Regular paper

Pseudomonas aeruginosa induces spatio-temporal secretion of IL-1β, TNFα, proMMP-9, and reduction of epithelial E-cadherin in human alveolar epithelial type II (A549) cells

Paulina Fuentes-Zacarías1, Diego Armando Arzate-Castañeda1, Irma Elena Sosa-González2, Graciela Villeda-Gabriel2, Iyari Morales-Méndez1, Mauricio Osorio-Caballero3,
Addy Cecilia Helguera-Repetto1, Néstor Fabián Díaz4, Guadalupe García-López4,
Oscar Flores-Herrera5, Francisco Arenas-Huertero6, Carlos Eslava-Campos7, Oscar Díaz-Ruíz8 and Héctor Flores-Herrera1✉

1Department of Inmunobioquímica, Instituto Nacional de Perinatología “Isidro Espinoza de los Reyes” (INPerIER), Ciudad de México, México; 2Department of Inmunología e Infectología, INPerIER, Ciudad de México, México; 3Department of Salud Sexual y Reproductiva, INPerIER, Ciudad de México, México; 4Department of Fisiología y Desarrollo Celular, INPerIER, Ciudad de México, México; 5Department of Bioquimica, Escuela de Medicina, UNAM, Ciudad de México, México; 6Department of Patología Experimental, Hospital Infantil de México “Federico Gómez”, Ciudad de México, México; 7Laboratory of Patogenicidad Bacteriana, Unidad de Hemato-Oncología e Investigación, Hospital Infantil de México “Federico Gómez”, Ciudad de México, México; 8Department of Pharmacology, Emory University School of Medicine, Atlanta, GA 30322, USA

Pseudomonas aeruginosa, is an opportunistic bacterium with a high prevalence in diverse pulmonary infections. Although several genes are involved in the system of resistance and evasion of the immunological response of the host, little is known about the inflammatory, degradative, and cell-binding response induced by P. aeruginosa in human lung alveolar epithelial cells. The purpose of this study was to determine the cytokine expression (IL-1β and TNFα), pro matrix metalloproteinases activation (proMMP-2 and proMMP-9), and the effects on the cell-binding adhesion protein (E-cadherin) in an in vitro model of human lung alveolar epithelial cells. A549 cells were stimulated with a different number of colony-forming units of P. aeruginosa for 3, 6, and 24 hours. Subsequently, the culture medium was collected, IL-1β and TNFα levels were evaluated by ELISA; proMMP-2 and -9 levels were determined by substrate gel zymography, and the MMP-9 and E-cadherin assessed by immunostaining of A549 cells. Our results demonstrated that P. aeruginosa induces mainly the secretion of TNFα, increases actMMP-9 level, and significantly reduces the level of E-cadherin in the A549 cells. In summary, the inflammatory/degradative process induced by P. aeruginosa modulates the expression of the E-cadherin protein. The probable clinical implications of this study suggest the use of inhibitors that reduce the degradative activity of proMMP-9 which will be further explored in the next phase of this study.

Keywords: E-cadherin, human lung alveolar epithelial cells, matrix metalloproteinase, Pseudomonas aeruginosa, proinflammatory cytokine.

Received: 03 October, 2020; revised: 20 January, 2021; accepted:
20 January, 2021; available on-line: 04 May, 2021

✉e-mail: h.flores@inper.gob.mx

Acknowledgments of Financial Support: This work was supported by a grant from the National Institute of Perinatology “Isidro Espoinosa de los Reyes” (number 212250-22661 assigned to HFH), Mexico City. Paulina Fuentes-Zacarías was a fellowship recipient from Programas de Becas de Inicio a la Investigación Cientifica por la Comisión Coordinadora de Institutos Nacionales de Salud y Hospitales de Alta Especialidad (2013-2015) and from the Escuela Nacional de Ciencias Biológicas del Instituto Politécnico Nacional (2016-2017).

Abbreviations: MMP, matrix metalloproteinases; E-cadherin, epithelial cadherin; IL-1β, interleukin-1beta; CFU, colony-forming units; TNF-α, tumor necrosis factor-alpha; NFκβ, nuclear factor-kappa B

INTRODUCTION

Pseudomonas aeruginosa is an opportunistic Gram-negative bacterium that has been associated with chronic infections in airways (Beaudoin et al., 2012), cystic fibrosis (Holm et al., 2013) and pulmonary inflammation (Park et al., 2013). The pathogenicity of P. aeruginosa is mediated by several factors, including the production of diffusible molecules controlled by a mechanism known as quorum sensing (Chugani et al., 2012; Kownatzki et al., 1987; Perez et al., 2013; Rada and Leto, 2013). It was shown that lipopolysaccharides of P. aeruginosa induce in the alveolar and bronchial epithelium the secretion of nitric oxide (Pitt & St Croix, 2002), inflammatory cytokines (Wong & Johnson, 2013) and production of matrix metalloproteinases (MMPs) (Frisdal et al., 2001; Okamoto et al., 2002; Yao et al., 1996). MMPs are a family of zinc neutral endopeptidases produced in several pathological conditions (Churg et al., 2007; Holm et al., 2013) by a wide variety of cell types, including neutrophils (Bradley et al., 2012; Louhelainen et al., 2010), alveolar macrophages (Churg et al., 2007), and bronchial epithelial cells (Yao et al., 1996). MMPs induce degradations of various structural components of the extracellular matrix including collagen type I, IV, V, VII, X, fibronectin, elastin, proteoglycan (Woessner, 1991), basement membrane (Kargozaran et al., 2007) as well as cell-binding adhesion proteins (Allport et al., 2002; Nawrocki-Raby et al., 2003). Although the secretion of MMPs is well known in various lung diseases: bronchopulmonary dysplasia (Mizikova & Morty, 2015), adenocarcinomas (Canete-Soler et al., 1994), and chronic obstructive pulmonary disease (Louhelainen et al., 2010), the secretion profile of proMMP-2 and -9 produced by human pneumocytes secretory type II cells during infection with Pseudomonas aeruginosa is unknown. We chose the A549 cell line as it is a model of human lung alveolar epithelium which plays an important role in the immune response. We hypothesized that an increase in IL-1β and TNFα concentrations would be accompanied by a parallel increase in collagenolytic activity of MMP-2 and -9 in the culture medium, and thereby would induced changes in epithelial cadherin (E-cadherin) in A549 cells during transient P. aeruginosa stimulation.

MATERIALS AND METHODS

Antibodies and reagents

3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide, Mitochondrial uncoupler carbonyl cyanide m-chlorophenylhydrazone, Hoechst 33258, and 4´,6-diamidino-2-phenylindole were obtained from Sigma-Aldrich (St Louis, MO, USA). IL-1β and TNFα were purchased from R&D Systems (Minneapolis, MN, USA). Anti-MMP-9 antibodies were purchased from Calbiochem (Darmstadt, Germany). Anti-human E-cadherin antibody was purchased from BD Bioscience (San Jose. CA, USA).

Cell lines and culture

A549 cell line (American Type Culture Collections, Rockville, MD, USA) was obtained and its genetic profile corroborated by the amplification of 21 specific markers. The result showed a complete match with the A549 line (ATTC, CCL-185). A549 cells were cultivated on 12 well plates (Corning, Darmstadt, Germany) in RPMI 1640 medium (Roswell Park Memorial Institute; Gibco, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (FBS), an antibiotic-antimycotic solution (penicillin 100 U/mL, streptomycin 100 µg/mL; Gibco) and incubated at 37°C in 5% CO2. After reaching 95% of confluence, A549 cells were washed twice with sterile saline solution to remove RPMI-FBS, and 1 mL of RPMI with 0.2% lactoalbumin hydrolyzated (RPMI-LHA; Gibco) was added with subsequent incubation at 37°C in 5% CO2.

Bacterial strain and preparation

Prior to the stimulation experiments, we confirmed P. aeruginosa (ATCC 27853, Rockville, MD, USA) identity through the following screening methods: morphology, production of pigments (pyocyanin and fluorescein), and disk method to assess susceptibility-resistance for penicillins (piperacillin, carbenicillin), β-lactam-β-lactamase inhibitors combinations (piperacillin-tazobactam), cephems (ceftriaxone, cefoperazone, cefepime, and ceftazidime), carbapenems (meropenem), monobactams (aztreonam), aminoglycosides (gentamicin, and amikacin), fluoroquinolones (ciprofloxacin, norfloxacin). These analyses confirmed that P. aeruginosa strain maintains all its characteristics. For the stimulation assays P. aeruginosa was grown in 5% Blood Agar Base (Becton Dickison, USA) and harvested in calf medium.

Cell stimulation

After reaching 95% confluence, A549 cells were washed twice with sterile saline solution to remove RPMI-FBS and 1 mL of RPMI with 0.2% lactoalbumin hydrolysate (RPMI-LHA; Gibco) was added before incubation at 37°C in 5% CO2. Next A549 cells were infected with live P. aeruginosa in serial dilutions (102, 104, 105, and 106 colony-forming units (CFU/mL)). The CFU numbers were based on a turbidity equivalent to 0.5 McFarland standard. After the infection, A549 cells were cultured for 3, 6 or 24 hours. At the end of the incubation time, the medium was collected and samples were centrifugated at 1400 rpm at 4°C for 5 min, the supernatants were collected and stored at -70°C until further processing.

Cell viability assay

To evaluate A549 cells viability after incubation with P. aeruginosa we used the colorimetric assay of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MMT) as previously described by Zeng et al. (2017). Cells were washed twice with sterile saline solution to remove RPMI-LHA and P. aeruginosa, and then cultured for 3 hours in presence of 20 µl (5 mg/mL) of MMT in 5% CO2 at 37°C. Subsequently, 150 µl of Dimethyl sulfoxide (DMSO; Merck KGaA, Darmstadt, Germany) was added into each well (Zeng et al., 2017). For negative control, a mitochondrial uncoupler carbonyl cyanide m-chlorophenylhydrazone (CCCP) was dissolved in dimethylsulfoxide at a concentration of 80 µM (Chaudhari et al., 2008) and added to the cells before the incubation at 37°C with 5% CO2, 95 % air. Blue formazan product in the culture medium from A549 cells was analyzed by spectrophotometric absorbance reading at 570 nm in Benchmark microplate (model 550; BioRad. Hercules, CA, USA). Five independent experiments were performed, each in duplicate.

Measurement of proinflammatory cytokines

To quantify IL-1β and TNFα secreted to the culture medium of A549 cells after each period of incubation with P. aeruginosa we used a specific DouSet enzyme-linked immunosorbent assay (ELISA) (R&D Systems, Minneapolis, MN, USA) following the manufacturer´s instructions. This procedure was previously reported by our research group (Flores-Herrera et al., 2012; Osorio-Caballero et al., 2015). For IL-1β (DY201; R&D Systems) and TNF (DY210; R&D Systems), a standard curve was created from 4 to 260 pg/mL and 15 to 960 pg/mL, with a sensitivity of 2.0 and 5.0 pg/mL, respectively. The concentration of IL-1β and TNFα were expressed as pg/mL. The ELISA assay was performed in eight independent experiments.

Zymography gel activity

To evaluate the secretion of proMMP-2 and proMMP-9 into the culture media of A549 cells, SDS-polyacrylamide gels with porcine gelatin (1 mg/mL) were used as described previously (Flores-Herrera et al., 2012). Each well was loaded with 0.75 µg of protein and the activity band was determined by optical density using NIH ImageJ. We used a culture medium from, promyelocyte cells as a control of electrophoretic mobility (U937, ATCC, CRL-1593.2; Manassas, VA, USA). The gel activity assay was performed in eight independent experiments.

Inmunodetection of MMP-9 and E-cadherin in the A549 cells

To localize MMP-9 in A549 cells after infection with P. aeruginosa we used immunefluorescence as described previously (Flores-Herrera et al., 2012). After fixing the cells with 4% paraformaldehyde for 10 minutes, a primary mouse anti-MMP9 antibody (clone 56-2A4; Calbiochem Darmstadt, Germany) was added at 1:50 dilution. An appropriate fluorescent-labeled secondary antibody (Molecular Probes, USA) was used. The nucleus was stained with 1 ng/ml of Hoechst 33258 (Sigma-Aldrich). In another set of experiments, E-cadherin was immunodetected using mouse anti-human E-cadherin (clone NCH-38) antibody at a 1:100 dilution. The nucleus was stained using 4’,6-diamidino-2-phenylindole (DAPI) for 7 minutes. Negative controls consisted of cells without primary antibody, and, as expected, they did not exhibit any staining (not shown). The immunostaining was analyzed using an epi-fluorescence microscope (Olympus, IX-81, Tokyo, Japan) and photographed with a CCD camera (Hamamatsu, ORCA-Flash 2.8, Tokio, Japan).

Statistical analysis

Data were analyzed by one-way ANOVA with multiple comparisons followed by Tukey´s test using SigmaPlot version 11.0 (San Jose, CA, USA). Results are expressed as mean ± S.E.M. p<0.05 was considered significant. Immunostainings of proMMP-9 and E-cadherine were performed five times.

RESULTS

Effects of P. aeruginosa on A549 cells viability

Figure 1 shows the viability of A549 cells with and without P. aeruginosa stimulation after 3 (1A), 6 (1B) and 24 hours (1C). The viability was not affected by the different doses of P. aeruginosa when compared to the control group (p=0.65). In the same experiments, we included the mitochondrial inhibitor (CCCP), which significantly reduced the viability of A549 cells in comparison to the control group (p<0.05). Finally, MMT was not metabolized by P. aeruginosa (Fig. 1). These experiments demonstrated that infection with P. aeruginosa did not affect the viability of A549 cells. We then assessed the effect of P. aeruginosa on the secretion of IL-1β and TNFα.

Secretion of proinflammatory cytokines by A549 cells

IL-1β

Figure 2 shows that the stimulation of A549 cells with P. aeruginosa significantly increased the secretion of IL-1β in a dose-dependently manner. After 3 hours of stimulation with P. aeruginosa at 105, and 106 CFU/mL, A549 cells significantly increased the secretion of IL-1β by 1.2- and 1.6-fold, respectively, in comparison to the control (2.3±0.7; p≤0.05, Fig. 2A). A similar secretion profile was observed after 6 hours of stimulation (Fig. 2B). Maximal secretion of IL-1β was detected after 24 hours of stimulation with P. aeruginosa at 102, 104, 105, and 106 CFU/mL, with 3.8-, 5.1-, 6.7-, and 8.1-fold increase, respectively, when compared to the control (2.7±0.216; p≤0.05, Fig. 2C).

TNFα

Figure 3 shows that stimulation of A549 cells with P. aeruginosa increased the secretion profile of TNFα in a dose-dependent manner. After 3 hours of stimulation with P. aeruginosa at 104, 105, and 106 CFU/mL, A549 cells significantly increased the secretion of TNFα by 1.4-, 1.5-, and 1.6-fold, respectively when compared with the control (6.4±0.4; p≤0.05, Fig. 3A). A similar secretion profile was observed after 6 hours of stimulation (Fig. 3B). Maximal secretion was observed after 24 hours of stimulation with P. aeruginosa at 102, 104, 105, and 106 CFU/mL with 3.8-, 5.1-, 6.7-, and 8.1-fold increase, respectively, when compared to the control (6.7±1.1; p≤0.05, Fig. 3C).

Interestingly, it was shown that IL-1β and TNFα induce the secretion of MMPs (Roomi et al., 2013). Therefore, our next step was to determine the effect of the inflammatory responses induced by P. aeruginosa on the secretion of extracellular matrix metalloproteases into the culture medium from A549 cells. Lysis bands for proMMP-2 and -9 were identified by taking the mobility of U937 standard as a reference point, as previously reported and validated by our research group (Flores-Herrera et al., 2012).

Production of metalloproteinases by A549 cells

Figure 4 shows the lysis bands of proMMP-2 and -9 secreted by A549 cells after stimulation with P. aeruginosa for 3 (4A), 6 (4E), and 24 hours (4I). The relative densitometric analysis indicated that after 3 (Fig. 4B) and 6 hours (Fig. 4F) of stimulation, significantly higher levels of proMMP-2 were detected when compared to the control. Maximal secretion of proMMP-2 was observed after 24 hours of stimulation with P. aeruginosa at 105, and 106 CFU/mL, with a 1.3-fold increase compared to the control (48.7±2.8; p≤0.05 Fig. 4J). After the same period of stimulation, we observed a band of 66-KDa that corresponded to the MMP-2 active form (Fig. 4J).

The relative densitometric analysis indicated that after 3 (Fig. 4B) and 6 hours (Fig. 4F) of stimulation with P. aeruginosa, A549 cells secreted proMMP-9 in a dose-dependent manner. Maximal secretion was detected after incubation with 104, 105 and 106 CFU/mL with 1.8-, 1.7-, and 2.0-fold increase, respectively, in comparison to the control (21.7±2.4; p≤0.05, Fig. 4F). Interestingly, after 24 hours of stimulation, we did not detect the lysis band corresponding to proMMP-9 (Fig. 4J).

Consistent with these findings, we observed morphological changes in A549 cells characterized by an increase in the number of spherical cells (Fig. 4L), when compared to the control group (Fig. 4K). This finding, together with the absence of the proMMP-9 band in activity gels, suggested that this enzyme can be located in the extracellular matrix of A549 cells, as previously reported under other pathological conditions (Flores-Herrera et al., 2012; Nawrocki-Raby et al., 2003). To explore this hypothesis, we performed immunolocalization with specific antibodies.

proMMP-9 detection in A549 cells by immunofluorescence

As shown in Fig. 5, proMMP-9 was immunodetected in the extracellular matrix of A549 cells after stimulation with 106 CFU/mL of P. aeruginosa. We observed a significant increase in immunoreactivity after 3, 6 and 24 hours compared to the respective controls (Fig. 5). As it was previously demonstrated in another cellular system, the active isoform of MMP-9 is able to degrade different support components, including collagen type I, IV, V, XI, elastin, and proteoglycan of the extracellular matrix (Morrison et al., 2009; Woessner, 1991), as well as cell-binding proteins such as vascular endothelial-cadherin (Allport et al., 2002) and E-cadherin (Nawrocki-Raby et al., 2003). After observing a change in the morphology of A549 cells, a reduction in the number of adhered cells (data not shown), and a decrease of proMMP-9 immunoreactivity, we complemented our approach by analyzing E-cadherin using immunodetection.

proMMP-9 reduces E-cadherin immunofluorescence in A549 cells

A549 cells incubated for 24 hours with P. aeruginosa showed very low immunostaining intensity for E-cadherin compared to the respective controls (Fig. 6). Immunoreactivity was located around the cells and the nuclei.

DISCUSSION

Several in vivo and in vitro models of infection are able to release a diverse set of molecules that are associated with cellular stress (Osorio-Caballero et al., 2015), and the reduction of chemotactic (Henriquez et al., 2015) and proinflammatory cytokines (Keyel, 2014; van de Veerdonk et al., 2011), which are involved in the next phase of the inflammatory response through the secretion of degradatives enzymes, such as proMMPs (Flores-Herrera et al., 2012). In in vitro models, the induction of the degradative response affects cell integrity by decreasing the expression of cell-cell adhesion proteins, like E-cadherin and vascular endothelial-cadherin (Allport et al., 2002; Nawrocki-Raby et al., 2003). However, little evidence is available on the effect of Pseudomonas aeruginosa on the inflammatory-degradative response in human lung alveolar epithelial type II (A549 line) cells.

Our results showed that P. aeruginosa was able to increase the secretion of 1) the proinflammatory cytokines IL-1β and TNFα; and 2) the prodegradative enzyme MMP-9 in a time- and concentration-dependent manner. This proinflammatory/prodegradative environment compromised cell viability through changes in cell morphology and decrease of E-cadherin expression in the A549 cells.

IL-1β is a pivotal cytokine in several second messenger signaling pathways. It is involved in the activation of the inflammatory response (Chen et al., 2017; Ledesma et al., 2004), acts as a modulator of the specialized cells of the immune system (Gabay et al., 2010; Rubartelli et al., 1990), and induces the expression of MMPs (Eberhardt et al., 2000; Nam & Kwon, 2014). The production of IL-1β by alveolar macrophages and epithelial cells is induced by different bacterial components that interact with Toll-like receptors 4 (TLR4). Interestingly, this receptor has high homology with the IL-1R receptor which amplifies the inflammatory response and promotes the activation of transcription factors, such as nuclear factor kappa-beta (NFĸβ) and activator protein (AP-1), inducing the expression of genes related to the inflammatory response (Armstrong et al., 2004; Parker et al., 2016). Wong and others (Wong et al., 2012) showed that alveolar type I cells obtained from rats that were stimulated with LPS from E. coli for 18 hours, show high expression of TNFα and IL-1β, but a low expression of IL-6 (Wong & Johnson, 2013). Similarly, in our experiments A549 cells stimulated during with P. aeruginosa for 24 hours showed a 10-fold increase in secretion of TNFα (Fig. 3C) in comparison to IL-1β (Fig. 2C).

Saperstein and others (Saperstein et al., 2009) and Thorley and others (Thorley et al., 2007), demonstrated that the IL-1β signaling pathways modulate TNFα secretion. They used mouse lung epithelial type II and primary human alveolar type II cells to show that increase of TNFα can be reversed by using small interfering RNA and by neutralizing IL-1β with a specific antibody, respectively.

Recently, Jayaraman and others (Jayaraman et al., 2013) proposed a hypothetical mechanism by which IL-1β increases the secretion of TNFα via interacting with the type-1 form of the TNF receptor (TNFR1) and increasing the secretion of the soluble form of TNFα (Jayaraman et al., 2013; MacEwan, 2002). However, a alternative mechanisms mediated by nuclear factor kappa-beta (NFĸβ) could also explain the link between IL-1β and TNFα (Fig. 7). NFĸβ plays an important role in the immunological pathway (Tak & Firestein, 2001), and mutations of cellular NFκβ induced changes in this immunological response (Picard et al., 2011; Sung et al., 2014). NFĸβ and mitogen-activated protein kinases (MAPKs) knockout mice displayed an altered inflammatory response of chemokines and cytokines after LPS stimulation (Picard et al., 2011; Sung et al., 2014).

The next phase of the inflammatory response promoted by IL-1β/TNFα is the expression and secretion of MMPs (Fang et al., 2006; Flores-Herrera et al., 2012). Our results suggest that an infectious and inflammatory process modulates the secretion of proMMP-2 and -9 in a dose-dependent manner and in relation to the stimulation time (Fig. 4).

There is evidence of the mechanism through which IL-1β (Eberhardt et al., 2000; Mon et al., 2017; Ruhul Amin et al., 2003) and TNFα (Fang et al., 2006; Jayaraman et al., 2013; Mon et al., 2006; Tsai et al., 2014) increase the activity of MMP-9 (Fig. 7). Recently, Mon et al. (2017) demonstrated that IL-1β activates MMP-9 through a series of intracellular signals initiated by the activation of the proto-oncogene tyrosine-protein kinase Src (Src) which phosphorylates two tyrosines (Y397 and Y925), activating the system mediated by the growth factor receptor-bound protein 2 (Grb2) and Ras-dependent MAPK protein. This complex activates the MMP-9 (Mon et al., 2017). In addition, it was also shown by Mon et al. (2006) that TNFα interacts with the focal adhesion kinase (FAK) directly involved in the MMP-9 expresion. FAK activation is mediated by the TNFR2 receptor in two tyrosine (Y398, and Y925). These findings were confirmed using an antibody against TNFR2, which inhibited FAK phosphorylation and by using FAK-/- cells, which prevented the degradative activity of MMP-9 (Mon et al., 2006).

Finally, after 24 hours of stimulation with P. aeruginosa we observed a 72 KDa band corresponding to proMMP-2 and a 62-KDa band corresponding to its active form (Fig. 4E). Unfortunately, the activity of MMP-2 could not be determined. Furthermore, proMMP-9 (92 KDa) could not be clearly identified in the activity gels (Fig. 4E and F), but it was clearly detected in the extracellular matrix of A549 cells using a specific antibody (Fig. 5). Alterations in the morphology of A549 cells were also evident (Fig. 6). Frisdal et al. (Frisdal et al., 2001) and Jackson and others (Jackson et al., 2010) have shown higher expression of MMP-2 and -9 during pulmonary pathological. During physiological development, MMPs are secreted into the extracellular space in the form of proMMPs and are bound to specific tissue inhibitors (TIMPs), as well as to the membrane-type metalloproteases (MT-MMP) (Somerville et al., 2003). Their activation is triggered by the removal of the peptides associated with the active site of the proMMP-2 (72 KDa) and proMMP-9 (92 KDa), inducing conformational change (Defawe et al., 2005; Koo et al., 2012; Somerville et al., 2003) (Fig. 7). Moreover, evidence from different sources suggests that in pathological processes, actMMP-9 degrades the E-cadherin involved in cell-cell adhesion (Allport et al., 2002; Nawrocki-Raby et al., 2003). Using immunohistochemistry, Shaco-Levy et al. (2008) showed that an increase in the secretion of actMMP-9 reduces the level of E-cadherin and intracellular β-catenin protein. Our results showed a reduction of the E-cadherin with relation to the concentration of P. aeruginosa used for stimulation and the time of stimulation (Fig. 7). Carayol et al. (2002) and Kim et al. (2018) used human nasal epithelial cell to demonstrated the association between an increase of MMP-9 expression and a decrease in E-cadherin levels. Interestingly, the activity of MMP-9 was inhibited by preincubation with dexamethasone which was accompanied by increased levels of E-cadherin (Carayol et al., 2002; Kim et al., 2018).

Although in this study we did not examine the expression of NFĸβ, we are planning to do it as part of our research project in order to explore the potential links between inflammasome (IL-1β/TNFα) and NFĸβ.

The studies reported here demonstrated that P. aeruginosa induces mainly the secretion of TNFα, increasing the actMMP-9, and significantly reduces the level of E-cadherin in the A549 cells.

Conflicts of Interest

The authors declare no financial or commercial conflict of interest.

REFERENCES

Allport JR, Lim YC, Shipley JM, Senior RM, Shapiro SD, Matsuyoshi N, Vestweber D, Luscinskas FW (2002) Neutrophils from MMP-9- or neutrophil elastase-deficient mice show no defect in transendothelial migration under flow in vitro. J Leukoc Biol 71: 821–828

Armstrong L, Medford AR, Uppington KM, Robertson J, Witherden IR, Tetley TD, Millar AB (2004) Expression of functional toll-like receptor-2 and -4 on alveolar epithelial cells. Am J Respir Cell Mol Biol 31: 241–245. https://doi.org/10.1165/rcmb.2004-00780C

Beaudoin T, Lafayette S, Nguyen D, Rousseau S (2012) Mucoid Pseudomonas aeruginosa caused by mucA mutations result in activation of TLR2 in addition to TLR5 in airway epithelial cells. Biochem Biophys Res Commun 428: 150–154. https://doi.org/10.1016/j.bbrc.2012.10.030

Bradley LM, Douglass MF, Chatterjee D, Akira S, Baaten BJ (2012) Matrix metalloprotease 9 mediates neutrophil migration into the airways in response to influenza virus-induced toll-like receptor signaling. PLoS Pathog 8(4):e1002641. https://doi.org/10.1371/journal.ppat1002641

Canete-Soler R, Litzky L, Lubensky I, Muschel RJ (1994) Localization of the 92 kd gelatinase mRNA in squamous cell and adenocarcinomas of the lung using in situ hybridization. Am J Pathol 144: 518–527

Carayol N, Vachier I, Campbell A, Crampette L, Bousquet J, Godard P, Chanez P (2002) Regulation of E-cadherin expression by dexamethasone and tumour necrosis factor-alpha in nasal epithelium. Eur Respir J 20: 1430–1436. https://doi.org/10.1183/09031936.02.00013602

Chaudhari AA, Seol JW, Kang SJ, Park SY (2008). Mitochondrial transmembrane potential and reactive oxygen species generation regulate the enhanced effect of CCCP on TRAIL-induced SNU-638 cell apoptosis. J Vet Med Sci 70: 537–542. https://doi.org/10.1292/jvms.70.537

Chen AC, Xi Y, Carroll M, Petsky HL, Gardiner SJ, Pizzutto SJ, Yerkovich ST, Baines KJ, Gibson PG, Hodge S, Masters IB, Buntain HM, Chang AB, Upham JW (2017) Cytokine responses to two common respiratory pathogens in children are dependent on interleukin-1beta. ERJ Open Res 3: 00025–2017. https://doi.org/10.1183/23120541.00025-2017

Chugani S, Kim BS, Phattarasukol S, Brittnacher MJ, Choi SH, Harwood CS, Greenberg EP (2012) Strain-dependent diversity in the Pseudomonas aeruginosa quorum-sensing regulon. Proc Natl Acad Sci USA 9: 109: E2823–E2831. https://doi.org/10.1073(pnas.1214128109

Churg A, Wang X, Wang RD, Meixner SC, Pryzdial EL, Wright JL (2007) Alpha1-antitrypsin suppresses TNF-alpha and MMP-12 production by cigarette smoke-stimulated macrophages. Am J Respir Cell Mol Biol 37: 144–151. https://doi.org/10.1165/rcmb.2006-0345OC

Defawe OD, Kenagy RD, Choi C, Wan SY, Deroanne C, Nusgens B, Sakalihasan N, Colige A, Clowes AW (2005) MMP-9 regulates both positively and negatively collagen gel contraction: a nonproteolytic function of MMP-9. Cardiovasc Res 66: 402–409. https://doi.org/10.1016/j.cardiores.2004.11.025

Eberhardt W, Huwiler A, Beck KF, Walpen S, Pfeilschifter J (2000) Amplification of IL-1 beta-induced matrix metalloproteinase-9 expression by superoxide in rat glomerular mesangial cells is mediated by increased activities of NF-kappa B and activating protein-1 and involves activation of the mitogen-activated protein kinase pathways. J Immunol 165: 5788–5797. https://doi.org/10.4049/jimmunol.165.10.5788

Fang Q, Liu X, Al-Mugotir M, Kobayashi T, Abe S, Kohyama T, Rennard SI (2006). Thrombin and TNF-alpha/IL-1beta synergistically induce fibroblast-mediated collagen gel degradation. Am J Respir Cell Mol Biol 35: 714–721. https://doi.org/10.1165/rcmb.2005-0026OC

Flores-Herrera H, Garcia-Lopez G, Diaz NF, Molina-Hernandez A, Osorio-Caballero M, Soriano-Becerril D, Zaga-Clavellina V (2012) An experimental mixed bacterial infection induced differential secretion of proinflammatory cytokines (IL-1beta, TNFalpha) and proMMP-9 in human fetal membranes. Placenta 33: 271–277. https://doi.org/10.1016/j.placenta.2012.01.007

Frisdal E, Gest V, Vieillard-Baron A, Levame M, Lepetit H, Eddahibi S, Lafuma C, Harf A, Adnot S, Dortho MP (2001) Gelatinase expression in pulmonary arteries during experimental pulmonary hypertension. Eur Respir J 18: 838–845. https://doi.org/10.1183/09031936.01.00084601

Gabay C, Lamacchia C, Palmer G (2010) IL-1 pathways in inflammation and human diseases. Nat Rev Rheumatol 6: 232–241. https://doi.org/10.1038/nrrheum.2010.4

Henriquez KM, Hayney MS, Xie Y, Zhang Z, Barrett B (2015) Association of interleukin-8 and neutrophils with nasal symptom severity during acute respiratory infection. J Med Virol 87: 330–337. https://doi.org/10.1002/jmv.24042

Holm JP, Hilberg O, Noerskov-Lauritsen N, Bendstrup E (2013) Pseudomonas aeruginosa in patients without cystic fibrosis is strongly associated with chronic obstructive lung disease. Dan Med J 60: A4636.

Jackson PL, Xu X, Wilson L, Weathington NM, Clancy JP, Blalock JE, Gaggar A (2010) Human neutrophil elastase-mediated cleavage sites of MMP-9 and TIMP-1: implications to cystic fibrosis proteolytic dysfunction. Mol Med 16: 159–166. https://doi.org/10.2119/molmed.2009.00109

Jayaraman P, Sada-Ovalle I, Nishimura T, Anderson AC, Kuchroo VK, Remold HG, Behar SM (2013) IL-1beta promotes antimicrobial immunity in macrophages by regulating TNFR signaling and caspase-3 activation. J Immunol 190: 4196–4204. https://doi.org/10.4049/jimmunol.1202688

Kargozaran H, Yuan SY, Breslin JW, Watson KD, Gaudreault N, Breen A, Wu MH (2007) A role for endothelial-derived matrix metalloproteinase-2 in breast cancer cell transmigration across the endothelial-basement membrane barrier. Clin Exp Metastasis 24: 495–502. https://doi.org/10.1007/s10585-007-9086-6

Keyel PA (2014) How is inflammation initiated? Individual influences of IL-1, IL-18 and HMGB1. Cytokine 69: 136–145. https://doi.org/10.1016/j.cyto.2014.03.007

Kim B, Lee HJ, Im NR, Lee DY, Kang CY, Park IH, Lee SH, Lee SH, Baek SK, Kim TH (2018) Effect of matrix metalloproteinase inhibitor on disrupted E-cadherin after acid exposure in the human nasal epithelium. Laryngoscope 128: E1–E7. https://doi.org/10.1002./lary.26932

Koo BH, Kim YH, Han JH, Kim DS (2012) Dimerization of matrix metalloproteinase-2 (MMP-2): functional implication in MMP-2 activation. J Biol Chem 287: 22643–22653. https://doi.org/10.1074/jbc.M111.337949

Kownatzki R, Tummler B, Doring G (1987) Rhamnolipid of Pseudomonas aeruginosa in sputum of cystic fibrosis patients. Lancet 1: 1026–1027. https://doi.org/10.1016/s0140-6736(87)92286-0

Ledesma E, Martinez I, Cordova Y, Rodriguez-Sosa M, Monroy A, Mora L, Soto I, Ramos G, Weiss B, Santiago Osorio E (2004) Interleukin-1 beta (IL-1beta) induces tumor necrosis factor alpha (TNF-alpha) expression on mouse myeloid multipotent cell line 32D cl3 and inhibits their proliferation. Cytokine 26: 66–72. https://doi.org/10.1016/j.cyto.2003.12.009

Liu T, Zhang L, Joo D, Sun SC (2017) NF-ĸB signaling in inflammation. Signal Transduct Target Ther 23: 17023. https://doi.org/10.1038/sigtrans.2017.23

Louhelainen N, Stark H, Mazur W, Rytila P, Djukanovic R, Kinnula VL (2010) Elevation of sputum matrix metalloproteinase-9 persists up to 6 months after smoking cessation: a research study. BMC Pulm Med 10: 13. https://doi.org/10.1186/1471-2466-10-13

MacEwan DJ (2002) TNF ligands and receptors – a matter of life and death. Br J Pharmacol 135: 855–875. https://doi.org/10.1038/sj.bjp.0704549

Mizikova I, Morty RE (2015) The extracellular matrix in bronchopulmonary dysplasia: target and source. Front Med (Lausanne) 2: 91. https://doi.org/10.3389/fmed.2015.00091

Mon NN, Hasegawa H, Thant AA, Huang P, Tanimura Y, Senga T, Hamaguchi M (2006) A role for focal adhesion kinase signaling in tumor necrosis factor-alpha-dependent matrix metalloproteinase-9 production in a cholangiocarcinoma cell line, CCKS1. Cancer Res 66: 6778–6784. https://doi.org/10.1158/0008-5472.CAN-054159

Mon NN, Senga T, Ito S (2017) Interleukin-1beta activates focal adhesion kinase and Src to induce matrix metalloproteinase-9 production and invasion of MCF-7 breast cancer cells. Oncol Lett 13: 955–960. https://doi.org/10.3892/ol.2016.5521

Morrison CJ, Butler GS, Rodriguez D, Overall CM (2009) Matrix metalloproteinase proteomics: substrates, targets, and therapy. Curr Opin Cell Biol 21: 645–653. https://doi.org/10.1016/j.ceb.200906.006

Nam SI, Kwon TK (2014) Dexamethasone inhibits interleukin-1beta-induced matrix metalloproteinase-9 expression in cochlear cells. Clin Exp Otorhinolaryngol 7: 175–180. https://doi.org/10.3342/ceo.2014.7.3.175

Nawrocki-Raby B, Gilles C, Polette M, Martinella-Catusse C, Bonnet N, Puchelle E, Foidart JM, Van Roy F, Birembaut P (2003) E-Cadherin mediates MMP down-regulation in highly invasive bronchial tumor cells. Am J Pathol 163: 653–661. https://doi.org10.1016/S0002-9440(10)63692-9/

Okamoto T, Valacchi G, Gohil K, Akaike T, van der Vliet A (2002) S-nitrosothiols inhibit cytokine-mediated induction of matrix metalloproteinase-9 in airway epithelial cells. Am J Respir Cell Mol Biol 27: 463–473. https://doi.org/10.1165/rcmb.2002-0039OC

Osorio-Caballero M, Perdigon-Palacio C, Garcia-Lopez G, Flores-Herrera O, Olvera-Sanchez S, Morales-Mendez I, Sosa-Gonzalez I, Acevedo JF, Guzman-Grenfell AM, Molina-Hernandez A, Diaz NF, Flores-Herrera H (2015) Escherichia coli-induced temporal and differential secretion of heat-shock protein 70 and interleukin-1beta by human fetal membranes in a two-compartment culture system. Placenta 36: 262–269. https://doi.org/10.1016/j.placenta.2014.12011

Park KS, Lee J, Jang SC, Kim SR, Jang MH, Lotvall J, Kim YK, Gho YS (2013) Pulmonary inflammation induced by bacteria-free outer membrane vesicles from Pseudomonas aeruginosa. Am J Respir Cell Mol Biol 49: 637–645. https://doi.org/10.1165/rcmb.2012-0370OC

Parker D, Ahn D, Cohen T, Prince A (2016) Innate immune signaling activated by MDR bacteria in the airway. Physiol Rev 96: 19–53. https://doi.org/10.1152/physrev.00009.2015

Perez LR, Machado AB, Barth AL (2013) The presence of quorum-sensing genes in Pseudomonas isolates infecting cystic fibrosis and non-cystic fibrosis patients. Curr Microbiol 66: 418–420. https://doi.org/10.1007/s00284-012-0290-5

Picard C, Casanova JL, Puel A (2011) Infectious diseases in patients with IRAK-4, MyD88, NEMO, or IkappaBalpha deficiency. Clin Microbiol Rev 24: 490–497. https://doi.org/10.1128/CMR.00001-11

Pitt BR, St Croix CM (2002) Complex regulation of iNOS in lung. Am J Respir Cell Mol Biol 26: 6–9. https://doi.org/10.1165/ajrcmb.26.1.f224

Rada B, Leto TL (2013) Pyocyanin effects on respiratory epithelium: relevance in Pseudomonas aeruginosa airway infections. Trends Microbiol 21: 73–81. https://doi.org/10.1016/j.tim.2012.10.004

Roomi MW, Kalinovsky T, Monterrey J, Rath M, Niedzwiecki A (2013) In vitro modulation of MMP-2 and MMP-9 in adult human sarcoma cell lines by cytokines, inducers and inhibitors. Int J Oncol 43: 1787–1798. https://doi.org/10.3892/ijo.2013.2113

Rubartelli A, Cozzolino F, Talio M, Sitia R (1990) A novel secretory pathway for interleukin-1 beta, a protein lacking a signal sequence. EMBO J 9: 1503–1510

Ruhul Amin AR, Senga T, Oo ML, Thant AA, Hamaguchi M (2003) Secretion of matrix metalloproteinase-9 by the proinflammatory cytokine, IL-1beta: a role for the dual signalling pathways, Akt and Erk. Genes Cells 8: 515–523. https://doi.org/10.1046/j.1365-2443.2003.00652.x

Saperstein S, Chen L, Oakes D, Pryhuber G, Finkelstein J (2009) IL-1beta augments TNF-alpha-mediated inflammatory responses from lung epithelial cells. J Interferon Cytokine Res 29: 273–284. https://doi.org/10.1089/jir.2008.0076

Shaco-Levy R, Sharabi S, Benharroch D, Piura B, Sion-Vardy N (2008) Matrix metalloproteinases 2 and 9, E-cadherin, and beta-catenin expression in endometriosis, low-grade endometrial carcinoma and non-neoplastic eutopic endometrium. Eur J Obstet Gynecol Reprod Biol 139: 226–232. https://doi.org/10.1016/j.ejogrb.2008.01.004

Somerville RP, Oblander SA, Apte SS (2003) Matrix metalloproteinases: old dogs with new tricks. Genome Biol 4: 216. https://doi.org/10.1186/gb-2003-4-6-216

Sung MH, Li N, Lao Q, Gottschalk RA, Hager GL, Fraser ID (2014). Switching of the relative dominance between feedback mechanisms in lipopolysaccharide-induced NF-kappaB signaling. Sci Signal 7: ra6. https://doi.org/10.1126/scisignal.2004764

Tak PP, Firestein GS (2001) NF-kappaB: a key role in inflammatory diseases. J Clin Invest 107: 7–11. https://doi.org/10.1172/JCI11830

Thorley AJ, Ford PA, Giembycz MA, Goldstraw P, Young A, Tetley TD (2007) Differential regulation of cytokine release and leukocyte migration by lipopolysaccharide-stimulated primary human lung alveolar type II epithelial cells and macrophages. J Immunol 178: 463–473. https://doi.org/10.4049/jimmunol.178.1.463

Tsai CL, Chen WC, Hsieh HL, Chi PL, Hsiao LD, Yang CM (2014). TNF-alpha induces matrix metalloproteinase-9-dependent soluble intercellular adhesion molecule-1 release via TRAF2-mediated MAPKs and NF-kappaB activation in osteoblast-like MC3T3-E1 cells. J Biomed Sci 21: 12. https://doi.org/10.1186/1423-0127-21-12

van de Veerdonk FL, Netea MG, Dinarello CA, Joosten LA (2011) Inflammasome activation and IL-1beta and IL-18 processing during infection. Trends Immunol 32: 110–116. https://doi.org/10.1016/j.it.2011.01.003

Woessner JF Jr (1991) Matrix metalloproteinases and their inhibitors in connective tissue remodeling. FASEB J 5: 2145–2154

Wong MH, Johnson MD (2013) Differential response of primary alveolar type I and type II cells to LPS stimulation. PLoS One 8: e55545. https://doi.org/10.1371/journal.pone.0055545

Yao PM, Buhler JM, d’Ortho MP, Lebargy F, Delclaux C, Harf A, Lafuma C (1996) Expression of matrix metalloproteinase gelatinases A and B by cultured epithelial cells from human bronchial explants. J Biol Chem 271: 15580–15589. https://doi.org/10.1074/jbc.271.26.15580

Zeng H, Fu R, Yan L, Huang J (2017) Lycorine induces apoptosis of A549 cells via AMPK-mammalian target of rapamycin (mTOR)-S6K signaling pathway. Med Sci Monit 23: 2035–2041. https://doi.org/10.12659/msm.900742