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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-mediated inhibition of IFN-γ induction of CXCL11 and CIITA decreases expression of protein products.A. BALB/c BMDM were treated with 10 ng/ml Pam3CSK4 for 8 h followed by 20 ng/ml IFN-γ for 4, 8, and 12 h. Culture supernatants were collected and assayed for CXCL11 protein by ELISA. *, p<0.01, **, p<0.05 comparing IFN-γ alone with Pam3CSK4 and IFN-γ treated samples (as determined by two-tailed t-test). B and C. BMDM were treated with 10 ng/ml Pam3CSK4 for 12–15 h prior to stimulation with 20 ng/ml IFN-γ for 24 h. Cells were stained with Alexa 647-conjugated anti-mouse I-A/I-E and analyzed by flow cytometry. Data shown are fluorescence intensity vs. cell number (B) and mean I-A/I-E fluorescence (C). Results are expressed as means±SEM from two independent experiments (A) and are representative of at least five independent experiments (B and C).
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pone-0006329-g002: TLR2-mediated inhibition of IFN-γ induction of CXCL11 and CIITA decreases expression of protein products.A. BALB/c BMDM were treated with 10 ng/ml Pam3CSK4 for 8 h followed by 20 ng/ml IFN-γ for 4, 8, and 12 h. Culture supernatants were collected and assayed for CXCL11 protein by ELISA. *, p<0.01, **, p<0.05 comparing IFN-γ alone with Pam3CSK4 and IFN-γ treated samples (as determined by two-tailed t-test). B and C. BMDM were treated with 10 ng/ml Pam3CSK4 for 12–15 h prior to stimulation with 20 ng/ml IFN-γ for 24 h. Cells were stained with Alexa 647-conjugated anti-mouse I-A/I-E and analyzed by flow cytometry. Data shown are fluorescence intensity vs. cell number (B) and mean I-A/I-E fluorescence (C). Results are expressed as means±SEM from two independent experiments (A) and are representative of at least five independent experiments (B and C).

Mentions: Since transcription of CXCL11 and CIITA was significantly reduced in TLR2 stimulated macrophages, we determined whether inhibition was reflected by reduction of the protein products of these genes. To examine the effect on CXCL11, we treated BALB/c BMDM with Pam3CSK4 for 8 h followed by IFN-γ for 4, 8, and 12 h. Culture supernatants were harvested and assayed for CXCL11 by ELISA. Stimulation with IFN-γ resulted in secretion of CXCL11 after 8 and 12 h of treatment (Fig. 2A). However, prior TLR2 stimulation inhibited IFN-γ-induced CXCL11 protein levels by over 80% at both of these time points.


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

Benson SA, Ernst JD - PLoS ONE (2009)

TLR2-mediated inhibition of IFN-γ induction of CXCL11 and CIITA decreases expression of protein products.A. BALB/c BMDM were treated with 10 ng/ml Pam3CSK4 for 8 h followed by 20 ng/ml IFN-γ for 4, 8, and 12 h. Culture supernatants were collected and assayed for CXCL11 protein by ELISA. *, p<0.01, **, p<0.05 comparing IFN-γ alone with Pam3CSK4 and IFN-γ treated samples (as determined by two-tailed t-test). B and C. BMDM were treated with 10 ng/ml Pam3CSK4 for 12–15 h prior to stimulation with 20 ng/ml IFN-γ for 24 h. Cells were stained with Alexa 647-conjugated anti-mouse I-A/I-E and analyzed by flow cytometry. Data shown are fluorescence intensity vs. cell number (B) and mean I-A/I-E fluorescence (C). Results are expressed as means±SEM from two independent experiments (A) and are representative of at least five independent experiments (B and C).
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pone-0006329-g002: TLR2-mediated inhibition of IFN-γ induction of CXCL11 and CIITA decreases expression of protein products.A. BALB/c BMDM were treated with 10 ng/ml Pam3CSK4 for 8 h followed by 20 ng/ml IFN-γ for 4, 8, and 12 h. Culture supernatants were collected and assayed for CXCL11 protein by ELISA. *, p<0.01, **, p<0.05 comparing IFN-γ alone with Pam3CSK4 and IFN-γ treated samples (as determined by two-tailed t-test). B and C. BMDM were treated with 10 ng/ml Pam3CSK4 for 12–15 h prior to stimulation with 20 ng/ml IFN-γ for 24 h. Cells were stained with Alexa 647-conjugated anti-mouse I-A/I-E and analyzed by flow cytometry. Data shown are fluorescence intensity vs. cell number (B) and mean I-A/I-E fluorescence (C). Results are expressed as means±SEM from two independent experiments (A) and are representative of at least five independent experiments (B and C).
Mentions: Since transcription of CXCL11 and CIITA was significantly reduced in TLR2 stimulated macrophages, we determined whether inhibition was reflected by reduction of the protein products of these genes. To examine the effect on CXCL11, we treated BALB/c BMDM with Pam3CSK4 for 8 h followed by IFN-γ for 4, 8, and 12 h. Culture supernatants were harvested and assayed for CXCL11 by ELISA. Stimulation with IFN-γ resulted in secretion of CXCL11 after 8 and 12 h of treatment (Fig. 2A). However, prior TLR2 stimulation inhibited IFN-γ-induced CXCL11 protein levels by over 80% at both of these time points.

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