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Published ahead of print on April 10, 2008, doi:10.1165/rcmb.2007-0458OC
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American Journal of Respiratory Cell and Molecular Biology. Vol. 39, pp. 305-311, 2008
© 2008 American Thoracic Society
DOI: 10.1165/rcmb.2007-0458OC

DNA Vector Augments Inflammation in Epithelial Cells via EGFR-Dependent Regulation of TLR4 and TLR2

Kenneth Liu1, Gary P. Anderson1,2 and Steven Bozinovski1

Departments of 1 Pharmacology and 2 Medicine, The University of Melbourne, Melbourne, Victoria, Australia

Correspondence and requests for reprints should be addressed to Steven Bozinovski, Ph.D., Department of Pharmacology, The University of Melbourne, Melbourne, Victoria, Australia 3010. E-mail: bozis{at}unimelb.edu.au


   Abstract
Top
 Abstract
CLINICAL RELEVANCE
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 
Gene delivery applications to treat lung diseases are, in some instances, suboptimal due to deleterious host inflammatory reactions. Current DNA plasmids (pDNA) exert toxicity in part via unmethylated CpG motifs that stimulate Toll-like receptor (TLR)9-expressing leukocytes; however, the airway epithelial response has not been well defined. Bronchial epithelial cells (BEAS-2B) were exposed to pDNA complexes and inflammatory mediators were measured. As patients with inflammatory lung disease are susceptible to infectious exacerbations, we also evaluated the reciprocal inflammatory response to pDNA and bacterial components lipopolysaccharide (LPS) and lipoteichoic acid (LTA), recognized by TLR4 and TLR2, respectively. Cells primed with pDNA synergistically expressed IL-8 mRNA and protein in response to LPS and LTA (3- to 5-fold). A similar induction was also observed for IL-1β, IL-6, colony-stimulating factor (CSF)-1, and granulocyte macrophage–CSF. Their synergistic elevation was associated with an increase in TLR4 and TLR2 levels. Methylation of pDNA only partially reduced (25–30%) IL-8 release; hence, signaling occurs via CpG/TLR9-dependent and -independent modules. As epidermal growth factor receptor (EGFR) signaling has been implicated in bronchial IL-8 expression, we assessed whether pDNA priming events were coordinated via EGFR. AG1478 (EGFR inhibitor) restored normal TLR4/2 levels and also suppressed synergistic release of IL-8. The extracellular signal–regulated kinase (Erk) mitogen-activated protein kinase inhibitor also blocked IL-8 release, implicating Erk as a key mediator of EGFR signaling. Our findings identify a novel EGFR-dependent mechanism for regulating TLR, and show that targeted disruption of EGFR signaling ameliorates the airway epithelial inflammatory response to pDNA. Targeting the EGFR system may improve the efficiency, tolerability, and safety of gene therapy strategies.

Key Words: inflammation • epidermal growth factor receptor • gene therapy • bronchial epithelium



   CLINICAL RELEVANCE
Top
 Abstract
 CLINICAL RELEVANCE
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 
Inflammatory mechanisms responsible for poor gene transfer therapy in chronic lung disease are not well defined. We show that targeting the epidermal growth factor receptor system may improve the efficiency, tolerability, and safety of gene therapy strategies.

 
Gene transfer therapy is of great interest not only to monogenetic lung diseases such as cystic fibrosis (CF), but also asthma and chronic obstructive pulmonary disease (COPD), since the airway epithelium is accessible to noninvasive delivery modes (1). It is also technically feasible with current methods to formulate and deliver such aerosolized agents to the upper or lower airways as required (2). Viral vectors are an attractive delivery vehicle as they can transfect terminally differentiated cells and typically result in robust gene expression required for a clinical benefit. While host humoral antibody responses are not as limiting as initially believed, gene therapy vectors may still elicit deleterious inflammatory responses that may lessen their therapeutic utility (3–5).

Plasmid DNA (pDNA) vectors are considered to be a safer and less immunogenic gene delivery agent, as they generally induce a weaker host response. Lipid-mediated transfer of pDNA has successfully been introduced into human airways (6), and green fluorescent protein–tagging experiments in murine lung have identified the respiratory epithelium as the major cellular target (7). Unfortunately, the current generation of cationic lipid–DNA delivery agents tested in CF airways have not resulted in any clinical benefit, as expression of the target gene in the conducting airways is suboptimal (6). Lipid–DNA complexes delivered via nasal or lung routes were reported to elicit significant side effects, including transient flu-like symptoms associated with elevated IL-6 levels induced by the host's immune response (6, 8, 9). The presence of unmethylatated CpG motifs in DNA vectors are thought to drive pDNA-mediated inflammatory events (10). CpG motifs are common in the prokaryotic genome and are recognized as a pathogen-associated molecular pattern (PAMP). The recognition of PAMPs is facilitated by a family of evolutionarily conserved pathogen recognition receptors known as Toll-like receptors (TLRs) (11). There are at least 10 human TLRs; each recognizes unique microbial PAMPs. Upon PAMP engagement, TLRs trigger multiple signaling cascades that activate the transcriptional factors, NF-{kappa}B and AP-1, which promote expression of key inflammatory mediators. The neutrophil chemokine IL-8 cooperates with cytokines like TNF-{alpha} and IL-6 to coordinate recruitment of neutrophils, which typically destroy the invading pathogen via potent proteases and oxidative free radicals.

Discrete cellular distribution and localization of specific TLRs are thought to limit the deleterious effects associated with acute inflammatory responses. TLR2 recognizes numerous PAMPs, including lipoteichoic acid (LTA) expressed on gram-positive bacteria, and TLR4 is the stereotypic lipopolysaccharide (LPS) receptor engaged by gram-negative bacteria. TLR9 is responsible for recognizing unmethylated CpG motifs and, like TLR4 (12), is thought to be localized in intracellular endocytic compartments to avoid persistent activation by luminal commensal bacteria (13). Although alveolar macrophages have traditionally been viewed as the primary professional airway innate immune cell type, the respiratory epithelium expresses a comprehensive suite of TLRs, including TLR1–6 and 9 (14, 15). Importantly, targeted disruption of NF-{kappa}B signaling in the airway epithelium prevents lung inflammation and injury resulting from local or systemic administration of LPS (16). Since CF and emphysema airways are typically colonized with bacteria normally absent in healthy lungs, and patients with chronic inflammatory lung diseases are highly susceptible to exacerbations triggered by infection, TLR interactions may contribute to the pathogenesis of these conditions. TLR reciprocation has been implicated in augmented inflammatory responses, as co-stimulation of macrophages or dendritic cells (DC) with LPS and CpG-DNA synergize the release of inflammatory cytokines (17–19). CpG-DNA and IL-1β also synergistically elevate IL-8 release from bronchial epithelial cells (20).

The cytokine response to cationic lipid–DNA complexes is not well characterized in airway epithelial cells. This response occurs rapidly, promotes recruitment of phagocytic cells to the airways, and contributes to reduced transgene expression. In diseased airways, inflammation is compounded by the persistence of colonizing bacterial strains. Since the bronchial epithelium is capable of mounting TLR-dependent inflammatory responses, targeted disruption of epithelial signaling may improve transgene expression and reduce the overall inflammatory burden associated with chronic lung diseases. The signaling modules responsible for NF-{kappa}B–driven cytokine production in respiratory epithelium are cell type specific (21) and refractory to conventional anti-inflammatory agents such as glucocorticosteroids (22). Recently, LPS induced IL-8 production was shown to be regulated by a novel airway epithelial signaling cascade that requires ligand-mediated activation of the epidermal growth factor receptor (EGFR) (21). EGFR is typically expressed in intercellular lateral epithelial junctions (23) and is transiently induced at sites of damage to coordinate epithelial repair mechanisms (24). EGFR may also regulate neutrophil recruitment through IL-8 to clear cellular and pathogenic debris. However, EGFR expression appears to be deranged in CF, emphysema, and asthma airways due to their chronic nature, and may contribute to persistent pathogenic processes (25–27). Indeed, a positive correlation between EGFR, IL-8, and submucosal neutrophils in asthmatic bronchial biopsies has been demonstrated (28).

In this study we investigated inflammatory mechanisms triggered by pDNA in BEAS-2B bronchial epithelial cells. We found that lipid–pDNA complexes were modestly pro-inflammatory. However, when combined with LPS or LTA, pDNA primed epithelial cells responded by synergistically increasing IL-8 production. Signaling mechanisms were evaluated using selective pharmacologic inhibitors to EGFR (AG1478), extracellular signal–regulated kinase (Erk) (U0126), and p38 (SB203580) mitogen-activated protein kinases (MAPKs), identifying EGFR as a central signaling pathway responsible for exaggerated inflammation in pDNA-primed bronchial cells. Furthermore, lipid–pDNA complexes augment cytokine and chemokine responses by up-regulation of TLR4 and TLR2 expression via EGFR-dependent mechanisms. We suggest that inhibition of the EGFR system may be useful to improve the efficacy, tolerability, and safety of gene transfer to the lung and potentially other cells and tissues.


   MATERIALS AND METHODS
Top
 Abstract
 CLINICAL RELEVANCE
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 References
 
Cell Culture
Human BEAS-2B cells were obtained from ATCC (Rockville, MD) and were cultured in a 1:1 mixture of keratinocyte–serum-free medium supplemented with 50 mg/ml of bovine pituitary extract and 5 µg/ml of epidermal growth factor, and Minimum Essential Medium (MEM) supplemented with 10% fetal calf serum (FCS), 2 mM L-glutamine, 1.0 mM sodium pyruvate, 0.1 mM nonessential amino acids, and 1.5 g/L sodium bicarbonate and antibiotics (25 µg/ml gentamicin, 100 µg/ml penicillin, 100 µg/ml streptomycin). All cell culture medium and supplements were purchased from Invitrogen (Carlsbad, CA).

Plasmid DNA Treatment and Methylation of Vector
pcDNA1/Amp vector (Invitrogen) was purified using Endofree plasmid Giga kit (Qiagen, Valencia, CA). BEAS-2B cells were plated out in 6-well multidishes for 24 hours before treatment. Cells were treated with liposome-pDNA (0.8 µg pDNA) using Effectene (Qiagen) in accordance to the manufacturer's instruction. Lipid–pDNA exposure was performed in Dulbecco's modified Eagle's medium (DMEM) supplemented with 5% FCS and 25 µg/ml gentamicin. Where indicated, pDNA was also methylated at CpG sites using 4 U of CpG methylase (SssI; New England Biolabs, Ipswich, MA) per microgram of pDNA in a reaction containing 160 µM S-adenylmethionine as a methyl donor for 2 hours at 37°C. At the end of the reaction, the enzyme was removed using Wizard DNA clean-up system (Promega, Madison, WI) following manufacturer's instructions, and resuspended in endotoxin-free TE buffer. To confirm methylation, pDNA was digested with HpaII restriction enzyme, which digests only unmethylated CpG sequences.

LPS and LTA Challenge after Lipid–pDNA Exposure and Use of Selective Pharmacologic Inhibitors
For secondary challenge, cells were first rinsed with PBS and then stimulated with LPS (1 ng/ml, from Salmonella enterica Re 595) and LTA (1 µg/ml, from Staphylococcus aureus) (Sigma-Aldrich, St. Louis, MO) in DMEM supplemented with 1% FCS and 25 µg/ml gentamycin. In indicated experiments, cells were treated with the EGFR inhibitor AG1478 (10 µM; Biomol, Plymouth Meeting, PA), the Erk 1/2 inhibitor U0126 (10 µM; Cell Signaling Technology, Danvers, MA), and the p38 inhibitor SB203580 (10 µM; Sigma). The granulocyte macrophage colony-stimulating factor (GM-CSF)–neutralizing antibody (0.5 µg/ml; R&D Systems, Minneapolis, MN) and corresponding IgG isotype were also used.

Quantification of IL-8 Protein
Cell-free supernatants were collected and IL-8 concentration was measured using commercially available paired antibodies and standards, following the manufacturer's instructions (BD PharMingen, Franklin Lakes, NJ).

RNA Extraction and Quantitative Real-Time PCR
Total RNA was isolated from individual samples, according to the manufacturer's instructions, using the RNeasy kit (Qiagen). The purified RNA was used as a template to generate first-strand cDNA synthesis using SuperScript III (Invitrogen) as previously described (29). QPCR was performed using the ABI PRISM 7900HT sequence detection system (Applied Biosystems, Foster City, CA). For mRNA quantitation, Taqman probe/primer combinations were used. A total volume of 10 µl was used for Taqman PCR, using AmpliTaq Gold polymerase and universal master mix (Applied Biosystems). The threshold cycle numbers were calculated using the {Delta}{Delta}CT relative value method and normalized to 18S rRNA.


   RESULTS
Top
 Abstract
 CLINICAL RELEVANCE
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 References
 
pDNA Primes Bronchial Epithelial Inflammatory Responses
The lipid–pDNA complex induced a 7-fold increase in IL-8 secretion above the no pDNA control in vehicle-treated cells as assessed by enzyme-linked immunosorbent assay (Figures 1A and 1C, P < 0.05, t test). IL-8 protein secretion was associated with an increase in IL-8 transcription measured by QPCR (Figures 1B and 1D). In the absence of pDNA priming, LPS stimulated IL-8 secretion by approximately 25-fold. When cells were pretreated with the lipid–pDNA complex, LPS responses were synergistically elevated 5-fold above LPS alone (Figure 1A). IL-8 protein expression is transcriptionally regulated as a similar profile for IL-8 mRNA was observed (Figure 1B). In the absence of pDNA, LTA (TLR2 agonist) induced a 15-fold increase in IL-8 secretion above vehicle-treated cells. In pDNA-primed cells, the LTA response was further enhanced 3- to 4-fold (Figure 1C); with a similar fold increase observed in IL-8 mRNA (Figure 1D).


Figure 1
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  		Figure 1. Lipid–pDNA complex enhances lipopolysaccharide (LPS)- and lipoteichoic acid (LTA)-induced IL-8 expression. Cells were exposed to lipid–pDNA complex for 24 hours (Dulbecco's modified Eagle's medium [DMEM] + 5% fetal calf serum [FCS]), and subsequently stimulated with LPS (1 ng/ml) or LTA (1 µg/ml) in DMEM + 1% FCS. At 3 hours after secondary stimulation (B and D), cells were collected for analysis by quantitative real-time PCR (QPCR), and at 24 hours after stimulation (A and C), cell-free supernatants were collected and assayed for secreted IL-8 by enzyme-linked immunosorbent assay (ELISA). (*P < 0.05, t test, n = 4). (A and B) Open bars, Veh; solid bars, LPS. (C and D) Open bars, Veh; solid bars, LTA.

 
Synergistic IL-8 Release Occurs Predominantly through CpG-Independent Mechanisms
Since methylation of CpG motifs almost completely abolishes its immune activation properties, methylase (SssI) was used to modify the pDNA. Methylation of pDNA was confirmed by restriction enzyme (HpaII) digestion that selectively targets the unmethylated sequence (Figure 2A). To assess if CpG motifs in pDNA were exclusively responsible for the synergistic release of IL-8, cells were pretreated with various liposome–pDNA combinations (Figure 2B). Liposome (Effectene) alone did not induce an inflammatory response and did not alter LPS-induced IL-8 release. pDNA alone modestly induced IL-8 secretion; however, when combined with LPS, no synergistic increase in IL-8 was observed. In contrast, cells pretreated with the liposome–pDNA complex clearly enhanced IL-8 release mediated by LPS. Methylation of pDNA only modestly reduced the synergistic release of IL-8 release from LPS-stimulated cells by approximately 25 to 30%, implicating both CpG-dependent and -independent stimulatory events.


Figure 2
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  		Figure 2. Synergistic release of IL-8 is mediated by CpG-dependent and -independent mechanisms. To determine if the inflammatory response was mediated by unmethylated CpG motifs, (A) pDNA was methylated using SssI methylase and the completion of the methylation was tested by HpaII digestion, which is subsequently visualized using agarose gel electrophoresis. (B) Cells were treated with the indicated liposome–pDNA combinations, and IL-8 secretion was assessed by ELISA 24 hours after stimulation (P < 0.05 t test, #no liposome complex versus liposome–pDNA and *liposome–pDNA versus liposome–methpDNA, n = 4). Open bars, Veh; solid bars, LPS.

 
Liposome–pDNA Complexes Augment Expression of Other Inflammatory Mediators by Increasing TLR2 and TLR4 Levels
We next evaluated a panel of inflammatory cytokines and growth factors critical to leukocyte recruitment, maturation, and survival. Consistent with IL-8 expression, lipid–pDNA induced basal IL-6 (5-fold), CSF-1 (1.8-fold), and GM-CSF (14-fold), whereas no significant change in basal IL-1β was observed (Figures 3A–3D). LPS-induced transcript levels were synergistically increased in pDNA-primed cells, ranging from 1.5- to 6-fold above LPS alone. Since GM-CSF is an integral mediator of lung inflammation and is known to regulate TLR4 expression (29, 30), we postulated that enhanced basal GM-CSF may contribute to elevated basal TLR4 expression. Hence, we measured TLR4 and TLR2 levels by QPCR and observed a significant increase in basal TLR4 (3.5-fold) and TLR2 (2-fold) expression in lipid–pDNA primed cells, as shown in Figure 4. Blocking GM-CSF bioactivity from pDNA primed cells with a neutralizing antibody did not restore normal basal TLR4 or 2 expression (data not shown), implicating novel mechanisms of pDNA-mediated TLR up-regulation. Hence, pharmacologic inhibitors of EGFR, Erk, and p38 MAPKs were evaluated. Here, epithelial cells were treated with the EGFR (AG1478), Erk (U0126), or p38 (SB203580) inhibitors for 3 hours after pDNA exposure and TLR4 and TLR2 expression was assessed by QPCR. The EGFR and Erk inhibitor reduced TLR4 and TLR2 expression to basal levels as seen in untreated cells, whereas p38 inhibition selectively reduced TLR2 levels only (Figures 5A and 5B).


Figure 3
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  		Figure 3. Lipid–pDNA complex augments expression of other inflammatory mediators. Cells were treated with lipid–pDNA complex for 24 hours, followed by stimulation with LPS (1 ng/ml; solid bars) or vehicle (DMEM + 1% FCS; open bars) for 3 hours. Cell pellets were retained and subjected to QPCR using validated Taqman probe-primer combinations for (A) IL-1β, (B) IL-6, (C) CSF-1, and (D) GM-CSF (* P < 0.05, t test, n = 3).

 

Figure 4
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  		Figure 4. Lipid–pDNA priming of airway epithelial cells increases Toll-like receptor (TLR)2 (solid bars) and TLR4 (open bars) expression. Cells were treated with lipid–pDNA complex for 24 hours, at which time-point cell pellets were retained for measurement of TLR4 (open bars) and TLR2 (solid bars) by QPCR using Taqman assays and normalized to 18S expression. (*P < 0.05, t test, n = 5).

 

Figure 5
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  		Figure 5. Induction of TLR4 and TLR2 is suppressed by EGFR (AG1478) and Erk (U0126) inhibitors. BEAS-2B cells exposed to the lipid–pDNA complex for 24 hours were incubated with AG1478 (10 µM), U0126 (10 µM), and SB203580 (10 µM) in DMEM + 1% FCS for 3 hours. Cell pellets were then harvested and subjected to QPCR measurement of TLR4 and TLR2 using Taqman assays. Dotted line represents TLR4 and TLR2 levels in cells that were not exposed to pDNA. Data were expressed as a percentage of the DMSO control (*P < 0.05, t test versus DMSO control, n = 4).

 
Blocking Elevated TLR4 and TLR2 Expression Correlates with Normalized IL-8 Expression
To establish if reversal of augmented TLR4 and TLR2 expression by pharmacologic intervention impacted on cellular inflammatory responses, we next measured IL-8 responses in the presence of specific inhibitors. Enhanced IL-8 expression in pDNA-primed and LPS- or LTA-challenged cells was significantly suppressed by the EGFR inhibitor (AG1478) and the Erk blocker, U0126 (Figures 6A and 6B). The p38 inhibitor SB203580 was also effective at reducing augmented IL-8 production attributed to pDNA priming. Since the p38 inhibitor did not alter TLR4 expression, our results suggest that p38 MAPK signaling is not regulating TLR4 levels but an important downstream mediator of TLR-driven inflammatory processes. This is consistent with previous reports that implicate p38 as an integral regulator of post-transcriptional mechanisms responsible for IL-8 mRNA stability and protein expression (31).


Figure 6
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  		Figure 6. Suppression of pDNA primed TLR responses by inhibition of EGFR, Erk, and p38 MAPK signaling. Cells were treated with lipid–pDNA complex for 24 hours, followed by stimulation with (A) LPS (1 ng/ml), (B) LTA (1 µg/ml), or vehicle (DMEM supplemented with 1% FCS). Before LPS and LTA challenge, cells were exposed to the indicated inhibitors for 30 minutes and retained in media during TLR agonist challenge. Twenty-four hours after LPS/LTA challenge, cell-free supernatants were collected for IL-8 measurement by ELISA. Dotted line represents IL-8 production in cells that were not pretreated with pDNA. Data were expressed as a percentage of the DMSO-LPS control (*P < 0.05, t test versus DMSO control, n = 4).

 

Figure 7
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  		Figure 7. Concept diagram of pDNA primed inflammatory events. The TLR receptor network normally (right) responds to pathogen-associated molecular patterns (PAMPS) and host-derived damage or intrinsic damage-associated molecular patterns (DAMPS) by inducing self-limiting inflammatory signaling pathways associated with activation of the transcription factors AP-1 and NF-{kappa}B. Our findings show that prior exposure to pDNA–lipid vectors augments TLR4 and TLR2 expression via EGFR-dependent mechanisms, leading to an amplified inflammatory response after receptor engagement. This would intensify inflammation triggered by PAMPS and might also intensify responses to TLR ligands released from damaged tissue (DAMPS).

 

   DISCUSSION
Top
 Abstract
 CLINICAL RELEVANCE
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
References
 
Acute inflammatory responses are known to significantly reduce transgene expression, an event that becomes more apparent with secondary applications (4). Suppression of transgene expression is caused by induction of inflammatory cytokines known to attenuate promoters/enhancers common to viral and nonviral vectors (32). Current strategies to limit this inflammatory response include the construction of plasmid sequences with reduced CpG content, which significantly dampens innate-immune–mediated inflammation (33, 34). However, CpG depletion in typical promoter regions can reduce transgene expression, which limits the utility of such applications. Likewise, global methylation of CpG-containing vectors reduce TLR9-mediated inflammatory events (35); however, since most promoters are negatively regulated by methylation, reduced transgene expression is typically prevalent. Furthermore, our results show that methylation of pDNA–liposome complexes only partially reduce IL-8 release from bronchial epithelial cells. Previous reports also describe CpG/TLR9-independent induction of inflammatory cytokines (36, 37), and TLR9-deficient mice display significant toxicity to lipid–pDNA complexes, although the overall inflammatory burden was reduced (38). BEAS-2B cells also express low TLR9 levels and hence respond to CpG oligonucleotides very modestly (15). Since we observed a 7-fold induction in IL-8 release, pDNA appears to be targeting an alternative inflammatory cascade in BEAS-2B cells.

Another approach to reducing pDNA-mediated immunity involves the use of anti-inflammatory drugs such as glucocorticosteroids. These agents reduce the toxicity associated with systemic administration of lipid–pDNA complexes and, importantly, can improve transgene expression when administered prophylactically (39). A novel approach to gene delivery involves the co-administration of steroids encapsulated within the liposome–pDNA complex, thereby localizing both vector and steroid to the cellular targets (40). However, a likely caveat of treating lung diseases with such an approach is that lung inflammatory events can be particularly refractory to conventional steroid treatment (41). Intravenous delivery of high dose steroids may improve their efficacy, but are associated with significant side effects. Hence, the benefit of encapsulating steroids within aerosolized liposome–DNA complexes may be compromised in the chronically inflamed CF and COPD lung micro-environment. Our findings also describe the cooperative nature of pathogen recognition receptors, since bronchial epithelial cells primed with pDNA synergistically express key inflammatory mediators in response to TLR4 and TLR2 agonists. This observation highlights the difficulty of introducing gene delivery agents to CF or COPD lungs that are readily colonized by pathogenic bacteria, and may provide novel insights into the pathogenesis of these chronic lung diseases. For example, since pDNA can act as a surrogate for bacterial DNA, which is abundantly present in CF and COPD sputum, infectious exacerbations may be worsened by bacterial DNA priming events. These acute episodes are associated with an increased local and systemic inflammatory burden that is poorly tolerated in individuals with reduced lung reserve capacity (42). Hence, the persistence of bacterial DNA fragments in CF and COPD airways may contribute to excessive inflammatory responses to new bacterial pathogens by priming TLR signaling pathways that control cytokine and chemokine gene programs.

In this study, we identified EGFR signaling as a novel mechanism responsible for pDNA priming of bronchial epithelial cells as AG1478 completely inhibited pDNA-mediated up-regulation of TLR4/2 levels and associated IL-8 release in response to LPS and LTA (summarized in Figure 7). Transactivation of EGFR is controlled by ligand engagement, which promotes formation of homodimeric and heterodiomeric receptor complexes (EGFR/HER2) (43). Upon activation, EFGR is phosphorylated at specific intracellular residues that become docking sites for the recruitment of downstream signaling molecules. There are three major pathways engaged by EGFR transactivation (43). Erk MAPK is activated through a series of intermediate kinases and docking modules such as the Grb/Sos complex. Once activated, Erk promotes inflammatory gene expression via the AP-1 transcription factor complex consisting of Jun, Fos, and ATF-2 dimers that are regulated by MAPK-mediated phosphorylation. To assess the role of Erk MAPK in pDNA primed inflammatory processes, the selective inhibitor U0126 was used, which displayed an inhibitory profile similar to that of the EGFR blocker. In contrast, p38 MAPK did not suppress TLR4 expression, and hence may coordinate inflammation via stabilization of transcription and protein expression (31). A secondary pathway involves the STAT transcription factor family, which also induces gene expression (44). To evaluate STAT activation, we transiently expressed kinase-inactive EGFR (K721R), which is selectively deficient in STAT signaling while retaining Erk and Akt activation (45, 46). Overexpression of the EGFR-K721R mutant did not interfere with pDNA priming responses (data not shown). The PI3K/Akt pathway is also engaged during EGFR transactivation. Akt is a pleiotropic kinase that regulates cell survival, glucose metabolism, protein synthesis, and NF-{kappa}B–mediated gene expression in a cell type– and stimuli-specific manner. However, the PI3K inhibitor, Wortmannin, did not suppress the IL-8 release from LPS or LTA cells (data not shown). Consistent with our findings, Nakanaga and colleagues also describe a novel EGFR-dependent signaling pathway responsible for IL-8 release in bronchial epithelial cells (21).

In summary, we have shown that lipid–pDNA complexes signal via EGFR-Erk to induce expression of TLR4 and TLR2. Hence, secondary exposure to their respective agonists results in the synergistic release of key inflammatory mediators responsible for recruiting and maintaining leukocytes to inflamed tissue. We show that targeted inhibition of EGFR signaling suppresses pDNA-mediated inflammatory responses. Furthermore, as EGFR is also an important regulator of airway mucous production (21), inhibition of EGFR signaling may significantly improve transgene lung delivery by (1) dampening primary and secondary inflammatory events attributed to pDNA exposure in airway epithelium and (2) reducing mucous content that acts as a physical barrier to vector administration. We suggest that targeting the EGRF system may improve the efficacy, tolerability, and safety of gene transfer strategies.


   Footnotes
 
This work was supported by NHMRC and CRC-CID, Australia.

Originally Published in Press as DOI: 10.1165/rcmb.2007-0458OC on April 10, 2008

Conflict of Interest Statement: None of the authors has a financial relationship with a commercial entity that has an interest in the subject of this manuscript.

Received in original form December 19, 2007

Accepted in final form February 1, 2008


   References
Top
 Abstract
 CLINICAL RELEVANCE
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 

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