Two new papers describing a recently discovered mechanism of CD4 T cell death in HIV infection are receiving extensive press coverage. Both are from the laboratory of Warner Greene at the Gladstone Institutes, and follow up on their prior work published in the journal Cell in 2010. The papers have simultaneously appeared in two high profile journals, Nature and Science, prompting the slew of publicity. Unfortunately, the media is largely failing to convey the subtleties and uncertainties associated with the research. The most common narrative being offered is that the cause of CD4 T cell depletion in HIV infection has remained a mystery, which the new studies have now solved. This is not just an oversimplification; it is not true.
The impetus for the studies by the Greene laboratory came from observations using a system that attempts to mimic the conditions found in human lymphoid tissues (the main tissues where interactions between immune system cells take place). Designated the ex vivo human lymphoid aggregate culture (HLAC) system, it is created using either fresh human tonsil or spleen tissues. Studies using HLAC have shown that, when they are infected with HIV isolates that gain entry to CD4 T cells via the CXCR4 (X4) co-receptor, there is a massive depletion of uninfected “bystander” CD4 T cells. It is these data, as confirmed by the Greene lab, that are being referred to in the media stories that discuss bystander CD4 T cell death and cite that “over 95%” of dying CD4 T cells are uninfected. This “over 95%” figure is also cited in the abstract of the Nature paper. In the prior Cell paper and the new work, Greene and colleagues show that this bystander cell death is attributable to abortive infection of the cells by X4-using HIV. The virus enters via the X4 receptor (which is expressed by most CD4 T cells in lymphoid tissues), but cannot replicate because the cells are in a quiescent non-activated state. What happens, however, is that the cells sense the presence of HIV DNA and this initiates a particularly inflammatory form of cellular suicide called pyroptosis. The Science paper identifies a protein named IFI16 as responsible for the sensing of HIV DNA, and the Nature paper identifies an extant experimental anti-epilepsy drug (VX-765) that appears able to inhibit this pathway and prevent pyroptosis.
This is all potentially important science and the call for an exploration of the effects of VX-765 in HIV infection is well justified. But there is a big caveat. Most HIV isolates utilize the CCR5 co-receptor to enter CD4 T cells. HIV capable of using X4 emerges in later stages of infection in some, but not all, HIV-positive individuals, and is very rarely transmitted. With some HIV subtypes, X4-using viruses are hardly ever seen. Importantly, in the studies in the HLAC system that led to this research, R5-using HIV isolates caused very little death of bystander CD4 T cells, because few of the quiescent CD4 T cells express the CCR5 co-receptor. So the claim that “over 95%” of CD4 T cell death in HIV infection involves uninfected bystander cells is highly misleading, because it only applies to X4-using viruses, and most untreated HIV-positive people progress to AIDS without ever showing evidence of the emergence of X4-using variants. This caveat also undermines the claim that CD4 T cell death by pyroptosis is central to HIV pathogenesis. The Nature paper does include data indicating that R5-using HIV isolates can cause pyroptosis by abortively infecting quiescent CD4 T cells expressing CCR5, but notes "the small number of target CCR5-expressing cells" in the lymphoid tissues (the researchers had to artificially bolster their numbers using a cell line in order to perform the experiment). The percentage of CD4 T cell death that occurs in bystander cells when R5-using HIV isolates are employed in the HLAC system is not reported. So the critical question of how much cell death results from this phenomenon in most people with HIV remains unanswered.
The Nature paper only addresses this question indirectly, and rather confusingly, by including images of lymph nodes from two HIV-positive individuals with relatively low CD4 counts (156 and 259, respectively). Neither is currently receiving antiretroviral therapy and both are infected with R5-tropic HIV. The lymph node images show evidence of caspase 1 activity in the areas where quiescent CD4 T cells typically reside, and because this enzyme is a critical part of the cascade of events that causes the sensing of HIV DNA to induce pyroptosis, the authors interpret these data as consistent with their results in the HLAC system. They write: “these data closely correlate with the findings in HIV-infected HLACs where the 95% of the CD4 T cells are non-productively infected CD4 T cells and show activation of intracellular caspase 1” but do not clarify that the HLAC findings they are referring to were obtained with an X4-using virus, and do not explain how they think the effect is being duplicated in individuals reported to be infected with R5-using HIV. The lymph node images are not stained for CCR5 expression to evaluate whether there is any overlap with the staining for caspase 1.
Overall, the failure of the researchers to clearly address or discuss the large difference in the numbers of bystander CD4 T cells that are depleted by X4 vs. R5-using HIV isolates is disconcerting, arguably even disingenuous. This aspect of the work has certainly not featured in any of the news stories. On a more encouraging note, the identification of the drug VX-765 as an inhibitor of HIV-induced pyroptosis means that future studies of this intervention will be able to shed light on the importance of this mechanism of cell death in HIV infection. It remains to be seen if it will turn out to be as important as Warner Greene and colleagues are suggesting.
In terms of the attendant misconceived media narrative that suggests that CD4 depletion in HIV remains a “mystery” (to quote one headline), it’s important to appreciate that there has been enormous progress in understanding HIV pathogenesis since the days of simplistic theories about HIV infecting and killing CD4 T cells in a manner akin to the now ancient Pac-Man video game. Over the past 10-15 years, the evidence has become overwhelming—and widely accepted—that immune activation drives the progression of HIV infection and ultimately leads to AIDS. Persistent activation of the immune system increases the turnover and death rates of not just CD4 T cells but also CD8 T cells, B cells and other immune system components (such as dendritic cells). Many of these cells are uninfected and have been shown to die as a normal consequence of activation (activation-induced cell death). Activation-related scarring damage to lymphoid tissue, called fibrosis, has also been linked to CD4 depletion, as healthy tissue is needed to promote CD4 T cell survival. These are just examples to highlight that mechanisms of CD4 T cell death in HIV infection are far from entirely mysterious. There are certainly many important issues in HIV pathogenesis that remain to be resolved, often due to the still limited understanding of the intricate workings of the human immune system, but for news articles to suggest that there has been little progress on this front does a great disservice to the research effort.
Nature (2013) doi:10.1038/nature12940
Received 21 August 2013 Accepted 05 December 2013 Published online 19 December 2013
Gilad Doitsh, Nicole L. K. Galloway, Xin Geng, Zhiyuan Yang, Kathryn M. Monroe, Orlando Zepeda, Peter W. Hunt, Hiroyu Hatano, Stefanie Sowinski, Isa Muñoz-Arias & Warner C. Greene
The pathway causing CD4 T-cell death in HIV-infected hosts remains poorly understood although apoptosis has been proposed as a key mechanism. We now show that caspase-3-mediated apoptosis accounts for the death of only a small fraction of CD4 T cells corresponding to those that are both activated and productively infected. The remaining over 95% of quiescent lymphoid CD4 T cells die by caspase-1-mediated pyroptosis triggered by abortive viral infection. Pyroptosis corresponds to an intensely inflammatory form of programmed cell death in which cytoplasmic contents and pro-inflammatory cytokines, including IL-1β, are released. This death pathway thus links the two signature events in HIV infection—CD4 T-cell depletion and chronic inflammation—and creates a pathogenic vicious cycle in which dying CD4 T cells release inflammatory signals that attract more cells to die. This cycle can be broken by caspase 1 inhibitors shown to be safe in humans, raising the possibility of a new class of ‘anti-AIDS’ therapeutics targeting the host rather than the virus.
Science DOI: 10.1126/science.1243640
Published Online December 19 2013
Kathryn M. Monroe,* Zhiyuan Yang,* Jeffrey R. Johnson, Xin Geng, Gilad Doitsh, Nevan J. Krogan, Warner C. Greene
The progressive depletion of quiescent “bystander” CD4 T cells, which are non-permissive to HIV infection, is a principal driver of the acquired immunodeficiency syndrome (AIDS). These cells undergo abortive infection characterized by the cytosolic accumulation of incomplete HIV reverse transcripts. These viral DNAs are sensed by an unidentified host sensor that triggers an innate immune response, leading to caspase-1 activation and pyroptosis. Using unbiased proteomic and targeted biochemical approaches, as well as two independent methods of lentiviral short hairpin RNA–mediated gene knockdown in primary CD4 T cells, we identify interferon-gamma–inducible protein 16 (IFI16) as a host DNA sensor required for CD4 T cell death due to abortive HIV infection. These findings provide insights into a key host pathway that plays a central role in CD4 T cell depletion during disease progression to AIDS.