Background:  HIV-1 evades the immune system in part by inducing the destruction of both infected and uninfected immune cells. Apoptosis plays a crucial role in depletion of CD4⁺ T cells and destruction of lymphoid tissue architecture. The thymus is the primary lymphoid organ where T cells are generated during fetal and neonatal development. HIV-1 infection of the thymus, particularly of infants, has been documented and is frequently associated with rapid progression to AIDS. The mechanisms by which HIV-1 infection impedes thymic function and depletes thymocytes are not well understood.

Methods:  Thymocytes were infected with HIV-1 clone NL4-3 at high multiplicity to produce approximately 60% to 90% infection. RNA was isolated at different points post-infection and used to probe Affymetrix U95 chips. Apoptosis was detected by measuring activation of caspase-3, annexin V binding, and TUNEL assay by flow cytometry. Inhibitors were used to delineate the signals and pathways that induce apoptosis in HIV-1-infected thymocytes.

Results:  HIV-1 infection of thymocytes induced apoptosis and damage to genes, including tumor necrosis factor receptor, TRAIL-R2, FasR, Bax, NOD1, MALT-1, DRAK-1, CED4, GADD45, cyclin-dependent kinase inhibitor (p21, Cip1), Rad50, Rad51, and E2F-3. Anti-apoptotic genes, which were down-modulated, include Bcl-2 and survivin. We confirmed some of the induced genes by real-time quantitative polymerase chain reaction, Western blot, or flow-cytometry. Moreover, HIV-1 infection of thymocytes induced apoptosis when measured by any of 3 parameters: activation of caspase-3, annexin-V binding, and TUNEL assay. HIV-1-induced apoptosis was abrogated by treatment with cyclohexamide, suggesting that an active process is responsible for cell death in HIV-1-infected thymocytes. However, activation of caspase-3 was nonlinear as a function of internal p24, suggesting that more than one mechanism accounted for cell death. X4 HIV-1 induced apoptosis primarily among immature CD4⁺CD8⁺ (DP) thymocytes and could be inhibited by treatment with the caspase inhibitor z-VAD-fmk, the mitochondrial membrane-permeability inhibitor cyclosporine-A, or the MEK1 inhibitor PD98059.

Conclusions:  X4 HIV-1 infection induces apoptosis primarily in DP thymocytes, contributing to thymocyte depletion by killing cells both directly and indirectly. HIV-1 induces apoptosis in thymocytes through both intrinsic and extrinsic pathways and involves caspases.

Keywords: Apoptosis; Thymocyte; X4 HIV-1