Activation of the hypoxia inducible transcription factor HIF and the NF-ĸB pathway promotes inflammation-mediated tumor progression. The cellular transcription factor ZNF395 has repeatedly been found overexpressed in various human cancers, particularly in response to hypoxia, implying a functional relevance. To understand the biological activity of ZNF395, we identified target genes of ZNF395 through a genome-wide expression screen. Induced ZNF395 expression led to the upregulation of genes known to play a role in cancer as well as a subset of interferon (IFN)-stimulated genes (ISG) involved in antiviral responses such as IFIT1/ISG56, IFI44 and IFI16. In cells that lack ZNF395, the IFN-α-mediated stimulation of these factors was impaired, demonstrating that ZNF395 is required for the full induction of these antiviral genes. Transient transfections revealed that ZNF395-mediated activation of the IFIT1/ISG56 promoter depends on the two IFN-stimulated response elements within the promoter and on the DNA-binding domain of ZNF395, a so-called C-clamp. We also show that IĸBα kinase (IKK)-signaling is necessary to allow ZNF395 to activate transcription and simultaneously enhances its proteolytic degradation. Thus, ZNF395 becomes activated at the level of protein modification by IKK. Moreover, we confirm that the expression of ZNF395 is induced by hypoxia. Our results characterize ZNF395 as a novel factor that contributes to the maximal stimulation of a subset of ISGs. This transcriptional activity depends on IKK signaling further supporting a role of ZNF395 in the innate immune response. Given these results it is possible that under hypoxic conditions, elevated levels of ZNF395 may support inflammation and cancer progression by activating the target genes involved in the innate immune response and cancer.
Herpes simplex virus type 1 (HSV-1) infection results in lifelong chronic infection of trigeminal ganglion (TG) neurons, also referred to as neuronal HSV-1 latency, with periodic reactivation leading to recrudescent herpetic disease in some persons. HSV-1 proteins are expressed in a temporally coordinated fashion during lytic infection, but their expression pattern during latent infection is largely unknown. Selective retention of HSV-1 reactive T-cells in human TG suggests their role in controlling reactivation by recognizing locally expressed HSV-1 proteins. We characterized the HSV-1 proteins recognized by virus-specific CD4 and CD8 T-cells recovered from human HSV-1–infected TG. T-cell clusters, consisting of both CD4 and CD8 T-cells, surrounded neurons and expressed mRNAs and proteins consistent with in situ antigen recognition and antiviral function. HSV-1 proteome-wide scans revealed that intra-TG T-cell responses included both CD4 and CD8 T-cells directed to one to three HSV-1 proteins per person. HSV-1 protein ICP6 was targeted by CD8 T-cells in 4 of 8 HLA-discordant donors. In situ tetramer staining demonstrated HSV-1-specific CD8 T-cells juxtaposed to TG neurons. Intra-TG retention of virus-specific CD4 T-cells, validated to the HSV-1 peptide level, implies trafficking of viral proteins from neurons to HLA class II-expressing non-neuronal cells for antigen presentation. The diversity of viral proteins targeted by TG T-cells across all kinetic and functional classes of viral proteins suggests broad HSV-1 protein expression, and viral antigen processing and presentation, in latently infected human TG. Collectively, the human TG represents an immunocompetent environment for both CD4 and CD8 T-cell recognition of HSV-1 proteins expressed during latent infection. HSV-1 proteins recognized by TG-resident T-cells, particularly ICP6 and VP16, are potential HSV-1 vaccine candidates.
HSV-1 is an endemic human herpesvirus worldwide that establishes a lifelong latent infection of neurons in the trigeminal ganglion (TG), allowing intermittent reactivation resulting in recurrent disease in some persons. Studies in HSV-1 models suggest a central role of TG-infiltrating virus-specific CD8 T-cells to control reactivation. In humans, however, the functional properties and fine specificity of intra-TG T-cell responses remain enigmatic. The current study used molecular, immunological and in situ analysis platforms on human cadaveric TG obtained within hours after death to characterize the local HSV-1 specific T-cell response in latently infected human TG in detail. We identified that CD4 and CD8 T-cells were juxtaposed to TG neurons and expressed host transcripts and proteins consistent with in situ antigen recognition and antiviral function. The intra-TG T-cell response, involving both CD4 and CD8 T-cells, was directed to a limited set of HSV-1 proteins per person, which was not limited to a specific kinetic or structural class of viral proteins. Collectively, the data indicate that the human TG is an immunocompetent environment for CD4 and CD8 T-cell recognition of diverse HSV-1 proteins expressed during latent infection and that the viral antigens identified herein are rational candidates for HSV-1 subunit vaccines.
Pleural fibrosis and malignant mesotheliomas (MM) occur after exposures to pathogenic fibers, yet the mechanisms initiating these diseases are unclear.
We document priming and activation of the NLRP3 inflammasome in human mesothelial cells by asbestos and erionite that is causally related to release of IL-1β, IL-6, IL-8, and Vascular Endothelial Growth Factor (VEGF). Transcription and release of these proteins are inhibited in vitro using Anakinra, an IL-1 receptor antagonist that reduces these cytokines in a human peritoneal MM mouse xenograft model.
These novel data show that asbestos-induced priming and activation of the NLRP3 inflammasome triggers an autocrine feedback loop modulated via the IL-1 receptor in mesothelial cell type targeted in pleural infection, fibrosis, and carcinogenesis.
Asbestos; Mesothelioma; Mesothelium; Inflammasomes; NLRP3
ICP4 is the major transcriptional regulatory protein of herpes simplex virus (HSV). It is expressed in infected cells with immediate early kinetics and is essential for viral growth. ICP4 is also a structural component of the virion tegument layer. Herpesviral tegument proteins exert regulatory functions important for takeover of the host cell. Tegument ICP4 has not been well characterized. We examined the ICP4 present in HSV-1 virions that were either derived from wild type infected cells or from ICP4-expressing (E5) cells infected with ICP4 deletion virus d120. Limited proteolysis demonstrated that virion-associated ICP4 from particles derived from E5 cells was indeed an internal component of the virion. A similar subset of virion structural proteins was detected in viral particles regardless of the cellular origin of ICP4. Genotypically ICP4-negative virions complemented with tegument ICP4 entered cells via a proteasome-dependent, pH-dependent pathway similar to wild type virions. In infected cells, ICP4 was distributed predominantly in intranuclear replication compartments regardless of whether it was expressed from a transgene or from the HSV genome.
Therapeutic oligonucleotides including siRNA and immunostimulatory ligands of Toll-like receptors (TLR) or RIG-I like helicases (RLH) are a promising novel class of drugs. They are in clinical development for a broad spectrum of applications, e.g. as adjuvants in vaccines and for the immunotherapy of cancer. Species-specific immune activation leading to cytokine release is characteristic for therapeutic oligonucleotides either as an unwanted side effect or intended pharmacology. Reliable in vitro tests designed for therapeutic oligonucleotides are therefore urgently needed in order to predict clinical efficacy and to prevent unexpected harmful effects in clinical development. To serve this purpose, we here established a human whole blood assay (WBA) that is fast and easy to perform. Its response to synthetic TLR ligands (R848: TLR7/8, LPS: TLR4) was on a comparable threshold to the more time consuming peripheral blood mononuclear cell (PBMC) based assay. By contrast, the type I IFN profile provoked by intravenous CpG-DNA (TLR9 ligand) in humans in vivo was more precisely replicated in the WBA than in stimulated PBMC. Since Heparin and EDTA, but not Hirudin, displaced oligonucleotides from their delivery agent, only Hirudin qualified as the anticoagulant to be used in the WBA. The Hirudin WBA exhibited a similar capacity as the PBMC assay to distinguish between TLR7-activating and modified non-stimulatory siRNA sequences. RNA-based immunoactivating TLR7/8- and RIG-I-ligands induced substantial amounts of IFN-α in the Hirudin-WBA dependent on delivery agent used. In conclusion, we present a human Hirudin WBA to determine therapeutic oligonucleotide-induced cytokine release during preclinical development that can readily be performed and offers a close reflection of human cytokine response in vivo.
Theiler's virus is a neurotropic picornavirus responsible for chronic infections of the central nervous system. The establishment of a persistent infection and the subsequent demyelinating disease triggered by the virus depend on the expression of L*, a viral accessory protein encoded by an alternative open reading frame of the virus. We discovered that L* potently inhibits the interferon-inducible OAS/RNase L pathway. The antagonism of RNase L by L* was particularly prominent in macrophages where baseline oligoadenylate synthetase (OAS) and RNase L expression levels are elevated, but was detectable in fibroblasts after IFN pretreatment. L* mutations significantly affected Theiler's virus replication in primary macrophages derived from wild-type but not from RNase L-deficient mice. L* counteracted the OAS/RNase L pathway through direct interaction with the ankyrin domain of RNase L, resulting in the inhibition of this enzyme. Interestingly, RNase L inhibition was species-specific as Theiler's virus L* protein blocked murine RNase L but not human RNase L or RNase L of other mammals or birds. Direct RNase L inhibition by L* and species specificity were confirmed in an in vitro assay performed with purified proteins. These results demonstrate a novel viral mechanism to elude the antiviral OAS/RNase L pathway. By targeting the effector enzyme of this antiviral pathway, L* potently inhibits RNase L, underscoring the importance of this enzyme in innate immunity against Theiler's virus.
Theiler's virus is a murine picornavirus (same family as poliovirus) which has a striking ability to establish persistent infections of the central nervous system. To do so, the virus has to counteract the immune response of the host and particularly the potent response mediated by interferon. We observed that a protein encoded by Theiler's virus, the L* protein, inhibited the RNase L pathway, one of the best-characterized pathways mediating the antiviral IFN response. In contrast to previously identified viral antagonists of this pathway, L* was found to act directly on RNase L, the effector enzyme of the pathway. L* activity was found to be species-specific as it inhibited murine but not human RNase L. We confirmed the species-specificity and the direct interaction between L* and RNase L in vitro, using purified proteins. Acting at the effector step in the pathway allows L* to block RNase L activity efficiently. This suggests that RNase L is particularly important to control Theiler's virus replication in vivo. Another virus, mouse hepatitis virus (MHV), was recently shown to interfere with RNase L activation. Theiler's virus and MHV share a marked tropism for macrophages which may suggest that the RNase L pathway is particularly important in this cell type.
Persistent infections with hepatitis C virus (HCV) may result in life-threatening liver disease, including cirrhosis and cancer, and impose an important burden on human health. Understanding how the virus is capable of achieving persistence in the majority of those infected is thus an important goal. Although HCV has evolved multiple mechanisms to disrupt and block cellular signaling pathways involved in the induction of interferon (IFN) responses, IFN-stimulated gene (ISG) expression is typically prominent in the HCV-infected liver. Here, we show that Toll-like receptor 3 (TLR3) expressed within uninfected hepatocytes is capable of sensing infection in adjacent cells, initiating a local antiviral response that partially restricts HCV replication. We demonstrate that this is dependent upon the expression of class A scavenger receptor type 1 (MSR1). MSR1 binds extracellular dsRNA, mediating its endocytosis and transport toward the endosome where it is engaged by TLR3, thereby triggering IFN responses in both infected and uninfected cells. RNAi-mediated knockdown of MSR1 expression blocks TLR3 sensing of HCV in infected hepatocyte cultures, leading to increased cellular permissiveness to virus infection. Exogenous expression of Myc-MSR1 restores TLR3 signaling in MSR1-depleted cells with subsequent induction of an antiviral state. A series of conserved basic residues within the carboxy-terminus of the collagen superfamily domain of MSR1 are required for binding and transport of dsRNA, and likely facilitate acidification-dependent release of dsRNA at the site of TLR3 expression in the endosome. Our findings reveal MSR1 to be a critical component of a TLR3-mediated pattern recognition receptor response that exerts an antiviral state in both infected and uninfected hepatocytes, thereby limiting the impact of HCV proteins that disrupt IFN signaling in infected cells and restricting the spread of HCV within the liver.
Persistent hepatitis C virus (HCV) infection is an important cause of fatal cirrhosis and liver cancer in humans. While viral disruption of interferon (IFN) signaling pathways may contribute to the persistence of HCV, IFN-stimulated gene (ISG) expression is often prominent within the infected liver. We show here that this is due, at least in part, to Toll-like receptor 3 sensing of HCV mediated by class A scavenger receptor type 1 (MSR1)-dependent endocytosis and transport of extracellular viral double-stranded RNA (dsRNA) allowing it to be engaged by TLR3 in the late endosome. TLR3 expressed within uninfected cells is capable of sensing HCV infection in neighboring infected cells in a process that is dependent upon the dsRNA-scavenging activity of MSR1, resulting in the induction of a localized functional antiviral response. This contributes to the ISG expression that typifies the chronically-infected liver, as it occurs within cells that do not express HCV proteins that disrupt IFN signaling. TLR3 signaling thus limits the spread of virus within the liver, potentially explaining why only a small fraction of hepatocytes are infected with HCV in vivo.
Background & Aims
Our previous results showed that the knockdown of woodchuck hepatitis virus (WHV) by RNA interference (RNAi) led to upregulation of interferon stimulated genes (ISGs) in primary hepatocytes. In the present study, we tested the hypothesis that the cellular signaling pathways recognizing RNA molecules may be involved the ISG stimulation by RNAi.
Primary murine hepatocytes (PMHs) from wild type mice and WHV transgenic (Tg) mice were prepared and treated with defined siRNAs. The mRNA levels of target genes and ISGs were detected by real-time RT-PCR. The involvement of the signaling pathways including RIG-I/MDA5, PKR, and TLR3/7/8/9 was examined by specific inhibition and the analysis of their activation by Western blotting.
In PMHs from WHV Tg mice, specific siRNAs targeting WHV, mouse β-actin, and GAPDH reduced the levels of targeted mRNAs and increased the mRNA expression of IFN-β, MxA, and IP-10. The enhanced ISG expression by siRNA transfection were abolished by siRNA-specific 2′-O-methyl antisense RNA and the inhibitors 2-AP and chloroquine blocking PKR and other TLR-mediated signaling pathways. Furthermore, Western blotting revealed that RNAi results in an increase in PKR phosphorylation and nuclear translocation of IRF3 and NF-êB, indicating the possible role of IRF3 in the RNAi-directed induction of ISGs. In contrast, silencing of RIG-I and MDA5 failed to block RNAi-mediated MxA induction.
RNAi is capable of enhancing innate immune responses through the PKR- and TLR-dependent signaling pathways in primary hepatocytes. The immune stimulation by RNAi may contribute to the antiviral activity of siRNAs in vivo.
The type III interferons (IFNs), comprising IFN-λ1, IFN-λ2, and IFN-λ3, behave similarly to IFN-α in eliciting antiviral, antitumor, and immune-modulating activities. Due to their more restricted cellular targets, IFN-λs are attractive as potential alternatives to existing therapeutic regimens based on IFN-αs. We have applied the DOCK-AND-LOCK™ method to improve the anti-proliferative potency of IFN-λ1 up to 1,000-fold in targeted cancer cell lines by tethering stabilized Fab dimers, derived from hRS7 (humanized anti-Trop-2), hMN-15 (humanized anti-CEACAM6), hL243 (humanized anti-HLA-DR), and c225 (chimeric anti-EGFR), to IFN-λ1 site-specifically, resulting in novel immunocytokines designated (E1)-λ1, (15)-λ1, (C2)-λ1, and (c225)-λ1, respectively. Targeted delivery of IFN-λ1 via (15)-λ1 or (c225)-λ1 to respective antigen-expressing cells also significantly increased antiviral activity when compared with non-targeting (C2)-λ1, as demonstrated in human lung adenocarcinoma cell line A549 by (15)-λ1 against encephalomyocarditis virus (EC50 = 22.2 pM versus 223 pM), and in human hepatocarcinoma cell line Huh-7 by (c225)-λ1 against hepatitis C virus (EC50 = 0.56 pM versus 91.2 pM). These promising results, which are attributed to better localization and stronger binding of IFN-λ1 to antibody-targeted cells, together with the favorable pharmacokinetic profile of (E1)-λ1 in mice (T1/2 = 8.6 h), support further investigation of selective prototypes as potential antiviral and antitumor therapeutic agents.
The DExD/H box RNA helicases retinoic acid-inducible gene-I (RIG-I) and melanoma differentiation associated gene-5 (mda-5) sense viral RNA in the cytoplasm of infected cells and activate signal transduction pathways that trigger the production of type I interferons (IFNs). Laboratory of genetics and physiology 2 (LGP2) is thought to influence IFN production by regulating the activity of RIG-I and mda-5, although its mechanism of action is not known and its function is controversial. Here we show that expression of LGP2 potentiates IFN induction by polyinosinic-polycytidylic acid [poly(I:C)], commonly used as a synthetic mimic of viral dsRNA, and that this is particularly significant at limited levels of the inducer. The observed enhancement is mediated through co-operation with mda-5, which depends upon LGP2 for maximal activation in response to poly(I:C). This co-operation is dependent upon dsRNA binding by LGP2, and the presence of helicase domain IV, both of which are required for LGP2 to interact with mda-5. In contrast, although RIG-I can also be activated by poly(I:C), LGP2 does not have the ability to enhance IFN induction by RIG-I, and instead acts as an inhibitor of RIG-I-dependent poly(I:C) signaling. Thus the level of LGP2 expression is a critical factor in determining the cellular sensitivity to induction by dsRNA, and this may be important for rapid activation of the IFN response at early times post-infection when the levels of inducer are low.
Chronic hepatitis C virus (HCV) infection is a leading cause of liver disease. Liver inflammation underlies infection-induced fibrosis, cirrhosis and liver cancer but the processes that promote hepatic inflammation by HCV are not defined. We provide a systems biology analysis with multiple lines of evidence to indicate that interleukin-1β (IL-1β) production by intrahepatic macrophages confers liver inflammation through HCV-induced inflammasome signaling. Chronic hepatitis C patients exhibited elevated levels of serum IL-1β compared to healthy controls. Immunohistochemical analysis of healthy control and chronic hepatitis C liver sections revealed that Kupffer cells, resident hepatic macrophages, are the primary cellular source of hepatic IL-1β during HCV infection. Accordingly, we found that both blood monocyte-derived primary human macrophages, and Kupffer cells recovered from normal donor liver, produce IL-1β after HCV exposure. Using the THP-1 macrophage cell-culture model, we found that HCV drives a rapid but transient caspase-1 activation to stimulate IL-1β secretion. HCV can enter macrophages through non-CD81 mediated phagocytic uptake that is independent of productive infection. Viral RNA triggers MyD88-mediated TLR7 signaling to induce IL-1β mRNA expression. HCV uptake concomitantly induces a potassium efflux that activates the NLRP3 inflammasome for IL-1β processing and secretion. RNA sequencing analysis comparing THP1 cells and chronic hepatitis C patient liver demonstrates that viral engagement of the NLRP3 inflammasome stimulates IL-1β production to drive proinflammatory cytokine, chemokine, and immune-regulatory gene expression networks linked with HCV disease severity. These studies identify intrahepatic IL-1β production as a central feature of liver inflammation during HCV infection. Thus, strategies to suppress NLRP3 or IL-1β activity could offer therapeutic actions to reduce hepatic inflammation and mitigate disease.
Hepatitis C virus (HCV) causes chronic infection of the liver and is a leading cause of liver inflammation, cirrhosis and liver cancer in nearly 200 million people worldwide. Importantly, hepatic inflammation during chronic HCV infection is considered to be the primary catalyst for progressive liver disease and development of liver cancer. However, the underlying molecular mechanism(s) of HCV-mediated hepatic inflammation are not well understood. The goal of this study was to determine the mechanisms of HCV-induced inflammation. We found that serum IL-1β levels are elevated in chronic hepatitis C patients. Furthermore, we found that hepatic macrophages or Kupffer cells are the major IL-1β-producing cell population within HCV infected livers. Our studies, using the THP1 cell culture model of HCV exposure, reveal that exposure of macrophages to HCV induces IL-1β through a process of infection-independent phagocytic virus uptake that triggers signaling through MyD88/TLR7 and NLRP3 inflammasome pathways to drive IL-1β expression and maturation/secretion, respectively. RNA sequencing (RNA-seq) analysis of patient liver biopsies shows that viral triggering of these signaling pathways drives an inflammatory response linked with liver disease in patients with chronic hepatitis C. Our results identify HCV-induced IL-1β production by hepatic macrophages as a critical and central process that promotes liver inflammation and disease.
Human metapneumovirus (hMPV) is a recently identified RNA virus belonging to the Paramyxoviridae family. It is a common cause of respiratory tract infections in children, adults, and immunocompromised patients, for which no specific treatment or vaccine is available. Recent investigations in our lab identified hMPV glycoprotein G as an important virulence factor, as a recombinant virus lacking the G protein (rhMPV-ΔG) exhibited enhanced production of important immune and antiviral mediators, such as cytokines, chemokines and type I interferon (IFN) in airway epithelial cells, and expression of G protein alone inhibits cellular signaling dependent on retinoic induced gene (RIG)-I, a RNA helicase with a fundamental role in initiating hMPV-induced cellular responses. In this study, we have further investigated the mechanism underlying the inhibitory role of hMPV G protein on RIG-I-dependent signaling. We found that the interaction of hMPV G with RIG-I occurs primarily through the CARD domains of RIG-I N-terminus, preventing RIG-I association with the adaptor protein MAVS (mitochondrial antiviral signaling protein), recruitment of RIG-I to mitochondria, as well as the interaction between mitochondria and mitochondria-associated membrane (MAM) component of the endoplasmic reticulum (ER), which contains STINGS, an important part of the viral-induced RIG-I/MAVS signaling pathway, leading in the end to the inhibition of cytokine, chemokine and type I IFN expression. Mutagenesis analysis showed that hMPV G protein cytoplasmic domain played a major role in the observed inhibitory activity, and recombinant viruses expressing a G protein with amino acid substitution in position 2 and 3 recapitulated most of the phenotype observed with rhMPV-ΔG mutant upon infection of airway epithelial cells.
Plasmacytoid Dendritic Cells (pDCs) represent a key immune cell in the defense against viruses. Through pattern recognition receptors (PRRs), these cells detect viral pathogen associated molecular patterns (PAMPs) and initiate an Interferon (IFN) response. pDCs produce the antiviral IFNs including the well-studied Type I and the more recently described Type III. Recent genome wide association studies (GWAS) have implicated Type III IFNs in HCV clearance. We examined the IFN response induced in a pDC cell line and ex vivo human pDCs by a region of the HCV genome referred to as the HCV PAMP. This RNA has been shown previously to be immunogenic in hepatocytes, whereas the conserved X-region RNA is not. We show that in response to the HCV PAMP, pDC-GEN2.2 cells upregulate and secrete Type III (in addition to Type I) IFNs and upregulate PRR genes and proteins. We also demonstrate that the recognition of this RNA is dependent on RIG-I-like Receptors (RLRs) and Toll-like Receptors (TLRs), challenging the dogma that RLRs are dispensable in pDCs. The IFNs produced by these cells in response to the HCV PAMP also control HCV replication in vitro. These data are recapitulated in ex vivo pDCs isolated from healthy donors. Together, our data shows that pDCs respond robustly to HCV RNA to make Type III Interferons that control viral replication. This may represent a novel therapeutic strategy for the treatment of HCV.
Hepatitis C Virus (HCV) is the most common bloodborne pathogen for which no vaccine is available. Infection with the virus often leads to persistent (or chronic) infection. Patients with chronic HCV infection can develop progressive liver disease and liver failure, leading to the need for a transplant. It is not fully understood why some people clear the virus and others develop persistent infection. Understanding differences in how patients respond to the virus in the early phases of infection may lead to better treatment of HCV. Here, we use a highly conserved region of the HCV genome to examine innate immunological responses to HCV. We found that plasmacytoid dendritic cells, innate cells keyed to respond with anti-viral interferon proteins, recognize the virus. Additionally, we show that pDCs use RIG-I in the recognition of this virus, which was previously thought to be dispensable in pDCs. The proteins secreted by these cells can control viral replication in a cell-based laboratory system. In cells isolated from healthy donors, we found that fresh human cells can respond in the same manner to the virus as the laboratory strain of cells, and there was a correlation with genetic differences. Our study offers novel insight to how the body recognizes HCV during early infection and host-virus interactions that mediate viral control of this common infection.
Both NK cells and CTLs kill virus-infected and tumor cells. However, the ways by which these killer cells recognize the infected or the tumorigenic cells are different, in fact almost opposite. CTLs are activated through the interaction of the TCR with MHC class I proteins. In contrast, NK cells are inhibited by MHC class I molecules. The inhibitory NK receptors recognize mainly MHC class I proteins and in this regard practically all of the HLA-C proteins are recognized by inhibitory NK cell receptors, while only certain HLA-A and HLA-B proteins interact with these receptors. Sophisticated viruses developed mechanisms to avoid the attack of both NK cells and CTLs through, for example, down regulation of HLA-A and HLA-B molecules to avoid CTL recognition, leaving HLA-C proteins on the cell surface to inhibit NK cell response. Here we provide the first example of a virus that through specific down regulation of HLA-C, harness the NK cells for its own benefit. We initially demonstrated that none of the tested HSV-2 derived microRNAs affect NK cell activity. Then we show that surprisingly upon HSV-2 infection, HLA-C proteins are specifically down regulated, rendering the infected cells susceptible to NK cell attack. We identified a motif in the tail of HLA-C that is responsible for the HSV-2-meduiated HLA-C down regulation and we show that the HLA-C down regulation is mediated by the viral protein ICP47. Finally we show that HLA-C proteins are down regulated from the surface of HSV-2 infected dendritic cells (DCs) and that this leads to the killing of DC by NK cells. Thus, we propose that HSV-2 had developed this unique and surprising NK cell-mediated killing strategy of infected DC to prevent the activation of the adaptive immunity.
Approximately 20% of all humans are latently and asymptomatically infected with HSV-2. This suggests that the virus developed mechanisms to avoid immune cell detection; many of which are still unknown. Infected cells are killed mainly by two lymphocyte populations; NK cells and CTLs that belong to the innate and the adaptive immunity, respectively. While the killing machinery of these two cell types is similar, almost identical, the ways by which they discriminate between infected and uninfected cells is different. CTLs are activated, primarily by DCs, to become effector cells. They then recognize virus-derived peptides in the groove of MHC class I molecules and eliminate the virally infected cells. In contrast, NK cells recognize infected cells through several NK cell activating receptors, while the recognition of MHC class I proteins by NK cells leads to inhibition of NK cell killing. Viruses, such as HIV, developed mechanisms to interfere with the function of both NK cells and CTLs via targeting of specific MHC class I proteins. Here we show that HSV-2 developed a MHC class I-dependent mechanism in which the virus, through specific targeting of HLA-C by the viral protein ICP47, harness the NK cells for its own benefit, probably to avoid the activation of adaptive immune response.
There has been increased interest in the possible role of human cytomegalovirus (HCMV) in carcinogenesis during the last decade. HCMV seroprevalence was enhanced in patients with hepatocellular carcinoma (HCC) but a possible relationship between HCC and HCMV infection remained to be assessed. The aim of this work was to investigate the pro-tumor influence of HCMV on primary human hepatocytes (PHH) and HepG2 cells.
Following infection of PHH and HepG2 cells by two different strains of HCMV, we measured the production of IL-6 in culture supernatants by ELISA and the protein levels of STAT3, pSTAT3, JAK, cyclin D1, survivin, p53, p21, and Mdm2 by western Blotting in infected and uninfected cells. Cell proliferation and transformation were investigated using Ki67Ag expression measurement and soft-agar colony formation assay respectively.
Infection of HepG2 cells and PHH by HCMV resulted in the production of IL-6 and the subsequent activation of the IL-6R-JAK-STAT3 pathway. HCMV increased the expression of cyclin D1 and survivin. Cell proliferation was enhanced in HepG2 and PHH infected with HCMV, despite a paradoxical overexpression of p53 and p21. More importantly, we observed the formation of colonies in soft agar seeded with PHH infected with HCMV and when we challenged the HepG2 cultures to form tumorspheres, we found that the HCMV-infected cultures formed 2.5-fold more tumorspheres than uninfected cultures.
HCMV activated the IL-6-JAK-STAT3 pathway in PHH and HepG2 cells, favored cellular proliferation, induced PHH transformation and enhanced HepG2 tumorsphere formation. Our observations raise the possibility that HCMV infection might be involved in the genesis of hepatocellular carcinoma.
Primary cells are often used to study viral replication and host-virus interactions as their antiviral pathways have not been altered or inactivated; however, their use is restricted by their short lifespan. Conventional methods to extend the life of primary cultures typically utilize viral oncogenes. Many of these oncogenes, however, perturb or inactivate cellular antiviral pathways, including the interferon (IFN) response. It has been previously shown that expression of the telomerase reverse transcriptase (TERT) gene extends the life of certain cell types. The effect that TERT expression has on the innate antiviral response to RNA- and DNA-containing viruses has not been examined. In the current study, we introduced the human TERT (hTERT) gene into a primary human embryonic lung (HEL-299) cell strain, which is known to respond to the type I IFN, IFN-β. We show that the resulting HEL-TERT cell line is capable of replicating beyond 100 population doublings without exhibiting signs of senescence. Treatment with IFN-β resulted in the upregulation of four model IFN stimulated genes (ISGs) in HEL-299 and HEL-TERT cells. Both cell lines supported the replication of herpes simplex virus type 1 (HSV-1) and vesicular stomatitis virus (VSV) and impaired the replication of both viruses upon IFN-β pretreatment. Introduction of the viral oncoprotein, simian virus 40 (SV40) large T-antigen, which is frequently used to immortalize cells, largely negated this effect. Taken together, our data indicate that expression of hTERT does not alter type 1 IFN signaling and/or the growth of two viruses, making this cell line a useful reagent for studying viral replication and virus-cell interactions.
Presently there is limited research to suggest efficacious interventions for infants at-risk for autism. Pivotal response treatment (PRT) has empirical support for use with preschool children with autism, but there are no reports in the literature utilizing this approach with infants. In the current study, a developmental adaptation of PRT was piloted via a brief parent training model with three infants at-risk for autism. Utilizing a multiple baseline design, the data suggest that the introduction of PRT resulted in increases in the infants’ frequency of functional communication and parents’ fidelity of implementation of PRT procedures. Results provide preliminary support for the feasibility and utility of PRT for very young children at-risk for autism.
Early intervention; Pivotal response treatment; Parent education; Infant siblings
In myeloid cells the inflammasome plays a crucial role in innate immune defenses against pathogen- and danger-associated patterns such as crystalline silica. Respirable mineral particles impinge upon the lung epithelium causing irreversible damage, sustained inflammation and silicosis. In this study we investigated lung epithelial cells as a target for silica-induced inflammasome activation.
A human bronchial epithelial cell line (BEAS-2B) and primary normal human bronchial epithelial cells (NHBE) were exposed to toxic but nonlethal doses of crystalline silica over time to perform functional characterization of NLRP3, caspase-1, IL-1β, bFGF and HMGB1. Quantitative RT-PCR, caspase-1 enzyme activity assay, Western blot techniques, cytokine-specific ELISA and fibroblast (MRC-5 cells) proliferation assays were performed.
We were able to show transcriptional and translational upregulation of the components of the NLRP3 intracellular platform, as well as activation of caspase-1. NLRP3 activation led to maturation of pro-IL-1β to secreted IL-1β, and a significant increase in the unconventional release of the alarmins bFGF and HMGB1. Moreover, release of bFGF and HMGB1 was shown to be dependent on particle uptake. Small interfering RNA experiments using siNLRP3 revealed the pivotal role of the inflammasome in diminished release of pro-inflammatory cytokines, danger molecules and growth factors, and fibroblast proliferation.
Our novel data indicate the presence and functional activation of the NLRP3 inflammasome by crystalline silica in human lung epithelial cells, which prolongs an inflammatory signal and affects fibroblast proliferation, mediating a cadre of lung diseases.
Silica; NLRP3 inflammasome; Caspase-1; IL-1β; HMGB1; bFGF
The innate immune response plays a critical role in the host defense against invading pathogens, and TLR2, a member of the Toll-like receptor (TLR) family, has been implicated in the immune response and initiation of inflammatory cytokine secretion against several human viruses. Previous studies have demonstrated that infectious and ultraviolet-inactivated herpes simplex virus 1 (HSV-1) virions lead to the activation of nuclear factor kappa B (NF-κB) and secretion of proinflammatory cytokines via TLR2. However, except for the envelope glycoprotein gH and gL, whether there are other determinants of HSV-1 responsible for TLR2 mediated biological effects is not known yet. Here, we demonstrated that the HSV-1-encoded envelope glycoprotein gB displays as molecular target recognized by TLR2. gB coimmunoprecipitated with TLR2, TLR1 and TLR6 in transfected and infected human embryonic kidney (HEK) 293T cells. Treatment of TLR2-transfected HEK293T (HEK293T-TLR2) cells with purified gB results in the activation of NF-κB reporter, and this activation requires the recruitment of the adaptor molecules myeloid differentiation primary-response protein 88 (MyD88) and tumor necrosis factor receptor-associated factor 6 (TRAF6) but not CD14. Furthermore, activation of NF-κB was abrogated by anti-gB and anti-TLR2 blocking antibodies. In addition, the expression of interleukin-8 induced by gB was abrogated by the treatment of the human monocytic cell line THP-1 with anti-TLR2 blocking antibody or by the incubation of gB with anti-gB antibody. Taken together, these results indicate the importance and potency of HSV-1 gB as one of pathogen-associated molecular patterns (PAMPs) molecule recognized by TLR2 with immediate kinetics.
It is becoming increasingly clear that many diseases are the result of infection from multiple genetically distinct strains of a pathogen. Such multi-strain infections have the capacity to alter both disease and pathogen dynamics. Infection with multiple strains of human cytomegalovirus (HCMV) is common and has been linked to enhanced disease. Suggestions that disease enhancement in multi-strain infected patients is due to complementation have been supported by trans-complementation studies in mice during co-infection of wild type and gene knockout strains of murine CMV (MCMV). Complementation between naturally circulating strains of CMV has, however, not been assessed. In addition, many models of multi-strain infection predict that co-infecting strains will compete with each other and that this competition may contribute to selective transmission of more virulent pathogen strains. To assess the outcome of multi-strain infection, C57BL/6 mice were infected with up to four naturally circulating strains of MCMV. In this study, profound within-host competition was observed between co-infecting strains of MCMV. This competition was MCMV strain specific and resulted in the complete exclusion of certain strains of MCMV from the salivary glands of multi-strain infected mice. Competition was dependent on Ly49H+ natural killer (NK) cells as well as the expression of the ligand for Ly49H, the MCMV encoded product, m157. Strains of MCMV which expressed an m157 gene product capable of ligating Ly49H were outcompeted by strains of MCMV expressing variant m157 genes. Importantly, within-host competition prevented the shedding of the less virulent strains of MCMV, those recognized by Ly49H, into the saliva of multi-strain infected mice. These data demonstrate that NK cells have the strain specific recognition capacity required to meditate within-host competition between strains of MCMV. Furthermore, this within-host competition has the capacity to shape the dynamics of viral shedding and potentially select for the transmission of more virulent virus strains.
Infection of the host with multiple strains of a pathogen is common and occurs with the herpesvirus, human cytomegalovirus (HCMV). However the effects of multi-strain infection on the host and the pathogen remain poorly studied. Here we show, in a mouse model, that infection of C57BL/6 mice with multiple strains of murine CMV (MCMV) results in profound within-host competition. Competition between the strains of MCMV is dependent on Ly49H+ natural killer (NK) cells. The NK cell activation receptor Ly49H receptor targets certain genotypes of the viral protein, m157. During multi-strain infection, strains of MCMV encoding an m157 capable of binding Ly49H are excluded from the salivary gland and the saliva of C57BL/6 mice, allowing for the shedding of only non-Ly49H binding strains of MCMV in the saliva. This within-host competition could therefore have significant impacts on the circulation of MCMV strains, as only the most virulent MCMV strains were present in the saliva.
Viruses are strictly dependent on cells to propagate and many incorporate host proteins in their viral particles, but the significance of this incorporation is poorly understood. Recently, we performed the first comprehensive characterization of the mature herpes simplex virus type 1 (HSV-1) in which up to 49 distinct cellular proteins were identified by mass spectrometry. In the present study, we sought to identify if these cellular factors are relevant for the HSV-1 life cycle. To this end, we performed a small interfering RNA functional screen and found that 15 of these host proteins altered HSV-1 proliferation in cell culture, without any significant effect on cell viability. Moreover, the siRNA used had no negative consequences for Adenovirus type 5 propagation (with one exception) indicating that the modulation was specific for HSV-1 and not merely due to unhealthy cells. The positive host proteins include several Rab GTPases and other intracellular transport components as well as proteins involved in signal transduction, gene regulation and immunity. Remarkably, in most cases when virions were depleted for one of the above proteins, they replicated more poorly in subsequent infections in wild type cells. This highlights for the first time that both the cellular and virion-associated pools of many of these proteins actively contribute to viral propagation. Altogether, these findings underscore the power and biological relevance of combining proteomics and RNA interference to identify novel host-pathogen interactions.
Recent studies investigating possible causes of male subfertility have largely focused on how lifestyle or environmental factors impact on the process of spermatogenesis. Markedly, fewer studies have investigated those risk factors that result in reduced sperm quality, such as poor sperm motility. The speed at which sperm swim is a major predictor of fertility and is extremely variable in human populations. It has been hypothesized that offspring sex may be adaptively manipulated to maximize the offspring's reproductive fitness (e.g., parents with genes for good male fertility traits, such as high sperm speed, would produce primarily sons and fewer daughters because the offspring will inherit advantageous male fertility genes). Conversely, parents with poor male fertility genes would produce primarily daughters. We tested whether there was an association between how fast a man's sperm swam and the sex bias of his siblings in a sample of men attending clinic for fertility investigations with their partner and with a wide range of semen characteristics, including sperm speed. We found that the sex bias of a man's siblings is associated with his sperm speed; men with female-biased siblings had significantly slower sperm (judged using computer-assisted sperm analysis (CASA)) than men from male-biased sibships. This observation suggests family composition is an important factor that needs to be considered in future epidemiological and clinical studies of human fertility.
computer-assisted sperm analysis (CASA); fertility; intralocus sexual conflict; male; offspring; sex ratio; sexual antagonism
After HSV infection, some trigeminal ganglion neurons support productive cycle gene expression, while in other neurons the virus establishes a latent infection. We previously demonstrated that HSV-1 and HSV-2 preferentially establish latent infection in A5+ and KH10+ sensory neurons, respectively, and that exchanging the latency-associated transcript (LAT) between HSV-1 and HSV-2 also exchanges the neuronal preference. Since many viral genes besides the LAT are functionally interchangeable between HSV-1 and HSV-2, we co-infected HSV-1 and HSV-2, both in vivo and in vitro, to determine if trans-acting viral factors regulate whether HSV infection follows a productive or latent pattern of gene expression in sensory neurons. The pattern of HSV-1 and HSV-2 latent infection in trigeminal neurons was no different following co-infection than with either virus alone, consistent with the hypothesis that a trans-acting viral factor is not responsible for the different patterns of latent infection of HSV-1 and HSV-2 in A5+ and KH10+ neurons. Since exchanging the LAT regions between the viruses also exchanges neuronal preferences, we infected transgenic mice that constitutively express 2.8 kb of the LAT region with the heterologous viral serotype. Endogenous expression of LAT did not alter the pattern of latent infection after inoculation with the heterologous serotype virus, demonstrating that the LAT region does not act in trans to direct preferential establishment of latency of HSV-1 and HSV-2. Using HSV1-RFP and HSV2-GFP in adult trigeminal ganglion neurons in vitro, we determined that HSV-1 and HSV-2 do not exert trans-acting effects during acute infection to regulate neuron specificity. Although some neurons were productively infected with both HSV-1 and HSV-2, no A5+ or KH10+ neurons were productively infected with both viruses. Thus, trans-acting viral factors do not regulate preferential permissiveness of A5+ and KH10+ neurons for productive HSV infection and preferential establishment of latent infection.
We hypothesized that normal human mesothelial cells acquire resistance to asbestos-induced toxicity via induction of one or more epidermal growth factor receptor (EGFR)–linked survival pathways (phosphoinositol-3-kinase/AKT/mammalian target of rapamycin and extracellular signal–regulated kinase [ERK] 1/2) during simian virus 40 (SV40) transformation and carcinogenesis. Both isolated HKNM-2 mesothelial cells and a telomerase-immortalized mesothelial line (LP9/TERT-1) were more sensitive to crocidolite asbestos toxicity than an SV40 Tag-immortalized mesothelial line (MET5A) and malignant mesothelioma cell lines (HMESO and PPM Mill). Whereas increases in phosphorylation of AKT (pAKT) were observed in MET5A cells in response to asbestos, LP9/TERT-1 cells exhibited dose-related decreases in pAKT levels. Pretreatment with an EGFR phosphorylation or mitogen-activated protein kinase kinase 1/2 inhibitor abrogated asbestos-induced phosphorylated ERK (pERK) 1/2 levels in both LP9/TERT-1 and MET5A cells as well as increases in pAKT levels in MET5A cells. Transient transfection of small interfering RNAs targeting ERK1, ERK2, or AKT revealed that ERK1/2 pathways were involved in cell death by asbestos in both cell lines. Asbestos-resistant HMESO or PPM Mill cells with high endogenous levels of ERKs or AKT did not show dose-responsive increases in pERK1/ERK1, pERK2/ERK2, or pAKT/AKT levels by asbestos. However, small hairpin ERK2 stable cell lines created from both malignant mesothelioma lines were more sensitive to asbestos toxicity than shERK1 and shControl lines, and exhibited unique, tumor-specific changes in endogenous cell death–related gene expression. Our results suggest that EGFR phosphorylation is causally linked to pERK and pAKT activation by asbestos in normal and SV40 Tag–immortalized human mesothelial cells. They also indicate that ERK2 plays a role in modulating asbestos toxicity by regulating genes critical to cell injury and survival that are differentially expressed in human mesotheliomas.
mesothelioma; asbestos; toxicity; epidermal growth factor receptor; protein kinase B/AKT
Viral infections are detected in most cases by the host innate immune system through pattern-recognition receptors (PRR), the sensors for pathogen-associated molecular patterns (PAMPs), which induce the production of cytokines, such as type I interferons (IFN). Recent identification in mammalian and teleost fish of cytoplasmic viral RNA sensors, RIG-I-like receptors (RLRs), and their mitochondrial adaptor: the mitochondrial antiviral signaling (MAVS) protein, also called IPS-1, highlight their important role in the induction of IFN at the early stage of a virus infection. More recently, an endoplasmic reticulum (ER) adaptor: the stimulator of interferon genes (STING) protein, also called MITA, ERIS and MPYS, has been shown to play a pivotal role in response to both non-self-cytosolic RNA and dsDNA. In this study, we cloned STING cDNAs from zebrafish and showed that it was an ortholog to mammalian STING. We demonstrated that overexpression of this ER protein in fish cells led to a constitutive induction of IFN and interferon-stimulated genes (ISGs). STING-overexpressing cells were almost fully protected against RNA virus infection with a strong inhibition of both DNA and RNA virus replication. In addition, we found that together with MAVS, STING was an important player in the RIG-I IFN-inducing pathway. This report provides the demonstration that teleost fish possess a functional RLR pathway in which MAVS and STING are downstream signaling molecules of RIG-I. The Sequences presented in this article have been submitted to GenBank under accession numbers: Zebrafish STING (HE856619); EPC STING (HE856620); EPC IRF3 (HE856621); EPC IFN promoter (HE856618).