Per2 Upregulation in Circulating Hematopoietic Progenitor Cells During Chronic HIV Infection

Abstract

Chronic HIV infection accelerates immune aging and is associated with abnormal hemato-lymphopoiesis, but the relationship between HIV-induced aging and Hematopoietic Progenitor Cells (HPC) function is not well-defined. In the context of aging, it has been demonstrated using a murine model that Per2 (Period circadian clock 2) is a negative regulator of HPC survival and lineage potential. A possible involvement of Per2 modulation on hematopoietic failure during HIV infection has not yet been investigated. The aim of this study was to analyze whether Per2 is differently expressed and regulated on HPC during HIV infection, possibly providing a therapeutic target to restore lymphoid potential in the HPC compartment. To this purpose, Per2 expression in circulating HPC was compared in 69 chronic HIV infected patients under successful ART and in matched 30 uninfected healthy donors (HD). HPC aging was assessed by measuring relative telomere length (RTL), and HPC functionality was evaluated by Colony Forming Cell (CFC) assay from both ex vivo HIV+ patients and in vitro Per2 overexpressing donors. Our results showed a lower RTL in HPC and a decrease of white progenitor colonies from HIV+ patients with lower CD4 respect to those with higher CD4 T cell count (<500 respect to >500 CD4 T cell/mmc). Interestingly, we found that the frequency of Per2-expressing HPC is higher in HIV+ patients than in HD and correlated to RTL of CFC derived cells, highlighting a relationship between low proliferative rate and Per2 expression. Indeed, the in vitro overexpression of Per2 resulted in a significant decrease of white progenitor colonies respect to control cells. Finally, we showed that the deacetylase Sirtuin 1, a negative regulator of Per2, was downregulated in HPC from HIV+ patients, and the peripheral blood treatment with resveratrol (Sirtuin 1 inducer) determined a decrease of Per2 expressing HPC. Altogether, these results suggest that during HIV infection, Per2 is involved in the regulation of HPC expansion and differentiation and its overexpression may impair the immune reconstitution. These data support the rationale to explore the role of this regulatory mechanism during aged-associated hemato-lymphopoiesis impairment in HIV infection.

Keywords: HIV; Sirtuin 1; hematopoietic progenitor cells; period circadian clock 2; senescence; telomere length.

Figure 1

Relative telomere length (RTL) and expansion/differentiation of circulating Hematopoietic Progenitor Cells (HPC) from HD, and HIV-infected patients (<500 and >500 CD4 T cell count). (A) Colony forming cell assay (CFC) showing the numbers of white and red colonies (left and right panel) generated from circulating HPC from HD (n = 8) and from HIV-infected patients (n = 15). (B) Correlation between relative telomere length and numbers of CFC white colonies in HD and HIV-infected subjects. Mann–Whitney test, *p < 0.05.

Figure 2

Evaluation of Per2 expression in circulating HPC. (A) Representative flow cytometry contour plots of CD34+CD45+/low cells from whole blood of HIV+ patients. In the square the percentage of CD34+Per2+ cells is shown. To set Per-2 positive/negative gate, we used a mix of antibodies containing CD34–APC and the secondary antibody Alexa 488 without Per2 primary antibody. (B) Histograms show the median and range percentage of CD34+Per2+ cells of HD and HIV-infected patients. Mann–Whitney test, *p < 0.05; HD, healthy donors (n = 32), HIV + patients with CD4 T cells count <500 (n = 27), HIV + patients with CD4 T cells count >500 (n = 26). (C) Linear regression analysis between RTL of CFC derived cells and % CD34+Per2+ cells (r = 0.6, p = 0.04).

Figure 3

Circulating Per2 expressing-HPC differentiation features. (A) Representative flow cytometry contour plots of Per2 expression in HPC after lentiviral infection: in the square is indicated the percentage of CD34+Per2+ cells obtained after infection of PBMC with control lentiviral vector (GFP, left panel) or with Per2 specific lentiviral vector (right panel). (B) Colony Forming Cell assay (CFC) showing the absolute value of white and red colonies generated from circulating HPC of HD infected with Per2 specific lentiviral vector or control GFP lentiviral vector. Mann-Whitney test, *p < 0.05.

Figure 4

Sirt1 modulation. (A) Histograms show the median and range of Sirt1 MFI in CD34+ cells from HD and HIV-infected patients. Mann–Whitney test, *p < 0.05; HD, healthy donors (n = 7), <500, HIV+ patients with CD4 T cells count <500 (n = 8), >500, HIV+ patients with CD4 T cells count >500 (n = 7). (B) Relative Sirt1 expression after 24 h of whole blood stimulation with resveratrol (50, 25, and 10 μM concentration). The data refer to the mean of 2 pool (each pool consists of 4 samples). (C) Histograms show the median and range of the fold of decrease of CD34+Per2+ cells percentage after 24 h of whole blood stimulation with resveratrol (50, 25, and 10 μM concentration). Wilcoxon matched-pairs signed rank test, *p < 0.05.