Reduced levels of reactive oxygen species correlate with inhibition of apoptosis, rise in thioredoxin expression and increased bovine leukemia virus proviral loads

Abstract

Background: Bovine Leukemia virus (BLV) is a deltaretrovirus that induces lymphoproliferation and leukemia in ruminants. In ex vivo cultures of B lymphocytes isolated from BLV-infected sheep show that spontaneous apoptosis is reduced. Here, we investigated the involvement of reactive oxygen species (ROS) in this process.

Results: We demonstrate that (i) the levels of ROS and a major product of oxidative stress (8-OHdG) are reduced, while the thioredoxin antioxidant protein is highly expressed in BLV-infected B lymphocytes, (ii) induction of ROS by valproate (VPA) is pro-apoptotic, (iii) inversely, the scavenging of ROS with N-acetylcysteine inhibits apoptosis, and finally (iv) the levels of ROS inversely correlate with the proviral loads.

Conclusion: Together, these observations underline the importance of ROS in the mechanisms of inhibition of apoptosis linked to BLV infection.

Figure 1

Spontaneous apoptosis and ROS production in short term cultures. A) Peripheral blood mononuclear cells (PBMCs) from BLV-infected (n = 13) and control (n = 6) sheep were isolated and cultivated for 24 h. B cells were labeled using anti-IgM monoclonal antibody (clone 1H4) and FITC-conjugated rabbit anti-mouse and fixed in ethanol. After staining with PI, hypodiploid cells (sub-G1 population) considered to be apoptotic were quantified by flow cytometry. Data are presented as the means of apoptotic rates ± standard deviation. ** denotes the statistical significance according to the non-paired Student's t test p < 0.01. B) PBMCs from BLV-infected (n = 13) and non-infected sheep (n = 6) were seeded in 24-well plates at a density of 10⁶ cells/ml and incubated for 30 min at 37°C with 10 μM of CM-H₂DCFDA. After 24 h of culture, B cells were stained using anti-IgM monoclonal (clone Pig45) and Alexa Fluor 647-conjugated donkey anti-mouse antibodies. The intracellular ROS levels were determined by flow cytometry and are presented as the mean fluorescence intensities (± standard deviation) of cellular chloromethyldichlorofluorescein (CM-DCF) within B and non-B lymphocyte populations. *** denotes the statistical significance according to the non paired Student's t test p < 0.001. C) Correlation between apoptotic rates and ROS levels measured in ex vivo cultures. Apoptotic rates and ROS levels were determined by flow cytometry as described in panels A and B, respectively. A correlation coefficient R² = 0.4097 was calculated from the linear regression analysis on percentages of apoptotic B cells and means of ROS fluorescence intensities. p < 0.05 denotes the statistical significance according to the non parametric Spearman test.

Figure 2

Generation of ROS in B lymphocyte mitochondria. Confocal microscopy analysis of ROS production in B cells isolated from 3 BLV-infected and 3 control sheep. 24 h after culture, PBMCs were labeled with 10 nM of Red CM-XROS probe and stained with anti-IgM monoclonal antibody (clone Pig45) and Alexa Fluor 488 goat anti-mouse conjugate. A) Confocal microscopy photographs and profiles of one representative experiment showing the fluorescence intensities of ROS (red fluorescence) and IgM (green fluorescence). For comparison, arrows indicate ROS levels (red) in B cells (green) in PBMCs from infected and control sheep. B) Quantification of arbitrary fluorescence intensities in 20 B cells from 3 BLV- infected and 3 control sheep. Data are presented as the means of fluorescence intensities (± standard deviation). *** denotes the statistical significance according to the non paired Student's t test p < 0.001.

Figure 3

Analysis of DNA oxidative damage. After 24 h of culture of PBMCs from BLV-infected (n = 3) and control (n = 3) sheep, cells were stained for IgM and 8-hydroxy-2'-deoxyguanosine (8-OHdG) and analyzed by flow cytometry. Data are presented as the percentages of 8-OHdG positive B cells (± standard deviation) in the total B cell population. *** denotes the statistical significance according to the non paired Student's t test p < 0.001.

Figure 4

Analysis of thioredoxin expression. A) Western blot analysis of thioredoxin (TRX) expression in PBMCs isolated from BLV-infected (n = 5) and control (n = 5) sheep using a goat polyclonal antibody specific for TRX. Actin was analyzed in parallel as a loading control. B) Immunofluorescence staining of thioredoxin was performed with 10⁶ PBMCs/ml isolated from BLV-infected (n = 8) and control (n = 5) sheep. After B cell staining using anti-IgM monoclonal antibody (clone Pig45) and Alexa Fluor 647-conjugated donkey anti-mouse antibodies, cells were fixed in paraformaldehyde (4%) and permeabilised with PBS/TritonX-100 (0.5%). Cells were then incubated with anti-human TRX monoclonal antibody and Alexa Fluor 488 goat anti-mouse conjugate and analyzed by flow cytometry. Results are presented as the means of fluorescence intensities (± standard deviation) of TRX in B and non-B cells. ** denotes the statistical significance according to the non paired Student's t test p < 0.01.

Figure 5

Effect of VPA on apoptosis and ROS production. PBMCs from BLV-infected and control sheep were isolated and cultivated for 24 h in the absence (0) or the presence of 1 mM of VPA. A) Representative Western blot analysis using an antibody specific for the acetylated form of histone H3. Actin was analyzed in parallel as a loading control. B) The extent of B cell apoptosis was measured by determining the level of nuclear DNA fragmentation. B lymphocytes from 6 BLV-infected and control sheep were labeled using an anti-IgM monoclonal antibody (clone 1H4) and a FITC-conjugated rabbit anti-mouse antiserum (Becton Dickinson). After ethanol fixation and propidium iodide staining, hypodiploid B lymphocytes (i.e. in sub-G1) considered to be apoptotic were quantified by flow cytometry. ** denotes the statistical significance of the differences between 0 and 1 mM VPA, accordingly to the paired Student's t test p < 0.01. C) ROS levels were evaluated in parallel after incubation of PBMCs with 10 μM of CM-H₂DCFDA probe prior to VPA treatment. Data represent the mean fluorescence intensities (± standard deviation) of cellular chloromethyldichlorofluorescein (CM-DCF). * and ** denote the statistical significance of the differences between 0 and 1 mM VPA, accordingly to the paired Student's t test (p < 0.05 and p < 0.01, respectively). D and E) PBMCs from BLV-infected and control sheep were cultivated for 24 h in absence or presence of the free radical scavenger N-acetyl-L-cysteine (NAC) added 2 h prior VPA treatment (1 mM). Cells isolated from BLV-infected and control sheep were analyzed by flow cytometry to determine the levels of intracellular ROS (D) and the rates of apoptosis (E). ** and *** denote the statistical significances according to the paired Student's t test (p < 0.01 and p < 0.001, respectively).

Figure 6

Correlation between ROS levels and proviral loads. Proviral loads (expressed as numbers of viral copies in B cells) were measured by real-time PCR in PBMCs isolated from BLV-infected sheep (n = 13). ROS levels were measured in B cells cultivated for 24 h using the CM-H₂DCFDA probe. A correlation coefficient R² = 0.7764 was calculated. p < 0.001 was determined using the non parametric Spearman test.