Epigenetic Reprogramming of the Type III Interferon Response Potentiates Antiviral Activity and Suppresses Tumor Growth

<div><p>Type III interferon (IFN-λ) exhibits potent antiviral activity similar to IFN-α/β, but in contrast to the ubiquitous expression of the IFN-α/β receptor, the IFN-λ receptor is restricted to cells of epithelial origin. Despite the importance of IFN-λ in tissue-specific antiviral immunity, the molecular mechanisms responsible for this confined receptor expression remain elusive. Here, we demonstrate that the histone deacetylase (HDAC) repression machinery mediates transcriptional silencing of the unique IFN-λ receptor subunit (IFNLR1) in a cell-type-specific manner. Importantly, HDAC inhibitors elevate receptor expression and restore sensitivity to IFN-λ in previously nonresponsive cells, thereby enhancing protection against viral pathogens. In addition, blocking HDAC activity renders nonresponsive cell types susceptible to the pro-apoptotic activity of IFN-λ, revealing the combination of HDAC inhibitors and IFN-λ to be a potential antitumor strategy. These results demonstrate that the type III IFN response may be therapeutically harnessed by epigenetic rewiring of the IFN-λ receptor expression program.</p></div

IFNLR1 expression is silenced via HDAC-mediated repression and reactivated by 5azadC and MS-275 treatment.

<p>(A–C) ChIP analysis was performed on the <i>IFNLR1</i> promoter in Huh7 and U87 cells with AcH3, RNA Pol II, H3K27me3, and control IgG antibodies. (D) U87 cells were cultured with DMSO or 10 µM 5azadC for 72 h. For the latter, 2 µM SAHA, 1 µM MS-275, or 2 µM tubacin were added in the last 24 h. IFNLR1 and IL10RB expression was determined by RT-qPCR. (E) Primary astrocytes were cultured in the presence of DMSO or 10 µM 5azadC (72 h) and 1 µM MS-275 (24 h), and stimulated with PBS, 100 ng/ml IFN-λ1, or 500 U/ml IFN-α for 24 h. IFNLR1 and IL10RB expression was measured by RT-qPCR. In all panels, data represent the mean and SEM of at least three experiments.</p

<i>IFNLR1</i> promoter methylation negatively correlates with IFN-λ responsiveness.

<p>(A) Huh7 and U87 genomic DNA was digested with <i>McrBC</i> in the presence or absence of GTP and used as template for nested PCR with primers specific for the <i>IFNLR1</i> promoter. (B) Huh7, HepG2, U87, and U373 genomic DNA was subject to bisulfite conversion sequencing. Each circle represents one CpG dinucleotide, with filled circles indicating methylated motifs and open circles nonmethylated motifs. Each row represents an individual clone of the population. Lower numbers indicate relative distance to the TSS. (C) Quantification of the methylation status on both CpG islands in (B). (D) U87 cells were cultured in the presence of vehicle control DMSO, 3 µM, or 10 µM 5azadC for 72 h. IFNLR1 and IL10RB expression was examined by RT-qPCR. In all panels, data represent the mean and standard error of the mean (SEM) of at least three experiments.</p

IFN-λ protects U87 cells from VSV infection in the presence of small-molecule inhibitors.

<p>(A) U87 cells were treated with DMSO or inhibitors, stimulated with 100 ng/ml IFN-λ1 or 500 U/ml IFN-β for 24 h, and infected with VSV-GFP (MOI = 0.1) for 16 h. GFP expression was monitored by fluorescence microscopy (representative images are shown; 40× magnification). (B) Lysates from infected U87 cells following the indicated treatment were used for WB using indicated antibodies. G, glycoprotein; N, nucleoprotein; P, phosphoprotein; M, matrix protein. (C) Cytolytic effects were measured in U87 cells following the indicated treatment and infection with VSV (MOI = 1) for 24 h. Viable cells were stained with crystal violet. (D) VSV titers in infected U87 cells were determined by a standard plaque assay. In all panels, data represent the mean and SEM of at least three experiments.</p

Combination of MS-275 and IFN-λ inhibits cancer proliferation by elevated rate of apoptosis.

<p>(A) U87 cells were cultured in the presence of DMSO, 1 µM MS-275 alone, 100 ng/ml IFN-λ1 alone, or both for the course of 4 d. Cell numbers were manually determined by hemacytometer counting at the indicated time points. (B, F) Cell proliferation of U87 cells or U87 spheroids in 3D culture with indicated treatment were performed using the WST-1 assay, which measures active cellular metabolism. (C) U87 spheroid formation in 3D culture was photographed at day 14 in culture (representative images are shown; 200× magnification). (D–E) Quantification of the relative sizes and numbers of U87 spheroids in (C). (G) Cell cycle analysis was performed in U87 cells with indicated treatment using propidium iodide staining. Numbers in the histogram show fractions (percent) of sub-G1, N, 2N, and polyploidy from left to right. (H) U87 cells with indicated treatment were stained with Annexin V-FITC and 7-AAD. Numbers indicate the percentage of FITC-positive cells (upper left quadrant). FITC, fluorescein isothiocyanate; 7-AAD, 7-Aminoactinomycin. In all panels, data represent the mean and SEM of at least three experiments.</p

5azadC and MS-275 open <i>IFNLR1</i> promoter chromatin and increase IFN-λ sensitivity in nonresponsive cells.

<p>(A–C) ChIP analysis was performed on the <i>IFNLR1</i> promoter in 5azadC and MS-275-treated U87 cells with AcH3, RNA Pol II, NF-YC, and control IgG antibodies. (D) U87 cells were preincubated with DMSO or small-molecule inhibitors and stimulated with or without 10 ng/ml or 100 ng/ml of IFN-λ1/2/3 for 6 h. Lysates were used for WB using the indicated antibodies. (E–F) MX1 expression was measured by RT-qPCR in U87 cells and primary astrocytes following the indicated treatment. In all panels, data represent the mean and SEM of at least three experiments.</p

Molecular mechanism of differential IFNLR1 expression.

<p>Epigenetic silencing is the major mechanism that restricts IFNLR1 expression in IFN-λ nonresponsive cell types. In the low-expressing cells (red background), the <i>IFNLR1</i> promoter is repressed through DNA hypermethylation, histone hypoacetylation, and loss of affinity for TFs and RNA Polymerase II complexes. In contrast, the <i>IFNLR1</i> promoter is associated with DNA hypomethylation, histone acetylation, and binding of activating TF (represented by trimeric NF-Y) and RNA Polymerase II in high-expressing cells (green background). Importantly, sensitivity to IFN-λ can be regained by blocking DNMT and HDAC activities with 5azadC and MS-275, which epigenetically reconfigure the promoter chromatin structure. Gray line, <i>IFNLR1</i> promoter DNA; red filled circles, H3K27 trimethylation; green filled circles, histone H3 acetylation; black filled circle, methylated CpG dinucleotides; white filled circle, unmethylated CpG dinucleotides.</p

IFN-λ protects primary astrocytes from HSV-1 infection in the presence of small-molecule inhibitors.

<p>(A) Primary human astrocytes were treated with DMSO or inhibitors, stimulated with 100 ng/ml IFN-λ1 or 500 U/ml IFN-β for 24 h, and infected with HSV-1-GFP (MOI = 1). At 12 h postinfection, GFP expression was analyzed by flow cytometry. (B) Quantification of the percentage of GFP positive cells and mean fluorescence intensity (MFI) in (A). (C) Expression of HSV-1 genes, including ICP27 (immediate early), UL30 (early), VP16 (leaky-late), and UL36 (true-late), was measured by RT-qPCR. (D) Expression of MX1 and ISG15 was measured by RT-qPCR. (E) Lysates from infected astrocytes were used for WB using indicated antibodies. In all panels, data represent the mean and SEM of at least three experiments.</p

Macrophages transfer mitochondria to sensory neurons to resolve inflammatory pain

The current paradigm is that inflammatory pain passively resolves following the cessation of inflammation. Yet, in a substantial proportion of patients with inflammatory diseases, resolution of inflammation is not sufficient to resolve pain, resulting in chronic pain. Mechanistic insight into how inflammatory pain is resolved is lacking. Here, we show that macrophages actively control resolution of inflammatory pain remotely from the site of inflammation by transferring mitochondria to sensory neurons. During resolution of inflammatory pain in mice, M2-like macrophages infiltrate the dorsal root ganglia that contain the somata of sensory neurons, concurrent with the recovery of oxidative phosphorylation in sensory neurons. The resolution of pain and the transfer of mitochondria requires expression of CD200 receptor (CD200R) on macrophages and the non-canonical CD200R-ligand iSec1 on sensory neurons. Our data reveal a novel mechanism for active resolution of inflammatory pain