Autoregulation of CCL26 synthesis and secretion in A549 cells: a possible mechanism by which alveolar epithelial cells modulate airway inflammation

Eotaxins (CCL11, CCL24, CCL26) originating from airway epithelial cells and leukocytes have been detected in bronchoalveolar lavage of asthmatics. Although the alveolar epithelium is the destination of uncleared allergens and other inflammatory products, scanty information exists on their contribution to the generation and regulation of the eotaxins. We envisioned a state whereby alveolar type II cells, a known source of other inflammatory proteins, could be involved in both the production and regulation of CCL24 and CCL26. Herein, we demonstrated that all three eotaxins are constitutively expressed in A549 cells. IL-4 and IL-13 stimulated a concentration-dependent secretion of CCL24 and CCL26. The cytokines did not act synergistically. Cycloheximide and actinomycin D abrogated IL-4- and IL-13-dependent CCL26 but not CCL24 secretion. Both IL-13 and IL-4 stimulated CCL26 synthesis that was inhibited in a concentration-dependent manner by CCL26 but not CCL24. Only CCL26 reduced expression of CCR3 receptors by 30-40%. On the other hand, anti-CCR3 pretreatment reduced IL-4+IL-13-dependent CCL26 secretion, implying autoregulation. A CCR3-specific antagonist (SB-328437) significantly decreased IL-4-dependent synthesis and release of CCL26. Eosinophils treated with medium from IL-4-stimulated A549 cells preincubated with anti-CCL26 showed a marked decrease of superoxide anion production compared with anti-CCL24 treated. These results suggest that CCL26 is a major eotaxin synthesized and released by alveolar epithelial cells and is involved in autoregulation of CCR3 receptors and other eotaxins. This CCL26-CCR3 ligand-receptor system may be an attractive target for development of therapeutics that limits progress of inflammation in airway disease.     

Silencing of Paralemmin-3 Protects Mice from lipopolysaccharide-induced acute lung injury

Excessive inflammatory response induced by lipopolysaccharide (LPS) plays a critical role in the development of acute lung injury (ALI). Paralemmin-3 (PALM3) is a novel protein that can modulate LPS-stimulated inflammatory responses in alveolar epithelial A549 cells. However, it remains unclear whether it is involved in the progression of ALI in vivo. Therefore, we studied the role of PALM3 in the pathogenesis of ALI induced by LPS. ALI was induced by LPS peritoneal injection in C57BL/6J mice. Lentivirus-mediated small interfering RNA (siRNA) targeting the mouse PALM3 gene and a negative control siRNA were intranasally administered to the mice. We found that the expression of PALM3 was up-regulated in the lung tissues obtained from the mouse model of LPS-induced ALI. The LPS-evoked inflammatory response (neutrophils and the concentrations of proinflammatory cytokines [IL-6, IL-1β, TNF-α, MIP-2] in the bronchoalveolar lavage fluid [BALF]), histologic lung injury (lung injury score), permeability of the alveolar capillary barrier (lung wet/dry weight ratio and BALF protein concentration) and mortality rates were attenuated in the PALM3 siRNA-treated mice. These results indicate that PALM3 contributes to the development of ALI in mice challenged with LPS. Inhibiting PALM3 through the intranasal application of specific siRNA protected against LPS-induced ALI.     

Effects of esculetin on lipopolysaccharide (LPS)-induced acute lung injury via regulation of RhoA/Rho Kinase/NF-кB pathways in vivo and in vitro

The purpose of the present study was to investigate the protective effect of esculetin (ES) in lipopolysaccharide (LPS)-induced acute lung injury (ALI) and the lung epithelial A549 cells. Mice were intragastrically administered with ES (20 and 40 mg/kg) 1 h prior to LPS challenge. ES pretreatment at doses of 20 and 40 mg/kg effectively attenuated LPS-induced lung histopathological change, myeloperoxidase or MPO activity, inflammatory cells infiltration, pulmonary wet-to-dry weight ratio, and the generation of pro-inflammatory cytokines including tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and interleukin-6 (IL-6) in vivo and in vitro. Furthermore, we demonstrated that ES blocked the activation of NF-кB and RhoA/Rho kinase pathways in LPS-induced mice and A549 cells. The results suggested that ES exhibited protective effect on ALI and might attribute partly to the inhibition of NF-кB and RhoA/Rho kinase pathways in vivo and in vitro.     

Activation of MTOR in pulmonary epithelium promotes LPS-induced acute lung injury

MTOR (mechanistic target of rapamycin [serine/threonine kinase]) plays a crucial role in many major cellular processes including metabolism, proliferation and macroautophagy/autophagy induction, and is also implicated in a growing number of proliferative and metabolic diseases. Both MTOR and autophagy have been suggested to be involved in lung disorders, however, little is known about the role of MTOR and autophagy in pulmonary epithelium in the context of acute lung injury (ALI). In the present study, we observed that lipopolysaccharide (LPS) stimulation induced MTOR phosphorylation and decreased the expression of MAP1LC3B/LC3B (microtubule-associated protein 1 light chain 3 β)-II, a hallmark of autophagy, in mouse lung epithelium and in human bronchial epithelial (HBE) cells. The activation of MTOR in HBE cells was mediated by TLR4 (toll-like receptor 4) signaling. Genetic knockdown of MTOR or overexpression of autophagy-related proteins significantly attenuated, whereas inhibition of autophagy further augmented, LPS-induced expression of IL6 (interleukin 6) and IL8, through NFKB signaling in HBE cells. Mice with specific knockdown of Mtor in bronchial or alveolar epithelial cells exhibited significantly attenuated airway inflammation, barrier disruption, and lung edema, and displayed prolonged survival in response to LPS exposure. Taken together, our results demonstrate that activation of MTOR in the epithelium promotes LPS-induced ALI, likely through downregulation of autophagy and the subsequent activation of NFKB. Thus, inhibition of MTOR in pulmonary epithelial cells may represent a novel therapeutic strategy for preventing ALI induced by certain bacteria.     

Lipopolysaccharide-induced CCN1 production enhances interleukin-6 secretion in bronchial epithelial cells

Acute lung injury/acute respiratory distress syndrome (ALI/ARDS) is a clinical complication caused by primary or secondary lung injury, as well as by systemic inflammation. Researches regarding molecular pathophysiology of ALI/ARDS are immerging with an ultimate aim towards developing prognostic molecular biomarkers and molecule-based therapy. However, the molecular mechanisms concerning ALI/ARDS are still not completely understood. The purpose of the present study was to identify a crucial role of CCN1 in inflammatory microenvironment during ALI/ARDS and focus on a potential communication between CCN1 and interleukin-6 (IL-6) in the airway epithelial cells. Our data illustrated that the expression levels of CCN1 and IL-6 in bronchoalveolar lavage fluid (BALF) in a lipopolysaccharide (LPS)-induced ALI mouse model were significantly elevated and the pulmonary expression of CCN1 was restricted to bronchial epithelial cells. Interestingly, both endogenous and exogenous CCN1 stimulated IL-6 production in vitro. Furthermore, LPS-induced IL-6 production in a bronchial epithelial cell line was blocked by CCN siRNA whereas CCN1 induced by LPS was sensitive to PI3K inhibition. Together, our data indicate a linear signal pathway, LPS-CCN1-IL-6, existing in bronchial epithelial cells after LPS exposure. This finding may represent an additional mechanism and a novel target for development of therapy and biomarker on ALI/ARDS.     

Lipopolysaccharide-induced cytokine expression in alveolar epithelial cells: role of PKCζ-mediated p47phox phosphorylation

Chronic inflammation incited by bacteria in the saccular lung of premature infants contributes to the pathogenesis of bronchopulmonary dysplasia (BPD). LPS-mediated type II alveolar epithelial cell (AEC) injury induces the expression of pro-inflammatory cytokines that trigger pulmonary neutrophil influx, alveolar matrix degradation and lung remodeling. We hypothesized that NADPH oxidase (Nox)-dependent mechanisms mediate LPS-induced cytokine expression in AEC. We examined the role of p47phox in mediating LPS-dependent inflammatory cytokine expression in A549 cells (which exhibit phenotypic features characteristic of type II AEC) and elucidated the proximal signaling events by which Nox is activated by LPS. LPS-induced ICAM-1 and IL-8 expression was associated with increased superoxide formation in AEC. LPS-mediated oxidative stress and cytokine expression was inhibited by apocynin and augmented by PMA demonstrating that Nox-dependent redox signaling regulates LPS-dependent pro-inflammatory signaling in AEC. In LPS-treated cells, p47phox translocated from the cytoplasm to the perinuclear region and co-localized with gp91phox. LPS also induced a temporal increase in p47phox serine304 phosphorylation in AEC. While inhibition of classical PKC and novel PKC with calphostin and rottlerin did not inhibit ICAM-1 or IL-8 expression, the myristolyated PKCζ pseudosubstrate peptide (a specific inhibitor of PKCζ) inhibited LPS-induced cytokine expression in AEC. Inhibition of PKCζ also attenuated LPS-mediated p47phox phosphorylation and perinuclear translocation in AEC. Consistent with these data, LPS activated PKCζ in AEC as evidenced by increased threonine410 phophorylation. We conclude that PKCζ-mediated p47phox activation regulates LPS-dependent cytokine expression in AEC. Selective inhibition of PKCζ or p47phox might attenuate LPS-mediated inflammation and alveolar remodeling in BPD.     

Bacterial neuraminidase increases IL-8 production in lung epithelial cells via NF-κB-dependent pathway

Bacterial neuraminidase, a sialic acid-degrading enzyme, is one of the virulent factors produced in pathogenic bacteria like as other bacterial components. However little is known about whether bacterial neuraminidase can initiate or modify a cellular response, such as cytokine production, in epithelial cells at infection and inflammation. We demonstrate here that bacterial neuraminidase, but not heat-inactivated neuraminidase, up-regulates expression of interleukin-8 (IL-8) mRNA and protein in lung epithelial A549 and NCI-H292 cells. We also show that bacterial neuraminidase significantly up-regulates IL-8 promoter activity as well as nuclear factor-kappaB (NF-κB) reporter activity. Moreover, inhibition of NF-κB signaling suppressed IL-8 mRNA expression induced by bacterial neuraminidase. Taken together, desialylation-induced IL-8 production in lung epithelial cells may play an important role in infection-associated inflammatory events.     

The microRNA miR-17 regulates lung FoxA1 expression during lipopolysaccharide-induced acute lung injury

Acute lung injury (ALI) is a severe pulmonary disease that causes a high number of fatalities worldwide. Studies have shown that FoxA1 expression is upregulated during ALI and may play an important role in ALI by promoting the apoptosis of alveolar type II epithelial cells. However, the mechanism of FoxA1 overexpression in ALI is unclear. In this study, an in vivo murine model of ALI and alveolar type II epithelial cells injury was induced using lipopolysaccharide (LPS). LPS upregulated FoxA1 in the lung tissue of the in vivo ALI model and in LPS-challenged type II epithelial cells. In contrast, miR-17 was significantly downregulated in these models. After miR-17 antagomir injection, the expression of FoxA1 was significantly increased in ALI mice. MiR-17 mimics could significantly inhibit FoxA1 mRNA and protein expression, whereas the miR-17 inhibitor could significantly increase FoxA1 mRNA and protein expression in LPS-induced type II epithelial cells. Thus, our results suggest that the downregulation of miR-17 expression could lead to FoxA1 overexpression in ALI.     

Activation of epidermal growth factor receptor via CCR3 in bronchial epithelial cells

We have previously found that bronchial epithelial cells express CCR3 whose signaling elicits mitogen-activated protein (MAP) kinase activation and cytokine production. Several investigators have focused on the signaling crosstalk between G protein-coupled receptors (GPCRs) and epidermal growth factor receptor (EGFR) in cancer cells. In this study, we investigated the role of EGFR in CCR3 signaling in the bronchial epithelial cell line NCI-H292. Eotaxin (1-100 nM) induced dose-dependent tyrosine phosphorylation of EGFR in NCI-H292 cells. Pretreatment of the cells with the EGFR inhibitor (AG1478) significantly inhibited the MAP kinase phosphorylation induced by eotaxin. Eotaxin stimulated IL-8 production, which was inhibited by AG1478. The transactivation of EGFR through CCR3 is a critical pathway that elicits MAP kinase activation and cytokine production in bronchial epithelial cells. The delineation of the signaling pathway of chemokines will help to develop a new therapeutic strategy to allergic diseases including bronchial asthma.     

Modulation of eotaxin-3 (CCL26) in alveolar type II epithelial cells

Airway epithelial inflammation associated with emphysema, chronic bronchitis, chronic obstructive pulmonary disease (COPD) and asthma is regulated in part by alveolar type II cell chemokine signaling. Data suggest that resident lung cells use CCR3, CCR5 and CCR2 chemokine receptor/ligand systems to regulate the profile of leukocytes recruited in disease-associated inflammatory conditions. Thus studies were designed to test whether alveolar type II cells possess a Th1-activated CCR5-ligand system that modulates the Th2-activated CCR3/eotaxin-2 (CCL24), eotaxin-3 (CCL26) chemokine systems. The A549 alveolar type II epithelial-like cell culture model was used to demonstrate that alveolar type II cells constitutively express CCR5 which may be upregulated by MIP-1alpha (CCL3) whose expression was induced by the Th1 cytokines IL-1beta and IFN-gamma. Selective down-regulation of CCL26, but not CCL24, was observed in CCL3 and IL-4/CCL3 stimulated cells. Down-regulation was reversed by anti-CCR5 neutralizing antibody treatment. Thus, one mechanism through which Th1-activated CCCR5/ligand pathways modulate Th2-activated CCR3/ligand pathways is the differential down-regulation of CCL26 expression. Results suggest that the CCR3 and CCR5 receptor/ligand signaling pathways may be important targets for development of novel mechanism-based adjunctive therapies designed to abrogate the chronic inflammation associated with airway diseases.     

Modulation of eotaxin-3 (CCL26) in alveolar type II epithelial cells

Airway epithelial inflammation associated with emphysema, chronic bronchitis, chronic obstructive pulmonary disease (COPD) and asthma is regulated in part by alveolar type II cell chemokine signaling. Data suggest that resident lung cells use CCR3, CCR5 and CCR2 chemokine receptor/ligand systems to regulate the profile of leukocytes recruited in disease-associated inflammatory conditions. Thus studies were designed to test whether alveolar type II cells possess a Th1-activated CCR5-ligand system that modulates the Th2-activated CCR3/eotaxin-2 (CCL24), eotaxin-3 (CCL26) chemokine systems. The A549 alveolar type II epithelial-like cell culture model was used to demonstrate that alveolar type II cells constitutively express CCR5 which may be upregulated by MIP-1α (CCL3) whose expression was induced by the Th1 cytokines IL-1β and IFN-γ. Selective down-regulation of CCL26, but not CCL24, was observed in CCL3 and IL-4/CCL3 stimulated cells. Down-regulation was reversed by anti-CCR5 neutralizing antibody treatment. Thus, one mechanism through which Th1-activated CCCR5/ligand pathways modulate Th2-activated CCR3/ligand pathways is the differential down-regulation of CCL26 expression. Results suggest that the CCR3 and CCR5 receptor/ligand signaling pathways may be important targets for development of novel mechanism-based adjunctive therapies designed to abrogate the chronic inflammation associated with airway diseases.     

Protective effects of intratracheally administered quercetin on lipopolysaccharide-induced acute lung injury

Background

Acute respiratory distress syndrome (ARDS) can result in a life-threatening form of respiratory failure, and established, effective pharmacotherapies are therefore urgently required. Quercetin is one of the most common flavonoids found in fruits and vegetables, and has potent anti-inflammatory and anti-oxidant activities. Quercetin has been demonstrated to exhibit cytoprotective effects through the induction of heme oxygenase (HO)-1. Here, we investigated whether the intratracheal administration of quercetin could suppress lipopolysaccharide (LPS)-induced acute lung injury (ALI) in mice as well as the involvement of HO-1 in quercetin’s suppressive effects.

Methods

Mouse model of ALI were established by challenging intratracheally LPS. The wet lung-to-body weight ratio, matrix metalloproteinase (MMP)-9 activities, and pro-inflammatory cytokine productions, including tumor necrosis factor (TNF)-α, interleukin (IL)-1β, and IL-6 in bronchoalveolar lavage fluid (BALF) were examined in ALI mice with or without quercetin pretreatment. We also examined the effects of quercetin on LPS stimulation in the mouse alveolar macrophage cell line, AMJ2-C11 cells.

Results

Intratracheal administration of quercetin decreased the wet lung-to-body weight ratio. Moreover, quercetin decreased MMP-9 activity and the production of pro-inflammatory cytokines in BALF cells activated by LPS in advance. We determined the expression of quercetin-induced HO-1 in mouse lung, e.g., alveolar macrophages (AMs), alveolar and bronchial epithelial cells. When AMJ2-C11 cells were cultured with quercetin, a marked suppression of LPS-induced pro-inflammatory cytokine production was observed. The cytoprotective effects were attenuated by the addition of the HO-1 inhibitor SnPP. These results indicated that quercetin suppressed LPS-induced lung inflammation, and that an HO-1-dependent pathway mediated these cytoprotective effects.

Conclusions

Our findings indicated that quercetin suppressed LPS-induced lung inflammation, and that an HO-1-dependent pathway mediated these cytoprotective effects. Intratracheal administration of quercetin will lead to new supportive strategies for cytoprotection in these serious lung conditions.     

Effect of proinflammatory cytokines on interleukin-8 mRNA expression and protein production by isolated human alveolar epithelial cells type II in primary culture

Alveolar epithelial cells type II (AEC-II) are ideally situated to regulate the recruitment and activation of different types of cells through the production of chemokines in response to inflammatory stimulation from the alveolar space. We hypothesized that these cells are important producers of interleukin-8 (IL-8) in the lung. This lead us to investigate the capacity of isolated human AEC-II cells to release IL-8 and whether this IL-8 release is regulated by proinflammatory cytokines, i.e. IL-1 beta, TNF-alpha and IFN-gamma. We isolated AEC-II from tumor-free sections of human lungs obtained by pneumectomy and purified the cells by magnetic activated cell sorting. For control experiments the AEC-II-like cell line A549 was used. IL-8 concentration was measured by ELISA in supernatants of unstimulated and LPS-, IL-1 beta-, TNF-alpha- and IFN-gamma- stimulated cells. IL-8 mRNA expression was evaluated by RT-PCR. Spontaneous IL-8 mRNA expression and protein secretion by AEC-II were significantly higher in comparison with A549 cells. TNF-alpha increased both IL-8 mRNA expression and protein production, whereas IL-1 beta slightly increased IL-8 release but did not change mRNA expression in AEC-II. LPS and IFN-gamma did not influence IL-8 expression in AEC-II and A549 cells. These results show considerable differences between A549 cell and AEC-II. The latter are capable of producing IL-8 under the control of proinflammatory cytokines. Our findings demonstrate that the modulation of IL-8 release in AEC-II may have an important impact on the immunoreactivity of these cells during pulmonary inflammation in vivo.     

Lysophosphatidic acid receptor 1 modulates lipopolysaccharide-induced inflammation in alveolar epithelial cells and murine lungs

Lysophosphatidic acid (LPA), a bioactive phospholipid, plays an important role in lung inflammation by inducing the release of chemokines and lipid mediators. Our previous studies have shown that LPA induces the secretion of interleukin-8 and prostaglandin E(2) in lung epithelial cells. Here, we demonstrate that LPA receptors contribute to lipopolysaccharide (LPS)-induced inflammation. Pretreatment with LPA receptor antagonist Ki16425 or downregulation of LPA receptor 1 (LPA(1)) by small-interfering RNA (siRNA) attenuated LPS-induced phosphorylation of p38 MAPK, I-κB kinase, and I-κB in MLE12 epithelial cells. In addition, the blocking of LPA(1) also suppressed LPS-induced IL-6 production. Furthermore, LPS treatment promoted interaction between LPA(1) and CD14, a LPS coreceptor, in a time- and dose-dependent manner. Disruption of lipid rafts attenuated the interaction between LPA(1) and CD14. Mice challenged with LPS increased plasma LPA levels and enhanced expression of LPA receptors in lung tissues. To further investigate the role of LPA receptors in LPS-induced inflammation, wild-type, or LPA(1)-deficient mice, or wild-type mice pretreated with Ki16425 were intratracheally challenged with LPS for 24 h. Knock down or inhibition of LPA(1) decreased LPS-induced IL-6 release in bronchoalveolar lavage (BAL) fluids and infiltration of cells into alveolar space compared with wild-type mice. However, no significant differences in total protein concentration in BAL fluids were observed. These results showed that knock down or inhibition of LPA(1) offered significant protection against LPS-induced lung inflammation but not against pulmonary leak as observed in the murine model for lung injury.     

Inhaled nitric oxide attenuates acute lung injury via inhibition of nuclear factor-kappa B and inflammation.

The effect of inhaled nitric oxide (NO) on inflammatory process in acute lung injury (ALI) is unclear. The aims of this study were to 1) examine whether inhaled NO affects the biochemical lung injury parameters and cellular inflammatory responses and 2) determine the effect of inhaled NO on the activation of nuclear factor-kappa B (NF-kappa B) in lipopolysaccharide (LPS)-induced ALI. Compared with saline controls, rabbits treated intravenously with LPS showed increases in total protein and lactate dehydrogenase in the bronchoalveolar lavage (BAL) fluid, indicating ALI. LPS-treated animals with NO inhalation (LPS-NO) showed significant decreases in these parameters. Neutrophil numbers in the BAL fluid, the activity of reactive oxygen species in BAL cells, and the levels of interleukin (IL)-1 beta and IL-8 in alveolar macrophages were increased in LPS-treated animals. In contrast, neutrophil numbers and these cellular activities were substantially decreased in LPS-NO animals, compared with LPS-treated animals. NF-kappa B activation in alveolar macrophages from LPS-treated animals was also markedly increased, whereas this activity was effectively blocked in LPS-NO animals. These results suggest that inhaled NO attenuates LPS-induced ALI and pulmonary inflammation. This attenuation may be associated with the inhibition of NF-kappa B activation.     

Role of TLR4-p38 MAPK-Hsp27 signal pathway in LPS-induced pulmonary epithelial hyperpermeability

Background

The breakdown of alveolar barrier dysfunction contributes to Lipopolysaccharide stimulated pulmonary edema and acute lung injury. Actin cytoskeleton has been implicated to be critical in regulation of epithelial barrier. Here, we performed in vivo and in vitro study to investigate role of TLR4-p38 MAPK-Hsp27 signal pathway in LPS-induced ALI.

Methods

For in vivo studies, 6–8-week-old C57 mice were used, Bronchoalveolar lavage Fluid /Blood fluorescent ratio, wet-to-dry lung weight ratio, as well as protein concentrations and neutrophil cell counts in BALF were detected as either directly or indirectly indicators of pulmonary alveolar barrier dysfunction. And hematoxylin and eosin staining was performed to estimate pulmonary injury. The in vitro explorations of transepithelial permeability were achieved through transepithelial electrical resistance measurement and testing of FITC-Dextran transepithelial flux in A549. In addition, cytoskeletal rearrangement was tested through F-actin immunostaining. And SB203580 was used to inhibit p38 MAPK activation, while siRNA was administered to genetically knockdown specific protein.

Results

We showed that LPS triggered activation of p38 MAPK, rearrangement of cytoskeleton which resulted in severe epithelial hyperpermeability and lung edema. A549 pretreated with TLR4 siRNA、p38 MAPK siRNA and its inhibitor SB203580 displayed a lower permeability and fewer stress fibers formation after LPS stimulation, accompanied with lower phosphorylation level of p38 MAPK and Hsp27, which verified the involvement of TLR4-p38 MAPK-Hsp27 in LPS-evoked alveolar epithelial injury. Inhibition of p38 MAPK activity with SB203580 in vivo attenuated pulmonary edema formation and hyperpermeability in response to LPS.

Conclusions

Our study demonstrated that LPS increased alveolar epithelial permeability both in vitro and in vivo and that TLR4- p38 MAPK- Hsp27 signal pathway dependent actin remolding was involved in this process.

     

A crucial role of Flagellin in the induction of airway mucus production by Pseudomonas aeruginosa

Pseudomonas aeruginosa is an opportunistic pathogen involved in nosocomial infections. Flagellin is a P. aeruginosa virulence factor involved in host response to this pathogen. We examined the role of flagellin in P. aeruginosa-induced mucus secretion. Using a mouse model of pulmonary infection we showed that PAK, a wild type strain of P. aeruginosa, induced airway mucus secretion and mucin muc5ac expression at higher levels than its flagellin-deficient mutant (ΔFliC). PAK induced expression of MUC5AC and MUC2 in both human airway epithelial NCI-H292 cell line and in primary epithelial cells. In contrast, ΔFliC infection had lower to no effect on MUC5AC and MUC2 expressions. A purified P. aeruginosa flagellin induced MUC5AC expression in parallel to IL-8 secretion in NCI-H292 cells. Accordingly, ΔFliC mutant stimulated IL-8 secretion at significantly lower levels compared to PAK. Incubation of NCI-H292 cells with exogenous IL-8 induced MUC5AC expression and pre-incubation of these cells with an anti-IL-8 antibody abrogated flagellin-mediated MUC5AC expression. Silencing of TLR5 and Naip, siRNA inhibited both flagellin-induced MUC5AC expression and IL-8 secretion. Finally, inhibition of ERK abolished the expression of both PAK- and flagellin-induced MUC5AC. We conclude that: (i) flagellin is crucial in P. aeruginosa-induced mucus hyper-secretion through TLR5 and Naip pathways; (ii) this process is mediated by ERK and amplified by IL-8. Our findings help understand the mechanisms involved in mucus secretion during pulmonary infectious disease induced by P. aeruginosa, such as in cystic fibrosis.     

Diesel exhaust particles induce matrix metalloprotease-1 in human lung epithelial cells via a NADP(H) oxidase/NOX4 redox-dependent mechanism

Chronic exposure to particulate air pollution is associated with lung function impairment. To determine the molecular mechanism(s) of this phenomenon, we investigated, in an alveolar human epithelial cell line (A549), whether diesel exhaust particles (DEPs), a main component of particulate air pollution, modulates the expression and activity of the matrix metalloprotease (MMP)-1, a collagenase involved in alveolar wall degradation. Interaction of DEPs with cigarette smoke, which also produces structural and functional lung alterations, was also investigated. A noncytotoxic concentration of DEPs induced an increase in MMP-1 mRNA and protein expression and activity in A549 cells without modifying the expression of the MMP inhibitors TIMP-1 and -2. This effect was not potentiated when cells were coexposed to noncytotoxic concentrations of cigarette smoke condensate. DEP-induced MMP-1 was associated with increased ERK 1/2 phosphorylation and upregulation of expression and activity of the NADPH oxidase analog NOX4. Cell transfection with a NOX4 small interfering RNA prevented these phenomena, showing the critical role of a NOX4 ERK 1/2 pathway in DEP-induced MMP-1 expression and activity. Similar results to those observed in A549 cells were obtained in another human lung epithelial cell line, NCI-H292. Furthermore, experiments in mice intratracheally instilled with DEPs confirmed the in vitro findings, showing the induction of NOX4 and MMP-1 protein expression in alveolar epithelial cells. We conclude that alveolar alterations secondary to MMP-1 induction could explain lung function impairment associated with exposure to particulate pollution.