OF APOPTOSIS BY HIV IN THE THYMUS

Role of Cytokines in HIV-Mediated Perturbation of Thymocyte Apoptosis

Influence of IL-7 Levels

An overall increase in plasma of IL-7 levels was demonstrated during HIV infection.

Because HIV replication usually leads to the destruction of T cells, two mechanisms could explain the contradictory effects of IL-7, which increases CD4 cell numbers despite the fact that this cytokine also enhances HIV replication. First, IL-7 exerts an antiapoptotic effect on various thymocyte subsets by increasing Bcl-2 expression.⁹² A culture of CD4⁺ SP thymocytes in the absence of IL-7 results in a decrease in Bcl-2 levels, which is compensated for by the addition of IL-7 in the medium. IL-7R-deficient mice exhibit a severe block in thymocyte maturation, which is rescued by Bcl-2 overexpression.^93-96 Interestingly, HIV p24 protein was detected exclusively in Bcl-2- positive CD4⁺ thymocytes infected in vitro, suggesting that Bcl-2 expression protects infected thymocytes from apoptosis and allows HIV replication.⁷⁸ Therefore, the high levels of IL-7 maintain infected thymocyte viability and increase HIV replication, which is detrimental for thymic function.

Role of TNF-α

Thymocytes can be killed by TNF-α in vitro and in vivo,⁹⁸,⁹⁹ although this cytokine is not absolutely required for negative selection, as shown by a normal clonal deletion in the presence of anti-TNF neutralizing antibody.¹⁰⁰ However, TNFR-deficient mice exhibit thymic hypertrophy, with a 60% increase in total thymocytes but no alteration in the CD4/CD8 subset proportions.¹⁰¹ Therefore, thymic output is affected by TNF-α, which reduces thymic cellularity by triggering the extrinsic pathway. During the course of HIV infection, TNF-α plasma levels are elevated^102-104 and affect thymocyte function in three different ways. First, TNF-α produced in the bone marrow during HIV infection stimulates apoptosis in hematopoietic progenitors,¹⁰⁵ reducing the thymic input of lym­phoid precursors. Second, early T cell precursors such as DN thymocytes are sensitive to TNF-α- mediated apoptosis,¹⁰¹ and increased levels of TNF-α in HIV-infected patients are likely to affect the survival of this subset. Finally, TNF-α-mediated NF-κB activation in CD4⁺ ISP and CD4⁺ SP thymocytes increases HIV replication.⁶⁷,¹⁰⁶ Furthermore, IL-7 produced by TEC may amplify the negative effect of TNF-α by sustaining the expression of the p75 TNF receptor in thymocytes.⁶⁷ Therefore, the increase in systemic TNF-α levels after HIV infection leads to the deletion of several thymocyte subsets and enhances HIV replication in the thymus.

Perturbation of Thymic Function by IFN-α

As part of the natural innate immune response, triggering of the Toll-like receptors (TLR) by viruses such as HIV induces the secretion of type I interferons, including interferon-α (IFN-α) and interferon-β (IFN-β). Increased levels of serum IFN-α are observed during primary HIV infection.¹⁰⁷ Moreover, IFN-α levels increase in patients’ serum in parallel with disease progression and are associated with p24 antigen levels.¹⁰⁷ It was shown that the human thymus contains IFN-α- producing CD11c-negative plasmacytoid dendritic cells.¹⁰⁸ In an FTOC model as well as the SCID- hu Thy/Liv model, IFN-α is secreted in the thymus after HIV infection.¹⁰⁹ This secretion of IFN- α within the thymus may have detrimental consequences for thymopoiesis, as the administration of IFN-α to a mouse causes a severe thymic atrophy, a decrease in thymic cellularity, and an impairment in progenitor T cell development.¹¹⁰ As for its effects on thymic stroma, the addition of recombinant IFN-α, IFN-β, or IFN-γ to human thymic epithelial cell cultures is sufficient to induce differentiation and apoptosis of these cells.¹¹¹

Furthermore, HIV-induced IFN-α upregulates the expression of MHC class I (MHC-I) mole­cules on thymocytes as well as on thymic epithelium in the SCID-hu Thy/Liv model,¹⁰⁹ which may have important consequences on the threshold of negative and positive selection and, hence, on DP thymocyte apoptotic rate. HIV infection causes the generation of CD3⁺CD8^low thymocytes in SCID-hu Thy/Liv mice,¹¹² suggesting that the IFN-α-induced increase in MHC-I expression enhances the avidity of the TCR/MHC interaction and decreases the requirement for CD8 in thymocytes expressing MHC-I restricted TCR. IFN-α may also directly influence apoptosis by activating proapoptotic molecules involved in thymocyte depletion.

Influence of IL-10

Finally, IL-10 plasma levels also increase during the course of HIV infection and progression to AIDS.¹¹⁴ PBMCs from infected patients produce higher amounts of IL-10 following stimula­tion.¹¹⁵,¹¹⁶ This response is thought to contribute to the Th1/Th2 imbalance triggered by HIV infection. IL-10 expression is induced in HIV-1-infected thymus organs and may result in increased MHC-I expression on DP thymocytes by HIV.¹¹⁷ Overexpression of MHC-I molecules on DP thymocytes could interfere with thymocyte maturation and may contribute to HIV-1-induced thy­mocyte depletion.

Role of Glucocorticoids

Adrenal function is elevated following HIV infection. Compared with control asymptomatic patients, individuals suffering from lymphadenopathy have higher levels of adrenocorticotropic hormone (ACTH) and basal cortisol.¹¹⁸ In HIV-infected children, an increased cortisol secretion may be associated with specific central nervous system damage.¹¹⁹ Elevated GC levels in HIV- infected patients could hypothetically cause an increase in thymocyte death during HIV infection. However, GCs can also antagonize TCR-mediated apoptosis in thymocytes and may protect HIV- infected T cells from apoptosis. HIV may mimic this mechanism through the action of the Vpr protein on the GC receptor. After the entry of HIV into T cells, Vpr is able to trigger GC receptor translocation to the nucleus, through its interaction with a GC receptor-binding protein Rip-1,¹²⁰ and is able to act as a cofactor for GC-mediated gene expression.^121,122 Expression of Vpr in T cells antagonizes TCR-induced apoptosis by inhibition of NF-κB activity through a mechanism depen­dent on the function of the GC receptor.¹²³ Vpr-mediated GC receptor activation also increases HIV replication, because the HIV long terminal repeat (LTR) contains a functional GC response ele- ment,¹²⁴ and GCs were also shown to synergize with TNF-α for the induction of HIV expression.¹²⁵ Although these results were obtained in peripheral T cells, the presence of Vpr in infected thy­mocytes may allow a higher level of HIV replication, particularly in CD4⁺ SP cells, by antagonizing negative selection through a GC receptor-dependent mechanism.

Role of the CD95∕Fas Pathway

Peripheral CD4 T cell depletion during HIV infection occurs through several mechanisms, including sensitization of CD4⁺ T cells to Fas-mediated apoptosis through the extrinsic pathway. CD4 cross­linking by the HIV-envelope protein stimulates Fas expression and increases CD4⁺ T cell apoptosis after Fas cross-linking.^126-128 The Tat protein also causes CD4⁺ T cell death by upregulating the expression of Fas ligand in T cells.¹²⁹ Finally, HIV infection induces the production and release of Fas ligand by monocytic cells, which can kill peripheral T cells.¹³⁰ During the course of SIV infection in macaques, the expression of Fas and Fas ligand increases slightly in the thymus,⁷⁶ although there is no evidence that this response plays a role in thymocyte depletion. Moreover, some studies have revealed discrepancies between the sensitivity of human and murine thymocytes to Fas-mediated apoptosis. Murine DP thymocytes are sensitive to apoptosis induced by anti-Fas antibody in vitro³ and in FTOC,^131,132 as well as by Fas ligand in vivo.¹³³ However, human thymocytes exhibit a different expression pattern for Fas, which is found in DN thymocytes that are not susceptible to apoptosis induced by Fas cross-linking using antibodies or Fas ligand.¹³³ Some human thymocytes even express a Fas decoy receptor, lacking a death domain critical for its proapoptotic function and able to antagonize Fas-ligand function.¹³⁴ This lack of sensitivity of human thymocytes to Fas-mediated cell death certainly explains the lack of evidence so far that the Fas pathway plays a role in thymocyte depletion after HIV infection, whereas it is certainly involved in the deletion of peripheral T cells.

Role of HIV Proteins in Thymic Apoptosis Modifications

We described above how the HIV protein Vpr affects thymocyte development by activating the GC receptor and interfering with the apoptotic process.

The presence of HIV-infected cells could also trigger thymocyte cell death in a bystander mechanism. T cell clones expressing the HIV envelope protein gp160 induce normal DP thymocyte apoptosis in co-cultures by a CD4-dependent mechanism.¹³⁹ Moreover, addition of recombinant gp120 to FTOC results in an impairment of T cell maturation⁶⁶ (Figure 20.3). Another HIV protein could also exert a negative influence through stromal cells. Tat-expressing TECs have a decreased ability to drive the generation of DP thymocytes from DN precursors, as shown by gene transfer- mediated Tat expression in thymic stroma.¹⁴⁰ Finally, it was shown that the HIV protease can cleave and activate the proapoptotic caspase-8, which results in the cleavage of Bid, cytochrome c release from the mitochondria, and T cell death.¹⁴¹ Although this mechanism remains to be confirmed in thymocytes, it is possible that it contributes to thymocyte depletion during HIV replication. In summary, many HIV proteins, including Vpr, Nef, gp120, Tat, and the HIV protease, contribute to thymocyte apoptosis during HIV infection.