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. 2006 Aug;80(15):7578-89.
doi: 10.1128/JVI.02421-05.

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

Giada Frascaroli  1 , Stefania Varani, Barbara Moepps, Christian Sinzger, Maria Paola Landini, Thomas Mertens
Affiliations

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

Giada Frascaroli et al. J Virol. 2006 Aug.

Erratum in

  • J Virol. 2013 Dec;87(23):13082-3

Abstract

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.

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Figures

FIG. 1.
FIG. 1.
Monocytes can be infected by endotheliotropic strains of HCMV, but the infection is blocked at early stages of the viral cycle. (A) At 24 h after infection, the IE (IE1-2), early (p52), and early-late (phosphoprotein pp65 and glycoprotein gB) viral antigens were detected by immunofluorescence (green staining) in monocytes inoculated with TB40E, AD169, and UV-inactivated TB40E (UV-TB40E) at an MOI of 5. Mock-infected monocytes were the negative controls. All photographs are from 1 donor representative of 20 (original magnification, ×60). Insets show in detail the pattern of fluorescence for a single cell (original magnification, ×100). (B) The percentages of IE1-2, p52, gB, and pp65 antigen-positive cells were evaluated at different time points after TB40E infection as the ratio of the number of positive cells to the total number of cells. Values are the mean ± SD of 10 different microscopic fields. The kinetic analysis from one donor representative of 20 is shown. (C) At different time points after infection, the amount of infectious virus present in monocyte supernatants and cytoplasmic extracts was evaluated by titration. Monocytes (3 × 106) were inoculated at time t = 0 with 1.5 × 107 PFU of TB40E (corresponding to an MOI of 5), and at 12 h postinfection they were washed with acid buffer in order to remove the unabsorbed viral particles. The number of viable monocytes (line graph) was evaluated at each time point. Uninfected and TB40E-infected monocytes were similar in viability and morphology.
FIG. 2.
FIG. 2.
Endotheliotropic and clinical strains of HCMV specifically downregulate the expression of chemokine receptors on the monocyte cell surface. (A and B) At 24 h after infection, mock- and HCMV-infected monocytes (MOI = 5) were examined by FACS for the surface expression of immune antigens and chemokine receptors. When indicated, monocytes were incubated with UV-inactivated TB40E (UV-TB40E) under the same conditions as for TB40E. The thick solid lines represent staining with specific MAbs for the indicated molecules; the thin lines represent staining with isotype-matched control antibodies. (C) The percentages of cells expressing the indicated chemokine receptors were evaluated in uninfected, TB40E-infected, and UV-inactivated TB40E-infected monocytes and statistically analyzed. Values are the mean ± SD of 20 different blood donors. formula image, P ≤ 0.05 between mock- and TB40E-infected cells. (D) The expression of CCR1, CCR2, CCR5, and CXCR4 was compared in monocytes infected with TB40E, VHLE, a representative clinical isolate, and AD169. Mock-infected monocytes were used as a control. Values are the mean ± SD of five different blood donors. (E) With four different blood donors, the percentages of mock- (○) and TB40E-infected (•) monocytes expressing chemokine receptors were evaluated at different time points after infection. Values are the mean ± SD of four separate experiments.
FIG. 3.
FIG. 3.
TB40E infection induces chemokine receptor redistribution from the cell membrane to the cytoplasm. (A) At 24 h after infection, both the surface (intact cells; thick solid lines) and the total levels (permeabilized cells; gray-filled histograms) of chemokine receptors were evaluated in mock- and TB40E-infected monocytes by FACS. The dotted lines and the thin lines represent the isotype-matched control antibodies on the surface and total staining, respectively. One donor representative of 10 is shown. (B) Monocytes were mock infected or infected with TB40E, and 24 h after infection chemokine receptor mRNA expression was examined by Northern blotting. For CCR1 a single 3.0-kb transcript, for CCR2 a single 3.5-kb transcript, for CCR5 a single 4.4-kb transcript, and for CXCR4 a single 1.8-kb transcript was found in mock-infected (−) and TB40E-infected (+) monocytes. The results show monocytes from 2 representative donors of 10. The lower part of the panel shows the ethidium bromide-stained rRNA. (C) The cellular distribution of chemokine receptors was investigated by confocal microscopy at 24 h after infection. Intact and permeabilized cells were stained with specific antibodies for CCR1, CCR2, CCR5, and CXCR4. As controls, isotype-matched and anti-CD14 antibodies were used. The microphotographs (original magnification, ×60) show the localization of CD14 and CXCR4, as a representative example of chemokine receptors, in uninfected and TB40E-infected monocytes.
FIG. 4.
FIG. 4.
TB40E induces the block of chemokine-driven migration in monocytes. Monocyte chemotaxis toward inflammatory (CCL5, CCL2, and CX3CL1) and homeostatic (CCL19 and CXCL12) chemokines was evaluated with a Boyden chamber as described in Materials and Methods. The migration induced by the indicated chemokines (100 ng/ml) was evaluated by assessing each stimulus in triplicate. As a control, migration toward fMLP (10−8 M) was evaluated. (A) Monocytes were mock infected or incubated at an MOI of 5 with both replication-competent TB40E and UV-inactivated TB40E (UV-TB40E). At 24 h after infection, the net numbers of cells that migrated were obtained by subtracting the number of cells that migrated in response to medium alone from the number of cells that migrated in response to chemokines. Net migration values (mean ± SD) of 10 experiments performed with monocytes from 10 different donors are shown. formula image, P ≤ 0.05 between mock- and TB40E-infected cells. (B) Monocytes were infected with TB40E at different MOIs (5, 1, and 0.5 PFU/cell). At 24 h after infection, chemotaxis induced by CCL5 and CXCL12 was assessed. Values are means of three separate experiments. (C) In four different donors, the numbers of migrating monocytes were evaluated at different time points during HCMV infection. Values are the mean ± SD of four independent experiments; formula image, P ≤ 0.05 between mock- and TB40E-infected cells.
FIG. 5.
FIG. 5.
Viral particles and nonsoluble factors are required for inhibition of monocyte chemotaxis. (A) To determine if soluble factors in the viral stock were involved in the inhibition of monocyte migration, monocytes were treated with virus-free supernatants obtained by filtration (filtered viral stock) or by ultracentrifugation (UC-viral stock) of the viral stock and with purified viral particles (gradient-purified virions and DB). Mock-infected monocytes (mock) and monocytes infected with a viral stock of TB40E (viral stock) were used as controls. At 24 h after infection, migration in response to medium, fMLP (10−8 M), CCL2, CCL5, and CXCL12 (100 ng/ml) was assessed. The numbers of cells that migrated were obtained as the mean ± SD of separate experiments performed with monocytes from five different donors. **, P ≤ 0.005 between mock-treated monocytes and monocytes treated with a viral stock, gradient-purified virions, or DB. (B) Fresh monocytes were incubated for 24 h with conditioned medium obtained from uninfected (white bars) and TB40E-infected monocytes and tested for responsiveness to the indicated chemoattractants. Conditioned medium from TB40E-infected monocytes was filtered (gray bars) or not filtered (black bars) before the assay. Values are the mean ± SD of five experiments performed with monocytes from five different donors. *, P ≤ 0.05 between supernatants from uninfected and TB40E-infected cells.
FIG. 6.
FIG. 6.
TB40E-infected monocytes do not recruit other leukocytes. Leukocyte recruitment was evaluated with the supernatants produced by uninfected (white bars) and TB40E-infected (black bars) cells as chemoattractants. Cells were infected with TB40E at an MOI of 5, and after 24 h the supernatants were collected, filtered, and then seeded in triplicate in the lower wells of a Boyden chamber. Leukocyte subpopulations such as PBMC, monocytes (MO), and neutrophils (PMN) were resuspended at a concentration of 1.5 × 106 cells/ml in RPMI-1% FCS and allowed to migrate for 90 min (PBMC and monocytes) or 60 min (PMN). (A) Supernatants obtained from uninfected and TB40E-infected monocytes were tested for recruitment of PBMC, PMN, and monocytes. The numbers of cells that migrated were obtained as the mean ± SD of five independent experiments performed with five different blood donors. As a control for the ability of monocytes to produce chemoattractants, monocytes were stimulated with LPS (500 ng/ml; gray bars) for 6 h before supernatants were collected. formula image, P ≤ 0.05 between supernatants from uninfected and LPS-treated cells. (B) Supernatants obtained from uninfected (white bars) and TB40E-infected (black bars) HUVEC and HFF were tested for the recruitment of fresh monocytes. The numbers of cells that migrated were obtained as the mean ± SD of five independent experiments performed with different donors as a source of monocytes. formula image, P ≤ 0.05 between supernatants from mock- and TB40E-infected cells.
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