HIV-1 cellular and tissue replication patterns in infected humanized mice

Abstract

Humanized mice have emerged as a testing platform for HIV-1 pathobiology by reflecting natural human disease processes. Their use to study HIV-1 biology, virology, immunology, pathogenesis and therapeutic development has served as a robust alternative to more-well developed animal models for HIV/AIDS. A critical component in reflecting such human pathobiology rests in defining the tissue and cellular sites for HIV-1 infection. To this end, we examined the tissue sites for viral infection in bone marrow, blood, spleens, liver, gut, brain, kidney and lungs of human CD34+ hematopoietic stem cell engrafted virus-infected NOD.Cg-Prkdc(scid) Il2rg(tm1Wjl)/SzJ mice. Cells were analyzed by flow cytometry and sorted from species mixtures defined as CD34+ lineage negative progenitor cells, CD14+CD16+ monocyte-macrophages and central, stem cell and effector memory T cells. The cell distribution and viral life cycle were found dependent on the tissue compartment and time of infection. Cell subsets contained HIV-1 total and integrated DNA as well as multi-spliced and unspliced RNA in divergent proportions. The data support the idea that humanized mice can provide a means to examine the multifaceted sites of HIV-1 replication including, but not limited to progenitor cells and monocyte-macrophages previously possible only in macaques and human.

Figure 1. Experimental approaches for tissue and cellular target testing of HIV-1 infected humanized mice.

(A) New-born NSG mice were transplanted with human HSC from cord blood. (B) After three months of human cells transplantation, mice were infected with HIV-1. (C) At 5 weeks post HIV-1 infection, several tissues such as bone marrow, spleen, lung, gut, brain, kidney and liver were collected for RNA and DNA isolation for the detection of HIV-1 using a semi-nested qPCR method. In addition, at 11 weeks post infection, spleen and bone marrow cells were isolated and sorted for collection of Lin-CD34+, CD14+ CD16+, T_SCM, T_CM, T_EM and T_REG populations. Sorted cells were used for the extraction of RNA and DNA, and viral quantification in each cellular subsets was determined by semi-nested qPCR. Moreover, cell phenotyping was determine by flow cytometry for the duration of the study where blood, spleen and bone marrow were collected for the identification of, memory CD4+ T cells, regulatory CD4+ T cells, Lin- CD34+, monocytes/macrophages CD14+ CD16+ and dendritic cells CD14− CD16+, as explained in methods.

Figure 2. HIV-1 detection in humanized mice tissues.

Humanized NSG mice were infected with HIV-1_ADA; and after 5 week of infection, plasma viral load was assessed. First, (A) Percentages of CD4+ and CD8+ T cells were determined by flow cytometry from the total human CD45+ CD3+ gate. This analysis was performed at pre- (0) and post- infection (5 and 14 weeks) by flow cytometry. Then (B) total viral DNA and inDNA and (C) viral RNA (msRNA and usRNA) levels were determined by semi-nested real time PCR in bone marrow, spleen, lung, gut, brain, kidney and liver tissues. The figures represent nucleic acid viral copies, DNA or RNA, per 10⁶ cells normalized to human CD45+ cells, as described in methods.

Figure 3. Identification of immune cell subsets.

Multicolor flow cytometric analysis was performed on bone marrow, whole blood and spleen tissues. (A) Cells from each tissue were incubated with labelled antibodies for the identification of CD34, Lineage-1, CD19, HLA-DR, CD3, CD8, CD14 and CD16 cellular markers. Monocytes/Macrophages were identified as CD3−/CD20−/CD8−/HLA-DR+/CD14+/CD16+ and DC as CD3−/CD20−/CD8−/HLA-DR+/CD14−/CD16+. Lin- CD34+ progenitor cells were determined by the expression of CD34 marker, excluding all Lineage-1 cells, as shown in the histogram. (B) Representation of frequencies for human Lin- CD34+, monocytes/macrophages CD14+ CD16+ and dendritic cells CD14− CD16+ from bone marrow, blood and spleen origins. Cellular debris was excluded by their light-scattering characteristics, and dead cells were excluded by UV fluorescence using a live/dead fluorochrome (Life Technology). Data were analyzed with FlowJo software. (C) Frequencies of CD34+, monocytes/macrophages and dendritic cells during HIV-1 infection.

Figure 4. Frequencies of CD4+ memory and T_REG phenotypes during progressive HIV-1 infection in humanized mice.

The T cells immune phenotypes were evaluated during acute (5 weeks) and chronic (14 weeks) infections in blood, spleen and bone marrow tissues. (A) Cell suspensions were labelled with anti-human monoclonal antibodies (mAb) targeting the following cell-surface markers: CD45, CD3, CD19, CD4, CD8, CD25, CD127, CD45RA, CD45RO, CD95, CCR7 (all from BD Biosciences). Histograms show the strategy for identification and isolation of CD4+ memory T cells (T_SCM, T_NM, T_CM and T_EM) and T_REG. All acquisitions were performed on a LSRII flow cytometer (Beckman Coulter). Cellular debris was excluded by their light-scattering characteristics, and dead cells were excluded by UV fluorescence using a live/dead fluorochrome (Life Technology). (B) Schematic description of the frequencies of T_SCM, T_NM, T_CM and T_EM and T_REG during HIV-1 acute and chronic infection in a humanized mice. Frequencies of immune cells were detected by flow cytometry following the strategies for gating and detecting each specific population, as described above. All data were analyzed by FlowJo software. (C) Frequency of memory and T_REG populations in HIV-1 infected humanized mice.

Figure 5. Defining the levels and species of HIV-1 infection in immune subsets from bone marrow and spleen.

Tissues were collected and cells in suspension were incubated with anti-human CD45 magnetic beads for isolating human CD45+ cells previous to FACS. Specific antibodies were applied and cells were sorted following the same procedure as described for immunophenotyping. Sorted cells from eleven weeks HIV-1 infected humanized mice were used for detecting both total viral DNA and inDNA and RNA (msRNA and usRNA). (A) Sorted Lin- CD34+, Monocyte-macrophages CD14+ CD16+, CD4+ T_SCM, CD4+ T_CM, CD4+ T_EM, and CD4+ T_REG cells were processed for DNA and RNA isolation and examined by semi-nested qPCR. Samples were quantified by the standard curve method using serial dilutions of viral standards (ACH2+ cells). Detection of total HIV-1 DNA, inDNA, msRNA and usRNA were achieved by using specific primers and probes as described in methods and in supplementary table 1. Dots and colors are representations for the frequency of viral DNA and RNA in different cell populations from bone marrow and spleen tissues. (B) Viral DNA and RNA quantification in bone marrow and spleen of HIV-1 infected humanized mice. The numbers indicate nucleic acid viral copies, DNA or RNA, per 10⁴ cells normalized to human GAPDH+ cells, as described in methods. Numbers between parentheses indicate the SEM. (C) Bone marrow cells from HIV-infected humanized mice were stained for the detection of human CD34+ cells (red) and HIV-p24 (green) and examined by confocal microscopy. Upper panel represent a picture at 400× original magnification and lower panel represent a different picture captured at 600X magnification. Representative images are shown from two HIV-infected humanized mice. Images were captured using a Zeiss confocal microscope.