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Infection and Immunity, August 2005, p. 5262-5268, Vol. 73, No. 8
0019-9567/05/$08.00+0     doi:10.1128/IAI.73.8.5262-5268.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Similarity of Gene Expression Patterns in Human Alveolar Macrophages in Response to Pseudomonas aeruginosa and Burkholderia cepacia

Stefan Worgall,1,2 Adriana Heguy,1 Karsta Luettich,1 Timothy P. O'Connor,1 Ben-Gary Harvey,3 Luis E. N. Quadri,4 and Ronald G. Crystal1,3*

Department of Genetic Medicine,1 Department of Pediatrics,2 Division of Pulmonary and Critical Care Medicine,3 Department of Microbiology and Immunology, Weill Medical College of Cornell University, New York, New York4

Received 20 December 2004/ Returned for modification 9 February 2005/ Accepted 31 March 2005


   ABSTRACT
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To determine if differences in the severity of pulmonary infection in cystic fibrosis seen with late isolates of Pseudomonas aeruginosa and Burkholderia cepacia are associated with differences in the initial response of alveolar macrophages (AM) to these pathogens, we assessed gene expression changes in human AM in response to infection with a laboratory strain, early and late clinical isolates of P. aeruginosa, and B. cepacia. Analysis of gene expression changes at the RNA level using oligonucleotide microarrays, following exposure to laboratory P. aeruginosa strain PAK, showed significant (P < 0.01) >2.5-fold upregulation of 42 genes and >2.5-fold downregulation of 45 genes. The majority of the changes in gene expression involved genes as part of inflammatory pathways and signaling systems. Interestingly, similar responses were observed following exposure of AM to early and late clinical isolates of P. aeruginosa, as well as with B. cepacia, suggesting that the more severe clinical outcome of infections with late clinical isolates of P. aeruginosa or with B. cepacia cannot be explained by differences in the early interactions of these organisms with the human AM, as reflected by the similarity of gene expression changes in response to exposure of AM to these pathogens.


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Cystic fibrosis (CF), a hereditary disorder caused by mutations of the CF transmembrane conductance regulator (CFTR) gene, is characterized by chronic infection of the respiratory epithelium by Pseudomonas aeruginosa (6, 14, 33). While the early P. aeruginosa isolates from CF patients are planktonic, free-floating organisms, P. aeruginosa isolates from the respiratory tract later in the disease often produce a mucoid biofilm and are difficult to eradicate (6, 14, 16, 33). In some cases of CF, there is superinfection with Burkholderia cepacia, a pathogen with a distant relationship to P. aeruginosa (16, 37). B. cepacia superinfection is an ominous sign in CF, as it is invariably associated with a progressive downhill course and death (14, 16, 37).

Although the airway epithelial cells are the dominant cell type affected by CFTR dysfunction in the CF lung, the interaction of organisms with cells of the pulmonary host immune system may be important in determining the extent of the inflammatory responses to these pathogens and the lack of bacterial clearance from the airways that characterizes the disease (5, 14, 18, 20, 21, 30). Alveolar macrophages (AM), the resident mononuclear phagocytes in the respiratory tract, are the first line of cell-mediated defenses against inhaled organisms (4, 18, 25). Besides their scavenger function upon interaction with pathogens, AM release a variety of mediators that play a central role in the inflammatory response to organisms, such as the recruitment of polymorphonuclear phagocytes to the respiratory epithelium (4, 18, 25, 26). The phagocytic contribution of AM to the defense against P. aeruginosa is thought to be negligible (9), and studies with mice have shown that temporary elimination of AM did not affect the course of P. aeruginosa pulmonary or systemic infection; however, it affected cytokine and chemokine production (13). AM likely play a role in the control and maintenance of the pulmonary inflammatory response to P. aeruginosa (9, 20, 21, 23, 27, 41). As AM express only negligible amounts of the CFTR gene, mutations in CFTR associated with CF are unlikely to be associated with AM dysfunction per se (44). This has also been confirmed by studies with transgenic G551D mice expressing the human CFTR gene in lung epithelium or AM, with an increase in pulmonary clearance of P. aeruginosa and a decrease in inflammatory cytokines in epithelial cell-corrected, but not macrophage-corrected, mice (28). However, the epithelial abnormalities associated with CFTR mutations present the AM with the challenge of clearing a large, persistent burden of P. aeruginosa and, in some late cases, B. cepacia. AM isolated from patients with CF produce larger amounts of inflammatory cytokines compared to non-CF AM (21, 30). The inability of AM to efficiently clear organisms like P. aeruginosa and B. cepacia from the lung, in combination with an exacerbated inflammatory milieu, may be important factors in the interaction of these pathogens with the host defense system in CF (5, 7, 9, 14, 15, 20, 41). One clue to the inability of AM to efficiently clear P. aeruginosa is the observation that AM are more susceptible than epithelial cells to P. aeruginosa-induced apoptosis (11, 12, 17, 40, 42, 43).

Since the clinical outcome of infection with late strains of P. aeruginosa and with B. cepacia is more severe than with early isolates of P. aeruginosa, we hypothesized that these differences could be due to a differential response of the AM upon exposure to these different pathogens. To understand the interaction of human AM with P. aeruginosa and B. cepacia, we used an unbiased approach based on microarray technology to assess the early changes in human AM gene expression in response to short time points of exposure to two clinical strains of P. aeruginosa (one early and one late isolate) and a strain of B. cepacia.

Changes in gene expression in AM infected with P. aeruginosa strains and B. cepacia. The P. aeruginosa strains used included laboratory strain PAK (provided by A. Prince, Columbia University, New York, NY) and two clinical strains isolated from the sputum of an individual with CF, AD2A and AD15B (provided by J. Burns, University of Washington, Seattle). AD2A is an early clinical isolate, and AD15B is a late clinical isolate; both were derived from the same individual. The B. cepacia strain was J2315, genomovar 3 (kindly provided by John LiPuma, University of Michigan). Human AM were obtained by bronchoalveolar lavage of healthy, nonsmoking or smoking volunteers (36). AM represented >95% of the cells in all cases. Cell viability (always >90%) was determined by trypan blue exclusion. The yield of AM derived from individuals with a history of smoking was four to five times higher compared to nonsmokers. After 3 h of adherence, the cells were infected with the bacteria at a dose of 5 CFU/cell in RPMI 1640-25 mM HEPES, pH 7.4, for 4 h. The viability of the cells following 4 h of infection was >85% as assessed by trypan blue exclusion for all bacterial strains. All analyses were carried out with the Affymetrix HuGeneFL chip using the protocols from Affymetrix (Santa Clara, CA) and the GeneSpring software (Silicon Genetics, Redwood City, CA). As the yield of AM derived from nonsmoking individuals was not sufficient to allow assessment of more than two conditions (e.g., control and infection with PAK) and the preliminary analysis of gene expression changes in AM infected with PAK demonstrated that the results were independent of whether the AM were derived from smokers or nonsmokers (Fig. 1), all subsequent experiments were performed with AM derived from smokers, which by virtue of their increased numbers (2, 4, 38) allowed infection with PAK, AD2A, AD15B, and B. cepacia within the same experiment and sample.



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  		FIG. 1. Venn diagram representation of gene expression changes following infection with PAK in AM derived from smokers and nonsmokers. AM were obtained by bronchoalveolar lavage from smokers (n = 8) or nonsmokers (n = 4) and infected in vitro with laboratory P. aeruginosa strain PAK at 5 CFU/cell. Gene expression changes in PAK-infected AM were compared to uninfected AM. Differences were evaluated using a nonparametric test (P < 0.05) with Benjamini-Hochberg correction.

 
There were a total of 87 genes differentially expressed in PAK-infected cells versus uninfected controls, with a P value of <0.01 and a fold change of >2.5-fold (up- or downregulated). Gene expression changes in AM following exposure to PAK (the laboratory P. aeruginosa strain) for 4 h showed significant upregulation (P < 0.01, >2.5-fold change) of a total of 42 genes, which were categorized into the following five functional groups: secreted proteins, nuclear proteins, metabolism and enzymes, signal transduction and growth control, and cell surface proteins (Table 1). The category with the greatest number of upregulated genes (15 out of 42) was secreted proteins, including cytokines and chemokines known to be induced by bacterial stimulation of macrophages (25, 26, 34, 35). The strongest upregulation was seen for interleukin-6 (IL-6) and tumor necrosis factor alpha (TNF-{alpha}) (102- and 62-fold compared to controls, respectively). The second largest functional group consisted of genes coding for factors related to signal transduction and growth control (12 out of 42). The majority of these are known to be induced by cytokine signaling (e.g., TNF receptor-associated factor I{kappa}B). Some of the upregulated genes in this category, such as Jagged, have not been previously reported as associated with bacterial stimulation of macrophages. A smaller group of upregulated genes codes for nuclear proteins involved in transcriptional regulation, including the enhancer of zeste homolog 2 (EZH2), also not previously associated with bacterial activation of macrophages.


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  		TABLE 1. Upregulation of genes in AM following infection with P. aeruginosa strains and B. cepacia

 
A total of 45 genes were significantly (P < 0.01) downregulated by >2.5-fold in PAK-infected AM samples (Table 2). In contrast to the genes upregulated following infection with PAK, none of these were genes coding for secreted proteins. The majority were in the category of genes coding for factors involved in signal transduction and growth control (11 out of 45), as well as metabolism and enzymes (12 out of 45). Another functional category with several members was that of genes coding for nuclear proteins (8 out of 45). Overall, decreases in gene expression levels following P. aeruginosa infection of AM were of lesser magnitude than the changes observed for upregulated genes. The two largest decreases in gene expression levels were for Lyl-1 and docking protein 1.


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  		TABLE 2. Downregulated genes in AM following infection with P. aeruginosa strains and B. cepacia

 
The n-fold change for the 87 genes up- or downregulated in AM in response to PAK infection were also assessed following infection of AM with P. aeruginosa clinical isolates AD2A and AD15B and with B. cepacia. The n-fold changes were calculated individually for each strain as the ratio of the average expression level in infected AM samples to that of uninfected controls (Tables 1 and 2). Similar changes were observed following infection with AD2A, AD15B, and B. cepacia. The 42 upregulated genes and the 45 downregulated genes selected by comparison of PAK-infected cells with uninfected controls were also upregulated or downregulated following exposure of AM to both P. aeruginosa clinical isolates, as well as to B. cepacia, and the extent of upregulation was similar for all of the strains. Infection with AD2A appeared to lead to higher induction levels for the upregulated genes, especially in the categories of nuclear proteins, metabolism and enzymes, and cell surface proteins (Table 1), and to a less pronounced decrease in all categories (Table 2). However, none of these differences were statistically significant. To visualize the similarities of the overall patterns of gene expression changes following infection with the different P. aeruginosa strains and B. cepacia, the n-fold gene expression changes for the genes up- or downregulated by more than 2.5-fold in AM following infection with PAK (ordered by gene identification number within each functional category) were plotted for all strains (Fig. 2A and B).



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  		FIG. 2. Gene expression changes in human AM following infection with laboratory P. aeruginosa strain PAK, clinical P. aeruginosa isolates AD2A (early) and AD15B (late), and B. cepacia. The ordinate shows the n-fold up- or downregulation in infected AM for all genes listed in Tables 1 and 2. The abscissa shows the individuals genes, ordered by gene identification number within each functional category for the genes increased (A) or decreased (B) following infection.

 
Relevant to CF, studies of the response of the host to the different forms of P. aeruginosa, as well as B. cepacia, have focused primarily on pathogen interaction with epithelial cells (19, 24). Differences in the induction of cytokine responses have been observed with different P. aeruginosa isolates or B. cepacia (31). B. cepacia is also thought to be located mostly intracellularly, in contrast to the extracellular location of P. aeruginosa (8). P. aeruginosa and B. cepacia also seem to differ in their abilities to induce cytolysis or apoptosis in cells with which they interact, important features in the creation of an inflammatory environment.

As an important component of the innate immune system in the lung, AM are in close contact with pulmonary pathogens such as P. aeruginosa and B. cepacia and are found in abundance in the inflamed local milieu of the CF lung (20, 21). Studies with mice suggest that AM are not directly involved in the clearance of P. aeruginosa (21). However, since the AM response to the bacteria contributes to the composition of the inflammatory milieu on the airway epithelial surface, it is relevant to ask whether there is variability of the response of AM to different P. aeruginosa strains or B. cepacia. Interestingly, the present study demonstrates that the human AM response to short-term exposure of various P. aeruginosa isolates or B. cepacia is relatively uniform. Although mRNA levels for a variety of cytokines and chemokines were rapidly induced following exposure of the AM to the various Pseudomonas strains and B. cepacia, the short-term response of AM to P. aeruginosa isolates and B. cepacia was, at least in vitro, relatively homogeneous. These homogenous responses are dominated by the induction of inflammatory cytokines and chemokines, which are important in the host responses that lead to elimination of the bacteria in the healthy host, with TNF-{alpha} and IL-6 showing the highest levels of induction (3, 26). Similarly, signal transduction pathways known to be related to cytokine activation, especially those mediated by transcription factor NF-{kappa}B (1, 3, 26), were rapidly induced to similar extents following exposure to the different P. aeruginosa isolates and B. cepacia. Future experiments need to identify the specificity of the gene expression changes with those observed with other, not lung or CF-related, gram-negative pathogens.

Confirmation by TaqMan real-time reverse transcription (RT)-PCR of selected genes. Most of the genes up- or downregulated in AM exposed to the P. aeruginosa strains or to B. cepacia corresponded to genes coding for inflammatory mediators or other proteins involved in pathways previously described as part of the response of AM to pathogens (25, 26, 34, 35). The agreement between the microarray data presented here and previous studies using other techniques to measure mRNA levels is in itself confirmation of the validity of the approach (10). Therefore, we chose to focus the validation of the results obtained by microarray analysis on four genes that had not been previously described in association with AM activation or response to pathogens: Jagged, EZH2, Lyl-1, and DOK1. The gene expression changes observed by microarray analysis were independently confirmed by TaqMan real-time RT-PCR as follows: Jagged was upregulated (PAK, 21.1-fold ± 6.7-fold; B. cepacia, 21.3-fold ± 3.6-fold; Fig. 3A), EZH2 was upregulated (PAK, 4.3-fold ± 0.4-fold; B. cepacia, 5.1-fold ± 0.6-fold; Fig. 3B), DOK1 was downregulated (PAK, 5.3-fold ± 0.4-fold; B. cepacia, 3.4-fold ± 0.8-fold; Fig. 3C), and Lyl-1 was downregulated (PAK, 4.1-fold ± 1.3-fold; B. cepacia, 4.8-fold ± 0.7-fold; Fig. 3D). Compared to the gene expression changes observed by microarray analysis, the magnitude of the changes was higher for the upregulated Jagged and EZH2 genes and lower for the downregulated Lyl-1 and DOK1 genes. Two-way analysis of variance confirmed that for each of the four genes there was a statistically significant effect of infection with PAK (P < 0.001) or B. cepacia (P < 0.001), but not methodology (P > 0.9 for PAK, P > 0.6 for B. cepacia).



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  		FIG. 3. Confirmation of microarray results by TaqMan real-time RT-PCR. Human AM were infected in vitro with PAK or B. cepacia (5 CFU/cell) for 4 h. RNA levels for four genes were measured by quantitative real-time RT-PCR. A, Jagged; B, EZH2; C, DOK1; D, Lyl-1. Relative expression levels in the infected samples were calculated using the {Delta}{Delta}Ct method, using 18S rRNA as an internal normalization control (Co), and the expression levels in the uninfected controls (naive) as the calibrator levels, set at 1. Shown is the mean ± the standard error of four independent experiments, using four individual AM samples, split for the three conditions used: uninfected, PAK infected, and B. cepacia infected.

 
To follow the gene expression of Jagged, EZH2, Lyl-1, and DOK1 at later time points, AM were infected for 4 h with PAK and then analyzed 0, 12, and 24 h following the initial infection by TaqMan real-time RT-PCR. The expression levels of Jagged and EZH2 were higher at all time points tested compared to the uninfected cells (Fig. 4A and B). The expression levels of Lyl-1 and DOK1 were decreased at all time points evaluated compared to the uninfected controls (Fig. 4C and D).



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  		FIG. 4. Time course of mRNA levels of Jagged, EZH2, Lyl-1, and DOK1 in AM following infection with P. aeruginosa. Human AM were infected in vitro with PAK (5 CFU/cell) for 4 h, medium was then changed, and gentamicin was added. RNA was analyzed by quantitative real-time PCR for the mRNA expression levels of Jagged (A), EZH2 (B), DOK1 (C), and Lyl-1 (D) and normalized to levels of 18S rRNA at 0, 12, and 24 h after the initial infection. Data are presented as the expression level of the sample divided by the expression of uninfected control AM within the same experiment. Expression levels of uninfected AM of each experiment were set as 1. Shown are representative results of three independent experiments.

 
Jagged, one of the ligands for the notch receptor, is known to influence hematopoietic cell fate decisions (22). The Jagged-notch interaction plays a role in the survival and differentiation of stem and T cells and the induction of antigen-specific regulatory T cells (45). EZH2, a member of the polycomb group of proteins, functions as a transcriptional repressor and plays a role as an epigenetic regulator in the development of B cells (39). Similarly to Jagged, upregulation of EZH2 in AM may play a role in the activation of pulmonary immune responses. Of the downregulated genes, docking protein 1, originally found constitutively tyrosine phosphorylated in hematopoietic progenitors isolated from patients with leukemia, has been postulated to be a critical substrate for p210(bcr/abl) and acts as an adaptor protein that links the activin receptors with Smad proteins, factors involved in apoptosis (29). Lyl-1 is a helix-loop-helix DNA binding protein with lineage and differentiation-specific properties (32). No known function related to bacterial activation of myeloid cells has been described so far. Docking protein 1 and Lyl-1 may both be related to apoptosis induced by P. aeruginosa, to which macrophages have been shown to be more susceptible too.

Taken together, relevant to the pathogenesis of Pseudomonas and Burkholderia infection in CF, the observations in the present study are consistent with the concept that the AM play an important role in contributing to the inflammatory milieu of the respiratory epithelium. However, while Pseudomonas clinical isolates late in the course and superinfection with Burkholderia are both associated with progressive deterioration (14, 37), the responses of human AM to different strains of Pseudomonas, as well as Burkholderia, seem to be remarkably similar, suggesting that AM are not responsible for the worsening of CF in association with different strains of bacteria.


   ACKNOWLEDGMENTS
 
We thank K. Martushova for technical help and N. Mohamed for help in preparing the manuscript.

These studies were supported, in part, by P01 HL51746, P01 HL59312, U01 HL66952, M01RR00047, the Will Rogers Memorial Fund (Los Angeles, CA), and the Cystic Fibrosis Foundation (Bethesda, MD).


   FOOTNOTES
 
* Corresponding author. Mailing address: Department of Genetic Medicine, Weill Medical College of Cornell University, 515 East 71st Street, S-1000, New York, NY 10021. Phone: (212) 746-2258. Fax: (212) 746-8383. E-mail: geneticmedicine{at}med.cornell.edu. Back

Editor: J. N. Weiser


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Infection and Immunity, August 2005, p. 5262-5268, Vol. 73, No. 8
0019-9567/05/$08.00+0     doi:10.1128/IAI.73.8.5262-5268.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.




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