The mechanism through which HIV-1 kills CD4 T cells, a hallmark of AIDS, has been a topic of vigorous research and one of the most pressing questions for the field over the last 28 years (Thomas, 2009
). In this study, we investigated the mechanism of HIV-1-mediated killing in lymphoid tissues, which carry the highest viral burdens in infected patients. We used HLACs formed with fresh human tonsil cells and an experimental strategy that clearly distinguishes between direct and indirect mechanisms of CD4 T-cell depletion. We now demonstrate that indirect cell killing involving abortive HIV infection of CD4 T-cells accounts for the vast majority of cell death occurring in lymphoid tissues. No more than 5% of the CD4 T cells are productively infected, but virtually all the remaining CD4 T cells are abortively infected ultimately leading to caspase-mediated cell death. Equivalent findings were observed in HLACs formed with fresh human spleen (Figure S6 B–C
), indicating this mechanism of CD4 T-cell depletion can be generalized to other lymphoid tissues.
The massive depletion of non-productively infected CD4 T cells is in contrast to their survival after infection of intact blocks of tonsillar tissue in human lymphoid histoculture (HLH) (Grivel et al., 2003
). This result probably reflects differences between the HLH and the HLAC experimental systems. In HLH, the complex three-dimensional spatial cellular organization of lymphoid tissue is preserved, but cellular movement and interaction are restricted, both of which are required for indirect killing. In HLAC, the tissue is dispersed, and cells are free to interact, resulting in a rapid and robust viral spread. While the mechanism triggering indirect CD4 T-cell death is certainly identical in both settings, HLH allows only a slow, nearly undetectable progression of indirect CD4 T-cell death. In HLAC, this process is accelerated, allowing the outcome to be detected in a few days. Interestingly, indirect killing was also less efficient when peripheral blood cells were tested (data not shown). It is possible that cellular factors specifically produced in lymphoid organs are required to accelerate indirect killing of peripheral blood CD4 T cells.
Several mechanisms have been proposed to explain indirect CD4 T-cell killing during HIV infection. Our finding that CD4 T-cell death is blocked by entry and fusion inhibitors but not by AZT, strongly suggested that such killing involves non-productive infection of CD4 T cells. Therefore, we focused on events that occur after HIV-1 entry. Our investigations demonstrate that abortive viral DNA synthesis occurring in nonpermissive, quiescent CD4 tonsil T cells, plays a key role in the cell death response. Conversely, in the small subset of permissive target cells, reverse transcription is not interrupted, minimizing the accumulation and subsequent detection of such reverse transcription intermediates ().
Consequences of Inhibiting Early Steps of HIV-1 Infection on CD4 T-cell Death
Interrupted or slowed reverse transcription may create persistent exposure to cytoplasmic DNA products that elicit an antiviral innate immune response coordinated by activation of type I IFNs (Stetson and Medzhitov, 2006
). Such activation, termed IFN-stimulatory DNA (ISD) response, may be analogous to the type I IFN response triggered by the RIG-I-like receptor (RLR) family of RNA helicases that mediate a cell-intrinsic antiviral defense (Rehwinkel and Reis e Sousa, 2010
). Our results suggest that abortive HIV-1 infection also stimulates activation of caspase-3, which is linked to apoptosis, and caspase-1, which promotes the processing and secretion of the proinflammatory cytokines like IL–1β. It is certainly possible that pyroptosis elicited in response to caspase-1 activation also contributes to the observed cytopathic response (Schroder and Tschopp, 2010
). The release of inflammatory cytokines during CD4 T-cell death could also contribute to the state of chronic inflammation that characterizes HIV infection. This inflammation may fuel further viral spread by recruiting uninfected lymphocytes to the inflamed zone. While this innate response was likely designed to protect the host, it is subverted in the case of HIV infection and importantly contributes to the immunopathogenic effects characteristic of HIV infection and AIDS.
Such antiviral pathways comprise an unrecognized cell-intrinsic retroviral detection system (Manel et al., 2010
; Stetson et al., 2008
). Viral RNA in infected cells is recognized by members of the RIG-I-like family of receptors that detect specific RNA patterns like uncapped 5′ triphosphate (Rehwinkel and Reis e Sousa, 2010
). Although uncapped RNA intermediates are generated by the HIV-1 RNase H, they contain a 5′ monophosphate and therefore may be not recognized by the RIG-I system (). In contrast to RNA receptors, intracellular sensing of viral DNA remains poorly understood. Consequently, it is unclear how HIV-1 DNA intermediates are detected in the cytoplasm of abortively infected CD4 T cells. AIM2 (absent in melanoma 2) was recently identified as a cytoplasmic dsDNA receptor that induces cell death in macrophages through activation of caspase-1 in imflammasomes (Hornung et al., 2009
). Our preliminary investigations have not supported a role for AIM2 in cell death induced by abortive HIV infection (not shown) suggesting the potential involvement of a different DNA-sensing mechanism. We also have not identified a role for TLR9 and MYD88 signaling in this form of cell death. Additional candidate sensors recognizing cytoplasmic HIV-1 DNA are now under study.
In summary, both productive and nonproductive forms of HIV infection contribute to the pathogenic effects of this lentivirus. The relative importance of these different cell death pathways might well vary with the stage of HIV infection. For example, direct infection and death might predominate during acute infection where CCR5-expressing memory CD4 T cells in gut-associated lymphoid tissue are effectively depleted. Conversely, the CXCR4-dependent indirect killing we describe in tonsil tissue may reflect later stages of HIV-induced disease where a switch to CXCR4 coreceptor usage occurs in approximately 50% of infected subjects. The current study demonstrates how a cytopathic response involving abortive viral infection of resting nonpermissive CD4 T cells can lead not only to CD4 T-cell depletion but also to the release of proinflammatory cytokines. The ensuing recruitment of new target cells to the site of inflammation may fuel a vicious cycle of continuing infection and CD4 T cell death centrally contributing to HIV pathogenesis.