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. 2014 Mar 12;15(3):351-62.
doi: 10.1016/j.chom.2014.02.002.

Cytomegalovirus hijacks CX3CR1(hi) patrolling monocytes as immune-privileged vehicles for dissemination in mice

Lisa P Daley-Bauer  1 , Linda J Roback  1 , Grace M Wynn  1 , Edward S Mocarski  2
Affiliations

Cytomegalovirus hijacks CX3CR1(hi) patrolling monocytes as immune-privileged vehicles for dissemination in mice

Lisa P Daley-Bauer et al. Cell Host Microbe. .

Abstract

Peripheral blood myelomonocytic cells are important for cytomegalovirus dissemination to distal organs such as salivary glands where persistent replication and shedding dictates transmission patterns. We find that this process is markedly enhanced by the murine cytomegalovirus (MCMV)-encoded CC chemokine, MCK2, which promotes recruitment of CX3CR1(hi) patrolling monocytes to initial infection sites in the mouse. There, these cells become infected and traffic via the bloodstream to distal sites. In contrast, inflammatory monocytes, the other major myelomonocytic subset, remain virus negative. CX3CR1 deficiency prevents patrolling monocyte migration on the vascular endothelium and interrupts MCMV dissemination to the salivary glands independent of antiviral NK and T cell immune control. In this manner, CX3CR1(hi) patrolling monocytes serve as immune-privileged vehicles to transport MCMV via the bloodstream to distal organs. MCMV commandeers patrolling monocytes to mediate systemic infection and seed a persistent reservoir essential for horizontal transmission.

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Figures

Figure 1
Figure 1
CX3CR1 deficiency disrupts MCMV dissemination to the salivary glands. (A) Viral titers in Cx3cr1gfp/+ (black) and Cx3cr1gfp/gfp (gray) mice at 14 days following systemic (ip) or FP inoculation. (B) Viral titers in salivary glands of Cx3cr1gfp/+ and Cx3cr1gfp/gfp mice at 1, 3, 5, 7 and 14 days following FP inoculation. Data points represent the mean viral titers ± SE for groups of five mice. Horizontal dashed lines indicate the limit of detection of the assay. Panels represent at least two independent experiments. *p < 0.05; **p < 0.01; ***p < 0.001; ns, not significant (p > 0.05).
Figure 2
Figure 2
Characterization of CX3CR1hi PMs in peripheral blood. (A -D) Gating strategy for PM (patrolling monocyte), IM (inflammatory monocyte), and, Neut (neutrophil) subpopulations. CD45+CD11b+ PBLs, gated to exclude T cells (TCRβ−) and NK cells (NK1.1−), were subjected to evaluation for Ly6C and CX3CR1 levels characteristic of these subpopulations. (E – M) Detection of monocyte subset-defining phenotypic markers on PMs (black, solid line), IMs (gray, dashed lines) and negative controls (filled histogram). PBLs were obtained from naïve Cx3cr1gfp/+ mice and phenotyped by flow cytometry following doublet exclusion. Plots are of data from one mouse representative of a group of four mice.
Figure 3
Figure 3
Viral MCK2 chemokine regulates PM recruitment to MCMV-inoculated FPs. (A − F) Initial cell recruitment patterns of CX3CR1+ PMs (A and B), NK cells (C and D) and IMs (E and F) shown as frequencies along with statistical comparison of groups at the same time points. (G) The second phase of MCK2-dependent PM recruitment shown as total PMs at 1, 3 and 5 dpi. CD45+ cells were isolated from Cx3cr1gfp/+ and Cx3cr1gfp/gfp mice inoculated with either Mck2+ or mutant virus and evaluated by flow cytometry. (H) FP swelling measured by digital caliper. Symbols show mean values ± SE for n = 5 mice. *p < 0.05; **p < 0.01; ***p < 0.001; n.s., not significant (p > 0.05). See also Figures S1 and S2.
Figure 4
Figure 4
PMs harbor infectious MCMV in blood. (A) Time course of PM levels in PBL of Cx3cr1gfp/+ and Cx3cr1gfp/gfp mice infected with Mck2+ (left panel) or mutant (right panel) virus. (B) Time course of PBL-associated viremia assessed by infectious centers assay. (C) Identification of PBL harboring infectious virus. PM, patrolling monocytes; IM, inflammatory monocytes; Neut, neutrophils. Mice were inoculated via the FP route and blood was collected at stated time points. PBLs or flow cytometry-sorted subsets were assessed by infectious centers assays. Symbols are mean values ± SE for n = 5 - 10 mice. (D – F) Infection of PM- or IM- derived macrophages with tissue culture (TC; D – F) or salivary gland (SG; D and E) stock virus. (E) Frequencies of IE1+ cells. (F) Virus replication. Data points are mean values ± SE. (G) Transfer of infection by CX3CR1gfp/+ CD45+ PBLs, PM-depleted CD45+ PBLs or sorted PMs to naïve Cx3cr1gfp/gfp recipient mice. *p < 0.05; **p < 0.01; ***p < 0.001; n.s., not significant (p > 0.05).
Figure 5
Figure 5
Impact of CX3CR1 on splenic PM and viral levels during acute infection as well as reactivation from latency. (A) Total PMs and (B) viral titers in spleens of Cx3cr1gfp/+ and Cx3cr1gfp/gfp mice during acute infection. Tissues were collected at 1, 3, 5, 7 and 14 dpi. Horizontal line represents assay threshold. PMs were enumerated by flow cytometry and viral titers were obtained by plaque assay. Symbols are mean values ± SE for groups of 5 – 8 mice. *p < 0.05; ***p < 0.001.(C) Reactivation of latent MCMV in explanted spleen collected at 63 dpi. Tissues were sectioned into three portions and supernatant sampled at weekly intervals were titered by plaque assay. Bars show the percentage of groups of 5 animals whose splenic tissue yielded virus.
Figure 6
Figure 6
CX3CR1 deficiency does not impair antiviral immune response. (A – D) NK cell levels (A and B) and viral titers (C and D) in inoculated FPs. Inset: IM numbers at 3 dpi. (E – H) NK cell levels (E and F) and viral titers (G and H) in draining popliteal lymph nodes. (I – L) Systemic CD8 T cell response in spleens at 7 dpi. Shown are (I) total CD8 T cells and, (J) the Ag-experienced population indentified as CD8αloCD11a+ cells. Ag-experienced cells were further characterized as effector T cells based (K) KLRG1 expression and (L) intracellular IFNγ and TNF following MCMV M45 peptide stimulation. Bars show mean values ± SE for n = 5 Cx3cr1gfp/+ (black) or Cx3cr1gfp/gfp (gray) mice subjected to mock (striped), Mck2+ (filled) or Mck2mut (open) virus infection. Cells were isolated from respective tissues and quantified by flow cytometry. Homogenized tissues were assessed by plaque assay for viral load. Data points are mean values ± SE for n = 5 mice and horizontal lines represent assay threshold. *p < 0.05; **p < 0.01; ***p < 0.001; n.s., not significant (p > 0.05).
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