Human Cytomegalovirus Activates Inflammatory Cytokine Responses via CD14 and Toll-Like Receptor 2

Human cytomegalovirus (CMV) is a ubiquitous opportunistic pathogen. Clinically, CMV disease correlates with immune suppression in which severe presentations are evident in neonates, persons with AIDS, and other immune-suppressed patient groups (38). CMV infection of neonates is associated with deafness, mental retardation, and mortality, whereas AIDS patients often suffer a blinding CMV retinitis, as well as pneumonia and gastrointestinal inflammation. In organ transplant recipients, a patient group hard hit by CMV infection, disease is associated with an increased frequency of graft rejection and is a major cause of posttransplant infection. The varied array of clinical disease correlates with the exceptionally broad tropism of this virus. Indeed, histological analysis of autopsy tissues obtained from patients with CMV disease has demonstrated infected cells in virtually all organs. At the cellular level, CMV can infect monocytes/macrophages, endothelial cells, epithelial cells, smooth muscle cells, fibroblasts, stromal cells, neuronal cells, neutrophils, and hepatocyes (13, 17, 35, 44, 48, 49, 61). In fact, CMV is a suspected pathogenetic agent in cardiovascular disease due to its ability to persist in large-vessel endothelial cells and to infect all cell types involved in cardiovascular lesions (24). Cells exposed to CMV undergo a number of physiological changes that are rendered upon the cell with extremely rapid kinetics (15). These events include changes in Ca2+ homeostasis (1) and activation of phospholipase C and phospholipase A2, as well as increased release of arachidonic acid and its metabolites (1, 59). All of these changes can be triggered by UV-inactivated virions, suggesting that structural components of the virus are responsible for the alterations in cell physiology and intracellular signaling that occur during virus-cell contact and/or virus entry. Virus-cell contact also results in the activation of the transcription factors NF-κB and SP-1, as well as mitogen-activated protein (MAP) kinase, ERK1/2, and p38 (7, 26, 63). Activation of transcription factors by CMV suggests that alterations in cellular transcription should occur in CMV-infected cells, and this is precisely the case. Several transcriptional-profiling studies reveal that cells infected with CMV exhibit profound reprogramming of gene expression (8, 47, 64, 65). Interestingly, the most strongly induced genes were indicators of innate immune activation. Antiviral genes belonging to the interferon-stimulated gene family (ISGs) and inflammatory genes, such as those for RANTES, interleukin 6 (IL-6), IL-7, IL-11, and cyclooxygenase 2 (COX-2), were all robustly induced in CMV-infected fibroblasts (8, 47, 64, 65). Induction of these innate immune markers did not require virus replication. Indeed, cells treated with only the primary ligand of CMV, glycoprotein B (gB) (6), exhibited a response very similar, but not wholly identical, to that of cells treated with intact virus (47). In particular, cells treated with gB strongly induced ISGs. Taken together, the findings suggest that a signal transduction pathway is activated by cell contact of CMV envelope proteins, resulting in numerous physiological changes that culminate, in part, with innate immune activation. The innate immune system is an ancient, universal host defense system. A limited number of evolutionarily conserved germ line receptors found in plants, Drosophila, and humans mediate certain innate immune responses. Termed pattern recognition receptors, these molecules form the basis of the primary host alarm system in response to pathogen-associated molecular patterns (PAMPs) (23). Toll-like receptors (TLRs) are now understood to play a major role in pathogen recognition. Stimulation of TLRs by pathogens activates signal transduction pathways that lead to induction of a range of antimicrobial genes and inflammatory cytokines (2, 23, 29). In addition, key costimulatory molecules, such as CD80 and CD86, which are important for activation of adaptive immunity, are also induced as a consequence of TLR signaling. At present, 10 TLR molecules have been described in humans and mice. Ligands ranging from lipopolysaccharide (LPS) of gram-negative bacteria, peptidoglycan of gram-positive bacteria, flagellin, CpG DNA, and various components from mycobacteria, yeast, and parasitic pathogens, are all detected by TLRs. Although the precise molecular patterns are not completely characterized, PAMPs are hypothesized to be macromolecular modifications unique to these organisms. Viruses have long been known to activate innate immune responses characterized by the induction of inflammatory cytokines and a comprehensive set of ISGs (45, 53). Until recently, the only identified molecular trigger of host innate responses was double-stranded RNA, a common replicative intermediate in the life cycle of many viruses. Double-stranded RNA can activate a key interferon transcription factor, interferon regulatory factor 3 (IRF-3), and induce synthesis of many antiviral genes. Generally, however, the mechanisms by which viruses activate innate immunity remain largely undefined. Since viruses are obligate intracellular parasites, virus-encoded proteins are synthesized by host machinery and ultimately bear protein modifications reflective of the host. Thus, it is not immediately obvious what PAMPs are displayed on viruses. Currently, however, data are clearly emerging showing that TLRs detect viruses and trigger inflammatory responses. Respiratory syncytcial virus (RSV) and mouse mammary tumor virus (MMTV) both signal through TLR4 (28, 42), the well-described LPS receptor. In the case of RSV, TLR4-deficient mice challenged with RSV exhibited a variety of impaired innate immune functions and an inability to clear the virus (20). These findings strongly suggest that Toll signaling pathways have an important role in the innate immunity to RSV. More recently, measles virus was reported to be detected by TLR2, a TLR with broad ligand recognition properties (5). The most striking common feature of these first three reports of virus detection by TLRs is that TLR responses were triggered by viral envelope glycoproteins. Specifically, the fusion protein of RSV, the envelope (env) protein of MMTV, and the hemagglutinin protein of measles virus were the identified triggers of the TLR responsiveness in their respective systems. Interestingly, all of these proteins play key roles in the virus entry pathway, such as attachment and fusion. These studies plainly point to a heretofore-unknown ability of the host to detect viruses during entry but prior to the onset of any replication events. Here, we report that TLR2 and CD14 form the central basis of the membrane detection machinery for CMV.