Limits...
Epigenetic regulation of human β-defensin 2 and CC chemokine ligand 20 expression in gingival epithelial cells in response to oral bacteria.

Yin L, Chung WO - Mucosal Immunol (2011)

Bottom Line: Pretreatment with trichostatin A and sodium butyrate, which increase acetylation of chromatin histones, significantly enhanced the gene expression of antimicrobial proteins human β-defensin 2 (hBD2) and CC chemokine ligand 20 (CCL20) in response to both bacterial challenges.Furthermore, we observed a differential pattern of protein levels of H3K4me3, which has been associated with chromatin remodeling and activation of gene transcription, in response to P. gingivalis vs.F. nucleatum.

View Article: PubMed Central - PubMed

Affiliation: Department of Oral Biology, University of Washington, Seattle, Washington, USA. leiyin@u.washington.edu

ABSTRACT
Gingival epithelia utilize multiple signaling pathways to regulate innate immune responses to various oral bacteria, but little is understood about how these bacteria alter epithelial epigenetic status. In this study we report that DNA methyltransferase (DNMT1) and histone deacetylase expression were decreased in gingival epithelial cells treated with oral pathogen Porphyromonas gingivalis and nonpathogen Fusobacterium nucleatum. Pretreatment with trichostatin A and sodium butyrate, which increase acetylation of chromatin histones, significantly enhanced the gene expression of antimicrobial proteins human β-defensin 2 (hBD2) and CC chemokine ligand 20 (CCL20) in response to both bacterial challenges. Pretreatment with DNMT inhibitor 5'-azacytidine increased hBD2 and CCL20 expression in response to F. nucleatum, but not to P. gingivalis. Furthermore, we observed a differential pattern of protein levels of H3K4me3, which has been associated with chromatin remodeling and activation of gene transcription, in response to P. gingivalis vs. F. nucleatum. This study provides a new insight into the bacteria-specific innate immune responses via epigenetic regulation.

Show MeSH

Related in: MedlinePlus

mRNA expression of innate immune markers human β-defensin 2 (hBD-2) are increased when histone deacetylase (HDAC) and DNA methyltransferase (DNMT) are inhibited. Gingival epithelial cells (GECs) were pretreated with trichostatin A (TSA; 9 and 45 m), sodium butyrate (SB; 0.5 and 2.0 m) or 5′-azacytidine (AZA; 1 and 10 μ) for 4 h, and subsequently exposed to Porphyromonas gingivalis (multiplicity of infection (MOI) 100:1) or Fusobacterium nucleatum (MOI 100:1) for 16 h. Gene expression of hBD2 was evaluated by quantitative real-time PCR (QRT-PCR) compared with unstimulated control after normalization with glyceraldehydes-3-phosphate dehydrogenase (GAPDH). Controls include unstimulated control, bacteria-alone treatment, and various inhibitors alone as indicated. No significant changes were found in the gene expression of hBD2 in GECs treated with inhibitor only: (a) SB, (b) TSA, (c) SB+TSA, and (d) AZA compared with unstimulated control. Data are expressed as means of fold change±s.e.m. from three donors evaluated in duplicate. Asterisks indicate statistically significant difference compared with unstimulated control (Ctl) (*P<0.05, **P<0.01).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3118861&req=5

fig4: mRNA expression of innate immune markers human β-defensin 2 (hBD-2) are increased when histone deacetylase (HDAC) and DNA methyltransferase (DNMT) are inhibited. Gingival epithelial cells (GECs) were pretreated with trichostatin A (TSA; 9 and 45 m), sodium butyrate (SB; 0.5 and 2.0 m) or 5′-azacytidine (AZA; 1 and 10 μ) for 4 h, and subsequently exposed to Porphyromonas gingivalis (multiplicity of infection (MOI) 100:1) or Fusobacterium nucleatum (MOI 100:1) for 16 h. Gene expression of hBD2 was evaluated by quantitative real-time PCR (QRT-PCR) compared with unstimulated control after normalization with glyceraldehydes-3-phosphate dehydrogenase (GAPDH). Controls include unstimulated control, bacteria-alone treatment, and various inhibitors alone as indicated. No significant changes were found in the gene expression of hBD2 in GECs treated with inhibitor only: (a) SB, (b) TSA, (c) SB+TSA, and (d) AZA compared with unstimulated control. Data are expressed as means of fold change±s.e.m. from three donors evaluated in duplicate. Asterisks indicate statistically significant difference compared with unstimulated control (Ctl) (*P<0.05, **P<0.01).

Mentions: β-Defensins and CCL20 have been known as critical markers of innate immune responses against microbial invasion.15, 16 We previously reported that P. gingivalis and F. nucleatum are all excellent inducers of hBD-213 and CCL20 (also known as MIP3α (macrophage inflammatory protein-3α)).17 In order to study the downstream response of innate immune markers following epigenetic modifications, we tested the effect of the inhibitors for chromatin-modifying enzymes followed by bacterial stimulation on the expression of the antimicrobial proteins hBD-2 and CCL20. GECs were pretreated with HDAC inhibitors sodium butyrate (SB) or trichostatin A (TSA), or DNMT inhibitor 5′-azacytidine (AZA) before bacterial exposure. Treatment of GECs with AZA demethylates DNA, leading to gene activation, whereas exposure to SB or TSA results in a higher level of histone acetylation. The gene expression of hBD2 and CCL20 was significantly increased in GECs in response to both P. gingivalis and F. nucleatum, which is consistent with our previous report (Figures 4 and 5).13, 18 Treatment with inhibitors alone did not significantly change expression of hBD2 and CCL20 in GECs. The inhibitor SB had no effect on the gene expression of hBD2 and CCL20 in response to these oral bacteria compared with relative bacterial treatment alone. Treatment with TSA, another HDAC inhibitor structurally unrelated to SB, resulted in significantly increased hBD2 and CCL20 gene expression in GECs in response to both oral bacteria (Figures 4b and 5b) compared with the bacterial treatment alone. Combining inhibitors SB and TSA synergistically enhanced CCL20 expression in GECs (Figure 5c). Treatment with HDAC inhibitors amplified hBD2 and CCL20 gene expression, thus suggesting that increasing histone acetylation will aid innate immune responses in GECs in the presence of oral bacteria. Treatment with AZA significantly increased gene expression of hBD2 and CCL20 in GECS in response to F. nucleatum, but not to P. gingivalis (Figures 4d and 5d). Our data suggest that the oral bridging microorganism F. nucleatum induced hBD2 and CCL20 via both demethylation and acetylation mechanisms, whereas the induction by oral pathogen P. gingivalis is only via acetylation mechanism. Thus, taken together, our data suggest that epithelial innate immune responses (hBD2 and CCL20) are regulated by epigenetic modifications, and these responses are bacteria specific.


Epigenetic regulation of human β-defensin 2 and CC chemokine ligand 20 expression in gingival epithelial cells in response to oral bacteria.

Yin L, Chung WO - Mucosal Immunol (2011)

mRNA expression of innate immune markers human β-defensin 2 (hBD-2) are increased when histone deacetylase (HDAC) and DNA methyltransferase (DNMT) are inhibited. Gingival epithelial cells (GECs) were pretreated with trichostatin A (TSA; 9 and 45 m), sodium butyrate (SB; 0.5 and 2.0 m) or 5′-azacytidine (AZA; 1 and 10 μ) for 4 h, and subsequently exposed to Porphyromonas gingivalis (multiplicity of infection (MOI) 100:1) or Fusobacterium nucleatum (MOI 100:1) for 16 h. Gene expression of hBD2 was evaluated by quantitative real-time PCR (QRT-PCR) compared with unstimulated control after normalization with glyceraldehydes-3-phosphate dehydrogenase (GAPDH). Controls include unstimulated control, bacteria-alone treatment, and various inhibitors alone as indicated. No significant changes were found in the gene expression of hBD2 in GECs treated with inhibitor only: (a) SB, (b) TSA, (c) SB+TSA, and (d) AZA compared with unstimulated control. Data are expressed as means of fold change±s.e.m. from three donors evaluated in duplicate. Asterisks indicate statistically significant difference compared with unstimulated control (Ctl) (*P<0.05, **P<0.01).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3118861&req=5

fig4: mRNA expression of innate immune markers human β-defensin 2 (hBD-2) are increased when histone deacetylase (HDAC) and DNA methyltransferase (DNMT) are inhibited. Gingival epithelial cells (GECs) were pretreated with trichostatin A (TSA; 9 and 45 m), sodium butyrate (SB; 0.5 and 2.0 m) or 5′-azacytidine (AZA; 1 and 10 μ) for 4 h, and subsequently exposed to Porphyromonas gingivalis (multiplicity of infection (MOI) 100:1) or Fusobacterium nucleatum (MOI 100:1) for 16 h. Gene expression of hBD2 was evaluated by quantitative real-time PCR (QRT-PCR) compared with unstimulated control after normalization with glyceraldehydes-3-phosphate dehydrogenase (GAPDH). Controls include unstimulated control, bacteria-alone treatment, and various inhibitors alone as indicated. No significant changes were found in the gene expression of hBD2 in GECs treated with inhibitor only: (a) SB, (b) TSA, (c) SB+TSA, and (d) AZA compared with unstimulated control. Data are expressed as means of fold change±s.e.m. from three donors evaluated in duplicate. Asterisks indicate statistically significant difference compared with unstimulated control (Ctl) (*P<0.05, **P<0.01).
Mentions: β-Defensins and CCL20 have been known as critical markers of innate immune responses against microbial invasion.15, 16 We previously reported that P. gingivalis and F. nucleatum are all excellent inducers of hBD-213 and CCL20 (also known as MIP3α (macrophage inflammatory protein-3α)).17 In order to study the downstream response of innate immune markers following epigenetic modifications, we tested the effect of the inhibitors for chromatin-modifying enzymes followed by bacterial stimulation on the expression of the antimicrobial proteins hBD-2 and CCL20. GECs were pretreated with HDAC inhibitors sodium butyrate (SB) or trichostatin A (TSA), or DNMT inhibitor 5′-azacytidine (AZA) before bacterial exposure. Treatment of GECs with AZA demethylates DNA, leading to gene activation, whereas exposure to SB or TSA results in a higher level of histone acetylation. The gene expression of hBD2 and CCL20 was significantly increased in GECs in response to both P. gingivalis and F. nucleatum, which is consistent with our previous report (Figures 4 and 5).13, 18 Treatment with inhibitors alone did not significantly change expression of hBD2 and CCL20 in GECs. The inhibitor SB had no effect on the gene expression of hBD2 and CCL20 in response to these oral bacteria compared with relative bacterial treatment alone. Treatment with TSA, another HDAC inhibitor structurally unrelated to SB, resulted in significantly increased hBD2 and CCL20 gene expression in GECs in response to both oral bacteria (Figures 4b and 5b) compared with the bacterial treatment alone. Combining inhibitors SB and TSA synergistically enhanced CCL20 expression in GECs (Figure 5c). Treatment with HDAC inhibitors amplified hBD2 and CCL20 gene expression, thus suggesting that increasing histone acetylation will aid innate immune responses in GECs in the presence of oral bacteria. Treatment with AZA significantly increased gene expression of hBD2 and CCL20 in GECS in response to F. nucleatum, but not to P. gingivalis (Figures 4d and 5d). Our data suggest that the oral bridging microorganism F. nucleatum induced hBD2 and CCL20 via both demethylation and acetylation mechanisms, whereas the induction by oral pathogen P. gingivalis is only via acetylation mechanism. Thus, taken together, our data suggest that epithelial innate immune responses (hBD2 and CCL20) are regulated by epigenetic modifications, and these responses are bacteria specific.

Bottom Line: Pretreatment with trichostatin A and sodium butyrate, which increase acetylation of chromatin histones, significantly enhanced the gene expression of antimicrobial proteins human β-defensin 2 (hBD2) and CC chemokine ligand 20 (CCL20) in response to both bacterial challenges.Furthermore, we observed a differential pattern of protein levels of H3K4me3, which has been associated with chromatin remodeling and activation of gene transcription, in response to P. gingivalis vs.F. nucleatum.

View Article: PubMed Central - PubMed

Affiliation: Department of Oral Biology, University of Washington, Seattle, Washington, USA. leiyin@u.washington.edu

ABSTRACT
Gingival epithelia utilize multiple signaling pathways to regulate innate immune responses to various oral bacteria, but little is understood about how these bacteria alter epithelial epigenetic status. In this study we report that DNA methyltransferase (DNMT1) and histone deacetylase expression were decreased in gingival epithelial cells treated with oral pathogen Porphyromonas gingivalis and nonpathogen Fusobacterium nucleatum. Pretreatment with trichostatin A and sodium butyrate, which increase acetylation of chromatin histones, significantly enhanced the gene expression of antimicrobial proteins human β-defensin 2 (hBD2) and CC chemokine ligand 20 (CCL20) in response to both bacterial challenges. Pretreatment with DNMT inhibitor 5'-azacytidine increased hBD2 and CCL20 expression in response to F. nucleatum, but not to P. gingivalis. Furthermore, we observed a differential pattern of protein levels of H3K4me3, which has been associated with chromatin remodeling and activation of gene transcription, in response to P. gingivalis vs. F. nucleatum. This study provides a new insight into the bacteria-specific innate immune responses via epigenetic regulation.

Show MeSH
Related in: MedlinePlus