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Chemical inhibition of DPP9 sensitizes the CARD8 inflammasome in HIV-1-infected cells

Nature Chemical Biology volume 19, pages 431–439 (2023)Cite this article

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Abstract

Non-nucleoside reverse transcriptase inhibitors (NNRTIs) induce pyroptosis of HIV-1-infected CD4+ T cells through induction of intracellular HIV-1 protease activity, which activates the CARD8 inflammasome. Because high concentrations of NNRTIs are required for efficient elimination of HIV-1-infected cells, it is important to elucidate ways to sensitize the CARD8 inflammasome to NNRTI-induced activation. We show that this sensitization can be achieved through chemical inhibition of the CARD8 negative regulator DPP9. The DPP9 inhibitor Val-boroPro (VbP) can kill HIV-1-infected cells without the presence of NNRTIs and act synergistically with NNRTIs to promote clearance of HIV-1-infected cells in vitro and in humanized mice. More importantly, VbP is able to enhance clearance of residual HIV-1 in CD4+ T cells isolated from people living with HIV (PLWH). We also show that VbP can partially overcome NNRTI resistance. This offers a promising strategy for enhancing NNRTI efficacy in the elimination of HIV-1 reservoirs in PLWH.

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Fig. 1: NNRTIs induce dose-dependent death of HIV-1-infected cells.
Fig. 2: DPP9 inhibition sensitizes the CARD8 inflammasome to HIV-1.
Fig. 3: VbP enhancement is CARD8- and HIV-1 protease dependent.
Fig. 4: CARD8 inflammasome sensitization overcomes NNRTI resistance.
Fig. 5: VbP enhances clearance of HIV-1-infected cells in mice.
Fig. 6: VbP enhances clearance of latent HIV-1.

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Data availability

Data supporting the findings of this study are available within the paper and its Supplementary Information files. Raw data generated with flow cytometry are available upon request from the corresponding author. Unprocessed western blots from the Supplementary figures are provided at the end of the Supplementary Information file. Source data are provided with this paper.

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Acknowledgements

We thank the volunteers who participated in this study, and L. Kessels, M. Klebert, A. Haile, T. Spitz and T. Minor for study subject recruitment. We thank Regeneron Pharmaceuticals and the Richard Flavell Laboratory at Yale University for generating the human cytokine knock-in mice. The following reagents were obtained through the AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH: rilpivirine, efavirenz, lopinavir, etravirine, nevirapine, maraviroc, T-20, tenofovir, raltegravir, pNL4-3-GFP, international HIV-1 isolates and HIV-1 p24 antibodies. Funding in support of this work was provided by NIH grants nos. R01AI162203 and R01AI155162 (L.S.) and F31AI165251 (K.M.C.). The organizations that supplied these funds had no part in the planning or execution of the work presented in this study.

Author information

Authors and Affiliations

  1. Division of Infectious Diseases, Department of Medicine, Washington University School of Medicine, St Louis, MO, USA

    Kolin M. Clark, Josh G. Kim, Qiankun Wang, Hongbo Gao, Rachel M. Presti & Liang Shan

  2. Andrew M. and Jane M. Bursky Center for Human Immunology and Immunotherapy Programs, Washington University School of Medicine, St Louis, MO, USA

    Liang Shan

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Contributions

L.S., K.M.C. and Q.W. designed the study. L.S. and K.M.C. analyzed the data and wrote the manuscript. J.G.K. edited the manuscript, performed experiments using dox-inducible CARD8 constructs and assisted in mouse and IUPM experiments. Q.W. conducted DPP9 knockdown experiments, provided CARD8- and Caspase 1-KO cell lines and generated dox-inducible CARD8 cell lines. H.G. conducted experiments of live/dead staining of GFP-sorted THP-1 cells. K.M.C. conducted all other experiments. R.M.P. supervised the studies using clinical samples.

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Correspondence to Liang Shan.

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Extended data

Extended Data Fig. 1 Cell killing measurement.

a, Scheme of the killing assays for single-round HIV-1 reporter viruses. Primary CD4+ T cells were infected with HIV-1 reporter viruses for 3 days before NNRTI treatment for 2 days. b, c, Representative flow cytometry plots are shown for one replicate of one donor. Percent killing is calculated as the percent infection of the treatment condition divided by the percent infection of DMSO control. Percent infection was determined by GFP (b) or intracellular p24 (c). d, Percent killing calculated from c. Unpaired two-sided t test. **** p < 0.0001. Error bars show mean values with SEM (n = 3). e, The log fold increase in EC50 calculated from the treatment of three donors of CD4+ T cells with EFV, RPV and ETR with or without the presence of 50% human serum (HS) in the culture media. Data from Fig. 1f–h were used.

Extended Data Fig. 2 DPP9 inhibition enhances NNRTI-mediated killing in THP−1 cells.

a, Two shRNA constructs delivered by lentiviral vectors for knockdown of DPP9 are confirmed by immunoblotting (experiment repeated three times with similar results). b, Killing of HIV-1-infected THP−1 cells by RPV. THP-1 cells were transduced with lentiviruses carrying DPP9-specific or scramble shRNA. Two-way ANOVA with Dunnett’s multiple comparison test (n = 3). c, Killing of HIV infected THP-1 cells treated for two days with DMSO, EFV, 1g244, or combination (n = 3). One-way ANOVA with Tukey’s multiple comparison test. * p < 0.05 and **** p < 0.0001. Error bars show mean values with SEM. d, Log fold changes in EC50 due to VbP. e, Combination treatment denotes a synergistic relationship as evidenced by Loewe’s additivity model calculated with SynergyFinder2.0. Areas in red denote increased synergy. In (d, e), data from Fig. 2b were used for the analysis.

Source data

Extended Data Fig. 3 CARD8 inflammasome activation is dependent on Gag-Pol expression.

a, Representative flow cytometry plots of CARD8-KO or Cas9 control THP-1 cells treated with DMSO, EFV (3 µM), VbP (1 µM), or combination. Heatmap plots are colored according to mean fluorescent intensity (MFI) of GFP with higher MFI colored in red and lower MFI in green. b, Mean fluorescent intensity of three replicates from (a). Two-way ANOVA with Dunnett’s multiple comparison test. c, Frequency of HIV-1 DNA+ cells measured by qPCR. CARD8-KO or Cas9 control THP-1 cells were infected with HIV-1 for 3 days and treated with DMSO, EFV (3 µM), VbP (1 µM), or combination for 2 days. HIV-1 gag levels were normalized to POLR2A to determine the frequency of HIV-1 DNA+ cells. The frequency in the treatment groups was measured as the percent of the DMSO control (n = 3). Two-way ANOVA with Tukey’s multiple comparison test. * p < 0.05, ** p < 0.01 and **** p < 0.0001. Error bars show mean values with SEM.

Extended Data Fig. 4 VbP cytotoxicity is CARD8 and HIV-1 specific.

a, VbP cytotoxicity. THP-1 cells were treated for two days with EFV with or without VbP. Cell viability was determined by the MTS assay denoted by the heatmap. b, VbP toxicity is CARD8 dependent. CARD8-KO and Cas9 control THP-1 cells were treated with VbP for 4 days. Cell viability was determined by the MTS assay. Two-way ANOVA with Dunnett’s multiple comparison test. c, d, VbP killing of THP-1 cells is HIV-1 and CARD8 dependent. CARD8-KO or Cas9 control THP-1 cells were infected with HIV-1 reporter viruses. On day 3 post infection, GFP+ and GFP- cells were purified by sorting before treatment with DMSO, EFV (3 µM), VbP (1 µM), or combination for 2 days. In c, representative flow cytometry plots of live dead staining. In (d), percent live cells normalized to DMSO control for Cas9 control (top) and CARD8-KO (bottom). One-way ANOVA with Tukey’s multiple comparison test. e, f, Time course of live/dead staining of CD4+ T cells treated with DMSO, EFV (3 µM), VbP, or combinations. Primary CD4+ T cells were co-stimulated with CD3 and CD28 antibodies for 3 days before EFV and VbP treatment. Percent live were normalized to DMSO control. In (e), representative FACS plots for the 48 hr time point. Error bars show mean values with SEM (n = 3). * p < 0.05, *** p < 0.001 and **** p < 0.0001.

Extended Data Fig. 5 VbP sensitizes the CARD8 inflammasome to HIV-1.

a, VbP overcomes reduced RPV killing efficacy by human serum. Dose response curves for killing of HIV-1-infected CD4+ T cells treated with RPV or combo (VbP 1 µM) with or without the presence of 50% human serum. Zero values of RPV were plotted at 0.1 nM to allow for log transformation. Error bars show mean values with SEM (n = 3). b–e, Time course treatment of three donors of HIV-1 infected primary CD4+ T cells (b, c) or THP-1 cells (d, e). Cells were treated with DMSO, EFV (0.5 µM), VbP (1 µM), or combo. Fold change enhancement of combination treatment in comparison to EFV alone treatment was shown in (c) and (e).

Extended Data Fig. 6 Characterization of VbP enhancement of CARD8 activation.

a, VbP enhancement and killing is HIV-1 protease dependent. Percent killing of HIV-1-infected THP-1 cells treated for two days with DMSO, EFV, VbP, or combination with or without protease inhibitor LPV (1 µM). One-way ANOVA with Tukey’s multiple comparison test. Error bars show mean values with SEM (n = 3). b, CARD8 cleavage in transfected HEK293T cells. HEK293T cells were co-transfected with a CARD8-expressing plasmid and the HIV-1 plasmid pNL4-3-GFP with the presence of indicated drugs. EFV: 3 µM. VbP: 1 µM. LPV: 1 µM. Cell lysates were collected 24hrs post transfection for western blot analysis. This experiment was repeated an additional two times and provided similar results. c, CARD8 cleavage in HIV-1-infected MT4 cells. MT4 cells were stably transduced with lentiviral vectors expressing WT or FAFA CARD8. Cells were infected for three days prior to treatment with DMSO, EFV (3 µM), VbP (1 µM), or EFV and VbP combination in the presence of a proteasome inhibitor MG132 (5 µM) to block degradation of the neo-C-fragment. Cell lysates were collected six hours post treatment for western blot analysis. This experiment was repeated an additional two times and provided similar results. d, Percent killing of CARD8-KO THP-1 cells replete with Dox-inducible CARD8 constructs for WT, S297A, or FAFA. Conditions shown were treated as in Fig. 3E but were not dox induced. One-way ANOVA with Dunnett’s multiple comparison test. **** p < 0.0001.

Source data

Extended Data Fig. 7 VbP enhancement and killing is Caspase 8 independent.

a, Two sgRNA constructs for knockout of Caspase 8 are confirmed by immunoblotting (experiment repeated three times with similar results). b, Percent killing of Cas9 Control, Casp8-KO, or CARD8-KO THP-1 cells infected with HIV-1 reporter viruses. Infected cells were treated with EFV (3 µM) with or without VbP for two days. Two-way ANOVA with Dunnett’s multiple comparison test. Error bars show mean values with SEM (n = 3). **** p < 0.0001.

Source data

Extended Data Fig. 8 Killing of cells infected with NNRTI RAMs by VbP.

Primary CD4+ T cells were infected with HIV-1 reporter viruses carrying various NNRTI RAMs for 3 days before treatment with 1 µM VbP for two days. Error bars show mean values with SEM (n = 3). One-way ANOVA with Dunnett’s multiple comparison test. ** p < 0.01 and **** p < 0.0001.

Extended Data Fig. 9 Combination treatment effects in vivo.

Primary CD4+ T cells were infected with the HIV-1 reporter virus NL4-3-Pol. Three days post infection, these cells were transfused into mice (5–10 million cells per mouse). EFV and VbP were provided by IV injection immediately after cell infusion. Remaining infected cells were measured by flow cytometry. a, Representative flow cytometry plots measuring remaining infected CD4+ T cells in blood 6hrs post treatment. b, c, blood samples were collected 6hrs and 24 hr post EFV and VbP treatment (n = 4) or control (n = 3). d, Lung tissues were collected 24 hrs post EFV and VbP treatment. Two-way ANOVA with Sidak’s multiple hypothesis test. e, f, Comparison of killing between IV and IP injection of EFV and VbP. Blood samples were collected 6hrs and 24 hr post EFV and VbP treatment. Two-way ANOVA with Sidak’s multiple hypothesis test. (IV sample sizes are the same as b-d, IP samples sizes are as follows: DMSO = 17, EFV = 12, combination = 20. g, CD4+ T cell counts from lung tissues of mice treated with control (n = 5), EFV (n = 4), VbP (n = 5), or combination (n = 5) after 24 hours. Two-way ANOVA with Dunnett’s multiple hypothesis test. h, CD4+ T cell counts from lung tissues from mice with control (n = 5), single-dose (n = 5), or multi-dose (n = 7) combination treatment regimens. Two-way ANOVA with Dunnett’s multiple hypothesis test. Error bars show mean values with SEM. ** p < 0.01, **** p < 0.0001.

Extended Data Fig. 10 DPP9 inhibition enhances clearance of HIV-1 clinical isolates.

a, b, CD4+ T cells were infected with HIV-LAI for four days and treated for one day with DMSO, EFV (1 µM), VbP (1 µM), or combination. In (a), representative flow cytometry plots. Heatmap plots are colored according to mean fluorescent intensity (MFI) of intracellular HIV-p24 (PE) with higher MFI colored in red and lower MFI in green. In (b), Mean fluorescent intensity of three replicates from (a). One-way ANOVA with Dunnett’s multiple hypothesis test. c–h, 1G244 enhances killing of primary CD4+ T cells infected with various HIV-1 clinical isolates. Cells were infected for 4–6 days before treated with DMSO, EFV, 1g244, or combination for one day prior to intracellular HIV-p24 staining. One-way ANOVA with Tukey’s multiple comparisons test. Error bars show mean values with SEM (n = 3). * p < 0.05, ** p < 0.01, ** p < 0.001 and **** p < 0.0001.

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Clark, K.M., Kim, J.G., Wang, Q. et al. Chemical inhibition of DPP9 sensitizes the CARD8 inflammasome in HIV-1-infected cells. Nat Chem Biol 19, 431–439 (2023). https://doi.org/10.1038/s41589-022-01182-5

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