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TLR2-dependent inhibition of macrophage responses to IFN-gamma is mediated by distinct, gene-specific mechanisms.

Benson SA, Ernst JD - PLoS ONE (2009)

Bottom Line: Surface expression of MHC class II and secretion of CXCL11 were greatly reduced as well, indicating that the reduction in transcripts had downstream effects.Histone H3 and H4 acetylation was reduced at the CIITA promoter but unaffected at the CXCL11 promoter.Taken together, these results indicate that TLR2-dependent inhibition of IFN-gamma-induced gene expression is mediated by distinct, gene-specific mechanisms that disrupt binding of the transcriptional machinery to the promoters.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, Division of Infectious Diseases, New York University School of Medicine, New York, New York, United States of America.

ABSTRACT
Mycobacterium tuberculosis uses multiple mechanisms to avoid elimination by the immune system. We have previously shown that M. tuberculosis can inhibit selected macrophage responses to IFN-gamma through TLR2-dependent and -independent mechanisms. To specifically address the role of TLR2 signaling in mediating this inhibition, we stimulated macrophages with the specific TLR2/1 ligand Pam(3)CSK(4) and assayed responses to IFN-gamma. Pam(3)CSK(4) stimulation prior to IFN-gamma inhibited transcription of the unrelated IFN-gamma-inducible genes, CIITA and CXCL11. Surface expression of MHC class II and secretion of CXCL11 were greatly reduced as well, indicating that the reduction in transcripts had downstream effects. Inhibition of both genes required new protein synthesis. Using chromatin immunoprecipitation, we found that TLR2 stimulation inhibited IFN-gamma-induced RNA polymerase II binding to the CIITA and CXCL11 promoters. Furthermore, TATA binding protein was unable to bind the TATA box of the CXCL11 promoter, suggesting that assembly of transcriptional machinery was disrupted. However, TLR2 stimulation affected chromatin modifications differently at each of the inhibited promoters. Histone H3 and H4 acetylation was reduced at the CIITA promoter but unaffected at the CXCL11 promoter. In addition, NF-kappaB signaling was required for inhibition of CXCL11 transcription, but not for inhibition of CIITA. Taken together, these results indicate that TLR2-dependent inhibition of IFN-gamma-induced gene expression is mediated by distinct, gene-specific mechanisms that disrupt binding of the transcriptional machinery to the promoters.

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TLR2 stimulation prevents binding of general transcriptional machinery to the CIITA and CXCL11 promoters.BMDM were treated with 10 ng/ml Pam3CSK4 for 8–9 h, then 20 ng/ml IFN-γ for 4 h. Cross-linked DNA was sheared and immunoprecipitated with anti-PolII (A) or anti-TBP (B) antibodies. Precipitated and input DNA for each sample were assayed by qPCR with primers specific for the transcriptional start site in the promoters of CXCL11, CIITA, and NOS2 (A) or the TATA box of the CXCL11 promoter (B). All values were normalized to GAPDH. Results are expressed as fold increase over untreated controls and are the mean of triplicate samples±SD. Statistical significance between IFN-γ alone samples and Pam3CSK4 prior to IFN-γ treated samples was determined by two-tailed t-test. Results are representative of at least two independent experiments.
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pone-0006329-g004: TLR2 stimulation prevents binding of general transcriptional machinery to the CIITA and CXCL11 promoters.BMDM were treated with 10 ng/ml Pam3CSK4 for 8–9 h, then 20 ng/ml IFN-γ for 4 h. Cross-linked DNA was sheared and immunoprecipitated with anti-PolII (A) or anti-TBP (B) antibodies. Precipitated and input DNA for each sample were assayed by qPCR with primers specific for the transcriptional start site in the promoters of CXCL11, CIITA, and NOS2 (A) or the TATA box of the CXCL11 promoter (B). All values were normalized to GAPDH. Results are expressed as fold increase over untreated controls and are the mean of triplicate samples±SD. Statistical significance between IFN-γ alone samples and Pam3CSK4 prior to IFN-γ treated samples was determined by two-tailed t-test. Results are representative of at least two independent experiments.

Mentions: To further characterize the mechanism of inhibition of transcriptional responses to IFN-γ by prior TLR2 stimulation, we determined whether RNA polymerase II (PolII) binds the promoters of the affected genes. We treated BMDM with Pam3CSK4 for 8 h then stimulated with IFN-γ for 4 h, followed by chromatin immunoprecipitation (ChIP) to assay binding of PolII to the CXCL11, CIITA, and NOS2 promoters. Primers that specifically amplified regions flanking the transcriptional start site of each gene were designed to detect initial binding of PolII to the promoters. IFN-γ stimulation induced binding of PolII to the CXCL11 and CIITA promoters, as indicated by a three-fold increase in pulldown of promoter fragments of these genes (Fig. 4A). However, stimulation of TLR2 prior to IFN-γ inhibited binding of PolII by 66% (CXCL11) and 76% (CIITA) compared with IFN-γ alone. The effect of TLR2 stimulation was not merely to delay IFN-γ-stimulated PolII binding at these promoters, as similar inhibition of binding was seen after 8 and 12 h of IFN-γ stimulation (data not shown). As a control, we assayed PolII binding at the NOS2 promoter, since TLR2 stimulation did not decrease NOS2 mRNA induction by IFN-γ (Fig. 1C). IFN-γ stimulation alone caused a two-fold increase in binding and prior Pam3CSK4 treatment resulted in a further increase in binding (Fig. 4A). This was similar to the effect observed at the level of transcription (Fig. 1C). These results indicate that TLR2 stimulation prevents PolII from binding to the CXCL11 and CIITA promoters, but does not affect binding to the NOS2 promoter. The effect of TLR2 stimulation on transcription of IFN-γ-responsive genes correlates with PolII binding at the respective promoters.


TLR2-dependent inhibition of macrophage responses to IFN-gamma is mediated by distinct, gene-specific mechanisms.

Benson SA, Ernst JD - PLoS ONE (2009)

TLR2 stimulation prevents binding of general transcriptional machinery to the CIITA and CXCL11 promoters.BMDM were treated with 10 ng/ml Pam3CSK4 for 8–9 h, then 20 ng/ml IFN-γ for 4 h. Cross-linked DNA was sheared and immunoprecipitated with anti-PolII (A) or anti-TBP (B) antibodies. Precipitated and input DNA for each sample were assayed by qPCR with primers specific for the transcriptional start site in the promoters of CXCL11, CIITA, and NOS2 (A) or the TATA box of the CXCL11 promoter (B). All values were normalized to GAPDH. Results are expressed as fold increase over untreated controls and are the mean of triplicate samples±SD. Statistical significance between IFN-γ alone samples and Pam3CSK4 prior to IFN-γ treated samples was determined by two-tailed t-test. Results are representative of at least two independent experiments.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2710511&req=5

pone-0006329-g004: TLR2 stimulation prevents binding of general transcriptional machinery to the CIITA and CXCL11 promoters.BMDM were treated with 10 ng/ml Pam3CSK4 for 8–9 h, then 20 ng/ml IFN-γ for 4 h. Cross-linked DNA was sheared and immunoprecipitated with anti-PolII (A) or anti-TBP (B) antibodies. Precipitated and input DNA for each sample were assayed by qPCR with primers specific for the transcriptional start site in the promoters of CXCL11, CIITA, and NOS2 (A) or the TATA box of the CXCL11 promoter (B). All values were normalized to GAPDH. Results are expressed as fold increase over untreated controls and are the mean of triplicate samples±SD. Statistical significance between IFN-γ alone samples and Pam3CSK4 prior to IFN-γ treated samples was determined by two-tailed t-test. Results are representative of at least two independent experiments.
Mentions: To further characterize the mechanism of inhibition of transcriptional responses to IFN-γ by prior TLR2 stimulation, we determined whether RNA polymerase II (PolII) binds the promoters of the affected genes. We treated BMDM with Pam3CSK4 for 8 h then stimulated with IFN-γ for 4 h, followed by chromatin immunoprecipitation (ChIP) to assay binding of PolII to the CXCL11, CIITA, and NOS2 promoters. Primers that specifically amplified regions flanking the transcriptional start site of each gene were designed to detect initial binding of PolII to the promoters. IFN-γ stimulation induced binding of PolII to the CXCL11 and CIITA promoters, as indicated by a three-fold increase in pulldown of promoter fragments of these genes (Fig. 4A). However, stimulation of TLR2 prior to IFN-γ inhibited binding of PolII by 66% (CXCL11) and 76% (CIITA) compared with IFN-γ alone. The effect of TLR2 stimulation was not merely to delay IFN-γ-stimulated PolII binding at these promoters, as similar inhibition of binding was seen after 8 and 12 h of IFN-γ stimulation (data not shown). As a control, we assayed PolII binding at the NOS2 promoter, since TLR2 stimulation did not decrease NOS2 mRNA induction by IFN-γ (Fig. 1C). IFN-γ stimulation alone caused a two-fold increase in binding and prior Pam3CSK4 treatment resulted in a further increase in binding (Fig. 4A). This was similar to the effect observed at the level of transcription (Fig. 1C). These results indicate that TLR2 stimulation prevents PolII from binding to the CXCL11 and CIITA promoters, but does not affect binding to the NOS2 promoter. The effect of TLR2 stimulation on transcription of IFN-γ-responsive genes correlates with PolII binding at the respective promoters.

Bottom Line: Surface expression of MHC class II and secretion of CXCL11 were greatly reduced as well, indicating that the reduction in transcripts had downstream effects.Histone H3 and H4 acetylation was reduced at the CIITA promoter but unaffected at the CXCL11 promoter.Taken together, these results indicate that TLR2-dependent inhibition of IFN-gamma-induced gene expression is mediated by distinct, gene-specific mechanisms that disrupt binding of the transcriptional machinery to the promoters.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, Division of Infectious Diseases, New York University School of Medicine, New York, New York, United States of America.

ABSTRACT
Mycobacterium tuberculosis uses multiple mechanisms to avoid elimination by the immune system. We have previously shown that M. tuberculosis can inhibit selected macrophage responses to IFN-gamma through TLR2-dependent and -independent mechanisms. To specifically address the role of TLR2 signaling in mediating this inhibition, we stimulated macrophages with the specific TLR2/1 ligand Pam(3)CSK(4) and assayed responses to IFN-gamma. Pam(3)CSK(4) stimulation prior to IFN-gamma inhibited transcription of the unrelated IFN-gamma-inducible genes, CIITA and CXCL11. Surface expression of MHC class II and secretion of CXCL11 were greatly reduced as well, indicating that the reduction in transcripts had downstream effects. Inhibition of both genes required new protein synthesis. Using chromatin immunoprecipitation, we found that TLR2 stimulation inhibited IFN-gamma-induced RNA polymerase II binding to the CIITA and CXCL11 promoters. Furthermore, TATA binding protein was unable to bind the TATA box of the CXCL11 promoter, suggesting that assembly of transcriptional machinery was disrupted. However, TLR2 stimulation affected chromatin modifications differently at each of the inhibited promoters. Histone H3 and H4 acetylation was reduced at the CIITA promoter but unaffected at the CXCL11 promoter. In addition, NF-kappaB signaling was required for inhibition of CXCL11 transcription, but not for inhibition of CIITA. Taken together, these results indicate that TLR2-dependent inhibition of IFN-gamma-induced gene expression is mediated by distinct, gene-specific mechanisms that disrupt binding of the transcriptional machinery to the promoters.

Show MeSH
Related in: MedlinePlus