Monocytes are primary targets for human CMV (HCMV) infection and are proposed to be responsible for hematogenous dissemination of the virus. Monocytes acquire different functional traits during polarization to the classical proinflammatory M1 macrophage or the alternative antiinflammatory M2 macrophage. We hypothesized that HCMV induced a proinflammatory M1 macrophage following infection to promote viral dissemination because, biologically, a proinflammatory state provides the tools to drive infected monocytes from the blood into the tissue. To test this hypothesis of monocyte conversion from a normal quiescent phenotype to an inflammatory phenotype, we used Affymetrix Microarray to acquire a transcriptional profile of infected monocytes at a time point our data emphasized is a key temporal regulatory point following infection. We found that HCMV significantly up-regulated 583 (5.2%) of the total genes and down-regulated 621 (5.5%) of the total genes ≥1.5-fold at 4 h postinfection. Further ontology analysis revealed that genes implicated in classical M1 macrophage activation were stimulated by HCMV infection. We found that 65% of genes strictly associated with M1 polarization were up-regulated, while only 4% of genes solely associated with M2 polarization were up-regulated. Analysis of the monocyte chemokinome at the transcriptional level showed that 44% of M1 and 33% of M2 macrophage chemokines were up-regulated. Proteomic analysis using chemokine Ab arrays confirmed the secretion of these chemotactic proteins from HCMV-infected monocytes. Overall, the results identify that the HCMV-infected monocyte transcriptome displayed a unique M1/M2 polarization signature that was skewed toward the classical M1 activation phenotype.

Human cytomegalovirus infection of monocytes stimulates a unique monocyte differentiation reprogramming resulting in polarization towards an M1 pro-inflammatory macrophage that simultaneously exhibits characteristics of an M2 anti-inflammatory macrophage. Our laboratory has previously shown that HCMV infection stimulates monocyte NF-κB and PI(3)K activities and now provides evidence that these cellular factors are essential for the HCMV-induced polarization of infected monocytes/macrophages. We find that the induction of NF-κB and PI(3)K activities following HCMV infection was required for the initiation of monocyte-to-macrophage differentiation. HCMV-infected monocytes treated with Bay11-7802 (an inhibitor of NF-κB activity) or LY294002 [an inhibitor of PI(3)K activity] prior to infection exhibited a small, round and monocyte-like undifferentiated morphology and the lack of CD68 upregulation (a macrophage differentiation marker). Detailed transcriptome analysis revealed 48%, 7% and 31% of HCMVinduced M1-associated genes were dependent on NF-κB, PI(3)K or both activities, respectively; while 100% of HCMV-induced M2-associated genes required both NF-κB and PI(3)K activities. Functionally, we demonstrated that NF-κB and PI(3)K activities were critical for the production of M1-and M2-associated cytokines/chemokines, in HCMV-induced differentiating monocytes. Supernatant from HCMV-infected monocytes pretreated with Bay11-7802 or LY294002 exhibited an 80% and 67% reduction in cell motility inducing activity. Overall, these data show that HCMV usurps monocyte NF-κB and PI(3)K signal transduction pathways to induce the unique polarization of HCMV-infected monocytes needed for the earliest steps in the viral dissemination and persistence strategy. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. Keywords NIH Public Access SummaryHuman cytomegalovirus is a ubiquitous β-herpesvirus that infects 50%-90% of the adult population (Mocarski et al., 2007). Infection of immunocompetent hosts by HCMV is generally asymptomatic or results in mild symptoms, although HCMV infection can cause mononucleosis (Mocarski et al., 2007) and is associated with chronic inflammatory diseases such as arthrosclerosis (Mueller et al., 2003;Westphal et al., 2006). In contrast, HCMV infection can have severe clinical consequences in immunosuppressed individuals including neonates, AIDS and organ transplant patients (Ho, 1977;Masur et al., 1996;Stagno et al., 1982) where, due to HCMV's broad cellular tropism, multiple organ system disease can develop (Toorkey and Carrigan, 1989).Systemic spread occurs ...

Respiratory viruses cause asthma exacerbations. Because eosinophils are the prominent leukocytes in the airways of 60-70% of patients with asthma, we evaluated the effects of eosinophils on a common respiratory virus, parainfluenza 1, in the lung. Eosinophils recruited to the airways of wild-type mice after ovalbumin sensitization and challenge significantly decreased parainfluenza virus RNA in the lungs 4 days after infection compared with nonsensitized animals. This antiviral effect was also seen in IL-5 transgenic mice with an abundance of airway eosinophils (NJ.1726) but was lost in transgenic eosinophil-deficient mice (PHIL) and in IL-5 transgenic mice crossed with eosinophil-deficient mice (NJ.1726-PHIL). Loss of the eosinophil granule protein eosinophil peroxidase, using eosinophil peroxidase-deficient transgenic mice, did not reduce eosinophils' antiviral effect. Eosinophil antiviral mechanisms were also explored in vitro. Isolated human eosinophils significantly reduced parainfluenza virus titers. This effect did not involve degradation of viral RNA by eosinophil granule RNases. However, eosinophils treated with a nitric oxide synthase inhibitor lost their antiviral activity, suggesting eosinophils attenuate viral infectivity through production of nitric oxide. Consequently, eosinophil nitric oxide production was measured with an intracellular fluorescent probe. Eosinophils produced nitric oxide in response to virus and to a synthetic agonist of the virus-sensing innate immune receptor, Toll-like receptor (TLR) 7. IFNγ increased expression of eosinophil TLR7 and potentiated TLR7-induced nitric oxide production. These results suggest that eosinophils promote viral clearance in the lung and contribute to innate immune responses against respiratory virus infections in humans.

Infected peripheral blood monocytes are proposed to play a key role in the hematogenous dissemination of human cytomegalovirus (HCMV) to tissues, a critical step in the establishment of HCMV persistence and the development of HCMV-associated diseases. We recently provided evidence for a unique strategy involved in viral dissemination: HCMV infection of primary human monocytes promotes their transendothelial migration and differentiation into proinflammatory macrophages permissive for the replication of the original input virus. To decipher the mechanism of hematogenous spread, we focused on the viral dysregulation of early cellular processes involved in transendothelial migration. Here, we present evidence that both phosphatidylinositol 3-kinase [PI(3)K] and NF-B activities were crucial for the HCMV induction of monocyte motility and firm adhesion to endothelial cells. We found that the ␤ 1 integrins, the ␤ 2 integrins, intracellular adhesion molecule 1 (ICAM-1), and ICAM-3 were upregulated following HCMV infection and that they played a key role in the firm adhesion of infected monocytes to the endothelium. The viral regulation of adhesion molecule expression is complex, with PI(3)K and NF-B affecting the expression of each adhesion molecule at different stages of the expression cascade. Our data demonstrate key roles for PI(3)K and NF-B signaling in the HCMV-induced cellular changes in monocytes and identify the biological rationale for the activation of these pathways in infected monocytes, which together suggest a mechanism for how HCMV promotes viral spread to and persistence within host organs.

Human cytomegalovirus induces a proinflammatory monocyte following infection, and we have evidence that NF-B and phosphatidylinositol 3-kinase [PI(3)K] are key mediators in this early activation. To begin to address how these signaling pathways are responsible for the rapid activation of infected monocytes, we examined the role that these pathways played in the transcriptome of infected monocytes. Global transcriptional profiling using cDNA microarrays revealed that a significant number of genes, including inflammatory genes, were regulated in an NF-B-and/or PI(3)K-dependent manner, identifying the NF-B and PI(3)K pathways as key cellular control points in the conversion of monocytes to an activated proinflammatory state following HCMV infection.

CD40L is essential for the development of adaptive immune responses. It is generally thought that CD40L expression in CD4+ T cells is regulated transcriptionally and made from new mRNA following antigen recognition. However, imaging studies show that the majority of cognate interactions between effector CD4+ T cells and APCs in vivo are too short to allow de novo CD40L synthesis. We previously showed that Th1 effector and memory cells store preformed CD40L (pCD40L) in lysosomal compartments and mobilize it onto the plasma membrane immediately after antigenic stimulation, suggesting that primed CD4+ T cells may use pCD40L to activate APCs during brief encounters. Indeed, our recent study showed that pCD40L is sufficient to mediate selective activation of cognate B cells and trigger DC activation in vitro. In this study, we show that pCD40L is present in Th1 and follicular helper T cells developed during infection with lymphocytic choriomeningitis virus, Th2 cells in the airway of asthmatic mice, and Th17 cells from the CNS of animals with experimental autoimmune encephalitis (EAE). pCD40L is nearly absent in both natural and induced Treg cells, even in the presence of intense inflammation such as occurs in EAE. We also found pCD40L expression in CD4 single positive thymocytes and invariant NKT cells. Together, these results suggest that pCD40L may function in T cell development as well as an unexpectedly broad spectrum of innate and adaptive immune responses, while its expression in Treg cells is repressed to avoid compromising their suppressive activity.

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