The thalidomide analogue CC-3052 inhibits HIV-1 and tumour necrosis factor-alpha (TNF-alpha) expression in acutely and chronically infected cells in vitro

Abstract

We investigated the in vitro effect of the water-soluble, highly stable thalidomide analogue CC-3052 on HIV-1 expression and TNF-alpha production in latently infected promonocytic U1 cells, acutely infected T cells and monocyte-derived human macrophages (MDM), and in mitogen-stimulated ex vivo cultures from patients with primary acute HIV-1 infection. HIV-1 expression was assessed by Northern blot analysis of RNAs, and ELISA for p24 antigen release and reverse transcriptase (RT) activity. TNF-alpha expression was evaluated by RT-polymerase chain reaction (PCR)-ELISA for mRNA and ELISA for protein secretion. We demonstrated that CC-3052 is able to inhibit HIV-1 expression, as evaluated by mRNA, p24 release and RT activity, in phorbol myristate acetate (PMA)- and cytokine-stimulated U1 cells. Furthermore, CC-3052 inhibited HIV-1 expression, as evaluated by p24 and RT activity, in acutely infected MDM and T cells. As far as TNF-alpha is concerned, CC-3052 significantly reduced TNF-alpha mRNA and protein secretion in PMA-stimulated U937 and U1 cells, and in PMA-stimulated uninfected and acutely infected MDM. Consistently, the addition of CC-3052 reduced TNF-alpha production in phytohaemagglutinin (PHA) and lipopolysaccharide (LPS)-stimulated whole blood cultures from patients during the primary acute phase of HIV-1 infection. Since TNF-alpha is among the most potent enhancers of HIV-1 expression, the effect of CC-3052 on TNF-alpha may account for its inhibitory activity on HIV-1 expression. Given the well documented immunopathological role of TNF-alpha and its correlation with viral load, advanced disease and poor prognosis, CC-3052 could be an interesting drug for the design of therapeutic strategies in association with anti-retroviral agents.

Fig. 1

Effect of 10 μm CC-3052 on HIV-1 mRNA expression in phorbol myristate acetate (PMA)- and TNF-α-stimulated U1 cells. Densitometric analysis (a) and Northern blot autoradiograpy (b) of a typical experiment are shown.

Fig. 2

Effect of 10 μm CC-3052 (▪), compared with control (□) on TNF-α protein production (upper panels) and mRNA expression (lower panels) in unstimulated and phorbol myristate acetate (PMA)-stimulated U1 and U937 cells (a), and human monocyte-derived macrophages (MDM) either uninfected or HIV-1-infected (b). Results of protein secretion are expressed as pg/ml, mean ± s.e.m. of seven independent experiments, and those of mRNA expression as pg × 10⁻³ normalized for glyceraldehyde-3-phosphate dehydrogenase (GAPDH), mean ± s.e.m. of three independent experiments. P values of statistically significant comparisons are shown.

Fig. 3

Effect of CC-3052 on HIV-1 expression in acutely infected human monocytes and T lymphocytes. (a) Kinetic and dose–response in monocytes infected with 1:100 HIV-1_BAL without (○) and with 10 (▴), 25 (•) and 50 (▪) μm CC-3052. (b) Increased inhibitory effect of 25 μm CC-3052 (▪), compared with control (□) on HIV-1 expression in acutely infected T cells (14 days post-infection) as a function of increasing dilutions of HIV-1_IIIB. Values are expressed as the mean ± s.e.m. of four experiments with three replicates per data point. P values of statistically significant comparisons and percentages of inhibition are shown.

Fig. 4

Effect of CC-3052 on TNF-α production in whole blood cultures of acute HIV-1 patients and controls. Whole blood cultures were stimulated with lipopolysaccharide (LPS; 1 μg/ml) and phytohaemagglutinin (PHA; 2 μg/ml) alone (□) or in the presence of 10 μm CC-3052 (▪) for 24 h. Results are expressed as the mean ± s.e.m. of six patients and seven controls. P values of comparisons and percentages of inhibition are shown.