Having recently criticized a widely publicized paper on HIV-induced pyroptosis for not providing much information relating to CCR5-tropic HIV infection, I’m duty bound to highlight a new study that includes some data addressing this topic. Pyroptosis is an inflammatory form of cellular suicide that has been shown by Gilad Doitsh from Warner Greene’s laboratory to result from abortive HIV infection of resting “bystander” CD4 T cells in lymphoid tissue, leading to a massive depletion of these cells. The effect is dramatic with CXCR4-tropic HIV isolates, because the majority of resting CD4 T cells express the CXCR4 receptor. In a paper published in Science earlier this year, Doitsh’s research group identified the human IFI16 protein as the sensor that detects HIV genetic material in these cells, leading to pyroptosis. Doitsh has also demonstrated that the phenomenon can be induced by CCR5-tropic HIV isolates in CCR5-expressing CD4 T cells, but because CCR5 is most commonly expressed on activated cells and is very rarely present on resting CD4 T cells, the number of bystander cells that might die as a result of pyroptosis in the setting of CCR5-tropic HIV infection is as yet unclear. The new study, published in Retrovirology by Amanda Steele and colleagues, reports evidence that pyroptosis occurs in gut CD4 T cells after exposure to CCR5-tropic HIV, despite the fact that most of these cells appear to be activated (and therefore potentially susceptible to productive, rather than just abortive, infection).
Unlike the work of Doitsh et al, which primarily used a lymphoid tissue culture system derived from tonsillar material (human lymphocyte aggregate culture or HLAC), Steele created a culture model from gut cells, naming it the Lamina Propria Aggregate Culture (LPAC). Infection of these cultures with a CCR5-tropic HIV isolate led to a statistically significant depletion of CD4 T cells after six days. Further analyses showed that the extent of the depletion correlated with the magnitude of productive HIV infection (as measured by p24 antigen expression), but that the majority of dying CD4 T cells were uninfected and undergoing necrosis associated with expression of caspase 1, an indication of pyroptosis (as opposed to apoptosis, the less inflammatory type of cellular suicide seen with productive infection). Approximately 63% of the dying CD4 T cells were negative for p24 and displayed a necrotic phenotype, while 13% were positive for p24 and apoptotic. Inhibition of caspase 1 did not affect productively infected cells but was able to reduce the extent of CD4 T cell depletion in the cultures from 36% to 18%, providing evidence that pyroptosis was contributing substantially to CD4 T cell death.
To try and model how HIV infection might interact with microbial translocation (the leakage of gut bacteria into the tissues and systemic circulation), the researchers conducted similar experiments with the addition of a commensal gut bacterial species, Escherichia coli. The presence of E. coli significantly enhanced CD4 T cell depletion, but also, surprisingly, seemed to cause a profound shift in the type of cell death, from pyroptosis (associated with expression of caspase 1) to apoptosis (associated with expression of caspase 3). This was established by testing the effects of caspase inhibitors on the extent of CD4 T cell death in the cultures; in the presence of E. coli, a caspase 1 inhibitor no longer had an effect, while a caspase 3 inhibitor significantly reduced CD4 T cell depletion. The authors suggest that perhaps the threshold for apoptosis is reduced in the presence of microbial products, and they propose a biphasic model of gut HIV pathogenesis in which pyroptosis dominates early on, contributing to the breakdown of gut wall integrity, followed by the onset of microbial translocation and increased HIV replication and CD4 T cell apoptosis.
One possible question pertaining to the interpretation of these studies is whether the involvement of caspase 1 is always synonymous with pyroptosis caused by abortive HIV infection. In one study cited by Steele and collagues, which measured caspase 1 expression in CD4 and CD8 T cells from HIV-positive individuals, a significant sixfold upregulation was documented in CD8 T cells, and a non-significant increase of threefold in CD4 T cells. Not having any expertise in caspase 1 biology (to say the least), I’m not clear how these differences might be explained or how they relate to the use of caspase 1 as a marker of pyroptosis (it seems unlikely that the caspase 1 upregulation documented in CD8 T cells was due to abortive HIV infection). Additional research is likely to clarify current uncertainties, as this has clearly become an important and expanding area of investigation.
In another development linked to the work of the Greene laboratory on pyroptosis, a separate paper published in Clinical & Experimental Immunology by Sara Nissen and colleagues reports that the expression of IFI16, the DNA-sensing protein, is significantly elevated in untreated HIV-positive people. Levels of IFI16 expression were found to correlate positively with viral load and the activation of central memory and effector memory CD4 T cells (measured by the CD38 marker). There was also an inverse correlation between IFI16 and CD4 T cell count. The researchers note these findings are consistent with the proposed role of IFI16 in HIV-induced CD4 T cell death, and indicate a relationship between IFI16 and immune activation (although further work is needed to discern if elevated IFI16 expression is a cause or consequence of immune activation—or both).
Retrovirology. 2014 Feb 4;11(1):14. doi: 10.1186/1742-4690-11-14.
Steele AK, Lee EJ, Manuzak JA, Dillon SM, Beckham JD, McCarter MD, Santia ML, Wilson CC.
BACKGROUND: Early HIV-1 infection causes massive CD4+ T cell death in the gut and translocation of bacteria into the circulation. However, the programmed cell death (PCD) pathways used by HIV-1 to kill CD4+ T cells in the gut, and the impact of microbial exposure on T cell loss, remain unclear. Understanding mucosal HIV-1 triggered PCD could be advanced by an ex vivo system involving lamina propria mononuclear cells (LPMCs). We therefore modeled the interactions of gut LPMCs, CCR5-tropic HIV-1 and a commensal gut bacterial species, Escherichia coli. In this Lamina Propria Aggregate Culture (LPAC) model, LPMCs were infected with HIV-1BaL by spinoculation and cultured in the presence or absence of heat killed E.coli. CD4+ T cell numbers derived from flow cytometry and viable cell counts were reported relative to mock infection. Viable cells were identified by viability dye exclusion (AqVi), and intracellular HIV-1 Gag p24 protein was used to identify infected cells. Annexin V and AqVi were used to identify apoptotic versus necrotic cells. Caspase-1 and Caspase-3 activities were blocked using specific inhibitors YVAD and DEVD, respectively.
RESULTS: CD4+ T cell depletion following HIV-1 infection was reproducibly observed by 6 days post infection (dpi). Depletion at 6 dpi strongly correlated with infection frequency at 4 dpi, was significantly blocked by Efavirenz treatment, and was primarily driven by p24-negative cells that were predominantly necrotic. HIV-1 infection significantly induced CD4+ T-cell intrinsic Caspase-1 activity, whereas Caspase-1 inhibition, but not Caspase-3 inhibition, significantly blocked CD4+ T cell depletion. Exposure to E.coli enhanced HIV-1 infection and CD4+ T depletion, and significantly increased the number of apoptotic p24+ cells. Notably, CD4+ T cell depletion in the presence of E.coli was partially blocked by Caspase-3, but not by Caspase-1 inhibition.
CONCLUSIONS: In the LPAC model, HIV-1 induced Caspase-1 mediated pyroptosis in bystander CD4+ T cells, but microbial exposure shifted the PCD mechanism toward apoptosis of productively infected T cells. These results suggest that mucosal CD4+ T cell death pathways may be altered in HIV-infected individuals after gut barrier function is compromised, with potential consequences for mucosal inflammation, viral dissemination and systemic immune activation.
Clin Exp Immunol. 2014 Mar 5. doi: 10.1111/cei.12317. [Epub ahead of print]
Nissen SK1, Højen JF, Andersen KL, Kofod-Olsen E, Berg RK, Paludan SR, Ostergaard L, Jakobsen MR, Tolstrup M, Mogensen TH.
The innate immune system has been recognized to play a role in the pathogenesis of HIV infection, both by stimulating protective activities, and through contribution to chronic immune activation, the development of immunodeficiency, and progression to AIDS. A role for DNA sensors in HIV recognition has recently been suggested, and the aim of the present study was to describe the influence of HIV infection on expression and function of intracellular DNA sensing. Here we demonstrate impaired expression of interferon-stimulated genes in responses to DNA in PBMCs from HIV positive individuals, irrespective of whether patients receive antiretroviral treatment. Furthermore, we show that expression levels of the DNA sensors IFI16 and cGAS were increased in treatment-naïve patients, and for IFI16 expression was correlated with high viral load and low CD4 cell count. Finally, our data demonstrate a correlation between IFI16 and CD38 expression, a marker of immune activation, in CD4+ central and effector memory T cells, which may indicate that IFI16-mediated DNA sensing and signaling contributes to chronic immune activation. Altogether, the present study demonstrates abnormal expression and function of cytosolic DNA sensors in HIV patients, which may have implications for control of opportunistic infections, chronic immune activation, and T cell death.