Shedding light on the elusive role of endothelial cells in cytomegalovirus dissemination

Cytomegalovirus (CMV) is frequently transmitted by solid organ transplantation and is associated with graft failure. By forming the boundary between circulation and organ parenchyma, endothelial cells (EC) are suited for bidirectional virus spread from and to the transplant. We applied Cre/loxP-mediated green-fluorescence-tagging of EC-derived murine CMV (MCMV) to quantify the role of infected EC in transplantation-associated CMV dissemination in the mouse model. Both EC- and non-EC-derived virus originating from infected Tie2-cre ⁺ heart and kidney transplants were readily transmitted to MCMV-naïve recipients by primary viremia. In contrast, when a Tie2-cre ⁺ transplant was infected by primary viremia in an infected recipient, the recombined EC-derived virus poorly spread to recipient tissues. Similarly, in reverse direction, EC-derived virus from infected Tie2-cre ⁺ recipient tissues poorly spread to the transplant. These data contradict any privileged role of EC in CMV dissemination and challenge an indiscriminate applicability of the primary and secondary viremia concept of virus dissemination.     

The major virus-producing cell type during murine cytomegalovirus infection, the hepatocyte, is not the source of virus dissemination in the host

The course of systemic viral infections is determined by the virus productivity of infected cell types and the efficiency of virus dissemination throughout the host. Here, we used a cell-type-specific virus labeling system to quantitatively track virus progeny during murine cytomegalovirus infection. We infected mice that expressed Cre recombinase selectively in vascular endothelial cells or hepatocytes with a murine cytomegalovirus for which Cre-mediated recombination would generate a fluorescently labeled virus. We showed that endothelial cells and hepatocytes produced virus after direct infection. However, in the liver, the main contributor to viral load in the mouse, most viruses were produced by directly infected hepatocytes. Remarkably, although virus produced in hepatocytes spread to hepatic endothelial cells (and vice versa), there was no significant spread from the liver to other organs. Thus, the cell type producing the most viruses was not necessarily the one responsible for virus dissemination within the host.     

Human cytomegalovirus (HCMV) infection of endothelial cells promotes naive monocyte extravasation and transfer of productive virus to enhance hematogenous dissemination of HCMV

Human cytomegalovirus (HCMV) pathogenesis is dependent on the hematogenous spread of the virus to host tissue. While data suggest that infected monocytes are required for viral dissemination from the blood to the host organs, infected endothelial cells are also thought to contribute to this key step in viral pathogenesis. We show here that HCMV infection of endothelial cells increased the recruitment and transendothelial migration of monocytes. Infection of endothelial cells promoted the increased surface expression of cell adhesion molecules (intercellular cell adhesion molecule 1, vascular cell adhesion molecule 1, E-selectin, and platelet endothelial cell adhesion molecule 1), which were necessary for the recruitment of na?ve monocytes to the apical surface of the endothelium and for the migration of these monocytes through the endothelial cell layer. As a mechanism to account for the increased monocyte migration, we showed that HCMV infection of endothelial cells increased the permeability of the endothelium. The cellular changes contributing to the increased permeability and increased na?ve monocyte transendothelial migration include the disruption of actin stress fiber formation and the decreased expression of lateral junction proteins (occludin and vascular endothelial cadherin). Finally, we showed that the migrating monocytes were productively infected with the virus, documenting that the virus was transferred to the migrating monocyte during passage through the lateral junctions. Together, our results provide evidence for an active role of the infected endothelium in HCMV dissemination and pathogenesis.     

Systemic immune deficiency necessary for cytomegalovirus invasion of the mature brain

Cytomegalovirus (CMV) is a significant opportunistic pathogen associated with AIDS and immunosuppressive therapy. Infection of the mature central nervous system (CNS) can cause significant pathology with associated neurological deficits, mental disorders, and cognitive impairment and may have potentially fatal consequences. Using genetically immunocompromised mice, we studied mechanisms of CMV invasion into, and behavior within, the CNS. Adult immunodeficient (nude and SCID) and control mice were peripherally infected with recombinant mouse CMV expressing a green fluorescent protein reporter gene. Control mice actively eliminated acute peripheral infection and were resistant to invasion of CMV into the brain. In contrast, virus infected brains of immunodeficient mice but only after a minimum of 21 days postinoculation. After inoculation, CMV was found in circulating leukocytes (MAC-3/CD45⁺) and in leukocytes within the brain, suggesting these cells as a possible source of CMV entry into the CNS. CNS infection was observed in many different cell types, including neurons, glial cells, meninges, ependymal cells, and cells of cerebral vessels. Infection foci progressively expanded locally to adjacent cells, resulting in meningitis, choroiditis, encephalitis, vasculitis, and necrosis; clear indication of axonal transport of CMV was not found. Regional distribution of CMV was unique in each brain, consisting of randomly distributed, unilateral foci. Testing whether CMV gained access to brain through nonspecific vascular disruption, vascular injections of a tracer molecule revealed no obvious disruption of the blood brain barrier in mice with CMV in the brain. Results indicate the importance of host adaptive immunity (particularly T cells) in controlling entry and dissemination of CMV into the brain and are consistent with the view that virus may be carried into the brain by circulating mononuclear cells that traffic through the blood brain barrier.     

Strain-Specific Differences in LFA-1 Induction on Measles Virus-Infected Monocytes and Adhesion and Viral Transmission to Endothelial Cells

Measles virus (MV) infection of monocytes induces leukocyte function-associated antigen-1 (LFA-1), an integrin that mediates intercellular adhesion to the endothelium. Thus, an increase in LFA-1 expression could lead to enhanced monocyte adherence and virus dissemination to endothelial cells (ECs) and potentially be an important means of distinction between MV strains. We identified both vaccine and wild-type strains that induced LFA-1 and others that failed to induce. Although adhesion of MV-infected monocytes and viral transmission to ECs was demonstrated, strain-specific differences were not correlated with LFA-1 induction. MV infection of ECs was dramatically reduced in the absence of cell contact, suggesting virus dissemination by cell-cell transmission.     

Dissemination of rat cytomegalovirus through infected granulocytes and monocytes in vitro and in vivo

The role of leukocytes in the in vivo dissemination of cytomegalovirus was studied in this experiment. Rat cytomegalovirus (RCMV) could be transferred to rat granulocytes and monocytes by cocultivation with RCMV-infected fibroblasts in vitro. Intravenous injection of purified infected granulocytes or monocytes resulted in a systemic infection in rats, indicating that our model is a powerful tool to gain further insight into CMV dissemination and the development of new antivirals.     

Nipah virus uses leukocytes for efficient dissemination within a host

Nipah virus (NiV) is a recently emerged zoonotic paramyxovirus whose natural reservoirs are several species of Pteropus fruit bats. NiV provokes a widespread vasculitis often associated with severe encephalitis, with up to 75% mortality in humans. We have analyzed the pathogenesis of NiV infection, using human leukocyte cultures and the hamster animal model, which closely reproduces human NiV infection. We report that human lymphocytes and monocytes are not permissive for NiV and a low level of virus replication is detected only in dendritic cells. Interestingly, despite the absence of infection, lymphocytes could efficiently bind NiV and transfer infection to endothelial and Vero cells. This lymphocyte-mediated transinfection was inhibited after proteolytic digestion and neutralization by NiV-specific antibodies, suggesting that cells could transfer infectious virus to other permissive cells without the requirement for NiV internalization. In NiV-infected hamsters, leukocytes captured and carried NiV after intraperitoneal infection without themselves being productively infected. Such NiV-loaded mononuclear leukocytes transfer lethal NiV infection into naïve animals, demonstrating efficient virus transinfection in vivo. Altogether, these results reveal a remarkable capacity of NiV to hijack leukocytes as vehicles to transinfect host cells and spread the virus throughout the organism. This mode of virus transmission represents a rapid and potent method of NiV dissemination, which may contribute to its high pathogenicity.     

Endothelial cells elicit immune-enhancing responses to dengue virus infection

Dengue viruses cause two severe diseases that alter vascular fluid barrier functions, dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS). Preexisting antibodies to dengue virus disposes patients to immune-enhanced edema (DSS) or hemorrhagic (DHF) disease following infection by a discrete dengue virus serotype. Although the endothelium is the primary vascular fluid barrier, direct effects of dengue virus on endothelial cells (ECs) have not been considered primary factors in pathogenesis. Here, we show that dengue virus infection of human ECs elicits immune-enhancing EC responses. Our results suggest that rapid early dengue virus proliferation within ECs is permitted by dengue virus regulation of early, but not late, beta interferon (IFN-β) responses. The analysis of EC responses following synchronous dengue virus infection revealed the high-level induction and secretion of immune cells (T cells, B cells, and mast cells) as well as activating and recruiting cytokines BAFF (119-fold), IL-6/8 (4- to 7-fold), CXCL9/10/11 (45- to 338-fold), RANTES (724-fold), and interleukin-7 (IL-7; 128-fold). Moreover, we found that properdin factor B, an alternative pathway complement activator that directs chemotactic anaphylatoxin C3a and C5a production, was induced 34-fold. Thus, dengue virus-infected ECs evoke key inflammatory responses observed in dengue virus patients which are linked to DHF and DSS. Our findings suggest that dengue virus-infected ECs directly contribute to immune enhancement, capillary permeability, viremia, and immune targeting of the endothelium. These data implicate EC responses in dengue virus pathogenesis and further rationalize therapeutic targeting of the endothelium as a means of reducing the severity of dengue virus disease.     

Human cytomegalovirus UL131-128 genes are indispensable for virus growth in endothelial cells and virus transfer to leukocytes

Human cytomegalovirus (HCMV), a ubiquitous human pathogen, is the leading cause of birth defects and morbidity in immunocompromised patients and a potential trigger for vascular disease. HCMV replicates in vascular endothelial cells and drives leukocyte-mediated viral dissemination through close endothelium- leukocyte interaction. However, the genetic basis of HCMV growth in endothelial cells and transfer to leukocytes is unknown. We show here that the UL131-128 gene locus of HCMV is indispensable for both productive infection of endothelial cells and transmission to leukocytes. The experimental evidence for this is based on both the loss-of-function phenotype in knockout mutants and natural variants and the gain-of-function phenotype by trans-complementation with individual UL131, UL130, and UL128 genes. Our findings suggest that a common mechanism of virus transfer may be involved in both endothelial cell tropism and leukocyte transfer and shed light on a crucial step in the pathogenesis of HCMV infection.     

Persistent infection of human microvascular endothelial cells by coxsackie B viruses induces increased expression of adhesion molecules

Numerous studies indicate that enteroviruses, such as the Coxsackievirus (CV) group, are linked to autoimmune diseases. Virus tropism and tissue access are modulated by vascular endothelial cells (ECs), mainly at the level of the microvasculature. Data on the permissiveness of ECs to CV are, however, scanty and derived from studies on large vessel ECs. To examine the susceptibility of microvascular ECs to infection of group B CV (CVB), human dermal microvascular ECs (HMEC-1) were infected with three CVB strains, and the immunological phenotype of the infected cells was analyzed. All CVB persistently infected the EC cultures without producing overt cytopathic effects. Infected ECs retained endothelial characteristics. Release of infectious particles in cell supernatants persisted for up to 3 mo of culture. Infection up-regulated expression of the adhesion molecules ICAM-1 and VCAM-1, with the highest values detected during the first 30 days of infection (p < 0.05 vs uninfected HMEC-1). CVB infection increased production of the proinflammatory cytokines, IL-6, IL-8, and TNF-alpha, which may account for the enhanced expression of adhesion molecules. Parallel infection of macrovascular HUVEC had less evident effects on induction of ICAM-1 and did not significantly increase expression of VCAM-1. Moreover, mononuclear cell adhesion to CVB-infected HMEC-1 monolayers was increased, compared with uninfected monolayers. These results provide evidence that small vessel ECs can harbor a persistent viral infection, resulting in quantitative modification of adhesion molecule expression, which may contribute to the selective recruitment of subsets of leukocytes during inflammatory immune responses. Furthermore, our data confirm that the behavior against a viral challenge of ECs in large vessels and microvessels may differ.     

Quantitative In vitro Assay to Measure Neutrophil Adhesion to Activated Primary Human Microvascular Endothelial Cells under Static Conditions

The vascular endothelium plays an integral part in the inflammatory response. During the acute phase of inflammation, endothelial cells (ECs) are activated by host mediators or directly by conserved microbial components or host-derived danger molecules. Activated ECs express cytokines, chemokines and adhesion molecules that mobilize, activate and retain leukocytes at the site of infection or injury. Neutrophils are the first leukocytes to arrive, and adhere to the endothelium through a variety of adhesion molecules present on the surfaces of both cells. The main functions of neutrophils are to directly eliminate microbial threats, promote the recruitment of other leukocytes through the release of additional factors, and initiate wound repair. Therefore, their recruitment and attachment to the endothelium is a critical step in the initiation of the inflammatory response. In this report, we describe an in vitro neutrophil adhesion assay using calcein AM-labeled primary human neutrophils to quantitate the extent of microvascular endothelial cell activation under static conditions. This method has the additional advantage that the same samples quantitated by fluorescence spectrophotometry can also be visualized directly using fluorescence microscopy for a more qualitative assessment of neutrophil binding.     

Human cytomegalovirus subverts the functions of monocytes, impairing chemokine-mediated migration and leukocyte recruitment

Despite their role in innate and adaptive immunity, during human cytomegalovirus (HCMV) infection, monocytes are considered to be an important target of infection, a site of latency, and vehicles for virus dissemination. Since chemokine receptors play crucial roles in monocyte activation and trafficking, we investigated the effects of HCMV on their expression and function. By using endotheliotropic strains of HCMV, we obtained high rates (roughly 50%) of in vitro-infected monocytes but only restricted viral gene expression. At 24 h after infection, while the chemokine receptors CX3CR and CCR7 were unaffected, CCR1, CCR2, CCR5, and CXCR4 were downmodulated on the cell surface and retained intracellularly. Structural components of the viral particles, but not viral gene expression or soluble factors released from infected cells, accounted for the changed localization of the receptor molecules and for the block of chemokine-driven migration. HCMV-infected monocytes indeed became unresponsive to inflammatory and homeostatic chemokines, although the basal cell motility and responsiveness to N-formyl-Met-Leu-Phe were unaffected or slightly increased. The production of inflammatory mediators responsible for the recruitment of other immune cells was also hampered by HCMV. Whereas endothelial and fibroblast cells infected by HCMV efficiently recruited leukocytes, infected monocytes were unable to recruit lymphocytes, monocytes, and neutrophils. Our data further highlight the complex level of interference exerted by HCMV on the host immune system.     

Human cytomegalovirus latent infection of myeloid cells directs monocyte migration by up-regulating monocyte chemotactic protein-1

Following primary infection, human cytomegalovirus (HCMV) establishes a latent infection in hematopoietic cells from which it reactivates to cause serious disease in immunosuppressed patients such as allograft recipients. HCMV is a common cause of disease in newborns and transplant patients and has also been linked with vascular diseases such as primary and post-transplant arteriosclerosis. A major factor in the pathogenesis of vascular disease is the CC chemokine MCP-1. In this study, we demonstrate that granulocyte macrophage progenitors (GMPs) latently infected with HCMV significantly increased expression of MCP-1 and that this phenotype was dependent on infection with viable virus. Inhibitors of a subset of G(alpha) proteins and PI3K inhibited the up-regulation of MCP-1 in latently infected cultures, suggesting that the mechanism underlying this phenotype involves signaling through a G-protein coupled receptor. In GMPs infected with the low passage viral strain Toledo, up-regulated MCP-1 was restricted to a subset of myeloid progenitor cells expressing CD33, HLA-DR, and CD14 but not CD1a, CD15, or CD16, and the increase in MCP-1 was sufficient to enhance migration of CD14(+) monocytes to latently infected cells. Latent HCMV-mediated up-regulation of MCP-1 provides a mechanism by which HCMV may contribute to vascular disease during the latent phase of infection or facilitate dissemination of virus upon reactivation from latency.     

Measles virus-induced block of transendothelial migration of T lymphocytes and infection-mediated virus spread across endothelial cell barriers

In order to analyze whether measles virus (MV) is transported via transmigrating leukocytes across endothelial barriers or whether virus spreads via infection of endothelial cells and basolateral release, we investigated the migratory behavior of infected human primary T lymphocytes across polarized cell layers of human brain microvascular endothelial cells. We found that the capacity of lymphocytes to migrate through filter pores was only slightly affected by wild-type MV infection, whereas their capacity to migrate through endothelial barriers was drastically reduced. MV infection stimulated the expression and activation of the leukocyte integrins LFA-1 and VLA-4, mediating a strong adherence to the surface of endothelial cells. Furthermore, the formation of engulfing membrane protrusions by endothelial cells, so-called transmigratory cups, was induced, but transmigration was impaired. As a consequence of this close cell-cell contact, MV infection was transmitted from lymphocytes to the endothelium. MV envelope proteins were expressed on the apical and basolateral surfaces of infected polarized endothelial cells, and virus was released from both sides. Wild-type MV infection did not induce the formation of syncytia, suggesting virus spread from cell to cell via cell processes and contacts. Our data indicate that transendothelial migration of infected T cells is strongly inhibited, whereas virus can cross endothelial barriers by productive infection of the endothelium and subsequent bipolar virus release.     

Immediate-early antigen expression and modulation of apoptosis after in vitro infection of polymorphonuclear leukocytes by human cytomegalovirus

Polymorphonuclear leukocytes (PMNL) are a major carrier of human cytomegalovirus (CMV) in viremic immunodepressed patients. We transmitted infectious virions and viral components to PMNL by coculturing these cells with infected human embryonic lung fibroblasts (HELF) or human umbilical vein endothelial cells (HUVEC). Quantitative time-course analysis of viral DNA and protein expression in PMNL, after functional separation from infected donor cells, indicated the initiation of viral cycling, with immediate-early protein expression. No viral replication or early or late gene expression was observed, but infected PMNL were able to infect naive fibroblasts more than 48 h after the end of co-culture. PMNL apoptosis was significantly delayed during co-culture with infected or uninfected HUVEC, and this phenomenon did not require contact between the two cell populations. The increased production of IL-8 in the same culture conditions that protect PMNL from apoptosis, associated with the reversion of this protection by inhibiting or depleting this factor in the culture media, targets this cytokine as a likely candidate for this protective effect. These data suggest that PMNL play a key role in virus dissemination in vivo, through their interactions with infected endothelial cells.     

Highly pathogenic H5N1 influenza virus causes coagulopathy in chickens

Severe hemorrhage at multiple organs is frequently observed in chickens infected with highly pathogenic avian influenza (HPAI) A viruses. In this study we examined whether HPAI virus infection leads to coagulation disorder in chickens. Pathological examinations showed that the fibrin thrombi were formed in arterioles at the lung, associated with the viral antigens in endothelial cells of chickens infected intravenously with HPAI virus. Hematological analyses of peripheral blood collected from the chickens revealed that coagulopathy was initiated at early stage of infection when viral antigens were detected only in the endothelial cells and monocytes/macrophages. Furthermore, gene expression of the tissue factor, the main initiator of blood coagulation, was upregulated in the spleen, lung, and brain of HPAI virus-infected chickens. These results suggest that dysfunction of endothelial cells and monocytes/macrophages upon HPAI virus infection may induce hemostasis abnormalities represented by the excessive blood coagulation and consumptive coagulopathy in chickens.     

Contribution of vascular endothelial growth factor in the neovascularization process during the pathogenesis of herpetic stromal keratitis

This report analyzes the role of vascular endothelial growth factor (VEGF)-induced angiogenesis in the immunoinflammatory lesion stromal keratitis induced by ocular infection with herpes simplex virus (HSV). Our results show that infection with replication-competent, but not mutant, viruses results in the expression of VEGF mRNA and protein in the cornea. This a rapid event, with VEGF mRNA detectable by 12 h postinfection (p.i.) and proteins detectable by 24 h p.i. VEGF production occurred both in the virus-infected corneal epithelium and in the underlying stroma, in which viral antigens were undetectable. In the stroma, VEGF was produced by inflammatory cells; these initially were predominantly polymorphonuclear leukocytes (PMN), but at later time points both PMN and macrophage-like cells were VEGF producers. In the epithelium, the major site of VEGF-expressing cells in early infection, the infected cells themselves were usually negative for VEGF. Similarly, in vitro infection studies indicated that the cells which produced VEGF were not those which expressed virus. Attesting to the possible role of VEGF-induced angiogenesis in the pathogenesis of herpetic stromal keratitis were experiments showing that VEGF inhibition with mFlt(1-3)-immunoglobulin G diminished angiogenesis and the severity of lesions after HSV infection. These observations are the first to evaluate VEGF-induced angiogenesis in the pathogenesis of stromal keratitis. Our results indicate that the control of angiogenesis represents a useful adjunct to therapy of herpetic ocular disease, an important cause of human blindness.     

Increased production of 12/15 lipoxygenase eicosanoids accelerates monocyte/endothelial interactions in diabetic db/db mice

Atherosclerosis is a major complication of diabetes. Up to 16 weeks of age, the db/db mouse is insulin-resistant and hyperglycemic and is a good model of Type 2 diabetes. After approximately 16 weeks of age, the mice develop pancreatic beta cell failure that can progress to a Type 1 diabetes phenotype. We have previously shown that glucose increases production of endothelial 12/15 lipoxygenase (12/15LO) products in vitro. In young 10-week-old Type 2 diabetic db/db mice, we found significant elevations in levels of urinary 12/15LO products, 12S-hydroxyeicosatetraenoic acid (12S-HETE) and 13S-hydroxyoctadecaenoic acid (13S-HODE) in vivo compared with C57BLKS/J mice. Using isolated primary aortic endothelial cells (ECs) from db/db mice and WEHI78/24 mouse monocyte cells in static adhesion assays, we found increased WEHI monocyte adhesion to db/db ECs (14 +/- 2 monocytes/field for db/db ECs versus 4 +/- 1 monocytes/field for C57BLKS/J ECs, p < 0.002). Thus, ECs from db/db mice appear to be "pre-activated" to bind monocytes. Analysis of db/db ECs revealed a 2-fold elevation in 12/15LO protein compared with C57BLKS/J EC. To determine that 12/15LO products were responsible for the increased monocyte adhesion observed with db/db ECs, we inhibited expression of murine 12/15LO using either an adenovirus expressing a ribozyme to 12/15LO (AdRZ) or with the 12/15LO inhibitor cinnamyl-3,4-dihydroxy-alpha-cyanocinnamate. Treatment of db/db ECs for 48 h with AdRZ or 4 h with 10 microm cinnamyl-3,4-dihydroxy-alpha-cyanocinnamate significantly reduced monocyte adhesion to db/db endothelium (p < 0.009). Thus, inhibition of the murine 12/15LO in db/db mice significantly reduced monocyte/endothelial interactions. We also found that adhesion of monocytes to diabetic db/db ECs was mediated by interactions of alpha4beta1 integrin on monocytes with endothelial vascular cell adhesion molecule 1 and connecting segment 1 fibronectin and interactions of beta2 integrins with endothelial intercellular adhesion molecule 1. In summary, regulation of the 12/15LO pathway is important for mediating early vascular changes in diabetes. Modulation of the 12/15LO pathway in the vessel wall may provide therapeutic benefit for early vascular inflammatory events in diabetes.     

Detection of murine cytomegalovirus DNA in circulating leukocytes harvested during acute infection of mice.

We used virus assay and in situ hybridization with a cloned fragment of the murine cytomegalovirus (MCMV) genome to study MCMV infection of circulating leukocytes harvested from 3-week-old BALB/c, C57BL/6, and C3H mice infected with MCMV intraperitoneally. Infectious virus or MCMV DNA was detected in leukocytes on days 1 through 21 of infection in BALB/c mice and on days 3 through 7 in C57BL/6 mice. On days 5 and 7, MCMV DNA or infectious virus was detected in the leukocytes of 17 (94%) of 18 BALB/c mice and 10 (59%) of 17 C57BL/6 mice. In both strains infection peaked on days 5 and 7, when as many as 0.01 to 0.1% of the circulating leukocytes contained MCMV DNA. In C3H mice, however, infectious virus was rarely recovered from leukocyte fractions and MCMV DNA was detected in the circulating leukocytes of only one animal. Circulating leukocytes may have an important role in the dissemination of CMV infections in susceptible hosts.