Characterization of HCV Interactions with Toll-Like Receptors and RIG-I in Liver Cells

The aim of this study was to examine the mechanisms of IFN induction and viral escape. In order to accomplish the goal we compared our new hepatoma cell line LH86, which has intact TLR3 and RIG-I expression and responds to HCV by inducing IFN, with Huh7.5 cells which lack those features.The initial interaction of LH86 cells, Huh7.5 cells or their transfected counter parts (LH86 siRIG-I, siTLR3 or siTLR7 and Huh7.5 RIG-I, TLR3 or TLR7) after infection with HCV (strain JFH-1) was studied by measuring the expression levels of IFNβ, TRAIL, DR4, DR5 and their correlation to viral replication.HCV replicating RNA induces IFN in LH86 cells. The IFN induction system is functional in LH86, and the expression of the RIG-I and TLR3 in LH86 is comparable to the primary hepatocytes. Both proteins appear to play important roles in suppression of viral replication. We found that innate immunity against HCV is associated with the induction of apoptosis by RIG-I through the TRAIL pathway and the establishment of an antiviral state by TLR3. HCV envelope proteins interfere with the expression of TLR3 and RIG-I.These findings correlate with the lower expression level of PRRs in HCV chronic patients and highlight the importance of the PRRs in the initial interaction of the virus and its host cells. This work represents a novel mechanism of viral pathogenesis for HCV and demonstrates the role of PRRs in viral infection

Bone Marrow Mononuclear Cells Up-Regulate Toll-Like Receptor Expression and Produce Inflammatory Mediators in Response to Cigarette Smoke Extract

Several reports link cigarette smoking with leukemia. However, the effects of cigarette smoke extract (CSE) on bone marrow hematopoiesis remain unknown. The objective of this study was to elucidate the direct effects of cigarette smoke on human bone marrow hematopoiesis and characterize the inflammatory process known to result from cigarette smoking. Bone marrow mononuclear cells (BMCs) from healthy individuals when exposed to CSE had significantly diminished CFU-E, BFU-E and CFU-GM. We found increased nuclear translocation of the NF-κB p65 subunit and, independently, enhanced activation of AKT and ERK1/2. Exposure of BMCs to CSE induced IL-8 and TGF-β1 production, which was dependent on NF-κB and ERK1/2, but not on AKT. CSE treatment had no effect on the release of TNF-α, IL-10, or VEGF. Finally, CSE also had a significant induction of TLR2, TLR3 and TLR4, out of which, the up-regulation of TLR2 and TLR3 was found to be dependent on ERK1/2 and NF-κB activation, but not AKT. These results indicate that CSE profoundly inhibits the growth of erythroid and granulocyte-macrophage progenitors in the bone marrow. Further, CSE modulates NF-κB- and ERK1/2-dependent responses, suggesting that cigarette smoking may impair bone marrow hematopoiesis in vivo as well as induce inflammation, two processes that proceed malignant transformation

Primers sets used for Real time RT-PCR(D-Lux primers show fluorochrome in the sequence).

<p>Primers sets used for Real time RT-PCR(D-Lux primers show fluorochrome in the sequence).</p

The expression of TLR3 and RIG-I is affected by intact virion proteins in hepatocytes

<p>A) IFNβ mRNA expression in Huh7.5 TLR3 or TLR7 stable cell lines co-transfected with either PKR or RIG-I after infection with HCV MOI = 0.1. Expression was calculated by the ΔΔCt method where uninfected cells were the experimental control and the housekeeping gene GAPDH was the internal control. Error bars represent the SEM of three separate experiments. B) TLR3 and RIG-I mRNA expression levels 7 days after infection with different HCV dilutions (methodology as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0021186#pone-0021186-g001" target="_blank">figure 1C</a>). C) TLR3 and RIG-I gene expression levels 7 days after infection with normal, heated or UV-treated virus calculated as described in part A.</p

A strong initial IFN response is induced an IRF-3 response by TLR3 but is not enough to clear HCV.

<p>A) Viral replication in Huh7.5 cells stably transfected with TOPO (control) TLR3 or TLR7 infected with HCV MOI of 0.1 and collected every 2–3 days for RNA isolation (total 75 days). The HCV copy numbers from each time points were calculated by real time RT-PCR and compared against an HCV standard curve. B) IFNβ mRNA expression of the experiment described in part A. Expression was calculated by the ΔΔCt method where uninfected cells were the experimental control and the housekeeping gene GAPDH was the internal control. Error bars represent the SEM of three separate experiments. C) IP-10 and RANTES mRNA expression of the experiment described in part A. Methodology as described for part B.</p

Envelope proteins affect the response to non-HCV responses through RIG-I receptor but only temporarily through TLR3.

<p>A) IFNβ gene expression of stably transfected LH86 cells expressing Core, E1E2 or NS3/4A 4 days after HCV infection. Expression was calculated by the ΔΔCt method where uninfected cells were the experimental control and the housekeeping gene GAPDH was the internal control. Error bars represent the SEM of three separate experiments. B) TLR3 mRNA expression 7 days after infection of stably transfected LH86 cells carrying the HCV proteins Core, E1/E2 or NS3/4A. Expression determined as described in part A. E) RIG-I mRNA expression 7 days after infection of stably transfected LH86 cells carrying HCV proteins Core, E1/E2 or NS3/4A. Expression determined as described in part A. D) IFNβ mRNA expression of LH86 cells or LH86 stably transfected with E1/E2 treated with 50 µg/µL transfected Poly I:C. Expression was calculated as described in part A. E) IFNβ mRNA expression of LH86 cells or LH86 stably transfected with E1/E2 treated with 50 µg/µL extracellular Poly I:C. Expression determined as described in part A.</p

Both TLRs and RIG-I prevent viral replication and are needed for the induction of IFN.

<p>A) IFNβ mRNA expression at day 4 post-infection in LH86 cells after transfecting with a control siRNA, or siRNA against TLR3 or TLR7. Expression was calculated by the ΔΔCt method where uninfected cells were the experimental control and the housekeeping gene GAPDH was the internal control. Error bars represent the SEM of three separate experiments. No virus indicates cells were cultured with the same volume in uninfected Huh7. 5 supernatant, HCV is the supernatant from infected Huh7.5 cells as described in the methods section (MOI = 0.1). B) IFNβ mRNA expression at day 4 post-infection in Huh7.5 cells expressing TLR3 or TLR7. Expression was calculated by the ΔΔCt method as described in part A and bars represent the SEM of three separate experiments. C) Viral replication in Huh7.5 cells expressing TLR3 or TLR7 at day 7 after infection. HCV copy numbers were calculated by real time RT-PCR run with an HCV standard curve. D) IFNβ mRNA expression at day 4 post-infection in LH86 cells with silenced RIG-I. Methodology as described in part A. E) IFNβ mRNA expression at day 4 post-infection in Huh7.5 cells transfected with RIG-I. Methodology as described in part A. F) Viral replication in Huh7.5 cells transfected with RIG-I after 7 days of culture. Methodology as described in part C.</p

IFN response is dependent on viral replication.

<p>A) IFNβ mRNA expression was measured daily from the total RNA of LH86 cells treated with an MOI of 0.1 of HCV/JFH-1. The expression was calculated by the ΔΔCt method where uninfected cells were the experimental control and the housekeeping gene GAPDH was the internal control. Error bars represent the SEM of three separate experiments. The “No virus” control indicates cells that were cultured with uninfected Huh7.5 supernatant, “HCV” is the supernatant from infected Huh7.5 cells as described in the methods section (MOI = 0.1), “heated HCV” is the same supernatant as “HCV” but heated for 15 minutes at 72°C and “UV-treated HCV” was exposed to UV light for 15 minutes. B) IFNβ mRNA expression of different dilutions of virus (1∶1 MOI = 0.1) from day 0 and day 4 post infection calculated by the ΔΔCt method (determined as in part A). C) IFNβ mRNA expression of LH86 cells electroporated with <i>in vitro</i> transcribed HCV/JFH-1 or its non-replicating counterpart HCV/JFH-GND (day 0 is the day of the electroporation). Expression was calculated as described for part A.</p

Inactivation of DAP12 in PMN inhibits TREM1-mediated activation in rheumatoid arthritis.

Rheumatoid arthritis (RA) is an autoimmune disease characterized by dysregulated and chronic systemic inflammatory responses that affect the synovium, bone, and cartilage causing damage to extra-articular tissue. Innate immunity is the first line of defense against invading pathogens and assists in the initiation of adaptive immune responses. Polymorphonuclear cells (PMNs), which include neutrophils, are the largest population of white blood cells in peripheral blood and functionally produce their inflammatory effect through phagocytosis, cytokine production and natural killer-like cytotoxic activity. TREM1 (triggering receptor expressed by myeloid cells) is an inflammatory receptor in PMNs that signals through the use of the intracellular activating adaptor DAP12 to induce downstream signaling. After TREM crosslinking, DAP12's tyrosines in its ITAM motif get phosphorylated inducing the recruitment of Syk tyrosine kinases and eventual activation of PI3 kinases and ERK signaling pathways. While both TREM1 and DAP12 have been shown to be important activators of RA pathogenesis, their activity in PMNs or the importance of DAP12 as a possible therapeutic target have not been shown. Here we corroborate, using primary RA specimens, that isolated PMNs have an increased proportion of both TREM1 and DAP12 compared to normal healthy control isolated PMNs both at the protein and gene expression levels. This increased expression is highly functional with increased activation of ERK and MAPKs, secretion of IL-8 and RANTES and cytotoxicity of target cells. Importantly, based on our hypothesis of an imbalance of activating and inhibitory signaling in the pathogenesis of RA we demonstrate that inhibition of the DAP12 signaling pathway inactivates these important inflammatory cells