type-I IFN Response Research Articles

An HD-domain phosphodiesterase mediates cooperative hydrolysis of c-di-AMP to affect bacterial growth and virulence.

The nucleotide cyclic di-3',5'- adenosine monophosphate (c-di-AMP) was recently identified as an essential and widespread second messenger in bacterial signaling. Among c-di-AMP-producing bacteria, altered nucleotide levels result in several physiological defects and attenuated virulence. Thus, a detailed molecular understanding of c-di-AMP metabolism is of both fundamental and practical interest. Currently, c-di-AMP degradation is recognized solely among DHH-DHHA1 domain-containing phosphodiesterases. Using chemical proteomics, we identified the Listeria monocytogenes protein PgpH as a molecular target of c-di-AMP. Biochemical and structural studies revealed that the PgpH His-Asp (HD) domain bound c-di-AMP with high affinity and specifically hydrolyzed this nucleotide to 5'-pApA. PgpH hydrolysis activity was inhibited by ppGpp, indicating a cross-talk between c-di-AMP signaling and the stringent response. Genetic analyses supported coordinated regulation of c-di-AMP levels in and out of the host. Intriguingly, a L. monocytogenes mutant that lacks c-di-AMP phosphodiesterases exhibited elevated c-di-AMP levels, hyperinduced a host type-I IFN response, and was significantly attenuated for infection. Furthermore, PgpH homologs, which belong to the 7TMR-HD family, are widespread among hundreds of c-di-AMP synthesizing microorganisms. Thus, PgpH represents a broadly conserved class of c-di-AMP phosphodiesterase with possibly other physiological functions in this crucial signaling network.

Type-I interferon response affects an inoculation dose-independent mortality in mice following Japanese encephalitis virus infection.

BackgroundThe laboratory mouse model is commonly employed to study the pathogenesis of encephalitic flaviviruses such as Japanese encephalitis virus (JEV). However, it is known that some strains of these viruses do not elicit a typical mortality dose response curve from this organism after peripheral infection and the reason for it has not yet been fully understood. It is suggested that induction of more vigorous Type-I IFN (IFN-I) response might control early virus dissemination following increasing infectious challenge doses of the virus. Thus, the objective of this study was to examine this suggested role of IFN-I in the mortality of mice infected with various doses of JEV.MethodsInbred 129 mice and their IFNAR KO (A129) mice were subcutaneously inoculated with 100, 102, 104 or 106 pfu of JaOArS982 strain of JEV. Mice were weighed daily and observed for clinical signs. Virus titers in the brains and spleens of JEV-infected mice were determined by plaque forming assays. The upregulated mRNA levels of genes related to IFN-I response of mice were examined by real-time PCR.ResultsThe mortality rates of 129 mice infected with JaOArS982 did not significantly increase despite the increase in inoculation dose and no significant difference of viral loads was observed between their brains. However, there was clear elevation of the mRNA levels of interferon regulatory factor (IRF)3, IRF7, IRF9, MDA5 and RIG-I at 24 hours post-infection depending on the inoculation dose. In A129 mice, length of survival days and the viral loads of spleen and brain were observed to be inoculation dose-dependent.ConclusionsFrom these results, it is suggested that early IFN-I response elicited by high inoculation doses of JEV provides an anti-viral effect during the early phase of infection. Accordingly, virus replication is counteracted by IFN-I response at each increasing inoculation dose resulting in the interference of impending severe disease course or fatal outcome; hence, this might explain the inoculation dose-independent mortality in mice caused by Japanese encephalitis virus.

IL-7/Lymphopenia enhanced Type-I IFN response by modulating STAT-1 levels: its impact in CD4 T cell homeostasis (HUM8P.338)

Abstract HIV infection is associated with chronic immune activation and alteration of T cell homeostasis (CD4 T cell depletion and CD8 T cell expansion). While the acute CD4 depletion observed in the initial phase of HIV infection is likely due to direct cytopatic effects of the virus, the mechanism/s underlying the steady decline of the CD4 T cell pool during the chronic phase of infection are unclear and are felt to be associated with “immune activation”. In the current study, we addressed the impact of the combination of two distinct forces: homeostatic (CD4 T cell depletion) and inflammatory (HIV-driven IFN-α) on T cell homeostasis. We present evidences that IL-7 and lymphopenia enhanced CD4 T cell responsiveness to IFN-α by modulating expression of the Signal Transducers and Activators of Transcription (STAT) 1, 2 and 3. In a murine model, CD4 T cell depletion and CD8 T cell expansion were observed in a lymphopenic host chronically treated with IFN-α. These findings suggest that a synergistic interaction between lymphopenia and IFN-α may play a role in the pathogenesis of HIV infection. The analysis of this pathway may contribute to the development of new strategies to reverse the dysregulation of the T cell pools seen in patients with HIV infection.

Short-Term Intratumoral Interleukin-12 Expressed from an Alphaviral Vector Is Sufficient to Induce an Efficient Antitumoral Response Against Spontaneous Hepatocellular Carcinomas

Interleukin-12 (IL-12) is an immunostimulatory cytokine that has shown strong antitumor effects in animal models of liver cancer. In order to overcome the severe toxicity associated with its systemic administration, we had previously tested different strategies based on IL-12 gene transfer to tumor cells or to the surrounding liver tissue. We obtained promising results both with a recombinant Semliki Forest virus (SFV) vector expressing high levels of IL-12 (SFV-IL-12) after intratumoral injection and with a plasmid vector [pTonL2(T)-mIL12] that allows liver-specific and inducible IL-12 expression. The aim of the present study was to compare the antitumor responses induced by both systems in a clinically relevant animal model of hepatocellular carcinoma (HCC) developed in L-PK/c-myc transgenic mice. These animals overexpress the c-myc oncogene in their livers, giving rise to spontaneous hepatic tumors with latency, histopathology, and genetic characteristics similar to human HCCs. We observed that intratumoral inoculation of SFV-IL-12 induced growth arrest in most tumors, providing 100% survival rate, in contrast to no survival in control animals. Similar results were obtained with hydrodynamic injection of pTonL2(T)-mIL12 after long-term induction of IL-12 expression in the liver. However, tumor arrest was less evident in plasmid-treated mice and the survival rate was slightly lower, despite higher and more sustained levels of IL-12 and IFN-γ in serum. The fact that SFV-IL-12 was able to induce both apoptosis and a type-I IFN response specifically in the tumor could explain why short-term IL-12 expression from this vector was sufficient to mediate an antitumoral response comparable with long-term IL-12 expression driven by pTonL2(T)-mIL12. Since SFV-IL-12 could reduce the possible toxicity associated with long-term IL-12 expression, we believe that this vector could have a potential application for HCC gene therapy.

IFN-α suppresses HTLV-1 expression via PKR in infected cells and renders them susceptible to AZT through p53 activation in AZT/IFN-α treatment

HTLV-1 expression is maintained at low levels in vivo through unknown mechanisms. Interferon-a (IFN-a) has been used in combination with zidovudin (AZT) to treat adult T-cell leukemia/lymphoma (ATL) patients with favorable effects, although the mechanism is also unclear. We previously reported that HTLV-1 gene expression could be markedly suppressed by stromal cells through a type-I IFN response in IL-2-dependent HTLV-1-infected T-cells (ILTs) derived from ATL patients. Here, we found that treatment with IFN-a alone produced suppressive effects on HTLV-1 gene expression in ILTs and also on the spontaneous HTLV-1 induction in short-term cultured primary ATL cells. Following IFN-a treatment, the levels of intracellular Tax protein decreased earlier than those of HTLV-1 mRNA in ILTs. An RNA-dependent protein kinase (PKR) inhibitor reversed IFN-a-mediated suppression of Tax in ILTs, suggesting the involvement of a post-transcriptional mechanism mediated by PKR in the suppression. IFN-a also induced cell cycle arrest at the G0/G1 phase in ILTs. We further found that AZT combined with IFN-a induced cell apoptosis in ILTs, associated with phosphorylation of p53 and enhanced expression of genes responsive to p53, whereas AZT alone did not affect cell viability or viral expression. Our results suggest that ILTs and ATL cells retain susceptibility to type-I IFNs, which suppress HTLV-1 gene expression primarily at a posttranscriptional level and potentially allow for signaling through the p53 pathway in combination with AZT. These findings would partly explain how HTLV-1 gene expression is regulated in vivo, and how AZT/IFN-a produces therapeutic effects in ATL.

Modulation of type-I IFN response by equine herpesvirus-1

Modulation of type-I IFN response by equine herpesvirus-1

CS02-3 In vivo IL-7 exposure of CD4 T cells during HIV induced lymphopenia leads to an enhanced Type-I IFN response

CS02-3 In vivo IL-7 exposure of CD4 T cells during HIV induced lymphopenia leads to an enhanced Type-I IFN response

RNA Interference Shows Critical Requirement for NF-κB p50 in the Production of IL-12 by Human Dendritic Cells

Specific NF-kappaB/Rel proteins regulate murine dendritic cell (DC) survival, differentiation, and activation, but little is known of their role in human cells because of limited loss-of-function analyses. RNA interference (RNAi) is a mechanism to effectively silence gene expression via sequence-specific double-stranded small interfering RNAs (siRNAs). RNAi was used to assess the role of the p50 (NF-kappaB1) protein in the maturation and activation of cultured human monocyte-derived DC (MoDC). Transfection of cultured MoDC with siRNAs reduced p50 mRNA and protein levels in a specific, dose-dependent, and time-dependent manner. Basal or maturation-induced expression of HLA-DR and costimulatory molecules were not affected, whereas transcription of the IL-12 p40 gene and the secretion of IL-12alphabeta were reduced. Such MoDC induced less IFN-gamma production by T cells in MLR. This is the first report of RNAi-induced phenotype in human primary DC with a method that caused no measurable toxicity or type-I IFN response. siRNAs appear useful for the study of signaling pathways in immune cells, revealing a pivotal requirement for p50 in MoDC for IL-12 production and induction of optimal type-1 immune responses.