Cutting Edge: STAT4 promotes Bhlhe40 induction to drive protective IFN-γ from natural killer cells during viral infection

Abstract

NK cells represent a cellular component of the mammalian innate immune system, and mount rapid responses against viral infection, including the secretion of the potent anti-viral effector cytokine IFN-γ. Following mouse cytomegalovirus (MCMV) infection, Bhlhe40 was the most highly induced transcription factor in NK cells among the basic helix-loop-helix family. Bhlhe40 upregulation in NK cells depended upon IL-12 and IL-18 signals, with the promoter of Bhlhe40 enriched for STAT4 and the permissive histone H3K4me3, and STAT4-deficient NK cells showing an impairment of Bhlhe40 induction and diminished H3K4me3. Transcriptomic and protein analysis of Bhlhe40-deficient NK cells revealed a defect in IFN-γ production during MCMV infection, resulting in diminished protective immunity following viral challenge. Finally, we provide evidence that Bhlhe40 directly promotes IFN-γ by binding throughout the Ifng loci in activated NK cells. Thus, our study reveals how STAT4-mediated control of Bhlhe40 drives protective IFN-γ secretion by NK cells during viral infection.

Introduction

Natural Killer (NK) cells are innate lymphocytes able to respond to stress, transformed, or infected cells without prior sensitization. However, resting NK cells can become more potent effector cells through activation by germ-line-encoded surface receptors and proinflammatory cytokines, including interleukin (IL)-12, IL-18, and type I interferon (IFN) produced during viral infection (1). These activating signals will induce a robust transcriptional and epigenetic response in NK cells, leading to their effector programs (2),including the production of the potent anti-viral cytokine IFN-γ.

During MCMV infection, IL-12 is produced and binds to a heterodimeric receptor leading to a signal cascade via Janus kinase (JAK)-mediated phosphorylation and homodimerization of signal transducer and activator of transcription 4 (STAT4) in the cytoplasm. In NK cells, STAT4 activation causes it to translocate to the nucleus where it induces the transcription of a multitude of genes by targeting both promoter and non-promoter regions (3). Previous studies have demonstrated that an IL-12/STAT4 signaling axis in NK cells can drive a plethora of effector functions, including cytokine production and proliferation (4) through transcription factors Zbtb32, IRF8, STAT5, CBFb and Runx factors (5–8), among others. However, many additional genes targeted by STAT4 in NK cells during MCMV infection have not been characterized.

In our current study, we discovered that Bhlhe40 was highly induced in activated NK cells during MCMV infection, among the family of basic helix-loop-helix (bHLH) transcription factors. Bhlhe40, also known as Bhlhb2, Dec1, and Stra13, consists of a basic DNA-binding domain, a helix-loop-helix domain, which facilitates dimerization, and a protein-protein interaction orange domain (9, 10). Members of the bHLH family (specifically factors in the E clade including Bhlhe40) have been shown to regulate numerous physiological processes including differentiation, proliferation, and apoptosis in multiple cell types due to its ability to act as both transcriptional activator and repressor in a context dependent manner (11). Although Bhlhe40 was initially described as a regulator of circadian rhythm and cellular differentiation, emerging evidence suggests that it is also acting as a regulator of the innate and the adaptive immune systems (11).

In T cells, Bhlhe40 is rapidly expressed following TCR activation (12) and plays a number of critical roles to support T cell identity and effector functions. In the context of autoimmunity and aging, Bhlhe40 promotes GM-CSF expression while suppressing IL-10 production (13), and maintains regulatory T cell function/identity, respectively (14, 15). Additionally, Bhlhe40 is also required for the function of tissue-resident memory CD8⁺ and CD4⁺ T cells, and tumor-infiltrating lymphocytes, by maintaining their mitochondrial fitness and epigenetic states (16, 17). More recently, Bhlhe40 was shown to be a cell-intrinsic negative regulatory of both activated B cells and follicular helper T cells in germinal center responses (18). Within the innate immune system, Bhlhe40 controls the self-renewal and maintenance of alveolar and large peritoneal macrophages, and their proliferation in response to IL-4 (19, 20). Here, we investigated mechanisms of Bhlhe40 induction and consequences of its deletion on NK cell activation and effector function in response to viral infection.

Materials and Methods

Mice

Mice were bred at Memorial Sloan Kettering Cancer Center in accordance with the guidelines of the Institutional Animal Care and Use Committee. The following strains were used in this study: C57BL/6 (CD45.2), B6.SJL (CD45.1), Stat4^−/−, Il12rb2^−/−, Il18r1^−/−, Bhlhe40^−/−, and Klra8^−/− (Ly49h^−/−). Experiments were conducted using age- and gender-matched mice in accordance with approved institutional protocols. Adoptive transfer studies and generation of mixed bone marrow chimeric mice were performed as previously described (21).

Viral infection

MCMV (Smith) was passaged through BALB/c mice three times, followed by preparation of salivary gland viral stocks using a homogenizer for dissociating the salivary glands (of BALB/c mice 3 weeks after infection). MCMV stocks were diluted prior to injection, and 10³ plaque-forming units (PFU) were delivered in adoptive transfer studies, and 10⁴ PFU in all other experiments.

Flow cytometry

Single cell suspensions were prepared from indicated organs and indicated fluorophore-conjugated antibodies (Biolegend, Tonbo, eBioscience) used to stain lymphocytes. Flow cytometry was performed on a BD Biosciences LSR II or Cytek Biosciences Aurora, and data were analyzed with FlowJo software.

NK cell stimulation

NK cells were enriched ex vivo using a no-touch bead enrichment protocol as previously described (21), and plated at 10⁵ per well with mouse IL-2, IL-12, or IL-15 (R&D Systems) at 20 ng/ml, IL-18 (MBL) at 10 ng/ml, or IFN-α (R&D Systems) at 100 IU. Intracellular staining for IFN-γ was performed as previously described (21).

ChIP-Seq, RNA-seq, ATAC-seq, and bioinformatic analysis

The following datasets and analyses were performed on NK cells as previously described by our lab and can be found under the GEO as a SuperSeries under GSE106139 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE106138): H3K4me3 ChIP-seq, and ATAC-seq following cytokine stimulation, RNA-seq from MCMV-infected Il12rb2^–/–, Il18r1^–/–, Stat4^–/– mice, RNA-seq on days 0, 2, 4, 7, 35 after MCMV infection, RNA-seq on 3 hour and overnight in vitro cytokine stimulations (2, 6, 22, 23).

For new datasets generated in this study, RNA-seq was performed as previously described (2). Differential analysis was executed with DESeq2 (v.1.22.2). For all differential analyses, genes were considered differential if they showed an FDR-adjusted P value (FDR) ≤ 0.05 by contrast using DESeq2 model. For all sequencing counts shown, values were normalized using sizeFactors calculated by the DESeq2 software or Saccharomyces cerevisiae spike-in DNA. Paired analysis was performed between wildtype (WT) versus Bhlhe40^–/– NK cells for d0 versus d2 PI, where calculations for generalized linear models were fitted with a design that controlled for each group. RNA-seq data comparing WT versus Bhlhe40^–/– NK cells before and after MCMV infection are available under GEO accession number GSE235608 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE235608).

Cleavage Under Targets & Release Using Nuclease (CUT&RUN)

For the Stat4 and Bhlhe40 CUT&RUN, 500,000 sorted NK cells were washed 3X with PBS, resuspended in Buffer 1 (1X eBioscience Perm/Wash Buffer, 1X Roche cOmplete EDTA-free Protease Inhibitor, 0.5 uM Spermidine in H2O) and incubated with BHLHE40 (anti-DEC1, Novus NB100–1800) or STAT4 (Invitrogen 71–4500) antibodies at 1:100 dilution in Antibody Buffer (Buffer 1 + 2uM EDTA) in 96 well V-bottom plate at 4°C overnight. Upon antibody incubation, cells were washed twice with Buffer 1 and resuspended in 50ul of Buffer 1 + 1X pA/G-MNase (Cell Signaling #57813) and incubated on ice for 1 hr and washed with Buffer 2 (0.05% w/v Saponin, 1X Roche cOmplete EDTA-free Protease Inhibitor, 0.5 uM Spermidine in 1X PBS) three times. After washing, Calcium Buffer (Buffer 2 + 2uM of CaCl2) was used to resuspend the cells for 30 mins on ice to activate the pA/G-MNase reaction, and equal volume of 2X STOP Buffer (Buffer 2 + 20uM EDTA + 4uM EGTA) was added along with 1pg of Saccharomyces cerevisiae spike-in DNA (Cell Signaling #29987). Samples were incubated for 15 mins at 37°C and DNA was isolated and purified using Qiagen MinElute Kit according to manufacturer’s protocol and subjected to library amplification.

Immunoprecipitated DNA was quantified by PicoGreen and the size was evaluated by Agilent BioAnalyzer. When possible, fragments between 100 and 600 bp were size selected using aMPure XP beads (Beckman Coulter catalog # A63882) and Illumina sequencing libraries were prepared using the KAPA HTP Library Preparation Kit (Kapa Biosystems KK8234) according to the manufacturer’s instructions with 0.001–0.5ng input DNA and 14 cycles of PCR. Barcoded libraries were run on the NovaSeq 6000 in a PE100 run, using S4 kit v1.5 with XP mode (Bhlhe40 CUT&RUN) or NovaSeq X in a PE100 run using 10B reagent kit (200 Cycles, Illumina, STAT4 CUT&RUN). An average of 23 million paired reads were generated per sample. CUT&RUN data are available under GEO accession number GSE242900 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE242900) and GSE242911 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE242911).

CUT&RUN data processing

Paired reads were trimmed for adaptors and removal of low-quality reads by using Trimmomatic (v0.39) and aligned to the mm10 reference genome using Bowtie2 (v2.4.1). Upon alignment, peaks were called using MACS2 (v2.2.7.1) with input samples as control using narrow peak parameters --cutoff-analysis -p 1e-5 --keep-dup all -B --SPMR. For each sample groups, irreproducible discovery rate (IDR) calculations using scripts provided by the ENCODE project (https://www.encodeproject.org/software/idr/; v.2.0.4.2). Reproducible peaks showing an IDR value of 0.05 or less in each conditions were aggregated across the experiment and merged via union to the peak atlas, annotated with UCSC Known Gene model. Peak atlas consisted of 316 peaks for BHLHE40 CUT&RUN and 2654 for STAT4 CUT&RUN.

Statistical analyses

Data are shown as mean ± s.e.m in all graphs and statistical differences, and unless noted otherwise, were calculated using a two-tailed unpaired Student’s t-test. P values ≤ 0.05 were considered significant. In these figures, * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, **** P ≤ 0.0001. For data showing summary of normalized counts from RNA-seq and differential analysis from various sequencing experiments, statistics were calculated using DESeq2 method as noted above. For these data, FDR adjusted P values (padj) ≤ 0.05 were considered statistically significant. In these figures, * P ≤ 0.05. All statistical analyses and plots were produced in GraphPad Prism or R.

Results and Discussion

Induction of Bhlhe40 expression in NK cells during infection is dependent on IL-12

Basic helix-loop-helix (bHLH) transcription factors are widely expressed in different immune cells (11). To assess the regulation of basic helix-loop-helix transcription factors in NK cells during MCMV infection, we performed comparative transcriptome analysis by RNA sequencing (RNA-seq) during their immune response. At day 2 post-infection (PI), a time point when NK cells are robustly exhibiting their innate immune functions (1), Bhlhe40 transcript was the most significantly upregulated among the E family bHLH transcription factors in NK cells (Fig. 1A–B).

FIGURE 1. Early induction of Bhlhe40 in NK cells during MCMV infection is driven by IL-12 and IL-18.

(A) Dot plot of RNA-seq log₂ fold change comparing expression of select bHLH family E clade transcription factors in NK cells at day 2 PI compared to day 0. Black points refer to genes with FDR ≤ 0.05 in day 2 PI to day 0 differential expression contrast.

(B) RNA-seq normalized counts of Bhlhe40 from mouse Ly49H⁺ NK cells during MCMV infection.

(C-D) RNA-seq data showing Bhlhe40 from sorted NK cells stimulated with indicated cytokines for 3 hours (C) or 16 hours (D) in vitro. * FDR ≤ 0.05 in differential expression contrast against unstimulated samples.

(E-F) RNA-seq normalized counts of Bhlhe40 from WT versus IL-12R-deficient (E) or IL-18R-deficient (F) NK cells at day 0 and day 2 PI. * FDR ≤ 0.05 in differential expression contrast against both day 0 PI sample groups or against indicated deficient sample.

Given that homeostatic and proinflammatory cytokines (IL-2, IL-12, IL-15, IL-18 or type I IFN) and their downstream STAT molecules (STAT1, STAT4, or STAT5) are strongly signalling in NK cells at day 2 PI (3), we analyzed Bhlhe40 expression in NK cells stimulated with various cytokines for 3 hours or overnight. We found that NK cells stimulated with IL-12 + IL-18, but not other cytokines, showed the greatest induction of Bhlhe40 (Fig. 1C–D). Consistent with this observation, the induction of Bhlhe40 was largely or partially ablated in NK cells deficient for the IL-12 receptor or IL-18 receptor, respectively, during MCMV infection (Fig. 1E–F). Together, these data suggest that Bhlhe40 induction in NK cells early after viral infection is mainly dependent on IL-12 and to a lesser extent IL-18 signals.

Bhlhe40 is induced in a STAT4-dependent manner in NK cells

Given that IL-12 signals via the transcription factor STAT4, we investigated whether STAT4 controls transcription of Bhlhe40 in NK cells during MCMV infection. Indeed, STAT4-deficiency resulted in a significant loss of Bhlhe40 induction (Fig. 2A) regardless of activating receptor Ly49H expression, suggesting Bhlhe40 is critically dependent on STAT4 in activated NK cells. To determine whether STAT4 directly targets the Bhlhe40 loci, we performed STAT4 CUT&RUN on NK cells stimulated with IL-12 + IL-18 (2). Significantly increased differential STAT4 binding was detected at the promoter of Bhlhe40 in stimulated NK cells when contrasted with unstimulated or cytokine-stimulated Stat4^−/− NK cells (log₂ fold change of 3.17 and 2.26, and FDR of 8.95e-40 and 1.74e-17, respectively) (Fig. 2B). Although we did not observe large changes in chromatin accessibility (by ATAC-seq) at the promoter region of Bhlhe40 in NK cells stimulated with IL-12 + IL-18 (Fig. 2C), we observed significant increase in the permissive histone mark H3K4me3 following stimulation (Fig. 2D). Furthermore, we determined that the increase in H3K4me3 at the Bhlhe40 locus in stimulated NK cells is dependent on an IL-12/STAT4 signalling axis (Fig. 2E). Altogether, we provide evidence that STAT4 regulates the expression of Bhlhe40 in activated NK cells during MCMV infection via deposition of the permissive histone mark H3K4me3.

FIGURE 2. Bhlhe40 upregulation is controlled by Stat4 in activated NK cells.

(A) RNA-seq showing Bhlhe40 from WT versus STAT4-deficient Ly49H⁺ or Ly49H⁻ NK cells at day 0 and day 2 PI. Every permutation of contrasts for differential expression analysis show FDR ≤ 0.05 except WT day 0 versus STAT4-deficient day 0 in both Ly49H⁺ or Ly49H⁻ dataset, but only WT versus STAT4-deficient at day 2 is shown.

(B) STAT4 CUT&RUN tracks show STAT4 binding as mean normalized fragment pileup (y-axis) plotted by genome position (x-axis) from unstimulated or IL-12 + IL-18-stimulated WT and STAT4-deficient NK cells with bin length of 10bp. Signals normalized using spike-in DNA were averaged per bin in each sample condition group (WT groups n=3, KO n=2). Dotted box indicates range for peak called for differential binding analysis.

(C-D) ATAC-seq and ChIP-seq TSS-centered meta-peak plots show chromatin accessibility (C) and H3K4me3 binding signal (D), respectively at the TSS of the Bhlhe40 locus in unstimulated NK cells (grey line) or NK cells stimulated with IL-12 + IL-18 (black line). ATAC-seq signals are normalized using size factors calculated by DESeq2 and averaged in each sample condition group (n=3).

(E) H3K4me3 ChIP-seq signal tracks at the Bhlhe40 locus in WT or STAT4-deficient NK cells stimulated with IL-12 + IL-18.

Bhlhe40 is required for IFN-γ production and host protection

Since Bhlhe40 is strongly induced in NK cells early after viral infection, we investigated whether Bhlhe40 may be driving the specific NK cell effector functions of cytokine secretion, killing, and proliferation. To test the latter using a well-established adoptive transfer model, we injected an equal number of WT (CD45.1) and Bhlhe40^−/− (CD45.2) NK cells into Ly49H-deficient recipient mice, infected with MCMV, and tracked the Ly49H⁺ NK cell responses in the recipient. No detectable difference was observed in the magnitude of NK cell expansion following MCMV infection, as WT and Bhlhe40^−/− counterparts not only proliferated and contracted similarly, but also generated a comparable pool of memory cells (Supplemental Fig. 1A–1B), suggesting NK cell proliferation and “adaptive” features are not impacted by loss of Bhlhe40. Additionally, their maturation/activation profiles were similar and commensurate with effector NK cells at day 7 PI (Supplemental Fig. 1C). Furthermore, WT and Bhlhe40^−/− NK cells were indistinguishable in their ability to produce granzyme B and upregulate CD69 (Supplemental Fig. 1D), suggesting that cytolytic capacity and activation of NK cells remained intact in the absence of Bhlhe40.

In transcriptomic analysis, among the genes dysregulated in Bhlhe40^−/− NK cells compared to WT during MCMV infection were Ifng, Cxcr4, and Bhlhe40 (lower in Bhlhe40^−/−), and Il21r, Miga1, and Cnbd2 (elevated in Bhlhe40^−/−) (Fig. 3A). Confirming that Ifng mRNA was downregulated, Bhlhe40^−/− NK cells produced less IFN-γ protein compared to WT NK cells following infection (Fig. 3B). Moreover, Bhlhe40^−/− NK cells similarly produced diminished IFN-γ compared to their WT counterparts following in vitro stimulation with proinflammatory cytokines (Fig. 3C). Finally, we observed that Bhlhe40^−/− mice were more susceptible to MCMV challenge than WT mice, and most Bhlhe40^−/− mice succumbed at a similar rate and with similar kinetics to NK cell-depleted WT mice (Fig. 3D).

FIGURE 3. Bhlhe40 control of IFN-γ in NK cell-mediated host protection.

(A) Volcano plot of RNA-seq log₂ fold change comparing WT versus Bhlhe40^−/− NK cells at day 2 PI compared to day 0. Black dots show differentially expressed genes (FDR < 0.05), and genes of note are bolded. Horizontal dashed line indicates p-value = 0.05, and vertical dashed lines indicate absolute log₂ fold change = 0.5.

(B) Representative histogram (left) shows WT versus Bhlhe40^−/− NK cells from mixed bone marrow chimeric mice infected with MCMV were assessed for IFN-γ secretion at day 2 PI and compared against NK cells from uninfected mice. Paired dot plot (right) shows IFN-γ MFI of paired WT and Bhlhe40^−/− NK cells from individual mice. Paired two-tailed t-test was performed for right plot. **** p < 0.0001.

(C) Representative histogram (left) of IFN-γ production between WT and Bhlhe40^−/− NK cells following stimulation with IL-12 + IL-18, and percentage of NK cells producing IFN-γ measured (right). Unpaired two-tailed t-test was performed for right graph between WT and Bhlhe40^−/− within each stimulation condition. *** p < 0.001.

(D) Kaplan-Meier survival curves for WT versus Bhlhe40^−/− mice, and mice depleted of NK cells following MCMV challenge. Log-rank (Mantel-Cox) test was performed between each contrast. *** p < 0.001. All data shown are representative of 2–3 experiments (n=3–5 per experiment).

To determine the direct binding sites of Bhlhe40, we performed CUT&RUN on NK cells stimulated with IL-12 + IL-18. We identified a total of 316 Bhlhe40 binding sites, where most of these peaks were called in the stimulated condition (n=279, Fig. 4A–4B). Through global differential binding analysis, we found that the majority of these sites were significantly enriched upon IL-12 + IL-18 stimulation (Fig. 4C). Notably, the top differential binding site of Bhlhe40 was within the Ifng locus (Fig. 4D). Among the significant differentially binding sites, most were annotated as promoter peaks (74%, 208 out of 281, Fig. 4E). Motif analysis with differentially binding peaks revealed de novo motifs that suggest potential interactions with families including ATF1 and IRF6, and since the bHLH family is present within the motif database used, the top match includes TFEC, a bHLH family member (Fig. 4F). Taken together, our data indicate that Bhlhe40 is critical for host protection against viral infection by acting as a direct driver of the proinflammatory/immunoregulatory cytokine IFN-γ in NK cells.

FIGURE 4. Bhlhe40 binds the Ifng loci of NK cells following IL-12 plus IL-18 stimulation.

(A) Mean normalized counts heatmap of Bhlhe40 binding in NK cells without stimulation or following IL-12 + IL-18 for 3 hours, peak-centered and flanked 1kb each in 5’ and 3’ directions (n=316) with bin length of 50bp. Signals normalized using spike-in DNA were averaged per bin between each sample condition replicates (n=3).

(B) Venn diagram of peaks called within the peak atlas between the two sample conditions (n=316).

(C) Volcano plot of differential binding analysis between IL-12 + IL-18 stimulation and unstimulated samples. Red points represent significant differentially binding peaks (FDR < 0.05). Horizontal dashed line shows guide for intercept at −log₁₀(0.05) and vertical for −1 and 1. Select peaks and their annotated gene names are highlighted and labeled.

(D) Bhlhe40 CUT&RUN tracks show normalized signal pileup for the Ifng locus. Signals normalized using spike-in DNA were averaged per bin (bin length=10bp) between each sample condition replicates (n=3).

(E) Pie chart of annotated peak type composition for peaks that significantly increase binding upon IL-12 + IL-18 stimulation (n=281). 208 (74.0%) peaks were assigned promoter, 41 (14.6%) peaks intergenic, 27 (9.6%) peaks intron, and 5 (1.8%) peaks exon.

(F) Enriched de novo motifs found by HOMER on peaks that significantly increase binding following IL-12 + IL-18 stimulation. Top 4 motifs ranked by P-value that exceeded HOMER motif score of 0.7 are shown.

Previous studies have identified that IL-12 and STAT4 signalling can drive multiple effector functions in NK cells during MCMV infection, including the secretion of IFN-γ and the prolific expansion of anti-viral “adaptive” NK cells (1). How these different processes are controlled by a single transcription factor STAT4, and whether these specific functions can be segregated in their regulation, remained incompletely understood. We previously identified that STAT4-mediated expression of Zbtb32, Runx1, Runx3, and IRF8 could specifically impact clonal proliferation but not IFN-γ secretion by antiviral NK cells (5, 6, 8), whereas STAT4-mediated upregulation of STAT5 could impact both processes (7). Here, we have discovered a cell-intrinsic role for the transcription factor Bhlhe40 in specifically controlling IFN-γ production but not proliferation of NK cells following viral infection, highlighting a mechanism that decouples STAT4-driven innate functions from STAT4-driven adaptive features.

Control of cytokine secretion by Bhlhe40 has also been observed in other cell types, but whether Bhlhe40 was directly regulated by STAT transcription factors in these settings were not elucidated (13, 24–28). Through epigenetic studies, we find that STAT4 is inducing the permissive histone H3K4me3 mark at the Bhlhe40 locus rather than mediating changes in chromatin accessibility, in contrast to many other genetic loci targeted by STAT4 (3). This distinct role for STAT4 in modulating the chromatin landscape at the Bhlhe40 locus critically drives its expression and subsequently protective IFN-γ from NK cells during viral infection. Thus, the mode of STAT4 activity can potentially show specificity dependent upon the range of co-transcription factors it induces in activated NK cells (given that STAT4 also binds to the Ifng loci (29)) and understanding such underlying mechanisms can aid in the goal of harnessing these potent effector cells in our battle against infectious diseases.

KEY POINTS:

• The transcription factor Bhlhe40 is highly induced in NK cells during MCMV infection.

• Bhlhe40 upregulation is driven by IL-12 via STAT4 binding of the Bhlhe40 promoter.

• Bhlhe40 is critical for protection against infection by promoting IFN-γ production.