HIV protease inhibitors do not cause the accumulation of prelamin A in PBMCs from patients receiving first line therapy: the ANRS EP45 "aging" study

Abstract

Background: The ANRS EP45 "Aging" study investigates the cellular mechanisms involved in the accelerated aging of HIV-1 infected and treated patients. The present report focuses on lamin A processing, a pathway known to be altered in systemic genetic progeroid syndromes.

Methods: 35 HIV-1 infected patients being treated with first line antiretroviral therapy (ART, mean duration at inclusion: 2.7±1.3 years) containing boosted protease inhibitors (PI/r) (comprising lopinavir/ritonavir in 65% of patients) were recruited together with 49 seronegative age- and sex-matched control subjects (https://ichgcp.net/clinical-trials-registry/NCT01038999" title="See in ClinicalTrials.gov">NCT01038999). In more than 88% of patients, the viral load was <40 copies/ml and the CD4+ cell count was >500/mm³. Prelamin A processing in peripheral blood mononuclear cells (PBMCs) from patients and controls was analysed by western blotting at inclusion. PBMCs from patients were also investigated at 12 and 24 months after enrolment in the study. PBMCs from healthy controls were also incubated with boosted lopinavir in culture medium containing various concentrations of proteins (4 to 80 g/L).

Results: Lamin A precursor was not observed in cohort patient PBMC regardless of the PI/r used, the dose and the plasma concentration. Prelamin A was detected in PBMC incubated in culture medium containing a low protein concentration (4 g/L) but not in plasma (60-80 g/L) or in medium supplemented with BSA (40 g/L), both of which contain a high protein concentration.

Conclusions: Prelamin A processing abnormalities were not observed in PBMCs from patients under the PI/r first line regimen. Therefore, PI/r do not appear to contribute to lamin A-related aging in PBMCs. In cultured PBMCs from healthy donors, prelamin A processing abnormalities were only observed when the protein concentration in the culture medium was low, thus increasing the amount of PI available to enter cells. ClinicalTrials.gov NCT01038999 https://ichgcp.net/clinical-trials-registry/NCT01038999.

Conflict of interest statement

Competing Interests: The authors have the following interests. PC was a recipient of grants from GlaxoSmithKline and from Boehringer Mannheim. There are no patents, products in development or marketed products to declare. This does not alter the authors' adherence to all the PLOS ONE policies on sharing data and materials, as detailed online in the guide for authors.

Figures

Figure 1. Lack of prelamin A in PBMCs from compliant patients receiving the 2NRTI+1PI/r regimen.

(A) Plasma concentrations of lopinavir, atazanavir and ritonavir from ANRS EP45 “Aging” patients reported at M0 (circles), M12 (square) and M24 (triangle). Colors indicate the durations between last drug uptake and venipuncture (Cmax, red; Cmin, green, Cintermediate, brown) and PI therapeutic concentration range (Cmax, red; Cmin, green). The majority of patients receiving lopinavir treatment were assayed at Cmin and remained within the therapeutic range. Samples from patients being treated with the atazanavir regimen were mainly assayed at mid-dose. (B) Representative western blots of PBMCs from patients receiving lopinavir or atazanavir treatment using three different lamin A/C-specific antibodies and one prelamin A-specific antibody. No prelamin A was detected in PBMCs from patients. Control PBMCs from healthy controls incubated with ZoPra were used as positive control cells. PI plasma concentrations in the patients shown: lopinavir 10.7 µM (M0), 12.3 µM (M12) and 9.3 µM (M24); atazanavir 1.5 µM (M0), 0.7 µM (M12) and 0 µM (M24).

Figure 2. The protein concentration of PI incubation medium influences the effect of PI on prelamin A processing in PBMC.

PBMC were incubated for 24 hours with increasing concentrations of lopinavir (0, 2, 20, 40, 200 µM) diluted in plasma (total protein concentration: 60–80 g/L), in RPMI culture medium supplemented with 10% FBS and 2 mM L-glutamine (total protein concentration: 4 g/L), or in the RPMI culture medium supplemented with BSA (total protein concentration: 40 g/L). (A) Percentage of viable cells in plasma (white), culture medium (black), and BSA supplemented culture medium (gray) (error bars = SD, n ≥3, at least 200 cells counted in each experiment). **p<0.01. No viability changes were observed when PBMC were incubated in plasma containing lopinavir. A decrease in cell viability was apparent when PBMC were incubated in culture medium containing 20 µM or 40 µM PI. Only a slight decrease in viability was observed in cells incubated in BSA supplemented culture medium containing 200 µM PI. (B) Western blotting experiments. Both plasma and BSA supplemented culture medium containing 2 to 40 µM lopinavir had no effect on prelamin A maturation in PBMC. Prelamin A (anti-lamin A/C H110) was detected (*) in culture medium containing 20 µM and 40 µM lopinavir, and in BSA supplemented culture medium containing 200 µM lopinavir. Fibroblasts were cultured in the presence or absence of either 20 µM lopinavir (farnesylated prelamin A positive control) or 60 µM ZoPra (unfarnesylated prelamin A positive control) for 72 hours. GAPDH was used as total cellular protein loading control. (A) Western blot comparing the three lamin A/C antibodies used (N18, sc6215; H110, sc20681; Jol2, MAB3211). All antibodies recognized both lamin A and lamin C. Different amounts of farnesylated prelamin A were detected by N18 and H110, as shown by the ratio of prelamin A reported to the total prelamin A+lamin A signal. In the same conditions, Jol2 did not detect prelamin A. (B) Western blot comparing the two prelamin A antibodies tested (sc6214, ANT0045). The sc6214 antibody exhibited a higher affinity for both farnesylated and unfarnesylated prelamin A than the ANT0045 antibody. (C) Prelamin A, lamin A and lamin C protein domains and antibody epitopes. Lamin A/C N18 (sc6215, green); lamin A/C H110 (sc20681, blue); lamin A/C Jol2 (MAB3211, purple); prelamin A sc6214 (pink); prelamin A ANT0045 (orange).