Expression of Werner and Bloom syndrome genes is differentially regulated by in vitro HIV-1 infection of peripheral blood mononuclear cells

Abstract

In HIV infection, continuous immune activation leads to accelerated ageing of the adaptive immune system, similar to that observed in elderly people. We investigated the expression of WRN and BLM (genes involved in disorders characterized by premature ageing, genomic instability and cancer predisposition) in peripheral blood mononuclear cells (PBMC) activated in vitro with phytohaemagglutinin (PHA) and infected with different HIV-1 strains. The steady state levels of mRNA were analysed by reverse transcription-polymerase chain reaction (RT-PCR), and protein expression was assayed using immunocytochemistry and Western blot techniques. In uninfected PBMC, PHA stimulation induced an increase in BLM mRNA and protein expression, while WRN expression remained virtually unchanged. When PBMC were infected in vitro with a lymphotropic HIV-1 strain, the level of BLM mRNA showed a peak at 24 h of infection, followed by a decline to uninfected culture levels. A similar result failed to be seen using an R5-tropic HIV-1 strain. In accordance with mRNA expression, in HIV-infected cultures PBMC were stained more frequently and more intensely by a BLM-specific antibody as compared to uninfected cultures, staining peaking at 24. Conversely, WRN expression was not modulated by HIV-1. The proportion of cells showing BLM up-regulation, established by immunocytochemical staining, was much greater than the proportion of productively infected PBMC, as established by proviral DNA measurement. This result indicates that BLM up-regulation is probably a result of an indirect bystander cell effect. Activation of the BLM gene in infected PBMC suggests that premature ageing could be a further immunopathogenetic mechanism involved in HIV-induced immunodeficiency, and points to a possible new candidate target for innovative therapeutic intervention.

Fig. 1

Effect of PHA activation on the expression of WNR and BLM genes in normal PBMC. (a) Steady state levels of WRN, BLM and β-actin mRNA in PBMC exposed or not to PHA for 24 and 48 h. Total RNA has been retrotranscribed and amplified with selected primers in semiquantitative RT-PCR. Serial 1 : 10 dilutions of cDNA, starting from a dilution corresponding to 20 000 cells were amplified. The amplicons were revealed by Southern blotting using specific radiolabelled probes. (b) Relative levels of mRNA for WRN and BLM in PBMC after different times of induction with PHA. Quantitative data were obtained as described in the text and expressed as a ratio to β-actin mRNA. Mean values and standard deviations of seven different experiment are shown. Empty squares and circles represent the time-course of PHA-activated PBMC for WRN and BLM, respectively. Filled squares and circles represent the time-course of untreated PBMC for WRN and BLM, respectively. (c) Immunocytochemical staining of PBMC stimulated or not for 48 h with PHA. Polyclonal IgG raised in rabbits anti-BLM and anti-WRN were used as primary antibodies, as described in Materials and methods. The immunoreaction was revealed using DAB as chromogen substrates. Cells were counterstained in Mayer's acid haemalum.

Fig. 2

Effect of HIV-1 infection on the expression of WNR and BLM genes in PHA-activated PBMC. (a,d) p24 antigen production in PHA-stimulated PBMC infected with the X4 lymphotropic HIV-1 primary isolate Is13 and the R5 macrophage-tropic strain BaL, respectively. (b,e) WNR expression, PBMC infected with X4 and R5 strains, respectively. (c,f) BLM expression, PBMC infected with X4 and R5 strains, respectively. Empty symbols: uninfected PBMC. Filled symbols: HIV-1-infected PBMC. Relative levels of mRNA for WRN and BLM before PHA stimulation (−48), at the time of infection with HIV-1 (0) and after infection (4, 24, 48 h), expressed as a ratio to β-actin mRNA. After retrotranscription, serial 1 : 10 dilutions of cDNA, starting from a dilution corresponding to 20 000 cells, were amplified, revealed by Southern blotting analysis and hybridized with specific radiolabelled probes. Quantitative data were obtained as described in the Materials and methods section. Mean values and standard deviations of five different experiments are shown.

Fig. 3

Expression of WNR and BLM proteins in PHA-stimulated PBMC infected with HIV-1. PBMC were stimulated with PHA for 48 h and then infected (or not) with HIV-1 Is13. Immunostaining for WRN and BLM was performed at the time of infection and 24 h postinfection. The figure shows one representative of five independent experiments. (a,d) WRN and BLM proteins expression in PBMC at the time of infection, i.e.at 48 h of PHA stimulation. (b,e) WRN and BLM proteins expression 24 h later without infection. (c,f) WRN and BLM proteins expression in HIV-infected PBMC, 24 h postinfection.