α-Cyano-4-hydroxycinnamic acid (α-CHCA) (C2020) was obtained from Sigma (St. Louis, MO, USA). BAY 11-7085 (HY-10257) was purchased from MedChemExpress, and U0126 (9903) was from Cell Signaling Technology (Danvers, MA, USA). Medium and fetal bovine serum (FBS) were purchased from Thermo Scientific (Kalamazoo, MI, USA). LDH (ab102526) was purchased from Abcam. Trizol was from Invitrogen (Invitrogen, Thermo Fisher Scientific). Antibodies were obtained from Abclonal. All-in-one™ first-strand cDNA synthesis kit and All-in-one™ qPCR mix were from Genecopoeia™ (Rockville, MD, USA). Other chemical reagents were from Sigma (St. Louis, MO, USA).

As described in Wang et al.’s study (20), CaCO2 and HT29 cell have served as in vitro model to delineate potential function of MCT4 in intestinal enterocytes (characterized by expression of the brush-border enzymes IAP and SI, villin, and Keratin 20 (KRT20) in CaCO2 and HT-29 cells) (24–26), goblet cells (characterized by the increased MUC2 expression in HT29) (27, 28), and Paneth cells as shown by the induction of LYZ in HT29 cells (29). HT-29 and CaCO2 cells were purchased from China Center for Type Culture Collection (Beijing, China) and maintained in DMEM (Thermo Scientific) with 10% fetal bovine serum (FBS) (Gibco, USA) in a humidified 5% CO₂ incubator.

As described in our pervious study (23), lentivirus of MCT4 overexpression was purchased from GenePharma (Shanghai, China) and used to infect HT-29 and CaCO2 cells to establish stable cell lines. Puromycin was from Sigma and used to select for stable cell line.

Cells were harvested and reseeded into 96-well plate. After 24 h, the LDH level was assessed by LDH assay. The optical absorbance at 490 nm was detected. The absorbance value of RPMI 1640 medium served as benchmark, and the absorbance value of completely lysed cells was regarded as the maximal LDH release. The practical value detected by LDH assay was corrected to indicate cytotoxicity.

As described in our pervious study (23), the level of IL-1β was measured IL-18 in the serum and in the supernatants from HT-29 and CaCO2 cells with or without MCT4 overexpression which were collected and quantitated by sandwich ELISA according to the manufacturer’s instructions (Elabscience Biotechnology).

As described in our previously study (30), total RNA was extracted in indicated cells using trizol reagent (Invitrogen) according to the manufacturer’s protocol, and All-in-One First-Strand cDNA Synthesis Kit was used to convert mRNA into cDNA. The relative gene expression was performed for detection using an All-in-One qPCR Mix on an Applied Biosystems StepOnePlus system. Sequences of the primers used in this study are as follows: IL-1β:Forward: 5′-ATGATGGCTTATTACAGTGGCAA-3′, Reverse: 5′-GTCGGAGATTCGTAGCTGGA-3′; IL-18: Forward: 5′-TCTTCATTGACCAAGGAAATCGG-3′, Reverse: 5′-TCCGGGGTGCATTATCTCTAC-3′; GSDMD: Forward: 5′-GTGTGTCAACCTGTCTATCAAGG-3′, Reverse: 5′-CATGGCATCGTAGAAGTGGAAG-3′; NLRP3: Forward: 5′-GATCTTCGCTGCGATCAACAG-3′, Reverse: 5′-CGTGCATTATCTGAACCCCAC-3′; ASC: Forward: 5′-TGGATGCTCTGTACGGGAAG-3′, Reverse: 5′-CCAGGCTGGTGTGAAACTGAA-3′; Caspase-1: Forward: 5′-CCTTAATATGCAAGACTCTCAAGGA-3′’, Reverse: 5′-TAAGCTGGGTTGTCCTGCACT-3′; UBC: Forward: 5′-ATTTGGGTCGCGGTTCTTG-3′ and reverse, 5′-TGCCTTGACATTCTCGATGGT-3′.

Protein extraction and western blot analysis were performed as described (23). Proteins were separated by 10% SDS-PAGE and transferred to PVDF membrane (Millipore, USA) after blocking in PBS with 5% bovine serum albumin (BSA) for 1 h at room temperature. Primary antibodies were used to probe blots against indicated protein, which were incubated overnight at 4°C. HRP-conjugated secondary antibodies, including anti-rabbit and anti-mouse antibodies, were incubated for another 1 h at room temperature subsequently. The bands were visualized using a chemiluminescent imaging system.

Immunofluorescence was performed as in our pervious study (23); intestinal tissues were drawn from patients diagnosed with IBD and fixed in 10% paraformaldehyde. Briefly, the slides were dewaxed and rehydrated in distilled water, sections were immersed in citrate buffer (C₆H₅Na₃O₇·2H₂O) and then microwaved for 20 min for antigen retrieval. Endogenous peroxidase activity was blocked with 0.5% (v/v) H₂O₂. Following blocking with 5% goat serum for 30 min, sections were incubated overnight (4°C) with different primary antibodies. After washing for three times, the section was incubated with appropriate fluorescent secondary antibodies and mounted using DAPI and imaged by fluorescent microscopy.

As described in the study (31), Image J software was used to calculate mean fluorescence intensity. To perform the image segmentation, the histogram of each image was first extended to saturate the gray scale. Then, the images were filtered using a median filter (r = 0.5). Afterwards 12-bit images were converted to an 8-bit gray scale. An algorithm then attributed pixels to the appropriate class of luminosity (255 steps of luminosity) which was arbitrarily divided into: class 1: 1 to 24; class 2: 25 to 50; class 3: 51 to 75; class 4: (the brightest): 76 to 100.

All statistical analysis and graphing were finished using the GraphPad Prism 8 software. The t test was used to determine the significance of differences in the qPCR assay, ELISA assays and LDH assay. One-way ANOVA was performed to determine the significance among groups. A p value of less than 0.05 was considered statistically significant.

We previously showed that overexpression of MCT4 in IECs destroyed intestinal barrier function and induced intestinal inflammation to aggravate IBD (23). To further address whether MCT4 is involved in the cell pyroptosis, we performed WB, real-time PCR, and ELISA assay to analyze the effect of MCT4 on the executor and production of pryoptosis, including GSDMD, IL-1β, and IL-18. The results showed that overexpression of MCT4 in IECs significantly increased relative cell death as evidenced by LDH assay ( Figure 1A ), and real-time PCR assay showed that GSDMD, IL-1β, and IL-18 expression at mRNA level ( Figure 1B ). In line with this, the results from WB further confirmed that ectopic expression of MCT4 drastically increased cleaved GSDMD, mature IL-1β and IL-18 expression in HT-29 and CaCO2 cells ( Figure 1C ). In addition, the ELISA result also demonstrated that ectopic expression of MCT4 in HT-29 and CaCO2 cells led to a significantly enhanced IL-1β and IL-18 release ( Figure 1D ). These results suggested MCT4 could promote cell pyroptosis in HT-29 and CaCO2 cells.

The above results suggested MCT4 has a critical role in cell pyroptosis, which focused us to test the possible change of NLRP3 inflammasomes in response to MCT4 stimulation. As expected, real-time PCR assay revealed that overexpression of MCT4 in HT-29 and CaCO2 cells resulted in a significantly increased NLRP3, ASC, and caspase-1 mRNA ( Figure 2A ); in line with this, WB and intensity quantified results also showed that overexpression of MCT4 enhanced NLRP3, ASC, and caspase-1 expression at the protein level ( Figures 2B–D ), which in turn processed and released the inflammatory cytokines IL-1β and IL-18 by directly promoting pyroptosis through cleavage of the pore-forming protein GSDMD. What’s more, the slight change of cleaved caspase-4 and caspase-5 expression ( Figures 2E, F ) was observed. Taken together, our results showed MCT4 triggered cell pyroptosis in a caspase-1-dependent pathway.

Our previous study have demonstrated that MCT4 expression was increased in intestinal mucosal tissue of IBD (22), and overexpression of MCT4 in IECs destroyed CREB-mediated ZO-1 expression and promoted NF-κB-induced IL-6 expression (23). While α-Cyano-4-hydroxycinnamic acid (α-CHCA), a classical MCT inhibitor, has been reported to inhibit proliferation and induce p38-mediated apoptosis in murine pancreatic adenocarcinoma cell line 6606PDA and 7265PDA (32), which focused us to explore the potential mechanism through which MCT4 regulated cell pyroptosis. Our results showed that MCT4 overexpression in CaCO2 cell significantly increased phosphorylation of ERK1/2(p44/42) and NF-κB p65(Ser536) ( Figure 3A ), while α-CHCA treatment caused an inhibition of ERK1/2 and NF-κB activity ( Figure 3B ).

To further determine that phosphorylation of ERK1/2 and NF-κB was involved in MCT4-mediated pyroptosis, we analyzed whether inhibition of ERK1/2 and NF-κB activation could rescue the effect of MCT4 on pyroptosis. As shown in Figures 3C, D , NF-κB inhibitor BAY 11-7085 and U0126, an inhibitor of ERK1/2, could reverse the promotion effect of MCT4 on IL-1β expression, which is attributed to the disruption of NLRP3 inflammasomes’ form.

The above results suggested the critical role of MCT4 in cell pyroptosis, so we further focused on exploring the clinical relationship between MCT4 and pyroptosis-related proteins. As shown in Table 1 , we have collected 45 subjects, including 30 patients with IBD and 15 healthy controls with aged between 1 and 16 years in Guangzhou Women and Children’s Medical Center. The study was divided into 15 healthy control subjects, consisting of 11 boys and four girls, and 30 patients with IBD, including 13 boys and 17 girls, respectively. Detailed clinical characteristics of the subjects, which are not public, could be available upon reasonable request.

We detected MCT4 and Caspase-1 expression in a set slide tissue by IF to analyze their possible relationship with in IBD. The results showed that both MCT4 and Caspase-1 expression levels were increased in the intestinal epithelial cells labeled with E-cadherin in patients with IBD ( Figure 4A ). Further analysis of fluorescence intensity showed that MCT4 is positively correlated with Caspase-1 ( Figure 4B ). Also, we have detected GSDMD expression, a substrate of Caspase-1, in healthy control and IBD. The results showed that GSDMD is upregulated in IBD compared with that in healthy control ( Figure 4C ). Taken together, these findings suggested that both MCT4 and cell pyroptosis related protein, including caspase-1 and GSDMD, were increased in IBD, and MCT4 contributed to cell pyroptosis through caspase-1-mediated GSDMD activation.