Cell-to-cell transmission of HIV-1 is required to trigger pyroptotic death of lymphoid-tissue-derived CD4 T cells

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Highlights

  • The mode of HIV-1 spread determines the outcome form of cell death
  • Cell-to-cell spread is required to deplete non-permissive CD4 T cells via pyroptosis
  • Free HIV-1 particles, even in large quantities, are unable to trigger pyroptosis
  • The fundamental “killing units” of CD4 T cells are infected cells, not the virus

Summary

The progressive depletion of CD4 T cells underlies clinical progression to AIDS in untreated HIV-infected subjects. Most dying CD4 T cells correspond to resting nonpermissive cells residing in lymphoid tissues. Death is due to an innate immune response against the incomplete cytosolic viral DNA intermediates accumulating in these cells. The viral DNA is detected by the IFI16 sensor, leading to inflammasome assembly, caspase-1 activation, and the induction of pyroptosis, a highly inflammatory form of programmed cell death. We now show that cell-to-cell transmission of HIV is obligatorily required for activation of this death pathway. Cell-free HIV-1 virions, even when added in large quantities, fail to activate pyroptosis. These findings underscore the infected CD4 T cells as the major killing units promoting progression to AIDS and highlight a previously unappreciated role for the virological synapse in HIV pathogenesis.

Introduction

The primary cause of AIDS in subjects is the progressive loss of CD4 T cells due to HIV infection (Thomas, 2009). The depletion of these cells has often been studied using cell-free virions infections of activated blood-derived CD4 T cells because of their ready availability and capacity to support productive viral infection (Cooper et al., 2013). However, the cytopathic response to HIV is not restricted to productively infected cells. Indeed, most dying CD4 T cells in lymphoid tissues are resting cells that cannot support productive infection and instead become abortively infected (Doitsh et al., 2010). We have used an ex vivo human lymphoid aggregate culture (HLAC) system formed with fresh human tonsil tissues to study CD4 T cell death during HIV infection (Glushakova et al., 1995). HLACs can be infected with a small number of viral particles in the absence of exogenous mitogens, allowing analysis of HIV-1 cytopathicity in a natural and preserved lymphoid microenvironment (Eckstein et al., 2001). Infection of HLACs with HIV-1 produces extensive loss of CD4 T cells––less than 5% of the cells die as a result of productive viral infection, while >95% of them die as a consequence of abortive infection (Doitsh et al., 2010). Due to the nonpermissive nature of these quiescent cells, the viral life cycle attenuates during the chain elongation phase of reverse transcription, giving rise to incomplete transcripts of cytosolic viral DNA. These intermediates are sensed by interferon gamma inducible protein 16 (IFI16) (Monroe et al., 2014), which activates caspase-1 in inflammasomes, leading in turn to pyroptosis, a highly inflammatory form of programmed cell death (Doitsh et al., 2014).

Retroviruses disseminate between susceptible cells either by cell-free infection or by direct cell-to-cell spread (Sattentau, 2010). The advantage of cell-to-cell spread on viral infectivity has been recognized for two decades (Jolly and Sattentau, 2004, Lehmann et al., 2011, Phillips, 1994, Sato et al., 1992,Sourisseau et al., 2007). For HIV-1, the infectivity of virus-producing cells, as measured in co-culture systems, is ∼102 to 103 times higher than the infectivity of cell-free particles from the same infected cells (Jolly, 2011). However, in the context of pathogenesis, it was unclear whether transfer of HIV-1 through cell-to-cell contact triggers the same innate immune responses as cell-free particles in resting CD4 T cells, the predominant target cells depleted by HIV in lymphoid tissues.

Results

The mode of HIV-1 transfer markedly affects the death response in target lymphoid CD4 T cells

Most studies examining innate immune recognition of HIV-1 have utilized cell-free particles and characterized responses occurring in dendritic cells or macrophages (Gao et al., 2013, Hayashi et al., 2010, Jakobsen et al., 2013, Lahaye et al., 2013, Manel et al., 2010, Sun et al., 2013, Yan et al., 2010). More recently, attention has focused on resting CD4 T cells in lymphoid tissues, which are mostly non-permissive for productive HIV infection. We previously have shown that the massive death of lymphoid CD4 T cells that are abortively infected with HIV-1 requires close interaction between uninfected target and HIV-producing cells (Doitsh et al., 2010). These findings were consistent with in vitro (Garg et al., 2007, Holm and Gabuzda, 2005) and in vivo (Finkel et al., 1995) studies showing that dying non-productively infected cells in human lymph nodes often cluster near productively infected cells (Finkel et al., 1995). In contrast, we found that cell-free virions accumulating in the supernatants of HIV-infected HLACs, even at high concentrations, were much less efficient at inducing killing of resting target cells by abortive infection. One potential explanation for these differences was that transfer of cell-free particles may not generate sufficient incomplete reverse DNA transcripts to induce a cytopathic response in target CD4 T cells. Cell-to-cell spread increases infection kinetics by two to three orders of magnitude by directing virus assembly and obviating the rate-limiting step of extracellular diffusion required for cell-free virus to find and engage a susceptible target cell (Jolly, 2011, Martin and Sattentau, 2009, Sato et al., 1992, Sourisseau et al., 2007).

To test this hypothesis, we used spinoculation to emulate efficient cell-to-cell spread of virus (Geng et al., 2014). Spinoculation accelerates the binding of cell-free virions to target cells, facilitates synchronized delivery of a large number of particles into the cells (O’Doherty et al., 2000, Saphire et al., 2002), and enhances accumulation of cytoplasmic reverse DNA transcripts (Figure 1A) (O’Doherty et al., 2000, Pace et al., 2012). As expected, spinoculation of HLACs with free HIV-1 promoted high levels of HIV-1 fusion into target lymphoid CD4 T cells (Figure S1). Spinoculation also caused extensive and selective depletion of target CD4 T cells (Figure 1B). The relative proportion of CD8 T cells was unaltered. CD3+/CD8− T cells were similarly depleted, indicating that cell loss was not an artifact of downregulated surface expression of CD4 following direct infection (not shown). Consistent with our previous reports (Doitsh et al., 2010,Doitsh et al., 2014, Monroe et al., 2014), loss of CD4 T cells was prevented by addition of efavirenz, an NNRTI that allosterically inhibits HIV-1 reverse transcriptase, and by AMD3100, an entry inhibitor that blocks gp120 engagement of the CXCR4 coreceptor. However, unexpectedly and not in keeping with our previous reports, addition of raltegravir, an integrase inhibitor also blocked CD4 T cell death (Figures 1B and S5). Because cell death involves viral life cycle events occurring prior to viral integration, raltegravir should act too late to affect the abortive infection process that triggers the pyroptotic response.

To further investigate this surprising result, carboxyfluorescein succinimidyl ester (CFSE)-labeled target CD4 T cells were co-cultured with productively infected HLACs, and raltegravir was added at the time of mixing of productively infected and target CD4 T cells (Figure 1C). Under these conditions, raltegravir had no effect on target CD4 T cell death, while efavirenz and AMD3100 blocked the response (Figure 1D).

Free HIV-1 particles do not induce cell death of target lymphoid CD4 T cells

Based on these contrasting effects of raltegravir, we hypothesized that in the co-culture experiments involving mixing of HIV-infected HLACs with target cells, raltegravir had no effect on the ensuing death of target CD4 T cells that became abortively infected, because the culture already contained productively infected cells. Conversely, in the spinoculation experiments, raltegravir blocked cell death, because it prevented the establishment of a productively infected subset of cells needed for cell-to-cell spread of the virus to target CD4 T cells. To test this hypothesis, we spinoculated HLACs with either single-round or multiple-round viruses containing a GFP reporter (NLENG1I) (Levy et al., 2004). These viruses permit the dynamics of HIV-1 infection and T cell depletion to be simultaneously monitored in the spinoculated cultures. Four days after spinoculation, we observed a similar number of GFP-positive, productively infected cells with both the single-round and multiple-round viruses, indicating that viral spread was not required to establish an initial population of productively infected cells. However, we observed a massive loss of CD4 T cells only in cultures spinoculated with the multiple-round virus. Notably, spinoculation with an integrase-deficient GFP HIV-1 (NLENG1I-D116N) (Gelderblom et al., 2008) resulted in no productive infection and no CD4 T cell death (Figure 2A). These results suggested that viral spread from productively infected cells, but not spinoculation of cell-free virions, is promoting the death of non-permissive lymphoid CD4 T cells. In agreement with this conclusion, addition of the AMD3100 entry inhibitor 4 hr after spinoculation efficiently blocked the ensuing death response while not affecting the number of GFP-positive productively infected cells (Figure 2B). Moreover, treatment with the viral protease inhibitor saquinavir, which acts during the budding stage of HIV-1 replication, did not inhibit productive infection but prevented CD4 T cell death by newly released HIV-1 virions (Figure S2). These findings further indicated that CD4 T cell death occurs after establishment of productive infection, but not during infection with cell-free viruses.

Single-round and integrase-deficient HIV-1 clones are not competent for cell-to-cell dissemination following spinoculation with HLACs. To confirm that the mode of viral transfer influenced the death response of target CD4 T cells, we modified the infection system by overlaying HLACs on a monolayer of 293T cells that had been transfected with these single-round proviral clones (Figure 2C). Interestingly, when these single-round viruses were transferred to HLACs by direct interaction with virus-producing 293T cells, a massive killing of target lymphoid CD4 T cells was observed (Figure 2D). These results demonstrate that recapitulating the cell-to-cell mode of viral transfer is sufficient to restore the killing capacity of these single-round clones.

The death response involves cell adhesion molecules required for virological synapse formation

To further explore whether cell-cell contact was needed to induce death of CD4 T cells, we repeated the co-culture assay using productively infected and target CFSE-labeled HLACs. However, in this experiment, the cells were co-cultured under conditions of increasing surface area, thereby reducing the likelihood of cell-cell interactions. Using flow cytometry, we analyzed the levels of viable target CD4 T cells in the plates every 24 hr during 4 days of co-culture. The death of target CD4 T cells decreased as the surface area of the culture increased (Figure 3A), even in samples where the volume of culture medium remained constant (Figure 3B). These data suggest that the physical distance between HIV-producing and target cells directly affects the kinetics of CD4 T cell depletion and argue further against a role for free virions released into the medium in the death response.

Cell-to-cell spread of HIV-1 predominantly takes place across specialized contact-induced structures known as virological synapses (Agosto et al., 2015,Jolly et al., 2004, Jolly et al., 2007, Jolly and Sattentau, 2004). These synapses facilitate efficient transmission of virus toward the uninfected and engaged target cell. The synapse gains stability through actin-mediated recruitment of adhesion molecules, such as the integrin leukocyte function-association antigen 1 (LFA-1) and its cognate ligand ICAM-1 to the junction point of cellular interaction (Jolly et al., 2007). To examine whether virological synapse formation between HIV-infected and target cells is required to promote CD4 T cell death, productively infected and target CFSE-labeled HLACs were co-cultured in the presence of blocking antibodies against ICAM-1 or CD11a, the α-subunit of the LFA-1 heterodimer. Addition of either the anti-ICAM-1 (Figure 3C) or anti-CD11a (Figure 3D) antibodies, but not isotype-matched control antibodies, effectively blocked depletion of target CD4 T cells in the mixed cultures as efficiently as the antiviral drug efavirenz. These findings suggest that the death response involves adhesion molecules that are required for virological synapse formation, indicative of a requirement for close cell-cell contact in mediating pyroptotic cell death. Because cell-free virions also express LFA-1 and ICAM-1 (Bounou et al., 2002,Fortin et al., 1997), we cannot completely rule out an additional effect on HIV virions. However, when combined with all of the data (Figure S2), we conclude that cell-to-cell transmission is critical for the induction of pyroptosis.

Western blotting analysis of HLAC revealed high expression levels of ICAM-1 in B cells, but not in CD4 or CD8 T lymphocytes. However, activated CD4 T cells, which correspond to those that become productively infected with HIV-1, express high levels of this adhesion molecule (Figure 3E). In contrast to ICAM-1, CD11a expression levels were high in both resting and activated CD4 T cells (Figure 3F). Thus, synapse formation between activated CD4 T cells expressing ICAM-1 and target CD4 T cells (either activated or resting) expressing LFA-1 may occur regularly in lymphoid tissues, independently of viral infection.

Caspase-1 activation in abortively infected cells requires cell-to-cell spread of HIV-1

Most CD4 T cells in lymphoid tissues infected with HIV die by caspase-1-mediated pyroptosis triggered by abortive viral infection (Doitsh et al., 2014). To test whether caspase-1 is induced by cell-free HIV-1 particles or by cell-to-cell spread of HIV-1, we spinoculated HLACs with single-round or multiple-round clones of a DsRedExpress reporter virus (NLRX-IRES) (Gelderblom et al., 2008) and analyzed intracellular caspase-1 activity using cell-permeable fluorogenic caspase-1-specific substrates (CaspaLux1) (Komoriya et al., 2000, Packard and Komoriya, 2008). Consistent with our previous reports (Doitsh et al., 2014,Monroe et al., 2014), spinoculation with multiple-round HIV-1 particles triggered high levels of intracellular caspase-1 activity in target CD4 T cells. In contrast, spinoculation with single-round or integrase-deficient HIV-1 particles produced only background levels of caspase-1 activity (Figure 4A). Inhibition of cell-to-cell spread using the viral protease inhibitor saquinavir, or by treatment with AMD3100 4 hr after spinoculation, also markedly inhibited caspase-1 activation induced by multiple-round HIV-1 particles.

Consistent with pyroptosis as the pathway of programmed cell death, spinoculation with multiple-round HIV particles resulted in the release of the intracellular enzyme lactate dehydrogenase (LDH) (Fink and Cookson, 2005) (Figure 4B). Further, the release of LDH was completely blocked when AMD3100 was added 4 hr after spinoculation or when single-round or integrase-deficient HIV-1 particles were used for initial infection. Together, these findings indicate that infection with cell-free HIV-1 particles does not lead to caspase-1 activation, despite apparent abortive infection of lymphoid CD4 T cells. Rather, capsase-1 activation and the induction of pyroptosis require the generation of productively infected cells and successful cell-to-cell spread of HIV-1 to quiescent bystander lymphoid CD4 T cells.

Discussion

The life cycle of HIV-1 involves the release of particles into the extracellular space, followed by spread to distant susceptible cellular hosts. HIV-1 can also spread directly from productively infected cells to neighboring cells through virological synapses, a process that is 102– to 103-fold more efficient than infection with cell-free virions. Despite the high efficiency of this mode of viral transmission, most HIV-1 pathogenesis research has involved the study of cell-free viruses (Cummins and Badley, 2010, Février et al., 2011) in part because highly permissive cells, such as activated peripheral blood lymphocytes, have been used as cellular targets. These cells are fully permissive to HIV infection and give rise to new virions but then die primarily by caspase-3-mediated apoptosis (Cooper et al., 2013, Gougeon et al., 1996). However, in human lymphoid tissues such as tonsil and spleen, activated and permissive cells represent only 5% of the total CD4 T cell population. Far more commonly, HIV enters resting non-permissive cells that represent >95% of the CD4 T cell population (Doitsh et al., 2010, Eckstein et al., 2001, Moore et al., 2004). These non-permissive cells undergo abortive infection and ultimately die due to an innate immune response launched by the host against cytosolic viral DNA culminating in caspase-1-dependent pyroptosis, a highly inflammatory form of programmed cell death (Doitsh et al., 2014, Monroe et al., 2014).

Here, we explored the death of lymphoid CD4 T cells in HLACs using experimental strategies that unambiguously distinguish between cell-free and cell-to-cell modes of HIV-1 transmission. Using this system, we now demonstrate that the mode of HIV-1 spread determines the outcome form of cell death. Specifically, cell-to-cell spread of HIV-1 is required to deplete non-permissive lymphoid CD4 T cells via caspase-1-dependent pyroptosis. Free HIV-1 particles, even when added in large quantities, are unable to trigger innate immune recognition leading to pyroptosis. Conversely, infection with free HIV-1 particles does cause a small fraction of permissive cells in HLACs to become productively infected. It is these cells that mediate cell-to-cell spread culminating in the pyroptotic death of nonpermissive CD4 T cells. These findings suggest a radical change in the prevailing view of HIV pathogenesis where most of the pathogenic effects of HIV-1 are attributed to killing of CD4 T cells by circulating free virions. We propose that the fundamental “killing units” of CD4 T cells leading to CD4 T cell depletion and ultimately progression to AIDS are productively infected cells residing in lymphoid tissues that mediate cell-to-cell spread of the virus. Productive (“direct”) and abortive (“bystander”) infections are often viewed as independent pathways underlying the progressive depletion of CD4 T cells (Cooper et al., 2013, Doitsh et al., 2010, Doitsh et al., 2014). Our findings now show that productive and abortive infections are not independent cytopathic events but rather are linked in a single pathogenic cascade (Figure 5). Productively infected cells are obligatorily required to transmit the virus across the virological synapse formed with resting CD4 T cells. The productively infected cell ultimately dies by apoptosis, while the bystander resting cell dies by pyroptosis.

The interaction of the cognate adhesion molecules ICAM-1 and LFA-1 at the virological synapse is critically important for efficient HIV-1 spread between permissive effector and target CD4 T cells. Our findings in HLACs demonstrate the role of the virological synapse in viral infection and depletion of non-permissive CD4 T cell targets. Human lymphoid tissues predominantly consist of non-permissive CD4 T cells (Doitsh et al., 2010, Doitsh et al., 2014, Eckstein et al., 2001, Glushakova et al., 1995). Therefore, the interaction and formation of virological synapses between productively infected cells expressing ICAM-1 and non-permissive targets expressing LFA-1 likely occur at high frequency in the T cell zone found in HIV-infected lymphoid tissues and centrally contribute to the immunopathogenic effects of HIV-1. The interaction of LFA-1 on T cells with ICAM-1 also mediates the arrest and migration of T cells on surfaces of postcapillary venules at sites of infection or injury, as well as the ability of these cells to crawl out of the bloodstream between high endothelial venules and into lymph nodes (Girard et al., 2012). Importantly, interleukin-1β (IL-1β) increases the expression of adhesion molecules such as ICAM-1 on endothelial cells (Dinarello, 2009, Dustin et al., 2011, Hubbard and Rothlein, 2000). The release of IL-1β by dying pyroptotic CD4 T cells in HIV-infected lymphoid tissues likely attracts more cells from the blood into the infected lymph nodes to die and produce more inflammation. Thus, the interaction of LFA-1 with ICAM-1 contributes to a pathogenic cycle occurring during HIV infection by both promoting the depletion of CD4 T cells and facilitating a state of chronic inflammation, two key processes that propel clinical progression of disease ultimately culminating in AIDS (Deeks, 2011).

The molecular mechanisms that limit pyroptosis to virus transmission occurring via the cell-to-cell route are unknown. One possibility relates to TREX1, a cellular 3′ DNA exonuclease, and SLX4-associated MUS81-EME1 endonucleases that function as “cytoplasmic cleaners” that degrade single- and double-stranded DNA, respectively (Laguette et al., 2014, Stetson et al., 2008). Indeed, the intrinsic action of the TREX1- and SLX4-associated endonucleases in the cytoplasm may set a threshold level for reverse-transcribed DNA products needed for either productive infection in permissive cells or, alternatively, pyroptosis in abortively infected non-permissive cells (Laguette et al., 2014, Yan et al., 2010). Cell-to-cell spread across the virological synapse may overcome TREX1/SLX4-mediated restriction by rapidly transferring large quantities of viral nucleic acid to the apposing target cell. Ironically, while this mechanism likely evolved for efficient viral spread between permissive cells, it acts against HIV-1 in non-permissive targets, where it triggers abortive infection and pyroptosis that drives inflammation and disease in the host.

Author contributions

N.L.K.G. performed most of the studies and participated in writing the manuscript; G.D. identified the absolute requirement for cell-to-cell transmission in promoting pyroptosis of lymphoid CD4 T cells abortively infected with HIV-1, developed and designed most of the studies, collected the data, and wrote the manuscript; K.M.M. examined the effect of ICAM-1 and LFA-1 antibodies on CD4 T cell death; Z.Y. analyzed expression of cellular ICAM-1 and LFA-1; I.M.-A. explored the effect of integrase inhibitors on CD4 T cell depletion during spinoculation and provided reagents and tissues; D.N.L. developed and provided the reporter HIV-1 clones used in this study; and W.C.G. supervised the studies and participated in the preparation of the final manuscript.

Acknowledgments

The following reagents were obtained through the AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH: AMD3100, efavirenz, and raltegravir. We thank Jason Neidleman for assistance with HIV-1-based virion fusion assays, stimulating discussions, and technical advice. We thank Dr. Marielle Cavrois, Marianne Gesner, and Mekhala Maiti for assistance with flow cytometry. We also thank Gary Howard, Crystal Herron, Celeste Brennecka, and Anna Lisa Lucido for editorial assistance; John C.W. Carroll, Giovanni Maki, and Teresa Roberts for graphic arts; and Robin Givens and Sue Cammack for administrative assistance. This work was supported by NIH/NIAID grants R21 AI102782, 1DP1036502, and U19 AI096113 (W.C.G); the UCSF/Robert John Sabo Trust Award (Doitsh); and A.P. Giannini Foundation Postdoctoral Research Fellowship (Monroe). We also acknowledge support from NIH P30 AI027763 (UCSF-GIVI Center for AIDS Research) to Dr. Doitsh, Dr. Yang, and the Gladstone Flow Cytometry Core.

Supplemental information

Document S1. Supplemental Experimental Procedures and Figures S1–S5
Document S2. Article plus Supplemental Information

This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Received: January 5, 2015; Received in revised form: June 17, 2015; Accepted: August 4, 2015; Published Online: August 27, 2015
© 2015 The Authors. Published by Elsevier Inc.

 

This report was originally published on Cell Reports on 27th August 2015 and re-published under a Creative Commons License.

NAPWHA

Founded in 1989, The National Association of People with HIV Australia (NAPWHA) is Australia’s peak non-government organisation representing community-based groups of people living with HIV (PLHIV).

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