Human cytomegalovirus induces monocyte differentiation and migration as a strategy for dissemination and persistence

Abstract

Human cytomegalovirus (HCMV) pathogenesis is characterized by multiple organ system involvement due to viral spread to host organs after a cell-associated viremia. The cell type responsible for HCMV dissemination is unknown. Monocytes are the most likely candidate since they are the predominant cell type infected in the blood. However, monocytes are not productive for viral replication and are abortively infected. The results presented here provide a potential answer to this conundrum. We report that primary HCMV infection of monocytes induces transendothelial migration and monocyte-to-macrophage differentiation and that these HCMV-differentiated macrophages are productive for viral replication. Together, our data suggest a novel mechanism for HCMV pathogenesis; HCMV induces cellular changes in monocytes to promote viral replication and spread to host organs.

FIG. 1.

HCMV infection promotes monocyte-to-macrophage differentiation. (a) HCMV promotes differentiation independently of viral gene expression as defined by the gain of macrophage morphology. Mock-infected, HCMV-infected, and UV-inactivated HCMV-treated monocytes were cultured on fibronectin and collagen for 11 days postinfection. Magnification, ×200 for all panels shown. The data shown are representative of five independent experiments from separate human blood donors. (b and c) HCMV promotes differentiation independently of viral gene expression as defined by the gain of macrophage function. FITC-zymosan was added to mock-infected, HCMV-infected, and UV-inactivated virus-treated monocytes at the indicated times postinfection, followed by incubation for 1 h. (b) Total ingestion represents the total number of FITC-zymosan internalized by 50 cells. (c) The percentage of cells positive for phagocytosis represents the percentage of cells positive for the internalization of one or more yeast particles out of 50 cells. The results are plotted as means ± the SD of quadruplicate experiments from a single blood donor. The data shown are representative of five independent experiments from separate blood donors. HCMV-infected and UV-inactivated HCMV-treated groups are significantly different (P < 0.01) from mock-infected groups at 48, 72, 120, and 168 h postinfection.

FIG. 2.

HCMV infection results in the upregulation of macrophage markers. (a) Western blot analyses of HLA-DR and CD68 were performed by using equal protein loading of mock-infected and HCMV-infected monocyte cell lysates harvested at the indicated times postinfection. Bands were analyzed by densitometry to determine relative levels of HLA-DR (b) and CD68 (c) expression. T0′ (time zero) represents cell lysates harvested from freshly isolated monocytes. The results are representative of three independent experiments from separate human blood donors.

FIG. 3.

HCMV-induced macrophages become permissive for HCMV gene expression. Mock-infected and HCMV-infected monocytes were cultured on fibronectin-coated coverslips, and immunofluorescent staining for the IE viral gene product IE2 and the late viral gene product gH was performed at the indicated times postinfection. The data are presented as the percentage of cells positive for IE2 or gH expression. The results are representative of three independent experiments from separate human blood donors.

FIG. 4.

HCMV infection of monocytes promotes transendothelial migration. (a) HCMV infection of monocytes is associated with an increase in diapedesis at 24 h postinfection. Cell tracker green-labeled mock-infected, HCMV-infected, UV-inactivated HCMV-treated, and PMA-treated monocytes were added to cell culture inserts containing confluent monolayers of HMECs. The ratios of cells undergoing diapedesis to those cells that were stationary at the monolayer surface were determined by fluorescence microscopy after 24 h in culture. The results are plotted as means ± the SD of 10 random fields of view at ×100 magnification. The results are representative of five independent experiments from separate human blood donors. HCMV-infected, UV-inactivated HCMV-treated, and PMA-treated groups are significantly different (P < 0.01) from the mock-infected group. (b) HCMV infection of monocytes is associated with increased total migration through endothelial monolayers. The number of monocytes that had migrated through endothelial monolayers and the cell culture insert filter was determined by fluorescence microscopy after 96 h in culture. The results are plotted as means ± the SD of 10 random fields of view at ×200 magnification. The results are representative of five independent experiments from separate human blood donors. HCMV-infected, UV-inactivated HCMV-treated, and PMA-treated groups are significantly different (P < 0.01) from the mock-infected group. (c) The majority of HCMV-infected and UV-inactivated HCMV-treated monocytes added to the transwells underwent complete transendothelial migration. At 96 h after the addition of monocytes to the transwells, monocytes adhered to the bottom of the wells were removed by using EDTA and then counted with a hemocytometer to determine the percentage of monocytes having undergone total migration out of the total number (2.5 × 10⁴) of monocytes added per transwell. The results are plotted as means ± the SD of three wells per experimental arm. The results are representative of three independent experiments from separate blood donors. HCMV-infected, UV-inactivated HCMV-treated, and PMA-treated groups are significantly different (P < 0.01) from the mock-infected group.

FIG. 5.

HCMV infection of monocytes promotes cell motility and adhesion molecule expression. (a to d) HCMV-infected monocytes have a motile morphology, as illustrated by F-actin staining. HCMV-infected monocytes at 48 h (a) and 72 h (b) postinfection and mock-infected monocytes at 48 h (c) and 72 h (d) postinfection were stained with Alexa Fluor 594 phalloidin and DAPI (4′,6′-diamidino-2-phenylindole). The results are representative of three independent experiments from separate human blood donors. (e) HCMV infection of monocytes promotes cell motility independent of viral gene expression. Phagokinetic track motility assays were performed to determine the average area (in square arbitrary units) of colloidal gold cleared per monocyte by mock-infected, HCMV-infected, UV-inactivated HCMV-treated, and PMA-treated samples at 12 h postadhesion. The results are plotted as means ± the standard error of the mean of 50 cells. The results are representative of three independent experiments from separate human blood donors. HCMV-infected, UV-inactivated HCMV-treated, and PMA-treated groups are significantly different (P < 0.01) from the mock-infected group. (f) HCMV infection of monocytes upregulates the expression of adhesion molecules. Western blot analyses for the β₁ integrin subunit, occludin, and ZO-1 were performed by using equal protein loading of mock-infected and HCMV-infected monocyte cell lysates harvested at the indicated times postinfection. The results are representative of two independent experiments from separate human blood donors.

FIG. 6.

Model for HCMV infection and dissemination in the host. Epithelial cells of the host are infected by contact with HCMV-containing bodily fluids. HCMV then replicates and spreads to monocytes in the peripheral blood by an unknown mechanism. As indicated by the red arrow, our data suggest that primary infection of monocytes promotes both their migration into host organ tissue and their differentiation into permissive macrophages. These macrophages could become sites of persistent infection in the host tissue. The possibility also exists that HCMV-induced macrophages could migrate into the bone marrow and infect myeloid progenitor cells, which are believed to be sites of HCMV latency.