Epstein-Barr virus induces MCP-1 secretion by human monocytes via TLR2

Epstein-Barr virus (EBV) is a gammaherpesvirus infecting the majority of the human adult population in the world. TLR2, a member of the Toll-like receptor (TLR) family, has been implicated in the immune responses to different viruses including members of the herpesvirus family, such as human cytomegalovirus, herpes simplex virus type 1, and varicella-zoster virus. In this report, we demonstrate that infectious and UV-inactivated EBV virions lead to the activation of NF-kappaB through TLR2 using HEK293 cells cotransfected with TLR2-expressing vector along with NF-kappaB-Luc reporter plasmid. NF-kappaB activation in HEK293-TLR2 cells (HEK293 cells transfected with TLR2) by EBV was not enhanced by the presence of CD14. The effect of EBV was abrogated by pretreating HEK293-TLR2 cells with blocking anti-TLR2 antibodies or by preincubating viral particles with neutralizing anti-EBV antibodies 72A1. In addition, EBV infection of primary human monocytes induced the release of MCP-1 (monocyte chemotactic protein 1), and the use of small interfering RNA targeting TLR2 significantly reduced such a chemokine response to EBV. Taken together, these results indicate that TLR2 may be an important pattern recognition receptor in the immune response directed against EBV infection.     

TLR3 signaling in a hepatoma cell line is skewed towards apoptosis

Toll-like receptors (TLRs) recognize pathogen-associated molecular patterns (PAMPS) leading to the activation of the innate immune response and subsequently to the shaping of the adaptive immune response. Of the known human TLRs, TLR3, 7, 8, and 9 were shown to recognize nucleic acid ligands. TLR3 signaling is induced by double-stranded (ds)RNA, a molecular signature of viruses, and is mediated by the TRIF (TIR domain-containing adaptor-inducing IFNbeta) adaptor molecule. Thus, TLR3 plays an important role in the host response to viral infections. The liver is constantly exposed to a large variety of foreign substances, including pathogens such as HBV (hepatitis B virus) and HCV (hepatitis C virus), which frequently establish persistent liver infections. In this work, we investigated the expression and signaling pathway of TLR3 in different hepatoma cell lines. We show that hepatocyte lineage cells express relatively low levels of TLR3 mRNA. TLR3 signaling in HEK293 cells (human embryonic kidney cells) activated NF-kappaB and IRF3 (interferon regulatory factor 3) and induced IFNbeta (interferon beta) promoter expression, which are known to lead to pro-inflammatory cytokine secretion. In Huh7 cells, there was only a short-term IRF3 activation, and a very low level of IFNbeta expression. In HepG2 cells on the other hand, while no induction of pro-inflammatory factors was observed, signaling by TLR3 was skewed towards the induction of apoptosis. These results indicate preferential induction of the apoptotic pathway over the cytokine induction pathway by TLR3 signaling in hepatocellular carcinoma cells with potential implications for therapeutic strategies.     

Human TLR3 recognizes dengue virus and modulates viral replication in vitro

The elicitation of large amount inflammatory cytokine in serum has been developed as the cause of the plasma leakage in dengue fever (DF)/dengue haemorrhagic fever (DHF) infection. Virus recognition in innate immunity is the key. The Toll-like receptors (TLRs) play an important role in pathogen recognition towards cytokine induction among several viruses; however, the role of TLRs on innate immune recognition against DENV remains unclear. This study aims at the interaction between dengue virus (DENV) and human TLRs at the incipient stage of infection in vitro . Our experiment reveals that stably expression of TLR3, 7, 8 on HEK293 enables IL-8 secretion after DENV recognition. By the model of human monocytic cells U937, we demonstrated the trigger of IL-8 after viral recognition of human monocytic cell is primary through TLR3 following endosomal acidification. Silencing of TLR3 in U937 cells significantly blocks the DENV-induced IL-8 production. Besides, the interaction is further corroborated by colocalization of TLR3 and DENV RNA upon DENV internalization. Furthermore, in this study we found the expression of TLR3 can mediate strong IFN-α/β release and inhibit DENV viral replication significantly, thus limit the cytopathic effect.     

West Nile virus envelope protein inhibits dsRNA-induced innate immune responses

The immune response against viral infection relies on the early production of cytokines that induce an antiviral state and trigger the activation of immune cells. This response is initiated by the recognition of virus-associated molecular patterns such as dsRNA, a viral replication intermediate recognized by TLR3 and certain RNA helicases. Infection with West Nile virus (WNV) can lead to lethal encephalitis in susceptible individuals and constitutes an emerging health threat. In this study, we report that WNV envelope protein (WNV-E) specifically blocks the production of antiviral and proinflammatory cytokines induced by dsRNA in murine macrophages. This immunosuppressive effect was not dependent on TLR3 or its adaptor molecule Trif. Instead, our experiments show that WNV-E acts at the level of receptor-interacting protein 1. Our results also indicate that WNV-E requires a certain glycosylation pattern, specifically that of dipteran cells, to inhibit dsRNA-induced cytokine production. In conclusion, these data show that the major structural protein of WNV impairs the innate immune response and suggest that WNV exploits differential vector/host E glycosylation profiles to evade antiviral mechanisms.     

Proteolytic Processing Regulates Toll-like Receptor 3 Stability and Endosomal Localization

Toll-like receptors (TLRs) 3, 7, and 9 are innate immune receptors that recognize nucleic acids from pathogens in endosomes and initiate signaling transductions that lead to cytokine production. Activation of TLR9 for signaling requires proteolytic processing within the ectodomain by endosome-associated proteases. Whether TLR3 requires similar proteolytic processing to become competent for signaling remains unclear. Herein we report that human TLR3 is proteolytically processed to form two fragments in endosomes. Unc93b1 is required for processing by transporting TLR3 through the Golgi complex and to the endosomes. Proteolytic cleavage requires the eight-amino acid Loop1 within leucine-rich repeat 12 of the TLR3 ectodomain. Proteolytic cleavage is not required for TLR3 signaling in response to poly(I:C), although processing could modulate the degree of response toward viral double-stranded RNAs, especially in mouse cells. Both the full-length and cleaved fragments of TLR3 can bind poly(I:C) and are present in endosomes. However, although the full-length TLR3 has a half-life in HEK293T cells of 3 h, the cleaved fragments have half-lives in excess of 7 h. Inhibition of TLR3 cleavage by either treatment with cathepsin inhibitor or by a mutation in Loop1 decreased the abundance of TLR3 in endosomes targeted for lysosomal degradation.     

Antiviral signaling through pattern recognition receptors

Viral infection is detected by the host innate immune system. Innate immune cells such as dendritic cells and macrophages detect nucleic acids derived from viruses through pattern recognition receptors (PRRs). Viral recognition by PRRs initiates the activation of signaling pathways that lead to production of type I interferon and inflammatory cytokines, which are important for the elimination of viruses. Two types of PRRs that recognize viral nucleic acids, Toll-like receptors (TLR) and RIG-I-like RNA helicases (RLH), have been identified. Of the TLRs, TLR3 recognizes viral double-stranded (ds) RNA, TLR7 and human TLR8 identify viral single-stranded (ss) RNA and TLR9 detects viral DNA. TLRs are located in endosomal compartments, whereas RLH are present in the cytoplasm where they detect viral dsRNA or ssRNA. Here we review the role of TLRs and RLHs in the antiviral innate immune response.     

Establishment and maintenance of the innate antiviral response to West Nile Virus involves both RIG-I and MDA5 signaling through IPS-1

RIG-I and MDA5, two related pathogen recognition receptors (PRRs), are known to be required for sensing various RNA viruses. Here we investigated the roles that RIG-I and MDA5 play in eliciting the antiviral response to West Nile virus (WNV). Functional genomics analysis of WNV-infected fibroblasts from wild-type mice and RIG-I null mice revealed that the normal antiviral response to this virus occurs in two distinct waves. The initial response to WNV resulted in the expression of interferon (IFN) regulatory factor 3 target genes and IFN-stimulated genes, including several subtypes of alpha IFN. Subsequently, a second phase of IFN-dependent antiviral gene expression occurred very late in infection. In cells lacking RIG-I, both the initial and the secondary responses to WNV were delayed, indicating that RIG-I plays a critical role in initiating innate immunity against WNV. However, another PRR(s) was able to trigger a response to WNV in the absence of RIG-I. Disruption of both MDA5 and RIG-I pathways abrogated activation of the antiviral response to WNV, suggesting that MDA5 is involved in the host's defense against WNV infection. In addition, ablation of the function of IPS-1, an essential RIG-I and MDA5 adaptor molecule, completely disabled the innate antiviral response to WNV. Our data indicate that RIG-I and MDA5 are responsible for triggering downstream gene expression in response to WNV infection by signaling through IPS-1. We propose a model in which RIG-I and MDA5 operate cooperatively to establish an antiviral state and mediate an IFN amplification loop that supports immune effector gene expression during WNV infection.     

Toll-like receptor 3 has a protective role against West Nile virus infection

Protection against West Nile virus (WNV) infection requires rapid viral sensing and the generation of an interferon (IFN) response. Mice lacking IFN regulatory factor 3 (IRF-3) show increased vulnerability to WNV infection with enhanced viral replication and blunted IFN-stimulated gene (ISG) responses. IRF-3 functions downstream of several viral sensors, including Toll-like receptor 3 (TLR3), RIG-I, and MDA5. Cell culture studies suggest that host recognizes WNV in part, through the cytoplasmic helicase RIG-I and to a lesser extent, MDA5, both of which activate ISG expression through IRF-3. However, the role of TLR3 in vivo in recognizing viral RNA and activating antiviral defense pathways has remained controversial. We show here that an absence of TLR3 enhances WNV mortality in mice and increases viral burden in the brain. Compared to congenic wild-type controls, TLR3(-/-) mice showed relatively modest changes in peripheral viral loads. Consistent with this, little difference in multistep viral growth kinetics or IFN-alpha/beta induction was observed between wild-type and TLR3(-/-) fibroblasts, macrophages, and dendritic cells. In contrast, a deficiency of TLR3 was associated with enhanced viral replication in primary cortical neuron cultures and greater WNV infection in central nervous system neurons after intracranial inoculation. Taken together, our data suggest that TLR3 serves a protective role against WNV in part, by restricting replication in neurons.     

Host species-specific usage of the TLR4-LPS receptor complex

Recognition of LPS depends on the interaction of at least three molecules forming the LPS-receptor complex. The most important ones, CD14, MD2 and Toll-like receptor (TLR) 4 share a high degree of homology between species. In the present study, we investigated the importance of species-specific restriction on the recognition of LPS using stably transfected HEK293 cell lines expressing either human or bovine LPS-receptor complex components. Species-specific MD2 appeared to confer LPS recognition, whereas species-specific CD14 only appeared to play a minor role. In addition to the recognition of LPS, there is evidence that the fusion (F) protein of respiratory syncytial virus (RSV), which is the most common viral respiratory pathogen during infancy world-wide, interacts with TLR4, and plays an important role in the initiation of the innate immune response. Our findings suggest that human and bovine RSV may activate human and bovine TLR4 receptors, respectively, in the presence of both MD2 and CD14. However, no clear role for the RSV F protein of either human or bovine RSV alone in stimulating TLR4-dependent NF-kappaB activation was observed.     

MAPK Activation Is Essential for Waddlia chondrophila Induced CXCL8 Expression in Human Epithelial Cells

Background

Waddlia chondrophila (W. chondrophila) is an emerging agent of respiratory and reproductive disease in humans and cattle. The organism is a member of the order Chlamydiales, and shares many similarities at the genome level and in growth studies with other well-characterised zoonotic chlamydial agents, such as Chlamydia abortus (C. abortus). The current study investigated the growth characteristics and innate immune responses of human and ruminant epithelial cells in response to infection with W. chondrophila.

Methods

Human epithelial cells (HEp2) were infected with W. chondrophila for 24h. CXCL8 release was significantly elevated in each of the cell lines by active-infection with live W. chondrophila, but not by exposure to UV-killed organisms. Inhibition of either p38 or p42/44 MAPK significantly inhibited the stimulation of CXCL8 release in each of the cell lines. To determine the pattern recognition receptor through which CXCL8 release was stimulated, wild-type HEK293 cells which express no TLR2, TLR4, NOD2 and only negligible NOD1 were infected with live organisms. A significant increase in CXCL8 was observed.

Conclusions/Significance

W. chondrophila actively infects and replicates within both human and ruminant epithelial cells stimulating CXCL8 release. Release of CXCL8 is significantly inhibited by inhibition of either p38 or p42/44 MAPK indicating a role for this pathway in the innate immune response to W. chondrophila infection. W. chondrophila stimulation of CXCL8 secretion in HEK293 cells indicates that TLR2, TLR4, NOD2 and NOD1 receptors are not essential to the innate immune response to infection.     

Immunologic consequences of Francisella tularensis live vaccine strain infection: role of the innate immune response in infection and immunity

Francisella tularensis (Ft), a Gram-negative intracellular bacterium, is the etiologic agent of tularemia. Although attenuated for humans, i.p. infection of mice with <10 Ft live vaccine strain (LVS) organisms causes lethal infection that resembles human tularemia, whereas the LD50 for an intradermal infection is >10(6) organisms. To examine the immunological consequences of Ft LVS infection on the innate immune response, the inflammatory responses of mice infected i.p. or intradermally were compared. Mice infected i.p. displayed greater bacterial burden and increased expression of proinflammatory genes, particularly in the liver. In contrast to most LPS, highly purified Ft LVS LPS (10 microg/ml) was found to be only minimally stimulatory in primary murine macrophages and in HEK293T cells transiently transfected with TLR4/MD-2/CD14, whereas live Ft LVS bacteria were highly stimulatory for macrophages and TLR2-expressing HEK293T cells. Despite the poor stimulatory activity of Ft LVS LPS in vitro, administration of 100 ng of Ft LVS LPS 2 days before Ft LVS challenge severely limited both bacterial burden and cytokine mRNA and protein expression in the absence of detectable Ab at the time of bacterial challenge, yet these mice developed a robust IgM Ab response within 2 days of infection and survived. These data suggest that prior administration of Ft LVS LPS protects the host by diminishing bacterial burden and blunting an otherwise overwhelming inflammatory response, while priming the adaptive immune response for development of a strong Ab response.