Human and simian immunodeficiency viruses deregulate early hematopoiesis through a Nef/PPARgamma/STAT5 signaling pathway in macaques

Abstract

Infection of primates by HIV-1 and SIV induces multiple hematological abnormalities of central hematopoietic origin. Although these defects greatly contribute to the pathophysiology of HIV-1 infection, the molecular basis for altered BM function remains unknown. Here we show that when cynomolgus macaques were infected with SIV, the multipotent potential of their hematopoietic progenitor cells was lost, and this correlated with downregulation of STAT5A and STAT5B expression. However, forced expression of STAT5B entirely rescued the multipotent potential of the hematopoietic progenitor cells. In addition, an accessory viral protein required for efficient SIV and HIV replication and pathogenicity, "Negative factor" (Nef), was essential for SIV-mediated impairment of the multipotent potential of hematopoietic progenitors ex vivo and in vivo. This newly uncovered property of Nef was both conserved between HIV-1 and SIV strains and entirely dependent upon the presence of PPARgamma in targeted cells. Further, PPARgamma agonists mimicked Nef activity by inhibiting STAT5A and STAT5B expression and hampering the functionality of hematopoietic progenitors both ex vivo and in vivo. These findings have extended the role of Nef in the pathogenicity of HIV-1 and SIV and reveal a pivotal role for the PPARgamma/STAT5 pathway in the regulation of early hematopoiesis. This study may provide a basis for investigating the potential therapeutic benefits of PPARgamma antagonists in both patients with AIDS and individuals with hematopoietic disorders.

Figure 1. SIV inhibits the clonogenic potential of hematopoietic progenitors through downregulation of STAT5.

(A) Evaluation of total progenitor cell counts in semisolid cultures of CD34⁺ BM cells collected from 5 animals before and after infection with SIV. Day 0 is the day of SIV injection. Horizontal lines indicate mean CFC numbers scored from all cell cultures from the 5 animals analyzed at the indicated time. Each symbol represents samples from a single animal. (B) STAT5B mRNA was evaluated by RT-PCR in CD34⁺ BM cells at various times following animal infection with SIV. Levels were normalized to GAPDH mRNA. Results were expressed relative to the average level of STAT5B mRNA in samples collected before SIV injection. Horizontal lines indicate the mean values for STAT5B mRNA in 5 animal samples at the indicated time. STAT5A mRNA was similarly evaluated and compared in noninfected control (NI) and chronically SIV-infected animals (SIV) (inset). (C) CD34⁺ BM cells from 3 noninfected control and 3 SIVmac251-infected macaques at 93 days after injection were lysed. Lysates were subjected to SDS-PAGE and western blotting. STAT5 and actin proteins were detected by coincubation with pan STAT5 and anti-actin antibodies. (D) CD34⁺ BM cells from 4 control or 4 chronically SIV-infected macaques were transduced with or without a lentiviral vector encoding simian STAT5B. They were processed for CFC assays. Experiments were repeated twice. Horizontal lines indicate the mean number of colonies scored in each condition. Each symbol represents samples from a single animal.

Figure 2. Nef mimics SIV actions on hematopoietic progenitors.

(A–C) CFC assays were performed with CD34⁺ cells isolated from 3–4 control animals following incubation for 48 hours with (A) plasma from noninfected animals in the absence (control) or presence of infectious or heat-inactivated SIVmac251 particles (1 × 10² particles/ml), (B) plasma from noninfected animal (control) or plasma from 4 chronically infected macaques without (–) or with (+) Nef immunodepletion, or (C) with various concentrations of recombinant SIVmac251 Nef. (D) CFC assays were performed with CD34⁺ BM cells isolated from SIVmac251-infected or noninfected macaques. Progenitors from uninfected animals were either left untreated or incubated for 48 hours with the viral isolate SIVmac251, molecular clones BK28-41 (BK) or BK28-41ΔNef (BKΔ) (1 × 10² infectious particles/ml), or with rNef (0.15 μM) before being processed for CFC assays. (E) Inhibitory activity of recombinant myristoylated HIV-1 Nef was assayed on CD34⁺ BM cells isolated from 2 healthy macaques. CD34⁺ BM cells were preincubated for 48 hours with the indicated concentration of myristoylated HIV-1 Nef before CFC assays. Horizontal lines and the diagonal line in C indicate mean of CFC numbers scored from all cell cultures from the animals analyzed. Each kind of symbol represents samples from a single animal.

Figure 3. HIV/SIV-related hematopoietic failure is dependent on a central Pro-rich motif of Nef.

(A) CD34⁺ cells from control animals were incubated for 48 hours with rNef (0.15 μM) or the indicated pools of Nef peptides (100 nM). (B) Alternatively, CD34⁺ cells were incubated with either wild-type or mutated (P₇₂, P₇₅, P₇₈) peptide B (100 nM). Cells were then processed for CFC assays.

Figure 4. SIV impairs LTC-IC.

The left panel shows LTC-IC assay of CD34⁺ BM cells from 5 uninfected and 5 SIV-infected macaques. Various numbers of progenitor cells were initially seeded as indicated. The right panel shows LTC-IC assay of CD34⁺ BM cells from 5 control animals that were preincubated for 48 hours with plasma from noninfected animals supplemented with SIVmac251 (SIV), BK28-41ΔNef (SIVΔ) (1 × 10² infectious particles/ml), or rNef (0.15 μM).

Figure 5. Nef action on hematopoietic cells depends on the presence of PPARγ and its downregulation of STAT5.

(A) Clonogenic capacities of CD34⁺ BM cells were assayed following preincubation for 2 days with culture medium alone (control) or supplemented with SIV-rNef (rNef, 0.15 μM) or PPARγ agonists troglitazone (T) or rosiglitazone (R) or PGJ₂ (25 μM) in the presence or absence of PPARγ antagonist GW9662 (antag, 10 μM). Samples from 5 macaques were analyzed, each represented by 1 symbol. The number of colonies scored was expressed as percentage of control values. (B) Clonogenic capacities of CD34⁺ BM cells were assayed on cells transfected with or without PPARγ siRNA or irrelevant siRNA (ir) and incubated for 2 days in culture medium in the absence or presence of rNef before CFC assays. Samples from 5 macaques were analyzed, each represented by 1 symbol. CFC numbers were expressed relative to the CFC scored in the absence of siRNA PPARγ and rNef. (C) Clonogenic capacities of hematopoietic progenitors isolated from mice (n = 8, left) or macaques (n = 2, right), pretreated with or without rosiglitazone (agonist) during 2 weeks. (D) Real-time RT-PCR of STAT5B mRNA from murine BM sca-1⁺ c-kit⁺ CD3^– cells or macaques CD34⁺ BM cells, treated with or without rosiglitazone (agonist) during 2 weeks. STAT5B mRNA were normalized to GAPDH mRNA. (E) K562 cell proliferation, evaluated by [³H]thymidine incorporation, in cells incubated for 7 days in the presence of various concentrations of rNef or troglitazone. (F) Real-time RT-PCR of STAT5B mRNA from K562 cells incubated with or without siRNA directed against PPARγ and troglitazone (25 μM) or rNef (0.15 μM). STAT5B mRNA were normalized to GAPDH mRNA and were expressed relative to untreated cells. Efficiency of the siRNA transfection has assessed with a fluorescent control siRNA (25 nM) was over 98%; inhibition of PPARγ mRNA was 98% ± 2% (n = 5).