Effects of extrinsic and intrinsic factors on age-associated decline in natural killer cell activity during primary influenza infection

The fastest growing segment of the U.S. population is the group of individuals over the age of 65 years. Aging is accompanied by numerous changes in immune function and is associated with an increased morbidity as well as mortality to virus infections. Influenza is a major public health concern. Each year seasonal influenza disease occurs with a number of hospitalizations. In addition, influenza and its secondary complications rank as the 4th leading cause of deaths among the elderly. An age-associated decrease and delay in cell-mediated response, a reduction in the proliferative T cell response, and an altered cytokine production in response to influenza vaccination or infection have been reported. These are parameters of adaptive immune response. However, study of innate immune response against primary influenza infection has lacked attention. Natural killer (NK) cells are an integral part of innate immunity and play a key role in controlling viral infections and foreign pathogens. Here we report that NK cells are essential for controlling influenza virus replication early during infection independent of age. Importantly, there is an age-associated impairment in influenza-inducible NK cytotoxicity and a lack of increase in NK cells at the site of infection. In addition, aged mice produced less interleukin (IL)-15 and interferon (IFN)-γ compared to young mice following influenza infection. While enhancement of NK cytotoxicity in response to stimulation with IL-2 or IL-15 in vitro was comparable in young and aged mice, aged mice demonstrated significantly lower enhancement of granzyme B expression in response to IL-2 or IL-15 and of IFN-γ production in response to IL-15. These results demonstrate that age-associated impairment in influenza-inducible NK cytotoxicity may be caused by a combination of extrinsic influences, i.e. limited NK stimulating cytokines in the aged environment, and intrinsic defects, i.e. a limited inability of NK cells of aged mice to respond to cytokines and possibly other stimuli. These findings suggest that NK cells may be therapeutic targets for an intervention to restore or boost inducible NK cell cytotoxicity during primary viral infection.Ph.D., Biology -- Drexel University, 201

The interferon-inducible antiviral protein Daxx is not essential for interferon-mediated protection against avian sarcoma virus

BACKGROUND: The antiviral protein Daxx acts as a restriction factor of avian sarcoma virus (ASV; Retroviridae) in mammalian cells by promoting epigenetic silencing of integrated proviral DNA. Although Daxx is encoded by a type I (α/β) interferon-stimulated gene, the requirement for Daxx in the interferon anti-retroviral response has not been elucidated. In this report, we describe the results of experiments designed to investigate the role of Daxx in the type I interferon-induced anti-ASV response. FINDINGS: Using an ASV reporter system, we show that type I interferons are potent inhibitors of ASV replication. We demonstrate that, while Daxx is necessary to silence ASV gene expression in the absence of interferons, type I interferons are fully-capable of inducing an antiviral state in the absence of Daxx. CONCLUSIONS: These results provide evidence that Daxx is not essential for the anti-ASV interferon response in mammalian cells, and that interferons deploy multiple, redundant antiviral mechanisms to protect cells from ASV

Structure Guided Design of Potent and Selective Ponatinib-Based Hybrid Inhibitors for RIPK1

SummaryRIPK1 and RIPK3, two closely related RIPK family members, have emerged as important regulators of pathologic cell death and inflammation. In the current work, we report that the Bcr-Abl inhibitor and anti-leukemia agent ponatinib is also a first-in-class dual inhibitor of RIPK1 and RIPK3. Ponatinib potently inhibited multiple paradigms of RIPK1- and RIPK3-dependent cell death and inflammatory tumor necrosis factor alpha (TNF-α) gene transcription. We further describe design strategies that utilize the ponatinib scaffold to develop two classes of inhibitors (CS and PN series), each with greatly improved selectivity for RIPK1. In particular, we detail the development of PN10, a highly potent and selective “hybrid” RIPK1 inhibitor, capturing the best properties of two different allosteric RIPK1 inhibitors, ponatinib and necrostatin-1. Finally, we show that RIPK1 inhibitors from both classes are powerful blockers of TNF-induced injury in vivo. Altogether, these findings outline promising candidate molecules and design approaches for targeting RIPK1- and RIPK3-driven inflammatory pathologies

Interferon Impedes an Early Step of Hepatitis Delta Virus Infection

Hepatitis delta virus (HDV) infects hepatocytes, the major cell type of the liver. Infection of the liver may be either transient or chronic. The prognosis for patients with chronic HDV infection is poor, with a high risk of cirrhosis and hepatocellular carcinoma. The best antiviral therapy is weekly administration for at least one year of high doses of interferon alpha. This efficacy of interferon therapy has been puzzling in that HDV replication in transfected cell lines is reported as insensitive to administration of interferon alpha or gamma. Similarly, this study shows that even when an interferon response was induced by transfection of poly(IC) into a cell line, HDV RNA accumulation was only modestly inhibited. However, when the HDV replication was initiated by infection of primary human hepatocytes, simultaneous addition of interferons alpha or gamma at 600 units/ml, a concentration comparable to that achieved in treated patients, the subsequent HDV RNA accumulation was inhibited by at least 80%. These interferon treatments were shown to produce significant time-dependent increases of host response proteins such as for Stat-1, phosphoStat-1, Mx1/2/3 and PKR, and yet interferon pretreatment of hepatocytes did not confer an increased inhibition of HDV replication over interferon treatment at the time of (or after) infection. These and other data support the interpretation that interferon action against HDV replication can occur and is largely mediated at the level of entry into primary human hepatocytes. Thus in vivo, the success of long-term interferon therapy for chronic HDV, may likewise involve blocking HDV spread by interfering with the initiation of productive infection of naïve hepatocytes

Distinct Roles for the NF-κB RelA Subunit during Antiviral Innate Immune Responses▿ †

Production of type I interferons (IFNs; prominently, IFN-α/β) following virus infection is a pivotal antiviral innate immune response in higher vertebrates. The synthesis of IFN-β proceeds via the virus-induced assembly of the transcription factors IRF-3/7, ATF-2/c-Jun, and NF-κB on the ifnβ promoter. Surprisingly, recent data indicate that the NF-κB subunit RelA is not essential for virus-stimulated ifnβ expression. Here, we show that RelA instead sustains autocrine IFN-β signaling prior to infection. In the absence of RelA, virus infection results in significantly delayed ifnβ induction and consequently defective secondary antiviral gene expression. While RelA is not required for ifnβ expression after infection, it is nonetheless essential for fully one-fourth of double-stranded RNA (dsRNA)-activated genes, including several mediators of inflammation and immune cell recruitment. Further, RelA directly regulates a small subset of interferon-stimulated genes (ISGs). Finally, RelA also protects cells from dsRNA-triggered RIP1-dependent programmed necrosis. Taken together, our findings suggest distinct roles for RelA in antiviral innate immunity: RelA maintains autocrine IFN-β signaling in uninfected cells, facilitates inflammatory and adaptive immune responses following infection, and promotes infected-cell survival during this process

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

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

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

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