Type I interferon-mediated immune response against influenza A virus is attenuated in the absence of p53

Influenza A virus (IAV) infection induces secretion of type I interferon (IFN) and activation of p53, which play essential roles in the host defense against tumor development and viral infection. In this study, we knocked down p53 expression by RNA interference. The expression levels of IFN-stimulated genes (ISGs) including IFN regulatory factor (IRF) 5, IRF9, ISG15, ISG20, guanylate-binding protein 1, retinoic acid-inducible gene-I and 2′-5′-oligoadenylate synthetase 1 were significantly attenuated in response to IAV infection and IFN-α stimulation in p53-knockdown cells. This attenuated expression of ISGs was associated with enhanced replication of IAV. Pretreatment of p53-knockdown cells with IFN-α failed to inhibit IAV replication, indicating impaired antiviral activity. These findings indicate that p53 plays an essential role in the enhancement of the type I IFN-mediated immune response against IAV infection.     

Interplay between interferon-stimulated gene 15/ISGylation and interferon gamma signaling in breast cancer cells

Interferon-stimulated gene 15 (ISG15) is a ubiquitin-like protein that conjugates to its target proteins to modify them through ISGylation, but the relevance of ISG15 expression and its effects have been not completely defined. Herein, we examined the interplay between ISG15/ISGylation and the interferon-gamma (IFN-γ) signaling pathway in mammary tumors and compared it with that in normal mammary tissues. Our results indicated that mammary tumors had higher levels of ISG15 mRNA and ISG15 protein than the adjacent normal mammary tissue. Furthermore, the expression of IFN-γ signaling components was altered in breast cancer. Interestingly, IFN-γ treatment induced morphological changes in MCF-7 and MDA-MB-231 breast cancer cell lines due to cytoskeletal reorganization. This cellular process seems to be related to the increase in ISGylation of cytoplasmic IQ Motif Containing GTPase Activating Protein 1 (IQGAP1). Interactome analysis also indicated that IFN-γ signaling and the ISGylation system are associated with several proteins implicated in cytoskeletal remodeling, including IQGAP1. Thus, ISG15 may present a potential biomarker for breast cancer, and IFN-γ signaling and protein ISGylation may participate in the regulation of the cytoskeleton in breast cancer cells.     

乳腺癌雌激素受体β不同亚型表达载体的构建及其转录活性分析

目的：构建人乳腺癌中雌激素受体B（ERβ）3种亚型ERβ1、ERβ2和ERβ5的真核表达载体,测定不同亚型ERβ的转录活性。方法：以乳腺癌细胞MCF7的cDNA为模板,PCR扩增ERβ1、ERβ2和ERβ5万基因,分别克隆到pXJ40-Mye载体,Western印迹检测克隆载体在293T细胞内的表达;将上述载体与含雌激素应答元件（ERE）的萤光素酶（Luc）报告基因载体（ERE—luc）共转293T细胞,测定各亚型ER／3的转录活性。结果：构建了Myc—ERβ1、Myc—ERβ2和Mye—ERβ5表达载体,转录活性结果显示上述表达载体均具有活性,雌激素可升高ERβ5的转录活性,不能升高Eβ2和ER5的转录活性。结论：该研究为进-步探讨不同亚型ERβ在乳腺癌中的功能奠定了基础。     

Psoralidin,a coumestan analogue,as a novel potent estrogen receptor signaling molecule isolated from Psoralea corylifolia

A novel biological activity of psoralidin as an agonist for both estrogen receptor (ER)α and ERβ agonist has been demonstrated in our study. Psoralidin has been characterized as a full ER agonist, which activates the classical ER-signaling pathway in both ER-positive human breast and endometrial cell lines as well as non-human cultured cells transiently expressing either ERα or ERβ. The estrogenic activity was determined using the relative expression levels of either reporter or the endogenous genes dependent on the agonist-bound ER to the estrogen response element (ERE). Psoralidin at 10 μM was able to induce the maximum reporter gene expression corresponding to that of E₂-treated cells and such activation of the ERE-reporter gene by psoralidin was completely abolished by the cotreatment of a pure ER antagonist, implying that the biological activities of psoralidin are mediated by ER. Psoralidin was also able to induce the endogenous estrogen-responsive gene, pS2, in human breast cancer cells MCF-7. It was observed that activation of the classical ER-signaling pathway by psoralidin is mediated via induction of ER conformation by psoralidin and direct binding of the psoralidin–ER complex to the EREs present in the promoter region of estrogen-responsive genes, as shown by chromatin immunoprecipitation assay results. Finally, molecular docking of psoralidin to the ligand binding pocket of the ERα showed that psoralidin is able to mimic the binding interactions of E₂, and thus, it could act as an ER agonist in the cellular environment.     

Evidence of vitamin D and interferon-β cross-talk in human osteoblasts with 1α,25-dihydroxyvitamin D3 being dominant over interferon-β in stimulating mineralization

It is well established that 1α-25-dihydroxyvitamin D3 (1,25D3) regulates osteoblast function and stimulates mineralization by human osteoblasts. The aim of this study was to identify processes underlying the 1,25D3 effects on mineralization. We started with gene expression profiling analyses of differentiating human pre-osteoblast treated with 1,25D3. Bioinformatic analyses showed interferon-related and -regulated genes (ISG) to be overrepresented in the set of 1,25D3-regulated genes. 1,25D3 down-regulated ISGs predominantly during the pre-mineralization period. This pointed to an interaction between the vitamin D and IFN signaling cascades in the regulation of osteoblast function. Separately, 1,25D3 enhances while IFNβ inhibits mineralization. Treatment of human osteoblasts with 1,25D3 and IFNβ showed that 1,25D3 completely overrules the IFNβ inhibition of mineralization. This was supported by analyses of extracellular matrix gene expression, showing a dominant effect of 1,25D3 over the inhibitory effect of IFNβ. We identified processes shared by IFNβ- and 1,25D3-mediated signaling by performing gene expression profiling during early osteoblast differentiation. Bioinformatic analyses revealed that genes being correlated or anti-correlated with interferon-induced protein with tetratricopeptide repeats 1 (IFIT1) were associated with osteoblast proliferation. In conclusion, the current study demonstrates a cross talk between 1,25D3 and IFNβ in osteoblast differentiation and bone formation/mineralization. The interaction is complex and depends on the process but importantly, 1,25D3 stimulation of mineralization is dominant over the inhibitory effect of IFNβ. These observations are of potential clinical relevance considering the impact of the immune system on bone metabolism in conditions such as rheumatoid arthritis.     

Major differences in the responses of primary human leukocyte subsets to IFN-beta

Treatment of cell lines with type I IFNs activates the formation of IFN-stimulated gene factor 3 (STAT1/STAT2/IFN regulatory factor-9), which induces the expression of many genes. To study this response in primary cells, we treated fresh human blood with IFN-β and used flow cytometry to analyze phosphorylated STAT1, STAT3, and STAT5 in CD4(+) and CD8(+) T cells, B cells, and monocytes. The activation of STAT1 was remarkably different among these leukocyte subsets. In contrast to monocytes and CD4(+) and CD8(+) T cells, few B cells activated STAT1 in response to IFN-β, a finding that could not be explained by decreased levels of IFNAR2 or STAT1 or enhanced levels of suppressor of cytokine signaling 1 or relevant protein tyrosine phosphatases in B cells. Microarray and real-time PCR analyses revealed the induction of STAT1-dependent proapoptotic mRNAs in monocytes but not in B cells. These data show that IFN-stimulated gene factor 3 or STAT1 homodimers are not the main activators of gene expression in primary B cells of healthy humans. Notably, in B cells and, especially in CD4(+) T cells, IFN-β activated STAT5 in addition to STAT3, with biological effects often opposite from those driven by activated STAT1. These data help to explain why IFN-β increases the survival of primary human B cells and CD4(+) T cells but enhances the apoptosis of monocytes, as well as to understand how leukocyte subsets are differentially affected by endogenous type I IFNs during viral or bacterial infections and by type I IFN treatment of patients with multiple sclerosis, hepatitis, or cancer.     

Indoleamine 2,3-dioxygenase mediates the antiviral effect of gamma interferon against hepatitis B virus in human hepatocyte-derived cells

Alpha interferon (IFN-α) is an approved medication for chronic hepatitis B. Gamma interferon (IFN-γ) is a key mediator of host innate and adaptive antiviral immunity against hepatitis B virus (HBV) infection in vivo. In an effort to elucidate the antiviral mechanism of these cytokines, 37 IFN-stimulated genes (ISGs), which are highly inducible in hepatocytes, were tested for their ability to inhibit HBV replication upon overexpression in human hepatoma cells. One ISG candidate, indoleamine 2,3-dioxygenase (IDO), an IFN-γ-induced enzyme catalyzing tryptophan degradation, efficiently reduced the level of intracellular HBV DNA without altering the steady-state level of viral RNA. Furthermore, expression of an enzymatically inactive IDO mutant did not inhibit HBV replication, and tryptophan supplementation in culture completely restored HBV replication in IDO-expressing cells, indicating that the antiviral effect elicited by IDO is mediated by tryptophan deprivation. Interestingly, IDO-mediated tryptophan deprivation preferentially inhibited viral protein translation and genome replication but did not significantly alter global cellular protein synthesis. Finally, tryptophan supplementation was able to completely restore HBV replication in IFN-γ- but not IFN-α-treated cells, which strongly argues that IDO is the primary mediator of IFN-γ-elicited antiviral response against HBV in human hepatocyte-derived cells.     

Actively replicating West Nile virus is resistant to cytoplasmic delivery of siRNA

Background

Recent studies have shown that gamma interferon (IFN-γ) synergizes with the innate IFNs (IFN-α and IFN-β) to inhibit herpes simplex virus type 1 (HSV-1) replication in vitro. To determine whether this phenomenon is shared by other herpesviruses, we investigated the effects of IFNs on human cytomegalovirus (HCMV) replication.

Results

We have found that as with HSV-1, IFN-γ synergizes with the innate IFNs (IFN-α/β) to potently inhibit HCMV replication in vitro. While pre-treatment of human foreskin fibroblasts (HFFs) with IFN-α, IFN-β or IFN-γ alone inhibited HCMV plaque formation by ~30 to 40-fold, treatment with IFN-α and IFN-γ or IFN-β and IFN-γ inhibited HCMV plaque formation by 163- and 662-fold, respectively. The generation of isobole plots verified that the observed inhibition of HCMV plaque formation and replication in HFFs by IFN-α/β and IFN-γ was a synergistic interaction. Additionally, real-time PCR analyses of the HCMV immediate early (IE) genes (IE1 and IE2) revealed that IE mRNA expression was profoundly decreased in cells stimulated with IFN-α/β and IFN-γ (~5-11-fold) as compared to vehicle-treated cells. Furthermore, decreased IE mRNA expression was accompanied by a decrease in IE protein expression, as demonstrated by western blotting and immunofluorescence.

Conclusion

These findings suggest that IFN-α/β and IFN-γ synergistically inhibit HCMV replication through a mechanism that may involve the regulation of IE gene expression. We hypothesize that IFN-γ produced by activated cells of the adaptive immune response may potentially synergize with endogenous type I IFNs to inhibit HCMV dissemination in vivo.     

Activation of interferon-stimulated genes by gamma-ray irradiation independently of the ataxia telangiectasia mutated-p53 pathway

The ataxia telangiectasia mutated (ATM)-p53 pathway is a well-known main signal transduction pathway for cellular responses, which is activated by γ-ray irradiation. Microarray analysis showed changes in the expressions of IFN-stimulated genes (ISG) in γ-ray-irradiated Balb/cA/Atm-deficient mouse embryonic fibroblasts (MEF) (ATM-KO), indicating that another pathway for cellular responses besides the ATM-p53 pathway was activated by γ-ray irradiation. The basal expression levels of Irf7 and Stat1 in ATM-KO and p53-deficient MEFs (p53-KO) were higher than those in Atm-wild-type MEFs (ATM-WT) and p53-wild-type MEFs (p53-WT), respectively. Irradiation stimulated the expressions of Irf7 and Stat1 in ATM-KO, p53-KO, ATM-WT, and p53-WT, indicating that upregulation of Irf7 and Stat1 expressions by irradiation did not depend on the ATM-p53 pathway. When conditioned medium (CM) obtained from irradiated ATM-WT or ATM-KO was added to nonirradiated MEFs, the expressions of Irf7 and Stat1 increased. We predicted that gene activation in nonirradiated MEFs was caused by IFN-α/β. Unexpectedly, significant amount of IFN-α/β could not be detected in the CM from irradiated ATM-WT or ATM-KO. Meanwhile, increased expression of Ccl5 (RANTES) protein was detected in the CM from irradiated MEFs. These results indicate that ISGs were activated by γ-ray irradiation independently of the ATM-p53 pathway and gene activation was caused by radiation-induced soluble factors.