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. 2014 Jul 31;10(7):e1004291.
doi: 10.1371/journal.ppat.1004291. eCollection 2014 Jul.

Plasmacytoid dendritic cells suppress HIV-1 replication but contribute to HIV-1 induced immunopathogenesis in humanized mice

Guangming Li  1 , Menglan Cheng  2 , Jun-Ichi Nunoya  3 , Liang Cheng  3 , Haitao Guo  3 , Haisheng Yu  2 , Yong-Jun Liu  4 , Lishan Su  5 , Liguo Zhang  2
Affiliations

Plasmacytoid dendritic cells suppress HIV-1 replication but contribute to HIV-1 induced immunopathogenesis in humanized mice

Guangming Li et al. PLoS Pathog. .

Abstract

The role of plasmacytoid dendritic cells (pDC) in human immunodeficiency virus type 1 (HIV-1) infection and pathogenesis remains unclear. HIV-1 infection in the humanized mouse model leads to persistent HIV-1 infection and immunopathogenesis, including type I interferons (IFN-I) induction, immune-activation and depletion of human leukocytes, including CD4 T cells. We developed a monoclonal antibody that specifically depletes human pDC in all lymphoid organs in humanized mice. When pDC were depleted prior to HIV-1 infection, the induction of IFN-I and interferon-stimulated genes (ISGs) were abolished during acute HIV-1 infection with either a highly pathogenic CCR5/CXCR4-dual tropic HIV-1 or a standard CCR5-tropic HIV-1 isolate. Consistent with the anti-viral role of IFN-I, HIV-1 replication was significantly up-regulated in pDC-depleted mice. Interestingly, the cell death induced by the highly pathogenic HIV-1 isolate was severely reduced in pDC-depleted mice. During chronic HIV-1 infection, depletion of pDC also severely reduced the induction of IFN-I and ISGs, associated with elevated HIV-1 replication. Surprisingly, HIV-1 induced depletion of human immune cells including T cells in lymphoid organs, but not the blood, was reduced in spite of the increased viral replication. The increased cell number in lymphoid organs was associated with a reduced level of HIV-induced cell death in human leukocytes including CD4 T cells. We conclude that pDC play opposing roles in suppressing HIV-1 replication and in promoting HIV-1 induced immunopathogenesis. These findings suggest that pDC-depletion and IFN-I blockade will provide novel strategies for treating those HIV-1 immune non-responsive patients with persistent immune activation despite effective anti-retrovirus treatment.

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Conflict of interest statement

I have read the journal's policy and the authors of this manuscript have the following competing interests: pending patent application for the pDC depleting monoclonal antibody. This does not alter our adherence to all PLOS policies on sharing data and materials.

Figures

Figure 1
Figure 1. HIV-1 replication and pathogenesis in HIV-1-infected humanized mice.
HIV-R3A replication and pathogenesis in humanized mice are summarized. (A) Plasma viral load of mice intravenously inoculated with 1 ng p24/mouse of R3A (n = 10). Viral RNA copy numbers were measured using real-time PCR quantification. (B) The production of IFN-a2 in plasma from uninfected (n = 3) and infected (n = 3) humanized mice measured by Luminex. (C) The relative level of Mx1 and TRIM22 gene expression in huCD45+ cell in spleen (n = 3) detected by real time PCR. (D) Summary data of the percentages of HLA-DR+CD38+ on CD8 T cells (CD3+CD4−CD8+) in peripheral blood and spleen measured by FACS. (E) Summary data of the percentages of CD4+ T cells of CD3+ cells. (F) Comparison of absolute CD4 T-cell, CD8 T-cell and huCD45+ cell numbers in spleen from uninfected controls (n = 3) and R3A-infected mice (n = 10). (B–F) Mice were analyzed at 8 days post infection. All bars in dot graphs indicate median value. Error bars indicate standard deviations (SD). * and ** indicate p<0.05 and p<0.01, respectively.
Figure 2
Figure 2. Specific depletion of human pDC in lymphoid organs in vivo with a human pDC-reactive monoclonal antibody.
Humanized Mice were treated with either 15B or isotype control (iso) antibody for 3 times on days -5, -3, -1 prior to termination. Percentages of pDC (Lin−CD4+CD123+) in total human leukocytes (CD45+) are analyzed. (A) Representative FACS plots and summarized data show relative pDC frequencies before and after antibody treatment in the peripheral blood (n = 7). (B) Representative FACS plots and summarized data show pDC depletion by 15B in mesenteric lymph nodes (mLN) and spleen (SP, isotype n = 4; 15B n = 5). All bars in dot graphs indicate median value. Error bars indicate standard deviations (SD). * and ** indicate p<0.05 and p<0.01, respectively. (C) Summarized percentages of CD3−CD19+ B cells in different lymphoid tissues. (D) Summarized percentages of CD3+CD19− T cells in different lymphoid tissues. (E) Summarized percentages of CD3−CD11c+ mDC cells in different lymphoid tissues. All bars in dot graphs indicate median value.
Figure 3
Figure 3. Pre-infection depletion of pDC abolishes IFN-I induction during acute HIV-1 infection in humanized mice.
(A) Summarized data of pDC percentages in total human leukocytes (CD45+) from humanized mice are shown mice. Mice were treated with either 15B or isotype control (iso) antibody. After pDC depletion, mice were infected with HIV-R3A and terminated on 8 days post infection (dpi) for analysis. Mock infected mice, n = 6; isotype+R3A infected mice, n = 9; 15B+R3A infected mice, n = 12. (B) Plasma levels of IFN-α2 from mock, HIV-1 infected and 15B or isotype mAb treated mice were quantified by Luminex assays. Mock, n = 3; isotype+R3A, n = 5; 15B+R3A, n = 5. (C) The mRNA expression of major type I IFN genes in purified human cells (CD45+) from mouse spleens was measured by real-time PCR. (D) ISGs (Mx1 and TRIM22) expression in purified human cells (CD45+) from mouse spleens was measured by real-time PCR. Mock, n = 3; isotype+R3A, n = 5; 15B+R3A, n = 5. Mice were analyzed at 8 days post infection. Error bars in graphs indicate median value. Error bars indicate standard deviations (SD). * and ** indicate p<0.05 and p<0.01, respectively.
Figure 4
Figure 4. Pre-depletion of pDC leads to higher levels of HIV-1 replication.
(A) Plasma HIV-1 RNA levels from HIV-R3A infected mice (genome copy#×log10/ml) are summarized. isotype+R3A, n = 9; 15B+R3A, n = 12. (B) Plasma HIV-1 RNA levels from JR-CSF infected mice at 2 wpi are summarized. isotype+JR-CSF, n = 12; 15B+JR-CSF, n = 12. (C) Immunohistochemistry staining for p24 positive cells in the spleen of mock or HIV-R3A infected mice. (D) Representative FACS plots for p24 positive CD4 T cells in the spleen of mock or JRCSF infected mice at 3 wpi. (E) Summarized data of D. isotype+JR-CSF, n = 7; 15B+JR-CSF, n = 7. Bars in dot graphs indicate median value. * indicates p<0.05.
Figure 5
Figure 5. Pre-depletion of pDC reduces HIV-R3A induced death of human leukocytes.
(A) CD4 T cell (CD3+CD8−) counts in peripheral blood (PBL) and spleens (SP) of mock or HIV-R3A infected mice. (B) CD8 T cell (CD3+CD4−CD8+) counts in PBL and SP. (C) Human CD45+ leukocyte counts in PBL and SP. (D) Representative histograms show percentages of dead CD4 T cells, CD8 T cells and huCD45+ cells in spleens of mice infected with mock or R3A at 8 dpi. (E) Summarized data of D. mock, n = 6; isotype+R3A, n = 9; 15B+R3A, n = 12. Bars in all graphs indicate median value. * and ** indicate p<0.05 and p<0.01, respectively.
Figure 6
Figure 6. Depletion of pDC during chronic HIV-1 infection increases HIV-1 replication associated with reduced IFN-I expression.
Humanized mice were infected HIV-JRCSF and were treated weekly with 15B or control at 11 weeks post-infection and terminated at 21 weeks post-infection (mock, n = 6; JR-CSF+control, n = 9; JR-CSF+15B, n = 9). (A) Plasma HIV-1 RNA levels (genome copy#×log10/ml) at each time point were analyzed by real-time PCR. (B) Representative FACS histograms and summarized data (C) show percentages of HIV p24-positive CD4 T cells (CD3+CD8−) in the spleen. (D) The production of IFNα2 in the plasma, from mock infected (n = 4), JR-CSF+PBS (n = 5) and JR-CSF+15B (n = 5) at either 11 wpi (pre-) or 21 wpi (post-), was measured by Luminex. (E) The mRNA levels of IFN-I genes or ISGs Mx1 and TRIM22 (F) in purified human CD45+ cells from spleens (n = 5). All bars in dot graphs indicate median value. Error bars indicate standard deviations (SD). * and ** indicate p<0.05 and p<0.01, respectively. Relative ISGs expression in human CD45+ cells or in CD8 T cells (CD3+CD4−CD8+) in spleens are summarized in Figure S5.
Figure 7
Figure 7. Depletion of pDC during chronic infection reduces HIV-1 induced immunopathogenesis.
Humanized mice were infected HIV-JRCSF and were treated weekly with 15B or control at 11 weeks post-infection and terminated at 21 weeks post-infection (mock, n = 6; JR-CSF+control, n = 9; JR-CSF+15B, n = 9). (A–C) Summarized data show relative numbers in PBL and SP of (A) CD4 T cells (CD3+CD8−), (B) CD8 T cells (CD3+CD4−CD8+), and (C) human CD45+ leukocytes. (D) Immunohistochemistry staining for human CD45+ cells in spleens of mock or HIV-1 infected mice. (E) FACS plots of cell death in human CD4+ T cells, CD8+ T cells and huCD45+ leukocytes from spleens. (F) Summary data show percentages of cell death in CD4+ T cells, CD8+ T cells and huCD45+ leukocytes. Bars in dot graphs indicate median value. * and ** indicate p<0.05 and p<0.01, respectively.
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