Cell death by pyroptosis drives CD4 T-cell depletion in HIV-1 infection

Abstract

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.

Figure 1

Host permissivity determines the CD4 T-cell death pathway employed following HIV infection. a. Kinetics of spreading viral infection versus depletion of CD4 T cells after infection of HLACs with a replication-competent HIV reporter virus encoding GFP. The relative proportion of CD8 T cells was not altered (not shown). Consistent with our previous report, HIV-infected HLACs contain a small number of productively infected cells, while almost all of the dying cells are abortively infected . b. Abortively infected CD4 T cells exclusively activate caspase-1. Nigericin induces abundant caspase-1 activation in uninfected cells. c. Productively-infected CD4 T cells activate caspase-3, but not caspase-1. (b) and (c) represent cells from the same infected tonsil culture. Efavirenz and AMD3100 were added to the indicated cultures prior to HIV infection. These data are representative of four independent experiments performed with tonsil cells isolated from four different donors.

Figure 2

Lymphoid CD4 T cells are primed to mount an inflammatory response and constitutively express high-levels of pro-IL-1β. a. A secondary inflammatory stimulus by nigericin induces lymphoid CD4 T cells to process and release bioactive IL-1β. Supernatants from cell cultures were filtered to remove all remaining cells and subjected to SDS-PAGE immunoblotting analyses for bioactive 17-kDa IL-1β. b. High levels of constitutive pro-IL-1β are selectively expressed in lymphoid CD4 T cells. Levels of intracellular pro-IL-1β were assessed in HLACs from fresh tonsils or spleen tissue from different donors. Asterisks indicate samples in which dead cells were removed. CD4 T cells were positively isolated from HLAC. Cells were lysed and analyzed for pro-IL-1β expression. c. Nearly all bioactive IL-1β produced by HIV-infected lymphoid CD4 T cells is released from CCR5-expressing cells. Indicated CD4 T-cell populations were isolated from HLAC and infected with HIV-1. Supernatants of cultures were filtered and analyzed for bioactive 17-kDa IL-1β. d. HLACs were characterized for expression of memory and activation markers by flow cytometry. The majority of CCR5-expressing CD4 T lymphocytes exhibit a memory phenotype. All CCR5-expressing CD4 T cells co-express the CXCR4 receptor.

Figure 3

Death of HIV-infected lymphoid CD4 T cells and release of bioactive IL-1β are controlled by caspase-1. a, b. Caspase-1 inhibitors are sufficient to prevent CD4 T-cell death in HIV-infected HLACs. Viable CD4 T cells were counted by flow cytometry, and supernatants were analyzed for levels of cytoplasmic LDH enzyme release. c, Infection with CCR5-dependent HIV-1 induces pyroptosis of lymphoid CD4 T cells. Death of CCR5-expressing CD4 T cells is prevented by caspase-1 inhibitors and TAK779, but not buy the CXCR4 antagonist, AMD3100. Due to the small number of target CCR5-expressing cells, this experiment was performed by overlaying tonsil cells on a monolayer of 293T cells that had been transfected with an R5-tropic proviral HIV-1 clone, as previously described. The co-culture conditions for the R5 virus experiment induced no activation of the overlaid cells. d. Efficient repression of target genes by shRNA-coding lentiviral vectors. e. shRNA LV designed to silence either caspase-1 or ASC, key components of the pyroptotic pathway, protect lymphoid CD4 T cells from death by nigericin or HIV-1 infection. To specifically assess non-productively infected cells, cultures were treated with AZT before infections with HIV-1. f. Caspase-1 cleavage in HIV-infected CD4 T cells is blocked by specific caspase-1 inhibitors. g. Inhibitors of caspase-1, but not NLRP3, prevent release of bioactive IL-1β from HIV-infected lymphoid CD4 T cells. Error bars represent SD/√n of at least three independent experiments utilizing tonsil cells from at least three different donors. Protein analyses represent results from three independent experiments utilizing tonsillar CD4 T cells from three different donors.

Figure 4

Distinct regions of caspase-1 and caspase-3 activity in lymph node of a patient chronically infected with R5-tropic HIV. Inguinal lymph node was collected from a 50-year-old immunosuppressed HIV-1 infected subject during the chronic phase of disease. The patient was first identified with HIV in 1985, has not been on anti-retroviral therapy and displayed CD4 count of 156 cells/μl and viral load of 85,756 copies/ml at the time of lymph node resection. (See also Extended Data Figure 8). GC, germinal center; MZ, mantle zone; PC, paracortical zone; E, epithelium.

Figure 5

Targeting caspase-1 via an orally bioavailable and safe drug prevents lymphoid CD4 T-cell death by HIV-1. (a, b). VX-765 efficiently blocks CD4 T-cell death in HIV-infected tonsillar and splenic lymphoid tissues. No toxicity was observed at any of these drug concentrations. Error bars represent SD/√n of three independent experiments utilizing tonsil or spleen cells from three different donors. c. Pyroptosis in HIV-infected lymphoid tissues may ensue a chronic cycle of CD4 T-cell death and inflammation that ultimately contributes to disease progression and tissue damage. Inhibitors of caspase-1 such as VX-765 may inhibit pyroptosis in a manner that both preserves CD4 T cells and reduces inflammation.