Cigarette smoke attenuation of poly I:C-induced innate antiviral responses in human PBMC is mainly due to inhibition of IFN-beta production

The cellular response to dsRNA or its synthetic analog polyinosinic-polycytidylic acid (poly I:C) results in IRF-3-, IRF-7- and NF-kB-mediated activation of type 1 IFNs and pro-inflammatory cytokines critical for innate antiviral immune responses. To investigate whether cigarette smoke compromises type 1 IFN signaling in humans, peripheral blood mononuclear cells (PBMCs) from non-smoking individuals were treated with smoke-conditioned media (SCM) and stimulated with poly I:C. We observed a marked attenuation of IRF-3 and NF-kB activation in PBMCs exposed to SCM compared to control PBMCs. Similarly, PBMCs from smokers or splenocytes from smoke-exposed mice also displayed marked reduction of poly I:C-induced antiviral responses compared with either non-smokers or sham-exposed mice. Cigarette smoke was found to block the production of type I IFNs following poly I:C treatment and inhibit subsequent STAT1 activation. Finally, we confirmed that inhibition of IFN-beta, but not IFN-alpha, predominantly contributes to the cigarette smoke-mediated suppression of innate antiviral responses. These findings provide novel mechanistic insights to the susceptibility of cigarette smokers to viral infections.     

Rabies viral mechanisms to escape the IFN system: the viral protein P interferes with IRF-3, Stat1, and PML nuclear bodies.

Interferons (IFNs) are a family of secreted proteins with antiviral, antiproliferative, and immunomodulatory activities. The different biologic actions of IFN are believed to be mediated by the products of specifically IFN-stimulated genes (ISG) in the target cells. The IFN response is the first line of defense against viral infections. Viruses, which require the cellular machinery for their replication, have evolved different ways to counteract the action of IFN by inhibiting IFN production or Jak-Stat signaling or by altering ISG products. This review focuses on the role of viral proteins from the RNA virus family, particularly rabies P protein. P protein mediates inhibition of the IFN system by different pathways: it inhibits IFN production by impairing IFN regulatory factor-3 (IRF-3) phosphorylation and IFN signaling by blocking nuclear transport of Stat1 and alters promyelocytic leukemia (PML) nuclear bodies by retaining PML in the cytoplasm.     

The role of interferon regulatory factors in the cardiac response to viral infection

Reovirus-induced murine myocarditis provides an excellent model for the human disease. Cardiac tissue damage varies between reovirus strains, and is caused by a direct viral cytopathogenic effect. One determinant of virus-induced cardiac tissue damage is the cardiac interferon-beta (IFN-beta) response to viral infection. Nonmyocarditic reoviruses induce more IFN-beta and/or are more sensitive to the antiviral effects of IFN-beta in cardiac cells than myocarditis reoviruses. The roles of interferon regulatory factors (IRFs) in the cardiac response to viral infection are reviewed, and results suggest possible cardiac-specific variations in IRF-3 and IRF-1 function. In addition, data are presented indicating that the role of IRF-2 in regulation of IFN-beta expression is cell type-specific and differs between skeletal and cardiac muscle cells. Together, results suggest that the heart may provide a unique environment for IRF function, critical for protection against virus-induced cardiac damage.     

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.     

Impairment of human NK cell cytotoxic activity and cytokine release by cigarette smoke

NK cells play essential roles in innate host defense against microbial infections and tumor surveillance. Although evidence suggests that smoking has adverse effects on the immune system, little is known about whether smoking compromises NK cell effector functions. In this study, we show that cigarette smoke-conditioned medium (SCM) dose-dependently inhibits in vitro IFN-gamma production by polyinosinic:polycytidylic acid (poly I:C)-activated PBMC and NK cells isolated from nonsmoking individuals. Similarly, SCM attenuated poly I:C-induced TNF-alpha production by PBMC and NK cells. The inhibitory effect of cigarette smoke on TNF-alpha production was reversible. PBMC and NK cells isolated from smokers displayed significant reduction of IFN-gamma and TNF-alpha secretions compared with nonsmokers in response to poly I:C activation. We further observed that SCM attenuated NK cell cytotoxic activity, which was associated with decreased up-regulation of perforin expression. Attenuated cytotoxic activity was also observed in PBMCs isolated from smokers. Finally, anti-IL-12 mAb-blocking data revealed that an attenuation of IFN-gamma production by PBMC was indirect, likely via attenuation of IL-12 production, and the effect on NK cells was IL-12-independent. Our data indicate that cigarette smoke compromises function of human NK cells. This may contribute to a higher incidence of viral infections and cancer among smokers.     

Functional evolution of the TICAM-1 pathway for extrinsic RNA sensing

Summary:  The type I interferon (IFN) is a host defense factor against microbial pathogens in vertebrates. In mammals, retinoic acid-inducible gene I (RIG-I) and melanoma differentiation-associated gene 5 (MDA5) in the cytoplasm are regarded as sensors for double-stranded RNA (dsRNA) and trigger IFN regulatory factor-3 (IRF-3) activation followed by type I IFN induction through the mitochondrial antiviral signaling (MAVS) adapter. This intrinsic pathway appears to link the main protective responses against RNA virus infection in mammals. On the other hand, human Toll-like receptor 3 (TLR3) is localized in the endosomal membrane or cell surface and signals the presence of extrinsic dsRNA. In response to RNA stimulation, TLR3 recruits the Toll-interleukin 1 receptor domain (TIR)-containing adapter molecule 1 (TICAM-1) adapter and induces IRF-3 activation followed by IFN-β promoter activation. Human TLR3 is localized limitedly extent in myeloid dendritic cells, fibroblasts, and epithelial cells. The TICAM-1 and cytoplasmic MAVS pathways converge at the IRF-3-activating kinase in human cells. The reason for the involvement of this extrinsic mode of IFN-inducing pathways in the dsRNA response remains unknown. In fish, two TLRs, i.e. endoplasmic TLR3 and cell surface TLR22, participate in teleost IFN production without the activation of IRF-3. TLR22 is distinct from mammalian TLR3 in terms of cellular localization, ligand selection, and tissue distribution. TLR22 may be a functional substitute for human cell surface TLR3 and may serve as a surveillance molecule for detecting dsRNA virus infection and alerting the immune system for antiviral protection in fish. In this review, we discuss the fundamentals of the extrinsic dsRNA recognition system, which has evolved to induce cellular effectors to cope with dsRNA virus infection across different vertebrate species.     

Poly I:CLC induction of the interferon system in mice: an initial study of four detection methods.

Induction of a large number of the components of the interferon (IFN) system (IFN genes, their mRNAs, IFN proteins, IFN receptors, IFN signaling molecules, the IFN response genes, and their effector proteins) has been studied. Less well studied is the comparative induction of these components in vivo. Induction of IFN by double-stranded RNA (dsRNA) treatment mimics certain aspects of viral infection and induces the components of the IFN system. To determine the comparative sensitivity of detection of induction in mice, we initially studied the limiting concentrations of polyribinosinic-polyribocytidylic acid, polylysine complex (poly I:CLC, a synthetic dsRNA preparation), for induction of four representative components of the IFN system: (1) IFN in serum, (2) the IFN response gene mRNA ISG54 in spleen and liver, (3) the IFN-beta mRNA in spleen, and (4) resistance of mice to Banzi viral infection. The results of this initial study showed that resistance to infection was 7-fold more sensitive for detection of the IFN response than was ISG54 mRNA and 70-fold more sensitive than either IFN-beta mRNA or IFN production in serum. In comparison, mouse cells in vitro treated with poly I:CLC were 3-10-fold less sensitive to its antiviral action than is the mouse. The results demonstrate that in the four tests in mice, the most sensitive indicator of poly I:CLC induction of the IFN system was protection against Banzi viral infection, followed by ISG54 mRNA levels, IFN-beta mRNA, and IFN protein levels. It is hypothesized that the highest sensitivity of mouse protection may be due to priming by the initial poly I:CLC-induced IFN of the subsequent Banzi virus-induced IFN, resulting in rapid and high concentrations of IFN at the local site of viral replication. Future studies are needed to study other molecular components of the IFN system to identify those that cause the unanticipated high sensitivity of mice to protection against Banzi virus.     

Human neutrophils produce macrophage inhibitory protein-1beta but not type i interferons in response to viral stimulation.

Several cell types have been shown to produce type I interferons (IFN). Of human leukocytes, monocytes and especially type 2 dendritic cell precursors (pDC2) seem to be the main producers and also have a wide spectrum of cytokine production. However, neutrophils seem to have a limited capacity for cytokine production but possess efficient defense mechanisms vs. bacterial infection by phagocytosis and degranulation. To determine whether they also have antiviral functions, IFN-alpha and IFN-beta were measured in preparations of pure neutrophils. The capacity of neutrophils to produce type I IFN is controversial. Additionally, macrophage inflammatory protein-1alpha (MIP-1alpha) and MIP-1beta were measured, as they are described to have indirect or direct antiviral activity. As stimulants, active and inactivated Newcastle disease virus (NDV), Sendai virus, and granulocyte colony-stimulating factor (G-CSF) were used. Peripheral blood mononuclear cells (PBMC) from the same donors were highly reactive to viral stimulation, whereas neutrophils failed to produce IFN but produced MIP-1beta in response to NDV. We conclude that neutrophils fail to prevent viral infection by IFN production but probably possess alternative mechanisms, such as secreting MIP-1beta in response to viruses.     

Soluble G protein of respiratory syncytial virus inhibits Toll-like receptor 3/4-mediated IFN-beta induction

Monocyte-derived dendritic cells (mDCs) recognize viral RNA extrinsically by Toll-like receptor (TLR) 3 on the membrane and intrinsically retinoic acid-inducible gene I (RIG-I)/melanoma differentiation-associated gene 5 (MDA5) in the cytoplasm to induce type I IFNs and mDC maturation. When mDCs were treated with live or UV-irradiated respiratory syncytial virus (RSV), early ( approximately 4 h) induction of IFN-beta usually occurs in other virus infections was barely observed. Live RSV subsequently replicated to activate the cytoplasmic IFN-inducing pathway leading to robust type I IFN induction. We found that RSV initial attachment to cells blocked polyI:C-mediated IFN-beta induction, and this early IFN-beta-modulating event was abrogated by antibodies against envelope proteins of RSV, demonstrating the presence of a IFN-regulatory mode by early RSV attachment to host cells. By IFN-stimulated response element (ISRE) reporter analysis in HEK293 cells, polyI:C- or LPS-mediated ISRE activation was dose dependently inhibited by live and inactive RSV to a similar extent. Of the RSV envelope proteins, simultaneously expressed or exogenously added RSV G or soluble G (sG) proteins inhibited TLR3/4-mediated ISRE activation in HEK293 cells. sG proteins expressed in cells did not affect the RIG-I/MDA5 pathway but inhibited the TLR adaptor TRIF/TICAM-1 pathway for ISRE activation. Finally, extrinsically added sG protein suppressed the production of IFN-beta in mDCs. Although the molecular mechanism of this extrinsic functional mode of the RSV G glycoprotein (G protein) remains undetermined, G proteins may neutralize the fusion glycoprotein function that promotes IFN-mediated mDC modulation via TLR4 and may cause insufficient raising cell-mediated immunity against RSV.     

Bax-dependent mitochondrial membrane permeabilization enhances IRF3-mediated innate immune response during VSV infection

An effective type I interferon (IFN-alpha/beta) response is critical for the control of many viral infections. Using an oncolytic strain of vesicular stomatitis virus, we have examined the cross-talk between virus-induced apoptosis and initiation of innate immune response. The intrinsic apoptotic cascade, specifically the Bax-Bcl-2-Caspase-9 cascade, was revealed as the primary pathway of VSV-induced apoptosis. Cell death was significantly reduced in BaxBak(-/-) murine embryonic fibroblasts (MEFs) and in human A549 epithelial cells treated with siRNA against Bax. Although inhibition of apoptosis resulted in enhanced virus replication in the BaxBak(-/-) MEFs as compared to wild-type cells, induction of the IFN antiviral response and expression of cytokine genes were attenuated in virus-infected cells. Moreover, Bax but not Bak pro-apoptotic protein was required for IRF-3 phosphorylation and activation, further substantiating a role for the intrinsic mitochondrial pathway in the innate immune response. Therefore, virus-induced apoptosis through a Bax-dependent mitochondrial pathway appears to enhance the full development of the IRF-3 mediated IFN antiviral response.     

Toll-like receptor 3 agonist poly(I:C)-induced antiviral response in human corneal epithelial cells

The objective of this study was to examine the expression of Toll-like receptor 3 (TLR3) by human corneal epithelial cells (HCECs) and to determine whether exposure to the TLR3 agonist polyinosinic-polycytidylic acid [poly(I:C)] induces an antiviral response in these cells. Fluorescence-activated cell sorter (FACS) analysis revealed TLR3 to be constitutively expressed and distributed intracellularly in HCECs. Stimulation of HCECs with the TLR3 agonist poly(I:C) induced the activation of nuclear factor (NF)-kappaB and production of the proinflammatory cytokine interleukin (IL)-6 and the chemokine IL-8. Upon exposure to poly(I:C), HCECs initiated a potent antiviral response resulting in an increase of interferon (IFN)-beta mRNA expression (7-fold). Poly(I:C) stimulation also up-regulated mRNA expression of the antiviral chemokine IFN-gamma inducible protein 10 (IP10), myxovirus resistance gene A and 2',5'-oligoadenylate synthetase (5-, 10- and 9-fold, respectively), and secretion of IP10. These responses were also induced by exogenously added type 1 IFNs, but could not be blocked by pretreatment of the cells with anti-TLR3 monoclonal antibody, suggesting that the receptor was not expressed on the cell surface. Furthermore, incubation of HCECs with an endosomal acidification inhibitor, chloroquine, markedly inhibited poly(I:C)-mediated IFN-beta expression in HCECs. These results suggest that corneal epithelial cells are important sentinels of the corneal innate immune system against viral infection, and that stimulation of TLR3 can induce the expression of key proinflammatory cytokines and chemokines and antiviral genes that help in the defence of the cornea against viral infection.