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Integrated microRNA-mRNA-analysis of human monocyte derived macrophages upon Mycobacterium avium subsp. hominissuis infection.

Sharbati J, Lewin A, Kutz-Lohroff B, Kamal E, Einspanier R, Sharbati S - PLoS ONE (2011)

Bottom Line: We furthermore outlined a regulatory network of potential interactions between microRNAs and mRNAs.This study provides a theoretical concept for unveiling how distinct mycobacteria could manipulate host cell response.In addition, functional relevance was confirmed by uncovering the control of major caspases 3 and 7 by let-7e and miR-29a, respectively.

View Article: PubMed Central - PubMed

Affiliation: Institute of Veterinary Biochemistry, Freie Universitaet Berlin, Berlin, Germany. sharbati@zedat.fu-berlin.de

ABSTRACT

Background: Many efforts have been made to understand basal mechanisms of mycobacterial infections. Macrophages are the first line of host immune defence to encounter and eradicate mycobacteria. Pathogenic species have evolved different mechanisms to evade host response, e.g. by influencing macrophage apoptotic pathways. However, the underlying molecular regulation is not fully understood. A new layer of eukaryotic regulation of gene expression is constituted by microRNAs. Therefore, we present a comprehensive study for identification of these key regulators and their targets in the context of host macrophage response to mycobacterial infections.

Methodology/principal findings: We performed microRNA as well as mRNA expression analysis of human monocyte derived macrophages infected with several Mycobacterium avium hominissuis strains by means of microarrays as well as quantitative reverse transcription PCR (qRT-PCR). The data revealed the ability of all strains to inhibit apoptosis by transcriptional regulation of BCL2 family members. Accordingly, at 48 h after infection macrophages infected with all M. avium strains showed significantly decreased caspase 3 and 7 activities compared to the controls. Expression of let-7e, miR-29a and miR-886-5p were increased in response to mycobacterial infection at 48 h. The integrated analysis of microRNA and mRNA expression as well as target prediction pointed out regulative networks identifying caspase 3 and 7 as potential targets of let-7e and miR-29a, respectively. Consecutive reporter assays verified the regulation of caspase 3 and 7 by these microRNAs.

Conclusions/significance: We show for the first time that mycobacterial infection of human macrophages causes a specific microRNA response. We furthermore outlined a regulatory network of potential interactions between microRNAs and mRNAs. This study provides a theoretical concept for unveiling how distinct mycobacteria could manipulate host cell response. In addition, functional relevance was confirmed by uncovering the control of major caspases 3 and 7 by let-7e and miR-29a, respectively.

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Temporal CASP3/CASP7 activity upon MAH infection of MDMs.MDMs were infected with bacteria and the luminescence signal (relative light units, RLU) proportional to the activity of CASP3/CASP7 was measured. Columns represent the mean of quintuplicate measurements while error bars show the standard deviation. Asterisks indicate statistical significance according to paired t test (***: P<0.001).
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pone-0020258-g003: Temporal CASP3/CASP7 activity upon MAH infection of MDMs.MDMs were infected with bacteria and the luminescence signal (relative light units, RLU) proportional to the activity of CASP3/CASP7 was measured. Columns represent the mean of quintuplicate measurements while error bars show the standard deviation. Asterisks indicate statistical significance according to paired t test (***: P<0.001).

Mentions: In order to assess the ability of MAH strains to inhibit apoptosis, CASP3/CASP7 assays were performed. As shown in figure 3, at 6 and 24 h p.i. significantly decreased caspase activity was observed in all infected samples compared to the non-infected controls. However, at 48 h p.i. MDMs infected with all three MAH strains showed significantly decreased caspase activity compared to the non-infected as well as E. coli K12 stimulated controls (P<0.001, paired t test), while no significant difference was detected between the latter. Accordingly, lower caspase expression was detected in qRT-PCR experiments (figure 4 A). Based on the observation that the employed MAH strains are able to inhibit apoptosis, we considered mainly genes in the qRT-PCR experiments being relevant to apoptosis and cytokine-cytokine receptor interaction. For visualisation of the comprehensive and individual expression data of selected genes, heat maps of log 2 ratios of infected samples and non-treated controls were created for each point in time. As shown in figure 4 A, MAH infections caused a unique response of MDMs compared to the E. coli K12 stimulation. The detailed expression analysis of selected genes showed on the one hand some donor specific differences in response to the MAH infection. For example, all MAH strains caused increased expression of IFNGR1 in donor 2 right after 6 h compared to the other samples (P<0.002, unpaired t test). The up-regulation of IFNGR1 appeared after 24 h in all samples followed by decreased expression after 48 h only in donor 3. Accordingly, we observed sustained and consistent induction of TNF receptor superfamily (TNFRSF) 6 and TNFRSF10C after 24 h by all MAH strains in donor 1 compared to the other donors (figure 4).


Integrated microRNA-mRNA-analysis of human monocyte derived macrophages upon Mycobacterium avium subsp. hominissuis infection.

Sharbati J, Lewin A, Kutz-Lohroff B, Kamal E, Einspanier R, Sharbati S - PLoS ONE (2011)

Temporal CASP3/CASP7 activity upon MAH infection of MDMs.MDMs were infected with bacteria and the luminescence signal (relative light units, RLU) proportional to the activity of CASP3/CASP7 was measured. Columns represent the mean of quintuplicate measurements while error bars show the standard deviation. Asterisks indicate statistical significance according to paired t test (***: P<0.001).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3101234&req=5

pone-0020258-g003: Temporal CASP3/CASP7 activity upon MAH infection of MDMs.MDMs were infected with bacteria and the luminescence signal (relative light units, RLU) proportional to the activity of CASP3/CASP7 was measured. Columns represent the mean of quintuplicate measurements while error bars show the standard deviation. Asterisks indicate statistical significance according to paired t test (***: P<0.001).
Mentions: In order to assess the ability of MAH strains to inhibit apoptosis, CASP3/CASP7 assays were performed. As shown in figure 3, at 6 and 24 h p.i. significantly decreased caspase activity was observed in all infected samples compared to the non-infected controls. However, at 48 h p.i. MDMs infected with all three MAH strains showed significantly decreased caspase activity compared to the non-infected as well as E. coli K12 stimulated controls (P<0.001, paired t test), while no significant difference was detected between the latter. Accordingly, lower caspase expression was detected in qRT-PCR experiments (figure 4 A). Based on the observation that the employed MAH strains are able to inhibit apoptosis, we considered mainly genes in the qRT-PCR experiments being relevant to apoptosis and cytokine-cytokine receptor interaction. For visualisation of the comprehensive and individual expression data of selected genes, heat maps of log 2 ratios of infected samples and non-treated controls were created for each point in time. As shown in figure 4 A, MAH infections caused a unique response of MDMs compared to the E. coli K12 stimulation. The detailed expression analysis of selected genes showed on the one hand some donor specific differences in response to the MAH infection. For example, all MAH strains caused increased expression of IFNGR1 in donor 2 right after 6 h compared to the other samples (P<0.002, unpaired t test). The up-regulation of IFNGR1 appeared after 24 h in all samples followed by decreased expression after 48 h only in donor 3. Accordingly, we observed sustained and consistent induction of TNF receptor superfamily (TNFRSF) 6 and TNFRSF10C after 24 h by all MAH strains in donor 1 compared to the other donors (figure 4).

Bottom Line: We furthermore outlined a regulatory network of potential interactions between microRNAs and mRNAs.This study provides a theoretical concept for unveiling how distinct mycobacteria could manipulate host cell response.In addition, functional relevance was confirmed by uncovering the control of major caspases 3 and 7 by let-7e and miR-29a, respectively.

View Article: PubMed Central - PubMed

Affiliation: Institute of Veterinary Biochemistry, Freie Universitaet Berlin, Berlin, Germany. sharbati@zedat.fu-berlin.de

ABSTRACT

Background: Many efforts have been made to understand basal mechanisms of mycobacterial infections. Macrophages are the first line of host immune defence to encounter and eradicate mycobacteria. Pathogenic species have evolved different mechanisms to evade host response, e.g. by influencing macrophage apoptotic pathways. However, the underlying molecular regulation is not fully understood. A new layer of eukaryotic regulation of gene expression is constituted by microRNAs. Therefore, we present a comprehensive study for identification of these key regulators and their targets in the context of host macrophage response to mycobacterial infections.

Methodology/principal findings: We performed microRNA as well as mRNA expression analysis of human monocyte derived macrophages infected with several Mycobacterium avium hominissuis strains by means of microarrays as well as quantitative reverse transcription PCR (qRT-PCR). The data revealed the ability of all strains to inhibit apoptosis by transcriptional regulation of BCL2 family members. Accordingly, at 48 h after infection macrophages infected with all M. avium strains showed significantly decreased caspase 3 and 7 activities compared to the controls. Expression of let-7e, miR-29a and miR-886-5p were increased in response to mycobacterial infection at 48 h. The integrated analysis of microRNA and mRNA expression as well as target prediction pointed out regulative networks identifying caspase 3 and 7 as potential targets of let-7e and miR-29a, respectively. Consecutive reporter assays verified the regulation of caspase 3 and 7 by these microRNAs.

Conclusions/significance: We show for the first time that mycobacterial infection of human macrophages causes a specific microRNA response. We furthermore outlined a regulatory network of potential interactions between microRNAs and mRNAs. This study provides a theoretical concept for unveiling how distinct mycobacteria could manipulate host cell response. In addition, functional relevance was confirmed by uncovering the control of major caspases 3 and 7 by let-7e and miR-29a, respectively.

Show MeSH
Related in: MedlinePlus