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Infection and Immunity, March 2005, p. 1879-1885, Vol. 73, No. 3
0019-9567/05/$08.00+0     doi:10.1128/IAI.73.3.1879-1885.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

GeneChip Analyses of Global Transcriptional Responses of Murine Macrophages to the Lethal Toxin of Bacillus anthracis

Jason E. Comer,1 Cristi L. Galindo,2 Ashok K. Chopra,2*,{dagger} and Johnny W. Peterson2*,{dagger}

Departments of Experimental Pathology,1 Microbiology and Immunology, The University of Texas Medical Branch, Galveston, Texas2

Received 10 September 2004/ Returned for modification 11 November 2004/ Accepted 16 November 2004


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ABSTRACT
 
We performed GeneChip analyses on RNA from Bacillus anthracis lethal toxin (LeTx)-treated RAW 264.7 murine macrophages to investigate global effects of anthrax toxin on host cell gene expression. Stringent analysis of data revealed that the expression of several mitogen-activated protein kinase kinase-regulatory genes was affected within 1.5 h post-exposure to LeTx. By 3.0 h, the expression of 103 genes was altered, including those involved in intracellular signaling, energy production, and protein metabolism.


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TEXT
 
Bacillus anthracis, the etiological agent of anthrax, causes fatal infections in humans and has gained notoriety as a bioterrorism weapon (10). B. anthracis produces two bipartite proteinaceous toxins, which are associated with virulence of the bacterium (6, 7, 41). Lethal toxin (LeTx) is comprised of protective antigen (PA), which is responsible for binding and entry into host cells (6), and lethal factor (LF), a zinc protease that cleaves mitogen-activated protein kinase kinases (MAPKKs) (11, 48, 63). LeTx is specifically cytotoxic for macrophages from certain inbred mouse strains (15, 55), which is likely physiologically relevant to human infection, since necropsy of anthrax inhalation victims revealed extensive macrophage apoptosis (22).

Resistance of some murine macrophages to LeTx-induced cytotoxicity was shown to be associated with alterations in the kinesin motor protein Kif1C (64), which was up-regulated in LeTx-sensitive macrophages and down-regulated in resistant cells (60). Further, it was shown by using DNA membrane arrays that genes under the regulation of glycogen synthase kinase 3ß (GSK-3ß) were down-regulated after LeTx treatment in macrophages (60). GSK-3ß is implicated in cell fate determination and differentiation and is also involved in energy metabolism. GSK-3ß is inhibited via phosphorylation by protein kinase A (PKA), PKB (also called Akt), PKC, and 90-kDa ribosomal subunit S6 kinase (p90RSK) (13, 31). LeTx-induced inhibition of MAPKKs blocked induction of certain NF-{kappa}B target genes, such as p38, allowing apoptosis of activated macrophages (13, 31).

Although an earlier study suggested that LeTx-mediated lethality of the host involved hyperproduction of cytokines by macrophages (24), more recently it was demonstrated that LeTx impaired host immune responses (12). Additionally, LeTx was reported to cause cell lysis via an increase in permeability to ions and rapid depletion of ATP (25), which might involve reactive oxygen species intermediates (26). Protein synthesis and proteasome activity are also required for LeTx cytotoxicity for macrophages, which suggests that degradation of survival factors might also be a potential mechanism of toxin-induced macrophage killing (4, 59).

To determine global host cell responses to intoxication, we employed Affymetrix Mouse Genome 430A 2.0 GeneChips (Santa Clara, Calif.) to investigate the expression profile of LeTx-treated macrophages and to identify potentially important genes involved in the mechanism of action of LeTx. GeneChips contained ~22,600 probe sets representing transcripts and variants from over 14,000 well-characterized mouse genes.

Expression profiles of LeTx-treated RAW 264.7 murine macrophages RAW 264.7 cells were grown to 60% confluence (19) and treated with 1.0 µg of PA/ml and 0.2 µg of LF/ml (List Biological Laboratories, Campbell, Calif.) for 0, 1.5, or 3.0 h in three independent experiments. This 5:1 ratio of PA to LF provided optimal cytotoxic responses in macrophages in a 4-h period (unpublished data). The toxin components were analyzed for lipopolysaccharide (LPS) contamination by using Pyrogent Plus (BioWhittaker, Walkersville, Md.); no LPS was detected in PA, and LPS contamination (1 ng/µg) of LF was noted. To compensate for the potential effect of LPS, a control experiment was performed to examine alterations in cellular transcription with 0.2 ng of LPS/ml, which corresponded to the concentration present in 0.2 µg of LF/ml. We also performed an experiment using 1 µg of PA/ml alone in order to determine the contribution, if any, of the toxin-binding moiety to macrophage gene expression. LF alone is nontoxic to host cells, as it requires PA to bind to eukaryotic cells (14).

There was no detectable cell death at 1.5 h in LeTx-treated macrophages; however, by 2 h we could detect cell lysis (20 to 30%), which increased to 80% by 4 h, as determined by the release of lactate dehydrogenase enzyme from host cells by using the CytoTox 96 nonradioactive cytotoxicity assay kit (Promega Corporation, Madison, Wis.) (data not shown). Therefore, these two time points (1.5 and 3.0 h) provided us with early and late samples, respectively, during the intoxication process. By 4 h, it was not possible to obtain enough RNA from macrophages to perform GeneChip analysis. After toxin challenge, RNA was isolated and applied to GeneChips and the data were analyzed using four separate techniques: GeneChip Operating Software (GCOS; Affymetrix), Significance of Analysis of Microarrays software (SAM; Stanford University, Palo Alto, Calif.), Spotfire DecisionSite 7.3 software (Spotfire Inc., Somerville, Mass.), and analysis of variance (ANOVA).

Principal component analysis with Spotfire DecisionSite 7.3 software was also employed to describe general trends in gene expression induced by LeTx in macrophages (19). Expression of 98.7% of the genes was not altered by LeTx treatment. A total of 1.2% of the genes (270 probe sets) showed altered expression between 1.5 and 3.0 h during the intoxication process, while the expression of only 0.1% of the genes (22 probe sets) was changed from 0 to 1.5 h (data not shown). These data indicated that the major effects of LeTx on macrophage gene transcription occurred just prior to cellular death.

We compared the 0-h time points to 1.5 and 3.0 h, and a change in gene expression was considered significant if it met the following criteria: at least twofold alteration compared to control samples, a P value of 0.05 or less, and the occurrence of the change for at least two out of the three independent experiments. We further expected that each significantly altered gene would be identified by all of the analysis techniques that were used. Based on these stringent criteria, 17 genes were up- or down-regulated at 1.5 h and included those involved in inflammation, intracellular signaling, and regulation of transcription. Inflammation-related genes were also up-regulated in LPS (0.2 ng/ml)-treated macrophages, suggesting that residual LPS contamination of LF might have been responsible for the induction of these genes. Therefore, we very cautiously interpreted our data and focused on genes that were not induced by LPS.

Two probe sets representing the gene coding for regulator of G-protein signaling 16 (Rgs16) were up-regulated by 1.5 h in LeTx-treated macrophages (Table 1). Rgs proteins inhibit signal transduction by increasing the GTPase activity of G protein {alpha} subunits, thereby driving them into their inactive GDP-bound form. Rgs16 specifically inhibits G{alpha}i3 and subsequently antagonizes RhoA signaling (30), which perturbs several intracellular signaling processes, including regulation of the cytoskeleton (47, 52), phospholipid metabolism (2, 51, 65), cell migration (18), and gene expression through c-fos serum response element sites (28). Rgs16 also preferentially attenuates the activation of p38 (66), which, in addition to LeTx-mediated cleavage of MKK3 and MKK6 (62), might contribute to negative regulation of MAPKK signaling.


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  		TABLE 1. Significant genes up- or down-regulated by LeTx in RAW 264.7 murine macrophages from 0 to 1.5 h as determined by four separate analysis methods (grouped by function)a

LeTx up-regulated prostaglandin E2 receptor, subtype 4 (EP4), by 1.5 h (Table 1). EP4 stimulates cyclic AMP (cAMP) formation and can activate extracellular signal-regulated kinases 1 and 2 (ERK1/2) by way of phosphatidylinositol 3-kinase (16). EP4 may also activate cAMP-independent signaling pathways, such as phosphatidylinositol 3-kinase-dependent stimulation of T-cell factor/lymphoid enhancer factor (Tcf/Lef) reporter activity (17), which is associated with agonist-dependent phosphorylation and inactivation of GSK-3 (8, 13, 16, 31, 57). LeTx-induced up-regulation of EP4 correlates with previous observations (i.e., LeTx-induced down-regulation of GSK-3ß target genes) (60).

Early growth response factor 2 (Egr2) was significantly up-regulated by 1.5 h in response to LeTx treatment of macrophages (Table 1). Egr2 is involved in the regulation of proliferation and differentiation of host cells (32). Egr2 plays a key role in the PTEN (phosphatase and tensin homolog on chromosome 10)-induced apoptotic pathway by altering the permeability of the mitochondrial membrane, thereby releasing cytochrome c and activating caspases 3, 8, and 9. Erg2 could also directly induce the expression of the proapoptotic proteins Bcl-2/E1B 19-kDa interacting protein-3-like (BNIP3L) and Bcl-2 homologous antagonist/killer (BAK) protein (61).

Genes that were down-regulated at 1.5 h were the gene that coded for dual-specificity phosphatase 6 (Dusp6), the FBJ osteosarcoma oncogene (c-Fos), and the Jun oncogene (c-Jun), with c-Fos having the greatest reduction in expression (Table 1). Dusp6 is a phosphatase that preferentially inactivates ERKs (43). The AP-1 transcription factor subunits c-Fos and c-Jun lie downstream of MAPK signaling, and since LeTx cleaves activators of MAPKs, their down-regulation was not surprising. Additionally, GSK-3ß activates transcription of c-Jun (33), so down-regulation of c-Jun, as determined by our GeneChip analyses, correlates with previous studies that demonstrated LeTx-induced down-regulation of GSK-3ß target genes (60).

By 3 h, expression of 103 genes was altered by at least twofold compared to the 0-h control as determined by the various statistical methods used (Table 2). Most of the up-regulated genes encoded inflammatory and signal transduction proteins, as well as several transcription factors. Of the 62 down-regulated genes, 35% were involved in protein metabolism, including genes that coded for 15 ribosomal subunits (represented by 19 probe sets). Several genes involved in the electron transport chain were also down-regulated, including ATP synthase epsilon chain, cytochrome c oxidase subunit VIIa polypeptide 2-like, NADH dehydrogenase (ubiquinone) 1 alpha subcomplexes 2 and 7, ubiquinol-cytochrome c reductase subunit, and ubiquinol-cytochrome c reductase complex 11-kDa protein, which indicated a decrease in energy production in intoxicated cells.


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  		TABLE 2. Significant genes up- or down-regulated by LeTx in RAW 264.7 murine macrophages from 0 to 3 h as determined by four separate analysis methods (grouped by function)a

There were several cytoskeletal genes that were down-regulated by toxin treatment at 3 h (Table 2): thymosin beta 10 (Tmsb10), thymosin beta 4 (Tmsb4x), troponin I skeletal fast 2 (Tnni2), and cytoplasmic dynein light chain. Tmsb10 and Tmsb4x are actin-binding proteins involved in cytoskeleton organization and biogenesis (45). Tnni2, another actin-binding protein, is a structural constituent of the cytoskeleton (44), and cytoplasmic dynein light chain is a molecular motor involved in intracellular transport along microtubules (21).

There were three immune response-associated genes that were specifically up- or down-regulated by LeTx and not by LPS or PA: CD137 ligand, Toll-like receptor 2 (TLR2), and plasminogen activator inhibitor type I (PAI-1) (Table 2). CD137 ligand (also called 4-IBB ligand) was down-regulated by the toxin. This protein is a tumor necrosis factor (TNF) receptor (ligand) superfamily member that stimulates both primary and secondary responses of CD4+ and CD8+ T cells (9). Additionally, CD137 is constitutively expressed on primary monocytes and, upon cross-linking with immobilized CD137 ligand, mediates adherence, activation, proliferation, survival, expression of proinflammatory cytokines, and down-regulation of the anti-inflammatory cytokine interleukin-10 (35-37, 40, 53, 56). Furthermore, it has also been demonstrated that CD137 ligand is required for initiation of proliferation of monocytes in response to LPS (34).

TLR2 was up-regulated by LeTx at 3 h (Table 2). TLR2 is a receptor that binds components of gram-positive bacteria and is essential for the recognition of peptidoglycan (58). Engagement of TLR2 has also been shown to result in activation of NF-{kappa}B, cytokine production, and apoptosis (1). Alternatively, recent data have also suggested that several pathogens might utilize TLR2 as an immune escape mechanism via the production of anti-inflammatory cytokines (3, 20, 46, 50, 54).

PAI-1, which was significantly up-regulated by LeTx (3.8- to 9.6-fold by 3 h; Table 2) is the main physiological inhibitor of tissue-type and urokinase-type plasminogen activators. Up-regulation of PAI-1 results in local tissue fibrin deposition during severe inflammation (29, 38) and massive imbalance of the coagulation and fibrinolytic systems during bacterial infection-induced sepsis, which ultimately leads to multiorgan failure (5, 49). In fact, the actual level of PAI-1 increase correlates with the severity of disseminated intravascular coagulation and sepsis, which is a 100% predictor of lethality (5, 42). Induction of PAI-1 and subsequent fibrinolysis could also interfere with migration of immune cells. LeTx-induced down-regulation of CD137 and up-regulation of TLR2 and PAI-1 could have profound consequences on host immune responses during B. anthracis infection and will be pursued in future.

In addition to the previously mentioned Dusp6, which dephosphorylates ERKs, there were three signaling molecules that were preferentially down-regulated by LeTx at 3 h but were not altered by LPS or PA treatment alone. These included G protein, gamma 2 subunit (Gng2); cAMP-dependent protein kinase inhibitor gamma (PKI{gamma}); and protein tyrosine phosphatase 4a2. On the other hand, protein tyrosine phosphatase non-receptor type 8 was up-regulated by LeTx (Table 2). The consequences of LeTx-mediated alteration in the expression of these genes are unclear but might be important for LeTx-mediated host responses to infection and will be further investigated.

There were several transcription factors that were up-regulated by LeTx by 3 h (Table 2), most notably activating transcription factor 3 (ATF3). ATF3 is a stress-inducible member of the ATF/CREB family of transcription factors (23), and induction of ATF3 is strongly associated with apoptosis (27). LeTx also induced alteration in the expression levels of zinc finger proteins, Kruppel-like factor 2, interleukin-3-regulated nuclear factor, orphan nuclear receptors, and TSC22-related transcription factor, which could contribute to LeTx-mediated host cell responses.

Confirmation of GeneChip data by real-time RT-PCR of selected genes In order to confirm the GeneChip data, we performed real-time reverse transcription-PCR (RT-PCR) (39) on selected genes using the ABI Prism 7000 sequence detection system. TaqMan reverse transcriptase (Applied Biosystems, Foster City, Calif.) was used to synthesize cDNA from total RNA isolated from LeTx-treated cells. Experiments were performed in parallel, and fold values were determined after normalization of each gene to glyceraldehyde-3-phosphate dehydrogenase by the comparative threshold method.

We chose nine representative genes from the functional groups affected by LeTx for confirmation (Table 3). The ribosomal subunit genes L13 and L22, as well as NADH dehydrogenase 1 alpha subcomplex 2, were confirmed to be down-regulated at 3 h (Table 3), reinforcing the GeneChip data showing that LeTx inhibited protein synthesis and energy production in intoxicated cells. Alterations in the transcription of genes involved in MAPK and other signaling pathways (G;protein gamma 2 subunit and protein tyrosine phosphatase non-receptor type 8) were also confirmed by this technique.


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  		TABLE 3. Real-time RT-PCR confirmation of altered expression levels of selected genes at 3.0 h postchallengea

According to GeneChip analysis and real-time RT-PCR confirmation, LeTx altered expression of genes involved in MAPK signaling cascades, including genes up- and downstream of the toxin's cleavage targets. LeTx also down-regulated electron transport chain genes and genes involved in protein synthesis. In our model, we were not able to confirm the observations of Tucker et al. (60) that expression of Kif1C was up-regulated in a LeTx-sensitive macrophage cell line. We did, however, observe decreases in c-Jun and c-Fos expression, confirming the suppression of GSK-3ß-regulated genes. Although the exact mechanism of LeTx-mediated macrophage cytolysis remains a mystery, these data provided for the first time a comprehensive expression profile of intoxicated cells. More studies are required to determine how the altered gene expression induced by LeTx causes macrophage-specific cytolysis.

Considered together, the data provided potential intracellular targets of LeTx-mediated macrophage signaling. Additionally, the results suggested avenues through which LeTx might induce macrophage cytotoxicity, resulting in apoptosis or necrosis, which, based on our analyses, appears to depend upon the energy status of host cells. There were several genes that were up- or down-regulated by LeTx that were not previously known to play a role in LeTx-mediated host cell responses. These newly discovered genes are prospects for future investigations of the mode of action of LeTx, which, given the lethality of anthrax and its potential as a biological weapon, are important for developing better diagnostic and treatment strategies.


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ACKNOWLEDGMENTS
 
This work was supported by a grant from the National Institutes of Health (U01AI5385802) and the U.S. Army (DAMD170210699). Jason E. Comer, a predoctoral fellow, was supported by the National Institutes of Health T32 Predoctoral Training Grant in Emerging and Tropical Infectious Disease (2T32A8007526) and in Biodefense (1T32AI060549). Cristi L. Galindo, also a predoctoral fellow, obtained funding from the National Science Foundation.

T. Wood from the Department of Human Biological Chemistry and Genetics, University of Texas Medical Branch, Galveston, provided facilities of his core laboratories for these studies. Michelle M. Guigneaux and Deborah J. Prusak from the Wood laboratory provided technical expertise for this project.


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FOOTNOTES
 
* Corresponding author. Mailing address: Department of Microbiology and Immunology, Medical Research Building, 301 University Blvd., Galveston, TX 77555-1070. For Ashok K. Chopra: Phone: (409) 747-0578. Fax: (409) 747-6869. E-mail: achopra{at}utmb.edu. For Johnny W. Peterson: Phone: (409) 772-4910. Fax: (409) 747-6869. E-mail: jpeterso{at}utmb.edu. Back

* Corresponding author. Mailing address: Department of Microbiology and Immunology, Medical Research Building, 301 University Blvd., Galveston, TX 77555-1070. For Ashok K. Chopra: Phone: (409) 747-0578. Fax: (409) 747-6869. E-mail: achopra{at}utmb.edu. For Johnny W. Peterson: Phone: (409) 772-4910. Fax: (409) 747-6869. E-mail: jpeterso{at}utmb.edu. Back

Editor: J. T. Barbieri

{dagger} A.K.C. and J.W.P. contributed equally to the manuscript. Back


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Infection and Immunity, March 2005, p. 1879-1885, Vol. 73, No. 3
0019-9567/05/$08.00+0     doi:10.1128/IAI.73.3.1879-1885.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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