Epigenetic Regulation of Host Defense Peptide Synthesis: Synergy Between Histone Deacetylase Inhibitors and DNA/Histone Methyltransferase Inhibitors

Abstract

Host defense peptides (HDPs) are an integral part of the innate immune system acting as the first line of defense. Modulation of HDP synthesis has emerged as a promising host-directed approach to fight against infections. Inhibition of histone deacetylation or DNA methylation is known to enhance HDP gene expression. In this study, we explored a possible synergy in HDP gene induction between histone deacetylase inhibitors (HDACi) and DNA/histone methyltransferase inhibitors (DNMTi/HMTi). Two chicken macrophage cell lines were treated with structurally distinct HDACi, HMTi, or DNMTi individually or in combinations, followed by HDP gene expression analysis. Each epigenetic compound was found to be capable of inducing HDP expression. To our surprise, a combination of HDACi and HMTi or HDACi and DNMTi showed a strong synergy to induce the expressions of most HDP genes. The HDP-inducing synergy between butyrate, an HDACi, and BIX01294, an HMTi, were further verified in chicken peripheral blood mononuclear cells. Furthermore, tight junction proteins such as claudin 1 were also synergistically induced by HDACi and HMTi. Overall, we conclude that HDP genes are regulated by epigenetic modifications. Strategies to increase histone acetylation while reducing DNA or histone methylation exert a synergistic effect on HDP induction and, therefore, have potential for the control and prevention of infectious diseases.

Keywords: DNA methyltransferase inhibitors; epigenetic modification; histone deacetylase inhibitors; histone methyltransferase inhibitors; host defense peptides.

Figure 1

A list of histone methyltransferase inhibitors (HMTi), DNA methyltransferase inhibitors (DNMTi), and histone deacetylase inhibitors (HDACi) used in this study. BIX01294 and A-366 are G9a-specific HMTi, while UNC1999 is a specific EZH1/2 HMTi.

Figure 2

Concentration- and time-dependent induction of AvBD9 mRNA expression by HMTi or DNMTi. (A) Fold changes in relative luciferase activity in stable chicken HTC/AvBD9-luc luciferase reporter cells in response to indicated concentrations (μM) of each compound for 24 h. (B) Fold changes in the AvBD9 mRNA expression level in chicken HTC cells stimulated with indicated concentrations of each compound for 24 h. (C) Fold changes in the AvBD9 mRNA expression level in chicken HTC cells stimulated with 10 µM of each compound for indicated lengths of time. AvBD9 mRNA expression was analyzed using RT-qPCR. Results are shown as means ± SEM of three independent experiments. The bars not sharing a common superscript are considered significantly different (P < 0.05) based on one-way analysis of variance and post hoc Tukey’s test.

Figure 3

Synergistic induction of luciferase activity in HTC/AvBD9-luc cells between butyrate and an HMTi or a DNMTi. Chicken HTC/AvBD9-luc luciferase reporter cells were stimulated in duplicate with indicated concentrations (μM) of BIX01294 (A), UNC1999 (B), or SGI-1027 (C) individually or in combination with 2 mM butyrate for 24 h, followed by luciferase assay for the AvBD9 gene promoter activity. Results are shown as means ± SEM of three independent experiments. The bars not sharing a common superscript are considered significantly different (P < 0.05) based on one-way analysis of variance and post hoc Tukey’s test.

Figure 4

Synergistic induction of the AvBD9 mRNA expression in HTC cells between HMTi and butyrate or between DNMTi and butyrate. Chicken HTC cells were stimulated in duplicate with indicated concentrations (μM) of BIX01294 (A), UNC1999 (B), SGI-1027 (C), or 5-azacytidine (D) individually or in combination with 2 mM butyrate for 24 h, followed by RT-qPCR analysis of AvBD9 mRNA expression. Results are shown as means ± SEM of three independent experiments. The bars not sharing a common superscript are considered significantly different (P < 0.05) based on one-way analysis of variance and post hoc Tukey’s test.

Figure 5

Synergistic induction of multiple HDP mRNA expression in HTC cells between BIX01294 and butyrate. Chicken HTC cells were stimulated in duplicate with indicated concentrations (μM) of BIX01294 with or without 2 mM butyrate for 24 h, followed by RT-qPCR analysis of different chicken HDP genes. Results are shown as means ± SEM of two independent experiments. The treatments not sharing a common superscript are considered significantly different (P < 0.05) based on one-way analysis of variance and post hoc Tukey’s test.

Figure 6

Synergistic induction of the HDP mRNA expression in HD11 cells and peripheral blood mononuclear cells (PBMCs) between butyrate and an HMTi. Chicken HD11 macropahges were stimulated in duplicate with 2 mM butyrate with or without either 5 μM BIX01294 or 5 μM UNC1999 for 24 h, followed by RT-qPCR analysis of the gene expression levels of AvBD9 (A), AvBD1 (B), AvBD4 (C), AvBD10 (D), and CATH2 (E). (F) Chicken PBMCs were stimulated in duplicate with indicated concentrations (μM) of BIX01294 in the presence or absence of 1 mM butyrate for 24 h, followed by RT-qPCR of AvBD9 expression. Results are shown as means ± SEM of 2-3 independent experiments. The treatments not sharing a common superscript are considered significantly different (P < 0.05) based on one-way analysis of variance and post hoc Tukey’s test.

Figure 7

Synergistic induction of chicken HDP mRNA expression in HTC cells between HMTi and HDACi or between DNMTi and HDACi. Chicken HTC cells were stimulated in duplicate with an HMTi (2.5 or 5 μM BIX01294) in the presence or absence of one of three HDACi (5 μM SAHA, 2 mM butyrate, or 2.5 μM mocetinostat) for 24 h, followed by RT-qPCR analysis of the expressions of AvBD9 (A) and other chicken HDP genes (B). (C) Chicken HTC cells were stimulated in duplicate with an HMTi (2 or 5 μM UNC1999 or 5 μM A-366) individually or in combination with an HDACi (2 mM butyrate or 2.5 μM mocetinostat) for 24 h, followed by RT-qPCR analysis of the expressions of AvBD9 gene. (D) Chicken HTC cells were stimulated in duplicate with a DNMTi (2.5 or 5 μM SGI-1027 or 5-azacytidine) individually or in combination with a HDACi (2 mM butyrate or 2.5 μM mocetinostat) for 24 h, followed by RT-qPCR analysis of AvBD9 expression. Results are shown as means ± SEM of three independent experiments. The bars not sharing a common superscript are considered significantly different (P < 0.05) based on one-way analysis of variance and post hoc Tukey’s test.

Figure 8

Regulation of the genes involved in barrier function, host defense, and inflammation in HTC cells by HMTi and HDACi. Chicken HTC cells were stimulated in duplicate with 5 μM BIX01294 and 2 mM butyrate individually or in combination for 24 h, followed by 3-h stimulation with 10 ng/ml lipopolysaccharide (LPS) for another 3 h. RT-qPCR was performed to analyzed the expressions of CLND1 (A) , MUC2 (B) , AvBD9 (C), IL-1β (D), and IL-8 (E). (F) Chicken HTC cells were stimulated in duplicate with 5 μM UNC1999 or in combination with 2 mM butyrate or 2.5 μM mocetinostat for 24 h, followed by RT-qPCR analysis of CLND1 expression. Results are shown as means ± SEM of three independent experiments. The bars not sharing a common superscript are considered significantly different (P < 0.05) based on one-way analysis of variance and post hoc Tukey’s test.