NLRP3-IN-2 (4-(2-(5-chloro-2-methoxybenzamido)ethyl)benzenesulfamide), JC124 (5-chloro-2-methoxy-N-(2-(4-(N-methylsulfamoyl)phenyl)-ethyl)benzamide), and MNS (3,4-methylenedioxy-β-nitrostyrene) (purity >99%) were purchased from MedChemExpress (New Jersey, United States).

IL-1β, IL-6, IL-12, and tumor necrosis factor-α (TNF-α) enzyme-linked immunosorbent assay (ELISA) kits were purchased from Thermo Fisher Scientific (Waltham, United States).

Antibodies against NLRP3 (ab270449), ASC (ab175449), IL-1β (ab254360), MUC-2 (ab272692), and HRP Anti-Rabbit IgG (ab288156) were purchased from Abcam Systems (Cambridge, United Kingdom). Antibodies against claudin1 (13050-1-AP) were purchased from Proteintech (Chicago, United States). Antibodies against caspase-1 (24232S), P65 (8242S), P-P65 (3033), β-actin (4970), GAPDH (5174), and Ly-6G (87048) were purchased from Cell Signaling Technology (Boston, United States).

Nigericin (tlrl-nig), ATP (tlrl-atp), MSU (tlrl-msu), and silica (tlrl-sio) were purchased from InvivoGen (San Diego, United States). Disuccinimidyl suberate (DSS) was purchased from MP (Waltham, United States).

Flow cytometry antibodies, FITC-CD45 (109806), Pe-Cy5-CD4 (100540), APC-F4/80 (123116), PE-IL-17A (506904), APC-IFN-γ (505809), PerCP-Cy5.5-CD11b (101228), and the Zombie dye kit (423106), were purchased from Biolegend (San Diego, United States). Fixation/permeabilization buffer and permeabilization buffer were purchased from BD Bioscience (Franklin Lake, United States).

Bone marrow cells were collected from C57BL/6 mice by flushing dissected tibias and femurs with aseptic PBS. The cells were cultured for 7 days in DMEM with 10% fetal bovine serum and 1% penicillin–streptomycin. 30% L929 cell-conditioned medium was added to promote bone marrow–derived macrophage (BMDM) differentiation. The cells were cultured in a humidified incubator with 5% CO₂ at 37°C.

BMDMs were plated in 96-well plates (5.0 × 10⁴ cells/well) before being incubated with NLRP3-IN-2, JC124, or MNS (0.5–20 μM) for 2 h and then assayed by a lactate dehydrogenase (LDH) assay kit (Beyotime Biotechnology, Shanghai, China).

BMDMs were seeded in 24-well plates (1.0 × 10⁶ cells/well) and primed with LPS (100 ng/ml) for 4 h. After that, the cells were incubated with inflammasome inhibitors for 1 h, and then, the cells were treated with nigericin (5 μg/ml) for 30 min or ATP (5 mM) for 2 h or silica (100 μg/ml) for 6 h or MSU (100 μg/ml) for 6 h.

The cell supernatant was collected after the activation of the inflammasome and centrifuged at 12,000 g for 1 min. Using relevant ELISA kits, IL-1β and TNF-α were assayed. Similar quality colon tissues were harvested from each group and incubated in DMEM. The culture supernatants were centrifuged for 1 min at 12,000 g and then IL-1β, IL-12p40, IL-6, and TNF-α levels were quantified by ELISA.

The cells were collected and lysed in lysis buffer. The sample lysates were sonicated and centrifuged at 12,000 g for 5 min, and the supernatant was collected. Protein samples were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then transferred to nitrocellulose membranes. The membranes were blocked in 5% skim milk in TBS + Tween 20 (TBST) for 1 h at room temperature. After that, the membranes were incubated with primary antibodies (caspase-1, IL-1β, ASC, NLRP3, P65, P-P65, and β-actin) overnight at 4°C. The next day, after washing in TBST, the membranes were incubated with peroxidase-conjugated secondary antibody for 1 h at room temperature. Protein bands were visualized with a high-signal enhanced chemiluminescence Western blotting substrate (Tanon, China) using a Tanon-Chemi imaging system (Tanon-5200).

BMDMs (6 × 10⁵ cells/well) were placed in 24-well plates. After inflammasome activation, the cells were fixed in 4% paraformaldehyde for 20 min at room temperature, washed with PBS three times, followed by permeabilization with 0.2% Triton X-100, and blocked with 10% goat serum in PBS for 60 min. After that, we poured out the blocking solution and incubated with anti-ASC antibody (1:100) diluted in 5% goat serum in PBS at 4°C overnight. Then, BMDMs were washed with PBS, incubated with Alexa Flour 488 goat anti-rabbit IgG (Invitrogen, Carlsbad, United States) diluted in PBS (1:1000) for 2 h, and nuclei were stained with DAPI (Beyotime Biotechnology, Shanghai, China) for 1 h. The ASC-speck formation was imaged under a positive fluorescence microscope (Nikon, Japan).

Male C57BL/6 mice (weighing 25–27 g at 8–10 weeks old) were purchased from Charles River Laboratories (Beijing, China). They were kept in a 12 h reverse light/dark cycle, with ad libitum access to food and water.

The animals were divided into three groups. The control group (n = 5) received no DSS or inhibitors. The DSS group (n = 5) was treated with 3% DSS to induce colitis but no inhibitors. Animals in the DSS + MNS group were simultaneously treated with 3% DSS and MNS (30 mg/kg/day) for 5 days. According to a previously described protocol (Zaki et al., 2010), the disease activity index (DAI) was used to record weight loss, fecal diarrhea, and the presence of fecal blood. After 7 days, the mice were humanely killed by cervical dislocation. The colon tissue was excised for subsequent analysis. The animal experiments were conducted in strict accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals, approved by the Ethics Committee of Ruijin Hospital, Shanghai Jiao Tong University School of Medicine.

Approximately 1 cm of colon tissue was fixed in 4% formalin. The samples were then dehydrated and embedded in paraffin. Histological sections were stained with hematoxylin and eosin (H&E) and observed under an optical microscope (Nikon, Japan). Colitis severity was evaluated using a lesion score system. 1) Inflammation severity of the intestinal wall (0.5 = extremely mild; 1 = mild; 2 = moderate; 3 = intense inflammation). 2) Lesion severity (1 = mucosal layer; 2 = mucosa; 3 = submucosa or transmural injury). 3) Crypt damage (0 = no significant damage; 1 = 1/3 crypt damage; 2 = 2/3 crypt damage; 3 = crypt loss but surface epithelium intact; and 4 = crypt and surface epithelium missing). The scores were summed up to generate the final score. The average score [mean ± standard error of the mean (SEM)] of each animal in each group was calculated, and t-tests were performed among different groups.

Neutrophil infiltration into inflamed bowel mucosa was determined by an MPO activity assay using the O-dianisidine method (Qian et al., 2020). The protein extracted from colonic tissue was used to evaluate the MPO level according to the manufacturer’s instructions (Beyotime Biotechnology, Shanghai, China).

Colon tissue sections were deparaffinized, rehydrated, and blocked. Then, the sections were incubated with the following primary antibodies at 4°C overnight: Ly-6G (1:800), claudin-1 (1: 400), and MUC-2 (1:1000) overnight at 4°C. After that, the sections were incubated with an HRP Anti-Rabbit IgG antibody (1:800) for 1 h at room temperature. Positively stained sites were visualized by incubating with peroxidase-labeled streptavidin-complex (Beyotime Biotechnology, Shanghai, China), and the nuclei were counterstained with hematoxylin. The immune-positive sites were recognized by yellowish-brown staining, and the blue or purple staining showed the nuclei. The images were captured with a Nikon confocal microscope (Tokyo, Japan).

The colon tissue was collected when mice were killed and immediately minced and digested with collagenase IV (Sigma-Aldrich, MO, United States) 300 U/ml and a hyaluronidase solution (Sigma-Aldrich, MO, United States) 200 U/ml in Roswell Park Memorial Institute 1640 medium supplemented with penicillin and streptomycin and incubated at 37°C for 1 h. The cell suspension was centrifuged at 12,000 g for 5 min and mechanically dissociated through a sterile nylon mesh filter. For intracellular cytokine analyses, the cells were stimulated in complete medium plus a Cell Activation Cocktail and Brefeldin A for 4 h at 37°C. The assay antibodies included anti–FITC-CD45, APC-F4/80, Pe-Cy5-CD4, PE-IL-17A, APC-IFN-γ, PerCP-Cy5.5-CD11b, and also a Zombie NIR dye. Data were acquired on an LSR-II system (BD Biosciences) and analyzed using FlowJo software (V10, Becton Dickinson, Ashland, OR, United States).

Statistical analyses were conducted using two-tailed Student’s tests for comparisons between two groups and a one-way analysis of variance (ANOVA) followed by Dunnett’s test for comparisons between three or more groups. The results were expressed as the mean ± standard error of the mean (SEM). p < 0.05 indicated statistical significance. All analyses were carried out by GraphPad Prism v8.0 (La Jolla, San Diego, United States).

To assess the toxicity of NLRP3-IN-2, JC124, and MNS (Figure 1A) on BMDM viability, LDH assays were performed. Treatment with any of the three compounds up to 20 μM did not generate any cytotoxicity (Figure 1B). Thus, in the subsequent experiments, we used inhibitors at concentrations of 1–10 μM, a safe range for BMDM viability. NLRP3-IN-2, JC124, and MNS were added before NLRP3 inflammasome activation to investigate their possible inhibition. Of them, MNS showed significant inhibitory effects toward IL-1β production in a concentration-dependent manner (Figure 1C). To further confirm the inhibitory effects of MNS on the NLRP3 inflammasome, we stimulated BMDMs with ATP, silica, and MSU (monosodium urate crystals) separately. IL-1β secretion was significantly increased after using these stimulants, while it was decreased with the treatment of MNS in a dose-dependent manner (Figure 1D). These evidences suggested that MNS could inhibit IL-1β secretion effectively.

The inflammasome contains multiple proteins, and the expression of these proteins affects its activity in turn (Martinon et al., 2002). Therefore, we examined the protein levels of NLRP3, ASC, pro–IL-1β, pro–caspase-1 in cell lysates, and those of caspase-1 and its substrate IL-1β in cell supernatants by immunoblotting. Caspase-1 and IL-1β were reduced, while NLRP3, ASC, pro–IL-1β, and pro–caspase-1 remained unchanged (Figure 2A). ASC specks were observed upon ASC oligomerization, which thereby reflected NLRP3 inflammasome activation (He et al., 2014). To investigate whether MNS suppressed IL-1β release by reducing ASC aggregation, we performed immunofluorescence on ASC specks and observed that nigericin triggered ASC speck formation in macrophages in the nigericin group. Compared to this, ASC specks decreased significantly in the MNS-treated group in an MNS dose-dependent manner (Figures 2B,C; Supplementary Figure S1). Therefore, MNS could inhibit ASC speck formation. We next examined the important proteins of nuclear factor-κB (NF-κB), p65 and P-p65, which are the primary stages of NLRP3 inflammasome formation (Hayden and Ghosh, 2008). Western blotting showed that these protein levels were unchanged (Figure 2D). In addition, the effect of MNS on LPS-induced TNF-α secretion levels was investigated, and a negligible change in TNF-α levels was observed at MNS concentrations of 0.5, 1, 5, and 10 μM (Figure 2E). Collectively, the inhibitory effects of MNS on NLRP3 inflammasome activation were pronounced, whereas NF-κB expression was unaffected.

To assess whether MNS protects against colitis in vivo, we designed an experiment using a DSS-induced colitis mouse model (Figure 3A). Both the DSS and DSS + MNS groups recorded weight loss on the fourth day, but with time, weight loss and disease activity index (DAI) in the DSS + MNS group improved compared with those in the DSS group (Figures 3B,C). We also observed a significant reduction in colonic length and splenomegaly in the DSS + MNS group compared to that in the DSS group (Figures 3D,E; Supplementary Figures S2A–C). In conclusion, our data suggest that MNS could significantly alleviate colitis symptoms of mice.

To observe the effects of MNS on intestinal tissue, we performed H&E staining on colon tissues from each group (Figure 4A). DSS treatment caused extensive colonic damage, accompanied by epithelium loss and crypt structure collapse compared with DSS + MNS treatment (Figures 4A,B). Myeloperoxidase (MPO) assay and immunohistochemical staining of Ly-6G were used to evaluate the recruitment and infiltration of neutrophils in mouse colon tissue. The results show that neutrophils significantly increased in the DSS group compared with the DSS + MNS group (Figures 4C,D). Colonic mucus is the first physical barrier that protects the colon against toxins and pathogenic invasion and is produced and maintained by glycosylated mucin-2 (MUC-2) and claudin-1 (Ambort et al., 2012; Citalán-Madrid et al., 2017; Chen et al., 2021). Immunohistochemistry staining revealed that the protein expression levels of MUC-2 and claudin-1 were significantly reduced in the DSS group, but the levels of these proteins in the DSS + MNS group were similar to those of the control (Figure 4E). Thus, MNS treatment retarded histopathological changes, protected the intestinal mucosa from damage, and maintained the intestinal barrier.

In patients with IBD, considerable macrophages usually infiltrate the intestine (Bernardo et al., 2018; Wright et al., 2021). To explore this further, we used flow cytometry to examine the number and proportion of F4/80+ and CD11b+ labeled macrophages in colon tissues. Compared with the DSS group, the MNS + DSS group displayed reduced intestinal macrophage infiltration (Figures 5A,B). Secretion of macrophage-derived inflammatory cytokines, such as IL-1β, IL-12p40, and TNF-α, is required for T helper 17 (Th17) and T helper 1 (Th1) cell differentiation (Conforti-Andreoni et al., 2011). IL-17A is the marker of Th17, and IFN-γ is the marker of Th1 (Harbour et al., 2015), so we investigated the expression of IL-17A and IFN-γ by flow cytometry. The results showed that IL-17A⁺IFN-γ^-CD4⁺ T cells (Th17) decreased slightly in the DSS + MNS group compared with those in the DSS group, but there was no significance (Figures 5C,D). However, IL-17A^-IFN-γ⁺CD4⁺ T cells (Th1) and IL-17A⁺IFN-γ⁺CD4⁺ T cells decreased significantly in the DSS + MNS group when compared with the DSS group (Figures 5C,D). In addition, the levels of TNF-α, IL-1β, IL-6, and IL-12p40 in colon tissue were measured by ELISA, which were all reduced in DSS + MNS animals, and significance was only observed in IL-1β and IL-12p40 (Figure 5E). These data show that MNS attenuated macrophage infiltration and IL-17A⁺IFN-γ⁺CD4⁺ T cell differentiation and IL-1β, IL-12, and IL-6 secretion.