Primary macrophages rely on histone deacetylase 1 and 2 expression to induce type I interferon in response to gammaherpesvirus infection

Abstract

Type I interferon is induced shortly following viral infection and represents a first line of host defense against a majority of viral pathogens. Not surprisingly, both replication and latency of gammaherpesviruses, ubiquitous cancer-associated pathogens, are attenuated by type I interferon, although the mechanism of attenuation remains poorly characterized. Gammaherpesviruses also target histone deacetylases (HDACs), a family of pleiotropic enzymes that modify gene expression and several cell signaling pathways. Specifically, we have previously shown that a conserved gammaherpesvirus protein kinase interacts with HDAC1 and -2 to promote gammaherpesvirus replication in primary macrophages. In the current study, we have used genetic approaches to show that expression of HDAC1 and -2 is critical for induction of a type I interferon response following gammaherpesvirus infection of primary macrophages. Specifically, expression of HDAC1 and -2 was required for phosphorylation of interferon regulatory factor 3 (IRF3) and accumulation of IRF3 at the beta interferon promoter in gammaherpesvirus-infected primary macrophages. To our knowledge, this is the first demonstration of a specific role for HDAC1 and -2 in the induction of type I interferon responses in primary immune cells following virus infection. Furthermore, because HDAC1 and -2 are overexpressed in several types of cancer, our findings illuminate potential side effects of HDAC1- and -2-specific inhibitors that are currently under development as cancer therapy agents. IMPORTANCE Gammaherpesviruses establish chronic infection in a majority of the adult population and are associated with several malignancies. Infected cells counteract gammaherpesvirus infection via innate immune signaling mediated primarily through type I interferon. The induction of type I interferon expression proceeds through several stages using molecular mechanisms that are still incompletely characterized. In this study, we show that expression of HDAC1 and -2 by macrophages is required to mount a type I interferon response to incoming gammaherpesvirus. The involvement of HDAC1 and -2 in the type I interferon response highlights the pleiotropic roles of these enzymes in cellular signaling. Interestingly, HDAC1 and -2 are deregulated in cancer and are attractive targets of new cancer therapies. Due to the ubiquitous and chronic nature of gammaherpesvirus infection, the role of HDAC1 and -2 in the induction of type I interferon responses should be considered during the clinical development of HDAC1- and -2-specific inhibitors.

FIG 1

Interferon-stimulated genes are not induced in primary macrophages depleted of HDAC1 and -2. Primary macrophages were cultured to induce depletion of HDAC1 and -2 and mock infected or infected at an MOI of 10 with MHV68. (A) Protein levels of viperin, total Stat1, ISG15, HDAC1, HDAC2, and β-actin were measured at 24 h postinfection. The image is representative of at least three independent experiments. Cellular transcripts were measured via qRT-PCR at 8, 24, and 48 hpi for viperin (B), GBP-1 (C), and Mx-1 (D) and normalized to GAPDH. Data were pooled from at least three independent experiments, including two repeats within each experiment. Black lines represent Cre-negative macrophages, and gray lines represent Cre-positive, HDAC1/2-depleted macrophages. Error bars represent 1 standard error of the mean.

FIG 2

ISGs are induced in response to exogenous IFN-β treatment in HDAC1- and -2-depleted primary macrophages. Primary macrophages were generated as for Fig. 1 and treated for 4 h with 5 U/ml of recombinant IFN-β. Transcripts for viperin (A), GBP-1 (B), and Mx-1 (C) were quantified by qRT-PCR, normalized to GAPDH, and further normalized to values of mock-treated cells that were set to 1. Data were pooled from three independent experiments, and error bars represent 1 standard error of the mean.

FIG 3

Type I interferons are not secreted or transcribed in HDAC1- and -2-depleted primary macrophages. Primary macrophages were depleted of HDAC1 and -2 as for Fig. 1 and mock infected or infected at an MOI of 10 with wt MHV68 or the N36S virus mutant. (A and B) Total secreted antiviral activity was measured at 4 and 24 h postinfection using EMCV bioassay and compared to IFN-β standards. Data are representative of two independent experiments. Transcript levels of IFN-α (C) and IFN-β (D) were measured at 4 h postinfection via qRT-PCR and normalized to corresponding GAPDH. (E) Primary macrophages were mock treated, infected with EMCV (TCID₅₀ = 10), or treated with 50 μg/ml of poly(I)·poly(C) (pI:pC) and 20 or 100 ng/ml of LPS. ISG15 and β-actin protein levels were measured at 8 h of treatment. (F) Viperin mRNA levels were measured at 4 h after treatment with 100 ng/ml of LPS or 50 μg/ml of pI:pC and at 24 h after infection with EMCV (TCID₅₀ = 10).

FIG 4

IRF3 does not localize to the IFN-β promoter and is not phosphorylated in HDAC1- and -2-depleted primary macrophages. (A) IRF-3 localization to the IFN-β promoter was measured via ChIP at 4 h postinfection. MHV68-infected IRF-3^−/− macrophages were used as specificity controls. Data were pooled from two independent experiments, with each condition performed in duplicate within each experiment. (B) IRF3 mRNA levels were measured via qRT-PCR at 4 h postinfection in Cre-positive and -negative macrophages. Data were pooled from 2 to 4 independent experiments, and errors bars represent 1 standard error of the mean. Enrichment of histone H3 (C and E) and AcH4 (D and F) at the GAPDH coding sequence (C and D) or IFN-β promoter (E and F) was measured by ChIP in Cre-negative and -positive macrophages following mock treatment or MHV68 infection (4 h, MOI = 10). Total IRF3 (H) and phosphorylated IRF3 (G) were measured at 4 h after MHV68 infection (MOI = 10) or LPS treatment (100 ng/ml). MHV68-infected IRF3^−/− macrophages were used to verify antibody specificity. Images are representative of four independent experiments.

FIG 5

MHV68 gene expression is not altered in HDAC1- and -2-depleted primary macrophages. Primary macrophages were mock infected or infected at an MOI of 10 with wt MHV68 or the N36S virus mutant. (A and B) mRNA levels of RTA were measured at 8 and 24 h postinfection using qRT-PCR. (C) orf57 mRNA levels were measured at 24 h postinfection. (D) orf6 ssDBP protein levels were measured at 24 h postinfection. (E to I) Expression of indicated MHV68 mRNA encoding essential DNA synthesis proteins was measured at 24 h postinfection. All transcription data were normalized to corresponding cellular GAPDH mRNA levels, with further normalization to the mRNA levels observed in wt MHV68-infected Cre-negative macrophages. Error bars represent 1 standard error of the mean. Data were pooled from 2 to 5 independent experiments.

FIG 6

IFN-β treatment of HDAC1- and -2-depleted primary macrophages reduces viral DNA accumulation in kinase-deficient MHV68 infection. Primary macrophages depleted (Cre+, hatched bars) or not depleted (Cre−) of HDAC1 and -2 were mock treated or treated with 5 U/ml of recombinant IFN-β immediately after infection with either wt MHV68 or the N36S mutant (MOI = 10). Total DNA was harvested at 48 h postinfection, and MHV68 DNA was measured by real-time PCR with subsequent normalization to the corresponding cellular GAPDH gene. Data are combined from two independent experiments. Error bars represent 1 standard error of the mean. **, P < 0.01. NS, not significant.

FIG 7

Working model. Upon infection of primary macrophages with MHV68, HDAC1 and -2 act upstream of IRF3 to promote its phosphorylation, recruitment to the IFN-β promoters, and subsequent type I IFN expression. In addition to antiviral functions of HDAC1 and -2, these two enzymes may engage other cellular signaling pathways to exert proviral functions that facilitate expression of viral genes, including RTA, and viral replication.