of CFTR directly alters the bone composition and metabolism of newborn pigs. Based on these findings, we speculate that bone defects in patients with CF could be a primary, rather than a secondary consequence of inflammation and infection.
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http://dx.doi.org/10.1016/j.jcf.2019.10.023DOI Listing
May 2020

Interleukin-7 protects against bacterial respiratory infection by promoting IL-17A-producing innate T-cell response.

Mucosal Immunol 2020 01 18;13(1):128-139. Epub 2019 Oct 18.

Centre d'Infection et d'Immunité de Lille, Inserm U1019, CNRS UMR 8204, University of Lille, CHU Lille- Institut Pasteur de Lille, 59000, Lille, France.

Interleukin-7 (IL-7) is a critical cytokine in B- and T-lymphocyte development and maturation. Recent evidence suggests that IL-7 is a preferential homeostatic and survival factor for RORγt innate T cells such as natural killer T (NKT) cells, γδT cells, and mucosal-associated invariant T (MAIT) cells in the periphery. Given the important contribution of these populations in antibacterial immunity at barrier sites, we questioned whether IL-7 could be instrumental in boosting the local host immune response against respiratory bacterial infection. By using a cytokine-monoclonal antibody approach, we illustrated a role for topical IL-7 delivery in increasing the pool of RORγt IL-17A-producing innate T cells. Prophylactic IL-7 treatment prior to Streptococcus pneumoniae infection led to better bacterial containment, a process associated with increased neutrophilia and that depended on γδT cells and IL-17A. Last, combined delivery of IL-7 and α-galactosylceramide (α-GalCer), a potent agonist for invariant NKT (iNKT) cells, conferred an almost total protection in terms of survival, an effect associated with enhanced IL-17 production by innate T cells and neutrophilia. Collectively, we provide a proof of concept that IL-7 enables fine-tuning of innate T- cell functions. This might pave the way for considering IL-7 as an innovative biotherapeutic against bacterial infection.
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http://dx.doi.org/10.1038/s41385-019-0212-yDOI Listing
January 2020

Lipoxygenase LoxA Contributes to Lung Infection by Altering the Host Immune Lipid Signaling.

Front Microbiol 2019 14;10:1826. Epub 2019 Aug 14.

INSERM, Centre d'Etude des Pathologies Respiratoires (CEPR), UMR 1100, Tours, France.

is an opportunistic bacteria and a major cause of nosocomial pneumonia. has many virulence factors contributing to its ability to colonize the host. LoxA is a lipoxygenase enzyme secreted by that oxidizes polyunsaturated fatty acids. Based on previous biochemical studies, several biological roles of LoxA have been hypothesized, including interference of the host lipid signaling, and modulation of bacterial invasion properties. However, the contribution of LoxA to lung pathogenesis remained unclear. In this study, we used complementary and approaches, clinical strains of as well as lipidomics technology to investigate the role of LoxA in lung infection. We found that several clinical isolates express LoxA. When secreted in the lungs, LoxA processes a wide range of host polyunsaturated fatty acids, which further results in the production of bioactive lipid mediators (including lipoxin A). LoxA also inhibits the expression of major chemokines (e.g., MIPs and KC) and the recruitment of key leukocytes. Remarkably, LoxA promotes persistence in lungs tissues. Hence, our study suggests that LoxA-dependent interference of the host lipid pathways may contribute to lung pathogenesis.
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http://dx.doi.org/10.3389/fmicb.2019.01826DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6702342PMC
August 2019

In a murine model of acute lung infection, airway administration of a therapeutic antibody confers greater protection than parenteral administration.

J Control Release 2019 06 11;303:24-33. Epub 2019 Apr 11.

INSERM, Centre d'Etude des Pathologies Respiratoires, U1100, F-37032 Tours, France; Université de Tours, F-37032 Tours, France. Electronic address:

Due to growing antibiotic resistance, pneumonia caused by Pseudomonas aeruginosa is a major threat to human health and is driving the development of novel anti-infectious agents. Preventively or curatively administered pathogen-specific therapeutic antibodies (Abs) have several advantages, including a low level of toxicity and a unique pharmacological profile. At present, most Abs against respiratory infections are administered parenterally; this may not be optimal for therapeutics that have to reach the lungs to be effective. Although the airways constitute a logical delivery route for biologics designed to treat respiratory diseases, there are few scientific data on the advantages or disadvantages of this route in the context of pneumonia treatment. The objective of the present study was to evaluate the efficacy and fate of an anti-P. aeruginosa Ab targeting pcrV (mAb166) as a function of the administration route during pneumonia. The airway-administered mAb166 displayed a favorable pharmacokinetic profile during the acute phase of the infection, and was associated with greater protection (relative to other delivery routes) of infected animals. Airway administration was associated with lower levels of lung inflammation, greater bacterial clearance, and recruitment of neutrophils in the airways. In conclusion, the present study is the first to have compared the pharmacokinetics and efficacy of an anti-infectious Ab administered by different routes in an animal model of pneumonia. Our findings suggest that local delivery to the airways is associated with a more potent anti-bacterial response (relative to parenteral administration), and thus open up new perspectives for the prevention and treatment of pneumonia with Abs.
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http://dx.doi.org/10.1016/j.jconrel.2019.04.005DOI Listing
June 2019

Tissue kallikrein regulates alveolar macrophage apoptosis early in influenza virus infection.

Am J Physiol Lung Cell Mol Physiol 2019 06 25;316(6):L1127-L1140. Epub 2019 Mar 25.

INSERM, U1100-Centre d'Etude des Pathologies Respiratoires , Tours , France.

Host cell proteases are involved in influenza pathogenesis. We examined the role of tissue kallikrein 1 (KLK1) by comparing wild-type (WT) and KLK1-deficient mice infected with influenza H3N2 virus. The levels of KLK1 in lung tissue and in bronchoalveolar lavage (BAL) fluid increased substantially during infection. KLK1 did not promote virus infectivity despite its trypsin-like activity, but it did decrease the initial virus load. We examined two cell types involved in the early control of pathogen infections, alveolar macrophages (AMs) and natural killer (NK) cells to learn more about the antiviral action of KLK1. Inactivating the gene or treating WT mice with an anti-KLK1 monoclonal antibody to remove KLK1 activity accelerated the initial virus-induced apoptotic depletion of AMs. Intranasal instillation of deficient mice with recombinant KLK1 (rKLK1) reversed the phenotype. The levels of granulocyte-macrophage colony-stimulating factor in infected BAL fluid were significantly lower in KLK1-deficient mice than in WT mice. Treating lung epithelial cells with rKLK1 increased secretion of this factor known to enhance AM resistance to pathogen-induced apoptosis. The recruitment of NK cells to the air spaces peaked 3 days after infection in WT mice but not in KLK1-deficient mice, as did increases in several NK-attracting chemokines (CCL2, CCL3, CCL5, and CXCL10) in BAL. Chronic obstructive pulmonary disease (COPD) patients are highly susceptible to viral infection, and we observed that the mRNA levels decreased with increasing COPD severity. Our findings indicate that KLK1 intervenes early in the antiviral defense modulating the severity of influenza infection. Decreased KLK1 expression in COPD patients could contribute to the worsening of influenza.
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http://dx.doi.org/10.1152/ajplung.00379.2018DOI Listing
June 2019

Inactivation of the interleukin-22 pathway in the airways of cystic fibrosis patients.

Cytokine 2019 01 28;113:470-474. Epub 2018 Oct 28.

Institut National de la Santé et de la Recherche Médicale, Centre d'Etude des Pathologies Respiratoires (CEPR), INSERM UMR 1100, 37032 Tours, France; Université de Tours, F-37032 Tours, France. Electronic address:

Interleukin (IL)-22 plays a critical role in regulating the maintenance of the mucosal barrier. As airway epithelial regeneration is abnormal in cystic fibrosis (CF), we investigated IL-22 integrity in CF. We first demonstrated, using Il-22-/- mice, that IL-22 is important to prevent lung damage induced by the CF pathogen Pseudomonas aeruginosa. Next, IL-22 receptor was found normally expressed at the airway epithelial surfaces of CF patients. In wound-healing assays, IL-22-treated CF cultures had higher wound-closure rate than controls, suggesting that IL-22 signaling per se could be functional in a CF context. However, persistence of neutrophil-derived serine-proteases is a major feature of CF airways. Remarkably, IL-22 was found altered in this protease-rich inflammatory microenvironment; the serine protease-3 being the most prone to fully degrade IL-22. Consequently, we suspect an acquired deficiency of the IL-22 pathway in the lungs of CF patients due to IL-22 cleavage by the surrounding neutrophil serine-proteases.
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http://dx.doi.org/10.1016/j.cyto.2018.10.015DOI Listing
January 2019

Ten-year trends in intensive care admissions for respiratory infections in the elderly.

Ann Intensive Care 2018 Aug 15;8(1):84. Epub 2018 Aug 15.

Université de Tours, Tours, France.

Background: The consequences of the ageing population concerning ICU hospitalisation need to be adequately described. We believe that this discussion should be disease specific. A focus on respiratory infections is of particular interest, because it is strongly associated with old age. Our objective was to assess trends in demographics over a decade among elderly patients admitted to the ICU for acute respiratory infections.

Methods: A cross-sectional study was performed between 2006 and 2015 based on hospital discharge databases in one French region (2.5 million inhabitants). Patients with acute respiratory infection were selected according to the specific ICD-10 diagnosis codes recorded, including acute exacerbation of chronic obstructive pulmonary disease (AECOPD) and community-acquired pneumonia (CAP). We also identified comorbid conditions based on any significant ICD-10 secondary diagnoses adapted from the Charlson and Elixhauser indexes.

Results: A total of 98,381 hospital stays for acute respiratory infection were identified among the 3,856,785 stays over the 10-year period. The number of patients 75 y/o and younger increased 1.6-fold from 2006 to 2015, whereas the numbers of patients aged 85-89 and ≥ 90 y/o increased by 2.5- and 2.1-fold, respectively. Both CAP and AECOPD hospitalisations significantly increased for all age groups over the decade. ICU hospitalisations for respiratory infection increased 2.7-fold from 2006 to 2015 (p = 0.0002). The greatest increases in the use of ICU resources were for the 85-89 and ≥ 90 y/o groups, which corresponded to increases of 3.3- and 5.8-fold. Indeed, the proportion of patients hospitalized for respiratory infection in ICU that were elderly clearly grew during the decade: 11.3% were ≥ 85 y/o in 2006 versus 16.4% in 2015 (p < 0.0001). This increase in ICU hospitalisation rate of ageing patients was not associated with significant changes in the level of care or ICU mortality except for patients ≥ 90 y/o (for whom ICU mortality dropped from 40.9 to 22.3%, p = 0.03).

Conclusion: We observed a substantial increase in acute respiratory infection diagnoses associated with hospitalisation between 2006 and 2015, with a growing demand for critical care services. Both the absolute number and the percentage of elderly patient ICU admissions increased over the last decade, with the greatest increases being observed for patients 85 years and older.
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http://dx.doi.org/10.1186/s13613-018-0430-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6093821PMC
August 2018

Treatment of Pseudomonas aeruginosa Biofilm Present in Endotracheal Tubes by Poly-l-Lysine.

Antimicrob Agents Chemother 2018 11 24;62(11). Epub 2018 Oct 24.

INSERM, Centre d'Etude des Pathologies Respiratoires, U1100, Tours, France

The endotracheal tube (ETT) is an essential interface between the patient and ventilator in mechanically ventilated patients. However, a microbial biofilm is formed gradually on this tube and is associated with the development of ventilator-associated pneumonia. The bacteria present in the biofilm are more resistant to antibiotics, and current medical practices do not make it possible to eliminate. is one of the leading pathogens that cause biofilm infections and ventilator-associated pneumonia. Poly-l-lysine (pLK) is a cationic polypeptide possessing antibacterial properties and mucolytic activity by compacting DNA. Here, we explored the antibiofilm activity of pLK to treat biofilms on ETTs while taking into consideration the necessary constraints for clinical translation in our experimental designs. First, we showed that pLK eradicates a biofilm formed on 96-well microplates. We further demonstrated that pLK alters bacterial membrane integrity, as revealed by scanning electron microscopy, and eventually eradicates biofilm formed either by reference or clinical strains of biofilms generated on ETTs. Second, we collected the ETT from patients with ventilator-associated pneumonia. We observed that a single dose of pLK is able to immediately disrupt the biofilm structure and kills more than 90% of bacteria present in the biofilm. Additionally, we did not observe any lung tolerance issue when the pLK solution was instilled into the ETT of ventilated pigs, an animal model particularly relevant to mimic invasive mechanical ventilation in humans. In conclusion, pLK appears as an innovative antibiofilm molecule, which could be applied in the ETT of mechanically ventilated patients.
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http://dx.doi.org/10.1128/AAC.00564-18DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6201101PMC
November 2018

Pseudomonas aeruginosa flagellum is critical for invasion, cutaneous persistence and induction of inflammatory response of skin epidermis.

Virulence 2018 ;9(1):1163-1175

a Laboratoire Inflammation Tissus Epithéliaux et Cytokines EA 4331 , Université de Poitiers , Poitiers , France.

Pseudomonas aeruginosa, an opportunistic pathogen involved in skin and lung diseases, possesses numerous virulence factors, including type 2 and 3 secretion systems (T2SS and T3SS) and its flagellum, whose functions remain poorly known during cutaneous infection. Using isogenic mutants deleted from genes encoding each or all of these three virulence factors, we investigated their role in induction of inflammatory response and in tissue invasiveness in human primary keratinocytes and reconstructed epidermis. Our results showed that flagellum, but not T2SS and T3SS, is involved in induction of a large panel of cytokine, chemokine, and antimicrobial peptide (AMP) mRNA in the infected keratinocytes. Chemokine secretion and AMP tissular production were also dependent on the presence of the bacterial flagellum. This pro-inflammatory effect was significantly reduced in keratinocytes infected in presence of anti-toll-like receptor 5 (TLR5) neutralizing antibody. Bacterial invasion of human epidermis and persistence in a mouse model of sub-cutaneous infection were dependent on the P. aeruginosa flagellum. We demonstrated that flagellum constitutes the main virulence factor of P. aeruginosa involved not only in early induction of the epidermis inflammatory response but also in bacterial invasion and cutaneous persistence. P. aeruginosa is mainly sensed by TLR5 during the early innate immune response of human primary keratinocytes.
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http://dx.doi.org/10.1080/21505594.2018.1480830DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6086312PMC
October 2018

Impact of the TAP-like transporter in antigen presentation and phagosome maturation.

Mol Immunol 2019 09 23;113:75-86. Epub 2018 Jun 23.

Institut National de la Sante et de la Recherche Médicale, Unité 1151; Université Paris Descartes, Faculté de médecine; Centre National de la Recherche Scientifique, UMR8253; 149 rue de Sèvres, 75743 Paris Cedex 15, France. Electronic address:

Cross-presentation is thought to require transport of proteasome-generated peptides by the TAP transporters into MHC class I loading compartments for most antigens. However, a proteasome-dependent but TAP-independent pathway has also been described. Depletion of the pool of recycling cell surface MHC class I molecules available for loading with cross-presented peptides might partly or largely account for the critical role of TAP in cross-presentation of phagocytosed antigens. Here we examined a potential role of the homodimeric lysosomal TAP-like transporter in cross-presentation and in presentation of endogenous peptides by MHC class II molecules. We find that TAP-L is strongly recruited to dendritic cell phagosomes at a late stage, when internalized antigen and MHC class I molecules have been degraded or sorted away from phagosomes. Cross-presentation of a receptor-targeted antigen in vitro and of a phagocytosed antigen in vivo, as well as presentation of a cytosolic antigen by MHC class II molecules, is not affected by TAP-L deficiency. However, accumulation in vitro of a peptide optimally adapted to TAP-L selectivity in purified phagosomes is abolished by TAP-L deficiency. Unexpectedly, we find that TAP-L deficiency accelerates phagosome maturation, as reflected in increased Lamp2b recruitment and enhanced proteolytic degradation of phagocytosed antigen and in vitro transported peptides. Although additional experimentation will be required to definitely conclude on the role of TAP-L in transport of peptides presented by MHC class I and class II molecules, our data suggest that the principal role of TAP-L in dendritic cells may be related to regulation of phagosome maturation.
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http://dx.doi.org/10.1016/j.molimm.2018.06.268DOI Listing
September 2019

Thymic Program Directing the Functional Development of γδT17 Cells.

Front Immunol 2018 8;9:981. Epub 2018 May 8.

INSERM, Centre d'Etude des Pathologies Respiratoires (CEPR), UMR 1100, Tours, France.

γδT cells comprise a unique T cell sublineage endowed with a wide functional repertoire, which allow them to play important-sometimes opposite-roles in many immune responses associated with infection, cancer, and inflammatory processes. This is largely dependent on the existence of pre-programmed discrete functional subsets that differentiate within the thymus at specific temporal windows of life. Since they represent a major early source of interleukin-17A in many models of immune responses, the γδT17 cell population has recently gained considerable interest. Thus, a better dissection of the developmental program of this effector γδT subset appears critical in understanding their associated immune functions. Several recent reports have provided new exciting insights into the developmental mechanisms that control γδT cell lineage commitment and differentiation. Here, we review the importance of thymic cues and intrinsic factors that shape the developmental program of γδT17 cells. We also discuss the potential future areas of research in γδT17 cell development especially in regards to the recently provided data from deep RNA sequencing technology. Pursuing our understanding into this complex mechanism will undoubtedly provide important clues into the biology of this particular T cell sublineage.
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http://dx.doi.org/10.3389/fimmu.2018.00981DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5951931PMC
July 2019

Exploration of the role of the virulence factor ElrA during Enterococcus faecalis cell infection.

Sci Rep 2018 01 29;8(1):1749. Epub 2018 Jan 29.

Micalis Institute, INRA, AgroParisTech, Université Paris-Saclay, 78350, Jouy en Josas, France.

Enterococcus faecalis, an organism generally not pathogenic for healthy humans, has the potential to cause disease in susceptible hosts. While it seems to be equipped to interact with and circumvent host immune defense, most of the molecular and cellular mechanisms underlying the enterococcal infectious process remain elusive. Here, we investigated the role of the Enterococcal Leucine Rich protein A (ElrA), an internalin-like protein of E. faecalis also known as a virulence factor. ElrA was previously shown to prevent adhesion to macrophages. We show that ElrA does not inhibit the basic phagocytic process, but is able to prevent sensing and migration of macrophages toward E. faecalis. Presence or absence of FHL2, a eukaryotic partner of ElrA, does not affect the ElrA-dependent mechanism preventing macrophage migration. However, we highlight a partial contribution of FHL2 in ElrA-mediated virulence in vivo. Our results indicate that ElrA plays at least a dual role of which anti-phagocytic activity may contribute to dissemination of extracellular E. faecalis during infection.
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http://dx.doi.org/10.1038/s41598-018-20206-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5788860PMC
January 2018

Th17 cytokines: novel potential therapeutic targets for COPD pathogenesis and exacerbations.

Eur Respir J 2017 10 12;50(4). Epub 2017 Oct 12.

Université de Lille, U1019 - UMR 8204, Lung Infection and Innate Immunity, Center for Infection and Immunity of Lille (CIIL), Lille, France

Chronic obstructive pulmonary disease (COPD) is a chronic inflammatory disease of the airways caused mainly by cigarette smoke exposure. COPD progression is marked by exacerbations of the disease, often associated with infections. Recent data show the involvement in COPD pathophysiology of interleukin (IL)-17 and IL-22, two cytokines that are important in the control of lung inflammation and infection. During the initiation and progression of the disease, increased IL-17 secretion causes neutrophil recruitment, leading to chronic inflammation, airways obstruction and emphysema. In the established phase of COPD, a defective IL-22 response facilitates pathogen-associated infections and disease exacerbations. Altered production of these cytokines involves a complex network of immune cells and dysfunction of antigen-presenting cells. In this review, we describe current knowledge on the involvement of IL-17 and IL-22 in COPD pathophysiology at steady state and during exacerbations, and discuss implications for COPD management and future therapeutic approaches.
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http://dx.doi.org/10.1183/13993003.02434-2016DOI Listing
October 2017

FPR2: A Novel Promising Target for the Treatment of Influenza.

Front Microbiol 2017 5;8:1719. Epub 2017 Sep 5.

Aix Marseille Univ, INSERM, INRA, NORT, UMR 1260/1062Marseille, France.

The Formyl-peptide receptor-2 (FPR2) is a seven transmembrane G protein-coupled receptor, which plays an important role in sensing of bacteria and modulation of immune responses. FPR2 is also used by viruses for their own profit. Annexin A1, one of the multiple ligands of FPR2, is incorporated in the budding virus membrane of influenza A viruses (IAV). Thereby, once IAV infect a host cell, FPR2 is activated. FPR2-signaling leads to an increase in viral replication, a dysregulation of the host immune response and a severe disease. Conversely, experiments using FPR2 antagonists in a preclinical model of IAV infections in mice showed that blocking FPR2 protects animals from lethal infections. Thus, FPR2 represents a very attractive host target against influenza. In this review we will give an overview on the pathogenesis of influenza with a focus on the role of FPR2 and we will discuss the advantages of using FPR2 antagonists to treat the flu.
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http://dx.doi.org/10.3389/fmicb.2017.01719DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5591951PMC
September 2017

Kallikrein-Related Peptidase 5 Contributes to H3N2 Influenza Virus Infection in Human Lungs.

J Virol 2017 08 27;91(16). Epub 2017 Jul 27.

INSERM U1100, Centre d'Etude des Pathologies Respiratoires, Faculté de Médecine, Tours, France

Hemagglutinin (HA) of influenza virus must be activated by proteolysis before the virus can become infectious. Previous studies indicated that HA cleavage is driven by membrane-bound or extracellular serine proteases in the respiratory tract. However, there is still uncertainty as to which proteases are critical for activating HAs of seasonal influenza A viruses (IAVs) in humans. This study focuses on human KLK1 and KLK5, 2 of the 15 serine proteases known as the kallikrein-related peptidases (KLKs). We find that their mRNA expression in primary human bronchial cells is stimulated by IAV infection. Both enzymes cleaved recombinant HA from several strains of the H1 and/or H3 virus subtype , but only KLK5 promoted the infectivity of A/Puerto Rico/8/34 (H1N1) and A/Scotland/20/74 (H3N2) virions in MDCK cells. We assessed the ability of treated viruses to initiate influenza in mice. The nasal instillation of only the KLK5-treated virus resulted in weight loss and lethal outcomes. The secretion of this protease in the human lower respiratory tract is enhanced during influenza. Moreover, we show that pretreatment of airway secretions with a KLK5-selective inhibitor significantly reduced the activation of influenza A/Scotland/20/74 virions, providing further evidence of its importance. Differently, increased KLK1 secretion appeared to be associated with the recruitment of inflammatory cells in human airways regardless of the origin of inflammation. Thus, our findings point to the involvement of KLK5 in the proteolytic activation and spread of seasonal influenza viruses in humans. Influenza A viruses (IAVs) cause acute infection of the respiratory tract that affects millions of people during seasonal outbreaks every year. Cleavage of the hemagglutinin precursor by host proteases is a critical step in the life cycle of these viruses. Consequently, host proteases that activate HA can be considered promising targets for the development of new antivirals. However, the specific proteases that activate seasonal influenza viruses, especially H3N2 viruses, in the human respiratory tract have remain undefined despite many years of work. Here we demonstrate that the secreted, extracellular protease KLK5 (kallikrein-related peptidase 5) is efficient in promoting the infectivity of H3N2 IAV and Furthermore, we found that its secretion was selectively enhanced in the human lower respiratory tract during a seasonal outbreak dominated by an H3N2 virus. Collectively, our data support the clinical relevance of this protease in human influenza pathogenesis.
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http://dx.doi.org/10.1128/JVI.00421-17DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5533929PMC
August 2017

FHL2 Regulates Natural Killer Cell Development and Activation during Infection.

Front Immunol 2017 13;8:123. Epub 2017 Feb 13.

INSERM, Centre d'Etude des Pathologies Respiratoires (CEPR), UMR 1100, Tours, France; Université François Rabelais, Tours, France.

Recent studies suggested that the transcription cofactor LIM-only protein FHL2 is a major transcriptional regulator of mouse natural killer (NK) cells. However, the expression and role of FHL2 in NK cell biology are unknown. Here, we confirm that FHL2 is expressed in both mouse and human NK cells. Using FHL2 mice, we found that FHL2 controls NK cell development in the bone marrow and maturation in peripheral organs. To evaluate the importance of FHL2 in NK cell activation, FHL2 mice were infected with . FHL2 mice are highly susceptible to this infection. The activation of lung NK cells is altered in FHL2 mice, leading to decreased IFNγ production and a loss of control of bacterial burden. Collectively, our data reveal that FHL2 is a new transcription cofactor implicated in NK cell development and activation during pulmonary bacterial infection.
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http://dx.doi.org/10.3389/fimmu.2017.00123DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5303898PMC
February 2017

Prolonged pharmacological inhibition of cathepsin C results in elimination of neutrophil serine proteases.

Biochem Pharmacol 2017 05 11;131:52-67. Epub 2017 Feb 11.

INSERM U-1100, "Centre d'Etude des Pathologies Respiratoires" and Université François Rabelais, Tours, France; Department of Pathology, University of Washington, Seattle, WA, USA. Electronic address:

Cathepsin C (CatC) is a tetrameric cysteine dipeptidyl aminopeptidase that plays a key role in activation of pro-inflammatory serine protease zymogens by removal of a N-terminal pro-dipeptide sequence. Loss of function mutations in the CatC gene is associated with lack of immune cell serine protease activities and cause Papillon-Lefèvre syndrome (PLS). Also, only very low levels of elastase-like protease zymogens are detected by proteome analysis of neutrophils from PLS patients. Thus, CatC inhibitors represent new alternatives for the treatment of neutrophil protease-driven inflammatory or autoimmune diseases. We aimed to experimentally inactivate and lower neutrophil elastase-like proteases by pharmacological blocking of CatC-dependent maturation in cell-based assays and in vivo. Isolated, immature bone marrow cells from healthy donors pulse-chased in the presence of a new cell permeable cyclopropyl nitrile CatC inhibitor almost totally lack elastase. We confirmed the elimination of neutrophil elastase-like proteases by prolonged inhibition of CatC in a non-human primate. We also showed that neutrophils lacking elastase-like protease activities were still recruited to inflammatory sites. These preclinical results demonstrate that the disappearance of neutrophil elastase-like proteases as observed in PLS patients can be achieved by pharmacological inhibition of bone marrow CatC. Such a transitory inhibition of CatC might thus help to rebalance the protease load during chronic inflammatory diseases, which opens new perspectives for therapeutic applications in humans.
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http://dx.doi.org/10.1016/j.bcp.2017.02.009DOI Listing
May 2017

The Pig: A Relevant Model for Evaluating the Neutrophil Serine Protease Activities during Acute Pseudomonas aeruginosa Lung Infection.

PLoS One 2016 16;11(12):e0168577. Epub 2016 Dec 16.

INSERM, Centre d'Etude des Pathologies Respiratoires, UMR 1100, Tours cedex, France.

The main features of lung infection and inflammation are a massive recruitment of neutrophils and the subsequent release of neutrophil serine proteases (NSPs). Anti-infectious and/or anti-inflammatory treatments must be tested on a suitable animal model. Mice models do not replicate several aspects of human lung disease. This is particularly true for cystic fibrosis (CF), which has led the scientific community to a search for new animal models. We have shown that mice are not appropriate for characterizing drugs targeting neutrophil-dependent inflammation and that pig neutrophils and their NSPs are similar to their human homologues. We induced acute neutrophilic inflammatory responses in pig lungs using Pseudomonas aeruginosa, an opportunistic respiratory pathogen. Blood samples, nasal swabs and bronchoalveolar lavage fluids (BALFs) were collected at 0, 3, 6 and 24 h post-insfection (p.i.) and biochemical parameters, serum and BAL cytokines, bacterial cultures and neutrophil activity were evaluated. The release of proinflammatory mediators, biochemical and hematological blood parameters, cell recruitment and bronchial reactivity, peaked at 6h p.i.. We also used synthetic substrates specific for human neutrophil proteases to show that the activity of pig NSPs in BALFs increased. These proteases were also detected at the surface of lung neutrophils using anti-human NSP antibodies. Pseudomonas aeruginosa-induced lung infection in pigs results in a neutrophilic response similar to that described for cystic fibrosis and ventilator-associated pneumonia in humans. Altogether, this indicates that the pig is an appropriate model for testing anti-infectious and/or anti-inflammatory drugs to combat adverse proteolytic effects of neutrophil in human lung diseases.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0168577PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5161375PMC
July 2017

Pseudomonas aeruginosa proteolytically alters the interleukin 22-dependent lung mucosal defense.

Virulence 2017 08 28;8(6):810-820. Epub 2016 Oct 28.

a Institut National de la Santé et de la Recherche Médicale, Center d'Etude des Pathologies Respiratoires (CEPR), INSERM UMR 1100 , Tours , France.

The IL-22 signaling pathway is critical for regulating mucosal defense and limiting bacterial dissemination. IL-22 is unusual among interleukins because it does not directly regulate the function of conventional immune cells, but instead targets cells at outer body barriers, such as respiratory epithelial cells. Consequently, IL-22 signaling participates in the maintenance of the lung mucosal barrier by controlling cell proliferation and tissue repair, and enhancing the production of specific chemokines and anti-microbial peptides. Pseudomonas aeruginosa is a major pathogen of ventilator-associated pneumonia and causes considerable lung tissue damage. A feature underlying the pathogenicity of this bacterium is its capacity to persist and develop in the host, particularly in the clinical context of nosocomial lung infections. We aimed to investigate the ability of P. auruginosa to disrupt immune-epithelial cells cross-talk. We found that P. aeruginosa escapes the host mucosal defenses by degrading IL-22, leading to severe inhibition of IL-22-mediated immune responses. We demonstrated in vitro that, protease IV, a type 2 secretion system-dependent serine protease, is responsible for the degradation of IL-22 by P. aeruginosa. Moreover, the major anti-proteases molecules present in the lungs were unable to inhibit protease IV enzymatic activity. In addition, tracheal aspirates of patients infected by P. aeruginosa contain protease IV activity which further results in IL-22 degradation. This so far undescribed cleavage of IL-22 by a bacterial protease is likely to be an immune-evasion strategy that contributes to P. aeruginosa-triggered respiratory infections.
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http://dx.doi.org/10.1080/21505594.2016.1253658DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5626239PMC
August 2017

Neutrophils can disarm NK cell response through cleavage of NKp46.

J Leukoc Biol 2017 01 1;101(1):253-259. Epub 2016 Sep 1.

Université François Rabelais de Tours, Tours, France;

Polymorphonuclear neutrophils (PMNs) can contribute to the regulation of the host immune response by crosstalk with innate and adaptive leukocytes, including NK cells. Mechanisms by which this immunoregulation process occurs remain incompletely understood. Here, we focused on the effect of human neutrophil-derived serine proteases on NKp46, a crucial activating receptor expressed on NK cells. We used flow cytometry, Western blotting, and mass spectrometry (MS) analysis to reveal that cathepsin G [CG; and not elastase or proteinase 3 (PR3)] induces a time- and concentration-dependent, down-regulatory effect on NKp46 expression through a restricted proteolytic mechanism. We also used a functional assay to demonstrate that NKp46 cleavage by CG severely impairs NKp46-mediated responses of NK cells, including IFN-γ production and cell degranulation. Importantly, sputa of cystic fibrosis (CF) patients, which have high concentrations of CG, also alter NKp46 on NK cells. Hence, we have identified a new immunoregulatory mechanism of neutrophils that proteolytically disarms NK cell responses.
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http://dx.doi.org/10.1189/jlb.3AB0316-140RRDOI Listing
January 2017

In vitro and in vivo evidence for an inflammatory role of the calcium channel TRPV4 in lung epithelium: Potential involvement in cystic fibrosis.

Am J Physiol Lung Cell Mol Physiol 2016 09 5;311(3):L664-75. Epub 2016 Aug 5.

Inserm U1100, Centre d'Etude des Pathologies Respiratoires, Tours, France; Université François Rabelais, Tours, France;

Cystic fibrosis (CF) is an inherited disease associated with chronic severe lung inflammation, leading to premature death. To develop innovative anti-inflammatory treatments, we need to characterize new cellular and molecular components contributing to the mechanisms of lung inflammation. Here, we focused on the potential role of "transient receptor potential vanilloid-4" (TRPV4), a nonselective calcium channel. We used both in vitro and in vivo approaches to demonstrate that TRPV4 expressed in airway epithelial cells triggers the secretion of major proinflammatory mediators such as chemokines and biologically active lipids, as well as a neutrophil recruitment in lung tissues. We characterized the contribution of cytosolic phospholipase A2, MAPKs, and NF-κB in TRPV4-dependent signaling. We also showed that 5,6-, 8,9-, 11,12-, and 14,15-epoxyeicosatrienoic acids, i.e., four natural lipid-based TRPV4 agonists, are present in expectorations of CF patients. Also, TRPV4-induced calcium mobilization and inflammatory responses were enhanced in cystic fibrosis transmembrane conductance regulator-deficient cellular and animal models, suggesting that TRPV4 is a promising target for the development of new anti-inflammatory treatments for diseases such as CF.
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http://dx.doi.org/10.1152/ajplung.00442.2015DOI Listing
September 2016

Altered expression of the CCN genes in the lungs of mice in response to cigarette smoke exposure and viral and bacterial infections.

Gene 2016 Jul 11;586(1):176-83. Epub 2016 Apr 11.

INSERM U1100, Centre d'Etude des Pathologies Respiratoires, Faculté de Médecine, Tours, France; Université François Rabelais, Tours, France. Electronic address:

The CCN proteins are key signaling and regulatory molecules involved in many biological functions and contribute to malignant and non-malignant lung diseases. Despite the high morbidity and mortality of the lung respiratory infectious diseases, there is very little data related to the expression of the CCNs during infection. We investigated in mice the pulmonary mRNA expression levels of five CCNs (1 to 5) in response to influenza A virus (IAV) and bacterial agents (Nontypeable Haemophilus influenzae (NTHi), lipopolysaccharide (LPS) and lipoteichoic acid (LTA)). IAV, NTHi, LPS or LTA were instilled intranasally into mice. Mice were also exposed for 4days or 8weeks to cigarette smoke alone or prior infection to IAV in order to determine if CS modifies the CCN response to a viral infection. All challenges induced a robust inflammation. The mRNA expression of CCN1, CCN2 and CCN3 was decreased after short exposure to CS whereas prolonged exposure altered the expression of CCN1, CCN3 and CCN4. Influenza A virus infection increased CCN1, 2, 4 and 5 mRNA levels but expression of CCN3 was significantly decreased. Acute CS exposure prior infection had little effect on the expression of CCN genes but prolonged exposure abolished the IAV-dependent induction. Treatment with LPS or LTA and infection with NTHi revealed that both Gram-positive and Gram-negative bacteria rapidly modulate the expression of the CCN genes. Our findings reveal that several triggers of lung inflammation influence differently the CCN genes. CCN3 deserves special attention since its mRNA expression is decreased by all the triggers studied.
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http://dx.doi.org/10.1016/j.gene.2016.04.022DOI Listing
July 2016