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Published ahead of print on May 14, 2003, doi:10.1165/rcmb.2002-0310OC
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American Journal of Respiratory Cell and Molecular Biology. Vol. 29, pp. 545-551, 2003
© 2003 American Thoracic Society
DOI: 10.1165/rcmb.2002-0310OC

Apoptosis Genes in Human Alveolar Macrophages Infected with Virulent or Attenuated Mycobacterium tuberculosis

A Pivotal Role for Tumor Necrosis Factor

Avrum Spira, J. David Carroll*, Gang Liu, Zeeshan Aziz, Vishal Shah, Hardy Kornfeld and Joseph Keane{dagger}

Pulmonary Center, Boston University School of Medicine, Boston, Massachusetts; and Dublin Molecular Medicine Centre, Trinity College, St. James's Hospital, Dublin, Ireland

Address correspondence to: Dr. J. Keane, St. James's Hospital, James's St., Dublin 3, Ireland. E-mail: jkeane{at}stjames.ie


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Tumor necrosis factor (TNF)-{alpha}–dependent apoptosis of alveolar macrophages (AM) after infection with avirulent Mycobacterium tuberculosis (Mtb) results in bacillary death and the destruction of a growth niche for the pathogen. This response is minimized after infection with virulent strains of Mtb. To study the genetic control of Mtb-induced apoptosis, we used microarrays to interrogate the expression profile of infected human AM. Although we found variation in gene expression between different donors of AM, a set of genes were constant for each condition. A group of proapoptotic genes were downregulated after infection by virulent Mtb strain H37Rv, whereas infection with avirulent Mtb H37Ra led to a gene expression profile that would favor macrophage apoptosis. Neutralizing TNF in macrophage cultures infected with H37Ra changed the gene expression profile to one that resembled the profile of macrophages infected with H37Rv. These data reveal that apoptosis-related genes are regulated differently by virulent or attenuated Mtb strains, and are consistent with the hypothesis that virulent Mtb interfere with TNF death signaling. Given the importance of TNF in host defense against tuberculosis, the ability to repress the expression of genes activated by TNF may constitute a bacillary virulence mechanism.

Abbreviations: alveolar macrophages, AM • anti-tumor necrosis factor-{alpha} antibody, anti-TNF Ab • M. tuberculosis H37Ra, H37Ra • M. tuberculosis H37Rv, H37Rv • multiplicity of infection, MOI • Mycobacterium tuberculosis, Mtb • tumor necrosis factor-{alpha}, TNF-{alpha}


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Mycobacterium tuberculosis (Mtb) is a leading cause of death from bacterial infection worldwide (1). Following transmission by droplet inhalation, bacilli are engulfed by alveolar macrophages (AM) in the airways of the lung. These host defense cells are capable of killing most inhaled microbes, but they are relatively ineffective at eliminating intracellular infection by Mtb. On the contrary, pathogenic mycobacteria have evolved to use AM as a preferred growth environment necessary for optimal infection (2). Intracellular survival of mycobacterial pathogens may be due in part to their interference with phagosomal acidification and fusion with lysosomes (3, 4). In this intracellular environment, bacilli are, to an extent, protected from host immunity, permitting them to establish progressive or latent infection (5) where they may use fatty acids as a carbon source through the use of isocitrate lyase (6).

Host cell apoptosis is a common defense strategy against intracellular pathogens, including viruses and certain protozoa and bacteria (7–10). We previously reported that human AM respond to Mtb infection by undergoing programmed cell death (11). Naive AM are resistant to the cytotoxic effects of tumor necrosis factor (TNF)-{alpha} but may become primed for TNF death signaling after Mtb infection. Infected and primed cells then undergo apoptosis in response to endogenous TNF in an autocrine or paracrine manner. There is an inverse correlation of mycobacterial virulence and infected macrophage apoptosis. After low-dose infection (~ 10 bacilli or less per macrophage), virulent strains induce little or no apoptosis over background. In contrast, avirulent strains strongly induce apoptosis. This relationship is observed consistently with a variety of virulent and attenuated strains (12). Together with evidence that macrophage apoptosis is linked to killing of internalized bacilli (13), these findings suggest the existence of mycobacterial virulence determinants acting on the host to avoid the apoptosis response to intracellular infection. This paradigm is well established in the case of virus–host interactions (7).

DNA microarrays offer a global view of transcriptional events that underlie the host response to microbial pathogens. This information has the potential to identify host defense strategies, the mechanisms by which they are regulated, and to identify targets of microbial virulence factors (14). We applied DNA microarrays to investigate the regulation of genes related to apoptosis in primary human AM infected with Mtb. Our study focused on the differential effect of virulent and avirulent strains, and the role of TNF. Although other studies have characterized gene expression in macrophage cell lines and blood derived macrophages after Mtb infection (15, 16), ours is the first to use primary human AM and to focus on apoptosis-related genes. AM are stable primary cells that do not replicate like cell lines or require mitogens for maturation like monocytes. They are also the cells that first encounter inhaled bacilli and may be a model more readily applicable to human infection.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Human AM
AM were obtained from bronchoalveolar lavage fluid of healthy nonsmoking volunteers using standard techniques, with their informed consent under a protocol approved by the Institutional Review Board of the Boston University Medical Center. Lavage fluid was filtered through sterile gauze, centrifuged (450 x g, 10 min), and the cell pellet was suspended in RPMI 1640 medium (Life Technologies, Gaithersburg, MD) with 10% FCS and cefotaxime 50 µg/ml. Cells were plated, and nonadherent cells were removed by washing at 24 h. Differential counts were performed on cyto-centrifuged preparations using the Leuko Stat Stain Kit (Fisher, Pittsburgh, PA). Viability of adherent AM was assessed by trypan blue dye.

Mycobacteria
M. tuberculosis H37Rv and H37Ra were purchased from American Type Culture Collection (Manassas, VA). Before inoculation of AM, mycobacteria were grown to log phase in small volumes of 7H9 broth supplemented with 10% (wt/vol) OADC. After centrifugation, bacilli were vortexed, and then sonicated (15 s, 500 W) in a bath sonicator (Laboratory Supplies, Hicksville, NY). After sonication, bacterial suspensions were allowed to stand (10 min), and the upper 500 µl were removed for use in experiments. For each experiment, the adequacy of dispersion and the multiplicity of infection (MOI) were checked by acid-fast stain of infected AM at 4 h. Ten high-power fields were counted to provide an equivalent MOI of 5–10 bacilli per cell for each strain examined. We saw variation in clumping between bacterial strains and experiments, so sonication and stand times were altered to ensure a uniform inoculum between experiments. The success of altered sonication times and inoculum doses at rendering a reproducible viable MOI was rechecked by AFB stain of infected AM before each array experiment. This method has been shown to correlate with bacillary numbers as measured by the Bactec viability assay (12, 17). Each bronchoscopy rendered 9–11 million normal AM, which were divided into groups of 3 million AM in T75 tissue culture flasks and infected under different conditions. AM were infected with H37Rv or H37Ra, and additional conditions included uninfected AM, AM infected with H37Ra and anti-TNF antibody (5 µg/ml; R&D Systems, Minneapolis, MN) and AM infected with H37Ra and isotype control antibody (5 µg/ml; R&D Systems). This number of AM retrieved at bronchoscopy prevented each and every condition to be performed on the same donor cells. The dose of antibody chosen has been shown to prevent TNF-mediated apoptosis of Mtb-infected AM (11). (After 4 h, cells were washed to remove any free bacilli and fresh media with or antibody was added back.) At 48 h, total RNA was prepared from the infected cells for array studies. RNA from two conditions can be compared on the same microarray (i.e., H37Ra-infected cells versus control uninfected cells, H37Rv-infected cells versus control uninfected cells, and H37Ra-infected cells versus H37Rv-infected cells). For each of these three types of comparative array studies, three microarray experiments were performed using AM obtained from three different donors. The anti-TNF experiment compared RNA derived from cells infected with Ra in the presence of anti-TNF Ab compared with H37Ra-infected cells with isotype control Ab, and was performed twice with AM obtained from two different donors. For each of the four comparative studies mentioned above, a replicate experiment from one of the donors was performed with reversal of the Cy3 and Cy5 fluorochrome labeling.

Microarray Data Acquisition
At 48 h after infection, RNA was isolated from both groups of human AM using the Qiagen RNeasy Midi RNA purification kit (Qiagen, Valencia, CA) as per the manufacturer protocol. A 3DNA Submicro detection kit (Genisphere, Hatfield, PA) was then used to convert mRNA to cDNA, with the two comparative samples being labeled with the Cy3 and Cy5 fluorochromes, respectively. The labeled cDNAs were hybridized to a DNA microarray containing triplicates of 374 known human apoptosis related genes (Human Array 1.0; Operon, Alameda, CA). The list of genes represented on this chip are listed on the website at http://pulm.bumc.bu.edu/tbdb/. After overnight hybridization, the microarray was washed and scanned on a GSILumonics ScanArray 4,000 reader (GSI Lumonics, Farmington Hill, MI). This scanner uses red and green Helium-Neon lasers operating at 633nm and 543nm to excite Cy5 and Cy3, respectively. Data from each fluorescence channel were collected and stored as a 16-bit TIF image.

Micorarray Data Analysis
Mean integrated fluorescent intensities for each spot and its local background were calculated in both the Cy3 and Cy5 channels by Quantarray software (GSI Lumonics). After background subtraction, total intensity normalization was performed to correct for differences in fluorochrome efficiencies and the amount of mRNA between experiment and control group. To filter out arrays of poor quality, several quality control measures on each array were assessed including a review of the scanned image for significant artifacts, background measurements that differ significantly from other arrays, and presence or absence of the positive and negative controls on the Operon array.

After normalization, the data were analyzed to identify genes that were differentially expressed. Normalized data from each array were imported into GeneSpring software (Silicon Genetics, Redwood City, CA), where a mean and standard deviation for the Cy3/Cy5 ratio for each gene (present in triplicate on each array) was calculated. Using the GeneSpring software, a t test was performed on the replicate measures for each gene on each array, with the resulting P value reflecting the likelihood of obtaining the variation for the triplicate intensities of each gene by chance (P < 0.05 considered significant). Mean Cy3/Cy5 ratios were converted to log2. Genes with P values < 0.05 and a log2 (mean Cy3/Cy5 ratio) of greater than 1 or less than -1 (corresponding to 2-fold up- or downregulation) were considered differentially expressed between comparative groups. In addition, we calculated the mean fold change across different donors for each gene under each experimental condition, as well as the standard deviation and range for that gene (data available at http://pulm.bumc.bu.edu/tbdb/). After application of a variation filter, hierarchical clustering of the data with average linkage clustering and a Pearson correlation (uncentered) similarity metric was performed in Cluster and Treeview software programs obtained at http://rana.lbl.gov/EisenSoftware.htm. Multidimensional scaling of samples was performed using the Partek 5.0 software (www.partek.com).

Quantitative Real-Time Polymerase Chain Reaction
Quantitative real-time polymerase chain reaction (QRT-PCR) was used to confirm the differential expression of select genes between comparative groups. Primer sequences were designed with Primer Express software (Applied Biosystems, Foster City, CA) based on alignments of candidate gene sequences. RNA samples were treated with DNA-free (Ambion, Austin, TX) as per the manufacturer protocol, to remove contaminating genomic DNA. Total RNA was reverse transcribed using SuperScript II (Gibco, Carlsbad, CA). Five microliters of the reverse transcription reaction was added to 45 µl of SYBR green PCR master mix (Applied Biosystems). Forty cycles of amplification, data acquisition, and data analysis were performed in an ABI Prism 7,700 Sequence Detector (PE Applied Biosystems).

Supplemental Information
Additional information on the Operon Human Apoptosis Array 1.0, microarray protocol, QRT-PCR, and Cy3/Cy5 ratio for all the genes on all arrays is available at http://pulm.bumc.bu.edu/tbdb/.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Apoptosis-Related Gene Expression in Macrophages Infected with H37Rv or H37Ra
Normal AM were harvested from healthy donors. As detailed in MATERIALS AND METHODS, one half of the cells were infected with the virulent Mtb strain H37Rv at an MOI of 5–10, whereas the rest were cultured in identical conditions but without infection. At 48 h after infection, cDNA was prepared from infected AM and naive AM, labeled with Cy3 and Cy5 fluorochromes, and hybridized to microarrays representing 374 human apoptosis-related genes. Although replicate experiments with fluorochrome reversal yielded consistent sets of over-/underexpressed genes, there was variation in differentially expressed genes between experiments using AM from different donors. However, a set of genes was consistently over-/underexpressed, on average, between infected and uninfected cells when all donors were compared. This was reflected by a > 2-fold mean change and a relatively small SD for these genes across all donors. Four proapoptotic genes were downregulated > 2-fold (mean fold change across all three arrays) in AM infected with H37Rv, as compared with uninfected AM. These genes were RAD23 homolog B, apoptotic-related protein PCAR, elongation factor 1-{alpha}, and ZK1 mRNA for Kruppel-type zinc finger protein. A number of additional proapoptotic genes were downregulated between 1.8- and 2.0-fold (Table 1). The mean expression of bcl-w, an antiapoptotic member of the bcl family, was upregulated > 2-fold by H37Rv across all donors (Table 1).


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  		TABLE 1 Genes differentially expressed in three comparative experiments

 
Under conditions identical to those used for studies with H37Rv, normal human AM were infected with the avirulent Mtb strain H37Ra and apoptosis-related gene expression at 48 h post-infection was characterized using Operon microarrays. As compared with uninfected cells, AM infected with H37Ra demonstrated a 2-fold increase in the expression of a proapoptotic gene described as being "upregulated during campothecin-induced apoptosis of U937 cells" as well as the superoxide dismutase 2 mitochondrial gene (Table 1). In contrast to the response induced by H37Rv, no genes were downregulated after H37Ra infection of AM.

Direct Comparison of H37Rv and H37Ra
The cDNA microarray technology is designed to compare two different experimental conditions on a single chip, identified by the signals from red and green fluorochromes. Having evaluated gene expression changes between naive AM and those infected with virulent H37Rv or avirulent H37Ra, we next directly compared the gene expression pattern in AM from single donors infected with either H37Rv or H37Ra. These experiments revealed five proapoptotic genes downregulated > 2-fold by H37Rv compared with H37Ra. They included ZK1, FIP-3, RAD23 homolog B, superoxide dismutase 2 mitochondrial, and EF-1-{alpha} (Table 1). The same set of genes was downregulated in H37Rv-infected AM compared with uninfected AM (Table 1).

Real-time PCR performed on four of the differentially expressed genes in H37Rv- and H37Ra-infected AM confirmed the findings of the microarray experiments (Table 2). Fold changes in expression were similar for the EF-1 and ZK1 genes, whereas RAD23 and SOD2 had larger fold changes on PCR, but in a direction consistent with the microarray experiment.


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  		TABLE 2 QRT-PCR confirmation of microarray data

 
Role of TNF Signaling in Apoptosis-Related Gene Expression
In previous functional studies, we determined that purified human AM infected with H37Ra undergo apoptosis in a TNF-dependent manner (10). We next investigated the role of TNF in the apoptosis-related gene expression pattern of AM infected with H37Ra. Microarray studies were performed comparing AM infected with H37Ra in the presence of neutralizing anti-TNF mAb at a dose sufficient to abrogate infection-induced apoptosis, or an isotype control mAb. In these experiments, two genes were downregulated > 2-fold in H37Ra plus anti-TNF condition, compared with H37Ra plus isotype control; TSIP-3, and superoxide dismutase 2 mitochondrial. Additionally, four other proapoptotic genes were downregulated 1.8- to 2.0-fold in the anti-TNF condition (Table 3). Of interest, all four of these genes as well as superoxide dismutase 2 were similarly downregulated in the experiment comparing AM infected with H37Rv versus H37Ra. No genes were upregulated in the comparison between H37Ra plus anti-TNF Ab versus H37Ra plus isotype control.


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  		TABLE 3 Downregulated genes in Ra + anti-TNF versus Ra + isotype antibody

 
Data Analysis
Hierarchical clustering of samples using genes that had passed the variation filter revealed clustering of the H37Ra plus anti-TNF versus H37Ra + isotype control samples with the H37Rv versus H37Ra and H37Rv versus uninfected samples (Figure 1) . In addition, multidimensional scaling of all samples according to the expression of differentially expressed genes (10 dimensional space) showed grouping together of the H37Ra plus anti-TNF versus H37Ra + isotype control samples with the H37Rv versus the H37Ra samples (Figure 2) . Together, these analyses reveal that the pattern of apoptosis-related gene expression in AM infected with avirulent H37Ra is changed by the addition of neutralizing anti-TNF mAb to a pattern similar to that of AM infected with virulent H37Rv.



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  		Figure 1. Hierarchical clustering of all arrays according to genes that passed the variation filter. We applied a variation filter to the data to remove genes showing minimal variation across the samples being analyzed. We included only those genes whose maximum–minimum log2ratio across all array experiments was > 2.5. Anti-TNF, H37Ra-infected AM + anti-TNF versus H37Ra-infected AM + isotype antibody; Rv versus uninf, H37Rv-infected AM versus uninfected AM; Ra versus uninf, H37Ra-infected AM versus uninfected AM; Rv versus Ra, H37Rv-infected AM versus H37Ra-infected AM. Red, higher ratio; green, lower ratio. Genbank accession numbers are listed in parentheses.

 


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  		Figure 2. Multidimensional scaling plot of all arrays according to the expression of all 10 genes identified as differentially expressed in Tables 1 and 3. TNF, H37Ra-infected AM + anti-TNF versus H37Ra-infected AM+ isotype antibody; Rv versus uninfect, H37Rv-infected AM versus uninfected AM; Ra versus uninfect, H37Ra-infected AM versus uninfected AM; Rv versus Ra, H37Rv-infected AM versus H37Ra-infected AM.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Data from this DNA microarray study support the hypothesis that the expression of certain apoptosis-related genes in human AM is consistently altered after infection with Mtb. Furthermore, the data indicate that Mtb elicits differential expression of apoptosis-related genes in a strain-specific pattern correlating with microbial virulence. Infection with the virulent Mtb strain H37Rv is associated with reduced expression of several proapoptotic genes and increased expression of the antiapoptotic gene bcl-w, when compared with uninfected AM . The biological significance of any or all of these individual genes in determining the fate of infected AM will only be revealed by functional genetic studies.

A member of the bcl-2 family, bcl-w has been shown to exert an antiapoptotic effect in leukocytes. Enforced expression of bcl-w rendered lymphoid and myeloid cells refractory to several cytotoxic conditions (18), but its potential role in regulating the fate of macrophages infected with Mtb is unknown. Interestingly, Klingler and coworkers (19) reported that the antiapoptotic gene bcl-2 is downregulated and apoptosis is increased in macrophages after infection with M. bovis BCG. Another bcl-2 family member, A1, has also been shown to be regulated by mycobacterial infection of monocytes (20). Altered expression of bcl-w has not previously been described with Mtb infection, but bcl-w has been shown to suppress Sindbis virus-induced apoptosis (21).

Among the proapoptotic genes downregulated by H37Rv, ZK1 is an early response gene to ionizing radiation and may function in radiation-induced apoptotic cell death of hematopoietic cells (22). The leucine zipper protein FIP-3 inhibits both basal and induced transcriptional activity of nuclear factor-{kappa}B and causes a late-appearing apoptosis (23). Brockstedt and colleagues (24) demonstrated that the ultraviolet excision repair protein, RAD23 homolog B, is an apoptosis-associated protein in a human Burkitt lymphoma cell line. EF-1{alpha} has a role in expediting the execution of the apoptotic program in response to oxidative stress, although upregulation of EF-1{alpha} in this setting is believed to be regulated at a post-transcriptional level (25).

Perhaps of greater importance than the expression levels of discrete genes, the experiments with neutralizing anti-TNF Ab suggest that interference with TNF signaling in macrophages is a feature of virulent Mtb infection. The pattern of apoptosis-related gene expression was different in AM infected with H37Ra, compared with H37Rv. However, when TNF was neutralized in H37Ra-infected cultures, the resulting gene expression pattern most closely resembled H37Rv infection rather than the pattern of naive AM. Given that there are no consistent differences in the levels of TNF released by AM infected with either strain (11), results from our study support the existence of mycobacterial virulence determinants acting on the host cell to influence death signals mediated by this crucial host defense cytokine. We found that addition of excess TNF to H37Rv-infected AM partly overcame this apoptosis prevention (11), which suggests that H37Rv's interference with the apoptosis pathway is not irreversible. A number of studies have conclusively demonstrated the importance of TNF in defense against Mtb in animal models (26) and in human disease (27, 28), although the mechanism whereby TNF deficiency promotes morbidity and mortality is not well understood (17, 29). It is reasonable to theorize that apoptosis of infected AM mediated by TNF constitutes one of its contributions to innate defense and that virulent bacilli have acquired the means to neutralize this effect of TNF. A precedent for mycobacterial interference with host defense cytokine signal pathways has been established in previous studies of interferon (30).

Although studying primary AM (as opposed to cell lines) provides a unique opportunity to assess host–apoptotic response to Mtb infection, there are a number of important limitations to this study design. Given the limited sample size, fold change was used as a threshold to identify differentially expressed genes. Although this threshold has been applied in prior microarray studies (31–33), fold changes do not provide any means to measure the statistical relevance of the results (34). In addition, there is no consensus as to the threshold level for fold change, which typically varies between 2 and 3 but can be as low as 1.7 (35). To perform a statistical analysis, an unfeasibly large number of samples would have been necessary to address the multiple comparison problem inherent in microarray experiments (36). The presence of triplicate probe sets for each gene on the microarrays did provide us with a statistical measure of consistency for the Cy3/Cy5 ratio for each gene on each array. In addition, we were able to confirm the differential expression of selected number of genes using an alternative technology (QRT-PCR).

By using primary AM obtained from human volunteers, microarray experiments in our study are subject to the variability of the donor pool, and by the asynchronous nature of the biological process we are studying. This is reflected by the large degree of variability in differentially expressed genes between different donors, and resulted in limiting our analysis to genes differentially expressed consistently in AM from all host donors. However, our finding a consistent pattern of changes in the expression of this limited set of genes in spite of the inherent variability in this approach supports the potential significance of the identified changes. The 48-h time point after infection was chosen to capture transcriptional events leading to apoptosis, which is apparent by 5 d after infection (11). A multiple time point experiment would help to characterize the kinetics of the transcriptional response to Mtb. Finally, microarrays may be limited in their ability to characterize the regulation of apoptotic machinery in the cell, as some genes and proteins involved in the signaling and execution of apoptosis are constitutively expressed and regulated by post-translational mechanisms (37–39). Bacterial products have been shown to influence apoptosis through transcription independent events such as Caspase binding and Bad phosphorlyation (40, 41). In this regard, H37Rv infection may interfere with the TNF-dependent apoptosis pathway components in a way that would not be detected by microarrays, as this assay depends on significant transcription level changes. Despite these limitations, the results of this study provide insight into the host AM response to infection with Mtb, demonstrating changes in apoptosis-related gene expression consistent with previous functional data and suggesting a novel virulence mechanism for Mtb based on the manipulation of TNF signaling pathways.


   Acknowledgments
 
This work was supported by a National Heart, Lung, and Blood Institute grant HL-03964, the Massachusetts Thoracic Society, and the American Lung Association of Massachusetts. A.S. is supported by a Doris Duke Charitable Foundation Clinical Scientist Development Award.


   Footnotes
 
* J.D.C. is currently at the Department of Microbiology and Immunology, University of Arkansas for Medical Sciences, Little Rock, Arkansas. Back

{dagger} H.K. is currently at the Department of Medicine, University of Massachusetts Medical School, Worcester, Massachusetts. Back

Received in original form December 20, 2002

Received in final form March 4, 2003


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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