Johnson et al. Cell Death and Disease (2020) 11:628 https://doi.org/10.1038/s41419-020-02865-4 Cell Death & Disease ARTICLE Open Access DPP8/9 inhibitors activate the CARD8 inflammasome in resting lymphocytes Darren C. Johnson1, Marian C. Okondo2,ElizabethL.Orth1,SahanaD.Rao1, Hsin-Che Huang1,DanielP.Ball2 and Daniel A. Bachovchin 1,2,3 Abstract Canonical inflammasomes are innate immune signaling platforms that are formed in response to intracellular pathogen-associated signals and trigger caspase-1-dependent pyroptosis. Inflammasome formation and signaling is thought to mainly occur in myeloid cells, and in particular monocytes and macrophages. Here we show that small molecule inhibitors of dipeptidyl peptidases 8 and 9 (DPP8/9), which activate the related CARD8 and NLRP1 inflammasomes, also activate pyroptosis in human and rodent resting lymphocytes. We found that both CD4+ and CD8+ T cells were particularly sensitive to these inhibitors, although the sensitivity of T cells, like macrophages, varied considerably between species. In human T cells, we show that CARD8 mediates DPP8/9 inhibitor-induced pyroptosis. Intriguingly, although activated human T cells express the key proteins known to be required for CARD8-mediated pyroptosis, these cells were completely resistant to DPP8/9 inhibitors. Overall, these data show that resting lymphoid cells can activate at least one inflammasome, revealing additional cell types and states poised to undergo rapid pyroptotic cell death in response to danger-associated signals. 1234567890():,; 1234567890():,; 1234567890():,; 1234567890():,; Introduction NLRP1 and CARD8 are related human PRRs that form – A number of intracellular pathogen- and danger- inflammasomes6 9. NLRP1 and CARD8 have similar associated signals trigger the formation of multiprotein C-terminal ZU5, UPA, and CARD domains, but have complexes called inflammasomes1,2. In the “canonical” different N-terminal regions (Fig. 1a). NLRP1 and CARD8 inflammasome signaling pathway, an intracellular pattern both undergo autoproteolysis at the C-terminal end of recognition receptor (PRR) detects its specific signal, oli- their ZU5 domains, generating N- and C-terminal frag- gomerizes with the adaptor protein ASC, and recruits pro- ments that remain non-covalently associated10. Although caspase-1. Pro-caspase-1 undergoes proximity induced the pathogen-associated signal (or signals) that activates autoproteolysis and activation on the inflammasome3, and NLRP1 and CARD8 has not been identified, small mole- then in turn cleaves and activates the pore-forming protein cule inhibitors of the host serine proteases DPP8 and gasdermin D (GSDMD) and the proinflammatory cyto- DPP9 (DPP8/9), including the nonselective DPP inhibitor kines pro-IL-1β and pro-IL-184,5. The N-terminal frag- Val-boroPro (VbP), were recently discovered to activate – ment of GSDMD forms pores in the cellular membrane, both NLRP1 and CARD86,9,11 13. DPP8/9 inhibitors releasing the activated cytokines and inducing pyroptotic induce the proteasome-mediated destruction of the cell death. NLRP1 and CARD8 N-terminal fragments through an unknown mechanism6,14, releasing their C-terminal frag- ments from autoinhibition. The liberated NLRP1 UPA- Correspondence: Daniel A. Bachovchin ([email protected]) 1Tri-Institutional PhD Program in Chemical Biology, Memorial Sloan Kettering CARD indirectly recruits and activates pro-caspase-1 via Cancer Center, New York, NY, USA ASC, whereas the CARD8 UPA-CARD directly recruits 2 Chemical Biology Program, Memorial Sloan Kettering Cancer Center, New and activates pro-caspase-13. In addition, DPP8/9 binds York, NY, USA Full list of author information is available at the end of the article directly to NLRP1 and CARD8. DPP8/9 inhibitors disrupt Edited by L. Sun © The Author(s) 2020 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a linktotheCreativeCommons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. Official journal of the Cell Death Differentiation Association Johnson et al. Cell Death and Disease (2020) 11:628 Page 2 of 10 a Human NLRP1 c d CD3+ T cells (Donor 013) Autoproteolytic cleavage T cells -+-VbP (20 μM) + T cells T cells + + 8j (20 μM) kDa --+ N PYD NACHT ZU5 UPA CARD C -PARP (FL) LRR 100- (Donor 004) -PARP (CL) FIIND B cells (Donor 003) (Donor 005) (Donor 002) Monocytes Naive CD4 Naive CD8 Memory CD4 Natural killer cells (Donor 001) (Donor 010) 50- -GSDMD (FL) Human CARD8 kDa -+ -+-+-+-+ -+VbP (20 μM) 50- -Pro-CASP1 -GSDMD (p43) 37- Autoproteolytic 37- cleavage -GSDMD (p30) * N ZU5 UPA CARD C 25- 37- -GAPDH 20- -CASP1 (p20) FIIND NLRP1 (FL) b 150- * NLRP1 (CL) 150 DMSO 100- * VbP (20 μM) 125 -CARD8 (FL) * 100 ** 50- *** 75 37- * 50 *** Cell viability (%) *** -CARD8 (C-term) 25 *** 100- -PARP (FL) 0 -PARP (CL) 50- -GSDMD (FL) T cells T cells T cells B cells + + + -GSDMD (p43) (Donor 001) 37- -GSDMD (p30) (Donor 003) (Donor 005) (Donor 002) α (Donor 004) - -Actin Natural killer cells Naive CD4 Naive CD8 37- Memory CD4 Fig. 1 DPP8/9 inhibitors induce pyroptosis in primary lymphocytes. a Domain structures of human NLRP1 and CARD8. Note that the ZU5 and UPA sub-domains together are referred to as a FIIND. b, c The indicated primary cells were treated with VbP (24 h) before cell death was assessed by Cell-TiterGlo (b) and lysates were evaluated by immunoblotting (c). Data are means ± s.e.m. of four biological replicates. d CD3+ T cells were treated with VbP (20 μM, 24 h) or 8j (20 μM, 24 h) before lysates were evaluated by immunoblotting. Immunoblots are representative of three independent experiments. the NLRP1-DPP9, but not the CARD8-DPP9, interac- forty years20. In particular, the discovery that DPP4 is tion9,15, and this direct displacement also contributes to highly expressed on the surface of T cells sparked NLRP1 inflammasome formation. extensive research into the role of DPP4 in lymphocyte Typically, inflammasomes are expressed in, and there- activation21,22. In the 1980s and 1990s, DPP4 inhibitors fore studied in, monocytes and macrophages1. Indeed, we with unknown selectivities were reported to block and others have demonstrated that DPP8/9 inhibitors mitogen-induced lymphocyte proliferation using radio- induce pyroptotic cell death in many monocyte-derived active thymidine incorporation and IL-2 release cancer cell lines and primary bone marrow-derived assays21,23,24. Years later, selective inhibitors showed that macrophages (BMDMs), but not in cells derived from these effects were in fact due to DPP8/9 inhibition25, but many other lineages6,9,11,16,17. However, DPP8/9 the underlying mechanism was not investigated further. inhibitor-induced pyroptosis is not entirely restricted to In a separate set of assays in the late 1990s, VbP itself was monocytes and macrophages. For example, we found that discovered to induce a “novel apoptotic pathway” in + VbP induced cell death in human CD34 cord blood resting, but not activated, peripheral blood mononuclear + (hCD34 CB) cells, which are a mixture of hematopoietic cells (PBMCs)26. The key VbP target was shortly there- stem and progenitor cells, and in primary B-cell acute after determined to be “quiescent cell protease”27, which lymphoblastic leukemia cells6. Moreover, NLRP1 is is now referred to as DPP7. However, selective DPP7 expressed in human skin, and, consistent with this inhibitors developed years later did not trigger this expression, germline mutations in NLRP1 cause skin apoptotic pathway28. The response of primary lympho- inflammatory syndromes7,18,19 and DPP8/9 inhibitors cytes, in particular resting and activated T cells, to induce pyroptosis in keratinocytes9. selective DPP8/9 inhibitors has not yet been investigated. In addition to these recent results, many groups have Here we show that DPP8/9 inhibitors induce pyroptosis + reported intriguing biological effects of dipeptidyl pepti- in resting lymphocytes, including in naïve CD4 T cells, + + dase inhibitors in a number of cell types over the past memory CD4 T cells, naïve CD8 T cells, B cells, and Official journal of the Cell Death Differentiation Association Johnson et al. Cell Death and Disease (2020) 11:628 Page 3 of 10 natural killer (NK) cells. T cells were particularly sensitive T cells were activated for 48 h using Human T-Activator to DPP8/9 inhibitors, although T-cell sensitivity, like CD3/CD28 Dynabeads for T-Cell Expansion and Activation macrophage sensitivity, varied considerably between spe- (Gibco) according to manufacturer’sprotocol. cies. As in acute myeloid leukemia (AML) cancer cell lines, we found that CARD8, and not NLRP1, mediates Mouse and rat T-cell isolation and culture DPP8/9 inhibitor-induced pyroptosis in human T cells. All mouse and rat experiments were performed at the Strikingly, activated human T cells, despite expressing the MSKCC animal facility and were approved by the insti- proteins involved in CARD8-mediated pyroptosis, were tutional animal care and use committee. Mouse and rat completely resistant to DPP8/9 inhibitors. The mechan- T cells were obtained from spleens harvested from female istic basis of this resistance is unclear. Regardless, this and male animals between the ages of 7 and 12 weeks. work reveals that mammalian resting lymphocytes can Briefly, spleens were harvested from mice or rats (male respond to certain danger-associated signals via inflam- and female mixture), crushed with a syringe plunger, and masome activation and pyroptotic cell death.