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. 2009 Sep;83(17):8869-84.
doi: 10.1128/JVI.00870-09. Epub 2009 Jun 17.

Liver sinusoidal endothelial cells are a site of murine cytomegalovirus latency and reactivation

Christof K Seckert  1 , Angélique Renzaho, Hanna-Mari Tervo, Claudia Krause, Petra Deegen, Birgit Kühnapfel, Matthias J Reddehase, Natascha K A Grzimek
Affiliations

Liver sinusoidal endothelial cells are a site of murine cytomegalovirus latency and reactivation

Christof K Seckert et al. J Virol. 2009 Sep.

Abstract

Latent cytomegalovirus (CMV) is frequently transmitted by organ transplantation, and its reactivation under conditions of immunosuppressive prophylaxis against graft rejection by host-versus-graft disease bears a risk of graft failure due to viral pathogenesis. CMV is the most common cause of infection following liver transplantation. Although hematopoietic cells of the myeloid lineage are a recognized source of latent CMV, the cellular sites of latency in the liver are not comprehensively typed. Here we have used the BALB/c mouse model of murine CMV infection to identify latently infected hepatic cell types. We performed sex-mismatched bone marrow transplantation with male donors and female recipients to generate latently infected sex chromosome chimeras, allowing us to distinguish between Y-chromosome (gene sry or tdy)-positive donor-derived hematopoietic descendants and Y-chromosome-negative cells of recipients' tissues. The viral genome was found to localize primarily to sry-negative CD11b(-) CD11c(-) CD31(+) CD146(+) cells lacking major histocompatibility complex class II antigen (MHC-II) but expressing murine L-SIGN. This cell surface phenotype is typical of liver sinusoidal endothelial cells (LSECs). Notably, sry-positive CD146(+) cells were distinguished by the expression of MHC-II and did not harbor latent viral DNA. In this model, the frequency of latently infected cells was found to be 1 to 2 per 10(4) LSECs, with an average copy number of 9 (range, 4 to 17) viral genomes. Ex vivo-isolated, latently infected LSECs expressed the viral genes m123/ie1 and M122/ie3 but not M112-M113/e1, M55/gB, or M86/MCP. Importantly, in an LSEC transfer model, infectious virus reactivated from recipients' tissue explants with an incidence of one reactivation per 1,000 viral-genome-carrying LSECs. These findings identified LSECs as the main cellular site of murine CMV latency and reactivation in the liver.

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Figures

FIG. 1.
FIG. 1.
Reactivation of mCMV from liver explants of latently infected bone marrow chimeras. (A) Experimental regimen. Sex-mismatched allogeneic BMT was performed with male BALB/c mice (XY; sry+) as BMC donors and female BALB/c mice (XX; sry−) as BMC recipients that were immunocompromised by total-body gamma irradiation with a single dose of 6.5 Gy and infected with wild-type (WT) mCMV, strain Smith. Viral latency and reactivation were usually studied at a minimum of 8 months after acute infection, when the viral genome was naturally cleared from circulating leukocytes. Note that throughout this paper, individually tested bone marrow chimeras are numbered consecutively. (B) Quantitation of latent viral genomes and of donor-derived cells in the livers of three individual bone marrow chimeras (mice 1 to 3) at 9 months after BMT and infection. M55/gB- and sry-specific qPCRs were performed with DNA isolated from 25-mg pieces of the livers. Circles represent triplicate measurements of each sample DNA (see inset legend). Median values are marked. The viral genome load is shown normalized to 1 × 105 liver cells. Tapered wedges symbolize the proportions of donor (D)- and recipient (R)-derived cells. (C) Reactivation of infectious mCMV in 24 liver explant cultures from each of the same three individual mice for which latent viral DNA load and chimerism are shown in panel B. Bars represent numbers of positive liver explant cultures (left ordinate scale) and incidences of reactivation (right ordinate scale).
FIG. 2.
FIG. 2.
Phenotyping of liver cells that carry latent viral DNA. (A) The latent viral genome localizes to nonparenchymal liver cells. At 9.5 months after sex-mismatched BMT and infection, DNA was prepared from 25 mg of unseparated liver tissue from three individual bone marrow chimeras (mice 4 to 6), from 5 × 106 isolated hepatocytes (chimeras 7 to 9), and from NPLCs (chimeras 10 to 12). Note that techniques for optimizing the purity of hepatocytes and NPLCs are mutually exclusive. Latent viral DNA loads and chimerism were determined by qPCRs specific for the viral gene M55/gB and the allosomal cellular gene sry. (B) DNA load and chimerism in subsets of NPLCs. NPLCs were isolated from a pool of five livers, and CD4+, CD31+, CD106+, CD11b+, CD11c+, CD45R+, and CD146+ subsets thereof were purified by immunomagnetic cell sorting (MACS). DNA was prepared from 5 × 106 cells of each sorted subset, and genes M55/gB and sry were quantitated by qPCR for determining viral DNA load and chimerism. Throughout, dot symbols represent triplicate measurements from each sample and the short horizontal bars mark the median values. Negative data are indicated below the dotted line. Tapered wedges symbolize the proportions of donor (D)- and recipient (R)-derived cells.
FIG. 3.
FIG. 3.
Purification of CD31+ CD146+ LSECs by two-color cytofluorometric cell sorting. (A) At 10 months after sex-mismatched BMT and infection, density gradient-enriched NPLCs derived from livers of five individual mice were separated by cytofluorometric cell sorting into CD146+ CD31+ LSECs and all remaining non-LSEC NPLCs. Shown are two-dimensional dot plots of CD31-stained and CD146-stained NPLCs of one representative mouse out of five mice tested, before (Pre-sort) and after (Post-sort) cell sorting. The presort dot plot represents a total of 6,676 cells, with 2,345 cells (35%) contained within the indicated sort gate and 4,331 cells outside of the sort gate. Postsort dot plots reveal the enrichment (upper plot) and the depletion (lower plot) of CD31+ CD146+ LSECs, respectively. Percentages indicate the proportions of cells within the indicated electronic gates. (B) For the five individually tested mice (mice 13 to 17), latent viral DNA loads and chimerism were determined by qPCRs specific for M55/gB and sry, respectively, in all NPLCs before cell sorting (NPLC fraction), in CD31+ CD146+ LSECs (LSEC-enriched fraction within the electronic gate), and in non-LSEC NPLCs (LSEC-depleted fraction outside the electronic gate). For the meaning of symbols, see the legend for Fig. 2.
FIG. 4.
FIG. 4.
Functional analysis of LSECs. (A) Immunomagnetically enriched ex vivo LSECs, with no prior cultivation, were incubated for 30 min with Dil-conjugated AcLDL and counterstained with FITC-conjugated anti-LSEC (anti-CD146) Ab. Cells were analyzed by two-color flow cytometry for endocytotic uptake of AcLDL (FL-2, carbocyanine dye Dil) and expression of CD146 (FL-1, FITC). (Left panel) Physical properties of LSECs as defined by size (forward scatter [FSC]) and granularity (sideward scatter [SSC]). The live gate for excluding dead cells and debris is indicated. (Right panel) Two-dimensional dot plot of AcLDL and CD146 staining of 30,000 cells analyzed, with 4,600 cells displayed as dots. Percentages of cells located in the four quadrants are indicated and reveal the purity of the LSEC preparation in the upper right quadrant. (B) Immunomagnetically enriched CD146+ LSECs were cultured for 24 h in the presence of 1.5 μg BODIPY FL-labeled AcLDL. The endocytotic activity of LSECs was determined by the uptake of AcLDL visualized by fluorescence microscopy with green cytoplasmic staining for AcLDL and blue staining of cell nuclei by Hoechst dye 33342. The bar marker represents 20 μm.
FIG. 5.
FIG. 5.
The latent viral genome localizes to CD146+ MHC-II− cells of recipient origin. (A) Immunomagnetically enriched CD146+ cells derived from livers of latently infected bone marrow chimeras at 11 months after sex-mismatched BMT and infection were separated into CD146+ MHC-II− and CD146+ MHC-II+ subsets by two-color cytofluorometric cell sorting. Shown are two-dimensional dot plots of MHC-II-stained and CD146-stained LSECs of one representative mouse out of three mice tested, before (Pre-sort) and after (Post-sort) cell sorting. The presort dot plot represents a total of 7,524 cells, with 6,089 cells (81%) within gate 1 (G1) representing CD146+ MHC-II− cells and 752 cells (10%) within gate 2 (G2) representing CD146+ MHC-II+ cells. Postsort dot plots reveal the purity of the sorted subsets. Percentages reveal the proportions of cells contained within the indicated electronic gates. (B and C) Chimerism (B) and latent viral DNA loads (C) determined for three individually tested mice (mice 18 to 20) by qPCRs specific for sry and M55/gB, respectively, in all immunomagnetically enriched cells before cytofluorometric cell sorting (CD146+ fraction) as well as in sorted cells of G1 (CD146+ MHC-II− fraction) and G2 (CD146+ MHC-II+ fraction). For the meaning of symbols, see the legend for Fig. 2.
FIG. 6.
FIG. 6.
Detection of latent viral genomes in LSECs coexpressing CD146 and the murine homolog of L-SIGN. CD146+ SIGN-R1+ LSECs were purified from density gradient-enriched NPLCs, followed by two-color cytofluorometric cell sorting. NPLCs were derived from mouse livers at 12 months after sex-mismatched BMT, and cell sortings were performed individually for four latently infected chimeras (mice 21 to 24). Viral genomes were quantitated by M55/gB-specific qPCR in NPLCs prior to cell sorting (left panel) as well as in sorted CD146+ SIGN-R1+ LSECs derived thereof (right panel). Note that sorted cells were mostly of the sry− genotype and thus recipient derived (data not shown). Filled circles represent triplicate qPCR data for each sample DNA preparation normalized to 1 × 105 cells. Median values are indicated by short horizontal bars.
FIG. 7.
FIG. 7.
Viral gene expression in latently infected LSECs ex vivo and after cultivation. Immunomagnetically enriched CD146+ LSECs were separately prepared from livers of five latently infected mice (mice 25 to 29) at 11 months after sex-mismatched BMT and infection and were tested for viral transcripts either directly ex vivo (Ø cultivation) or after a 24-h period of cultivation (24h cultivation). Highly purified, DNA-free total RNA was prepared from 4 × 106 LSECs, and triplicate 1/25 aliquots of each sample were used for the absolute quantitation of IE1, IE3, E1, M55/gB, and M86/MCP transcripts by the respective gene-specific RT-qPCRs using corresponding synthetic transcripts as standards. The experimentally determined numbers of transcripts were normalized to 500 ng of total RNA. Dot symbols represent triplicate measurements for each sample, with the median values marked by short horizontal bars. Negative data are indicated below the dotted line. Since genes M55/gB and M86/MCP have no exon-intron structure, PCRs were performed also with no RT (open circles) to control for amplification from contaminating DNA and were found to be negative throughout.
FIG. 8.
FIG. 8.
Frequency of LSECs carrying latent viral genomes. Limiting dilution analysis of immunomagnetically enriched CD146+ LSECs by M55/gB-specific qPCR was used to estimate the frequency of LSECs that carry latent viral DNA. (A) Standard curve for the quantitation of M55/gB by qPCR using log10-graded numbers of linearized plasmid pDrive_gB_PTHrP_Tdy as templates. The y-axis interception (one DNA molecule) of the extrapolated regression line reveals a cutoff cycle threshold (Ct) value of 41 cycles. Accordingly, experimental samples are classified as samples negative for viral DNA when no signal was obtained after 41 amplification cycles in the qPCR. (B) Graded numbers of LSECs derived from a pool of livers of three latently infected BALB/c mice at 8 months after sex-mismatched BMT and infection were tested in 12 replicates for the presence of viral DNA. The frequency estimate is based on the experimentally determined fractions of negative replicates. The log-linear plot shows the Poisson distribution graph and its 95% CI (shaded area) calculated by the maximum-likelihood method. The MPN, which is the reciprocal of the frequency, is revealed as the abscissa coordinate (dashed arrow) of the point of intersection between 1/e and the calculated regression line. It gives us the number of cells containing, on average, one latently infected cell. P, probability value indicating the goodness of fit.
FIG. 9.
FIG. 9.
Virus reactivation from latently infected LSECs by cell transfer and tissue explantation. (A) Experimental regimen. Immunomagnetically LSEC (CD146+)-enriched and LSEC (CD146+)-depleted NPLCs derived from pooled livers of three latently infected BALB/c donor mice at 8 months after sex-mismatched BMT and infection were transferred intravenously (i.v.) into CMV-naïve, immunocompromised (6.5 Gy of gamma irradiation 24 h before cell transfer) female BALB/c recipients. Of each population, 2 × 107 cells were transferred per recipient mouse. At 24 h after cell transfer, recipients' lungs were each cut into 48 pieces and the explants were kept in culture for 8 weeks, with weekly monitoring for the occurrence of infectious virus in the culture supernatants. (B) Reactivation incidences in 96 and 192 explant cultures from two and four recipients of LSEC-enriched and LSEC-depleted NPLCs, respectively. (C1) Two-by-two contingency tables (observed-value table and table of values expected according to the null hypothesis of random distribution) for significance analysis using Fisher's exact test. (C2) Two-by-two contingency tables (observed and expected values) under the rebuttable presumption that reactivation in the LSEC-enriched fraction resulted exclusively from contaminating 5% (worst-case assumption) of non-LSEC NPLCs. Differences between observed- and expected-value tables are considered significant for a P of <0.05 (two-sided).
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