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STAT1-dependent signal integration between IFNγ and TLR4 in vascular cells reflect pro-atherogenic responses in human atherosclerosis.

Chmielewski S, Olejnik A, Sikorski K, Pelisek J, Błaszczyk K, Aoqui C, Nowicka H, Zernecke A, Heemann U, Wesoly J, Baumann M, Bluyssen HA - PLoS ONE (2014)

Bottom Line: The synergistic interactions between IFNγ and TLR4 also resulted in increased T-cell migration and impaired aortic contractility in a STAT1-dependent manner.Expression of the chemokines CXCL9 and CXCL10 correlated with STAT1 phosphorylation in vascular cells in plaques from human carotid arteries.Moreover, using data mining of human plaque transcriptomes, expression of a selection of these STAT1-dependent pro-atherogenic genes was found to be increased in coronary artery disease (CAD) and carotid atherosclerosis.

View Article: PubMed Central - PubMed

Affiliation: Department of Nephrology, Klinikum rechts der Isar, Technical University Munich, Munich, Germany; Department of Human Molecular Genetics, Institute of Molecular Biology and Biotechnology, Faculty of Biology, Adam Mickiewicz University Poznan, Poznan, Poland.

ABSTRACT
Signal integration between IFNγ and TLRs in immune cells has been associated with the host defense against pathogens and injury, with a predominant role of STAT1. We hypothesize that STAT1-dependent transcriptional changes in vascular cells involved in cross-talk between IFNγ and TLR4, reflect pro-atherogenic responses in human atherosclerosis. Genome-wide investigation identified a set of STAT1-dependent genes that were synergistically affected by interactions between IFNγ and TLR4 in VSMCs. These included the chemokines Cxcl9, Ccl12, Ccl8, Ccrl2, Cxcl10 and Ccl5, adhesion molecules Cd40, Cd74, and antiviral and antibacterial genes Rsad2, Mx1, Oasl1, Gbp5, Nos2, Batf2 and Tnfrsf11a. Among the amplified genes was also Irf8, of which Ccl5 was subsequently identified as a new pro-inflammatory target in VSMCs and ECs. Promoter analysis predicted transcriptional cooperation between STAT1, IRF1, IRF8 and NFκB, with the novel role of IRF8 providing an additional layer to the overall complexity. The synergistic interactions between IFNγ and TLR4 also resulted in increased T-cell migration and impaired aortic contractility in a STAT1-dependent manner. Expression of the chemokines CXCL9 and CXCL10 correlated with STAT1 phosphorylation in vascular cells in plaques from human carotid arteries. Moreover, using data mining of human plaque transcriptomes, expression of a selection of these STAT1-dependent pro-atherogenic genes was found to be increased in coronary artery disease (CAD) and carotid atherosclerosis. Our study provides evidence to suggest that in ECs and VSMCs STAT1 orchestrates a platform for cross-talk between IFNγ and TLR4, and identifies a STAT1-dependent gene signature that reflects a pro-atherogenic state in human atherosclerosis.

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Effect of STAT1 dependent signal integration on chemokine expression.WT and STAT1−/− VSMCs, HMECs or WT aortic ring segments were treated as described in Fig. 1. A, RNA from VSMCs was isolated and qRT-PCR for Ccl5, Cxcl9 using Gapdh as internal control was performed. B, On the medium remained after treatment of VSMCs ELISA for Ccl5 and Cxcl9 was performed. C, Expression of CXCL10, CXCL9 and CCL5 upon stimulation in ECs. D, RNA from incubated aortic rings was isolated and qRT-PCR for Cxcl10, Cxcl9 using Gapdh as internal control was performed. Data represent means of at least 3 independent biological experiments ±SEM and p<0.05 was considered as significant. Data were tested for significance by one-way ANOVA followed by post-hoc Tukey or unpaired two-tailed student T-test when appropriate. E, ChIP-qPCR analysis of the Cxcl10 promoter region containing NFκB and ISRE binding sites show the enrichment with STAT1, NFκB and IRF1 antibodies compared with IgG control in an IFNγ, LPS or IFNy+LPS-dependent manner in WT VSMCs. Immunoprecipitated DNA was quantified by qPCR and normalized to values obtained after amplification of unprecipitated (input) DNA. A representative experiment is shown.
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pone-0113318-g003: Effect of STAT1 dependent signal integration on chemokine expression.WT and STAT1−/− VSMCs, HMECs or WT aortic ring segments were treated as described in Fig. 1. A, RNA from VSMCs was isolated and qRT-PCR for Ccl5, Cxcl9 using Gapdh as internal control was performed. B, On the medium remained after treatment of VSMCs ELISA for Ccl5 and Cxcl9 was performed. C, Expression of CXCL10, CXCL9 and CCL5 upon stimulation in ECs. D, RNA from incubated aortic rings was isolated and qRT-PCR for Cxcl10, Cxcl9 using Gapdh as internal control was performed. Data represent means of at least 3 independent biological experiments ±SEM and p<0.05 was considered as significant. Data were tested for significance by one-way ANOVA followed by post-hoc Tukey or unpaired two-tailed student T-test when appropriate. E, ChIP-qPCR analysis of the Cxcl10 promoter region containing NFκB and ISRE binding sites show the enrichment with STAT1, NFκB and IRF1 antibodies compared with IgG control in an IFNγ, LPS or IFNy+LPS-dependent manner in WT VSMCs. Immunoprecipitated DNA was quantified by qPCR and normalized to values obtained after amplification of unprecipitated (input) DNA. A representative experiment is shown.

Mentions: The expression of the chemokines Ccl5, Cxcl9, Ccl12 and chemokine receptor Ccrl2 (not shown) was additionally examined by qPCR and ELISA (Fig. 3), and confirmed the microarray data. In agreement with Table 1, the response of this selected group of genes was severely abolished in STAT1−/−-VSMCs, confirming the importance of STAT1 in the signal integration between IFNγ and LPS. Because we were not able to isolate a homogeneous population of mouse aortic endothelial cells (data not shown), we instead used the human microvascular endothelial cell-line, HMEC [20]. Pre-treatment of HMECs with IFNγ for 4 h followed by LPS for another 4 h resulted in a similar amplification pattern of Ccl5, Cxcl9 and Cxcl10 (Fig. 3C) as in WT-VSMCs, providing evidence for a universal STAT1-dependent mechanism in vascular cells triggered by IFNγ and LPS. Similarly, we were able to observe a synergistic amplification after IFNγ and LPS treatment of Cxcl9 and Cxcl10 in ex vivo treated aortic rings of WT animals as compared to IFNγ or LPS alone (Fig. 3D)


STAT1-dependent signal integration between IFNγ and TLR4 in vascular cells reflect pro-atherogenic responses in human atherosclerosis.

Chmielewski S, Olejnik A, Sikorski K, Pelisek J, Błaszczyk K, Aoqui C, Nowicka H, Zernecke A, Heemann U, Wesoly J, Baumann M, Bluyssen HA - PLoS ONE (2014)

Effect of STAT1 dependent signal integration on chemokine expression.WT and STAT1−/− VSMCs, HMECs or WT aortic ring segments were treated as described in Fig. 1. A, RNA from VSMCs was isolated and qRT-PCR for Ccl5, Cxcl9 using Gapdh as internal control was performed. B, On the medium remained after treatment of VSMCs ELISA for Ccl5 and Cxcl9 was performed. C, Expression of CXCL10, CXCL9 and CCL5 upon stimulation in ECs. D, RNA from incubated aortic rings was isolated and qRT-PCR for Cxcl10, Cxcl9 using Gapdh as internal control was performed. Data represent means of at least 3 independent biological experiments ±SEM and p<0.05 was considered as significant. Data were tested for significance by one-way ANOVA followed by post-hoc Tukey or unpaired two-tailed student T-test when appropriate. E, ChIP-qPCR analysis of the Cxcl10 promoter region containing NFκB and ISRE binding sites show the enrichment with STAT1, NFκB and IRF1 antibodies compared with IgG control in an IFNγ, LPS or IFNy+LPS-dependent manner in WT VSMCs. Immunoprecipitated DNA was quantified by qPCR and normalized to values obtained after amplification of unprecipitated (input) DNA. A representative experiment is shown.
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pone-0113318-g003: Effect of STAT1 dependent signal integration on chemokine expression.WT and STAT1−/− VSMCs, HMECs or WT aortic ring segments were treated as described in Fig. 1. A, RNA from VSMCs was isolated and qRT-PCR for Ccl5, Cxcl9 using Gapdh as internal control was performed. B, On the medium remained after treatment of VSMCs ELISA for Ccl5 and Cxcl9 was performed. C, Expression of CXCL10, CXCL9 and CCL5 upon stimulation in ECs. D, RNA from incubated aortic rings was isolated and qRT-PCR for Cxcl10, Cxcl9 using Gapdh as internal control was performed. Data represent means of at least 3 independent biological experiments ±SEM and p<0.05 was considered as significant. Data were tested for significance by one-way ANOVA followed by post-hoc Tukey or unpaired two-tailed student T-test when appropriate. E, ChIP-qPCR analysis of the Cxcl10 promoter region containing NFκB and ISRE binding sites show the enrichment with STAT1, NFκB and IRF1 antibodies compared with IgG control in an IFNγ, LPS or IFNy+LPS-dependent manner in WT VSMCs. Immunoprecipitated DNA was quantified by qPCR and normalized to values obtained after amplification of unprecipitated (input) DNA. A representative experiment is shown.
Mentions: The expression of the chemokines Ccl5, Cxcl9, Ccl12 and chemokine receptor Ccrl2 (not shown) was additionally examined by qPCR and ELISA (Fig. 3), and confirmed the microarray data. In agreement with Table 1, the response of this selected group of genes was severely abolished in STAT1−/−-VSMCs, confirming the importance of STAT1 in the signal integration between IFNγ and LPS. Because we were not able to isolate a homogeneous population of mouse aortic endothelial cells (data not shown), we instead used the human microvascular endothelial cell-line, HMEC [20]. Pre-treatment of HMECs with IFNγ for 4 h followed by LPS for another 4 h resulted in a similar amplification pattern of Ccl5, Cxcl9 and Cxcl10 (Fig. 3C) as in WT-VSMCs, providing evidence for a universal STAT1-dependent mechanism in vascular cells triggered by IFNγ and LPS. Similarly, we were able to observe a synergistic amplification after IFNγ and LPS treatment of Cxcl9 and Cxcl10 in ex vivo treated aortic rings of WT animals as compared to IFNγ or LPS alone (Fig. 3D)

Bottom Line: The synergistic interactions between IFNγ and TLR4 also resulted in increased T-cell migration and impaired aortic contractility in a STAT1-dependent manner.Expression of the chemokines CXCL9 and CXCL10 correlated with STAT1 phosphorylation in vascular cells in plaques from human carotid arteries.Moreover, using data mining of human plaque transcriptomes, expression of a selection of these STAT1-dependent pro-atherogenic genes was found to be increased in coronary artery disease (CAD) and carotid atherosclerosis.

View Article: PubMed Central - PubMed

Affiliation: Department of Nephrology, Klinikum rechts der Isar, Technical University Munich, Munich, Germany; Department of Human Molecular Genetics, Institute of Molecular Biology and Biotechnology, Faculty of Biology, Adam Mickiewicz University Poznan, Poznan, Poland.

ABSTRACT
Signal integration between IFNγ and TLRs in immune cells has been associated with the host defense against pathogens and injury, with a predominant role of STAT1. We hypothesize that STAT1-dependent transcriptional changes in vascular cells involved in cross-talk between IFNγ and TLR4, reflect pro-atherogenic responses in human atherosclerosis. Genome-wide investigation identified a set of STAT1-dependent genes that were synergistically affected by interactions between IFNγ and TLR4 in VSMCs. These included the chemokines Cxcl9, Ccl12, Ccl8, Ccrl2, Cxcl10 and Ccl5, adhesion molecules Cd40, Cd74, and antiviral and antibacterial genes Rsad2, Mx1, Oasl1, Gbp5, Nos2, Batf2 and Tnfrsf11a. Among the amplified genes was also Irf8, of which Ccl5 was subsequently identified as a new pro-inflammatory target in VSMCs and ECs. Promoter analysis predicted transcriptional cooperation between STAT1, IRF1, IRF8 and NFκB, with the novel role of IRF8 providing an additional layer to the overall complexity. The synergistic interactions between IFNγ and TLR4 also resulted in increased T-cell migration and impaired aortic contractility in a STAT1-dependent manner. Expression of the chemokines CXCL9 and CXCL10 correlated with STAT1 phosphorylation in vascular cells in plaques from human carotid arteries. Moreover, using data mining of human plaque transcriptomes, expression of a selection of these STAT1-dependent pro-atherogenic genes was found to be increased in coronary artery disease (CAD) and carotid atherosclerosis. Our study provides evidence to suggest that in ECs and VSMCs STAT1 orchestrates a platform for cross-talk between IFNγ and TLR4, and identifies a STAT1-dependent gene signature that reflects a pro-atherogenic state in human atherosclerosis.

Show MeSH
Related in: MedlinePlus