Human Cytomegalovirus Entry into Dendritic Cells Occurs via a Macropinocytosis-Like Pathway in a pH-Independent and Cholesterol-Dependent Manner

Human cytomegalovirus (HCMV) is a ubiquitous herpesvirus that is able to infect fibroblastic, epithelial, endothelial and hematopoietic cells. Over the past ten years, several groups have provided direct evidence that dendritic cells (DCs) fully support the HCMV lytic cycle. We previously demonstrated that the C-type lectin dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN) has a prominent role in the docking of HCMV on monocyte-derived DCs (MDDCs). The DC-SIGN/HCMV interaction was demonstrated to be a crucial and early event that substantially enhanced infection in trans, i.e., from one CMV-bearing cell to another non-infected cell (or trans-infection), and rendered susceptible cells fully permissive to HCMV infection. Nevertheless, nothing is yet known about how HCMV enters MDDCs. In this study, we demonstrated that VHL/E HCMV virions (an endothelio/dendrotropic strain) are first internalized into MDDCs by a macropinocytosis-like process in an actin- and cholesterol-dependent, but pH-independent, manner. We observed the accumulation of virions in large uncoated vesicles with endosomal features, and the virions remained as intact particles that retained infectious potential for several hours. This trans-infection property was specific to MDDCs because monocyte-derived macrophages or monocytes from the same donor were unable to allow the accumulation of and the subsequent transmission of the virus. Together, these data allowed us to delineate the early mechanisms of the internalization and entry of an endothelio/dendrotropic HCMV strain into human MDDCs and to propose that DCs can serve as a "Trojan horse" to convey CMV from entry sites to other locations that may favor the occurrence of either latency or acute infection

LIMLE, a New Molecule Over-Expressed following Activation, Is Involved in the Stimulatory Properties of Dendritic Cells

International audienceDendritic cells are sentinels of the immune system distributed throughout the body, that following danger signals will migrate to secondary lymphoid organs to induce effector T cell responses. We have identified, in a rodent model of graft rejection, a new molecule expressed by dendritic cells that we have named LIMLE (RGD1310371). To characterize this new molecule, we analyzed its regulation of expression and its function. We observed that LIMLE mRNAs were rapidly and strongly up regulated in dendritic cells following inflammatory stimulation. We demonstrated that LIMLE inhibition does not alter dendritic cell maturation or cytokine production following Toll-like-receptor stimulation. However, it reduces their ability to stimulate effector T cells in a mixed leukocyte reaction or T cell receptor transgenic system. Interestingly, we observed that LIMLE protein localized with actin at some areas under the plasma membrane. Moreover, LIMLE is highly expressed in testis, trachea, lung and ciliated cells and it has been shown that cilia formation bears similarities to formation of the immunological synapse which is required for the T cell activation by dendritic cells. Taken together, these data suggest a role for LIMLE in specialized structures of the cytoskeleton that are important for dynamic cellular events such as immune synapse formation. In the future, LIMLE may represent a new target to reduce the capacity of dendritic cells to stimulate T cells and to regulate an immune response

Endosomal pH neutralization does not inhibit HCMV internalization or MDDC infection.

<p>A) Cells were pre-incubated with NH₄Cl-containing buffer (50, 5, 0.5 mM) or bafilomycin A1 (320, 32, 3.2 nM) and compared to the vehicle (DMSO; 1/100). The cells were processed as described in the legend for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034795#pone-0034795-g001" target="_blank">Figure 1D</a>. For NH₄Cl, n= 3 independent experiments with 3 different donors in total, for bafilomycin A1 n= 6 independent experiments with 8 different donors in total. ns: not significant (p=0,0939). B) TEM picture of (50 nM) bafilomycin A1 treated MDDCs incubated with HCMV (VHL/E; MOI=10). Black arrows indicate HCMV virions. C) Quantification of infectious HCMV particles by TEM immobilized at the plasma membrane (OUT, white bars) or internalized into vacuoles (IN, black bars) of (50 nM) BafA1-treated MDDCs incubated for two hours with VHL/E (MOI=10) (n=5-6 cells per condition). The results are displayed as the median values of the percentage (±SD) of plasma membrane-associated and internalized HCMV particles. These results are representative of two distinct experiments.</p

LIMLE mRNA expression in transplantation models, cell subtypes and organs.

<p>LIMLE mRNA expression in (A) cardiac grafts and PBMC of rat syngenic recipients (Syng), allograft recipients developing acute (AR) or chronic rejection (CR), or allograft tolerant recipients (Tol) at day 5 or 100 after transplantation (n = 5, *p<0.05 **p<0.01 ***p<0.001), (B) Splenic DCs, alveolar macrophage lineage cells NR8383 (Mac), B cells, T cells and EC, stimulated (stim) or not (NS) with LPS, IFNγ, CpG, anti-CD3/CD28 or IFNγ respectively (for 12 hours) (n = 3), (C) rat BMDCs stimulated or not (NS) with LPS, IFNγ, CpG, Poly(I:C) or IL-10 for 6, 12, 24 or 48 hours (n = 3, *p<0.05), (D) Human DCs stimulated or not (NS) with LPS for 12 hours (n = 3, **p<0.01) (E) mRNA expression of LIMLE in different organs from naive rats as indicated (n = 5). (A–E) Quantitative RT-PCR results were expressed in Arbitrary Units (AU) of LIMLE/HPRT transcript ratio ± SEM. Statistical evaluation was performed using Student's <i>t</i> test for unpaired data, and results were considered significant if <i>p</i> values were <0.05.</p

Cholesterol depletion is detrimental to the HCMV entry into MDDCs.

<p>A) Cells were pre-incubated with filipin (7.66, 1.5, 0.3 µM), nystatin (21.2, 4.3, 0.85 µM) or methyl-β-cyclodextrin (MβCD; 5, 1, 0.2 mM) or with vehicle (DMSO, 1/100) and were processed as described in the legend for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034795#pone-0034795-g001" target="_blank">Figure 1D</a>. For nystatin, n= 2 independent experiments with 2 different donors in total; for Filipin and MβCD n=4 independent experiments with 6 different donors in total. <i>ns</i>: not significant (p=0,0535).</p

HCMV internalization into MDDCs is impaired by macropinocytosis inhibitors.

<p>A) MDDCs were pre-incubated with drugs blocking macropinocytosis (Gö6983∶ 13, 7.3, 3.75 nM and amiloride, Ami: 100, 20 µM) or clathrin-mediated endocytosis (chlorpromazine, CPZ: 30, 6, 1.2 µM) or with vehicle (DMSO, 1/100) prior to be infected with HCMV (MOI=2) for two hours. Cells were extensively washed then subcultured for 48 hours. The cells were then prepared and analyzed as described in the legend for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034795#pone-0034795-g001" target="_blank">Figure 1D</a>. For Gö6983, n=2 independent experiments with 4 different donors in total, for Ami and CPZ n=5 independent experiments with 7 different donors in total. ns: not significant (p=0,0733) B) TEM picture of (500µM) amiloride-treated MDDCs incubated with HCMV (VHL/E; MOI=10). Black arrows indicate HCMV virions. C) Quantification of infectious HCMV particles by TEM immobilized at the plasma membrane (OUT, white bars) or internalized into vacuoles (IN, black bars) of (500 µM) amiloride-treated MDDCs incubated for two hours with VHL/E (MOI=10) (n=6 cells per conditions). The results are displayed as the median values of the percentage (±SD) of plasma membrane-associated and internalized HCMV particles.</p

Internalized HCMV virions partially co localize with EEA1 and not with LAMP2.

<p>Confocal imaging was performed on immobilized immature MDDCs incubated for two hours on ice with the VHL/E strain (MOI=2) and subsequently cultured at 37°C for three hours (A) and 24 hours (B). Cells were then fixed/permeabilized and immunostained as indicated above each column with anti-EEA1 or -LAMP2 antibodies (green) and with an anti-HCMV gB (red). Images were obtained on a 510 LSM Meta (Zeiss, Germany). Single confocal planes are presented. In merged panels, green and red staining intensities were directly analyzed along a virtual section indicated by a thin white line with the ImageJ RGB profiler plugin. The results are displayed in graphs on the right (X-axis=distance in pixels; Y-axis=fluorescence intensity). White arrowheads indicate small CMV aggregates or isolated particles. C) Between 7 and 10 x 10⁷ HCMV-infected MDDCs (VHL/E; MOI=10) were subjected to three consecutive subcellular fractionation steps on sucrose and Percoll gradients to harvest early and late endosome-enriched and lysosome-enriched fractions. Here early endosomes (EE) and late endosomes (LE) enriched fractions were first pooled (EE+LE) and the EE+LE and the lysosomes (Ly) enriched fractions were concentrated before being used in a western blot analysis using anti-EEA1, LAMP-2, HCMV gB and MCP antibodies. Recombinant HCMV gB (Biomérieux, France) was used as a positive control. The molecular weight of the gB is 160 kDa and 153 kDa for the MCP. PN means post nuclear fraction.</p

Cellular localization of LIMLE protein.

<p>Representative pictures of immuno-fluorescence staining of (A and B) COS or (C) BMDCs transfected with a plasmid encoding full length rat LIMLE protein and containing the V5 tag (red) with DAPI (blue) and and phalloidin (polymerized actin) (green). Original magnification ×1200 (Plan Apo N.A: 1.4 zoom 2.). (Bi,ii) Representative histograms of fluorescence intensity (AU) in COS cells detected in pixels with the indicated box, graphed with the linescan function of Metamorph Image Processing Software. (Ci) Representative picture of LIMLE overlapping with actin in DCs, processed with Metamorph Image Processing Software. Pictures were representative of three independent experiments.</p

MDDCs can mediate HCMV <i>trans</i>-infection through both plasma membrane-associated virions and the release of internalized virions.

<p>MDDCs, MDMs or monocytes from the same blood donor were obtained as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034795#s2" target="_blank">Materials and Methods</a> section. Cells were pretreated with 40 nM BafA1 (black bars) or the vehicle (DMSO) (white bars) prior to incubation with the VHL/E HCMV strain for two hours (MOI=2). Cells were then extensively washed with a low-pH buffer (glycine 0.2M, pH=2.8) or with PBS alone as indicated above each panel of the figure and were subcultured in close contact with HFFs. After 48 hours, fibroblasts were processed as previously described <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034795#pone.0034795-Halary1" target="_blank">[14]</a> to evaluate the infection rate due to <i>trans</i>-infection by HCMV-loaded cells (absolute number of IE/E antigen-positive fibroblasts among 10⁵ total cells). n= 4 independent experiments with four different donors in total.</p

CMV internalization into large uncoated vesicles in MDDCs is an active process that requires actin cytoskeleton polymerization.

<p>A) Transmission electron microscopy (TEM) picture is shown of day 6 immature MDDCs incubated with HCMV (VHL/E; MOI=10) for two hours then extensively washed with PBS. A scale bar is indicated (magnification x7,500). N=nucleus. B) Close-ups of different pictures are represented. Black arrows indicate HCMV virions. A scale bar is indicated for each picture. Cells were prepared as described in 1A. C) Kinetic analysis of HCMV internalization in MDDCs. The data represent the quantification of the mean number of particles per cell from the TEM pictures (n≥20 independent cells) of viral particles (VHL/E; MOI=10) immobilized at the plasma membrane (OUT, white bars) or internalized into vacuoles (IN, black bars) as function of time (30 minutes, two, six and 24 hours incubations). Cells were extensively washed with PBS after incubation with HCMV. These results are representative of two distinct experiments. D) MDDCs were pre-incubated with drugs (cytochalasin, cytD: 50-5-0.5 µM) or vehicle (DMSO: 1/100) prior to be infected with HCMV (MOI=2) for two hours. Cells were extensively washed then subcultured for 48 hours. The cells were coated onto poly-L-lysine-coated slides, fixed and permeabilized with acetone and were stained with mAbs against anti-IE and –E antigens (Argene Biosoft, France). Four distinct fields were digitalized and analyzed with ImageJ software to determine the percentage of I.E.A./E.A.+ MDDCs. n=6 independent experiments with eight different donors in total.</p