Chemicals and reagents
TNF-α (PHC3011), IFN-γ (PHC4033), TRAIL (PHC1634) were from Invitrogen-Gibco (Grand Island, New York, USA). sFasL (126-FL) and granulysin (3138-GN/CF) were from R&D (Minneapolis, MN, USA). Perforin (ab114201) was from Abcam (Cambridge, UK). Granzyme B (HY-P75157), Z-YVAD-FMK (HY-P1009), necrosulfonamide (HY-100573), disulfiram (HY-B0240) and DMF (HY-Y0345) were from MedChem Express (New Jersey, USA). Following primary antibodies were used: β-actin (4970), GSDMD-N (36425) were from Cell Signaling Technology (Danvers, MA, USA), AIM2 (ab93015) was from Abcam (Cambridge, UK), NLRP3 (DF7438), cleaved caspase-1 (AF4005), IL-18 (DF6252) were from Affinity Biosciences (Zhenjiang, China), NALP1 (sc-166368), ASC (sc-514414), GSDMD (sc-81868), IL-1β (sc-52012) were from Santa Cruz Biotechnology (Dallas, TX, USA), NLRC4 (NB100-56142) was from Novus Biologicals (Littleton, CO, USA), caspase-1 (22915-1-AP), caspase-8 (66093-1-Ig) were from Proteintech (Wuhan, China). Following secondary antibodies were used: goat anti-rabbit (A0208) and anti-mouse (A0216) IgG H&L-horseradish peroxidase (HRP) were from Beyotime Biotechnology (Shanghai, China), goat anti-rabbit (4412), anti-mouse (4408) IgG H&L (Alexa Fluor 488) and DAPI (4683) were from Cell Signaling Technology (Danvers, MA, USA). CD3-PerCP antibody (MA1-19764) was from Invitrogen (Carlsbad, CA, USA). Poly(dA:dT) (tlrl-patn-1) was from Invivogen (San Diego, CA, USA). Lipofectamine 3000 (L3000015) was from Invitrogen (Carlsbad, CA, USA). TUNEL Assay Kit-HRP-DAB (ab206386) was from Abcam (Cambridge, UK). Deadend Fluorometric TUNEL System Kit (G3250) was from Progema (Madison, WI, USA). Calcein-AM/PI Double Stain Kit (4074ES76) was from Yeasen Biotech (Shanghai, China). Annexin V-FITC Apoptosis Detection Kit (APOAF) was from Sigma-Aldrich (St. Louis, MO, USA). FAM-FLICA Caspase-1 (YVAD) Assay Kit (19T33) was from ImmunoChemistry Technologies (Bloomington, MN, NSA). Cytotoxicity LDH Assay Kit (CK12) and Cell Counting Kit-8 (CK04) were from Dojindo (Kumamoto, Japan). Human IL-1β (DLB50) and IL-18 (DL180) ELISA Kit were from R&D (Minneapolis, MN, USA).
Human samples collection
A total of 18 adults [Female/Male: 7/11; with a mean age of 49.44 ± 21.75 years] with diagnosis of SJS/TEN were included in our study for the period from January 2019 to January 2022. All patients presented with a progressive evolution within the 24h preceding admission and admitted to the intensive care unit of the Department of Dermatology of Huashan Hospital affiliated with Fudan University. Peripheral blood was taken immediately after admission and repeated during the recovery period (no new eruptions, Nikolsky sign turing negative and exudation improved). The biopsies were performed using 8mm sterile dermal biopsy punches from non-sun-exposed erythematous area and the normal-looking skin surrounding the lesion site. Control samples were collected from 20 patients [Female/Male: 10/10; with a mean age of 42.8 ± 13.64 years] undergoing resection of non-sun-exposed pigmented nevi for the same time period, skin samples were obtained from the healthy skin at 5mm from the edge of the pigmented nevi, and their peripheral blood samples were collected from the outpatient department. Discarded human foreskins for the isolation and culture of human primary keratinocytes were collected from 12 adult male patients (with a mean age of 26.08 ± 3.32 years) who underwent circumcision surgery at the department of Urology of Huashan Hospital between January 2019 to January 2022. All included patients signed informed consents. Our study protocol was in accordance with the Helsinki Declaration and was approved by the Ethics Committee of Fudan University, Affiliated Huashan Hospital, Shanghai, China (No. KY2019-289).
Histology and immunohistochemistry (IHC), in situ DNA fragmentation staining
Skin tissues were post-fixed in 4% paraformaldehyde (PFA) at 4°C by overnight immersion, then processed by embedding in paraffin and sectioned (4µm thickness). The tissue sections were deparaffinized, rehydrated and then some sections were stained with hematoxylin-erosin (HE). For IHC, antigen retrieval was performed in 10 mM citrate buffer (pH 6.0) for 15 min by microwaving slides at high power. Sections were stained with anti-AIM2 (1:200), anti-NALP1 (1:50), anti-NLRP3 (1:100), anti-NLRC4 (1:1000), anti-ASC (1:50), anti-cleaved caspase-1 (1:100), anti-GSDMD-N (1:500), anti-IL-1β (1:50), and anti-IL-18 (1:100) primary antibodies at 4°C overnight. Mouse and Rabbit Specific HRP/DAB (ABC) Detection IHC kit (ab64264) was used as previously described.(58) TUNEL assay was performed to examine DNA fragmentation in skin sections.(59) The nuclear counterstaining was performed with hematoxylin (for 5 minutes), then the sections were dehydrated, and finally coverslipped. The images were taken under a magnification of ×200 with the upright microscope Nikon 80i and NIS-Elements software (Nikon Corporation, Tokyo, Japan). And analyzed by ImageJ software (version 1.51j8, NIH, Bethesda, MD, USA). Average values of 5 visual fields were randomly selected for statistical analyses.
Transmission electron microscopy
Skin and human primary keratinocyte samples were fixed in 2.5% glutaraldehyde (pH = 7.4) for overnight at 4°C, followed by washed three times with phosphate buffer (5 min each), then post-fixed in 1% osmic acid at 37°C for 2 hours. After three washes with ddH2O, the samples were placed in a graded series of ethanol for dehydration, subsequently infiltrated through a series of acetone/Epon-Araldite resin, and finally embedded in resin at 60°C overnight. Semi-thin and ultra-thin sections were obtained, after being stained with 4% uranium acetate and 1% Reynolds lead citrate, ultra-thin sections were mounted on nickel grids and collected for microstructure analysis using H-7500 transmission electron microscope (Hitachi, Tokyo, Japan).
Western blotting analysis
The total protein lysates were extracted from skin tissues and cultured keratinocytes using cell lysis buffer (P0013B) for 20 min at 4°C, then the lysates were centrifuged at 13000 rpm for 20 min at 4°C. Total protein concentration were quantified with the BCA Protein Assay Kit (P0009). Equal amounts of protein (20µg) were mixed by loading buffer (P0015) and heated in a 99°C metal bath for 10 minutes. The above reagents were from Beyotime Biotechnology (Shanghai, China). The sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE) and western blotting were performed according to our previously described methods.(60) 4–20% gradient precast gels (180-9115H) from Tanon Science and Technology (Shanghai, China) were used for SDS-PAGE. The following antibodies were used: anti-β-actin (1:5000), anti-AIM2 (1:2000), anti-NALP1 (1:1000), anti-NLRP3 (1:2000), anti-NLRC4 (1:2000), anti-ASC (1:1000), anti-caspase-1 (1:2000), anti-caspase-8 (1:2000), anti-GSDMD (1:2000), anti-IL-1β (1:11000), anti-IL-18 (1:2000), goat anti-rabbit and anti-mouse IgG H&L-HRP (1:5000). All the blots were developed using the ECL western blotting chemiluminescent system (WBULS0500) from Millipore (Billerica, MA, USA). The expression levels of each protein were calculated as relative expression to β-actin with ImageJ software.
Immunofluorescence
After washed by PBS, human primary keratinocytes (on the cell climbing slices) were fixed in 4% PFA for 30 min followed by incubated with 0.5% TirtonX-100 in PBS at room temperature for 30 min. Subsequently, cells were blocked for 1 hour in 5% bovine serum albumin (BSA) (A8020) from Solabio (Beijing, China). Next, cells were incubated with primary antibodies at 4°C overnight as follow: anti-AIM2 (1:200), anti-ASC (1:50), anti-cleaved caspase-1 (1:50), anti GSDMD-N (1:100), anti-IL-1β (1:50), anti-IL-18 (1:50), then with goat anti-rabbit or anti-mouse IgG H&L (Alexa Fluor 488) (1:800) at room temperature for 1 hour. Finally, the slices were stained with Antifade Mounting Medium with DAPI (P0131) from Beyotime Biotechnology (Shanghai, China). The fluorescence images were visualized with a ZOE Fluorescent Cell Imager (Bio-Rad, Hercules, CA, USA) and analyzed by ImageJ software. Average values of 5 visual fields were randomly selected for statistical analyses.
RNA extraction and quantitative real-time RT-PCR
RNA extraction of cultured keratinocytes and skin samples were implemented with RNAiso Plus (9109), The extracted total mRNA (1µg) was then reverse transcribed to cDNA with PrimeScript RT Master Mix (RR036A) from Takara Biotechnology (Otsu, Shiga, Japan). Quantitative real-time polymerase chain reaction (qRT-PCR) based on SYBR Green fluorescence (RR420A) using the QuantStudio 6 Flex and data were analyzed by QuantStudio Real-Time PCR software (Thermo Fisher, Carlsbad, CA, USA). The 2-delta-delta CT method was used for analyze the data quantitatively. The sequences of all the primers were listed in Supplementary Table S1.
Cytosolic DNA fragments assay
Human primary keratinocytes (on the cell climbing slices) were fixed in 4% PFA for 30 min followed by permeabilized in 0.5% TirtonX-100 in PBS at room temperature for 30 min and pre-equilibrated. Nucleotide mix was used to label DNA strand breaks for 60 min at 37°C. The reaction was stopped by 2×SSC, and the slices were stained with Antifade Mounting Medium with DAPI. The fluorescence images were visualized with a ZOE Fluorescent Cell Imager. Average values of 5 visual fields were randomly selected for statistical analyses.
Keratinocyte death assay
Apoptotic and necrotic cell deaths in epidermis of SJS/TEN patients and cultured keratinocytes were assessed by Annexin V-FITC Apoptosis Detection Kit. The assay followed the instruction manual. Dilution: propidium (PI) (1:50), Annexin V-FITC (1:100). FAM-FLICA Caspase-1 (YVAD) Assay Kit was used to detect pyroptotic cell death by double staining of PI (1.25µg/mL) and cleaved caspase-1 labeled by FAM-YVAD-FMK (1:150) according to the manufacturer’s instruction. Flow cytometry evaluation of the apoptotic, necrotic and pyroptotic rates were performed by using a FACS Caliber flow cytometer (BD Biosciences, San Diego, CA, USA) and data was analyzed in FlowJo (version 10.6.2, BD Biosciences, Franklin Lakes, NJ, USA). Necrotic cell death in keratinocytes was also evaluated by Calcein-AM/PI Double Stain Kit, living cells were labeled by Calcein-AM (2µM) and dead /dying cells were marked by PI (4µM). The fluorescence images were visualized with a ZOE Fluorescent Cell Imager and analyzed using ImageJ. Average values of 5 visual fields were randomly selected for statistical analyses. Lactic dehydrogenase (LDH) release in the cell culture supernatant from dead keratinocytes was also measured by Cytotoxicity LDH Assay Kit. Cell viability was determined by the Cell Counting Kit-8 for 2 hour of incubation at 37°C.
Enzyme-linked immunosorbent assay (ELISA)
Plasma and cell culture supernatant IL-1β and IL-18 concentrations were measured by Quantikine Immunoassay according to the manufacturer’s recommendations. The wells were read on a Sunrise plate reader (Tecan, Crailsheim, Germany) and the optical densities (OD) were determined at 450/620 nm. The concentration of cytokines (IL-1β, IL-18) is proportional to the measured absorbance and was expressed in pg/mL, and calculated based on the samples and calibrator OD values. Three duplicated wells were set up for each sample and all the experiments were performed in duplicate.
Cell culture and transfection
Primary keratinocytes of SJS/TEN patients were isolated from erythematous area of the non-sun-exposed skin, and human primary epidermal keratinocytes were isolated from the foreskin. After the skin biopsy incubating with 2.5mg/mL dispase (D4693) from Sigma-Aldrich (St. Louis, MO, USA) for 12 hour at 4°C, epidermis was mechanically isolated from dermis. All the keratinocytes were cultured in Keratinocyte Serum-Free Medium (C-20011) from PromoCell (Heidelberg, Germany) supplemented with bovine pituitary extract and epidermal growth factor. Keratinocytes of passage two to five were utilized in our experiments. T cell clones derived from erythematous area or the normal-looking skin surrounding the lesion site of SJS/TEN patients were isolated as previously described.(61) Briefly, the whole skin biopsies were cultured with 1 mM sodium pyruvate, 2 mM glutamine, 0.05 mM 2-mercaptoethanol, 1% penicillin/streptomycin, 1% nonessential amino acids (all Invitrogen; RPMI complete), and 5% human serum (Sigma-Aldrich) supplemented with 60 U/ml IL-2 in RPMI 1640. 72 hours later, the cell suspension was collected and centrifuged at 500g for 5 mins at 4°C, the supernatant was aspirated and used to stimulate primary keratinocytes. For introducing DNA into cytoplasm, primary keratinocytes were transfected with 4µg/mL poly(dA:dT) with Lipofectamine 3000 for 24 hours according to the manufacturer’s instruction. To generate primary keratinocytes with gene knockdown, shRNAs targeting AIM2, Caspase-1, Caspase-8 and GSDMD sequences or a control sequence from PPL (Public Protein/Plasmid Library, China) were transfected into primary keratinocytes (2.5µg per well in six-well plates) with Lipofectamine 3000 for 72 hours. Knockdown efficiency was identified by detecting the level of protein or mRNA. For each gene, 2–3 shRNA sequences were designed, and the one had the highest knockdown efficiency was chosen for subsequent experiments. All of the shRNA sequences used for transfections were listed in Supplementary Table S2.
Statistical analysis
All the results are presented as the Mean ± standard error of mean (SEM) and compared by two-tailed t-tests, Wilcoxon ranksum tests (for comparison between two groups) or one-way ANOVA (for three or more groups). P-values were expressed as ns (no significant) when p༞0.05, and significant when *p༜0.05, **p༜0.01, ***p༜0.001. For the normally distributed variables, the correlations were estimated using Pearson Correlation analysis. All the data were analyzed by Graph Prism software (version 8.0.1, GraphPad Software, San Diego, CA, USA).