Respiratory Epithelial Cells Can Remember Infection: A Proof-of-Concept Study.

Human bronchial epithelial cells play a key role in airway immune homeostasis. We hypothesized that these sentinel cells can remember a previous contact with pathogen compounds and respond nonspecifically to reinfection, a phenomenon called innate immune memory. We demonstrated that their preexposure to Pseudomonas aeruginosa flagellin modify their inflammatory response to a second, nonrelated stimulus, including live pathogens or lipopolysaccharide. Using histone acetyltransferase and methyltransferase inhibitors, we showed that this phenomenon relied on epigenetic regulation. This report is a major breakthrough in the field of multimicrobial respiratory tract infections, wherein control of inflammatory exacerbations is a major therapeutic issue.

CHAC1 Is Differentially Expressed in Normal and Cystic Fibrosis Bronchial Epithelial Cells and Regulates the Inflammatory Response Induced by Pseudomonas aeruginosa.

In cystic fibrosis (CF), Pseudomonas aeruginosa (Pa) colonizes the lungs, leading to chronic inflammation of the bronchial epithelium. ChaC glutathione-specific γ-glutamylcyclotransferase 1 (CHAC1) mRNA is differentially expressed in primary human airway epithelial cells from bronchi (hAECBs) from patients with CF and healthy patients at baseline and upon infection with Pa. CHAC1 degrades glutathione and is associated with ER stress and apoptosis pathways. In this study, we examined the roles of CHAC1 in the inflammatory response and apoptosis in lung epithelial cells. First, we confirmed by reverse transcription quantitative polymerase chain reaction that CHAC1 mRNA was overexpressed in hAECBs from patients without CF compared with the expression in hAECBs from patients with CF upon Pa (PAK strain) infection. Moreover, the Pa virulence factors LPS and flagellin were shown to induce CHAC1 expression in cells from patients without CF. Using NCI-H292 lung epithelial cells, we found that LPS-induced CHAC1 mRNA expression was PERK-independent and involved ATF4. Additionally, using CHAC1 small interfering RNA, we showed that reduced CHAC1 expression in the context of LPS and flagellin stimulation was associated with modulation of inflammatory markers and alteration of NF-κB signaling. Finally, we showed that Pa was not able to induce apoptosis in NCI-H292 cells. Our results suggest that CHAC1 is involved in the regulation of inflammation in bronchial cells during Pa infection and may explain the excessive inflammation present in the respiratory tracts of patients with CF.

Human Bronchial Epithelial Cells Inhibit Aspergillus fumigatus Germination of Extracellular Conidia via FleA Recognition.

Aspergillus fumigatus is an environmental filamentous fungus that may act as an opportunistic pathogen causing a variety of diseases, including asthma or allergic bronchopulmonary aspergillosis, and infection, ranging from asymptomatic colonization to invasive pulmonary form, especially in immunocompromised patients. This fungus is characterized by different morphotypes including conidia which are the infective propagules able to germinate into hyphae. Due to their small size (2-3 µm), conidia released in the air can reach the lower respiratory tract. The objective of this study was to characterize the interactions between conidia and bronchial epithelial cells. To this end, we studied the role of bronchial epithelial cells, i.e., the BEAS-2B cell line and human primary cells, in conidial germination of a laboratory strain and three clinical strains of A. fumigatus. Microscopic observations and galactomannan measurements demonstrated that contact between epithelial cells and conidia leads to the inhibition of conidia germination. We demonstrated that this fungistatic process is not associated with the release of any soluble components nor internalization by the epithelial cells. We highlight that this antifungal process involves the phosphoinositide 3-kinase pathway on the host cellular side and the lectin FleA on the fungal side. Collectively, our results show that bronchial epithelial cells attenuate fungal virulence by inhibiting germination of extracellular conidia, thus preventing the morphological change from conidia to filaments, which is responsible for tissue invasion.

Bronchial Epithelial Cells from Cystic Fibrosis Patients Express a Specific Long Non-coding RNA Signature upon Pseudomonas aeruginosa Infection.

Pseudomonas aeruginosa (Pa) is the leading cause of chronic lung infection in Cystic Fibrosis (CF) patients. It is well recognized that CF epithelial cells fail to develop an appropriate response to infection, allowing bacterial colonization and a chronic inflammatory response. Since long non-coding RNAs (lncRNAs), are known to play a key role in regulating mammalian innate immune response, we hypothesized that CF cells exposed to Pa could express a specific lncRNA signature responsible of the maladaptative CF response. We analyzed transcriptomic datasets to compare the expression profiles of lncRNAs in primary CF and non-CF epithelial cells infected with Pa at 0, 2, 4, and 6 h of infection. Our analysis identified temporal expression signatures of 25, 73, 15, and 26 lncRNA transcripts differentially expressed at 0, 2, 4, and 6 h post-infection respectively, between CF and non-CF cells. In addition, we identified profiles specific to CF and non-CF cells. The differential expression of two candidate lncRNAs were independently validated using real-time PCR. We identified a specific CF signature of lncRNA expression in a context of Pa infection that could potentially play a role in the maladaptive immune response of CF patients.

IRAP+ endosomes restrict TLR9 activation and signaling.

The retention of intracellular Toll-like receptors (TLRs) in the endoplasmic reticulum prevents their activation under basal conditions. TLR9 is activated by sensing ligands in specific endosomal-lysosomal compartments. Here we identified IRAP+ endosomes as major cellular compartments for the early steps of TLR9 activation in dendritic cells (DCs). Both TLR9 and its ligand, the dinucleotide CpG, were present as cargo in IRAP+ endosomes. In the absence of the aminopeptidase IRAP, the trafficking of CpG and TLR9 to lysosomes and signaling via TLR9 were enhanced in DCs and in mice following bacterial infection. IRAP stabilized CpG-containing endosomes by interacting with the actin-nucleation factor FHOD4, which slowed the trafficking of TLR9 toward lysosomes. Thus, endosomal retention of TLR9 via the interaction of IRAP with the actin cytoskeleton is a mechanism that prevents hyper-activation of TLR9 in DCs.

A novel role for ß2-microglobulin: a precursor of antibacterial chemokine in respiratory epithelial cells.

We analyzed a panel of cationic molecules secreted in the culture medium of human respiratory epithelial cells (REC) upon activation by IL-1ß and different pathogen-associated molecular patterns. A 9 kDa fragment derived from ß2-microglobulin (B2M) was identified and named shed 9 kDa B2M (sB2M-9). The primary structure of sB2M-9 was revealed to increase its pI value that potentially could play an important role in innate defense. sB2M-9 exhibits antibacterial activity against Gram positive Staphylococcus aureus (SA) but not against Gram negative Klebsiella pneumonia (KP). Upon its binding to SA, sB2M-9 induces clumps, a phenomenon not observed with B2M. Migration of THP-1 monocytes exposed to SA clumps was significantly greater than that to SA without clumps. sB2M-9 binds to SA, more likely as a chemokine, to facilitate THP-1 migration. As a whole, we demonstrated that REC release a novel chemokine with antibacterial activity that is shed from B2M to facilitate THP-1 migration.

Contribution of the Ade Resistance-Nodulation-Cell Division-Type Efflux Pumps to Fitness and Pathogenesis of Acinetobacter baumannii.

UNLABELLED: Overexpression of chromosomal resistance-nodulation-cell division (RND)-type efflux systems with broad substrate specificity contributes to multidrug resistance (MDR) in Acinetobacter baumannii We have shown that modulation of expression of the structural genes for the efflux systems AdeABC and AdeIJK confers MDR and results in numerous alterations of membrane-associated cellular functions, in particular biofilm formation. However, the contribution of these RND pumps to cell fitness and virulence has not yet been studied. The biological cost of an antibiotic resistance mechanism is a key parameter in determining its stability and dissemination. From an entirely sequenced susceptible clinical isolate, we have generated a set of isogenic derivatives having single point mutations resulting in overexpression of each efflux system or with every pump deleted by allelic replacement. We found that overproduction of the pumps results in a significant decrease in fitness of the bacterial host when measured by competition experiments in vitro Fitness and virulence were also evaluated in vivo both in systemic and pulmonary infection models in immunocompetent mice. A diminished competitiveness of the AdeABC-overexpressing mutant was observed only after intraperitoneal inoculation, but not after intranasal inoculation, the latter mimicking the most frequent type of human A. baumannii infection. However, in mice infected intranasally, this mutant was more virulent and stimulated an enhanced neutrophil activation in the lungs. Altogether, these data account for the observation that adeABC overexpression is common in MDR A. baumannii frequently found in ventilator-associated pneumonia. IMPORTANCE: Overproduction of the RND AdeABC efflux system is observed with a high incidence in multidrug-resistant Acinetobacter baumannii and results in increased resistance to several antibiotics of choice for the treatment of infections caused by this nosocomial pathogen. It was therefore important to study the biological cost of the overexpression of the adeABC structural operon which is normally tightly regulated. Fitness diminution of an overexpressing mutant detected in vitro and in vivo in a model that mimics sepsis was not observed in a pulmonary infection model in which the mutant was more virulent. This points out that increased virulence can occur independently from prolonged persistence in the infected organ and can account for the elevated incidence of this resistance mechanism in clinical isolates. The study also indicates that transposon libraries will identify only virulence genes that are expressed under physiological conditions but not those that are tightly regulated.

Targeting host calpain proteases decreases influenza A virus infection.

Influenza A viruses (IAV) trigger contagious acute respiratory diseases. A better understanding of the molecular mechanisms of IAV pathogenesis and host immune responses is required for the development of more efficient treatments of severe influenza. Calpains are intracellular proteases that participate in diverse cellular responses, including inflammation. Here, we used in vitro and in vivo approaches to investigate the role of calpain signaling in IAV pathogenesis. Calpain expression and activity were found altered in IAV-infected bronchial epithelial cells. With the use of small-interfering RNA (siRNA) gene silencing, specific synthetic inhibitors of calpains, and mice overexpressing calpastatin, we found that calpain inhibition dampens IAV replication and IAV-triggered secretion of proinflammatory mediators and leukocyte infiltration. Remarkably, calpain inhibition has a protective impact in IAV infection, since it significantly reduced mortality of mice challenged not only by seasonal H3N2- but also by hypervirulent H5N1 IAV strains. Hence, our study suggests that calpains are promising therapeutic targets for treating IAV acute pneumonia.

Normal and Cystic Fibrosis Human Bronchial Epithelial Cells Infected with Pseudomonas aeruginosa Exhibit Distinct Gene Activation Patterns.

BACKGROUND AND AIMS: In cystic fibrosis (CF), Pseudomonas aeruginosa is not eradicated from the lower respiratory tract and is associated with epithelial inflammation that eventually causes tissue damage. To identify the molecular determinants of an effective response to P. aeruginosa infection, we performed a transcriptomic analysis of primary human bronchial epithelial cells from healthy donors (CTRL) 2, 4, and 6 h after induced P. aeruginosa infection. Compared to noninfected cells, infected cells showed changes in gene activity, which were most marked 6 h postinfection and usually consisted in upregulation. RESULTS: By comparing for each time point of infection, the transcriptomic response of epithelial cells from CF patients and healthy donors, we identified 851, 638, 667, and 980 differentially expressed genes 0, 2, 4, and 6 h postinfection, respectively. Gene selection followed by bioinformatic analysis showed that most of the differentially expressed genes, either up- or downregulated, were in the protein-binding and catalytic gene-ontology categories. Finally, we established that the protein products of the genes exhibiting the greatest differential upregulation (CSF2, CCL2, TNF, CSF3, MMP1, and MMP10) between CF patients and CTRL were produced in higher amounts by infected cells from CF patients versus CTRL. CONCLUSIONS: The differentially expressed genes in CF patients may constitute a signature for a detrimental inflammatory response and for an inefficient P. aeruginosa host-cell response.

Staphylococcus aureus Adenosine Inhibits sPLA2-IIA-Mediated Host Killing in the Airways.

Staphylococcus aureus is a common cause of bacterial infections in respiratory diseases. It secretes molecules to dampen host immunity, and the recently identified adenosine is one of these molecules. The type IIA secretory phospholipase A2 (sPLA2-IIA) is a host protein endowed with antibacterial properties, especially against Gram-positive bacteria such as S. aureus. However, the role of adenosine in sPLA2-IIA-mediated S. aureus killing by host is still unknown. The present studies showed that the S. aureus mutant lacking adenosine production (∆adsA strain) increased sPLA2-IIA expression in guinea pig airways and was cleared more efficiently, compared with the wild-type strain. S. aureus ∆adsA strain induced sPLA2-IIA expression by alveolar macrophages after phagocytic process via NOD2-NF-κB-dependent mechanism. However, S. aureus adenosine (wild-type and adsA-complemented strains) and exogenous adenosine downregulated S. aureus phagocytosis by alveolar macrophages, leading to inhibition of sPLA2-IIA expression. This occurred through inhibition of p38 phosphorylation via adenosine receptors A2a-, A2b-, and protein kinase A-dependent pathways. Taken together, our studies suggest that, in the airway, S. aureus escapes sPLA2-IIA-mediated killing through adenosine-mediated inhibition of phagocytosis and sPLA2-IIA expression.

Pseudomonas aeruginosa eradicates Staphylococcus aureus by manipulating the host immunity.

Young cystic fibrosis (CF) patients' airways are mainly colonized by Staphylococcus aureus, while Pseudomonas aeruginosa predominates in adults. However, the mechanisms behind this infection switch are unclear. Here, we show that levels of type-IIA-secreted phospholipase A2 (sPLA2-IIA, a host enzyme with bactericidal activity) increase in expectorations of CF patients in an age-dependent manner. These levels are sufficient to kill S. aureus, with marginal effects on P. aeruginosa strains. P. aeruginosa laboratory strains and isolates from CF patients induce sPLA2-IIA expression in bronchial epithelial cells from CF patients (these cells are a major source of the enzyme). In an animal model of lung infection, P. aeruginosa induces sPLA2-IIA production that favours S. aureus killing. We suggest that sPLA2-IIA induction by P. aeruginosa contributes to S. aureus eradication in CF airways. Our results indicate that a bacterium can eradicate another bacterium by manipulating the host immunity.

Cytosolic phospholipase A2α enhances mouse mortality induced by Pseudomonas aeruginosa pulmonary infection via interleukin 6.

Pseudomonas aeruginosa pulmonary infection is a leading cause of death in numerous diseases such as cystic fibrosis (CF). The host cytosolic phospholipase A2α (cPLA2α) releases lipid mediators that play an important role in the pathogenesis of diseases, but its role in lung injury induced by P. aeruginosa infection is still obscure. Using an animal model of P. aeruginosa lung infection, we showed that the CHA strain of P. aeruginosa was more potent than the PAK strain in inducing mouse mortality and lung injury, and that both mouse mortality and lung injury were reduced in cPLA2α(-/-) mice as compared to cPLA2α(+/+) mice. This was accompanied by decreased levels of IL6 but not other inflammatory cytokines (IL1ß, KC and TNFα) in the bronchoalveolar lavage fluids (BALFs) of cPLA2α(-/-) mice. Given that CFTR(-/-) mice exhibit increased cPLA2α activation in the lung, the role of cPLA2α was further examined in this lung infection model. Compared to littermates, P. aeruginosa infection caused increased mortality in CFTR(-/-) mice with high IL6 levels in BALFs, which was attenuated by pharmacological inhibition of cPLA2α. In addition, compared to IL6(-/-) mice, an enhanced mortality was also observed in P. aeruginosa infected IL6(+/+) mice. Since alveolar macrophages (AMs) are the primary inflammatory cytokine source in the lung, murine AMs cell line (MH-S) were used to investigate the signalling pathways involved in this process. Incubation of MH-S cells with P. aeruginosa induced IL6 production, which was mediated by MAPKs ERK/p38 and was abolished by cPLA2α inhibitors. Furthermore, among cPLA2 downstream signalling pathways, only 15-lipoxygenase (15-LOX) and cyclooxygenase-2 (COX-2) were proven to participate in this P. aeruginosa-induced IL6 expression. Based on all these observations, we conclude that cPLA2α enhances P. aeruginosa-induced animal lethality in part via IL6 induction and that MAPKs ERK/p38, 15-LOX and COX-2 signalling pathways were involved in this process.

Flagellin concentrations in expectorations from cystic fibrosis patients.

BACKGROUND: The aim was to measure flagellin concentrations in the expectorations of CF patients and to examine whether there are correlations with the level of respiratory insufficiency and inflammation. METHODS: Sputum samples from 31 adult patients chronically colonized with P. aeruginosa were collected and analysed for their content of flagellin and IL-8. Clinical data were extracted from patient files. RESULTS: Regardless of whether patients are colonized with mucoid strains or not, they carry clones of P. aeruginosa that express flagellin. While flagellin was present in airways of all of our CF patients, it is difficult to ascertain its contribution to inflammation (IL-8) and lung function deterioration. CONCLUSIONS: This is the first demonstration that flagellin is present in the sputum of patients. Thus, attempts to down regulate inflammation by the use of TLR5 (flagellin receptor) antagonists remain a possibility. However, this result needs to be extended to a larger number of patients to validate it for future research on this subject.

Flagellin/TLR5 signalling activates renal collecting duct cells and facilitates invasion and cellular translocation of uropathogenic Escherichia coli.

Uropathogenic Escherichia coli (UPEC) colonizing kidneys is the main cause of acute pyelonephritis. TLR5 that senses flagellin was shown to be highly expressed in the bladder and to participate in host defence against flagellated UPEC, although its role in kidneys still remains elusive. Here we show that TLR5 is expressed in renal medullary collecting duct (MCD) cells, which represent a preferential site of UPEC adhesion. Flagellin, like lipopolysaccharide, stimulated the production of the chemoattractant chemokines CXCL1 and CXCL2, and subsequent migration capacity of neutrophils in cultured wild-type (WT) and Tlr4(-/-) MCDs, but not in Tlr5(-/-) MCDs. UPEC can translocate across intact MCD layers without altering tight junctions. Strikingly, the invasion capacity and transcellular translocation of the UPEC strain HT7 were significantly lower in Tlr5(-/-) than in WT MCDs. The non-motile HT7ΔfliC mutant lacking flagellin also exhibited much lower translocation capacities than the HT7 isolates. Finally, Tlr5(-/-) kidneys exhibited less infiltrating neutrophils than WT kidneys one day after the transurethral inoculation of HT7, and greater delayed renal bacterial loads in the day 4 post-infected Tlr5(-/-) kidneys. Overall, these findings indicate that the epithelial TLR5 participates to renal antibacterial defence, but paradoxically favours the translocation of UPEC across intact MCD cell layers.

Toll-like receptor 9 deficiency protects mice against Pseudomonas aeruginosa lung infection.

Pseudomonas aeruginosa is an opportunistic pathogen involved in nosocomial infections. While a number of studies have demonstrated the roles of TLR2, TLR4 and TLR5 in host defense againt P. aeruginosa infection, the implication of TLR9 in this process has been overlooked. Here, we show that P. aeruginosa DNA stimulates the inflammatory response through TLR9 pathway in both a cell line and primary alveolar macrophages (AMs). This activation requires asparagine endopeptidase- and endosomal acidification. Interestingly, TLR9-/- mice resisted to lethal lung infection by P. aeruginosa, compared to WT C57BL/6 mice. The resistance of TLR9-/- mice to P. aeruginosa infection was associated with: (i) a higher ability of TLR9-/- AMs to kill P. aeruginosa; (ii) a rapid increase in the pro-inflammatory cytokines such as TNFα, IL-1ß and IL-6 production; and (iii) an increase in nitric oxide (NO) production and inductible NO synthase expression in AMs. In addition, inhibition of both IL-1ß and NO production resulted in a significant decrease of P. aeruginosa clearance by AMs. Altogether these results indicate that TLR9 plays a detrimental role in pulmonary host defense toward P. aeruginosa by reducing the AMs clearance activity and production of IL-1ß and NO necessary for bacteria killing.

Pseudomonas aeruginosa type-3 secretion system dampens host defense by exploiting the NLRC4-coupled inflammasome.

RATIONALE: Pseudomonas aeruginosa, a major problem pathogen responsible for severe infections in critically ill patients, triggers, through a functional type-3 secretion system (T3SS), the activation of an intracellular cytosolic sensor of innate immunity, NLRC4. Although the NLRC4-inflammasome-dependent response contributes to increased clearance of intracellular pathogens, it seems that NLRC4 inflammasome activation decreases the clearance of P. aeruginosa, a mainly extracellular pathogen. OBJECTIVES: We sought to determine the underlying mechanisms of this effect of the activation of NLRC4 by P. aeruginosa. METHODS: We established acute lung injury in wild-type and Nlrc4(-/-) mice using sublethal intranasal inocula of P. aeruginosa strain CHA expressing or not a functional T3SS. We studied 96-hour survival, lung injury, bacterial clearance from the lungs, cytokine secretion in bronchoalveolar lavage, lung antimicrobial peptide expression by quantitative polymerase chain reaction, and flow cytometry analysis of lung cells. MEASUREMENTS AND MAIN RESULTS: Nlrc4(-/-) mice showed enhanced bacterial clearance and decreased lung injury contributing to increased survival against extracellular P. aeruginosa strain expressing a functional T3SS. The mechanism involved decreased NLRC4-inflammasome-driven IL-18 secretion attenuating lung injury caused by excessive neutrophil recruitment. Additionally, in the lungs of Nlrc4(-/-) mice secretion of IL-17 by innate immune cells was increased and responsible for increased expression of lung epithelial antimicrobial peptides. Furthermore, IL-18 secretion was found to repress IL-17 and IL-17-driven lung antimicrobial peptide expression. CONCLUSIONS: We report a new role of the T3SS apparatus itself, independently of exotoxin translocation. Through NLRC4 inflammasome activation, the T3SS promotes IL-18 secretion, which dampens a beneficial IL-17-mediated antimicrobial host response.

Protective role of LGP2 in influenza virus pathogenesis.

Influenza A virus triggers a contagious respiratory disease that can cause considerable morbidity and mortality. Using an in vitro approach, we previously demonstrated that the pattern recognition receptor retinoic acid-inducible gene I (RIG-I) plays a key role in influenza A virus-mediated immune response. However, the importance of RIG-I signaling in vivo has not been thoroughly examined, because of the lack of an appropriate mouse models. To circumvent this issue, we generated a new transgenic mouse overexpressing LGP2 (hereafter, "LGP2 TG mice"), a major regulator of the RIG-I signaling pathway. The time course of several parameters was compared in infected wild-type and LGP2 TG mice. We found that LGP2 TG mice displayed significantly reduced inflammatory mediators and a lower leukocyte infiltration into the bronchoalveolar airspace. More importantly, LGP2 TG mice had a significant survival advantage. Hence, our in vivo study reveals that LGP2 is a major downregulator of the influenza A virus-triggered detrimental inflammatory response.

A soluble fucose-specific lectin from Aspergillus fumigatus conidia--structure, specificity and possible role in fungal pathogenicity.

Aspergillus fumigatus is an important allergen and opportunistic pathogen. Similarly to many other pathogens, it is able to produce lectins that may be involved in the host-pathogen interaction. We focused on the lectin AFL, which was prepared in recombinant form and characterized. Its binding properties were studied using hemagglutination and glycan array analysis. We determined the specificity of the lectin towards l-fucose and fucosylated oligosaccharides, including α1-6 linked core-fucose, which is an important marker for cancerogenesis. Other biologically relevant saccharides such as sialic acid, d-mannose or d-galactose were not bound. Blood group epitopes of the ABH and Lewis systems were recognized, Le(Y) being the preferred ligand among others. To provide a correlation between the observed functional characteristics and structural basis, AFL was crystallized in a complex with methyl-α,L-selenofucoside and its structure was solved using the SAD method. Six binding sites, each with different compositions, were identified per monomer and significant differences from the homologous AAL lectin were found. Structure-derived peptides were utilized to prepare anti-AFL polyclonal antibodies, which suggested the presence of AFL on the Aspergillus' conidia, confirming its expression in vivo. Stimulation of human bronchial cells by AFL led to IL-8 production in a dose-dependent manner. AFL thus probably contributes to the inflammatory response observed upon the exposure of a patient to A. fumigatus. The combination of affinity to human epithelial epitopes, production by conidia and pro-inflammatory activity is remarkable and shows that AFL might be an important virulence factor involved in an early stage of A. fumigatus infection.