CARD8 inflammasome activation triggers pyroptosis in human T cells
2020; Springer Nature; Volume: 39; Issue: 19 Linguagem: Inglês
10.15252/embj.2020105071
ISSN1460-2075
AutoresAndreas Linder, Stefan Bauernfried, Yiming Cheng, Manuel Albanese, Christophe Jung, Oliver T. Keppler, Veit Hornung,
Tópico(s)Streptococcal Infections and Treatments
ResumoArticle25 August 2020Open Access Source DataTransparent process CARD8 inflammasome activation triggers pyroptosis in human T cells Andreas Linder Andreas Linder Gene Center and Department of Biochemistry, Ludwig-Maximilians-Universität München, Munich, Germany Department of Medicine II, University Hospital, Ludwig-Maximilians-Universität München, Munich, Germany Search for more papers by this author Stefan Bauernfried Stefan Bauernfried Gene Center and Department of Biochemistry, Ludwig-Maximilians-Universität München, Munich, Germany Search for more papers by this author Yiming Cheng Yiming Cheng Gene Center and Department of Biochemistry, Ludwig-Maximilians-Universität München, Munich, Germany Search for more papers by this author Manuel Albanese Manuel Albanese Gene Center and Department of Biochemistry, Ludwig-Maximilians-Universität München, Munich, Germany Max von Pettenkofer Institute, Virology, Ludwig-Maximilians-Universität München, Munich, Germany Search for more papers by this author Christophe Jung Christophe Jung Gene Center and Department of Biochemistry, Ludwig-Maximilians-Universität München, Munich, Germany Search for more papers by this author Oliver T Keppler Oliver T Keppler Gene Center and Department of Biochemistry, Ludwig-Maximilians-Universität München, Munich, Germany Max von Pettenkofer Institute, Virology, Ludwig-Maximilians-Universität München, Munich, Germany Search for more papers by this author Veit Hornung Corresponding Author Veit Hornung [email protected] orcid.org/0000-0002-4150-194X Gene Center and Department of Biochemistry, Ludwig-Maximilians-Universität München, Munich, Germany Search for more papers by this author Andreas Linder Andreas Linder Gene Center and Department of Biochemistry, Ludwig-Maximilians-Universität München, Munich, Germany Department of Medicine II, University Hospital, Ludwig-Maximilians-Universität München, Munich, Germany Search for more papers by this author Stefan Bauernfried Stefan Bauernfried Gene Center and Department of Biochemistry, Ludwig-Maximilians-Universität München, Munich, Germany Search for more papers by this author Yiming Cheng Yiming Cheng Gene Center and Department of Biochemistry, Ludwig-Maximilians-Universität München, Munich, Germany Search for more papers by this author Manuel Albanese Manuel Albanese Gene Center and Department of Biochemistry, Ludwig-Maximilians-Universität München, Munich, Germany Max von Pettenkofer Institute, Virology, Ludwig-Maximilians-Universität München, Munich, Germany Search for more papers by this author Christophe Jung Christophe Jung Gene Center and Department of Biochemistry, Ludwig-Maximilians-Universität München, Munich, Germany Search for more papers by this author Oliver T Keppler Oliver T Keppler Gene Center and Department of Biochemistry, Ludwig-Maximilians-Universität München, Munich, Germany Max von Pettenkofer Institute, Virology, Ludwig-Maximilians-Universität München, Munich, Germany Search for more papers by this author Veit Hornung Corresponding Author Veit Hornung [email protected] orcid.org/0000-0002-4150-194X Gene Center and Department of Biochemistry, Ludwig-Maximilians-Universität München, Munich, Germany Search for more papers by this author Author Information Andreas Linder1,2, Stefan Bauernfried1, Yiming Cheng1, Manuel Albanese1,3, Christophe Jung1, Oliver T Keppler1,3 and Veit Hornung *,1 1Gene Center and Department of Biochemistry, Ludwig-Maximilians-Universität München, Munich, Germany 2Department of Medicine II, University Hospital, Ludwig-Maximilians-Universität München, Munich, Germany 3Max von Pettenkofer Institute, Virology, Ludwig-Maximilians-Universität München, Munich, Germany *Corresponding author. Tel: +49 (0) 89 2180 71110; E-mail: [email protected] The EMBO Journal (2020)39:e105071https://doi.org/10.15252/embj.2020105071 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Inflammasomes execute a unique type of cell death known as pyroptosis. Mostly characterized in myeloid cells, caspase-1 activation downstream of an inflammasome sensor results in the cleavage and activation of gasdermin D (GSDMD), which then forms a lytic pore in the plasma membrane. Recently, CARD8 was identified as a novel inflammasome sensor that triggers pyroptosis in myeloid leukemia cells upon inhibition of dipeptidyl-peptidases (DPP). Here, we show that blocking DPPs using Val-boroPro triggers a lytic form of cell death in primary human CD4 and CD8 T cells, while other prototypical inflammasome stimuli were not active. This cell death displays morphological and biochemical hallmarks of pyroptosis. By genetically dissecting candidate components in primary T cells, we identify this response to be dependent on the CARD8-caspase-1-GSDMD axis. Moreover, DPP9 constitutes the relevant DPP restraining CARD8 activation. Interestingly, this CARD8-induced pyroptosis pathway can only be engaged in resting, but not in activated T cells. Altogether, these results broaden the relevance of inflammasome signaling and associated pyroptotic cell death to T cells, central players of the adaptive immune system. Synopsis Inhibition of the dipeptidyl-peptidase DPP9 by the compound Val-boroPro induces activation of the CARD8 inflammasome in resting human T cells. Upon CARD8 activation, caspase-1 cleaves its pore-forming substrate GSDMD which executes pyroptosis. Dipeptidyl-peptidase inhibition triggers pyroptosis in primary human T cells CRISPR-Cas9 editing reveals a functional CARD8 inflammasome in human T cells A genetic drop-out approach shows that DPP9 restrains CARD8 activation T cell receptor stimulation subverts CARD8-triggered pyroptosis Introduction Inflammasomes function as cytosolic sensors of microbial infection as well as perturbation of cellular integrity. A variety of inflammasome sensor pathways have been identified that converge on the activation of caspase-1, which plays an important effector function in antimicrobial defense and sterile inflammatory responses (Broz & Dixit, 2016). Currently, known inflammasomes include the NLRP1, NLRP3, NAIP/NLRC4, NLRP6, NLRP12, AIM2, and the PYRIN inflammasome. NOD-like receptors (NLR)-based inflammasome sensors typically display a three-domain structure: an N-terminal homotypic interaction domain that is part of the death-fold domain family (e.g. CARD or a pyrin domain), a central nucleotide binding and oligomerization domain, and a series of C-terminal leucine-rich repeats (LRRs). NLRP1 varies from this canonical architecture, as it harbors an additional C-terminal extension (see below). AIM2 and PYRIN are not part of the NLR protein family, yet also rely on an N-terminal pyrin domain for signal transduction. The activation of these inflammasome sensors results in the formation of a platform that allows the homotypic interaction domains to recruit the universal adapter protein ASC or caspase-1 directly. Direct or ASC-dependent caspase-1 recruitment then facilitates its autoproteolytic activation. Beyond serving as a simple signaling adapter, ASC recruitment typically results in the assembly of large filamentous structures that serve to strongly amplify caspase-1 activation (Broz & Dixit, 2016). The NAIP/NLRC4 inflammasome directly senses components of the bacterial type III secretion system (T3SS) or flagellin, with NAIP serving as the direct receptor in this setting. While different NAIP proteins with varying ligand specificities exist in mice, the human system appears to encode for only one NAIP protein that is especially sensitive to T3SS needle proteins (Yang et al, 2013). Ligand binding by NAIP initiates the formation of a large oligomeric complex, in which the ligand-bound NAIP protein recruits NLRC4, causing a conformational change in NLRC4 which facilitates recruitment of other NLRC4 molecules. This results in the formation of a wheel-like structure, in which the N-terminal CARD domains of NLRC4 are solvent exposed, producing a caspase-1 recruitment platform. Dissimilar to the NAIP/NLRC4 inflammasome, NLRP3 depends on the adaptor protein ASC to activate caspase-1, although ASC can enhance the NAIP/NLRC4 signal (Broz et al, 2010). NLRP3 also differs in that it is activated by a broad array of different stimuli, many of which converge on the induction of potassium (K+) efflux along its physiological transmembrane gradient. This phenomenon is typically associated with the loss of membrane integrity in the context of cellular damage or certain forms of programmed cell death (Gaidt & Hornung, 2018). Given the low specificity of this process, NLRP3 is often engaged in the context of acute or chronic sterile inflammatory conditions. Analogous to NAIP/NLRC4, it is believed that NLRP3 assembles a wheel-shaped oligomer that serves as a seed to initiate inflammasome formation, yet structural insight is currently missing. The domain architecture of NLRP1 is homologous to other inflammasome-forming NLR proteins, while its C-terminus is extended with two additional domains: a FIIND (function to find domain) and a CARD. Its FIIND constitutes an autoproteolytic domain that leads to NLRP1 processing into an N- and C-terminal part that remain non-covalently associated with one another (Mitchell et al, 2019). Of note, this autoprocessing event is critical to ascertain NLPR1's functionality. Most mechanistic studies on NLRP1 activation have been carried out in the murine system. Here, B. anthracis infection has been identified to trigger inflammasome activation in an Nlrp1b—one of several Nlrp1 paralogs present in the murine system—dependent manner. Mechanistically, the B. anthracis-encoded lethal factor (LF) cleaves murine Nlrp1b close to its N-terminal region. This cleavage event exposes a neo-N-terminus of the large Nlrp1b fragment, which then constitutes an N-degron signal that is detected by E3 ubiquitin ligases of the N-end rule pathway, marking it for proteasomal degradation (Chui et al, 2019; Sandstrom et al, 2019). Removal of this fragment, however, allows the release of the non-covalently associated C-terminal portion of Nlrp1b, distal to the autoprocessed FIIND. This thereby-released fragment contains the signal-transducing CARD that then recruits and triggers caspase-1 activation. In addition to the cleavage-induced exposure of an N-degron and the associated detection by endogenous E3 ligases of the N-end rule pathway, direct modification of Nlrp1b by pathogen-encoded E3-ligases has also been identified to activate Nlrp1b (Sandstrom et al, 2019). In light of the pivotal role of proteasomal degradation in this response pathway, this mechanism of Nlrp1b activation has also been referred to as “functional degradation” (Sandstrom et al, 2019). Of note, the N-terminal regions of murine Nlrp1b and human NLRP1 are poorly conserved, and human NLRP1 lacks the cleavage site for anthrax toxin lethal factor. As such, it remains to be determined what microbial pathogen triggers the activation of the human NLRP1 inflammasome. Another mode of murine and human NLRP1 activation has been identified by characterizing the cell death-inducing activity of the non-selective DPP-inhibitor Val-boroPro (VbP, Talabostat) and related compounds. VbP is an inhibitor of post-proline-cleaving serine proteases that include FAP, DPP4, DPP7, DPP8, and DPP9. In various human and murine cell lines, VbP was found to trigger caspase-1-mediated pyroptosis (Okondo et al, 2017; Taabazuing et al, 2017). This was later identified to depend on Nlrp1b in murine macrophages (Okondo et al, 2018), as well as on NLRP1 in human keratinocytes (Zhong et al, 2018). Moreover, complementing NLRP1-deficient cell lines with NLRP1 or Nlrp1b expression constructs rendered these cells competent to VbP-induced pyroptosis. Interestingly, in the course of these studies it was also found that VbP treatment could engage CARD8 to trigger caspase-1 activation (Johnson et al, 2018; Zhong et al, 2018). CARD8 is structurally related to NLRP1, in that it is homologous to the C-terminal portion of NLRP1, consisting of a small N-terminal region, a FIIND, and a CARD domain. While present in primates, CARD8 is not found in the murine system. Indeed, in human myeloid cells, differently from human keratinocytes, CARD8 but not NLRP1 is responsible for pyroptosis induction upon VbP treatment (Johnson et al, 2018). The mechanism by which VbP triggers NLRP1 or CARD8 activation is not fully understood. Inferring from the activity of more specific inhibitors and genetic perturbation studies, it could be concluded that DPP8 and DPP9 inhibition, but not other DPPs, function upstream of pyroptosis (Okondo et al, 2017). While both enzymes have been shown to restrain NLRP1/CARD8 inflammasome activity, it appears that DPP9 seems to play a more dominant role in this activity (Okondo et al, 2017). Apart from functional data, binding studies have shown that DPP8 and DPP9 bind to NLRP1 and CARD8 (Zhong et al, 2018; Griswold et al, 2019) and based on these experiments it had been suggested that direct interaction of DPP8 or DPP9 with these sensors restrains their activity, while inhibition of their activity relieves this inhibition. However, while DPP9 inhibition disrupts the binding of DPP9 to NLRP1, the interaction of CARD8 and DPP9 is not affected by DPP9 inhibition. Instead, it appears that the inhibition of its catalytic activity, rather than its binding is of relevance for inflammasome activation (Griswold et al, 2019). As such, the exact mechanism of how the activity of DPP8 and DPP9 functions upstream of CARD8 or NLRP1 remains enigmatic. Nevertheless, it is interesting to note that VbP-induced CARD8 or NLRP1 activation is also completely dependent on a step that involves proteasomal degradation. Upon autoproteolytic activation, caspase-1 cleaves a select number of substrates that include the highly pro-inflammatory cytokine IL-1β as well as the pore-forming molecule GSDMD. Caspase-1-dependent GSDMD cleavage at Asp275 relieves the auto-inhibitory control of the C-terminal portion of GSDMD on its N-terminus. This allows the recruitment of the N-terminal fragment to the inner leaflet of the plasma membrane, which results in its cooperative assembly and pore formation. The GSDMD pore allows macromolecules such as IL-1β to pass through the membrane, yet it also dissipates the electrochemical ion gradients across the membrane allowing for uncontrolled water influx. When subject to additional mechanical forces, such destabilized cells can rupture and release further cytosolic content (Davis et al, 2019). This lytic form of cell death plays an important role in the passive release of IL-1β and potentially other pro-inflammatory mediators (Broz et al, 2020). Previous studies have shown that inflammasome components can play important roles in T-cell biology. From a mechanistic standpoint, two independent roles can be delineated: First, in line with their well-described role in myeloid or epithelial cells, inflammasome functionalities associated with caspase activation have been described for T cells. As such, it has been shown that resting tonsillar T cells undergo pyroptosis upon non-productive HIV infection (Doitsh et al, 2014). This phenomenon was then later attributed to IFI16 sensing HIV-derived nucleic acids, triggering the activation of an inflammasome complex (Monroe et al, 2014). Another study has found that T cell-intrinsic engagement of the NLRP3 inflammasome critically contributed to IFNγ production and Th1 differentiation (Arbore et al, 2016). Moreover, it has been shown that an NLPR3-ASC-CASP8 signaling axis exerts the maturation and release of IL-1β in Th17 cells, thus contributing to disease pathology in a model of experimental autoimmune encephalomyelitis (Martin et al, 2016). While these studies provided unconventional models for the engagement of inflammasome components (e.g. IFI16) or associated downstream biological effects (e.g. caspase-8), they are in line with the concept of caspase-1/8 exerting proteolytic activity downstream of inflammasome activation. An alternative second line of research proposes that inflammasome components or caspase-1 itself exert functions in T cells beyond their conventional roles. To this end, it has been shown that NLRP3 functions as a transcriptional regulator of Th2 differentiation as evidenced in a loss-of-function (Bruchard et al, 2015) or gain-of-function setting (Braga et al, 2019). Adding to this, a recent study suggests that caspase-1 plays a critical role in Th17 differentiation, independent of its catalytic activity (Gao et al, 2020). While it is difficult to reconcile these different studies into a plausible working model, it has to be considered that differences in cell type, ligands, species, and experimental readouts may account for these results. Nevertheless, it is reasonable to assume that a number of the here-proposed mechanisms require validation by a genetic loss-of-function approach, especially when extrapolating these results to the human system. To this end, we set out to explore whether inflammasome components are indeed functional in human T cells. To address this, we employed a number of tool-compounds in combination with CRISPR/Cas9-based genetic perturbations. Doing so, we identified a critical role for the CARD8 inflammasome in governing pyroptosis in human primary T cells. Results Dipeptidyl-peptidase inhibition triggers a lytic cell death in primary human T cells To elucidate whether human primary T cells are inflammasome-competent, we isolated naïve and memory CD4 and CD8 T-cell subsets from peripheral blood of healthy donors. At the same time, we also generated monocyte-derived macrophages (MDMs) from these donors. We then subjected these cells to different stimuli that are known to engage distinct inflammasome pathways in human myeloid cells: The K+ ionophore Nigericin was employed to stimulate the NLRP3 inflammasome. The anthrax toxin lethal factor fused with the Burkholderia T3SS needle protein (YscF) was complexed with protective antigen (PA) to activate the NAIP/NLRC4 inflammasome (NeedleTox). Moreover, the DPP-inhibitor Val-boroPro (VbP) was used to trigger NLRP1 or CARD8 inflammasome activation. As an additional control, we included the combination of the BCL2-inhibitor ABT737 and the MCL1-inhibitor S63845 to engage intrinsic apoptosis. With the availability of a specific NLRP3 inhibitor (MCC950), we also included MCC950 to infer NLRP3 inflammasome activation for the Nigericin-treated conditions. In light of the fact that human monocytes require priming for IL-1β production and sufficient NLRP3 engagement, we also included a short course of LPS priming for the MDM stimulation experiments. As a measure for inflammasome activation, we assessed pyroptosis using LDH as a proxy, as well as IL-1β and IL18 release into the supernatant. Nigericin and NeedleTox treatment led to the expected outcomes in human MDMs: Nigericin triggered pyroptosis, IL-1β and IL-18 release in LPS-primed, but not in unprimed cells, while this response was fully blocked by MCC950 (Fig 1A–C). NeedleTox resulted in pyroptosis and IL-18 release in both unprimed and primed cells and again IL-1β release was only seen upon LPS priming. VbP treatment, on the other hand, led only to a small increase in LDH and IL-18 release in human monocytes and also the IL-1β response in LPS-primed cells was substantially lower compared to Nigericin or NeedleTox-treated cells. Primary T cells treated with NeedleTox showed no signs of cell death, while Nigericin treatment resulted in LDH release in both naïve CD4 and CD8 T cells. However, unlike for MDMs, this Nigericin-dependent cell death could not be blocked by MCC950 and hence constituted an NLRP3-independent response. VbP, on the other hand, led to robust LDH release in both naïve and memory CD4 and CD8 T cells. Of note, no IL-1β or IL-18 release was observed in stimulated T cells for any of the conditions tested. Interestingly, ABT737/S63845 treatment also resulted in a substantial lytic cell death response in primary T cells, as inferred from LDH release. While the here-studied T-cell populations were highly pure, we wanted to exclude the possibility that impurities in the cell preparations impacted on the cell death-inducing activity of VbP. To this end, we subjected primary CD4 T cells to single cell cloning and tested these cells for VbP-induced cell death. These experiments confirmed that VbP triggered cell death in human T cells, while the overall responses in clonally expanded T cells were lower as compared to resting T cells (Fig EV1A and Fig 1A). In summary, these results suggested that primary T cells are highly sensitive to DPP inhibition by VbP, resulting in a lytic type of cell death that is suggestive of pyroptosis. Figure 1. Dipeptidyl-peptidase inhibition triggers a lytic cell death in primary human T cells A–C. FACS-sorted T-cell populations and Monocyte-derived Macrophages were treated with the indicated stimuli: Nigericin (Nig. 4 h), NeedleTox (4 h), Val-boroPro (VbP 22 h), and ABT737/S63845 (A/S 22 h). When indicated, cells were primed with LPS for 2 h prior to stimulation. When indicated, MCC950 was added to the media 30 min prior to the addition of Nigericin. LDH activity (A) and IL-1β and IL-18 concentration (B and C, respectively) in the supernatant were determined by LDH cytotoxicity assay and ELISA, respectively. Individual data points ± SEM from three independent donors are shown. Statistics indicate significance by two-way ANOVA: ***P ≤ 0.001; **P ≤ 0.01; *P ≤ 0.05; ns, not significant. P-values were corrected for multiple comparisons (Dunnett). Download figure Download PowerPoint Click here to expand this figure. Figure EV1. CD4 T cell clones retain susceptibility toward VbPNaïve CD4 T cells were activated with CD3/CD28 beads in the presence of IL-2. On day 2, cells were subjected to minimal dilution cloning. When colonies became visible, clones were collected, cell numbers assessed and cells re-plated for stimulation. Forty thousand cells of each clone were stimulated with PMA/Ionomycin for 24 h and concentration of IFNγ and IL-4 was determined by ELISA. Based on the cytokine-profile, clones were identified as Th1 or Th2. Th1 and Th2 clones from one donor were then pooled and subjected to the indicated treatments. Freshly isolated PBMCs from an unrelated donor were stimulated in parallel. Cytotoxicity was determined by LDH assay. Individual data points ± SEM of three biological replicates from one donor. Download figure Download PowerPoint VbP treatment triggers pyroptosis in T cells To further investigate the nature of the cell death elicited by these stimuli, we performed live-cell imaging, in which we compared primary CD4 T cells and MDMs over a period of 16 h (Fig 2A and B, Fig EV2A and B, Movies EV1–EV8). We included the nuclear DNA stain propidium iodide (PI) as a marker of cell membrane integrity. Within the first hour of stimulation, the NAIP/NLRC4-stimulus NeedleTox induced rapid membrane swelling and PI uptake in MDMs, the characteristic features of pyroptosis (Movie EV1, Fig EV2B). However, in line with the LDH measurements, CD4 T cells were completely unaffected by NeedleTox treatment (Movie EV2). Treatment of macrophages with VbP induced the same characteristic morphology as NeedleTox treatment. However, the individual cells did not die simultaneously and it took up to 12 h for the last cell to commit to pyroptosis (Movie EV3, Fig EV2B). Strikingly, T cells treated with VbP also displayed characteristic features of pyroptosis, showing uniform ballooning of their cytoplasm accompanied by immediate PI uptake (Movie EV4, Fig EV2B). Similar to the VbP-treated macrophages, this followed an asynchronous—albeit faster—kinetic. As expected, treatment of T cells with ABT737/S63845 induced hallmarks of apoptosis, such as cell shrinkage and membrane blebbing (Movie EV6). At later time points, apoptotic cells also turned PI positive (Fig 2A, Fig EV2B), consistent with the concept of secondary necrosis that was also documented by LDH release (Fig 1A). To further corroborate that VbP-treated T cells undergo pyroptosis, we analyzed the cleavage of caspase-1 and its substrate GSDMD by immunoblot over a period of 48 h (Fig 2C). These experiments showed that caspase-1 was cleaved in T cells upon VbP stimulation, but not following ABT737/S63845 treatment. Moreover, VbP led to the appearance of the N-terminal 30 kDa fragment of GSDMD, which is associated with its cleavage at Asp275 and subsequent pore formation. In contrast, induction of intrinsic apoptosis readily resulted in caspase-3 maturation and PARP cleavage, as well as the formation of a 45 kDa fragment of GSDMD. The latter finding is consistent with a previous study that has shown that GSDMD can be cleaved by caspase-3 at Asp87 during apoptosis, resulting in a non-productive N-terminal (p13) and C-terminal (p45) fragment (Taabazuing et al, 2017). While in macrophages NeedleTox and Nigericin induced caspase-1 and GSDMD cleavage in a priming independent and dependent manner respectively, these same treatments did not lead to the appearance of the pyroptotic GSDMD fragment in the lysate or processed caspase-1 in the supernatant of T cells (Fig EV2C). While expression data (see below) indicate that the resistance of T cells to NeedleTox is due to the absence of NLRC4 expression, we cannot formally exclude the possibility that the toxin cannot enter T cells. Nevertheless, previous work has shown that human T cells are in principle amenable to protective antigen-mediated protein uptake (Paccani et al, 2005). Interestingly, Nigericin treatment induced PARP cleavage in T cells, suggesting that the cell death in T cells induced by Nigericin is apoptosis with subsequent secondary necrosis rather than pyroptosis. In line with this concept, Nigericin also induced the production of the apoptotic GSDMD p45 fragment. In summary, these experiments suggested that T cells are capable of undergoing bonafide pyroptosis upon VbP treatment. Figure 2. VbP-treated T cells display hallmarks of pyroptosis A. Macrophages and CD4 T cells from the same donor were subjected to the indicated treatments and morphologic changes as well as PI uptake were monitored by live-cell imaging microscopy using a 25× objective. Representative images from indicated time points are shown. Cyan color coding is used for the fluorescent PI signal. One donor out of two is shown. B. Representative images were acquired with a 63× objective at the end of the experiment shown in (A) at 16 h. One donor out of two is shown. Scale bars: 25 μm. C. CD4 T cells were treated with VbP or ABT737/S63845. Lysate and supernatant samples were collected at the indicated time points. Samples were analyzed by immunoblotting. αSS and αLS indicate the use of a small subunit or large subunit-specific caspase-1 antibody respectively. Lys = lysate, SN = supernatant, FL = full length. One representative experiment out of three is shown. Source Data for Figure 2 [embj2020105071-sup-0011-SDataFig2.pdf] Download figure Download PowerPoint Click here to expand this figure. Figure EV2. Proteasome-inhibition blocks VbP-induced pyroptosis in CD4 T cells A. Uncropped and less magnified versions of the images displayed in Fig 2B. Scale bars: 100 μm. B. Quantification of PI-positive cells of data shown in Movies EV1–EV6 and Fig 2A and of an additional donor. Data were normalized by a sliding window covering 30 min. Data (mean ± SEM) from two independent donors are shown. C. CD4 T cells or MDMs from one donor were subjected to the indicated treatments: Nigericin (Nig. 4 h), NeedleTox (4 h), Val-boroPro (VbP 22 h), and ABT737/S63845 (A/S 22 h). When indicated, cells were primed with LPS for 2 h prior to stimulation. αSS and αLS indicate the use of a small subunit- and a large subunit-specific CASP1 antibody, respectively. Lys = lysate, SN = supernatant, FL = full-length protein, pro = proIL-1β, mat = mature IL-1β. Shown is one representative experiment out of three independent experiments. D. CD4 T cells were treated as indicated for 8 h and cytotoxicity was determined by LDH assay. Individual data points ± SEM from five independent donors are shown. Statistics indicate significance by two-way ANOVA: ***P ≤ 0.001; ns, not significant. P-values were corrected for multiple comparisons (Dunnett). Download figure Download PowerPoint The CARD8 inflammasome is functional in human T cells VbP treatment is known to either activate NLRP1 (e.g. in keratinocytes) or CARD8 (e.g. in myeloid leukemia cell lines; Johnson et al, 2018; Zhong et al, 2018). Studying the myeloid cell line THP-1, we confirmed that myeloid cells engage CARD8 downstream of DPP inhibition (Fig EV3A–C). Of note, in this setting we did not observe a role for CARD8 in negatively regulating the NLRP3 inflammasome in human myeloid cells, as it has previously been suggested (Ito et al, 2014; Mao et al, 2018). Gene expression profiles from a publicly available dataset indicated that T cells express similar amounts of NLRP1 and CARD8 as monocytes, while other inflammasome sensors such as NAIP, AIM2, NLRP3, PYRIN (MEFV), or NLRC4 were scarcely expressed in T cells (Fig 3A). Immunoblotting confirmed that human T cells express both CARD8 and NLRP1, even at higher levels than MDMs (Fig 3B). GSDMD and CASP1 were expressed at comparable levels as in macrophages, while ASC expression was higher in macrophages (Fig 3B). These findings
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