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Histone Deacetylase Inhibitors Impair the Host Immune Response Against Mycobacterium Tuberculosis Infection T

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Histone Deacetylase Inhibitors Impair the Host Immune Response Against Mycobacterium Tuberculosis Infection T Tuberculosis 118 (2019) 101861 Contents lists available at ScienceDirect Tuberculosis journal homepage: www.elsevier.com/locate/tube Immunological Aspects Histone deacetylase inhibitors impair the host immune response against Mycobacterium tuberculosis infection T Chuanzhi Zhua,1, Yi Caib,1, Jialou Zhub, Lanyue Zhanga, Aiying Xinga, Liping Pana, Hongyan Jiaa, ∗ ∗∗ Siwei Mob, Carl G. Fengb,c, Hongbo Shend, Xinchun Chenb, , Zongde Zhanga, a Laboratory of Molecular Biology, Beijing Key Laboratory for Drug Resistance Tuberculosis Research, Beijing Chest Hospital, Capital Medical University, Beijing Tuberculosis and Thoracic Tumor Research Institute, Beijing, 101149, China b Department of Pathogen Biology, Guangdong Provincial Key Laboratory of Regional Immunity and Diseases, Shenzhen University School of Medicine, Shenzhen, 518060, Guangdong, China c Immunology and Host Defense Group, Department of Infectious Diseases and Immunology, Sydney Medical School, The University of Sydney, Sydney, NSW, 2006, Australia d Unit of Anti-Tuberculosis Immunity, Institute Pasteur of Shanghai, Chinese Academy of Sciences, Shanghai, 200031, China ARTICLE INFO ABSTRACT Keywords: Histone deacetylase inhibitors (HDACi), a novel class of anti-cancer drug, have been recently reported to sup- HDACi press host immunity and increase susceptibility to infection. Tuberculosis, a leading infectious disease killer Trichostatin A caused by Mycobacterium tuberculosis (M.tb), is basically the product of the interaction between bacterial Autophagy virulence and host resistance. However, the effects of HDACi in host immunity against M.tb is largely unknown. Mycobacterium tuberculosis In this study, we found that HDACi including Trichostatin A (TSA) and suberoylanilide hydroxamic acid (SAHA) Susceptibility significantly impaired phagocytosis and killing activity of macrophage. In line with these findings, we noted that M.tb induced reactive oxygen species (ROS) production and autophagy are significantly suppressed by TSA. Transcriptome analysis revealed that the suppression of autophagy by TSA might due to its inhibiting autophagy- regulating genes such as CACNA2D3, which regulates intracellular Ca2+ levels. Finally, we confirmed that HDACi including TSA and SAHA significantly exacerbated the histopathological damage and M.tb load in the lung of M.tb infected mice. Taken together, our results indicated that HDACi at least TSA and SAHA significantly impaired macrophage immunity against M.tb and therefore increase susceptibility to TB, our findings raised the concern that the potential side effects of HDACi on latent TB reactivation should be considered in clinic. 1. Introduction suppress gene transcription by epigenetic mechanisms [3]. HDAC-11 is the only member of Class IV HDACs. Histone deacetylases (HDACs) play important roles in regulating HDAC inhibitors (HDACi) have been developed for cancer therapy epigenetic states and gene expression by catalyzing the removal of based on known HDAC expression patterns and pathogenic functions acetyl groups from histones and some non-histone proteins. Based on [4–6]. Among them, Trichostatin A (TSA) and suberoylanilide hydro- their homology with yeast, HDACs are grouped into four classes. xamic acid (SAHA), are first generation HDACi with a prototypical Specifically, class I includes HDAC-1, -2, -3 and -8, which are mainly broad-spectrum inhibitory effect on class I, II, and IV HDACs [5]. They localized to the nucleus and primarily implicated in innate immunity are also the first chemical HDAC tools available to study immune reg- and regulating inflammatory cytokine production [1]. Class II includes ulation in macrophages [2]. While HDACi represent a promising cate- HDAC-4, -5, -6, -7, -9 and -10, which shuttle between the nucleus and gory of anti-cancer drugs, early clinical observations have suggested cytoplasm and are involved in controlling innate and adaptive im- that SAHA inhibits cell-mediated immunity, raising the concern that munity by regulating antigen presentation, B and T-lymphocyte acti- HDACi might increase patient susceptibility to opportunistic and latent vation and development [1,2]. Class III HDACs, also known as Sirtuins, infections [7,8]. Indeed, numerous studies have shown that HDACi contain an NAD+-dependent catalytic domain that allows them to inhibit proinflammatory cytokine secretion and impair the innate ∗ Corresponding author. ∗∗ Corresponding author. E-mail addresses: [email protected] (X. Chen), [email protected] (Z. Zhang). 1 Chuanzhi Zhu and Yi Cai contributed equally to this study. https://doi.org/10.1016/j.tube.2019.101861 Received 22 January 2019; Received in revised form 19 July 2019; Accepted 26 August 2019 Available online 27 August 2019 1472-9792/ © 2019 Published by Elsevier Ltd. C. Zhu, et al. Tuberculosis 118 (2019) 101861 immune response against infection [9]. For example, macrophages and 2.4. Colony forming units (CFU) assays dendritic cells treated with TSA or SAHA showed suppressed produc- tion of TLR-induced cytokines and chemokines, including IL-12p40, THP-1-derived macrophages were infected with H37Ra at MOI of 10 IFN-β, IP-10, MCP5, inducible nitric oxide synthetase, guanylate- for 6 h in the presence or absence of HDACi treatment. At 6 h post- binding protein 2 and IL-12p35 [10]. LPS-induced TNF-α and IL-6 se- infection, complete medium containing HDACi (TSA, SAHA, Entinostat, cretion was also dramatically impaired upon prolonged pretreatment of Sodium butyrate, Valproic acid or Sirtinol)was added to the infected dendritic cells with TSA or SAHA [10]. Another study showed that TSA macrophages for 72 h, respectively. In the reciprocal experiment, THP- inhibited LPS-induced Ccl8, Ccl12, Cxcl10 and Nos2 mRNA expression 1-derived macrophages were treated with HDACi for 18 h before in- by macrophages [11]. fection with H37Ra at MOI of 10 for 6 h. Then the cells were cultured in Tuberculosis (TB) is the leading cause of human mortality caused by complete media without HDACi for 72 h. At 6 h and 72 h, the infected a single infectious agent, Mycobacterial tuberculosis (M.tb). Remarkably, macrophages were lysed in 200 μl sterile PBS with 0.1% SDS. The about 23% of the world's population carry a latent tuberculosis infec- viability of the intracellular Mycobacteria was quantified by counting tion (LTBI) [12]. Cytokines and chemokines are critical for the host CFU. defense against TB. For example, neutralizing the proinflammatory cytokine, TNF-α, during rheumatoid arthritis treatment can reactivate 2.5. Measurement of ROS production latent TB [13]. Clinical trials of several HDACi, such as SAHA, MS-275, valproate and ITF2357, in tumor patients have experienced episodes of After infection with H37Ra in the presence or absence of TSA at severe infection [8,14–18]. Thus, it is important to determine the in- MOI of 10 for 24 h, the cytoplasmic ROS (cROS) and mitochondrial ROS fluence of HDACi on host immune response to M.tb. (mROS) using 5- (and-6) - chloromethyl- 2′,7′- dichlorodihydro-fluor- In this study, we found that HDACi impaired M.tb macrophage- escein diacetate, acetyl ester (CM-H2DCFDA) (Invitrogen) or MitoSOX™ mediated phagocytosis and enhanced M.tb growth in vitro and in vivo. Red mitochondrial superoxide indicator (Invitrogen), according to the TSA significantly impaired cytoplasmic and mitochondrial ROS pro- manufacturer's instructions. All fluorescence intensities were measured duction, and inhibited autophagosome formation through down- using a FACSCalibur flow cytometer (BD) and the data were analyzed regulation of M.tb-induced host immune response related gene expres- using FlowJo version 6.1 (FlowJo, LLC). sion, i.e. autophagy-regulating genes such as CACNA2D3. 2.6. Western blotting 2. Materials and methods THP-1-derived macrophages were infected with H37Ra in the pre- 2.1. Cells and bacteria culture sence or absence of TSA for 24 h. Total proteins were extracted using a Minute™ total protein extraction kit (Invent). The primary antibodies The human monocyte cell line THP-1 cell was purchased from the anti-LC3 (Abcam), anti-P62 (Abcam), anti-Beclin-1 (Cell Signaling Cell Bank of the Chinese Academy of Sciences (Shanghai, China). GFP- Technology) and HRP-conjugated secondary antibodies (Cell Signaling LC3-THP-1 cell was generated previously by Hongbo Shen [19]. Prior to Technology) were used. Western blotting signals were detected using an infection, THP-1 cells were treated with 20 ng/ml phorbol 12-myristate Amersham Imager 600 (GE Healthcare) and auto-radiographic film ff 13-acetate (PMA, Sigma-Aldrich) for 48 h to di erentiate into macro- after incubation with SuperSignal West Femto substrate (Thermo phages. Virulent M. tb (H37Rv), attenuated M. tb (H37Ra) and GFP- Scientific). The densitometric analysis was determined by Image J. H37Ra were grown at 37 °C in DifcoTM Middlebrook 7H9 broth with 10% oleic acid-albumin-dextrose-catalase (OADC) enrichment (Becton Dickinson), 0.05% (v/v) Tween 80 (Sigma-Aldrich), and 0.2% (v/v) 2.7. Immunofluorescence and confocal microscopy glycerol (Thermo Fisher). GFP-LC3-THP-1-derived macrophages were infected with H37Ra at MOI of 10 containing TSA or not for 24 h. Images were acquired of cells 2.2. RNA extraction and analysis (selected at random) containing GFP-LC3 using the Micropoint System (Andor). GFP-H37Ra infected THP-1-derived macrophages were treated Total RNA was isolated from macrophages using a RNeasy kit with or without TSA for 24 h. After infection, complete media con- (Omega). High RNA integrity of each sample (three biological replicates
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