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Transcriptional profiling of mycobacterial antigen-induced responses in infants vaccinated with BCG at birth.

Fletcher HA, Keyser A, Bowmaker M, Sayles PC, Kaplan G, Hussey G, Hill AV, Hanekom WA - BMC Med Genomics (2009)

Bottom Line: We found that ex vivo stimulation of PBMC with PPD and BCG induced largely similar gene expression profiles, except that BCG induced greater macrophage activation.In contrast, up-regulation of genes associated with the classic, pro-inflammatory macrophage response was noted.Interestingly, more leukocyte genes were down-regulated than up-regulated.

View Article: PubMed Central - HTML - PubMed

Affiliation: Jenner Institute, ORCRB, University of Oxford, Churchill Hospital, Oxford, OX3 7DQ, UK. helen.fletcher@ndm.ox.ac.uk

ABSTRACT

Background: Novel tuberculosis (TB) vaccines recently tested in humans have been designed to boost immunity induced by the current vaccine, Mycobacterium bovis Bacille Calmette-Guérin (BCG). Because BCG vaccination is used extensively in infants, this population group is likely to be the first in which efficacy trials of new vaccines will be conducted. However, our understanding of the complexity of immunity to BCG in infants is inadequate, making interpretation of vaccine-induced immune responses difficult.

Methods: To better understand BCG-induced immunity, we performed gene expression profiling in five 10-week old infants routinely vaccinated with BCG at birth. RNA was extracted from 12 hour BCG-stimulated or purified protein derivative of tuberculin (PPD)-stimulated PBMC, isolated from neonatal blood collected 10 weeks after vaccination. RNA was hybridised to the Sentrix(R) HumanRef-8 Expression BeadChip (Illumina) to measure expression of >16,000 genes.

Results: We found that ex vivo stimulation of PBMC with PPD and BCG induced largely similar gene expression profiles, except that BCG induced greater macrophage activation. The peroxisome proliferator-activated receptor (PPAR) signaling pathway, including PPAR-gamma, involved in activation of the alternative, anti-inflammatory macrophage response was down-regulated following stimulation with both antigens. In contrast, up-regulation of genes associated with the classic, pro-inflammatory macrophage response was noted. Further analysis revealed a decrease in the expression of cell adhesion molecules (CAMs), including integrin alpha M (ITGAM), which is known to be important for entry of mycobacteria into the macrophage. Interestingly, more leukocyte genes were down-regulated than up-regulated.

Conclusion: Our results suggest that a combination of suppressed and up-regulated genes may be key in determining development of protective immunity to TB induced by vaccination with BCG.

No MeSH data available.


Related in: MedlinePlus

Heatmap of gene expression profiles with a >2 fold change in expression in response to BCG stimulation, compared with the unstimulated control. Genes were ordered according to their cluster determined by the k-means algorithm. Values increase from green to red, via black. The incubation conditions and the participant identifiers are given below the heatmap, e.g., "UNS.028" refers to PBMC incubated without any specific antigens (unstimulated) in participant 28.
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Figure 1: Heatmap of gene expression profiles with a >2 fold change in expression in response to BCG stimulation, compared with the unstimulated control. Genes were ordered according to their cluster determined by the k-means algorithm. Values increase from green to red, via black. The incubation conditions and the participant identifiers are given below the heatmap, e.g., "UNS.028" refers to PBMC incubated without any specific antigens (unstimulated) in participant 28.

Mentions: Five 10-week old infants, routinely vaccinated with BCG at birth, were enrolled. DNA micro-array analysis was performed using amplified RNA, purified from cryopreserved PBMC that were later thawed and incubated with BCG, PPD or medium only (unstimulated) for 12 hours. Using K-means clustering, by the 3 incubation conditions and using all genes, unstimulated samples clustered away from BCG and PPD stimulated samples (Figure 1). However, BCG and PPD stimulated samples were not separated by this analysis (Figure 1). Overall, BCG induced differential expression of 411 genes (p < 0.01, eBayes), compared with the unstimulated control. Of the 411 genes, 136 were upregulated and 275 downregulated (Additional file 2). PPD induced differential expression in 291 genes (p < 0.01, eBayes, Additional file 2). Of the 291 genes, 95 were upregulated and 196 were downregulated. Of the significantly differentially expressed genes, 74 and 73 genes were up-regulated >2-fold by BCG and PPD, respectively; 201 and 127 genes were down-regulated >2-fold by these respective conditions (Additional file 2). We concluded that gene expression profiles obtained by stimulating PBMC with BCG were not sufficiently different from expression profiles obtained by stimulating cells with PPD to enable the samples to fall into separate clusters.


Transcriptional profiling of mycobacterial antigen-induced responses in infants vaccinated with BCG at birth.

Fletcher HA, Keyser A, Bowmaker M, Sayles PC, Kaplan G, Hussey G, Hill AV, Hanekom WA - BMC Med Genomics (2009)

Heatmap of gene expression profiles with a >2 fold change in expression in response to BCG stimulation, compared with the unstimulated control. Genes were ordered according to their cluster determined by the k-means algorithm. Values increase from green to red, via black. The incubation conditions and the participant identifiers are given below the heatmap, e.g., "UNS.028" refers to PBMC incubated without any specific antigens (unstimulated) in participant 28.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2654906&req=5

Figure 1: Heatmap of gene expression profiles with a >2 fold change in expression in response to BCG stimulation, compared with the unstimulated control. Genes were ordered according to their cluster determined by the k-means algorithm. Values increase from green to red, via black. The incubation conditions and the participant identifiers are given below the heatmap, e.g., "UNS.028" refers to PBMC incubated without any specific antigens (unstimulated) in participant 28.
Mentions: Five 10-week old infants, routinely vaccinated with BCG at birth, were enrolled. DNA micro-array analysis was performed using amplified RNA, purified from cryopreserved PBMC that were later thawed and incubated with BCG, PPD or medium only (unstimulated) for 12 hours. Using K-means clustering, by the 3 incubation conditions and using all genes, unstimulated samples clustered away from BCG and PPD stimulated samples (Figure 1). However, BCG and PPD stimulated samples were not separated by this analysis (Figure 1). Overall, BCG induced differential expression of 411 genes (p < 0.01, eBayes), compared with the unstimulated control. Of the 411 genes, 136 were upregulated and 275 downregulated (Additional file 2). PPD induced differential expression in 291 genes (p < 0.01, eBayes, Additional file 2). Of the 291 genes, 95 were upregulated and 196 were downregulated. Of the significantly differentially expressed genes, 74 and 73 genes were up-regulated >2-fold by BCG and PPD, respectively; 201 and 127 genes were down-regulated >2-fold by these respective conditions (Additional file 2). We concluded that gene expression profiles obtained by stimulating PBMC with BCG were not sufficiently different from expression profiles obtained by stimulating cells with PPD to enable the samples to fall into separate clusters.

Bottom Line: We found that ex vivo stimulation of PBMC with PPD and BCG induced largely similar gene expression profiles, except that BCG induced greater macrophage activation.In contrast, up-regulation of genes associated with the classic, pro-inflammatory macrophage response was noted.Interestingly, more leukocyte genes were down-regulated than up-regulated.

View Article: PubMed Central - HTML - PubMed

Affiliation: Jenner Institute, ORCRB, University of Oxford, Churchill Hospital, Oxford, OX3 7DQ, UK. helen.fletcher@ndm.ox.ac.uk

ABSTRACT

Background: Novel tuberculosis (TB) vaccines recently tested in humans have been designed to boost immunity induced by the current vaccine, Mycobacterium bovis Bacille Calmette-Guérin (BCG). Because BCG vaccination is used extensively in infants, this population group is likely to be the first in which efficacy trials of new vaccines will be conducted. However, our understanding of the complexity of immunity to BCG in infants is inadequate, making interpretation of vaccine-induced immune responses difficult.

Methods: To better understand BCG-induced immunity, we performed gene expression profiling in five 10-week old infants routinely vaccinated with BCG at birth. RNA was extracted from 12 hour BCG-stimulated or purified protein derivative of tuberculin (PPD)-stimulated PBMC, isolated from neonatal blood collected 10 weeks after vaccination. RNA was hybridised to the Sentrix(R) HumanRef-8 Expression BeadChip (Illumina) to measure expression of >16,000 genes.

Results: We found that ex vivo stimulation of PBMC with PPD and BCG induced largely similar gene expression profiles, except that BCG induced greater macrophage activation. The peroxisome proliferator-activated receptor (PPAR) signaling pathway, including PPAR-gamma, involved in activation of the alternative, anti-inflammatory macrophage response was down-regulated following stimulation with both antigens. In contrast, up-regulation of genes associated with the classic, pro-inflammatory macrophage response was noted. Further analysis revealed a decrease in the expression of cell adhesion molecules (CAMs), including integrin alpha M (ITGAM), which is known to be important for entry of mycobacteria into the macrophage. Interestingly, more leukocyte genes were down-regulated than up-regulated.

Conclusion: Our results suggest that a combination of suppressed and up-regulated genes may be key in determining development of protective immunity to TB induced by vaccination with BCG.

No MeSH data available.


Related in: MedlinePlus