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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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IRF8 mediated cross-talk and functional activity of synergistically amplified chemokines.WT, STAT1−/− and IRF8−/− VSMCs and HMECs were treated as described in Fig. 1. A, RNA was isolated and qRT-PCR for IRF8 using GAPDH as internal control was performed in VSMCs (left panel) and ECs (right panel). B, Protein extracts were analyzed for IRF8, tyrosine-phosphorylated STAT1, total STAT1 and GAPDH. C, CCL5 mRNA expression (left panel) and protein presence in the medium (right panel) was measured. D, Expression profiles of Cxcl9 (left panel) and Cxcl10 (right panel) between VSMCs WT, and IRF8−/− were compared. E, Migration assay of CD45+/CD3+ performed on conditioned medium remained after treatment of VSMCs WT and STAT1−/−. 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.
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pone-0113318-g004: IRF8 mediated cross-talk and functional activity of synergistically amplified chemokines.WT, STAT1−/− and IRF8−/− VSMCs and HMECs were treated as described in Fig. 1. A, RNA was isolated and qRT-PCR for IRF8 using GAPDH as internal control was performed in VSMCs (left panel) and ECs (right panel). B, Protein extracts were analyzed for IRF8, tyrosine-phosphorylated STAT1, total STAT1 and GAPDH. C, CCL5 mRNA expression (left panel) and protein presence in the medium (right panel) was measured. D, Expression profiles of Cxcl9 (left panel) and Cxcl10 (right panel) between VSMCs WT, and IRF8−/− were compared. E, Migration assay of CD45+/CD3+ performed on conditioned medium remained after treatment of VSMCs WT and STAT1−/−. 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.

Mentions: Notably, the transcription factor IRF8, which was thought to be restricted to lymphoid-cell lineages such as B-, T-, dendritic cells and macrophages, was identified among the amplified genes. Indeed, gene (Fig. 4A left panel) and protein expression (Fig. 4B left panel) of IRF8 in WT and STAT1−/− VSMCs in response to IFNγ, LPS or IFNγ+LPS, confirmed the microarray data. Interestingly, pre-treatment of ECs with IFNγ for 4 h followed by LPS for another 4 h resulted in a similar amplification pattern of IRF8 RNA (Fig. 4A right panel) and protein expression (Fig. 4B right panel) as in WT-VSMCs. These results provide evidence for STAT1-dependent expression of IRF8 in VSMCs and ECs upon treatment with IFNγ, and confirm amplification of IRF8 upon stimulation with IFNγ and LPS in vascular cells.


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)

IRF8 mediated cross-talk and functional activity of synergistically amplified chemokines.WT, STAT1−/− and IRF8−/− VSMCs and HMECs were treated as described in Fig. 1. A, RNA was isolated and qRT-PCR for IRF8 using GAPDH as internal control was performed in VSMCs (left panel) and ECs (right panel). B, Protein extracts were analyzed for IRF8, tyrosine-phosphorylated STAT1, total STAT1 and GAPDH. C, CCL5 mRNA expression (left panel) and protein presence in the medium (right panel) was measured. D, Expression profiles of Cxcl9 (left panel) and Cxcl10 (right panel) between VSMCs WT, and IRF8−/− were compared. E, Migration assay of CD45+/CD3+ performed on conditioned medium remained after treatment of VSMCs WT and STAT1−/−. 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.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4257532&req=5

pone-0113318-g004: IRF8 mediated cross-talk and functional activity of synergistically amplified chemokines.WT, STAT1−/− and IRF8−/− VSMCs and HMECs were treated as described in Fig. 1. A, RNA was isolated and qRT-PCR for IRF8 using GAPDH as internal control was performed in VSMCs (left panel) and ECs (right panel). B, Protein extracts were analyzed for IRF8, tyrosine-phosphorylated STAT1, total STAT1 and GAPDH. C, CCL5 mRNA expression (left panel) and protein presence in the medium (right panel) was measured. D, Expression profiles of Cxcl9 (left panel) and Cxcl10 (right panel) between VSMCs WT, and IRF8−/− were compared. E, Migration assay of CD45+/CD3+ performed on conditioned medium remained after treatment of VSMCs WT and STAT1−/−. 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.
Mentions: Notably, the transcription factor IRF8, which was thought to be restricted to lymphoid-cell lineages such as B-, T-, dendritic cells and macrophages, was identified among the amplified genes. Indeed, gene (Fig. 4A left panel) and protein expression (Fig. 4B left panel) of IRF8 in WT and STAT1−/− VSMCs in response to IFNγ, LPS or IFNγ+LPS, confirmed the microarray data. Interestingly, pre-treatment of ECs with IFNγ for 4 h followed by LPS for another 4 h resulted in a similar amplification pattern of IRF8 RNA (Fig. 4A right panel) and protein expression (Fig. 4B right panel) as in WT-VSMCs. These results provide evidence for STAT1-dependent expression of IRF8 in VSMCs and ECs upon treatment with IFNγ, and confirm amplification of IRF8 upon stimulation with IFNγ and LPS in vascular cells.

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