The initial step in an immune response toward a viral infection is the induction of inflammatory cytokines. This innate immune response is mediated by expression of a variety of cytokines exemplified by TNF-α and IL-1β. A key signal for the recognition of intracellular viral infections is the presence of dsRNA. Viral infections and dsRNA treatment can activate several signaling pathways including the protein kinase R pathway, mitogen-activated protein kinase (MAPK) pathways, and NF-κB, which are important in the expression of inflammatory cytokines. We previously reported that activation of protein kinase R was required for dsRNA induction of TNF-α, but not for IL-1β. In this study, we report that activation of the p38 MAPK pathway by respiratory viral infections is necessary for induction of inflammatory cytokines in human bronchial epithelial cells. Inhibition of p38 MAPK by two different pharmacological inhibitors showed that expression of both TNF-α and IL-1β required activation of this signaling pathway. Interestingly, inhibition of NF-κB did not significantly reduce viral induction of either cytokine. Our data show that, during the initial infections of epithelial cells with respiratory viruses, activation of the p38 MAPK pathway is associated with induction of inflammation, and NF-κB activation may be less important than previously suggested.

Epithelial cells represent the initial site of respiratory viral entry and the first line of defense against such infections. This early antiviral response is characterized by an increase in the production of proinflammatory cytokines such as TNF-α and IL-1β. dsRNA, which is a common factor present during the life cycle of both DNA and RNA viruses, is known to induce TNF-α and IL-1β in a variety of cells. In this work we provide data showing that dsRNA treatment induces TNF-α and IL-1β in human lung epithelial cells via two different mechanisms. Our data show that dsRNA activation of dsRNA-activated protein kinase (PKR) is associated with induction of TNF-α but not IL-1β expression. An inhibitor of PKR activation blocked the dsRNA-induced elevations in TNF-α but not IL-1β mRNA in epithelial cells. Data obtained from infection of epithelial cells with a vaccinia virus lacking the PKR inhibitory polypeptide, E3L, revealed that PKR activation was essential for TNF-α but not for IL-1β expression. In this report, we provide experimental support for the differential regulation of proinflammatory cytokine expression by dsRNA and viral infections in human airway epithelial cells.

Secretion of inflammatory cytokines is the initial step of the immune response to viral infections. This innate immune response is mediated by the expression of a variety of cytokines, exemplified by tumor necrosis factor- alpha (TNF-alpha). The presence of dsRNA during viral infections is a key step in activation of several signaling pathways, including protein kinase R (PKR), toll-like receptor 3 (TLR3), mitogen-activated protein kinase (MAPK), activator protein-1 (AP-1), interferon regulatory factors (IRFs), and NF-kappaB pathways, which are all relevant in the expression of inflammatory cytokines. We previously reported that PKR and p38 MAPK were required for dsRNA and viral induction of inflammatory cytokines in epithelial cells. Here, we report that activation of c-Jun N-terminal kinase (JNK) during dsRNA treatment or respiratory syncytial viral (RSV) infection negatively regulates the induction of TNF-alpha in human epithelial cells. Inhibition of JNK by a pharmacologic inhibitor showed that expression of TNF-alpha increased following both dsRNA treatment and infection with RSV. Importantly, transfection of epithelial cells with a dominant-negative mutant of JNK significantly increased dsRNA induction of TNF-alpha. The mechanism by which JNK inhibition increases TNF-alpha induction appears to be through p38 MAPK activation. Our data show that JNK is a negative regulator of dsRNA and RSV induction of TNF-alpha expression and, thus, may act as a counterbalance to proinflammatory signals generated during viral infections.