Murine Epidermal Keratinocyte Isolation and mPEO Culture.
We collected telogen back skin from C57BL/6 mice. The tissues were digested with 2.5 U/mL dispase (Stemcell) for 1 h at 37 °C. The epidermis was carefully peeled off with tweezers and cut into small pieces. The minced tissue pieces were subsequently digested with 0.25% Trypsin (Gibco) for 20-30 min. After filtering through a 70 μm nylon cell strainer and centrifugation, we finally obtained single primary epidermal cells. Keratinocytes were plated at a density of 2,500 cells per 10 μL Matrigel. Cells were cultured in medium consisting of advanced DMEM/F12 (Gibco) supplemented with penicillin/streptomycin (100 U/L; Gibco), HEPES (10 mM; Gibco), GlutaMAX (1×; Gibco), B27 supplement (50× stock; Gibco), N-acetylcysteine-1 (1 mM; Sigma‒Aldrich), bovine serum albumin (0.1%; Sigma‒Aldrich), forskolin (10 μM; BioGems), EGF (50 ng/mL; Peprotech), Wnt3a (100 ng/mL; Peprotech), Y-27632 (10 μM; BioGems) and A83-01 (1 μM; Sellect) that was added after 2 days of culture.
mPEOs could be removed from Matrigel by incubating with 1 U/mL Dispase for 60 min at 37 ℃ and further dissociated into small clumps of cells or single cells using Tryple (Gibco). mPEOs were passaged at a 1:3–1:4 ratio every 7 days. mPEOs could be dissociated into small clumps or single cells, cryopreserved in serum-free cryopreservation medium (StemCell), placed in −80 °C or liquid nitrogen, and recovered with the optimized organoid medium.
Bacterial strain culture
Staphylococcus aureus strain USA300, a methicillin-resistant strain derived from adolescent patients with severe sepsis syndrome at Texas Children's Hospital, was purchased from ATCC (BAA-1717). USA300 were cultured in tryptic soy broth (TSB) for 3 h to log-phase with shaking at 220 rpm at 37 °C.
Hematoxylin & Eosin (H&E) and Immunohistochemistry staining
mPEOs could be removed from Matrigel by incubating with 1 U/mL Dispase for 60 min at 37 ℃. Organoids were centrifuged at 300×g for 5 minutes. The medium was aspirated and the organoids were resuspended in 4% paraformaldehyde (PFA) and incubated for 1 h at 37 °C. The fixed mPEOs were then embedded in 2% agarose. Subsequently, organoids were dehydrated and embedded in paraffin wax. Hematoxylin & eosin (H&E) and immunohistochemical staining were performed on 4-µm sections of paraffin-embedded organoids. Paraffin sections of organoids were dewaxed, and antigen retrieval was performed by either pretreatment with EDTA Antigen Retrieval Solution pH 9 (50×, Beyotime) or boiling in citrate buffer pH 6 (50×, Beyotime). Subsequently, slides were incubated in blocking buffer consisting of 0.1% Triton X-100 (Beyotime) and 3% bovine serum albumin (BSA; Sigma‒Aldrich) in PBS for 1 h at room temperature. Slides were incubated overnight at 4 °C with the following primary antibodies: rabbit anti-involucrin (AF0186, 1:500, Affbiotech), rabbit loricrin (ab85679, 1:200, Abcam), rabbit anti-KRT10 (ab76318, 1:400, Abcam), and mouse anti-KRT14 (abs131470-50 µg, 1:500, absin). After washing, the slides were incubated with an SABC-HRP Kit with anti-rabbit IgG (1:50, P0615, Beyotime) for an hour at room temperature, after which the slides were washed and developed using DAB (3,3′-diaminobenzidine tetrahydrochloride hydrate, P0203, Beyotime) development. Finally, neutral resin was used to seal the film.
Immunofluorescence staining
Following Hans Clevers 21, mPEOs were released from the Matrigel and incubated with cell recovery solution reagent (Corning) on a horizontal shaker at 4 °C (60 rpm) for 30–60 min. The pellet of organoids was gently resuspended in 1 ml of PFA and incubated at 4 °C for 45 min. Then, cold OWB (Organoid Washing Buffer, including 0.1% Triton X-100 + 0.2% BSA) was used to transfer the appropriate amounts of organoids per staining to a low-adherence/suspension 24-well plate and incubated at 4 °C for 15 min. Then, 200 µl of OWB with primary antibodies (2× concentration) was added to each well and incubated overnight at 4 °C with mild rocking/shaking (60 rpm on a horizontal shaker). The cells were incubated with 200 µl of OWB-diluted secondary antibody (2× concentration) for 1 h at room temperature in the dark. The OWB was removed and incubated with DAPI for 1 h at room temperature in the dark. The images were captured using a Nikon ECLIPSE Ti2. A complete list of the primary and secondary antibodies used is provided in Supplementary Table 1.
Transmission electron microscopy
The mPEOs were fixed with 2.5% glutaraldehyde at 4 °C overnight. Then, the cells were postfixed by incubation for 2 h with 1% osmium tetroxide/0.1 sodium cacodylate and dehydrated in a graded series of acetone solutions. The cells were embedded in Polybed 812 epoxy resin. Ultrathin sections were cut and collected on 50 mesh copper grids, stained with 4% aqueous uranyl for 15 min, and then with Reynolds’ lead citrate for 7 min. The stained sections were examined with a Hitachi HT7800 Series 120 kV transmission electron microscope (TEM).
RNA sequencing
According to the results of M.Wang, et al. 22, we released mPEOs cultured for 7 days from Matrigel using 1 U/mL Dispase at 37 °C for 1 h, and the derived tissue was stored in RNAlater (Thermo Fisher, AM7020). The total RNA of the mPEOs and tissues was extracted using TRIzol according to the manufacturer’s instructions. One microgram of total RNA was used for subsequent library preparation. Poly(A) mRNA isolation was performed using Oligo(dT) beads. mRNA fragmentation was performed using divalent cations and high temperatures. Priming was performed using Random Primers. First-strand cDNA and second-strand cDNA were synthesized. The purified double-stranded cDNA was then treated to repair both ends, and dA-tailing was added in one reaction, followed by T-A ligation to add adaptors to both ends. Size selection of adaptor-ligated DNA was then performed using DNA clean beads. Each sample was then amplified with PCR using P5 and P7 primers, and the PCR products were validated. Then, libraries with different indices were multiplexed and loaded on an Illumina HiSeq/Illumina NovaSeq/MGI2000 instrument for sequencing using a 2x150 paired-end (PE) configuration according to the manufacturer’s instructions.
Proteomic analysis method
① Protein extraction and concentration determination
mPEOs and tissues were collected, and an appropriate amount of RIPA lysis buffer (Biotechwell, WB0101) and protease inhibitors (Biotechwell, WB0122) was added and shaken thoroughly on a shaker, followed by lysis on ice for 1 h. The supernatant was collected by centrifugation at 13300 rpm for 15 min. The protein concentration was determined according to the standard protocol of the BCA protein assay kit (Biotechwell, WB0123).
② Protein tryptic digestion
Proteins were reduced in 5 mM dithiothreitol at 56 ℃ for 30 min and then alkylated in 15 mM iodoacetamide at room temperature for 30 min in darkness. The reaction was quenched with 30 mM cysteine at room temperature for an additional 30 min. Protein samples underwent trypsin digestion (enzyme-to-substrate ratio of 1:50 at 37 ℃ for 16 hours) followed by desalting through MonoSpin C18 cartridges and vacuum-drying by Speed Vac. The peptide residues were reconstituted in water containing 0.1% formic acid and centrifuged at 14,000 rpm for 10 min prior to nano-LC‒MS/MS analysis.
③ Nano-LC‒MS/MS
Peptide samples were analyzed on a nano-HPLC (nanoElute, Bruker Daltonics) onto 250 mm × 75 μm ID pulled emitter columns (IonOptiks) packed with 1.6 μm C18-particles and heated at 50 °C in a column oven. The mobile phases consisted of 0.1% (v/v) formic acid (FA) in water (phase A) and acetonitrile (phase B). Samples were separated by a 60 min stepped gradient ranging from 2 to 30% B at a flow rate of 400 nL/min. Peptides were detected on a timsTOF Pro instrument (Bruker Daltonics) operated in PASEF mode. TIMS accumulation times were fixed at 100 ms, while the ion mobility separation was fixed to 100 ms. The range of mobility values was 0.45−1.45 vs./cm 2 (1/K0), and the covered m/z range was 100−1700 m/z.
④ MS database searching
MS raw files generated by LC‒MS/MS were searched against the UniProt mouse proteome database (version 2021-01-01) using PaSER (version 1.0) software. The protease was trypsin. Up to 2 missed cleavages were allowed. Carbamidomethyl (C) was considered a fixed modification. The variable modifications were oxidation (M) and acetylation (protein N-term). The cutoff of the false discovery rate (FDR) using a target-decoy strategy was 0.01 for both proteins and peptides.
USA300 infection of mPEOs
Before infection, mPEOs were released from Matrigel by incubating with 1 U/mL Dispase for 1 h at 37 ℃. Released mPEOs were suspended in the culture medium and infected with USA300 at a multiplicity of infection (MOI) of 10 for 2, 4, 8, and 24 h at 37 °C in 5% CO2. Specifically, we collected the mPEO suspension and mixed it with a Pasteur pipette. A total of 1 mL was centrifuged at 300 ×g for 5 min, and the supernatant was aspirated. Organoids were resuspended in 2 mL TrypLE (Gibco) and incubated for 15 minutes at 37 °C. Subsequently, organoids were disrupted by pipetting up and down at least 10 times until the mPEOs were disrupted to single cells, accurately counting cells to estimate mPEOs. Then, 105 mPEOs and 106 CFU/mL USA300 were resuspended in 1 mL of Advanced DMEM/F12 per well in a 24-well plate. After infection, the mPEOs were washed twice with the basal medium for subsequent CFU count, drug testing or immunofluorescence analysis.
Gentamicin protection assay
After the infection time arrived, extracellular bacteria were killed by the addition of gentamicin (MCE, HY-A0276) to a final concentration of 500 μg/mL for 1 h at 37 °C. To quantify intracellular bacteria, mPEOs were disassociated with Tryple at 37 °C for 15 min, pipetting up and down more than 10 times, and dilutions were plated on LB agar plates for CFU enumeration.
Flow Cytometry
Following Hans Clevers 23, after infection, mPEOs were harvested from the culture in 1 mL of ice-cold (4 °C) DMEM and centrifuged for 5 min at 300×g, and the supernatant was discarded. Then, mPEOs were resuspended in TrypLE (Gibco) and incubated for 15 minutes at 37 °C. Subsequently, organoids were disrupted by pipetting up and down at least 10 times until the mPEOs were dissociated into single cells. The cells were resuspended in 100 μL PBS with 5 μL 7-ADD (BioGems, 61410-00-200) and incubated at room temperature for 15 min in the dark. Filter with a 40 μm cell strainer. The cells were run on a Beckman CytoFlex, and the ratio of 7-ADD-positive cells to total cells was assessed to establish mPEO viability.
Detection of IL-Iβ levels by ELISA
At the time point of infection for 2, 4, 8, and 24 h, mPEO culture supernatants were collected for IL-1β enzyme-linked immunosorbent assay (ELISA) (MULTI SCIENCES, EK201B/3-48). The operation followed the standard experimental protocol of the ELISA kit.
Minimum Inhibitory Concentration (MIC) Assay
The MIC was determined using the microdilution method according to Clinical and Laboratory Standards Institute (CLSI) guidelines. USA300 was diluted in Mueller-Hinton broth (MHB) and introduced into 96-well plates. After that, methicillin (MCE, HY-B0974) and vancomycin (Sigma‒Aldrich, V2002-100MG) prepared in 10 mM stock solutions were subjected to twofold serial dilution in a 96-well plate to final concentrations of 1280 to 1.25 μg/mL, in triplicate. The MIC was defined as the lowest drug concentration that completely inhibited visible bacterial growth.
Drug testing for USA300-infected mPEOs
1) Method A
The cell number assessment of mPEOs was the same as “USA300 infection to mPEOs”. In black 96-well plates, each well contained 100 µL Advanced DMEM/F12 resuspended 104 mPEOs, 105 USA300 and the indicated concentrations of drug (methicillin: 12.8 μg/mL; vancomycin: 1.6, 3.2 μg/mL) or DMSO control. After drug treatment for 24 h, 100 µL of CellTiter-Lumi™ Luminescent Cell Viability Assay Kit (CTL, Beyotime, C0065S) was added to each well and incubated at room temperature for 1 h in the dark, and luminescence parameters were detected on a microplate reader to evaluate the activity of mPEOs.
2) Method B
USA300 infection and drug treatment were the same as in Method A. The difference was that after drug treatment for 24 h, 80 µL of supernatant was removed (to avoid the loss of mPEOs) and supplemented with Advanced DMEM/F12 to a total volume of 100 µL, and mPEO viability assays were performed using CTL.
3) Method C
mPEOs were harvested from Matrigel and infected with USA300 at MOI = 10 for 24 h. Then, the mPEOs were washed twice with basal medium. Infected mPEOs were suspended with 5% Matrigel and dispensed onto 96-well plates as 20 μL droplets and overlaid with 100 μL of medium per well. Each well contained up to 10,000 cells. USA300-infected mPEOs were treated with DMSO, 12.8 μg/mL methicillin, or 1.6 and 3.2 μg/mL vancomycin immediately for 24 h after re-embedding. Finally, mPEO viability assays were performed using CTL.
LIVE cell immunofluorescence staining
At the end of drug treatment, mPEOs were washed once with PBS and stained for 2 h with a mixture of 2.5 µM calcein AM (eBioscience, 65-0853-39) and 1× Hoechst (Beyotime, C1028), and fluorescent imaging was performed.
Quantification and statistical analyses
All data are presented as the mean of at least two independent experiments with corresponding error bars of standard deviation (SD) or the standard error of the mean (SEM). Data analysis was performed using GraphPad Prism version 8.02 using one-way or two-way ANOVA with post hoc multiple comparison tests or Student’s t test, as specified in the figure legends. Statistical significance is indicated with *p < 0.05, **p < 0.01 and ***p < 0.001.