Interferon-stimulated genes response in endothelial cells following Hantaan virus infection

The regulation mechanism of interferon (IFN) and IFN-stimulated genes is a very complex procedure and is dependent on cell types and virus species. We observed molecular changes related to anti-viral responses in endothelial cells during Hantaan virus (HTNV) infection. We found that there are two patterns of gene expression, the first pattern of gene expression being characterized by early induction and short action, as in that of type I IFNs,' and the other being characterized by delayed induction and long duration, as those of IRF-7, MxA, and TAP-1/2. Even though there are significant differences in their induction folds, we found that all of IFN-alpha/beta, IRF- 3/7, MxA, and TAP-1/2 mRNA expressions reached the peak when the viral replication was most active, which took place 3 days of post infection (d.p.i.). In addition, an interesting phenomenon was observed; only one gene was highly expressed in paired genes such as IFN-alpha/beta' (3/277-folds), IRF-3/7 (2.2/29.4-folds), and TAP- 1/2 (26.2/6.1-folds). Therefore, IFN-beta, IRF-7, and TAP-1 seem to be more important for the anti-viral response in HTNV infection. MxA was increased to 296-folds at 3 d.p.i. and kept continuing 207-folds until 7 d.p.i.. The above results indicate that IFN-beta works for an early anti-viral response, while IRF7, MxA, and TAP-1 work for prolonged anti-viral response in HTNV infection.     

Excessive CpG 1668 stimulation triggers IL-10 production by cDC that inhibits IFN-alpha responses by pDC

Upon stimulation with a wide range of concentrations of CpG oligodeoxynucleotide 2216 (CpG 2216), plasmacytoid DC are induced to produce type I IFN (IFN-alpha/beta). In contrast, CpG 1668 shows a bell-shaped dose-response correlation, i.e. only intermediate but not high doses of CpG 1668 induce IFN-alpha/beta. Interestingly, high-dose CpG 1668 completely inhibited IFN-alpha responses induced by CpG 2216. Experiments using supernatant of high-dose CpG-1668-treated cells indicated that secreted inhibitor(s) mediated the IFN-alpha shut-off. Among modulating cytokines, IL-10 turned out to be one important negative regulator. In line with this, supernatants of IL-10-deficient DC cultures stimulated with high-dose CpG 1668 did not inhibit IFN-alpha production. Interestingly, high-dose CpG 1668 also inhibited IFN-alpha responses induced by the DNA-encoded mouse cytomegalovirus, whereas IFN-alpha responses induced by negative-strand RNA-encoded vesicular stomatitis virus were only marginally affected. Experiments with DC cultures devoid of TLR9 indicated that TLR9 was critically required to mediate stimulatory and modulatory signals by low and high concentrations of CpG 1668, respectively. Analysis of purified DC subsets showed that conventional DC were the main IL-10 producers, whereas plasmacytoid DC hardly produced any IL-10.     

TLR9 and STING agonists synergistically induce innate and adaptive type‐II IFN

Agonists for TLR9 and Stimulator of IFN Gene (STING) act as vaccine adjuvants that induce type‐1 immune responses. However, currently available CpG oligodeoxynucleotide (ODN) (K‐type) induces IFNs only weakly and STING ligands rather induce type‐2 immune responses, limiting their potential therapeutic applications. Here, we show a potent synergism between TLR9 and STING agonists. Together, they make an effective type‐1 adjuvant and an anticancer agent. The synergistic effect between CpG ODN (K3) and STING‐ligand cyclic GMP–AMP (cGAMP), culminating in NK cell IFN‐γ (type‐II IFN) production, is due to the concurrent effects of IL‐12 and type‐I IFNs, which are differentially regulated by IRF3/7, STING, and MyD88. The combination of CpG ODN with cGAMP is a potent type‐1 adjuvant, capable of inducing strong T_h1‐type responses, as demonstrated by enhanced antigen‐specific IgG2c and IFN‐γ production, as well as cytotoxic CD8⁺ T‐cell responses. In our murine tumor models, intratumoral injection of CpG ODN and cGAMP together reduced tumor size significantly compared with the singular treatments, acting as an antigen‐free anticancer agent. Thus, the combination of CpG ODN and a STING ligand may offer therapeutic application as a potent type‐II IFN inducer.     

Dendritic cells respond to influenza virus through TLR7- and PKR-independent pathways

Natural interferon-producing cells (IPC) secrete type I IFN (IFN-alpha and -beta) in response to influenza virus. This process is independent of viral replication and is mediated by Toll-like receptor 7 (TLR7), which recognizes single-stranded RNA (ssRNA). DC also express TLR7 but its function in DC response to influenza virus is unknown. To address this, we compared the DC and IPC responses to influenza virus and ssRNA oligoribonucleotides (ORN) that activate TLR7. When stimulated by ORN in vitro and in vivo, DC matured and produced inflammatory cytokines but not IFN-alpha. DC did secrete IFN-alpha in response to influenza virus. However, this response was independent of TLR7 signaling and required viral replication but not dsRNA-activated protein kinase (PKR). We conclude that DC and IPC are hard-wired to secrete IFN-alpha via different pathways, reflecting their complementary but distinct roles in anti-viral immunity.     

Effects of the hepatitis C virus core protein on innate cellular defense pathways.

The hepatitis C virus (HCV) core protein is thought to contribute to HCV pathogenesis through its interaction with various signal transduction pathways. In this study, we explored the interaction of the core protein with innate defense pathways (interferon [IFN] regulatory factor [IRF], Jak-Stat, and inducible nitric oxide synthase [iNOS]) in HeLa and Huh7 human cell lines. Expression of a patient-derived genotype 1b core protein activated human IRF-1 and guanylate-binding protein-2 (GBP-2) promoters, induced IRF-1 mRNA, but failed to induce IRF-3 phosphorylation. HCV core protein caused dose-dependent induction of the IFN-beta promoter and IFN-beta mRNA but not the IFN-alpha1 and IFN-alpha4 promoters. In the presence of IFN-alpha, core expression was associated with increased IFN-stimulated gene factor 3 (ISGF3) binding to the IFN-stimulated response element (ISRE) and tyrosine phosphorylation of Stat1. Core expression resulted in dose-dependent activation of the ISRE and gamma activated sequence (GAS) promoters, in both the absence and the presence of either IFN-alpha or IFN-gamma. Core stimulated the human iNOS promoter and induced iNOS protein. The data indicate that HCV core can modulate IRF, Jak-Stat, and iNOS pathways and suggest mechanisms by which core could affect HCV persistence and pathogenesis.     

Innate immune recognition of an AT-rich stem-loop DNA motif in the Plasmodium falciparum genome

Although Toll-like receptor 9 (TLR9) has been implicated in cytokine and type I interferon (IFN) production during malaria in humans and mice, the high AT content of the Plasmodium falciparum genome prompted us to examine the possibility that malarial DNA triggered TLR9-independent pathways. Over 6000 ATTTTTAC ("AT-rich") motifs are present in the genome of P.?falciparum, which we show here potently induce type I IFNs. Parasite DNA, parasitized erythrocytes and oligonucleotides containing the AT-rich motif induce type I IFNs via a pathway that did not involve the previously described sensors TLR9, DAI, RNA polymerase-III or IFI16/p204. Rather, AT-rich DNA sensing involved an unknown receptor that coupled to the STING, TBK1 and IRF3-IRF7 signaling pathway. Mice lacking IRF3, IRF7, the kinase TBK1 or the type I IFN receptor were resistant to otherwise lethal cerebral malaria. Collectively, these observations implicate AT-rich DNA sensing via STING, TBK1 and IRF3-IRF7 in P.?falciparum malaria.     

Activation of mast cells by double-stranded RNA: evidence for activation through Toll-like receptor 3

BACKGROUND: Although mast cells (MCs) have been clearly implicated in innate immune responses involving bacteria, their ability to respond to viral infection is less clear. OBJECTIVE: Given that MCs increase at sites of inflammation and are located at surfaces where exposure to invading viruses may occur, we explored the ability of MCs to produce cytokines including type I IFNs after exposure to viruses and to polyinosine-polycytidylic acid (polyI:C), a synthetic mimic of viral double-stranded RNA, and characterized the receptors involved, if any. METHODS: Human peripheral blood-derived cultured MCs and 2 MC lines, Laboratory of Allergic Disease MC line and human MC line 1, were stimulated with viruses and polyI:C, and cytokine production, degranulation, and signaling pathway activation were examined. Because polyI:C is a ligand for Toll-like receptor (TLR)-3, human MCs were also analyzed for TLR expression. RESULTS: Viruses and polyI:C induced IFN-alpha and IFN-beta production. PolyI:C did not induce TNF, IL-1beta, IL-5, or GM-CSF production, in contrast with other TLR ligands (LPS, peptidoglycan, CpG-A, or flagellin). IFN-alpha production involved nuclear factor-kappaB, p38, and C-Jun NH2-terminal kinase and mitogen-activated protein kinase. RT-PCR and Western blot analysis confirmed expression of TLR-3 by all MCs. Human cultured MCs also expressed TLR-1, TLR-2, TLR-4, TLR-5, TLR-6, TLR-7 and TLR-9. Antibodies to TLR-3 significantly decreased IFN-alpha production. Bone marrow-derived MCs from TLR-3 knockout mice showed an ablated response to polyI:C. CONCLUSIONS: Murine and human MCs produce type I IFNs after exposure to double-stranded RNA and/or virus, the former via specific interactions with TLR-3. These data suggest that MCs contribute to innate immune responses to viral infection via the production of type I IFNs.     

IFN-beta modulates the response to TLR stimulation in human DC: involvement of IFN regulatory factor-1 (IRF-1) in IL-27 gene expression

Type I IFN are cytokines which play a central role in host resistance to viral or microbial infections and are important components linking innate and adaptive immunity. We and others have previously demonstrated that the production of IFN-beta by DC following bacterial infections or TLR triggering influences, in an autocrine manner, their maturation. In this study, we investigated whether IFN-beta release modulates the phenotype of the immature DC and their response to a subsequent TLR stimulation. The induction of CD86, HLA-DR, CD38 and B7H1 and the absence of CCR7 and CD83 expression upon IFN-beta treatment suggest that IFN-beta-primed DC remain at the site of infection acquiring an activated phenotype. These results prompted us to investigate the response of IFN-beta-primed DC to TLR stimulation. While IFN-beta pretreatment increases slightly the expression of maturation markers in TLR2- or TLR4-stimulated DC, it is able to modulate selectively the secretion of inflammatory and immuno-regulating cytokines. Interestingly, IL-27p28 subunit was induced by IFN-beta alone or during LPS-induced maturation of DC in a type I IFN-dependent manner through IFN regulatory factor-1 (IRF-1) activation. Taken together, our results shed light on the capacity of IFN-beta to finely tune DC response to invading pathogens.     

Regulation of STAT pathways and IRF1 during human dendritic cell maturation by TNF-alpha and PGE2

Maturation of dendritic cells (DCs) by TLR ligands induces expression of IFN-beta and autocrine activation of IFN-inducible Stat1-dependent genes important for DC function. In this study, we analyzed the regulation of STAT signaling during maturation of human DCs by TNF-alpha and PGE2, which induced maturation of human DCs comparably with LPS but did not induce detectable IFN-beta production or Stat1 tyrosine phosphorylation. Consistent with these results, TNF-alpha and PGE2 did not induce Stat1 DNA binding to a standard Stat1-binding oligonucleotide. Instead, TNF-alpha and PGE2 increased Stat1 serine phosphorylation and Stat4 tyrosine phosphorylation and activated expression of the NF-kappaB and Stat1 target gene IFN regulatory factor 1 (IRF1), which contributes to IFN responses. TNF-alpha and PGE2 induced a complex that bound an oligonucleotide derived from the IRF1 promoter that contains a STAT-binding sequence embedded in a larger palindromic sequence, and this complex was recognized by Stat1 antibodies. These results suggest that TNF-alpha and PGE2 activate STAT-mediated components of human DC maturation by alternative pathways to the IFN-beta-mediated autocrine loop used by TLRs.     

Type I interferon regulates respiratory virus infected dendritic cell maturation and cytokine production

Activation of dendritic cells (DCs) by viruses is critical for both innate and adaptive immune responses. In this report, we investigated the role of type I interferon (IFN) in the activation of DCs by respiratory syncytial virus (RSV). Using DCs from type I IFNR-/- mice, these studies indicate that maturation, including upregulation of co-stimulatory molecules and optimal cytokine production, by RSV infection was dependent on type I IFN receptor signaling. Subsequently, studies using DCs from wild type mice demonstrate that continued production of type I IFN during later stages of DC maturation could alter their activation profiles. IFN-alpha and IFN-beta were upregulated in DCs grown from bone marrow of wild type mice after infection with RSV. In order to determine their function in competent DCs, blocking antibodies were used to specifically inhibit IFN-alpha/beta . The data demonstrate that production of IFN-beta, but not IFN-alpha, in RSV-infected wild type DCs promotes chemokine production and toll-like receptor (TLR) expression, while limiting IL-12 production. The inhibition of IL-12p70 by IFN-beta correlated with suppressed IL-12p40 expression levels. Furthermore, the addition of recombinant IFN-beta potently inhibited IL-12p40 expression in mature DC subsets during RSV infection, while only the highest dose of IFN-alpha had any inhibitory effect. Together, our studies provide insight into the complex regulation of DC maturation and IL-12 production co-ordinated by type I interferons in RSV-infected dendritic cells, and demonstrate that type I IFN has specific roles depending upon the stage of DC maturation.     

IRF‐5 and NF‐κB p50 co‐regulate IFN‐β and IL‐6 expression in TLR9‐stimulated human plasmacytoid dendritic cells

Synthetic oligonucleotides (ODN) expressing CpG motifs mimic the ability of bacterial DNA to trigger the innate immune system via TLR9. Plasmacytoid dendritic cells (pDCs) make a critical contribution to the ensuing immune response. This work examines the induction of antiviral (IFN‐β) and pro‐inflammatory (IL‐6) cytokines by CpG‐stimulated human pDCs and the human CAL‐1 pDC cell line. Results show that interferon regulatory factor‐5 (IRF‐5) and NF‐κB p50 are key co‐regulators of IFN‐β and IL‐6 expression following TLR9‐mediated activation of human pDCs. The nuclear accumulation of IRF‐1 was also observed, but this was a late event that was dependant on type 1 IFN and unrelated to the initiation of gene expression. IRF‐8 was identified as a novel negative regulator of gene activation in CpG‐stimulated pDCs. As variants of IRF‐5 and IRF‐8 were recently found to correlate with susceptibility to certain autoimmune diseases, these findings are relevant to our understanding of the pharmacologic effects of “K” ODN and the role of TLR9 ligation under physiologic, pathologic, and therapeutic conditions.