Phenylephrine (PE) dose response (percent vessel constriction) from 10⁻⁹M to 10⁻⁵M at zero (n = 5), three (n = 4), and seven (n = 4) days p.i. in TNFα^-/- mice in comparison to WT C57 BL/6 mice (n = 5) (A).

<p>Representative images of lymph node arterioles following at day zero p.i. prior to PE application and following superfusion of 10⁻⁹, 10⁻⁷, and 10⁻⁵M PE (B). In each image the arteriole is shown to the upper right of the LN venule and indicated by a green arrow (C). Representative images of H&E stained arteriole cross-sections in TNFα^-/- mice zero and seven days p.i. Percentage of vessel constriction in TNFα^-/- mice in response to perfusion of 100μM L-NAME at days zero (n = 5), three (n = 4), and seven (n = 4) following infection (D). Data represents mean vessel constriction (A, C) ± SEM where one arteriole was studied per animal. *p<0.05, **p<0.01, and ***p<0.001.</p

Resting and maximal (+Nifedapine) arteriole diameter at days zero and seven after i.vag. infection in eNOS^-/- mice with TK^-HSV-2 (A).

<p>Representative images of resting and maximal diameter eNOS^-/- arterioles at days zero and seven after infection where arterioles are indicated by green arrows (B). Resting and maximal (+SNP) arteriole diameter at days zero and seven after i.vag. infection with Tk^-HSV-2 in mice treated systemically with L-NAME (C). Resting and maximal vessel diameters (% of uninfected) of WT, eNOS^-/- and L-NAME treated mice. For eNOS^-/- n = 4 and 5 respectively for days zero and seven vessel diameters, and n = 4 for all PE data points (D). For L-NAME treatment, n = 4 for all data points. For WT n = 5 at day seven p.i.. In all cases, one arteriole was studied per animal. *p<0.05, **p<0.01, and ***p<0.001.</p

Resting and maximal (+30µM Nifedapine) arteriole diameter at zero (n = 4) and seven (n = 5) days after i.vag. infection in mast cell deficient mice (A).

<p>Comparison of maximal arteriole diameter in wild-type (C57 BL/6) (n = 5) and mast cell deficient mice (n = 5) at day seven following infection (B). Representative images of lymph nodes from mast cell deficient mice at days zero and seven following infection (C). Data represents mean vessel diameter (A, B) ± SEM where one arteriole was studied per animal. *p<0.05, **p<0.01, and ***p<0.001.</p

Representative images of isolated eNOS^-/- lymph nodes following infection at days zero, one, five, and seven after infection (A).

<p>Lymph node weights of isolated eNOS^-/- lymph nodes following infection at days zero and seven (B).</p

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 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

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