Impact of Chronic Pulmonary Infection with Pseudomonas aeruginosa on Transfection Mediated by Viral and Nonviral Vectors

Abstract

Pseudomonas aeruginosa plays a crucial role in the lung pathology of cystic fibrosis (CF). We showed that acute infection with P. aeruginosa has a substantial impact on gene transfer into lung epithelial cells mediated by polyplexes. As an extension of those studies we report here on the effect of chronic pulmonary infection with P. aeruginosa on transfection of lung epithelial cells by viral and nonviral vectors. As an in vivo model of the persistent chronic infection in patients with CF we used C57BL/6 mice intratracheally infected with P. aeruginosa encapsulated in agar beads. Two weeks after infection the presence of viable bacteria in the lungs was confirmed, mostly in the bronchial lumen. In lung tissue sections stained with hematoxylin and eosin, extensive inflammatory infiltrations were found. At that time point the mice received an intratracheal dose of luciferase gene complexed with either Lipofectamine (Lf ), a GL67 lipid mixture (GL67), or polyethylenimine (PEI) or with lentivirus (LV) as a carrier system. Luciferase activity was determined by a luminescence assay in supernatants of lung homogenates. The transfection level induced by PEI/DNA polyplexes complexed with serum albumin was decreased in infected mice. Lf-mediated transfection was almost completely blocked in infected mice. Transfection levels in mice treated with LV or plain PEI/DNA polyplexes were unchanged in infected animals as compared with control mice. The only carrier that displayed a clearly increased transfection level in infected mice was the GL67 lipid mixture, which is tentatively ascribed to the presence of polyethylene glycol in this carrier.

Introduction

W e presented in vitro and in vivo data demonstrating the impact of acute infection by Pseudomonas aeruginosa on gene transfer into lung epithelial cells mediated by polyethylenimine/DNA complexes (Rejman et al., 2007). Pseudomonas aeruginosa infections play a crucial role in the lung pathology of cystic fibrosis (CF), a monogenetic disease that affects several organs, including the lungs. It is caused by mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR), a protein forming a chloride channel located in the apical membrane of epithelial cells. Impaired ion transport through the airway epithelium leads to the accumulation of a thick mucous layer, facilitating infection and colonization of the respiratory tract by bacteria such as P. aeruginosa. At early stages of the disease, patients with CF suffer from transient P. aeruginosa infections, which are still relatively easy to eradicate. However, repetitive cycles of P. aeruginosa infections ultimately lead to permanent lung colonization causing continuous inflammation, lung damage, and ultimately life-threatening pulmonary failure. Because CF is caused by mutations in a single gene, it made this disease ideal for treatment by gene therapy. Both viral and nonviral delivery of CFTR cDNA have been shown to correct the impaired primary electrophysiological potential difference to physiological levels (Bragonzi and Conese, 2002; Griesenbach et al., 2004, 2006). Moreover, it has been reported that as little as 5% of normal CFTR function suffices to provide for physiological levels of airway epithelial activity (Johnson et al., 1992; Ramalho et al., 2002). On the other hand, there are reports of no successful correction of Cl⁻ secretion and Na⁺ absorption in CF mice (Rozmahel et al., 1997; Ostrowski et al., 2007; Griesenbach et al., 2009). In the present study we extended our earlier observations on the acute infection model to a chronic pulmonary infection model in order to enhance its relevance for the situation in patients with CF.

Materials and Methods

Materials

Dithiothreitol (DTT), ethylenediaminetetraacetic acid (EDTA), glycerol, branched 25-kDa polyethylenimine (PEI), murine serum albumin (MSA), 2,2,2-tribromoethanol (Avertin), and Triton X-100 were purchased from Sigma-Aldrich (St. Louis, MO), GL67 (Genzyme cationic lipid 67; N ⁴-spermine cholesteryl carbamate) was from Genzyme (Framingham, MA); Lipofectamine 2000 was obtained from Invitrogen (Carlsbad, CA). All reagents were of analytical grade.

Bacterial strains

Pseudomonas aeruginosa, RP73 clinical strain isolated from a patient with CF, was kindly provided by B. Tümmler (Klinische Forschergruppe, Medizinische Hochschule Hannover, Hannover, Germany). Strain genotypic and phenotypic data have been reported previously (Bragonzi et al., 2006).

Plasmid preparation

Plasmid DNA carrying the Photinus pyralis luciferase coding region was isolated from Escherichia coli with an EndoFree plasmid kit (Qiagen, Hilden, Germany). It was stored in TE buffer (100 mM NaCl, 10 mM Tris-HCl [pH 8]). Its purity and concentration were determined by measuring absorbance at 260 and 280 nm and by gel electrophoresis.

Bacterial infection of airways

The agar bead model of chronic P. aeruginosa infection described earlier was used (Cash et al., 1979). P. aeruginosa RP73 strain was cultured overnight at 37°C in trypticase soy broth (TSB; Oxoid, Basingstoke, UK). A starting number of 5 × 10⁹ bacteria was used for inclusion in the agar beads as previously described (Bragonzi et al., 2005, 2009). Briefly, the bacteria were centrifuged and resuspended in 1 ml of phosphate-buffered saline (PBS), pH 7.4. Subsequently they were mixed with 9 ml of 1.5% trypticase soy agar (TSA; Oxoid) and dropped into 150 ml of heavy mineral oil prewarmed to 50°C. This mixture was stirred vigorously for 6 min at room temperature, which was followed by cooling at 4°C (continuous stirring, 20 min). The number of P. aeruginosa colony-forming units (CFU) in the beads was determined by plating serial dilutions of the homogenized bacteria–bead suspension on TSA plates. The inoculum was prepared by diluting the bead suspension with PBS to 4 × 10⁷ CFU/ml. C57BL/6NCrlBR mice (20–22 g; Charles River, Calco, Italy) were anesthetized with 2,2,2-tribromoethanol. The trachea was visualized by midline incision.

A 50-μl inoculum (2 × 10⁶ CFU) of a bacterial suspension was instilled into the trachea through a sterile, flexible 22-gauge catheter (Becton Dickinson, Heidelberg, Germany) attached to a 1-ml syringe. Agar beads not containing bacteria were instilled into the trachea of control mice. After 14 days three animals were sacrificed and checked for bacteria loading in the lung. To that end the lungs were excised and homogenized in PBS. Serial dilutions were plated on TSA plates. The presence of bacteria was confirmed in the lungs of all tested mice.

Intratracheal administration of complexes

Two weeks after bacterial infection the mice were anesthetized with 2,2,2-tribromoethanol (Avertin). The trachea was exposed by a skin incision and intubated with a sterile flexible 22-gauge catheter just below the cricoid cartilage. A 100-μl volume of complex solution was administered directly into the respiratory tree with a 100-μl Hamilton syringe. Ten micrograms of DNA per mouse was complexed with each carrier. GL67:DOPE:DMPE–PEG₅₀₀₀/pDNA complexes (DOPE, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine; DMPE, 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine; PEG₅₀₀₀, polyethylene glycol 5000; pDNA, plasmid DNA) were prepared as described earlier (Xenariou et al., 2006). Lipofectamine/pDNA complexes were prepared according to the manufacturer's instructions. PEI/pDNA and PEI/pDNA/MSA (N/P ratio [molar ratio of nitrogen atom content in polymer to phosphorus atom content in DNA], 10; 10 μg of MSA per 1 μg of DNA) were prepared as described earlier (Di Gioia et al., 2008). Lentiviral vector, pseudotyped with the envelope glycoprotein G of vesicular stomatitis virus (VSV-G), carried luciferase as a reporter gene. The transfer vector (pRRL.sin.PPT.CMVluciferase.IRES.EMCVwt.GFP.wPRE) was kindly provided by L. Naldini (San Raffaele Telethon Institute for Gene Therapy [HSR-TIGET], Milan, Italy). Lentivirus was produced according to a procedure described earlier (Copreni et al., 2008). Mice were infected at a dose of 10⁷ transducing units (TU)/mouse.

Assay for luciferase activity in vivo

Mice were sacrificed and their lungs were excised. The lungs were homogenized in 1 ml of lysis buffer (25 mM Tris-HCl, 2 mM DTT, 2 mM EDTA, 10% glycerol, 1% Triton X-100 [pH 7]), using an Ultra-Turrax homogenizer (maximum speed, 40 sec; IKA, Staufen, Germany). The samples were then frozen in liquid nitrogen, allowed to thaw on ice, and centrifuged (13,000 rpm, 4°C, 15 min). Luciferase activity was measured in 50 μl of each supernatant in a Lumat LB 9507 instrument (Berthold Technologies, Bad Wildbad, Germany). A 100-μl volume of luciferase substrate (Promega, Madison, WI) were injected into each sample. Emitted light was measured over a period of 30 sec. The protein concentration in each sample was determined with a modified Lowry protein assay kit (Pierce Biotechnology, Rockford, IL). The results are expressed as relative light units (RLU) per milligram of protein.

Histology and immunofluorescence

Lung tissues were fixed in 4% paraformaldehyde (4°C, 24 hr) and then embedded in paraffin. Five-micrometer sections were cut and every third section was collected for analysis. Hematoxylin and eosin staining of sections was performed by standard techniques. Localization of P. aeruginosa was performed in deparaffinized lung sections according to standard procedures. Unspecific binding was blocked by incubation with 10% normal swine serum and 0.5% bovine serum albumin (BSA) in PBS. Detection of P. aeruginosa was done with an antibody kindly provided by G. Pier (Channing Laboratory, Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, MA) (Pier and Thomas, 1982). The antibody was diluted 1:50 and incubated with the samples for 1 hr at room temperature. The primary antibody was detected with Alexa 594-labeled goat anti-rabbit IgG (Molecular Probes/Invitrogen). The nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI). The slides were mounted with fluorescent mounting medium (Dako, Carpinteria, CA). The micrographs are representative of lung sections obtained from three mice. Staining was performed on several sections per lobe. Photographs were taken with a Zeiss Axioplan 2 microscope equipped with an AxioCam digital camera (Carl Zeiss, Oberkochen, Germany).

Statistical analysis

Results are presented as means ± SD. Statistical significance of differences was evaluated by two-tailed unpaired Student t test.

Results

Chronic model of respiratory infection with P. aeruginosa clinical RP73 strain

C57BL/6 mice were inoculated with the RP73 P. aeruginosa clinical strain, according to the agar beads mouse model of chronic infection. As previously demonstrated (Bragonzi et al., 2005, 2009), intratracheal inoculation of P. aeruginosa-laden agar beads mimics the persistent and progressive bronchopulmonary infection typical of patients with CF. Accordingly, with the data published for other clinical strains (Bragonzi et al., 2009), the P. aeruginosa RP73 strain establishes in all treated mice a chronic infection that lasts for weeks after challenge with no mortality. In fact, hematoxylin and eosin-stained lung tissue sections (Fig. 1) show extensive inflammatory cell infiltrations in the bronchial lumen and the alveolar region 2 weeks after inoculation. Indirect immunofluorescence with an antibody against P. aeruginosa further revealed that the bacteria were present mostly in the bronchial lumen (Fig. 2), with only scarce bacterial cells being detectable in the alveoli. As an additional control, we sacrificed three infected mice from the group and checked them for bacterial load in the lung homogenate, confirming the presence of viable bacteria in the airways of the animals tested (5.88 × 10⁵ ± 3.62 × 10⁵ CFU/animal).

FIG. 1.

(A and B) Evidence of extensive damage observed in lungs of mice inoculated with Pseudomonas aeruginosa RP73 strain. Original magnification: (A) × 2.5; (B) × 10.

FIG. 2.

Evidence of the presence of bacteria in the lungs of mice inoculated with P. aeruginosa RP73 strain. Detection of P. aeruginosa was done with a specific antibody (T7WB). (A) The primary antibody was detected with goat anti-rabbit IgG–Alexa 594. (B) The nuclei were stained with DAPI. (C) Merge picture. Original magnification: (A and B) × 20; (C) × 20.

Effect of P. aeruginosa chronic infection on transfection mediated by viral and nonviral vectors in vivo

Infection was established in all mice treated for 2 weeks before transfecting them intratracheally with various gene carriers. In our study we employed cationic lipids (GL67 and Lipofectamine), cationic polymers (PEI and PEI/MSA), and lentiviruses as carrier systems. All of them have been shown to be able to deliver therapeutic genes to airway epithelium in vivo (Alton et al., 1999; Bragonzi et al., 2000; Pringle et al., 2005; Xenariou et al., 2006; Kramer et al., 2007; Di Gioia et al., 2008). Optimal pDNA-to-vector ratios for each nonviral vector were taken from the literature (Bragonzi et al., 2000; Sanders et al., 2002; Pringle et al., 2005; Xenariou et al., 2006; Kramer et al., 2007; Di Gioia et al., 2008). Each vector carried 10 μg of pDNA. The lentiviral titer used in the study was 10⁷ TU/animal.

As is apparent from Fig. 3, the effects of chronic infection vary substantially for the different carrier systems. Transfections mediated by PEI/DNA polyplexes complexed with albumin and Lipofectamine lipoplexes were decreased in the infected mice (Fig. 3). There was no significant difference in levels of transfection between normal and infected mice transfected with lentivirus or plain PEI/DNA complexes. Interestingly, however, transfection mediated by GL67 was increased 2-fold in the lungs of infected animals.

FIG. 3.

Effect of chronic infection on in vivo transfection mediated by viral and nonviral vectors. Before transfection animals were infected intratracheally with empty agar beads (open columns) or agar beads containing P. aeruginosa RP73 strain (solid columns). Luciferase expression was evaluated 24 hr after transfection. GL67, GL67:DOPE:DMPE-PEG₅₀₀₀; LIPOFECT, Lipofectamine 2000; PEI, polyethylenimine 25K; PEI/MSA, polyethylenimine complexed with murine serum albumin; LV, lentivirus. Shown are means and SD. *p < 0.05; **p < 0.005 (versus control values, i.e., animals inoculated with empty agar beads).

Discussion

Since the cloning of the CFTR gene, 25 gene delivery-related clinical trials have already been completed. In general, these studies have shown that viral vectors are more efficient in gene delivery to the lung than are nonviral vectors. On the other hand, nonviral vectors have been shown to induce less immune response, which is crucial when repeated administration is required, as for CF treatment. Cells expressing viral proteins are normally recognized by cytotoxic (CD8⁺) T lymphocytes, which in consequence leads to elimination of transduced cells. Moreover, viral capsid proteins induce the production of neutralizing antibodies that will disable their interaction with target cells if readministered. Interestingly, the presence of preexisting antibodies has been shown to hinder transduction mediated by wild-type viruses (adenoviruses and adeno-associated viruses) (Perricone et al., 2000).

Chronic bacterial infection is the hallmark of CF lung disease. Epithelial cells react to bacterial infection by increased production of cytokines (interleukin [IL]-8 and IL-4), intercellular adhesion molecule-1 (ICAM-1), and granulocyte macrophage colony-stimulating factor (GM-CSF), which drive the recruitment and persistence of neutrophils at the inflammatory site (Berger, 2002). In response to bacteria, lung macrophages produce their own IL-8, tumor necrosis factor (TNF)-α, and IL-1β, which in turn induce production of proinflammatory molecules by epithelial cells. However, applying any gene delivery system to infected lung brings along a high risk of its rapid elimination by the already alerted immune system. Indeed, Bastonero and colleagues demonstrated that IL-4 and TNF-α inhibit expression of the CFTR gene in CF tracheal gland cells (Bastonero et al., 2005). Therefore, the immune response to bacteria in the airways should be taken into serious consideration within the context of CF gene therapy. Thus far, the impact of the presence of bacteria on gene delivery to the lung has not been studied in detail. In our previous work we reported the effect of acute P. aeruginosa infection on gene delivery mediated by PEI/DNA complexes. The simple model of in vivo transfection suffered, however, from one major drawback—it lacked some important aspects of the physiological situation in the CF lung. In the present study we employed a clinical P. aeruginosa strain that establishes chronic infection in the lungs of all infected animals. The presence of a bacterially induced immune response at the moment of gene delivery was confirmed (Fig. 1). Interestingly, in this model transfection mediated by various viral and nonviral systems either increased, decreased, or remained unchanged, depending on the particular carrier system applied, implying the importance of the carrier in the process of delivery to the infected lung. The only carrier whose transfection potency increased in the chronic infection model was GL67. This might be due to the presence of the PEG moiety in the lipid formulation. PEGylation of viral and nonviral systems has been shown to both stabilize the particle and shield it from undesired interactions, leading to its clearance from the lung (Chillon et al., 1998; O'Riordan et al., 1999).

In our earlier work we evaluated the impact of acute respiratory infection on in vivo gene transfer mediated by PEI polyplexes (Rejman et al., 2007). In this study mice were infected with various loads of P. aeruginosa PAO1 strain and transfected 2 days later with PEI polyplexes. Under those conditions transfection mediated by the polyplexes increased significantly. We provided evidence that the gain in transfection efficiency was caused by P. aeruginosa-induced disruption of tight junctions, which is a well-known phenomenon (Coraux et al., 2004; Lau et al., 2005; Zulianello et al., 2006). In our present study transfection mediated by PEI polyplexes in the lungs of chronically infected mice was not significantly different from that of control animals. Precise data concerning the P. aeruginosa RP73 strain and its ability to open up tight junctions are not available. Nonetheless, the presence of single bacteria in the alveolar region suggests that these bacteria are also able to cross the epithelial barrier. It means, at least in theory, that PEI polyplexes could also, in that case, exploit the route “prepared” by bacteria to reach the basolateral side of the epithelial cell, which is known to possess a much higher capacity to take up gene vectors than the apical side (Coyne et al., 2000; Man et al., 2000; Johnson et al., 2003). Even so, the abundant presence of immune system cells, especially macrophages, is likely to eliminate a major fraction of the polyplexes before these can reach their target cells/membranes.

Transfection by Lipofectamine was almost entirely blocked by the presence of bacteria. This result is in agreement with data published by Bastonero and colleagues, who used the same cationic lipid to transduce CF tracheal gland cells. These authors observed a sharp drop in gene expression when the cells were pretreated with IL-4 or TNF-α, both involved in the inflammatory response in the CF lung (Bastonero et al., 2005).

The impact of chronic Pseudomonas aeruginosa infection on adenovirus-mediated gene transfer was studied earlier (Van Heeckeren et al., 1998; Tosi et al., 2004). Both studies demonstrated that the efficiency of adenovirus-mediated gene transfer is significantly reduced by inflammation induced by Pseudomonas. Increased adenovirus-specific CD8⁺ cytotoxic T lymphocyte activity was considered by the authors to be one of the underlying mechanisms. These results, together with the data presented in our paper, point out the need to consider preexisting lung inflammation while optimizing gene delivery carriers for CF lung disease.

Footnotes

Author Disclosure Statement

The authors have nothing to disclose.

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