CCR5 is a required signaling receptor for macrophage expression of inflammatory genes in response to viral double-stranded RNA.

Double-stranded (ds) RNA, both synthetic and produced during virus replication, rapidly stimulates MAPK and NF-κB signaling that results in expression of the inflammatory genes inducible nitric oxide synthase, cyclooxygenase 2, and IL-1ß by macrophages. Using biochemical and genetic approaches, we have identified the chemokine ligand-binding C-C chemokine receptor type 5 (CCR5) as a cell surface signaling receptor required for macrophage expression of inflammatory genes in response to dsRNA. Activation of macrophages by synthetic dsRNA does not require known dsRNA receptors, as poly(inosinic:cytidylic) acid [poly(I:C)] activates signaling pathways leading to expression of inflammatory genes to similar levels in wild-type and Toll-like receptor 3- or melanoma differentiation antigen 5-deficient macrophages. In contrast, macrophage activation in response to poly(I:C) is attenuated in macrophages isolated from mice lacking CCR5. These findings support a role for CCR5 as a cell surface signaling receptor that participates in activation of inflammatory genes in macrophages in response to the viral dsRNA mimetic poly(inosinic:cytidylic) acid by pathways that are distinct from classical dsRNA receptor-mediated responses.

Ccr5 regulates inflammatory gene expression in response to encephalomyocarditis virus infection.

Encephalomyocarditis virus (EMCV) is capable of stimulating inflammatory gene expression by macrophages as a result of interactions between EMCV capsid proteins and cell surface receptors. In this study, biochemical and genetic approaches identified a role for Ccr5, a chemokine receptor, in transducing the signals of EMCV infection that result in the expression of inflammatory genes in macrophages. Antibody neutralization and gene knockout strategies were used to show that the presence of Ccr5 is required for EMCV-stimulated mitogen-activated protein (MAP) kinase and nuclear factor-kappa B (NF-κB) activation, and the subsequent expression of the inflammatory gene-inducible nitric oxide synthase (iNOS) and cyclooxygenase 2 (COX-2). Ccr5 appears to participate in the early control of virus replication: EMCV mRNA accumulates to sevenfold higher levels in Ccr5-deficient mice when compared to wild-type controls. These findings support a regulatory role for Ccr5 in the antiviral response to EMCV in which this chemokine receptor participates in regulation of inflammatory gene expression in response to virus infection.

Phosphatidylinositol 3-kinase regulates macrophage responses to double-stranded RNA and encephalomyocarditis virus.

Virus infection of macrophages stimulates the expression of proinflammatory and antiviral genes interleukin-1 (IL-1), inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2). In this study, we show that phosphatidylinositol 3-kinase (PI3K) is required for the inflammatory response of macrophages to virus infection. When macrophages are infected with encephalomyocarditis virus (EMCV) there is a rapid and transient activation of PI3K and phosphorylation of its downstream target Akt. Inhibitors of PI3K attenuate EMCV- and double-stranded RNA-induced iNOS, COX-2 and IL-1 beta expression in RAW264.7 cells and mouse peritoneal macrophages. The attenuation of inflammatory gene expression in response to PI3K inhibition correlates with the induction of macrophage apoptosis. The morphology of macrophages shifts from activation in response to EMCV infection to apoptosis in the cells treated with PI3K inhibitors and EMCV. These morphological changes are accompanied by the activation of caspase-3. These findings suggest that PI3K plays a central role in the regulation of macrophage responses to EMCV infection. When PI3K is activated, it participates in the regulation of inflammatory gene expression; however, if PI3K is inhibited macrophages are unable to mount an inflammatory antiviral response and die by apoptosis.

Role of MAPK in the regulation of double-stranded RNA- and encephalomyocarditis virus-induced cyclooxygenase-2 expression by macrophages.

In response to virus infection or treatment with dsRNA, macrophages express the inducible form of cyclooxygenase-2 (COX-2) and produce proinflammatory prostaglandins. Recently, we have shown that NF-kappaB is required for encephalomyocarditis virus (EMCV)- and dsRNA-stimulated COX-2 expression in mouse macrophages. The dsRNA-dependent protein kinase R is not required for EMCV-stimulated COX-2 expression, suggesting the presence of protein kinase R-independent pathways in the regulation of this antiviral gene. In this study, the role of MAPK in the regulation of macrophage expression of cyclooxygenase-2 (COX)-2 in response to EMCV infection was examined. Treatment of mouse macrophages or RAW-264.7 cells with dsRNA or infection with EMCV stimulates the rapid activation of the MAPKs p38, JNK, and ERK. Inhibition of p38 and JNK activity results in attenuation while ERK inhibition does not modulate dsRNA- and EMCV-induced COX-2 expression and PGE2 production by macrophages. JNK and p38 appear to selectively regulate COX-2 expression, as inhibition of either kinase fails to prevent dsRNA- or EMCV-stimulated inducible NO synthase expression by macrophages. Using macrophages isolated from TLR3-deficient mice, we show that p38 and JNK activation and COX-2 expression in response to EMCV or poly(IC) does not require the presence the dsRNA receptor TLR3. These findings support a role for p38 and JNK in the selective regulation of COX-2 expression by macrophages in response to virus infection.

Encephalomyocarditis virus induces PKR-independent mitogen-activated protein kinase activation in macrophages.

In this study, we provide evidence that the double-stranded RNA-dependent protein kinase (PKR) is not required for virus-induced expression of inducible nitric oxide synthase (iNOS) or the activation of specific signaling pathways in macrophages. The infection of RAW264.7 cells with encephalomyocarditis virus (EMCV) induces iNOS expression and nitric oxide production, which are unaffected by a dominant-negative mutant of PKR. EMCV infection also activates the mitogen-activated protein kinase, cyclic AMP response element binding protein, and nuclear factor kappaB (NF-kappaB) signaling cascades at 15 to 30 min postinfection in PKR+/+ and PKR-/- macrophages. Activation of these signaling cascades does not temporally correlate with PKR activity or the accumulation of EMCV RNA, suggesting that an interaction between a structural component of the virion and the cell surface may activate macrophages. Consistent with this hypothesis, empty EMCV capsids induced comparable levels of iNOS expression, nitrite production, and activation of these signaling cascades to those induced by intact virions. These findings support the hypothesis that virion-host cell interactions are primary mediators of the PKR-independent activation of signaling pathways that participate in the macrophage antiviral response of inflammatory gene expression.

Genetic and pharmacologic evidence that calcium-independent phospholipase A2beta regulates virus-induced inducible nitric-oxide synthase expression by macrophages.

Recent evidence supports a regulatory role for the calcium-independent phospholipase A2 (iPLA2) in the antiviral response of inducible nitric-oxide synthase (iNOS) expression by macrophages. Because two mammalian isoforms of iPLA2 (iPLA2beta and iPLA2gamma) have been cloned and characterized, the aim of this study was to identify the specific isoform(s) in macrophages that regulates the expression of iNOS in response to virus infection. Bromoenol lactone (BEL), a suicide substrate inhibitor of iPLA2, inhibits the activity of both isoforms at low micromolar concentrations. However, the R- and S-enantiomers of BEL display approximately 10-fold greater potency for inhibition of the enzymatic activity of iPLA2gamma and iPLA2beta, respectively. In this study, we show that the iPLA2beta-selective (S)-BEL inhibits encephalomyocarditis virus (EMCV)-induced iNOS expression, nitric oxide production, and iPLA2 enzymatic activity in macrophages in a concentration-related manner that closely resembles the inhibitory properties of racemic BEL. cAMP response element-binding protein (CREB) is one downstream target of iPLA2 that is required for the transcriptional activation of iNOS in response to virus infection, and consistent with the effects of BEL enantiomers on iNOS expression, (S)-BEL more effectively inhibits EMCV-induced CREB phosphorylation than (R)-BEL in macrophages. Using macrophages isolated from iPLA2beta-null mice, virus infection fails to stimulate iNOS mRNA accumulation and protein expression, thus providing genetic evidence that iPLA2beta is required for EMCV-induced iNOS expression. These findings provide evidence for a signaling role for iPLA2beta in virus-induced iNOS expression by macrophages.

Distinct stress responses of two functional laccases in Cryptococcus neoformans are revealed in the absence of the thiol-specific antioxidant Tsa1.

Laccases are thought to be important to the virulence of many fungal pathogens by producing melanin, a presumed oxygen radical scavenger. A laccase in Cryptococcus neoformans has been shown to synthesize melanin and contributes to the virulence and the survival in macrophages of this fungal pathogen. One C. neoformans laccase gene, LAC1, previously called CNLAC1, has been extensively studied, and we describe a homologous gene, LAC2, that is found 8 kb away from LAC1 in the genome. In this study we report a role for both laccases, in addition to the thiol peroxidase, Tsa1, in oxidative and nitrosative stress resistance mechanisms of C. neoformans. With use of real-time PCR, similar changes in expression of the two laccase genes occur in response to oxidative and nitrosative stresses, but only the regulation of the LAC2 gene during stress is influenced by Tsa1. Both laccases contribute to melanin production using L-dopa as a substrate and are differentially localized in the cell based on green fluorescent protein fusions. A single deletion of either LAC1 or LAC2 alone had no effect on sensitivity to H2O2 or nitric oxide. However, deletion of either LAC1 or LAC2 in combination with a TSA1 deletion resulted in a slight peroxide sensitivity, and a lac2Delta tsa1Delta deletion strain was sensitive to nitric oxide stress. In addition, the deletion of both laccases reduces survival of C. neoformans in primary macrophages. Based on our expression and functional analysis, we propose a novel model for the interaction of these two systems, which are both important for virulence.

ERK activation is required for double-stranded RNA- and virus-induced interleukin-1 expression by macrophages.

Double-stranded (ds) RNA, which accumulates during viral replication, activates the antiviral response of infected cells. In this study, we have identified a requirement for extracellular signal-regulated kinase (ERK) in the regulation of interleukin 1 (IL-1) expression by macrophages in response to dsRNA and viral infection. Treatment of RAW 264.7 cells or mouse macrophages with dsRNA stimulates ERK phosphorylation that is first apparent following a 15-min incubation and persists for up to 60 min, the accumulation of iNOS and IL-1 mRNA following a 6-h incubation, and the expression of iNOS and IL-1 at the protein level following a 24-h incubation. Inhibitors of ERK activation prevent dsRNA-induced ERK phosphorylation and IL-1 expression by macrophages. The regulation of macrophage activation by ERK appears to be selective for IL-1, as ERK inhibition does not attenuate dsRNA-induced iNOS expression by macrophages. dsRNA stimulates both ERK activation and IL-1 expression by macrophages isolated from dsRNA-dependent protein kinase (PKR)-deficient mice, indicating that PKR does not participate in this antiviral response. These findings support a novel PKR-independent role for ERK in the regulation of the antiviral response of IL-1 expression and release by macrophages.

Regulation of cyclooxygenase-2 expression by macrophages in response to double-stranded RNA and viral infection.

In this study the regulation of macrophage expression of cyclooxygenase-2 (COX-2) in response to dsRNA and virus infection was examined. Treatment of RAW 264.7 macrophages with dsRNA results in COX-2 mRNA accumulation and protein expression and the production of PGE(2). Similar to dsRNA, encephalomyocarditis virus (EMCV) infection of RAW 264.7 cells stimulates COX-2 expression and PGE(2) accumulation. The dsRNA-dependent protein kinase (PKR), which has been shown to participate in the regulation of gene expression in response to dsRNA and virus infection, does not appear to participate in the regulation of COX-2 expression by macrophages. Expression of dominant negative mutants of PKR in RAW 264.7 cells fails to attenuate dsRNA- and EMCV-induced COX-2 expression or PGE(2) production. Furthermore, dsRNA and EMCV stimulate COX-2 expression and PGE(2) accumulation to similar levels in macrophages isolated from wild-type and PKR-deficient mice. Recently, a novel PKR-independent role for the calcium-independent phospholipase A(2) (iPLA(2)) in the regulation of inducible NO synthase expression by macrophages in response to virus infection has been identified. The selective iPLA(2) suicide substrate inhibitor bromoenol lactone prevents dsRNA- and EMCV-stimulated inducible NO synthase expression; however, bromoenol lactone does not attenuate dsRNA- or EMCV-induced COX-2 expression by macrophages. In contrast, inhibition of NF-kappaB activation prevents dsRNA-stimulated COX-2 expression and PGE(2) accumulation by macrophages. These findings indicate that virus infection and treatment with dsRNA stimulate COX-2 expression by a mechanism that requires the activation of NF-kappaB and that is independent of PKR or iPLA(2) activation.

Novel role for calcium-independent phospholipase A(2) in the macrophage antiviral response of inducible nitric-oxide synthase expression.

The double-stranded (ds) RNA-dependent protein kinase (PKR) is a primary regulator of antiviral responses; however, the ability of dsRNA to activate nuclear factor-kappa B (NF-kappa B) and dsRNA + interferon gamma (IFN-gamma) to stimulate inducible nitric-oxide synthase (iNOS) expression by macrophages isolated from PKR(-/-) mice suggests that signaling pathways in addition to PKR participate in antiviral activities. We have identified a novel phospholipid-signaling cascade that mediates macrophage activation by dsRNA and viral infection. Bromoenol lactone (BEL), a selective inhibitor of the calcium-independent phospholipase A(2) (iPLA(2)), prevents dsRNA- and virus-induced iNOS expression by RAW 264.7 cells and mouse macrophages. BEL does not modulate dsRNA-induced interleukin 1 expression, nor does it affect dsRNA-induced NF-kappa B activation. Protein kinase A (PKA) and the cAMP response element binding protein (CREB) are downstream targets of iPLA(2), because selective PKA inhibition prevents dsRNA-induced iNOS expression, and the inhibitory actions of BEL on dsRNA-induced iNOS expression are overcome by the direct activation of PKA. In addition, BEL inhibits dsRNA-induced CREB phosphorylation and CRE reporter activation. PKR does not participate in iPLA(2) activation or iNOS expression, because dsRNA stimulates iPLA(2) activity and dsRNA + IFN-gamma induces iNOS expression and nitric oxide production to similar levels by macrophages isolated from PKR(+/+) and PKR(-/-) mice. These findings support a PKR-independent signaling role for iPLA(2) in the antiviral response of macrophages.