Temporally distinct roles for NF-κB in antivirus innate immune responses.

<p>(A) In uninfected cells, NF-κB cycles robustly through the nucleus to maintain constitutive expression of basal <i>ifnβ</i> and sustain sutocrine IFN-β signaling. (B) Early in an infection, NF-κB cooperates with ATF-2/c-Jun and IRF-3 to recruit the transcription co-activator CBP/p300 to the <i>ifnβ</i> enhancer. (C) Later in an infection, IRF-3/7 powers expression of <i>ifnβ</i>, and NF-κB is rendered redundant in the <i>ifnβ</i> enhanceosome. (D) NF-κB then switches to regulating a distinct subset of non-IFN genes, including those involved in inflammation and cell survival. The relative importance of each transcription factor in driving gene expression during a particular stage of the immune response is indicated by the intensity of its color, with darker shades representing essential functions, and lighter shades indicating reduced roles.</p

Time-dependent induction by interferons alpha and gamma of innate immune response proteins in primary hepatocytes.

<p>Hepatocytes were exposed to either interferons alpha or gamma at 600 units/ml. At time points out to 24 h, as indicated, total cell protein was extracted and analyzed by immunoblot to detect the indicated host proteins. Note that after 2 hours of treatment with interferon gamma, the pSTAT1 increases but not the total STAT1; this is because only a small fraction of the total STAT1 undergoes phosphorylation.</p

Representation of time-lines for exposure of primary hepatocytes to HDV and potential inhibitors.

<p>For the five time-lines shown the open box indicates the period of HDV exposure and the shaded box the exposure to inhibitor. The effects on HDV replication of such treatments with three different inhibitors are summarized in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0022415#pone-0022415-t001" target="_blank">Table 1</a>.</p

HDV replication is inhibited by application of interferons alpha or gamma.

<p>Times of addition of inhibitors are measured relative to 3-h of virus exposure. The preS1 peptide was present at 50 nM while the interferons were at 600 U/ml. All assays were performed in at least triplicate, and the data expressed relative to control hepatocytes that were not treated with inhibitors. The indicated standard error of the mean includes the errors in both the control and the treated cultures.</p

Dose response of HDV and VSV infections to interferon-alpha and preS1 peptide.

<p>For HDV infections the inhibitors were present from 24 h prior to infection and during the 16 h of exposure to virus. Replication was assayed at 6 days post-infection by realtime PCR, as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0022415#pone-0022415-g001" target="_blank">Fig. 1C</a>. The assays were performed in triplicate, and average values are expressed as a percentage relative to the untreated controls, with the indicated standard error of the mean. For the VSV infections, the interferon was only present during the 16 h of virus exposure. VSV replication was assayed at 16 h by counting GFP positive cells. The values are expressed relative to untreated controls and the errors indicate the standard deviation as the square root of the mean. Interferon-alpha concentrations are expressed here as molarities, with 600 units/ml = 0.12 nM. The preS1 peptide, like interferon-alpha, can inhibit HDV infection, but the needed molarity is 300-times more. Interferon-alpha can inhibit VSV infection but the concentration is 1,000-time less than that needed to inhibit HDV.</p

Meta-Analysis Identifies NF-κB as a Therapeutic Target in Renal Cancer

<div><p>Objective</p><p>To determine the expression patterns of NF-κB regulators and target genes in clear cell renal cell carcinoma (ccRCC), their correlation with von Hippel Lindau (VHL) mutational status, and their association with survival outcomes.</p> <p>Methods</p><p>Meta-analyses were carried out on published ccRCC gene expression datasets by RankProd, a non-parametric statistical method. DEGs with a False Discovery Rate of < 0.05 by this method were considered significant, and intersected with a curated list of NF-κB regulators and targets to determine the nature and extent of NF-κB deregulation in ccRCC.</p> <p>Results</p><p>A highly-disproportionate fraction (~40%; <i>p</i> < 0.001) of NF-κB regulators and target genes were found to be up-regulated in ccRCC, indicative of elevated NF-κB activity in this cancer. A subset of these genes, comprising a key NF-κB regulator (<i>IKBKB</i>) and established mediators of the NF-κB cell-survival and pro-inflammatory responses (<i>MMP9</i>, <i>PSMB9</i>, and <i>SOD2</i>), correlated with higher relative risk, poorer prognosis, and reduced overall patient survival. Surprisingly, levels of several interferon regulatory factors (IRFs) and interferon target genes were also elevated in ccRCC, indicating that an ‘interferon signature’ may represent a novel feature of this disease. Loss of VHL gene expression correlated strongly with the appearance of NF-κB- and interferon gene signatures in both familial and sporadic cases of ccRCC. As NF-κB controls expression of key interferon signaling nodes, our results suggest a causal link between VHL loss, elevated NF-κB activity, and the appearance of an interferon signature during ccRCC tumorigenesis.</p> <p>Conclusions</p><p>These findings identify NF-κB and interferon signatures as clinical features of ccRCC, provide strong rationale for the incorporation of NF-κB inhibitors and/or and the exploitation of interferon signaling in the treatment of ccRCC, and supply new NF-κB targets for potential therapeutic intervention in this currently-incurable malignancy.</p> </div

A subset of NF-κB regulators and target genes correlate with poor outcome in <b>ccRCC</b>.

<p>Kaplan-Meier survival curves for genes in the NF-κB signature whose increased expression levels significantly correlate with poorer overall survival outcome are shown. Individual gene expression profiles were dichotomized by median split into ‘high’ (red) or ‘low’ (blue) expression groups. Gene names are indicated above each graph. Please see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0076746#pone.0076746.s007" target="_blank">Table S5</a> for <i>p</i>-values.</p

RIPK3 Is Largely Dispensable for RIG-I-Like Receptor- and Type I Interferon-Driven Transcriptional Responses to Influenza A Virus in Murine Fibroblasts

<div><p>The kinase RIPK3 is a key regulator of cell death responses to a growing number of viral and microbial agents. We have found that influenza A virus (IAV)-mediated cell death is largely reliant on RIPK3 and that RIPK3-deficient mice are notably more susceptible to lethal infection by IAV than their wild-type counterparts. Recent studies demonstrate that RIPK3 also participates in regulating gene transcription programs during host pro-inflammatory and innate-immune responses, indicating that this kinase is not solely an inducer of cell death and that RIPK3-driven transcriptional responses may collaborate with cell death in promoting clearance of IAV. Here, we carried out DNA microarray analyses to determine the contribution of RIPK3 to the IAV-elicited host transcriptional response. We report that RIPK3 does not contribute significantly to the RLR-activated transcriptome or to the induction of type I IFN genes, although, interestingly, IFN-β production at a post-transcriptional step was modestly attenuated in IAV-infected <i>ripk3</i>^<i>-/-</i> fibroblasts. Overall, RIPK3 regulated the expression of <5% of the IAV-induced transcriptome, and no genes were found to be obligate RIPK3 targets. IFN-β signaling was also found to be largely normal in the absence of RIPK3. Together, these results indicate that RIPK3 is not essential for the host antiviral transcriptional response to IAV in murine fibroblasts.</p></div

VHL mutational status correlates with expression of NF-κB and IFN signatures.

<p>Heatmaps showing fold-changes in expression levels of NF-κB signature genes (a) or IFN signature genes (b) between VHL+/- samples from cases of familial VHL disease compared to <i>VHL</i> +/+ normal renal epithelium (column 1) ; VHL-/- cases of familial ccRCC compared to normal renal tissue (column 2) ; or VHL-/- cases of sporadic ccRCC compared to normal renal tissue (column 3). Heat bar = fold-change (log₂ scale).</p

Model linking pVHL loss to NF-κB and IFN gene signatures.

<p>(a) Heatmap showing basal expression of the IFN gene signature in RelA+/+ and RelA-/- MEFs. Heat bar = fold-change (log₂ scale). Expression levels in RelA+/+ were arbitrarily set to 1 (beige). Note that ~80% of IFN signature genes are expressed at lower basal levels in cells lacking RelA than in controls, indicative of a role for NF-κB in controlling ISG expression. (b) Schematic depicting the model presented in this study. ccRCC cells display elevated levels of NF-κB activity, perhaps as a result of result of constitutive EGFR or CARD9 activity stemming from loss of pVHL, that drives expression of cell-survival, pro-inflammatory, NF-κB regulatory genes, as well as genes encoding key nodes of type I interferon signaling (IFN-β, IRFs). Type I IFNs produced in this manner then function in an autocrine fashion to induce the expression of an IFN gene signature.</p