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Bacillus anthracis' lethal toxin induces broad transcriptional responses in human peripheral monocytes.

Chauncey KM, Lopez MC, Sidhu G, Szarowicz SE, Baker HV, Quinn C, Southwick FS - BMC Immunol. (2012)

Bottom Line: Using Affymetrix Human Genome U133 Plus 2.0 Arrays, we identified over 820 probe sets differentially regulated after LT treatment at the p <0.001 significance level, interrupting the normal transduction of over 60 known pathways.As expected, the MAPKK signaling pathway was most drastically affected by LT, but numerous genes outside the well-recognized pathways were also influenced by LT including the IL-18 signaling pathway, Toll-like receptor pathway and the IFN alpha signaling pathway.This study provides further insights into the mechanisms associated with the host immune system collapse during an anthrax infection, and suggests that anthrax LT may have additional downstream targets outside the well-known MAPK pathway.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Medicine, University of Florida College of Medicine, Gainesville, FL 32610, USA.

ABSTRACT

Background: Anthrax lethal toxin (LT), produced by the Gram-positive bacterium Bacillus anthracis, is a highly effective zinc dependent metalloprotease that cleaves the N-terminus of mitogen-activated protein kinase kinases (MAPKK or MEKs) and is known to play a role in impairing the host immune system during an inhalation anthrax infection. Here, we present the transcriptional responses of LT treated human monocytes in order to further elucidate the mechanisms of LT inhibition on the host immune system.

Results: Western Blot analysis demonstrated cleavage of endogenous MEK1 and MEK3 when human monocytes were treated with 500 ng/mL LT for four hours, proving their susceptibility to anthrax lethal toxin. Furthermore, staining with annexin V and propidium iodide revealed that LT treatment did not induce human peripheral monocyte apoptosis or necrosis. Using Affymetrix Human Genome U133 Plus 2.0 Arrays, we identified over 820 probe sets differentially regulated after LT treatment at the p <0.001 significance level, interrupting the normal transduction of over 60 known pathways. As expected, the MAPKK signaling pathway was most drastically affected by LT, but numerous genes outside the well-recognized pathways were also influenced by LT including the IL-18 signaling pathway, Toll-like receptor pathway and the IFN alpha signaling pathway. Multiple genes involved in actin regulation, signal transduction, transcriptional regulation and cytokine signaling were identified after treatment with anthrax LT.

Conclusion: We conclude LT directly targets human peripheral monocytes and causes multiple aberrant gene responses that would be expected to be associated with defects in human monocyte's normal signaling transduction pathways and function. This study provides further insights into the mechanisms associated with the host immune system collapse during an anthrax infection, and suggests that anthrax LT may have additional downstream targets outside the well-known MAPK pathway.

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Monocyte purity, apoptosis, and susceptibility to LT. Red = CD14+ monocytes. Green = CD14-lymphocytes. A.) Forward and side scatter analysis of purified fixed human monocytes showing the monocyte population as compared to total population. B.) CD14 Pacific Blue and forward scatter analysis of fixed purified human monocytes showing >85% monocytes. C.) PI and annexin-FITC analysis of CD14 + monocytes after a 4 h incubation showing 99.0% viable cells indicated in quadrant 3. D.) PI and annexin-FITC analysis of CD14 + monocytes after a 4 h LT treatment showing 99.1% viable cells indicated in quadrant 3. HeLa cells or human monocytes were left untreated or treated with 500 ng/mL LT for 4 h at 37°C. Samples were lysed, run on SDS-PAGE, transferred to PVDF membrane, and probed with indicated antibodies. Both MEK3 and MEK1 were cleaved by LT while control cells showed no MEK cleavage. β-actin loading controls show equivalent loading of both control and LT treated cells.
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Figure 1: Monocyte purity, apoptosis, and susceptibility to LT. Red = CD14+ monocytes. Green = CD14-lymphocytes. A.) Forward and side scatter analysis of purified fixed human monocytes showing the monocyte population as compared to total population. B.) CD14 Pacific Blue and forward scatter analysis of fixed purified human monocytes showing >85% monocytes. C.) PI and annexin-FITC analysis of CD14 + monocytes after a 4 h incubation showing 99.0% viable cells indicated in quadrant 3. D.) PI and annexin-FITC analysis of CD14 + monocytes after a 4 h LT treatment showing 99.1% viable cells indicated in quadrant 3. HeLa cells or human monocytes were left untreated or treated with 500 ng/mL LT for 4 h at 37°C. Samples were lysed, run on SDS-PAGE, transferred to PVDF membrane, and probed with indicated antibodies. Both MEK3 and MEK1 were cleaved by LT while control cells showed no MEK cleavage. β-actin loading controls show equivalent loading of both control and LT treated cells.

Mentions: In order to first determine monocyte cell purity, isolated cells were analyzed using flow cytometry and gated using forward and side scatter, along with the monocytic marker, CD14. It was found that monocytes were isolated with a >85% purity (Figure 1A and1B). Because previous reports have documented LT induced cell apoptosis, it was important to assure the transcriptional response of LT treated monocytes were independent of apoptosis. This was assured by the analysis of the necrosis and apoptosis markers, propidium iodide (PI) and annexin V, on human peripheral monocytes. Nearly all (99%) human peripheral monocytes showed no evidence of necrosis or apoptosis after a 4 h treatment of LT (Figure 1C and1D). There has been some conflicting data suggesting monocytes, along with monocyte-derived cells, are not susceptible to the actions of anthrax LT. One study utilized human monocytic cell lines and found that undifferentiated monocytic cells did not undergo LT-induced cytotoxicity, while the differentiated cells were susceptible[10]. Another study investigating human alveolar macrophages (AM) found that these cells were relatively resistant to the actions of LT. It was ascertained that LT failed to suppress human AM cytokine responses, cleave MEKs, and induce apoptosis[14].


Bacillus anthracis' lethal toxin induces broad transcriptional responses in human peripheral monocytes.

Chauncey KM, Lopez MC, Sidhu G, Szarowicz SE, Baker HV, Quinn C, Southwick FS - BMC Immunol. (2012)

Monocyte purity, apoptosis, and susceptibility to LT. Red = CD14+ monocytes. Green = CD14-lymphocytes. A.) Forward and side scatter analysis of purified fixed human monocytes showing the monocyte population as compared to total population. B.) CD14 Pacific Blue and forward scatter analysis of fixed purified human monocytes showing >85% monocytes. C.) PI and annexin-FITC analysis of CD14 + monocytes after a 4 h incubation showing 99.0% viable cells indicated in quadrant 3. D.) PI and annexin-FITC analysis of CD14 + monocytes after a 4 h LT treatment showing 99.1% viable cells indicated in quadrant 3. HeLa cells or human monocytes were left untreated or treated with 500 ng/mL LT for 4 h at 37°C. Samples were lysed, run on SDS-PAGE, transferred to PVDF membrane, and probed with indicated antibodies. Both MEK3 and MEK1 were cleaved by LT while control cells showed no MEK cleavage. β-actin loading controls show equivalent loading of both control and LT treated cells.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3475123&req=5

Figure 1: Monocyte purity, apoptosis, and susceptibility to LT. Red = CD14+ monocytes. Green = CD14-lymphocytes. A.) Forward and side scatter analysis of purified fixed human monocytes showing the monocyte population as compared to total population. B.) CD14 Pacific Blue and forward scatter analysis of fixed purified human monocytes showing >85% monocytes. C.) PI and annexin-FITC analysis of CD14 + monocytes after a 4 h incubation showing 99.0% viable cells indicated in quadrant 3. D.) PI and annexin-FITC analysis of CD14 + monocytes after a 4 h LT treatment showing 99.1% viable cells indicated in quadrant 3. HeLa cells or human monocytes were left untreated or treated with 500 ng/mL LT for 4 h at 37°C. Samples were lysed, run on SDS-PAGE, transferred to PVDF membrane, and probed with indicated antibodies. Both MEK3 and MEK1 were cleaved by LT while control cells showed no MEK cleavage. β-actin loading controls show equivalent loading of both control and LT treated cells.
Mentions: In order to first determine monocyte cell purity, isolated cells were analyzed using flow cytometry and gated using forward and side scatter, along with the monocytic marker, CD14. It was found that monocytes were isolated with a >85% purity (Figure 1A and1B). Because previous reports have documented LT induced cell apoptosis, it was important to assure the transcriptional response of LT treated monocytes were independent of apoptosis. This was assured by the analysis of the necrosis and apoptosis markers, propidium iodide (PI) and annexin V, on human peripheral monocytes. Nearly all (99%) human peripheral monocytes showed no evidence of necrosis or apoptosis after a 4 h treatment of LT (Figure 1C and1D). There has been some conflicting data suggesting monocytes, along with monocyte-derived cells, are not susceptible to the actions of anthrax LT. One study utilized human monocytic cell lines and found that undifferentiated monocytic cells did not undergo LT-induced cytotoxicity, while the differentiated cells were susceptible[10]. Another study investigating human alveolar macrophages (AM) found that these cells were relatively resistant to the actions of LT. It was ascertained that LT failed to suppress human AM cytokine responses, cleave MEKs, and induce apoptosis[14].

Bottom Line: Using Affymetrix Human Genome U133 Plus 2.0 Arrays, we identified over 820 probe sets differentially regulated after LT treatment at the p <0.001 significance level, interrupting the normal transduction of over 60 known pathways.As expected, the MAPKK signaling pathway was most drastically affected by LT, but numerous genes outside the well-recognized pathways were also influenced by LT including the IL-18 signaling pathway, Toll-like receptor pathway and the IFN alpha signaling pathway.This study provides further insights into the mechanisms associated with the host immune system collapse during an anthrax infection, and suggests that anthrax LT may have additional downstream targets outside the well-known MAPK pathway.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Medicine, University of Florida College of Medicine, Gainesville, FL 32610, USA.

ABSTRACT

Background: Anthrax lethal toxin (LT), produced by the Gram-positive bacterium Bacillus anthracis, is a highly effective zinc dependent metalloprotease that cleaves the N-terminus of mitogen-activated protein kinase kinases (MAPKK or MEKs) and is known to play a role in impairing the host immune system during an inhalation anthrax infection. Here, we present the transcriptional responses of LT treated human monocytes in order to further elucidate the mechanisms of LT inhibition on the host immune system.

Results: Western Blot analysis demonstrated cleavage of endogenous MEK1 and MEK3 when human monocytes were treated with 500 ng/mL LT for four hours, proving their susceptibility to anthrax lethal toxin. Furthermore, staining with annexin V and propidium iodide revealed that LT treatment did not induce human peripheral monocyte apoptosis or necrosis. Using Affymetrix Human Genome U133 Plus 2.0 Arrays, we identified over 820 probe sets differentially regulated after LT treatment at the p <0.001 significance level, interrupting the normal transduction of over 60 known pathways. As expected, the MAPKK signaling pathway was most drastically affected by LT, but numerous genes outside the well-recognized pathways were also influenced by LT including the IL-18 signaling pathway, Toll-like receptor pathway and the IFN alpha signaling pathway. Multiple genes involved in actin regulation, signal transduction, transcriptional regulation and cytokine signaling were identified after treatment with anthrax LT.

Conclusion: We conclude LT directly targets human peripheral monocytes and causes multiple aberrant gene responses that would be expected to be associated with defects in human monocyte's normal signaling transduction pathways and function. This study provides further insights into the mechanisms associated with the host immune system collapse during an anthrax infection, and suggests that anthrax LT may have additional downstream targets outside the well-known MAPK pathway.

Show MeSH
Related in: MedlinePlus