<em>De novo</em> Generation of Cells within Human Nurse Macrophages and Consequences following HIV-1 Infection

<div><p>Nurse cells are defined as those that provide for the development of other cells. We report here, that <em>in vitro,</em> human monocyte-derived macrophages can behave as nurse cells with functional capabilities that include <em>de novo</em> generation of CD4+ T-lymphocytes and a previously unknown small cell with monocytoid characteristics. We named these novel cells “self-renewing monocytoid cells” (SRMC), because they could develop into nurse macrophages that produced another generation of SRMC. SRMC were not detectable in blood. Their transition to nurse behavior was characterized by expression of CD10, a marker of thymic epithelium and bone marrow stroma, typically absent on macrophages. Bromodeoxyuridine labeling and immunostaining for cdc6 expression confirmed DNA synthesis within nurse macrophages. T-cell excision circles were detected in macrophages, along with expression of pre-T-cell receptor alpha and recombination activating gene 1, suggesting that genetic recombination events associated with generation of the T-cell receptor were occurring in these cells. SRMC expressed CCR5, the coreceptor for R5 HIV-1 isolates, and were highly susceptible to HIV-1 entry leading to productive infection. While expressing HIV-1, SRMC could differentiate into nurse macrophages that produced another generation of HIV-1-expressing SRMC. The infected nurse macrophage/SRMC cycle could continue <em>in vitro</em> for multiple generations, suggesting it might represent a mechanism whereby HIV-1 can maintain persistence <em>in vivo</em>. HIV-1 infection of nurse macrophages led to a decline in CD4+ T-cell production. There was severe, preferential loss of the CCR5+ CD4+ T-cell subpopulation. Confocal microscopy revealed individual HIV-1-expressing nurse macrophages simultaneously producing both HIV-1-expressing SRMC and non-expressing CD3+ cells, suggesting that nurse macrophages might be a source of latently infected CD4+ T-cells. Real-time PCR experiments confirmed this by demonstrating 10-fold more HIV-1-genome-harboring T-cells, than virus-expressing ones. These phenomena have far-reaching implications, and elicit new perspectives regarding HIV pathogenesis and T-cell and hematopoietic cell development.</p> </div

Characteristics of small monocytoid cells produced by nurse macrophages.

<p>(A) Immunophenotype of nonadherent small monocytoid cells (SMC) harvested from an 18-day-old MDM culture. White arrow (top left plot) identifies the gated population shown in other plots. (B) Forward scatter (FSC) versus side scatter (SSC) plot demonstrates that SMC (CD3negCD4dimCD14+) are smaller than resting lymphocytes. Cells from 17-day-old MDM. Analysis performed using Paint-a-Gate software. (C) Confocal microscopy demonstrating that SMC adhere following their exit from nurse macrophages. Shown here is an 18-day-old primary MDM culture. An SMC adhering to a large macrophage (green arrow) and another adhering to the culture dish (pink arrow) can be seen. Others with elongated processes are also visible (white arrows) and viewable in greater detail in the panel on the right. The pink color of the nuclei reflects cdc6 colocalization with DNA as identified by TOTO-3 staining. SMC nuclei are typically cdc6+ during and shortly after budding from nurse macrophages, reflecting recent DNA synthesis. Size bar: 50 µm.</p

Characteristics of nonadherent cells in primary MDM cultures.

a<p>NAC, nonadherent cells;</p>b<p>Small cells defined as those within the lymphocyte gate. Small monocytoid cells identified by size and the phenotype presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040139#pone-0040139-g005" target="_blank">Fig. 5A</a>.</p>c<p>MM, maturing macrophages, defined as large CD14+CD33+ cells with high forward and side scatter. Days 4 and 7 include residual PBMC. Remaining cells from harvested populations include CD8+ T-cells and B, NK and stem cells. Values shown are the percentages determined by flow cytometry for nonadherent cells from pools of six T-25 flasks.</p

DNA synthesis and compartmentalization within multinucleated macrophages.

<p>(A-D) DNA synthesis, as evidenced by BrdU labeling, is occurring in multiple nuclei within a macrophage with nurse cell morphology. The phase contrast view (panel A) illustrates the nurse-like morphology of the living cell, prior to fixation. The culture was exposed to BrdU for 48 hours. (E-I) Confocal microscopy images demonstrating BrdU labeling in multinucleated macrophages. Culture was exposed to BrdU for 48 hours. Note that quiescent nuclei (blue only) are also present within both cells actively replicating DNA. Also apparent in the upper, somewhat smaller cell, are BrdU-labeled globular structures of multiple sizes. These colocalize with the DNA staining, confirming that they are active sites of DNA replication and not artifact. Panel I shows an enlarged view of this cell. (J-M) Confocal microscopy images demonstrating colocalization of expression of cell division cycle 6 (cdc6) protein with globular structures of DNA. Panels K-M are enlargements of the cell shown in Panel J. A gallery view of this cell is presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040139#pone.0040139.s005" target="_blank">Figure S5</a>. (N-R) DNA configurations and compartmentalization in Wright’s- Giemsa stained cultured macrophages. Size bars: (A) 50 µm, (E) 20 µm, (K) 10 µm and (N) through (R) 25 µm.</p

Characteristics distinguishing CD4+ T-cells produced in primary macrophage cultures from those produced in EDTA/IL2-mac.

<p>(A) As was the case in cultures of replated EDTA-mac treated with IL-2, the vast majority of T-cells produced in primary MDM cultures were also CD4+ T-cells. This is depicted here. However, in contrast to IL-2 treated cultures, these CD4+ T-cells were almost exclusively CD45RO+ and lacked CD45RA. Cells shown here were harvested on day 13 of culture and gated based on small size and CD3 expression (red arrows). The forward versus side scatter plots on the left demonstrate the relative numbers of large macrophages and lymphocytes within the nonadherent cell population. The numbers shown within the quadrants represent percentages from among the gated populations. (B) In contrast to those recovered from EDTA/IL2-mac, as shown here, the CD4+ T-cells produced in primary macrophage cultures did not express CD71, the transferrin receptor, indicating that they were resting lymphocytes. The CD71+ cells apparent are small CD4dim/CD14+ monocytoid cells, not lymphocytes. Analyses performed using Paint-a-Gate software. (C) Also in contrast to the CD4+ T-cells produced in IL-2-treated cultures, as shown here, 40–50% of these cells produced in primary MDM cultures expressed CD195, and 30–35% were dual positive for CD195 and CD184. Gates for the dot plots shown were established based on CD4bright expression and low side scatter, as indicated in the left panels (red arrows). Small, CD4dim cells were excluded from the gates because these were small monocytoid cells, not T-cells. The numbers shown within the quadrants represent percentages from among the gated populations. Analyses performed using CellQuest software. For (B) and (C), the letter D refers to the age of the culture in days at the time of harvest.</p

Human macrophage phenotypes.

<p>(A) complex colony in MDM culture. (B, C) Wright’s-Giemsa-stained, large multinucleated cells, with atypical (B) and typical (C) morphology, recovered from the adherent population of a 31-day-old MDM culture. (D-G) Responses of EDTA-recovered replated macrophages to IL-2. Macrophages were detached on D31 of culture, replated at low density, and IL-2 added 2 days later, after extensive washing. Cells were photographed on day 8 (D-F) or day 6 (G) of IL-2 exposure. Arrows in (G) point to nurse macrophages within a cell cluster. (H, I) IL-2-mediated induction of dendritic processes on replated macrophages. To aid visualization, shown here are macrophages infected with an HIV-1 strain harboring the enhanced green fluorescent protein gene. Macrophages were infected with HIV-1 pSV162R3, subjected to EDTA removal 15 days following infection, and replated. IL-2 was added 4 days later, after washing, and the treated and untreated cultures photographed on day 5. Similar responses of uninfected cells to IL-2 are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040139#pone.0040139.s001" target="_blank">Fig. S1</a>. (J, K) Nurse macrophages exhibit reduced phagocytic capabilities. Macrophage cultures were established directly from PBMC in 24-well plates. (No Il-2 was used.) To assess phagocytosis, the cells were incubated with 6 mm plastic microspheres for 24 hours, then washed to remove excess spheres and scored. For scoring, NM were identified by size, morphology and the presence of multiple nuclei. The fluorescent dye Hoechst 33342 was added to visualize cell nuclei. (J) Quantitation of microspheres per cell. Fields were selected for scoring based on the presence of NM; 100 macrophages of each type were scored per experiment. Error bars depict SEM. Similar results were obtained in two additional experiments. (K) Apparent is a large NM lacking microspheres (arrow), surrounded by several microsphere-containing macrophages. Size bars: (A) and (E) 100 µm, (B) (C) and (F) 25 µm and (G) 50 µm. Magnification: (D) and (I) x100, (H) x200 and (K) x400.</p

T-cells are produced within nurse macrophages.

<p>(A) Immunophenotype of nonadherent cells harvested from cultures of replated EDTA-recovered macrophages after 112 hours of exposure to IL-2. The forward (FSC) versus side (SSC) scatter plot shown on the left indicates the gate used for the other plots. As can be seen, the vast majority of cells are CD4+ T-lymphocytes. The 7-AAD plot indicates that these cells were viable. (B) Confocal microscopy images showing that the CD3+ cells present within this large MHC II+ macrophage appear as an organized array. This culture of replated EDTA-recovered macrophages was photographed on day 10 of exposure to IL-2. Size bar: 50 µm. (C) Confocal microscopy images illustrating CD3+ cells within a CD68+ macrophage at day 10 of IL-2 exposure. Free CD3 antigen deposition is also apparent within the macrophage (white arrow, far left panel), as is budding of a CD3+ cell from the macrophage surface (green arrow, far right panel). Boxed insert in the center panel is an enlargement of the cell identified by the white arrow. Small pockets of CD68 antigen can be seen within this CD3+ cell near the top edge. Size bar: 20 µm. Z indicates the Z-stack level(s) for each image. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040139#pone.0040139.s004" target="_blank">Fig. S4</a> for a gallery view of this series. (D) Time-lapse video images of a lymphocyte budding from a macrophage in a replated culture exposed to IL-2 for 7 days. (The budding cell is a lymphocyte, based on its subsequent nonadherent post-bud behavior. Data not shown.) Numbers below the arrows refer to the time interval in minutes and/or seconds between the two images. Arrowheads identify the position of the budding cell, which first becomes apparent in the second panel.</p

Small monocytoid cells are susceptible to HIV-1 entry leading to productive infection.

<p>(A, B) EGFP expression, a marker of productive HIV-1 infection, in cultured MDM at day 5 post infection. Upper panels photographed using phase contrast, and lower panels, which depict identical fields, using fluorescence microscopy. Areas boxed in white in (A) correspond to enlargements shown in (B). The virus-expressing cells (arrows in B) are very small adherent cells at this time. (C) Cluster of tightly associated virus-expressing small cells (arrow) at day 12 post infection. (D) Virus-expressing large macrophage with nurse cell morphology (arrow) at day 21 post infection. (E) Small, HIV-1-expressing cell budding from an infected large macrophage (arrow). (F) EGFP expression in nonadherent small monocytoid cells harvested at day 23 post infection. Small EGFP+ cells were gated as shown in the upper left panel. The other plots were based on the gated population. As can be seen, these small cells express CD14 and CD33 and lack CD3, indicative of a macrophage lineage phenotype. In the upper center plot, most of the EGFP+CD3- cells hug the X-axis. (G) EGFP expression in maturing macrophages harvested at day 9 post infection. The gated population of large macrophages is identified in the upper left panel. Because large macrophages have high levels of autofluorescence, the data are shown with isotype controls. Magnification: (A) x100, (C) x200, (D, E) x400.</p

A model for nurse macrophage capabilities and role in HIV-1 pathogenesis.

<p><i>In vitro,</i> we observed that human blood monocytes differentiate into macrophages, and that a portion of these can further develop into what we term “nurse macrophages.” These nurse cells can generate within themselves, CD4+ T-lymphocytes, as well as a previously unknown small monocytoid cell (SRMC). The SRMC, itself, can develop into a nurse macrophage with the ability to give rise to another SRMC. The NM/SRMC cycle, then, represents a form of self-renewal. When cultures of human macrophages are exposed to HIV-1, the SRMC become productively infected, but virus expression does not kill them. Rather, they can differentiate into macrophages, including nurse macrophages, in the presence of virus expression. Thus, a NM/SRMC cycle is established that is characterized by ongoing HIV-1 expression. In the absence of HIV-1 infection, nurse macrophages release resting CD4+ T-cells. The development and release of these lymphocytes is enhanced by treatment with IL-2. When productively infected with HIV-1, the nurse macrophage retains, rather than releases the T-lymphocytes and as a consequence, it takes on the appearance of a multinucleated giant cell. We propose that these events may recapitulate events that occur <i>in vivo</i> and if so, this model offers explanations for HIV-1 persistence and latency.</p

HIV-1 infection dysregulates T-cell development in nurse macrophages.

<p>(A) Confocal microscopy images showing an HIV-1-expressing small monocytoid cell (CD36+) budding from the center of a virus-expressing nurse macrophage, as evidenced by colocalization of HIV-1p24 with CD36. Also apparent is that the developing and post-budded CD3+ cells are not expressing HIV-1. This MDM culture was infected with HIV-1 5E14BM, an isolate from bone marrow, and photographed on ∼day 21 post infection. Size bar: 20 µm (B) Latently infected CD4+ T-cells are released from nurse macrophages. Numbers of genome-positive cells were determined by real-time PCR using ACH-2 cells as standards, and then normalized to 100%. Numbers of HIV-1-expressing cells were determined by flow cytometry. The numbers of CD14+ cells harboring HIV genomes also exceeded the numbers expressing HIV-1, but the relative difference was less than that seen for CD4+ T-cells. (C) Fewer CD4+ T-cells are released from nurse macrophages in MDM cultures infected with HIV-1. Nonadherent cells were harvested from infected and uninfected cultures, counted and immunophenotyped using flow cytometry. Cells from 3 or more replicate flasks were pooled. For each harvest, CD4+ T-cell numbers from uninfected flasks were normalized to represent 100%, and the numbers recovered from infected flasks were normalized, proportionately, to represent percent relative to uninfected control. (D) Confocal microscopy illustrating an HIV-1p24-expressing multinucleated giant cell in an MDM culture at D6 following infection with HIV-1 Ba-L. This cell has more than 65 nuclei. The numbers of these cells increased with time. Size bar: 20 µm (E) Abundant BrdU labeling in an HIV-1 Ba-L-infected giant cell. This labeling colocalizes with DNA. Cells were exposed to BrdU for 48 hours, beginning at day 12 post infection. 20 µm. Z numbers indicate the composite Z-stack levels for the images.</p