Lucie Roussel

IL-17 Promotes p38 MAPK-Dependent Endothelial Activation Enhancing Neutrophil Recruitment to Sites of Inflammation

Neutrophilic inflammation plays an important role in lung tissue destruction occurring in many chronic pulmonary diseases. Neutrophils can be recruited to sites of inflammation via the action of the cytokine IL-17. In this study, we report that IL-17RA and IL-17RC mRNA expression is significantly increased in asthmatic bronchoscopic biopsies and that these receptors are not only expressed on epithelial and inflammatory cells but also on endothelial cells. IL-17 potently stimulates lung microvascular endothelial cells to produce chemoattractants (CXCL8 and derivatives of the 5-lipoxygenase pathway) that selectively drive neutrophil but not lymphocyte chemotaxis. Moreover, IL-17 promotes endothelial activation by inducing the expression of endothelial adhesion markers (E-selectin, VCAM-1, and ICAM-1) in a p38 MAPK-dependent manner. This increased expression of adhesion molecules stimulates the trans-endothelial migration of neutrophils, as well as the transmigration of HT-29 colon carcinoma cells, suggesting a further role in promoting lung metastasis. Finally, IL-17 increased neutrophil adhesion to the endothelium in vivo as determined by intravital microscopy of mice cremaster muscle. Overall, our results demonstrate that IL-17 is a potent activator of the endothelium in vivo leading to neutrophil infiltration. Therefore, preventing neutrophil recruitment by blocking the action of IL-17 on endothelial cells may prove to be highly beneficial in diseases in which neutrophilic inflammation plays a key role.

TH17 cytokines induce human airway smooth muscle cell migration

BACKGROUND Migration of airway smooth muscle cells (ASMCs) might contribute to increased airway smooth muscle mass in asthma. T(H)17 cells and T(H)17-associated cytokines are involved in the pathogenesis of asthma and might also contribute to airway remodeling. OBJECTIVE We sought to explore the possibility that migration of ASMCs might contribute to airway remodeling through the action of T(H)17-related cytokines. METHODS The effect of exogenous T(H)17 cytokines on ex vivo human ASMC migration was investigated by using a chemotaxis assay. The involvement of signaling pathways, including p38 mitogen-activated protein kinase (MAPK), extracellular signal-regulated kinase 1/2 MAPK, nuclear factor κB, and phosphoinositide 3-kinase, was also examined. RESULTS We demonstrated that IL-17A, IL-17F, and IL-22 promote migration in a dose-dependent manner. We further demonstrated that ASMCs express receptors for IL-17RA, IL-17RC, and IL-22R1. Using mAbs directed against these receptors, we confirmed that T(H)17-associated cytokine-induced migration was dependent on selective receptor activation. Moreover, IL-17A and IL-17F exert their effects through signaling pathways that are distinct from those used by IL-22. The p38 MAPK inhibitor BIRB0796 inhibited the migration induced by IL-17A and IL-17F. PS1145, an inhibitor of nuclear factor κB, abolished the IL-22-induced migration. CONCLUSION These data raise the possibility that T(H)17-associated cytokines promote human ASMC migration in vivo and suggest an important new mechanism for the promotion of airway remodeling in asthma.

Loss of cystic fibrosis transmembrane conductance regulator function enhances activation of p38 and ERK MAPKs, increasing interleukin-6 synthesis in airway epithelial cells exposed to Pseudomonas aeruginosa.

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 important to design better therapies for CF. We found in CF lung biopsies increased immunoreactivity for p38 MAPK activity markers. Moreover, when compared with their non-CF counterpart, airway epithelial cells expressing the most common mutation in CF (CFTRΔF508) were more potent at inducing neutrophil chemotaxis through increased interleukin (IL)-6 synthesis when challenged with Pseudomonas aeruginosa diffusible material. We then discovered that in CFTRΔF508 cells, the p38 and ERK MAPKs are hyperactivated in response to P. aeruginosa diffusible material, leading to increased IL-6 mRNA expression and stability. Moreover, although TLR5 contributes to p38 MAPK activation upon P. aeruginosa challenge, it only played a weak role in IL-6 synthesis. Instead, we found that the production of reactive oxygen species is essential for IL-6 synthesis in response to P. aeruginosa diffusible material. Finally, we uncovered that in CFTRΔF508 cells, the extracellular glutathione levels are decreased, leading to a greater sensitivity to reactive oxygen species, providing an explanation for the hyperactivation of the p38 and ERK MAPKs and increased IL-6 synthesis. Taken together, our study has characterized a mechanism whereby the CFTRΔF508 mutation in airway epithelial cells contributes to increase inflammation of the airways.

The Level of p38α Mitogen-Activated Protein Kinase Activation in Airway Epithelial Cells Determines the Onset of Innate Immune Responses to Planktonic and Biofilm Pseudomonas aeruginosa

Biofilm microcolonies of Pseudomonas aeruginosa chronically infect the airways of patients with cystic fibrosis and fuel ongoing destructive inflammation, yet the impact of the switch from planktonic to biofilm growth on host responses is poorly understood. We report that in airway epithelial cells a threshold of p38α mitogen-activated protein kinase (MAPK) activation was required to trigger neutrophil recruitment, which is influenced by extrinsic and intrinsic factors. Planktonic P. aeruginosa diffusible material (PsaDM) induced stronger p38α MAPK activation as compared to biofilm PsaDM. Biofilm PsaDM activated p38α MAPK in a Toll-like receptor-independent fashion via the lasI/lasR quorum-sensing system, but this activation was insufficient to recruit neutrophils. However, in airway epithelial cells from patients with cystic fibrosis with hypersensitivity to injurious stimuli, biofilm PsaDM activated p38α MAPK strongly enough to recruit neutrophils, which can contribute to lung injury.

P. aeruginosa drives CXCL8 synthesis via redundant toll-like receptors and NADPH oxidase in CFTR∆F508 airway epithelial cells

BACKGROUND Understanding the mechanisms underlying bacterial-driven inflammation and neutrophil recruitment is important to design better therapies for CF. CXCL8 is an important chemokine found elevated in the airways of CF patients that recruits neutrophil to sites of the inflammation. METHODS Airway epithelial cells (AECs) expressing wild-type CFTR or CFTR∆F508 were challenged with Pseudomonas aeruginosa diffusible material (PsaDM) and the synthesis of CXCL8 was measured by quantitative real-time PCR and ELISA in absence or presence of MAPK inhibitors, TLR antagonists, glutathione and a NADPH oxidase inhibitor. RESULTS CFTR∆F508 AECs secrete more CXCL8 in response to PsaDM than their wild type counterpart, which can be reversed by addition of extracellular glutathione or incubating AECs at 27°C to favour folding and expression of CFTR at the cell membrane. Moreover, in CFTR∆F508 AECs, TLR2, TLR4 and TLR5 act redundantly to drive CXCL8 synthesis via the activation of NADPH oxidase. DISCUSSIONS These results demonstrate that NADPH oxidase is necessary for CXCL8 synthesis in response to TLRs activation by P. aeruginosa.

Steroid-Insensitive ERK1/2 Activity Drives CXCL8 Synthesis and Neutrophilia by Airway Smooth Muscle

Severe or refractory asthma affects 5 to 15% of all patients with asthma, but is responsible for more than half of the health burden associated with the disease. Severe asthma is characterized by a dramatic increase in smooth muscle and airway inflammation. Although glucocorticoids are the mainstay of treatment in asthma, they are unable to fully control the disease in individuals with severe asthma. We found that airway smooth muscle cells (ASMCs) from individuals with severe asthma showed elevated activities of the ERK1/ERK2 and p38 MAPK pathways despite treatment with oral and inhaled glucocorticoids, which increased the expression of DUSP1, a phosphatase shown to limit p38 MAPK activity. In ex vivo ASMCs, TNF-α but not IL-17A induced expression of the neutrophil chemoattractant CXCL8. Moreover, TNF-α led to up-regulation of the ERK1/ERK2 and p38 MAPKs pathways, with only the latter being sensitive to pretreatment with the glucocorticoid dexamethasone. In contrast to epithelial and endothelial cells, TNF-α-stimulated CXCL8 synthesis was dependent on ERK1/ERK2 but not on p38 MAPK. Moreover, suppressing ERK1/ERK2 activation prevented neutrophil recruitment by ASMCs, whereas suppressing p38 MAPK activity had no impact. Taken together, these results highlight the ERK1/ERK2 MAPK cascade as a novel and attractive target in severe asthma because the activation of this pathway is insensitive to the action of glucocorticoids and is involved in neutrophil recruitment, contributing the to inflammation seen in the disease.

Gram negative bacteria increase non-small cell lung cancer metastasis via Toll-like receptor 4 activation and mitogen-activated protein kinase phosphorylation.

Surgery is required for the curative treatment of lung cancer but is associated with high rates of postoperative pneumonias predominantly caused by gram negative bacteria. Recent evidence suggests that these severe infectious complications may decrease long term survival after hospital discharge via cancer recurrence, but the mechanism is unclear. Lung cancer cells have recently been demonstrated to express Toll‐like receptors (TLR) that mediate pathogen recognition. We hypothesized that incubation of non‐small cell lung cancer (NSCLC) cells with heat‐inactivated Escherichia coli can augment cancer cell adhesion, migration and metastasis via TLR4 signaling. Incubation of murine and human NSCLC cells with E. coli increased in vitro cell adhesion to collagen I, collagen IV and fibronectin, and enhanced in vitro migration. Using hepatic intravital microscopy, we demonstrated that NSCLC cells have increased in vivo adhesion to hepatic sinusoids after coincubation with gram negative bacteria. These enhanced cell adhesion and migration phenotypes following incubation with E. coli were attenuated at three levels: inhibition of TLR4 (Eritoran), p38 MAPK (BIRB0796) and ERK1/2 phosphorylation (PD184352). Incubation of murine NSCLC cells in vitro with E. coli prior to intrasplenic injection significantly augmented formation of in vivo hepatic metastases 2 weeks later. This increase was abrogated by NSCLC TLR4 blockade using Eritoran. TLR4 represents a potential therapeutic target to help prevent severe postoperative infection driven cancer metastasis.

Vx-809/Vx-770 treatment reduces inflammatory response to Pseudomonas aeruginosa in primary differentiated cystic fibrosis bronchial epithelial cells

Cystic fibrosis patients exhibit chronic Pseudomonas aeruginosa respiratory infections and sustained proinflammatory state favoring lung tissue damage and remodeling, ultimately leading to respiratory failure. Loss of cystic fibrosis transmembrane conductance regulator (CFTR) function is associated with MAPK hyperactivation and increased cytokines expression, such as interleukin-8 [chemoattractant chemokine (C-X-C motif) ligand 8 (CXCL8)]. Recently, new therapeutic strategies directly targeting the basic CFTR defect have been developed, and ORKAMBI (Vx-809/Vx-770 combination) is the only Food and Drug Administration-approved treatment for CF patients homozygous for the F508del mutation. Here we aimed to determine the effect of the Vx-809/Vx-770 combination on the induction of the inflammatory response by fully differentiated primary bronchial epithelial cell cultures from CF patients carrying F508del mutations, following exposure to P. aeruginosa exoproducts. Our data unveiled that CFTR functional rescue with Vx-809/Vx-770 drastically reduces CXCL8 (as well as CXCL1 and CXCL2) transcripts and p38 MAPK phosphorylation in response to P. aeruginosa exposure through a CFTR-dependent mechanism. These results suggest that ORKAMBI has anti-inflammatory properties that could decrease lung inflammation and contribute to the observed beneficial impact of this treatment in CF patients.

Abstract 1394: Acute bacterial infection increases lung cancer metastasis via toll-like receptors 2 and 4

INTRODUCTION: Surgery is required for curative treatment of lung cancer but is associated with a high rate of bacterial infections such as pneumonia. There is emerging clinical evidence to suggest that infectious complications may decrease survival after cancer surgery, but the mechanism is unclear. Interactions between bacteria and host cells are mediated by specialized pathogen recognition receptors, of which the Toll-like receptors (TLRs) are best characterized. We and others have shown that cancer cells express TLRs, but their function is largely unknown. The aim of this study is to investigate the mechanisms of bacterial infection-facilitated lung cancer metastasis with a particular focus on the role of TLRs. METHODS: Two lung cancer cell lines (murine H-59 and human A549) were incubated a) directly with heat-inactivated bacteria (S.pneumonia or E.coli) or purified bacterial antigens (lipopolysaccharide or lipoteichoic acid), or b) indirectly with an in vitro pneumonia model using conditioned media from pulmonary bronchial epithelial cells incubated with heat-inactivated bacteria or bacterial antigens. Migratory and metastatic ability of thus treated cells were determined in vitro (adhesion, migration) and in vivo (hepatic intravital microscopy and intra-splenic liver metastasis). A clinically relevant in vivo model of severe infection, cecal ligation and puncture (CLP) or sham control, was employed in some animals. To assess the role of TLRs, we used transgenic TLR4 knockout mice, and small molecule inhibiting molecules or antibodies of TLRs. RESULTS: Direct and indirect (in vitro pneumonia) infection of H-59 and A549 increased adhesion to extracellular matrix components approximately 2-8 fold and 2-4 fold over negative control, respectively. In vitro pneumonia increased in vitro migration of H-59 approximately 2-5 fold. Direct and indirect incubation of H-59 with S.pneumonia and E.coli increased the in vivo adhesion to liver sinusoids 2-4 fold. CLP (in vivo infection model) enhanced the in vivo adhesion of H-59 3-fold and increased number of liver metastases at 2 weeks post-injection approximately 12 fold. These effects were attenuated in TLR4 -/- mice and with TLR2 and TLR4 inhibition. CONCLUSION: Activation of TLR2 or TLR4 on lung cancer cells, either directly by S.pneumonia and E.coli, indirectly though activated bronchial epithelial cells, or via an in vivo infection, increases cancer cell adhesion, migration and metastasis. TLRs thus represent a potential therapeutic target in bacterial infection facilitated cancer metastasis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1394. doi:1538-7445.AM2012-1394