Entinostat up-regulates the CAMP gene encoding LL-37 via activation of STAT3 and HIF-1α transcription factors

Erica Miraglia,Birgitta Agerberth,Susan Farmand,Frank Nylén,Katarina Johansson,Peter Bergman,Gudmundur H Gudmundsson,Elias Arnér,Roger Strömberg,Håkan Ottosson,Marcus Cebula

doi:10.1038/srep33274

Erica Miraglia, Birgitta Agerberth + Show 9 more

Open Access

https://doi.org/10.1038/srep33274

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Journal: Scientific Reports	Publication Date: Sep 16, 2016
Citations: 39	License type: CC BY 4.0

Affiliation: Karolinska Institutet

Abstract

Bacterial resistance against classical antibiotics is a growing problem and the development of new antibiotics is limited. Thus, novel alternatives to antibiotics are warranted. Antimicrobial peptides (AMPs) are effector molecules of innate immunity that can be induced by several compounds, including vitamin D and phenyl-butyrate (PBA). Utilizing a luciferase based assay, we recently discovered that the histone deacetylase inhibitor Entinostat is a potent inducer of the CAMP gene encoding the human cathelicidin LL-37. Here we investigate a mechanism for the induction and also find that Entinostat up-regulates human β-defensin 1. Analysis of the CAMP promoter sequence revealed binding sites for the transcription factors STAT3 and HIF-1α. By using short hairpin RNA and selective inhibitors, we found that both transcription factors are involved in Entinostat-induced expression of LL-37. However, only HIF-1α was found to be recruited to the CAMP promoter, suggesting that Entinostat activates STAT3, which promotes transcription of CAMP by increasing the expression of HIF-1α. Finally, we provide in vivo relevance to our findings by showing that Entinostat-elicited LL-37 expression was impaired in macrophages from a patient with a STAT3-mutation. Combined, our findings support a role for STAT3 and HIF-1α in the regulation of LL-37 expression.

Highlights

Innate immunity consists of a wide array of first line defences against invading pathogens
Since the HDAC-inhibitors butyrate (BA) and phenylbutyrate (PBA) as well as their analogues isovaleric and isobutyric acids are known to induce CAMP gene expression[13,14], we expanded on these findings and used the CampLuc reporter cell line[19] to screen additional histone deacetylases (HDAC) inhibitors as well as Entinostat and related compounds[20]
The HDAC inhibitor Entinostat is a potent inducer of the human antimicrobial peptide LL-37 expression[20], as shown in several cell types, and it works in synergy with the active form of vitamin D

Summary

Introduction

Innate immunity consists of a wide array of first line defences against invading pathogens. In mammals there are two major classes of AMPs, the defensins (alpha, beta and theta) and the cathelicidins[2,3], where LL-37 is the sole cathelicidin in humans and encoded by the CAMP gene These peptides are synthesized at the host/microbe interface, e.g. epithelial linings and in certain immune cells[1]. Mutations in the gene encoding STAT3 cause autosomal-dominant hyper-IgE syndrome, a primary immunodeficiency characterized by recurrent staphylococcal infections, eczema as well as skeletal and connective tissue abnormalities[26,27,28] Another transcription-factor related to AMP-expression is Hypoxia-inducible factor 1 (HIF-1), which is a master regulator of the cellular response to hypoxia. We set out to test this hypothesis by using a combination of chemical inhibitors, short hairpin RNA-mediated knock-down of STAT3/HIF1-αexpression and – – in macrophages from a STAT3-deficient patient

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Conclusion