1. Gsdmd expression was elevated in airways with type 2 inflammation in both humans and mice.
The Gsdmd protein has been widely reported to function in multiple cell types, including immune cells, such as macrophages/neutrophils, and stromal cells, such as nasal epithelial cells46, 47, 48, 49. However, the expression of GSDMD in human lung tissue and asthma has not yet been defined. To address this question, we collected bronchial samples from asthma patients and non-asthmatic people (Supplementary Fig. 1). With activated immune responses in the lungs, the asthma patients presented elevated inflammatory cell infiltration into lung tissues, increasing amounts of smooth muscle and hypertrophy of the mucous glands compared with the non-asthmatic controls (Fig. 1a). Interestingly, lung tissue sample analysis with immunohistochemical staining indicated that GSDMD was mainly expressed in the pulmonary airway epithelium and presented an upregulated expression pattern in the asthma patient group, especially in areas with mucous gland hyperplasia (Fig. 1a, b), indicating that GSDMD may play fundamental roles in the human airway epithelium. In mice, the expression of Gsdmd presented a pattern similar to that in humans. Gsdmd was weakly expressed in the normal murine airway epithelium. With the induction of asthmatic airway inflammation by HDM exposure, Gsdmd expression was upregulated significantly (Fig. 1c, d). Together, our statistical data revealed that the expression of Gsdmd was significantly increased in human and murine lung tissues with asthma-associated inflammation, which suggested that Gsdmd might function in the pathogenesis of asthma in the lungs
2. Deletion of Gsdmd dampened ILC2s activation in both HDM-induced chronic and papain-induced acute airway inflammation
To further investigate the potential function of Gsdmd in airway inflammation, HDM extracts were administered intranasally to induce chronic asthmatic airway inflammation in both genetically Gsdmd-deficient and wild-type (WT) mice (Fig. 2a). The mice were sacrificed 17 days after the first stimulation. The deletion of Gsdmd in mice dampened the HDM-induced airway inflammatory response, with less inflammatory infiltration into the bronchus and less mucus production in the airways (Fig. 2b). The secretion of inflammatory cytokines, such as IL-5 and IL-13, into the bronchoalveolar lavage fluid (BALF) was also significantly suppressed (Fig. 2c). The number of infiltrated eosinophils and proliferation of ILC2s in the lungs were also decreased in the Gsdmd-deficient mice (Fig. 2d, Supplementary Fig. 2a, b). In addition, Papain-induced acute airway inflammation was also evaluated in genetically Gsdmd-deficient mice (Fig. 2e). Similarly, Gsdmd deficiency alleviated acute airway inflammation in the mouse lungs, with less immune cell infiltration (Fig. 2f, g) and typically decreased alveolar eosinophil infiltration and ILC2s proliferation (Fig. 2g). The numbers of activated IL-5- or IL-13-producing ILC2s were also significantly inhibited (Fig. 2h). Secretion of the inflammatory cytokines IL-5 and IL-13 into the BALF was also obviously impaired (Fig. 2i). Together, the above data indicated that Gsdmd deficiency in mice alleviated both HDM-induced chronic and papain-induced acute airway inflammatory responses by limiting ILC2s activation and eosinophil infiltration.
3. Gsdmd deficiency impaired early IL-33 release in vivo.
Multiple inflammatory cytokines are important in the initiation and development of airway inflammation, among which epithelial cell-derived IL-33, IL-25, and TSLP are believed to play fundamental roles in the initiation of type 2 inflammation50, 51, 52. To identify the factors that dominate airway inflammation in this process, we evaluated the secretion of inflammatory cytokines into the BALF of mice exposed to papain. With an MSD multiple cytokine detection systems, we analyzed the levels of different inflammatory cytokines, including IFNγ, IL-13, IL-33, IL-25, and IL-1β, in the BALF 3 hours after papain stimulation. Among these cytokines, IL-33 displayed an extremely high abundance (Fig. 3a), which suggested that the IL-33 release event is fundamental in boosting inflammation at this early stage. To expand our knowledge on IL-33 secretion into the BALF, we monitored the secretion of IL-33 over time. The results showed that the secretion of IL-33 into the BALF reached a peak at 3 hours after papain exposure (Fig. 3b). In contrast, both IL-25 and TSLP displayed no significant change after papain exposure (Fig. 3c). Our observations suggest that in the early stage, IL-33 is responsible for the enhancement of lung inflammation after allergen protease stimulation in vivo.
The airway epithelium functions as a physical barrier, initiating multiple innate and adaptive immune responses by secreting alarmins, including IL-33, which respond rapidly to allergens, viruses, and environmental pollutant insults. To clarify whether Gsdmd is involved in the early IL-33 release event, under the same experimental conditions, we collected BALF from WT and Gsdmd-deficient mice 3 hours after papain exposure. The data showed that Gsdmd deficiency inhibited IL-33 release into the BALF in the early stage (Fig. 3d) but did not interfere with the IL-33 mRNA expression level in lung tissues (Fig. 3e). These results indicated that Gsdmd might function by controlling the secretion of IL-33 and imply a crucial function for Gsdmd in airway epithelial cells during airway inflammation.
4. Exogenous recombinant IL-33 supplementation blunted the alleviation of inflammation in Gsdmd-/- mice.
As Gsdmd deficiency impairs IL-33 release in the early stage, we speculated that Gsdmd deficiency constrained IL-33 within cells. To identify whether the functions of Gsdmd in blocking ILC2s activation and eosinophil infiltration in mice were dependent on IL-33 signaling, we intranasally challenged Gsdmd-deficient mice with recombinant murine IL-33 (rIL-33) (Supplementary Fig. 3a). As expected, activating airway inflammation through IL-33/ST2 axis directly with rIL-33 stimulation resulted in no obvious inflammatory immune responses differences in inflammatory cell infiltration, ILC2s activation, or cytokine secretion between Gsdmd-deficient and WT mice (Supplementary Fig. 3b-g). Indicating that Gsdmd might function upstream of IL-33 within cells before its secretion, thus blocking the activation of ST2-expressing effector cells, such as ILC2s
5. The protease activity of papain is needed for IL-33 release and Gsdmd cleavage in vitro.
As the pulmonary epithelium has been reported to be an important source of IL-3316, 53, 54, we further analyzed the expression of IL-33 in murine lungs with immunofluorescence staining. We observed a typical expression pattern of IL-33 in lung surfactant protein C (SPC)-positive airway epithelial type 2 (AT2) cells (Supplementary Fig. 4a). Both IL-33 and Gsdmd are preferentially expressed in murine airway epithelial cells. Considering that Gsdmd has been proved with the pore-forming ability on the cell membrane in regulating pyroptosis and mediating IL-1β release, we hypothesized that Gsdmd may function similarly in controlling IL-33 release in the airway epithelium. Endogenous IL-33-expressing murine AT2 mle12 cells were used for an in vitro functional study. A549 airway epithelial cells constitutively expressing C-terminal-eGFP-fusional HA-tagged human IL-33 (A549-IL-33-eGFP) and the smallest nuclear localization sequence (NLS) linked with eGFP (A549-NLS-eGFP) were also used for functional analysis (Supplementary Fig. 5a).
Epithelial cells were exposed to papain, and IL-33 secretion from the nucleus was recorded for 30 min with a live cell imaging recording system (Fig. 4a; Supplementary Videos 1 and 2). IL-33 responded rapidly to papain protease stimulation in epithelial cells, while NLS-eGFP did not show any decrease in the GFP fluorescence signal in the nucleus (Fig. 4a, b), suggesting that papain was able to induce IL-33 secretion without extensive nuclear leakage. Cells and cell culture supernatants were collected and analyzed by western blotting, and we found that papain-stimulated IL-33 release occurred with the generation of a p40 mNT-Gsdmd fragment in murine alveolar epithelial cells (Fig. 4c) and a p35 hNT-GSDMD fragment in human epithelial cells (Supplementary Fig. 5b-d). In addition, papain-stimulated IL-33 release and Gsdmd cleavage presented a dose- and time-dependent manner (Fig. 4c, d). Moreover, cells did not present obvious death morphology during this time series (Supplementary Videos 1 and 2). Even with a relatively high dose of papain stimulation, LDH release from cells into the supernatant was nearly undetectable, and neither apoptosis-associated caspase-3 cleavage nor necroptosis-associated Mlkl phosphorylation was detected in cells (Fig. 4e). These observations suggested that papain-induced Gsdmd cleavage and IL-33 release did not accompany by cell death. Taken together, our results revealed that papain stimulation did not activate classic cell death pathways, such as apoptosis or necroptosis, whereas the activation of a neo-form of the p40 mNT-Gsdmd fragment was found to be involved in this papain-stimulated IL-33 release event. In addition, directly activating p-Mlkl-mediated necroptosis or caspase-mediated apoptosis did not contribute to IL-33 release in mle12 airway epithelial cells, which implied that the p-Mlkl-mediated necroptosis pathway did not dominate nuclear IL-33 release.
Papain is a proteolytic enzyme with cysteine protease activity. To determine whether Gsdmd cleavage and the IL-33 secretion process are dependent on the protease activity of papain, the irreversible cysteine protease inhibitor E64 was incubated with papain before papain was added to cell culture supernatants. With the inhibition of cysteine protease activity, the activation of Gsdmd in cells and the release of IL-33 into the supernatant were suppressed (Fig. 4f). Similarly, after heat inactivation at 100 °C for 10 min, papain no longer possessed dose-dependent activating effects on Gsdmd cleavage and IL-33 secretion (Fig. 4g). These observations suggested that the protease activity of papain was a fundamental element for Gsdmd cleavage and IL-33 secretion in epithelial cells.
Constituted with active enzymatic components is a common feature of allergens, and different enzymatic components from various allergens have been proven to possess the ability to induce IL-33 release both in vitro and in vivo. Next, we wanted to address whether Gsdmd activation is a common phenomenon after cells were exposed to allergen protease stimuli. Allergen-derived active enzymatic components, including the fungus A. oryzae, the bacteria Bacillus licheniformis, HDM extracts and the purified natural HDM proteases Der p and Der f 1, were cultured with mle12 murine airway epithelial cells at different concentrations. After exposure for 30 min, we collected the cell pellets and detected Gsdmd cleavage with immunoblotting. The results showed that all these tested allergen proteases were able to activate the Gsdmd-p40 N-terminal fragment in a dose-dependent manner, which was similar to papain stimulation (Fig. 4j). This raised the possibility that allergen proteases share a common cellular protease stress-sensing pathway that leads to Gsdmd activation by producing one p40 fragment, which might be associated with IL-33 release from the nucleus at an early stage.
6. Papain-activated Gsdmd is independent of the inflammasome caspase 1/11 pathway
Gsdmd serves as an executor of pyroptosis and can be cleaved by inflammatory caspases-1/11 through the activation of canonical and non-canonical inflammasome pathways promoting IL-1β release in multiple cell types, typically macrophages55. Macrophages can also be an efficient source of IL-33 upon inflammatory stimulation27, 56. To compare Gsdmd cleavage events between inflammatory caspase and papain stimulation, we conducted papain stimulation in murine bone marrow-derived macrophages (BMMs). After pre-stimulation with bacterial LPS, papain was added as a second stimulatory signal, similar to ATP. LPS activated TLR4 signaling, which led to the production of pro-IL-1β in BMMs. After the second stimulation with ATP or nigericin (Nig), the NLRP3 inflammasome was activated, which promoted the generation of p20 caspase 1 and the 35-kDa fragment of mNT-Gsdmd. This p35 fragment of mNT-Gsdmd displayed a high binding affinity to cell membrane-associated lipids with pore-forming capability, resulting in the release of IL-1β and LDH (Fig. 5a-d). In contrast, papain stimulation did not promote the activation of p20 caspase 1; thus, no mature p18 IL-1β was generated and secreted into the cell supernatant (Fig. 5a, b). However, papain promoted the generation of the p40 mNT-Gsdmd fragment and IL-33 release in BMMs, which kept the same pattern as airway epithelial cells. Additionally, this process did not result in obvious LDH release (Fig. 5a). Indeed, BMMs with Gsdmd deficiency presented both impaired caspase 1-dependent Gsdmd cleavage and IL-1β secretion upon pyroptotic stimulation, as well as impaired IL-33 release with papain stimulation (Fig. 5a-c), which further supported the conclusion that Gsdmd processing is needed for sufficient IL-33 secretion. These results indicate that the inflammatory caspase 1-generated 1-276 aa mNT-Gsdmd fragment does not contribute to the release of IL-33 and that the generation of the p40 mNT-Gsdmd fragment is independent of the active p20 caspase 1 signaling pathway.
Since BMMs can respond to papain stimulation by generating the p40 mNT-Gsdmd fragment and secreting IL-33, we isolated caspase 1 and caspase 11 double-deficient BMMs and conducted the same stimulation to exclude the involvement of inflammatory caspases 1/11. As reported55, caspase 1/11 double deficiency blocked the generation of the pyroptotic mNT-Gsdmd fragment and the secretion of IL-1β (Fig. 5d, e). However, caspase 1/11 double deficiency in macrophages did not prevent the generation of p40 mNT-Gsdmd or secretion of IL-33 when stimulated with papain (Fig. 5d, f), which revealed that inflammatory caspase 1/11 did not participate in p40 mNT-Gsdmd cleavage. To further exclude the potential function of inflammatory caspases in Gsdmd cleavage and IL-33 release in airway epithelial cells, the pan-caspase inhibitor Z-VAD-FMK (Z-VAD) was pre-cultured with mle12 murine airway epithelial cells before papain stimulation (Fig. 5g). The results showed that neither Gsdmd cleavage nor IL-33 release could be limited by the inhibition of caspase activity, which implied that the caspase family does not contribute to Gsdmd cleavage upon papain stimulation. Thus, there may be other enzymes involved in this protease stress-sensing pathway that mediate Gsdmd cleavage. Taken together, these results indicated that activating the pyroptotic residue 1-276 mNT-Gsdmd fragment with activated inflammatory caspase 1 did not contribute to the release of IL-33. Instead, the papain-induced p40 mNT-Gsdmd fragment promoted IL-33 release via a caspase 1/11-independent pathway.
7. The truncated p40 mNT-Gsdmd fragment contributed to the release of IL-33.
As described above, we observed an association between Gsdmd cleavage and IL-33 secretion in both mle12 airway epithelial cells and BMMs. However, whether this generation of the p40 N-terminal fragment of Gsdmd directly contributes to IL-33 release is still unknown. As we have no specific information on what kind of enzyme contributes to the generation of the p40 Gsdmd fragment, the online pro-protein conversion prediction tool ProP-1.0 was used to predict the possible cleavage sites of functional Gsdmd57. Excluding those amino acid sites within the 1-276 aa fragment generated by caspase 1/8/11 cleavage, we obtained a list of six possible arginine(R) or lysine(K) cleavage sites, among which sites R311, K394, and K409 were chosen for a broad screen to narrow the range of the functional p40 cleavage site (Supplementary Fig. 6).
We constructed predicted possible functional murine Gsdmd fragments (aa 1-311, 1-394 and 1-409) with a Flag-tagged N terminus, and the full-length (FL) form of Gsdmd was used as a non-functional control (Fig. 6a). To further confirm their function in IL-33 release, these truncated fragments were co-transfected into HEK293T cells with C-terminal HA-tagged IL-33 expression vectors. Cell supernatants were collected for immunoblotting and ELISA, and cells were collected for immunoblotting. We observed that compared to full-length Gsdmd, the truncated 1-311aa mNT-Gsdmd fragment presented an extremely high efficiency in promoting nuclear IL-33 release, whereas the other predicted fragments did not exhibit this capability (Fig. 6b, c). Next, we evaluated whether this 1-311aa mNT-Gsdmd fragment also functions in promoting the secretion of the cytosolic mature form of IL-33 (without the N-terminal nuclear localization signal peptide). As predicted, the truncated 1-311aa mGsdmd fragment also worked robustly in promoting cytosolic mature IL-33 release from cells into the culture supernatant, making this fragment unique among these selected truncated forms of mGsdmd (Fig. 6d, e). Differential point mutations and deletions near 311aa were constructed and stably expressed in epithelial mle12 cells via lentiviral infection. Mle12 cells with differentially mutated Gsdmd overexpression were stimulated with papain as described. We found that the amino acid mutation or deletion of residues 308–313 (ELRQQ) in the Gsdmd sequence could efficiently block the generation of the p40 mNT-Gsdmd fragment and prevent IL-33 release in airway epithelial cells, which suggested that the amino acid 308–313 (ELRQQ) sequence is important for the recognition and generation of the functional p40 fragment.
To determine the specific cleavage site of the p35 hNT-GSDMD fragment in human cells, we carried out a similar prediction as described in murine Gsdmd. Three possible truncations, 1-290 aa, 1-320 aa, and 1-327 aa, were cloned into tetracycline-responsive expression vectors and infused with eGFP (Supplementary Fig. 7a). With a HEK293T expression system, we analyzed the functions of these truncated fragments in mediating both nuclear and cytosolic IL-33 release. The results revealed that the corresponding 35-kDa fragment (1-290 aa hNT-GSDMD) presented the unique ability to promote IL-33 release from both the nucleus and the cytosol after tetracycline-induced expression (Supplementary Fig. 7b, c). To confirm the specific cleavage site in hGSDMD, we constructed Flag-tagged hGSDMD expression vectors with several mutations around aa 290. These vectors were transfected into and expressed in hGSDMD-deficient HeLa cells following papain stimulation, and Flag-GSDMD cleavage was analyzed with immunoblotting. We found that the substitution of 5 amino acids around aa 290, i.e., mutating GLRAE into AAAAA, could efficiently block the generation of the p35 hNT-GSDMD fragment after papain exposure, which suggested that the functional p35 fragment cleavage site is located in this region. The site was further narrowed to L289-R290, as deletion of both arginine 290 and leucine 289 could also effectively prevent the generation of p35 hGSDMD (Fig. 7b, c). Together, the results revealed that the human 1-290 aa fragment could sufficiently promote the secretion of IL-33 into the supernatant and that the endogenous cleavage of hGSDMD following papain treatment could be blocked by deletion of L289-R290.
8. GSDMD expression is positively correlated with IL-33 in patients with asthma.
It has been reported that patients with asthma presented higher IL-33 protein expression in the lungs58. As we observed elevated expression of GSDMD in patients, we analyzed both GSDMD and IL-33 protein expression in lung tissues with immunohistochemically staining of serial bronchial sections from a total of 11 patients with asthma. Statistical analysis results showed that the expression of GSDMD and IL-33 was positively correlated in the asthma patients (Fig. 7a, b), which suggested that IL-33 and GSDMD might function through the same axis during type 2 inflammation and work closely together to regulate the pathogenesis and development of asthma.