Gasdermin D Promotes AIM2 Inflammasome Activation and Is Required for Host Protection against Francisella novicida

This article is featured in In This Issue, p.3477

Inflammasomes are multiprotein complexes formed by activation of innate immune sensors, including nucleotide-binding oligomerization domain–like receptors absent in melanoma 2 (AIM2) or pyrin, in response to pathogen-associated molecular patterns or danger-associated molecular patterns (1). AIM2 is a cytoplasmic sensor for dsDNA. Upon activation, AIM2 assembles the inflammasome complex with adaptor ASC to mediate caspase-1 activation, caspase-1–dependent pyroptosis, and release of proinflammatory cytokines IL-1β and IL-18 (2–5).

Gasdermin D (GSDMD) was recently discovered as an executor of pyroptotic cell death downstream of caspase-1 and caspase-11 (6–8). GSDMD belongs to the gasdermin family of proteins and is maintained in an autoinhibitory form under steady state. Inflammatory caspases, including caspase-1 and -11, cleave at a link region between the N- and C-terminal domains of GSDMD, releasing the N-terminal fragment from the inhibition by the C-terminal domain (6–8). The cleaved N-terminal end of GSDMD binds to phosphoinositides located in the inner leaflet of the plasma membrane and forms pores on the membrane, leading to cell lysis (9–13). Recent studies have further shown that pores generated by GSDMD can function as channels to allow secretion of IL-1β and IL-18 independently of pyroptosis (14, 15).

AIM2 recognizes dsDNA originated from Gram-negative bacterium Francisella, DNA viruses mouse CMV (MCMV) and vaccinia virus, and host cells, thereby exhibiting crucial roles in microbial infections and autoimmune diseases (16–22). Mice lacking AIM2, ASC, or caspase-1 are highly susceptible to infection by Francisella novicida, demonstrating a protective role for AIM2 inflammasome during the infection (18, 22–27). IL-1β and IL-18 have been further shown to mediate the beneficial effects of AIM2 in response to F. tularensis live vaccine strain (28, 29). However, the role of pyroptosis downstream of AIM2 inflammasome is unknown. In this study, we showed that mice lacking the effector molecule of pyroptotic cell death, GSDMD, are susceptible to F. novicida infection. We further found that although cleavage of GSDMD is dependent on AIM2 inflammasome, GSDMD itself is required for optimal caspase-1 activation. Interestingly, GSDMD is also required for caspase-1 activation triggered by other AIM2 inflammasome stimuli, including poly(dA:dT) transfection and MCMV infection.

Wild type (WT) C57BL/6J mice were purchased from The Jackson Laboratory. Aim2^−/− mice (22), Casp1^−/− mice (30), Il1r^−/−Il18^−/− mice (31), and Gsdmd^−/− mice (31) have been described previously. Six- to eight-week-old male and female mice were used in this study. Animal studies were conducted according to the protocols approved by the St. Jude Institutional Animal Care and Use Committee.

F. novicida strain U112 was grown overnight at 37°C in BBL Trypticase soy broth (Becton Dickinson) containing 0.2% l-cysteine and was 1:10 subcultured for 4 h. Mice were infected s.c. with 7.5 × 10⁴ CFUs (survival analysis) or 1.5 × 10⁵ CFUs (day 3 CFU analysis) of F. novicida in 200 μl PBS. For CFU analysis, homogenized liver and spleen tissues were plated onto Trypticase soy broth agar containing 0.2% l-cysteine and incubated overnight.

Cytokine levels were measured by performing multiplex ELISA (MilliporeSigma) or IL-18 ELISA (MBL International) according to the manufacturer’s instructions.

Bone marrow–derived macrophages (BMDMs) were cultured for 6 d in macrophage culture medium (i.e., DMEM supplemented with 10% FBS, 30% L929 conditioned medium, and 1% penicillin and streptomycin) and seeded at a concentration of 1 × 10⁶ cells per well onto 12-well plates. The next day, BMDMs were incubated in antibiotic-free media. F. novicida was added to BMDMs at an indicated multiplicity of infection (MOI) for 20 h. Fifty micrograms per milliliter gentamicin was added 4 h after the infection. The MCMV strain (K181) was obtained from E.S. Mocarski (32) (Emory University School of Medicine). MCMV was added at an MOI of 10 for 10 h. For poly(dA:dT) transfection, 1 μg of poly(dA:dT) (InvivoGen) was resuspended in PBS, mixed with 0.3 μl of Xfect polymer in Xfect reaction buffer (Takara Bio USA), and incubated for 10 min. DNA complexes were added to BMDMs in Opti-MEM and incubated for 3 h.

Bone marrow cells were harvested from mice and seeded on plates with supernatant of J2 Cre retrovirus and macrophage culture medium in the presence of polybrene. Three days later, the supernatant was spun down, resuspended in fresh macrophage culture medium, and transferred back on plates, where fresh J2 Cre supernatant supplemented with polybrene was added. Macrophages were then split accordingly. L929 conditioned media in the macrophage culture medium were decreased by half with each passage.

Gene-encoding GSDMD tagged with FLAG was cloned into retroviral plasmid pMSCV-puro. To get the desired retrovirus, 293T cells were transfected with pMSCV-puro–FLAG-GSDMD, pAME, and pVSV-G with a ratio of 2:2:1 using lipofectamine 2000. Supernatants were collected at 48 h after transfection, and the Gsdmd^−/− immortalized BMDM (iBMDM) cells were used for transduction. Positive cells were selected by puromycin at a concentration of 5 μg/ml.

For caspase-1 immunoblots, GAPDH immunoblots that are associated with caspase-1 immunoblots, and immunoblots in Fig. 2C, BMDMs and supernatant were lysed in radioimmunoprecipitation assay buffer supplemented with protease inhibitor (Sigma-Aldrich) and sample loading buffer containing SDS and 100 mM DTT. For immunoblotting of GSDMD and GAPDH in Fig. 4, cell supernatant was removed. BMDMs were washed with PBS and further lysed in RIPA buffer supplemented with protease and phosphatase inhibitor (Roche). Prepared samples were then separated by SDS-PAGE and transferred to polyvinylidene difluoride membranes (MilliporeSigma). Membranes were blocked by 5% milk and incubated overnight with primary Abs against caspase-1 (AG-20B-0042; AdipoGen Life Sciences), GSDMD (ab209845; Abcam), FLAG (F1804; Sigma-Aldrich), or GAPDH (no. 5174; Cell Signaling Technology). Membranes were then incubated with HRP-conjugated secondary Ab for 1 h. Proteins were further visualized using Luminata Forte Western HRP substrate (MilliporeSigma).

Cell supernatants were collected 20 h after F. novicida infection, 3 h after poly(dA:dT) transfection, or 10 h after MCMV infection. Lactate dehydrogenase (LDH) activity was measured using the Promega cytotoxicity kit according to manufacturer’s protocols.

BMDMs were seeded at a concentration of 1 × 10⁶ cells per well onto 12-well plates. The next day, BMDMs were stimulated and incubated with 100 nM cell-impermeable DNA-binding fluorescent dye SYTOX Green (S7020; Life Technologies) to stain dead cells. Cell death was monitored in real time using a two-color IncuCyte ZOOM in-incubator imaging system (Essen BioScience). Nuclear-ID (ENZ-52406; Enzo Life Sciences) was added at the last time point to stain live and dead cells for the total cell number (33). Resulting images were analyzed using IncuCyte S3 software. Cell death was presented as a ratio of SYTOX Green⁺ cells to the total cell number. Experiments were conducted with three replicates for each experimental condition and nine image fields per well.

MAbs CD11b (M1/70) from eBioscience and F4/80 (BM8) from BioLegend were used for flow cytometry analysis. Flow cytometry data were acquired on FACSCalibur (Becton Dickinson) and were analyzed with FlowJo software (FlowJo and Illumina).

GraphPad Prism 7.0 software was used for data analysis. Data are shown as mean ± SEM. Statistical significance was determined by performing t tests (two-tailed), one-way ANOVA, or log-rank tests. A p value < 0.05 was considered to be statistically significant.

Mice lacking AIM2 inflammasome components such as AIM2 (Aim2^−/−) or caspase-1 (Casp1^−/−) were previously reported to be susceptible to F. novicida infection (18, 22–27). Consistent with previous findings, we found that Aim2^−/− and Casp1^−/− mice were susceptible to F. novicida infection (Fig. 1A). Following assembly of the inflammasome, activated caspase-1 cleaves pro–IL-1β and pro–IL-18 into mature forms and mediates their release from the cell. We therefore tested the role of IL-1β and IL-18 during F. novicida infection. Similar to Aim2^−/− and Casp1^−/− mice, mice lacking both IL-1R and IL-18 (Il1r^−/−Il18^−/−) were also highly susceptible and succumbed to death within 3 d of the infection (Fig. 1A), highlighting a protective role for IL-1β and IL-18 downstream of inflammasome activation during F. novicida infection.

GSDMD mediates pyroptosis downstream of AIM2 inflammasome in response to DNA transfection (7). To test the role of pyroptosis during F. novicida infection, we infected WT mice and mice lacking GSDMD (Gsdmd^−/−) with F. novicida and monitored the survival. Whereas 70% of WT mice survived on day 14 postinfection, only 10% of Gsdmd^−/− mice survived during the course of infection (Fig. 1A). Furthermore, Gsdmd^−/− mice had significantly increased bacterial burden in the liver and spleen 3 d after F. novicida infection (Fig. 1B), further confirming a protective role for GSDMD. Membrane pores formed by GSDMD have been shown to mediate secretion of IL-1β and IL-18. Indeed, we observed reduced levels of circulating IL-18 in Gsdmd^−/− mice compared with WT mice 1 d postinfection (Fig. 1C).

Inflammasome activation is critical for host defense against F. novicida infection in vivo. To test whether inflammasome activation is affected in the absence of GSDMD, we generated BMDMs from WT, Gsdmd^−/−, and Aim2^−/− mice. The population of CD11b⁺F4/80⁺ macrophages was comparable between WT and Gsdmd^−/− BMDMs (Supplemental Fig. 1), suggesting that there is no difference in the extent of macrophage differentiation. We then infected BMDMs from WT, Gsdmd^−/−, and Aim2^−/− mice with F. novicida and measured cleavage of pro–caspase-1 into its active form (p20) following assembly of inflammasome. Interestingly, Gsdmd^−/− BMDMs showed decreased activation of caspase-1 compared with WT cells after F. novicida infection (Fig. 2A), highlighting the requirement of GSDMD for inflammasome activation in response to F. novicida. Caspase-1 activation was reduced in Gsdmd^−/− BMDMs postinfection with multiple MOIs of F. novicida (Fig. 2A, Supplemental Fig. 2A, 2B). To further confirm the role of GSDMD in caspase-1 activation during F. novicida infection, we reconstituted GSDMD in Gsdmd^−/− iBMDMs and found that complementation of the Gsdmd^−/− iBMDMs with FLAG-GSDMD increased the caspase-1 activation induced by F. novicida (Fig. 2B, 2C). In addition, we observed impaired IL-1β and IL-18 release in F. novicida–infected Gsdmd^−/− BMDMs, potentially because of reduced caspase-1 activation along with defective pore formation (Fig. 2D), whereas secretion of TNF was similar between the cells (Supplemental Fig. 3). To test the requirement of GSDMD in pyroptosis triggered by F. novicida infection, we first monitored the extent of cell death in WT, Gsdmd^−/−, and Aim2^−/− BMDMs after F. novicida infection using SYTOX Green staining and found that Gsdmd^−/− BMDMs exhibited lower numbers of SYTOX Green–positive cells than WT BMDMs over the course of the infection (Fig. 2E, 2G, Supplemental Fig. 2C). Consistent with the SYTOX Green staining, LDH release was also decreased in Gsdmd^−/− BMDMs 20 h postinfection, which further confirms that pyroptosis in BMDMs during F. novicida infection is dependent on GSDMD (Fig. 2F, 2G). Interestingly, GSDMD-independent cell death was also observed in Gsdmd^−/− BMDMs after F. novicida infection, suggesting that other molecules may exist to mediate F. novicida–induced pyroptosis (Fig. 2E–G, Supplemental Fig. 2C).

In addition to F. novicida infection, AIM2 inflammasome can also be activated by transfection of poly(dA:dT) or MCMV infection. We found that caspase-1 activation induced by transfection of dsDNA ligand poly(dA:dT) was partially reduced in Gsdmd^−/− BMDMs compared with WT cells (Fig. 3A). Gsdmd^−/− BMDMs exhibited compromised release of IL-18 but not TNF (Fig. 3B, Supplemental Fig. 3). Consistent with previous studies (7, 34), cell death was completely abolished in poly(dA:dT)-transfected Gsdmd^−/− BMDMs during the early phase of stimulation (Fig. 3C–E). Furthermore, MCMV-induced caspase-1 activation was dampened in Gsdmd^−/− BMDMs compared with WT BMDMs (Fig. 3F). Release of IL-1β and IL-18 in MCMV-infected Gsdmd^−/− BMDMs was impaired (Fig. 3G), whereas TNF secretion was comparable between WT and Gsdmd^−/− BMDMs (Supplemental Fig. 3). Altogether, these data demonstrate that GSDMD is necessary for optimal caspase-1 activation in response to AIM2 inflammasome triggers, including F. novicida and MCMV infection and, to a lesser extent, poly(dA:dT) transfection, and that GSDMD plays a critical role in mediating pyroptosis after AIM2 inflammasome activation.

Previous studies have shown that GSDMD is cleaved downstream of inflammasome activation. To test if AIM2 inflammasome is able to mediate GSDMD cleavage following F. novicida infection, we infected cells with F. novicida and observed impaired cleavage of GSDMD in BMDMs lacking AIM2 (Aim2^−/−) or ASC (Asc^−/−) (Fig. 4A), suggesting that AIM2 inflammasome is required for GSDMD cleavage. Type I IFN signaling is required for activating AIM2 inflammasome in macrophages during F. novicida infection via induction of transcription factor IFN regulatory factor 1 (IRF1), an IFN-stimulated gene (16, 18, 22–25, 35, 36). IRF1, in turn, promotes expression of various cell-autonomous immunity proteins, including guanylate-binding protein (GBP) 2, GBP5, and immunity-related GTPase B10 (IRGB10), which facilitate bacteriolysis and release of bacterial DNA (23–25). Because GBPs are critical for AIM2 inflammasome activation during F. novicida infection (24, 25), we used BMDMs from mice lacking multiple GBPs (including GBP2 and 5) encoded on chromosome 3 (Gbp^chr3-knockout [KO]). We found reduced cleavage of GSDMD in Gbp^chr3-KO BMDMs, further demonstrating the requirement of AIM2 inflammasome for GSDMD cleavage (Fig. 4B). Overall, these data demonstrate that although caspase-1 activation induced by AIM2 inflammasome triggers is largely dependent on GSDMD, GSDMD cleavage still requires AIM2 inflammasome.

Pyroptosis is a lytic form of cell death that is induced by inflammasomes, leading to release of mature IL-1β and IL-18 and other cellular components, including high-mobility group box 1 (HMGB1) and IL-1α. It has not been discovered until recently that GSDMD induces pyroptosis by forming pores on plasma membrane after being activated by caspase-1 or -11 (6–8). In this study, we demonstrated a protective role for GSDMD during F. novicida infection. Although caspase-1 processing is intact in the absence of GSDMD downstream of canonical NLRP3, NLRC4, or pyrin inflammasome (6, 7), we observed that activation of caspase-1 is impaired in Gsdmd^−/− BMDMs in response to AIM2 inflammasome stimuli. Given that a residual amount of caspase-1 activation was observed in Gsdmd^−/− BMDMs infected with F. novicida, it is intriguing to hypothesize that during the early phase of F. novicida infection, caspase-1 is activated in a GSDMD-independent manner. Activated caspase-1 may cleave GSDMD to allow the N terminus to target F. novicida in the cytosol, leading to bacteriolysis and release of bacterial DNA, which potentially amplifies activation of AIM2 inflammasome and further GSDMD cleavage at the later phase of F. novicida infection in a positive feedback manner. Indeed, GSDMD has been shown to be associated with phosphoinositides and cardiolipin (9–11), the latter of which is a component of F. novicida membrane as well as mitochondria (37). Moreover, incubation of the N-terminal fragment of GSDMD with Escherichia coli or Staphylococcus aureus in vitro leads to reduced bacterial CFU (11). Furthermore, higher CFU of Listeria monocytogenes is observed in GSDMD–knockdown BMDMs (11), revealing a bactericidal role for GSDMD during intracellular bacterial infection (10, 11). Consistent with this hypothesis, we also observed a higher bacterial burden in Gsdmd^−/− mice after F. novicida infection. Polymers that facilitate transfection of poly(dA:dT) or viral particles may be targeted by GSDMD similarly to F. novicida, which explains a reduction in caspase-1 activation in the absence of GSDMD in response to poly(dA:dT) transfection and MCMV infection. In addition to the possibility that GSDMD targets bacterial membrane, GSDMD-mediated pore formation on plasma membrane at the early phase of F. novicida infection may act in a feed-forward loop, giving an unidentified secondary signal for AIM2 inflammasome activation. It is also possible that GSDMD binds to cardiolipin, which is located at the inner membrane of mitochondria. Pores may form by GSDMD activity on mitochondria, inducing mitochondrial damage and release of mitochondrial DNA or other mitochondria-associated molecules into the cytosol, serving as a potential source for AIM2 inflammasome activation. Although both cell death and release of IL-1β and IL-18 are greatly reduced in Gsdmd^−/− BMDMs stimulated with AIM2 inflammasome triggers compared with WT cells, these are still higher than those in Aim2^−/− BMDMs, suggesting that additional mechanisms may mediate pyroptosis and pyroptosis-dependent IL-1β and IL-18 release downstream of AIM2 inflammasome. Consistent with our findings, GSDMD-independent cell death and cytokine release are also reported previously (6, 34). It is intriguing to hypothesize that other members of the GSDMD family, such as GSDME, can be activated by AIM2 to induce pore formation on cell membrane, leading to pyroptosis (38, 39). Altogether, our study highlights a critical role for GSDMD in mediating host defense against F. novicida infection and further underscores a requirement for GSDMD in promoting optimal caspase-1 activation following AIM2 inflammasome triggers.

We thank members of the Kanneganti Laboratory for comments. We thank V. M. Dixit (Genentech) for the mutant mice.

The authors have no financial conflicts of interest.