Simon Rousseau

Cystic fibrosis–adapted Pseudomonas aeruginosa quorum sensing lasR mutants cause hyperinflammatory responses

Cystic fibrosis–adapted Pseudomonas aeruginosa lasR quorum sensing mutants cause hyperinflammation contributing to chronic lung disease. Cystic fibrosis lung disease is characterized by chronic airway infections with the opportunistic pathogen Pseudomonas aeruginosa and severe neutrophilic pulmonary inflammation. P. aeruginosa undergoes extensive genetic adaptation to the cystic fibrosis (CF) lung environment, and adaptive mutations in the quorum sensing regulator gene lasR commonly arise. We sought to define how mutations in lasR alter host-pathogen relationships. We demonstrate that lasR mutants induce exaggerated host inflammatory responses in respiratory epithelial cells, with increased accumulation of proinflammatory cytokines and neutrophil recruitment due to the loss of bacterial protease–dependent cytokine degradation. In subacute pulmonary infections, lasR mutant–infected mice show greater neutrophilic inflammation and immunopathology compared with wild-type infections. Finally, we observed that CF patients infected with lasR mutants have increased plasma interleukin-8 (IL-8), a marker of inflammation. These findings suggest that bacterial adaptive changes may worsen pulmonary inflammation and directly contribute to the pathogenesis and progression of chronic lung disease in CF patients.

Lipopolysaccharide-induced toll-like receptor 4 signaling enhances the migratory ability of human esophageal cancer cells in a selectin-dependent manner.

BACKGROUNDnEsophageal cancer is an aggressive malignancy, and emerging data suggest that postoperative infections may promote cancer progression. Systemic exposure to lipopolysaccharide (LPS), a Gram-negative bacterial antigen involved in such infections, has been shown to increase cancer cell adhesion to the hepatic sinusoids inxa0vivo. We investigated the direct impact of LPS on the migratory ability of esophageal cancer cells via the LPS receptor toll-like receptor 4 (TLR4).nnnMETHODSnHuman esophageal squamous carcinoma cell lines and immortalized normal esophageal mucosa cells were tested for TLR4 surface expression by reverse transcription polymerase chain reaction (RT-PCR) and flow cytometry. TLR4 signaling in response to LPS stimulation was tested in these cells by measuring p38 MAP kinase phosphorylation on Western blot. The impact of TLR4 signaling was measured by static adhesion assays inxa0vitro and on early inxa0vivo migration by intravital microscopy of the liver.nnnRESULTSnUpon LPS stimulation, phosphorylation of p38 was detected in the human esophageal cancer cells HKESC-2. Also, LPS-stimulated HKESC-2 cells showed a twofold increased adhesion to fibronectin and to hepatic sinusoidal endothelium. These effects were abolished by TLR4 inhibition using the small-molecule inhibitor eritoran. Adhesion to fibronectin and hepatic sinusoidal endothelium was also diminished by blockade of p38 phosphorylation and inhibitors of selectin-selectin ligand binding.nnnCONCLUSIONnLPS can increase the migratory ability of human esophageal cancer cells by increasing their adhesive properties through TLR4 signaling and selectin ligands. TLR4, p38, and selectin blockade may therefore prove to be a new therapeutic strategy for this aggressive malignancy.

IL-17 primes airway epithelial cells lacking functional Cystic Fibrosis Transmembrane conductance Regulator (CFTR) to increase NOD1 responses.

In Cystic Fibrosis (CF), the absence of functional Cystic Fibrosis Transmembrane conductance Regulator (CFTR) translates into chronic bacterial infection, excessive inflammation, tissue damage, impaired lung function and eventual death. Understanding the mechanisms underlying this vicious circle of inflammation is key to better therapies for CF. In this manuscript, we have found that the presence of IL-17 in the airways of CF patients not only exacerbates inflammation through the recruitment of neutrophils via secretion of CXCL8, but also by priming airway epithelial cells lacking functional CFTR to increase response to the bacterial sensor NOD1. IL-17 stimulation of airway epithelial cells (AECs) lacking functional CFTR increased the expression of NOD1, NOD2, TLR4 and its own receptors IL-17RA and IL-17RC. Moreover, prior stimulation of AECs expressing the CFTRDeltaF508 mutant with IL-17 showed much greater CXCL8 secretion in response to a NOD1 agonist and Pseudomonas aeruginosa diffusible material. Taken together our results show that IL-17 primes AECs expressing CFTRDeltaF508 to increase host defence response to bacteria through the up-regulation of PRRs, and in particular of NOD1, and identifies another mechanism of action through which the CFTRDeltaF508 mutation leads to increase inflammation in response to bacterial ligands. Therefore preventing IL-17 function in CF may prove an important strategy in decreasing lung inflammation due to both direct and indirect effects.

Quorum-sensing inhibition abrogates the deleterious impact of Pseudomonas aeruginosa on airway epithelial repair.

Chronic Pseudomonas aeruginosa lung infections are associated with progressive epithelial damage and lung function decline. In addition to its role in tissue injury, the persistent presence of P. aeruginosa–secreted products may also affect epithelial repair ability, raising the need for new antivirulence therapies. The purpose of our study was to better understand the outcomes of P. aeruginosa exoproducts exposure on airway epithelial repair processes to identify a strategy to counteract their deleterious effect. We found that P. aeruginosa exoproducts significantly decreased wound healing, migration, and proliferation rates, and impaired the ability of directional migration of primary non–cystic fibrosis (CF) human airway epithelial cells. Impact of exoproducts was inhibited after mutations in P. aeruginosa genes that encoded for the quorum‐sensing (QS) transcriptional regulator, LasR, and the elastase, LasB, whereas impact was restored by LasB induction in ΔlasR mutants. P. aeruginosa purified elastase also induced a significant decrease in non‐CF epithelial repair, whereas protease inhibition with phosphoramidon prevented the effect of P. aeruginosa exoproducts. Furthermore, treatment of P. aeruginosa cultures with 4‐hydroxy‐2,5‐dimethyl‐3(2H)furanone, a QS inhibitor, abrogated the negative impact of P. aeruginosa exoproducts on airway epithelial repair. Finally, we confirmed our findings in human airway epithelial cells from patients with CF, a disease featuring P. aeruginosa chronic respiratory infection. These data demonstrate that secreted proteases under the control of the lasR QS system impair airway epithelial repair and that QS inhibitors could be of benefit to counteract the deleterious effect of P. aeruginosa in infected patients.—Ruffin, M., Bilodeau, C., Maillé, É., LaFayette, S.L., McKay, G.A., Trinh, N.T.N., Beaudoin, T., Desrosiers, M.‐Y., Rousseau, S., Nguyen, D., Brochiero, E. Quorum‐sensing inhibition abrogates the deleterious impact of Pseudomon asaeruginosa on airway epithelial repair. FASEBJ. 30, 3011–3025 (2016). www.fasebj.org

The aryl hydrocarbon receptor suppresses cigarette-smoke-induced oxidative stress in association with dioxin response element (DRE)-independent regulation of sulfiredoxin 1

The aryl hydrocarbon receptor (AhR) is a ubiquitously expressed receptor/transcription factor that mediates toxicological responses of environmental contaminants such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Emerging evidence indicates that the AhR suppresses apoptosis and proliferation independent of exogenous ligands, including suppression of apoptosis by cigarette smoke, a key risk factor for chronic obstructive pulmonary disease (COPD). As cigarette smoke is a potent inducer of oxidative stress, a feature that may contribute to the development of COPD, we hypothesized that the AhR prevents smoke-induced apoptosis by regulating oxidative stress. Utilizing primary lung fibroblasts derived from AhR(+/+) and AhR(-/-) mice as well as A549 human lung adenocarcinoma cells deficient in AhR expression (A549-AhR(ko)), we first show that AhR(-/-) fibroblasts and A549-AhR(ko) epithelial cells have a significant increase in cigarette smoke extract (CSE)-induced oxidative stress compared to wild type. CSE induced a significant increase in the mRNA expression of key antioxidant genes, including Nqo1 and Srxn1, predominantly in AhR(+/+) fibroblasts, with significantly less induction in AhR(-/-) cells. The induction of Srxn1, but not Nqo1, was independent of dioxin-response element (DRE) binding as AhR(DBD/DBD) lung fibroblasts, which express an AhR incapable of binding the DRE, increased Srxn1 to a degree similar to wild-type cells in response to CSE. There was no difference in Nrf2 expression or activation based on AhR expression. Lung fibroblasts derived from COPD subjects have significantly less AhR protein expression compared with both never-smokers (Normal) and smokers (At Risk). Consequently, COPD-derived fibroblasts were less robust in their induction of both Nqo1 and Srxn1 mRNA after exposure to CSE, which also failed to activate the AhR in the COPD fibroblasts. Taken together, these results support a new role for the AhR in regulating antioxidant defense in lung structural cells, such that low AhR expression may facilitate the development or progression of COPD.

TPL2 signalling: From Toll-like receptors-mediated ERK1/ERK2 activation to Cystic Fibrosis lung disease

Cystic Fibrosis (CF) is the most common lethal genetic recessive disorder, with a carrier frequency of 1 in 27 among North American Caucasians. Mitogen-activated protein kinases (MAPKs) and pro-inflammatory cytokines have crucial functions in the innate immune response of epithelial cells. They determine the inflammation status and the host response to pathogenic infections. However, in CF, bacterial-driven inflammation leads to tissue destruction, reduction in lung function and mortality. Recognition of invading pathogens is mediated in part by Toll-like receptors (TLR) activation of intracellular signalling cascade leading to cytokines synthesis. The protein kinase Tumour Progression Locus 2 (TPL2) is a key molecule in relaying inflammatory stimuli to ERK1/ERK2 MAPKs. In this review, we summarized the recent findings on TPL2 signalling and how TPL2 can contribute to the excessive inflammation found in CF. Pharmacologically targeting this kinase could have a significant benefit for CF patients dealing with chronic bacterial infections such as Pseudomonas aeruginosa. This article is part of a Directed Issue entitled: Cystic Fibrosis: From o-mics to cell biology, physiology, and therapeutic advances.

The protein kinases TPL2 and EGFR contribute to ERK1/ERK2 hyper-activation in CFTRΔF508-expressing airway epithelial cells exposed to Pseudomonas aeruginosa.

Excessive inflammation and Pseudomonas aeruginosa infection are two major characteristics of cystic fibrosis (CF) lung disease. In this manuscript, we describe a novel mechanism of ERK1/ERK2 activation and CXCL8 expression in AECs lacking functional CFTR. In both non-CF and CF airway epithelial cells (AECs), the protein kinase TPL2 is required for ERK1/ERK2 MAPK activation. However, we have found that EGFR is strongly phosphorylated in the airway epithelium of CF lung and contributes to ERK1/ERK2 MAPK activation in CF AECs exposed to P. aeruginosa diffusible material (PsaDM). Moreover, PsaDM stimulates the expression of the EGFR pro-ligand HB-EGF more strongly, and in a sustained manner, in CF AECs compared to non-CF cells. Finally, although both non-CF and CF AECs expresses CXCL8 in response to PsaDM, the levels of CXCL8 are higher and EGFR plays a more important role in regulating CXCL8 synthesis in CF AECs. Together, our finding shows that in addition to the TLR-mediated TPL2 activation of ERK1/ERK2, an additional pathway contributing to ERK1/ERK2 activation is triggered by infection of CF AECs: the EGFR signalling pathway. This second pathway may contribute to excessive inflammation observed in CF.

Gain-of-Function Mutations in the Toll-Like Receptor Pathway: TPL2-Mediated ERK1/ERK2 MAPK Activation, a Path to Tumorigenesis in Lymphoid Neoplasms?

Lymphoid neoplasms form a family of cancers affecting B-cells, T-cells, and NK cells. The Toll-Like Receptor (TLR) signaling adapter molecule MYD88 is the most frequently mutated gene in these neoplasms. This signaling adaptor relays signals from TLRs to downstream effector pathways such as the Nuclear Factor kappa B (NFκB) and Mitogen Activated Protein Kinase (MAPK) pathways to regulate innate immune responses. Gain-of-function mutations such as MYD88[L265P] activate downstream signaling pathways in absence of cognate ligands for TLRs, resulting in increased cellular proliferation and survival. This article reports an analysis of non-synonymous somatic mutations found in the TLR signaling network in lymphoid neoplasms. In accordance with previous reports, mutations map to MYD88 pro-inflammatory signaling and not TRIF-mediated Type I IFN production. Interestingly, the analysis of somatic mutations found downstream of the core TLR-signaling network uncovered a strong association with the ERK1/2 MAPK cascade. In support of this analysis, heterologous expression of MYD88[L265P] in HEK293 cells led to ERK1/2 MAPK phosphorylation in addition to NFκB activation. Moreover, this activation is dependent on the protein kinase Tumor Promoting Locus 2 (TPL2), activated downstream of the IKK complex. Activation of ERK1/2 would then lead to activation, amongst others, of MYC and hnRNPA1, two proteins previously shown to contribute to tumor formation in lymphoid neoplasms. Taken together, this analysis suggests that TLR-mediated ERK1/2 activation via TPL2 may be a novel path to tumorigenesis. Therefore, the hypothesis proposed is that inhibition of ERK1/2 MAPK activation would prevent tumor growth downstream of MYD88[L265]. It will be interesting to test whether pharmacological inhibitors of this pathway show efficacy in primary tumor cells derived from hematologic malignancies such as Waldenstroms Macroglobulinemia, where the majority of the cells carry the MYD88[L265P] mutation.

The TAK1→IKKβ→TPL2→MKK1/MKK2 Signaling Cascade Regulates IL-33 Expression in Cystic Fibrosis Airway Epithelial Cells Following Infection by Pseudomonas aeruginosa

In cystic fibrosis (CF), chronic respiratory infections result in an exaggerated and uncontrolled inflammatory response that ultimately lead to a decrease in pulmonary function. We have previously described the presence of the alarmin IL-33 in lung explants from CF patients. The signals regulating IL-33 expression in the airway epithelium following a gram-negative bacterial infection are currently unknown. Our objective was to characterize the pathways in CF airway epithelial cells (AECs) leading to an increase in IL-33 expression. We found that, in CF AECs expressing a deletion of a phenylalanine at position 508 of the gene coding for Cystic Fibrosis Transmembrane Conductance Regulator (CFTRdelF508), exposure to live Pseudomonas aeruginosa upregulates IL-33 via the TLR2 and TLR5 signaling pathways. This up-regulation can be partially or fully reverted by pre-incubating CFTRdelF508 AECs with a CFTR corrector (VX-809) and/or a CFTR potentiator (VX-770). Similarly, incubation with the CFTR corrector and/or the CFTR potentiator also decreased IL-8 expression in response to infection. Moreover, using different protein kinase inhibitors that target elements downstream of TLR signaling, we show that the TAK1→IKKβ→TPL2→MKK1/MKK2 pathway regulates IL-33 expression following an infection with P. aeruginosa. Our findings represent the first characterization of the signals regulating IL-33 expression in CF airway epithelial cells in response to a bacterial infection.

Exposure of airway epithelial cells to Pseudomonas aeruginosa biofilm-derived quorum sensing molecules decrease the activity of the anti-oxidant response element bound by NRF2

Chronic bacterial infections in cystic fibrosis lung disease are often characterized by Pseudomonas aeruginosa biofilms that are regulated by bacterial intercellular signals termed quorum sensing (QS), such as N-(3-oxododecanoyl)-l-homoserine lactone (3OC12-HSL). This study reports that biofilm-derived exoproducts decrease the transcriptional activity of the anti-oxidant response element in bronchial epithelial cells. In a live co-culture assay of BEAS-2B cells and P.xa0aeruginosa biofilm, the QS molecule 3OC12-HSL was an important but not sole contributor to the inhibition of basal NRF2 luciferase reporter activity. Moreover, biofilm-derived exoproducts and 3OC12-HSL decrease the expression of endogenous antioxidant response element-regulated genes hemeoxygenase-1 (HO-1) and NAD(P)H Quinone Dehydrogenase-1 (NQO-1) while they increase IL-8 expression. As previously reported, IL-8 expression is partially dependent on p38 MAPK activity, but the inhibitory effect of biofilm QS molecules on HO-1 and NQO-1 expression occurs independently of this protein kinase. Finally, the transfection of CFTRdelF508 but not its wild type counterpart decreases basal, planktonic PsaDM and sulforaphane-driven NRF2 luciferase reporter activity in BEAS-2B cells. Therefore, the presence of quorum sensing molecules derived from bacterial biofilms lowers the transcriptional activity of the anti-oxidant response element, which may contribute to the establishment of chronic bacterial infections, especially in the presence of mutated CFTR. Increasing NRF2 activity may thus be a promising strategy to potentiate anti-biofilm activity in cystic fibrosis lung disease.