Pyroptosis of oral keratinocyte contributes to energy metabolic reprogramming of T cells in oral lichen planus via OPA1-mediated mitochondrial fusion

Oral lichen planus (OLP) is a chronic inflammatory disease that is associated with an increased risk of carcinogenesis. The typical pathological features of OLP include submucosal T-cell banding, infiltration, and liquefactive degeneration of basal epithelial cells. However, the histological appearance of basal cell death cannot be explained by apoptosis of keratinocytes alone. The aim of this study was to explore a novel mechanism of epithelial cell death, pyroptosis, and its role in the development of OLP. The immunohistochemical results initially revealed pyroptosis in the epithelial cells of OLP. There was significant upregulation of pyroptosis-related inflammatory cytokines, specifically IL-1β. The expression of IL-1β is closely related to the severity of the patient’s condition. In vitro, the culture supernatant from epithelial cells and exogenous IL-1β significantly promote the proliferation and activation of T cells. This effect can be inhibited by neutralizing antibody or receptor inhibitor of IL-1β. Stimulation with exogenous IL-1β enhances both glycolysis and oxidative phosphorylation in T cells, with a more pronounced increase in glycolysis. This is due to the regulation of NAD+ availability and mitochondrial dynamics by IL-1β. IL-1β specifically stimulates the expression of optic atrophy 1 (OPA1), particularly L-OPA1, which promotes mitochondrial fusion and increases NAD+ availability. This process upregulated glycolysis in T cells. The knockdown of OPA1 reverses these changes by reducing the proliferation and activation of T cells. In this study, IL-1β promoted OPA1 transcription by activating the NF-κB pathway. The expression of OPA1 is inhibited by the inhibitor of NF-κB pathway. These results suggest that OLP keratinocytes undergo pyroptosis, which then secrete inflammatory factors that activate the NF-κB signaling pathway of T cells. This pathway regulates OPA1-mediated mitochondrial fusion and energy metabolism reprogramming in T cells, contributing to the development of OLP. These findings provide new insights into the mechanisms and therapeutic strategies for OLP.

(c) The glycolysis-related protein levels in OLP-T cells with or without knock down OPA1, under indicated treatment (upper), semi-quantitative analysis using Image J (lower).(e) The protein levels of glycolysis-related genes in OLP-T cells under indicated treatment, semiquantitative analysis using Image J.

Samples, Immunohistochemistry (IHC) analyses and histopathology
The acquisition and execution of the experimental materials were approved by the Committee on Human Research of the School and Hospital of Stomatology at Sun Yat-sen University (KQEC-2024-05-02).The study sample included 19 male and 11 female patients.Written informed consent was obtained from all the patients.Oral mucosal tissues were collected from 11 healthy individuals and 30 patients diagnosed with OLP, of whom 15 were non-erosive and erosive.Tissues were fixed and embedded in paraffin.5-µm sections were stained with haematoxylin and eosin.The formalin-fixed, paraffin-embedded oral mucosal tissues were prepared, sectioned at 4 micrometers and analyzed for the presence of indicated antigen on the basis of standard immunohistochemistry protocols.The tissue sections were incubated with the indicated antibodies overnight at 4 °C.Subsequently, HRP peroxidase-conjugated anti-rabbit or antimouse secondary antibodies were added and incubated for 1h at 37 °C.Images were acquired at 200× and 400× magnification using a Leica Aperio AT2 microscope (#8482, Leica, Germany).The staining results were evaluated semi-quantitatively based on the ratio of the staining intensity to the proportion of positive cells.
The positive signal appeared as brownish-yellow staining in the cell nuclear/cytoplasm.All slides were evaluated by two senior oral pathologists independently.The staining results were semiquantitatively evaluated, based on the ratio of the staining intensity and proportion of positive cells.The intensity score was defined as 0, negative; 1, weak; 2, moderate; or 3, strong, brown.The proportion score was defined as 0, negative; 1, < 10%; 2, 11-50%; 3, 51-80%; or 4, > 80% positive cells.The immunoreactive score was calculated as the intensity score × proportion score [1].
The inclusion of OLP cases was based on the diagnostic criteria of the World Health Organization, and the diagnosis of OLP was confirmed through pathological examination.Patients with any of the following conditions were excluded from consideration: (1) pregnant or breastfeeding; (2) individuals with drug allergies, including those allergic to anesthetics; (3) patients for whom a biopsy was not feasible due to various factors; (4) individuals with other systemic or autoimmune diseases; (5) heavy smokers and drinkers; (6) those with other diseases or infections related to the oral mucosa; (7) patients who had taken antibiotics or immunomodulatory drugs within the last three months.

Disease scores of patients with OLP
OLP was diagnosed as follows: (1) presence of bilateral, symmetric white striations or papules, with or without erythema/erosions and/or ulceration (ulceration was defined as yellow-white fibrin membrane), and (2) a biopsy read as OLP.Clinical signs of OLP were measured using a semiquantitative Reticulation/keratosis, erythema, and ulceration (REU) scoring system (Table.S1).In this scoring system, the oral cavity was divided into 10 sites: right buccal mucosa, left buccal mucosa, tongue dorsum, tongue ventrum, maxillary gingiva, mandibular gingiva, floor of mouth, hard palatal mucosa, soft palate and tonsil, and labial mucosa (upper and lower together).The severity of the lesion in each site was scored according to the following: presence of reticular/hyperkeratotic/white papular (R) lesions (0 = none, 1 = presence), presence of each erosive/erythematous (E) lesions and/or ulcerative (U) lesions (0 = none, 1 = lesions smaller than 1 cm 2 , 2 = lesions from 1 to 3 cm 2 , 3 = lesions larger than 3 cm 2) .Each REU score was totaled from all 10 areas and the total weighted score was a summation of reticulation score, erythematous score (weighted 1.5), and ulcerative score (weighted 2.0) [2].

Isolation of oral keratinocytes and CD3 + T cells
Biopsies of oral mucosa were collected from patients were diagnosed was obtained under sterile conditions and divided into two even parts for pathological examination or cell culture.Normal oral mucosal tissue is derived from normal oral mucosal tissue obtained from orthognathic surgery, periodontal surgery, implant surgery or mandibular third molar extraction at the Oral and Maxillofacial Surgery Department.Mucosal samples were placed in DK-SFM medium (10744019, Thermo fisher Scientific) containing penicillin (100 U/mL) and streptomycin (100 μg/mL) (15140122, Gibco USA).
Mucosal samples were transported to the laboratory on 4℃ and, under sterile conditions, rinsed with phosphate-buffered saline (PBS) containing 2% antibiotics to remove blood.The samples were digested with 0.25% Dispase II (4942078001-1, Roche) at 4°C for 12 hours or in a 37°C water bath for 2 hours to separate the epithelial and connective tissues.After digestion, epithelial and subepithelial layers were gently peeled apart under a microscope using microscopic surgical tweezers.The isolated epithelium was rinsed three times with PBS, followed by microsurgical cutting of the epithelial tissues while maintaining tissue moisture.Subsequently, the tissues were oscillated and digested with 0.025% trypsin without EDTA at 37°C for 5 minutes.Digestion was terminated with serum-containing medium, and after centrifugation, the supernatant was discarded [3].DK-SFM medium serves as an inhibitor of fibroblast growth, making it suitable for the extraction of epithelial cells that are prone to fibroblast contamination.
Peripheral blood mononuclear cells (PBMCs) were isolated from blood samples of healthy individuals and patients with OLP using human peripheral blood lymphocyte isolation fluid (LTS1077, TBD) by density gradient centrifugation.CD3 + T cells were isolated from PBMCs using the EasySep™ Human CD3 Positive Selection Kit II (17851, Stemcell), according to the manufacturer's instructions.In brief, collect the PBMCs obtained in the previous step and resuspend in PBS, move to a flow tube, mix well, centrifuge at 2000 rpm for 5 min, and discard the supernatant.Resuspend, add CD3 antibody and incubate at 37°C for 5 min, then add magnetic beads and mix gently, incubate at 37°C for 10 min, flicking the flow tube every 3 min.Add 2 ml PBS mixed and place the flow tube into the magnetic rack (18000, Stemcell) for 5 min.The lymphocytes will be attracted to one side wall by the magnetic rack, discard the supernatant, resuspend and add CD3 antibody and magnetic beads and incubate at 37°C for a total of 3 times.The isolated cells were resuspended and cultured with T cell medium.

Culture of Cells and drug treatments
The keratinocytes were resuspended in DK-SFM medium containing penicillin (100 U/mL) and streptomycin (100 μg/mL).The cultures were incubated in an incubator (Thermo fisher Scientific, USA) with 5 % CO2 and saturated humidity at 37 °C.Medium was replaced every 2 to 3 days.
The conditional co-culture system is achieved through indirect co-culture, in which half of the culture supernatant from keratinocytes was mixed with half of the culture medium from T cell.

Cell proliferation experiments and Flow Cytometry
For cell proliferation experiments, CD3 + T cells were labelled with CellTrace™ CFSE (Invitrogen), were activated or not activated on a 24-well plate pre-coated with 5 µg/ml anti-CD3 in T cell culture medium supplemented with 2 µg/ml anti-CD28.CFSE dilution was assessed by flow cytometry on day 5 post-activation.All samples were acquired with BD LSRFortessa flow cytometer (Becton, Dickinson and Company) and analyzed with FlowJo software (TreeStar).

Imaging analysis of mitochondrial structure
MitoTracker (M7512, Thermo Fisher Scientific) and Hoechst 33342(C1025, Beyotime) staining were performed according to the manufacturer's instructions.For live imaging of mitochondria in cells, T cells were purified and activated as indicated, transferred to glass bottom dishes (801002, NEST) precoated with poly-D-lysine (C3012, Beyotime) in complete medium, and acquired using Olympus Confocal Microscope FV3000 with a 100× objective lens.Cells were kept in a humidified incubation chamber at 37°C with 5% CO2 during acquisition.For nuclei visualization, Hoechst 33342 was added prior to acquisition.For the analysis of mitochondrial morphology in fixed cells, cells from the indicated genotypes were activated as indicated, stained with Mitotracker red 45min at 37 °C, allowed to settle for 30min at 37 °C into poly-D-lysine-coated coverslips, fixed 10min with 2.5% glutaraldehyde.At least 5-10 fields were randomly acquired per condition and biological replicate.Confocal images were analyzed using Image J [4].Mean of mitochondria number, mean area and perimeter, and parameters representing the mitochondrial network (branches, branch lengths, branch junctions) were analyzed quantitatively.
For the mitochondria images captured by confocal microscopy, each group randomly selected more than 15 random fields of view from three or more biological replicates.Each field of view includes several cells, with a total cell count of over 100 across all fields of view.The mean mitochondria in cells and mean mitochondrial area in cells refers to the average number and average area divided by the number cells in the field.Mean area and perimeter represent the area and perimeter averaged to a single mitochondrion in the field.The network structure of mitochondria is evaluated based on the number of branches, average branch length, and number of branch junctions' points.
For transmission electron microscopy (TEM), cells were fixed in Gluta fixative (for electron microscopy, 2.5% glutaraldehyde).Following fixation, the fixed cells were centrifuged.The 0.1 M PB (pH7.4) was added into the tube after supernatant was discarded, and then the precipitation was resuspended and washed in PBS for 3times, 3min each.The 1% agarose solution was prepared by heating and dissolving in advance.After being cooled, the agarose solution was added into the EP tube.Before agarose solidification, the precipitation was suspended with forceps and wrapped in the agarose.Agarose blocks with samples avoid light post fixed with 1% OsO4 in 0.1 M PBS for 2h at room temperature.After remove OsO4, the tissues are rinsed in 0.1 M PBS for 3 times, 15min each.Dehydrate at room temperature, Resin penetration and embedding, pour the pure EMBed 812 into the embedding models and insert the tissues into the pure EMBed.The embedding models with resin and samples were moved into 65℃ ovens to polymerize for more than 48h.The resin blocks were cut into 60-80nm thin on the ultra-microtome, and the tissues were fished out onto the 150 meshes cuprum grids with formvar film.2% uranium acetate saturated alcohol solution avoid light staining for 8 min, rinsed in 70% ethanol for 3 times and then rinsed in ultra-pure water for 3 times.2.6% Lead citrate avoid CO2 staining for 8 min, and then rinsed with ultra-pure water for 3 times.After dried by the filer paper, the cuprum grids were put into the grids board and dried overnight at room temperature.The cuprum grids are observed under Transmission Electron Microscope (HT7800/HT7700; HITACHI).

Metabolic assays
Seahorse XFe96 Extracellular Flux Analyzer was used to perform extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) measurements.The Seahorse XF ATP rate or Glycolytic Rate Assay Kit (Agilent Technologies) was used, according to the manufacturer's instructions.All materials and compounds were obtained from Agilent Technologies.
The specific protocol is as follows: On the day before the experiment, the probe plate was hydrated, and more than 20 mL of detection solution was prepared in centrifuge tubes and left overnight in a non-CO2 incubator with the probe plate.On the day of the experiment, the culture medium was prepared in advance by mixing 97 mL of medium with 1 mL each of glutamine, glucose, and pyruvate, all sourced from Agilent.T cells were seeded at a density of 0.4-3.0×10^5cells per well in the XF96 cell culture microplate, which was pre-coated with poly-D-lysine and pre-incubated in a 37°C incubator for 2 hours.
Cells were then incubated at 37°C in a non-CO2 incubator for 25 minutes while the assay kit reagents, including Rot/AA, 2-DG, or oligomycin, were prepared.After incubation, T cell adhesion and density were observed under a microscope, and each well was replenished with 180 μL of culture medium.The plate was then centrifuged at 200g for 1 minute in a horizontal centrifuge with gradual acceleration and deceleration.Following this, the plate was returned to the 37°C non-CO2 incubator for an additional 20-30 minutes before analysis.ECAR and OCR were measured during the basal stage (basal glycolysis + mitochondrial acidification), in response to Rot/AA (inhibitors of the mitochondrial electron transport chain, inducing compensatory glycolysis), and post-2-deoxy-D-glucose (a glucose analog) acidification.
The basal and compensatory glycolytic rates were calculated using the Seahorse Glycolytic Rate Assay Report Generator, and account for contribution of CO2 to extracellular acidification derived from mitochondrial respiration.The intracellular NAD + /NADH levels were measured using the NAD + /NADH assay kit with WST-8 (#S0175, Beyotime, China) according to the manufacturer's instructions.5× 10 6 cells were harvested by centrifugation at 200 g for 5 minutes at 4 °C, and the medium was subsequently removed.The cells were lysed in 200 μL of pre-cooled NAD + /NADH lysis buffer, followed by centrifugation at 12,000 g for 10 minutes at 4 °C.Subsequently, 50-100 μL of the supernatant was transferred to a new tube and incubated at 60 °C for 30 minutes, followed by centrifugation at 10,000 g for 5 minutes at 4 °C.A 20 μL aliquot of the supernatant was then transferred to 96-well plates, incubated for 10 minutes at 37 °C, and followed by the addition of 10 μL of the color-developing solution.The NAD + /NADH ratio was determined by measuring the absorbance of the mixture at 450 nm [5].

Cell transfection
RiboBio (Guangzhou, China) chemically synthesized siRNAs.A negative control siRNA (siRNA-NC) with random sequences targeting unknown mammalian genes was used.Transfection was carried out using Lipofectamine 3000 (L3000075, Thermo Fisher Scientific) for 8 h, and then the medium was changed by half.The efficacy was routinely checked by RT-qPCR 4d after transfection, cells were collected and used for indicated assays.
siRNA number name target sequence

Bioinformatics
The search for promoter sequences of human genes was carried out in the Eukaryotic Promoter Database (EPD) (database URL: https://epd.expasy.org/epd/accessed by the 1 October 2023).

Chromatin immunoprecipitation assay (ChIP)
The ChIP assay was performed using the ChIP Assay Kit (Beyotime, China) following the manufacturer's guidelines.Briefly, CRC cells were cross-linked with 37%formaldehyde solution for

Statistical analysis
Statistical analyses were performed using SPSS 24.0 (USA) and Microsoft Excel.Statistical significance was set at p < 0.05.Data are presented as mean ± SEM. (standard error of the mean).
Comparisons for two groups were calculated using unpaired two-tailed Student's t-tests (for two groups meeting the normal distribution criteria, according to the Shapiro-Wilk normality or Kolmogorov-Smirnov test) and Mann Whitney test (for two groups that do not meet the normal distribution criteria).
Comparisons of more than two groups and grouped data were calculated using one-way or two-way ANOVA (for groups meeting gaussian distribution) and corrected for multiple comparisons between groups or to a reference group using Dunnett's tests, or Kruskal-Walli's test (for groups do not meet gaussian distribution), Brown-Forsythe and Welch ANOVA tests were selected for ANOVA when the variance was not homogeneous.No statistical method was used to predetermine sample size, but a minimum of three samples were used per experimental group or condition.Experiments were not randomized.
supplementary tables 1-3 The higher the score, the greater the likelihood of predicted binding.

Fig. S2 .
Fig. S2.IL-1β regulates the metabolic reprogramming of T cells.(a) Seahorse ® analysis of the glycolysis rate in OLP-T cells showed variations in the total PER and glycolytic PER over time, and ECAR over time (b), quantification by Agilent Seahorse Wave.(c) The protein levels of glycolysis-related genes in OLP-T cells under indicated treatment, semiquantitative analysis using Image J. (d) Seahorse ® analysis of ATP production rate and source changes in OLP-T cells, quantification by Agilent Seahorse Wave.* P＜0.05.**P < 0.01.***P < 0.001.OLP-T cells, T cells from peripheral blood of patients with OLP; 2-DG, 2-deoxy-D-glucose; PER, Proton Efflux Rate; OCR, Oxygen Consumption Rate; ECAR, Extracellular Acidification Rate.

Fig. S3 .
Fig. S3.IL-1β upregulates OPA1 expression in OLP-T cells, siRNA knockdown of OPA1.(a-b) The protein levels of mitochondrial dynamic-related genes in OLP-T cells under indicated treatment, semi-quantitative analysis using Image J. (c) The mRNA levels of OPA1 in OLP-T cells under 3 different siRNAs to knockdown OPA1.

Fig. S4 .
Fig. S4.IL-1β promotes mitochondrial fusion and glycolytic activity in OLP-T cells.(a) Mitochondria in OLP-T cells with or without knock down OPA1, labelled with Mitotracker captured by confocal microscopy under indicated treatment, confocal images were analysed using Image J to assess the size and network structure of mitochondria.(b) Seahorse ® analysis of the glycolysis rate in OLP-T cells with or without knock down OPA1, under indicated treatment, quantification by Agilent Seahorse Wave.

Fig. S5 .
Fig. S5.IL-1β promotes mitochondrial fusion and glycolytic activity in OLP-T cells through the NF-kB pathway.(a) The protein levels of NF-κB pathway-related genes in OLP-T cells under indicated treatment, semiquantitative analysis using Image J. (b) Agarose gel electrophoresis after ChIP-qPCR analysis for revealing potential binding sites of RELA (p65) within the OPA1 promoter region.(c) Mitochondria in OLP-T cells labelled with Mitotracker captured by confocal microscopy under indicated treatment, confocal images were analysed using Image J to assess the size and network structure of mitochondria.(d) Seahorse ® analysis of the glycolysis rate in OLP-T cells under indicated treatment, quantification by Agilent Seahorse Wave.
10min at room temperature and quenched with 125mM glycine.DNA fragments ranging from 200 to 500 bp were obtained by ultrasonication.Then the lysate was immunoprecipitated with anti-RELA (p65) or IgG antibodies.Immunoprecipitated DNAs were analyzed by RT-qPCR.Four hypothesized RELA (p65) binding sites (P1-P4) within the OPA1 promoter were selected, and corresponding primers were designed and synthesized, the primers were:

Table S2 . OLP erosive/non-erosive patient clinical parameter scoring sheet
OLP, oral lichen planus; M, male; F, female.Difference was considered significant at P < 0.05