Anti-Cancer Drug HMBA Acts as an Adjuvant during Intracellular Bacterial Infections by Inducing Type I IFN through STING This information is current as of September 26, 2021. Akshamal Mihiranga Gamage, Kok-Onn Lee and Yunn-Hwen Gan J Immunol 2017; 199:2491-2502; Prepublished online 21 August 2017; doi: 10.4049/jimmunol.1602162 Downloaded from http://www.jimmunol.org/content/199/7/2491 Supplementary http://www.jimmunol.org/content/suppl/2017/08/19/jimmunol.160216 http://www.jimmunol.org/ Material 2.DCSupplemental References This article cites 60 articles, 25 of which you can access for free at: http://www.jimmunol.org/content/199/7/2491.full#ref-list-1 Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision by guest on September 26, 2021 • No Triage! 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The Journal of Immunology Anti-Cancer Drug HMBA Acts as an Adjuvant during Intracellular Bacterial Infections by Inducing Type I IFN through STING Akshamal Mihiranga Gamage,* Kok-Onn Lee,† and Yunn-Hwen Gan* The anti-proliferative agent hexamethylene bisacetamide (HMBA) belongs to a class of hybrid bipolar compounds developed more than 30 y ago for their ability to induce terminal differentiation of transformed cells. Recently, HMBA has also been shown to trigger HIV transcription from latently infected cells, via a CDK9/HMBA inducible protein-1 dependent process. However, the effect of HMBA on the immune response has not been explored. We observed that pretreatment of human peripheral blood mononuclear cells with HMBA led to a markedly increased production of IL-12 and IFN-g, but not of TNF-a, IL-6, and IL-8 upon subsequent infection with Burkholderia pseudomallei and Salmonella enterica. HMBA treatment was also associated with better intracellular Downloaded from bacterial control. HMBA significantly improved IL-12p70 production from CD14+ monocytes during infection partly via the induction of type I IFN in these cells, which primed an increased transcription of the p35 subunit of IL-12p70 during infection. HMBA also increased early type I IFN transcription in human monocytic and epithelial cell lines, but this was surprisingly independent of its previously reported effects on positive transcription elongation factor b and HMBA inducible protein-1. Instead, the effect of HMBA was downstream of a calcium influx, and required the pattern recognition receptor and adaptor STING but not cGAS. Our work therefore links the STING-IRF3 axis to enhanced IL-12 production and intracellular bacterial http://www.jimmunol.org/ control in primary monocytes. This raises the possibility that HMBA or related small molecules may be explored as therapeutic adjuvants to improve disease outcomes during intracellular bacterial infections. The Journal of Immunology, 2017, 199: 2491–2502. urkholderia pseudomallei is a facultative intracellular numbers and IFN-g production correlate with protection in patients pathogen and the causative agent of melioidosis, an in- (6–8). We have previously shown that Type 2 diabetic patients, who B fectious disease endemic in tropical regions. Treatment are at increased risk for developing acute melioidosis and severe of melioidosis is complicated by the intrinsic resistance of the disease (9), showed defective IL-12 and IFN-g production in re- pathogen to many antibiotics. Relapse after the initial infection sponse to B. pseudomallei infection (10). The production of IL-12 is common, even after antibiotic therapy (1). Overall mortality from infected monocytes induced IFN-g secretion from NK cells, by guest on September 26, 2021 ranges from around 15% in Singapore and Australia, to up to 60% and this in turn activated the microbicidal activity of monocytes to in Thailand (2, 3). The closely related yet avirulent B. thailandensis control intracellular bacterial numbers (11). This defective IL-12/ is often used in experimental studies to circumvent logistical dif- IFN-g axis in diabetic patients was linked to a lower glutathione ficulties in working with B. pseudomallei, which is designated as a (GSH) ratio. Although ex vivo addition of free GSH could boost Risk Group 3 pathogen. IL-12 production from monocytes upon infection (10), oral sup- Cell-mediated immunity has been shown to be important for the plementation with N-acetylcysteine (a GSH prodrug) in diabetic control of melioidosis in animal models (4, 5), and CD4 T cell patients could not reverse the cytokine defect when diabetic pe- ripheral blood mononuclear cells (PBMCs) were infected ex vivo (11). This prompted us to examine whether other molecules have *Department of Biochemistry, Yong Loo Lin School of Medicine, National Univer- the potential to increase IL-12 production independently of the sity of Singapore, Singapore 117597, Singapore; and †Department of Medicine, Yong Loo Lin School of Medicine, National University of Singapore, Singapore 119228, GSH ratio, during B. pseudomallei infection. Singapore Hexamethylene bisacetamide (HMBA) was discovered in the ORCIDs: 0000-0002-3040-1185 (K.-O.L.); 0000-0001-9881-6881 (Y.-H.G.). 1970s as an inducer of terminal differentiation in murine erythroid Received for publication December 23, 2016. Accepted for publication July 26, 2017. leukemia cells, and was the lead molecule in a library of polar This work was supported by Singapore National Medical Research Council NMRC/ compounds structurally related to DMSO (12). HMBA is known to CBRG12nov035 and NMRC/CG/013/2013. The funders had no role in study design, cause the growth arrest and differentiation of various transformed data collection and interpretation, or the decision to submit the work for publication. cells, a property linked to its bromodomain inhibitory activity (13). Address correspondence and reprint requests to Dr. Yunn-Hwen Gan, National Uni- After entering phase II trials for acute myelogenous leukemia, the versity of Singapore, MD7, 8 Medical Drive, 117597 Singapore. E-mail address: [email protected] drug was discontinued from further clinical development as the re- The online version of this article contains supplemental material. missions were transient, and the high effective plasma concentrations required led to several side effects in the patients (14). Further de- Abbreviations used in this article: CaM, calmodulin; ER, endoplasmic reticulum; GR, glucocorticoid receptor; GRE, glucocorticoid transcriptional response element; velopment led to the discovery of suberanilohydroxamic acid (SAHA), GSH, glutathione; HMBA, hexamethylene bisacetamide; ISG, IFN-stimulated which was over 100 times more potent than HMBA in inducing gene; MOI, multiplicity of infection; PBMC, peripheral blood mononuclear cell; PRR, pattern recognition receptor; P-TEFb, positive transcription elongation terminal differentiation of murine erythroid leukemia cells (15). factor B; R848, resiquimod; SAHA, suberanilohydroxamic acid; siRNA, small in- SAHA was found to possess histone-deacetylase–inhibiting activity, terfering RNA; UPR, unfolded protein response; w-7, N-(6-aminohexyl)-5-chloro- a property not shared by HMBA (16). 1-naphthalenesulfonamide hydrochloride. Recently, HMBAwas found to drive HIV transcription in latently Copyright Ó 2017 by The American Association of Immunologists, Inc. 0022-1767/17/$35.00 infected cells (17, 18). Increased viral transcription was due to the www.jimmunol.org/cgi/doi/10.4049/jimmunol.1602162 2492 HMBA AS ADJUVANT IN BACTERIAL INFECTIONS VIA IFNb AND STING release of free positive transcription elongation factor B (P-TEFb) Assay for calcium flux. PMA-differentiated THP-1 cells were detached by from its inactive complex upon HMBA treatment. Free P-TEFb trypsinization, resuspended in serum-free RPMI 1640, and loaded with 10 also upregulates expression of the regulatory protein HEXIM-1 by mM Indo-1 AM for 30 min at 37˚C. The cells were pelleted to remove excess dye, resuspended in complete RPMI 1640, and equilibrated at 37˚C binding to its promoter region (19). Newly synthesized HEXIM-1 for 15 min prior to measurement on a BD LSR Fortessa X-20 flow protein can negatively regulate P-TEFb activity by directly seques- cytometer. Each sample was first assayed untreated for 60–90 s, stimulated tering the free P-TEFb back into the inactive complex. HEXIM-1 is with 2 mM ionomycin or 10 mM HMBA, and then immediately reassayed. also capable of inhibiting several other transcription factors includ- The BUV395 filter set (379/28) was used to measure calcium-bound Indo- 1, and the BUV496 (515/30) filter set was used to measure free Indo-1. ing NF-kB, glucocorticoid receptor (GR), and the estrogen receptor Calcium flux was plotted as the ratio of bound/free Indo-1, and viewed on a (20–22). a time versus ratio plot. We discovered that HMBA has an unexpected immune adjuvant property, capable of potently increasing IL-12 and IFN-g pro-