Experimental model and content details
Animals
Female mice were used throughout (25–30 g, 5–8 weeks old). The following strains of mice were used: C57BL/6 J (Charles River, RRID: IMSR_JAX:000664), B6.Cg-Tg(Plp1-CreERT)3Pop/J mice (Plp-CreERT, RRID: IMSR_JAX:005975 Jackson Laboratory) expressing a tamoxifen-inducible Cre in Schwann cells (Plp1, proteolipid protein myelin 1). Both positive and negative mice for CreERT (Plp-CreERT+ or Plp-CreERT- (control) respectively) were treated with intraperitoneal (i.p.) 4-hydroxytamoxifen (4-OHT, 1 mg/100 μL in corn oil once a day consecutively for 3 days) before the infection with AAV for selective silencing of the different genes in Schwann cells.
To selectively delete Trpa1 in primary sensory neurons, homozygous 129S-Trpa1tm2Kykw/J (floxed Trpa1, Trpa1fl/fl, RRID:IMSR_JAX: 008649 Jackson Laboratory) mice were crossed with hemizygous Advillin-Cre mice (Adv-Cre). Mice positive or negative for Cre and homozygous for floxed Trpa1 (Adv-Trpa1 and control respectively) were used. The successful Cre-driven deletion of TRPA1 mRNA was confirmed using reverse transcription quantitative real-time PCR (RT-qPCR). Some Adv-Cre+ or Adv-Cre- (control) were used for the infection with AAV for selective gene silencing in primary sensory neurons.
To evaluate the involvement of macrophages, transgenic Macrophage Fas-Induced Apoptosis (MaFIA) mice (C57BL/6-Tg(Csf1r-EGFP-NGFR/FKBP1A/TNFRSF6)2Bck/J, stock No: 005070, RRID:IMSR_JAX:005070, Jackson Laboratories) were used. These transgenic mice express a mutant human FK506 binding protein 1A, 12kDa (FKBP12)-Fas inducible suicide/apoptotic system, driven by the mouse Csf1r promoter conjugated with a green fluorescent protein (GFP), which preferentially binds the B/B dimerizing agent (B/B-HmD, AP20187). Treatment of mice with AP20187 induces the dimerization of the suicide protein to activate the cytoplasmic FKBP12-Fas fragments, leading to the apoptosis of transgene‐expressing cells and consequent macrophage depletion 32.
The group size of n=8 mice for behavioral experiments was determined by sample size estimation using G Power [v3.1 53] to detect the size effect in a post-hoc test with type 1 and 2 error rates of 5% and 20%, respectively. Allocation concealment of mice into the vehicle(s) or treatment groups was performed using a randomization procedure (http://www.randomizer.org/). The assessors were blinded to the identity of the animals (genetic background) or allocation to treatment groups. None of the animals were excluded from the study. Mice were housed in a temperature- and humidity-controlled vivarium (12 h dark/light cycle, free access to food and water, 5 animals per cage). At least 1 h before behavioral experiments, mice were acclimatized to the experimental room and behavior was evaluated between 9:00 am and 5:00 pm. Animals were anesthetized with a mixture of ketamine and xylazine (90 mg/kg and 3 mg/kg, respectively, i.p.) and euthanized with inhaled CO2 plus 10-50% O2.
All behavioral experiments followed Animal Research: Reporting of In Vivo Experiments (ARRIVE) guidelines and were in accordance with European Union (EU) guidelines for animal care procedures and the Italian legislation (DLgs 26/2014) application of the EU Directive 2010/63/EU. The study was approved by the National Committee for the Protection of Animals used for Scientific Purposes of the Italian Ministry of Health (research permits # 452/2020-PR).
Human samples
The use of blood plasma samples collected from human individuals diagnosed with endometriosis and healthy control was approved by the Local Ethics Committee of the Florence University Hospital (Area Vasta Toscana Centro, CEAVC) (EndoTRP study, 15211.oss and subsequent amendments), according to the Helsinki Declaration and good clinical practice guidelines, and all patients gave their informed consent. Participants did not receive any form of compensation. The blood plasma samples derived from nineteen endometriosis diagnosed patients [female, median age 30 years (range 22-43)] and fifteen healthy individuals [female, median age 27 years (23-42)]. Considering the exploratory nature of the blood sampling analyses, the sample size was not determined based on statistical considerations but enrolling all consecutive outpatients and healthy controls.
Cell lines
Mouse Schwann cells (MSC). MSC were isolated from sciatic nerves of C57BL/6J mice 54. Briefly, the epineurium was removed, and nerve explants were cut into 1 mm segments and enzyme-dissociated in Hank's Balanced Salt Solution (HBSS, 2 hr, 37 °C) added with collagenase (0.05%) and hyaluronidase (0.1%). Cells were collected by centrifugation (150xg, 10 min, room temperature RT) and the pellet was resuspended and cultured in Dulbecco's Modified Eagle Medium (DMEM) containing fetal calf serum (10%), L-glutamine (2 mM), penicillin (100 U/ml), streptomycin (100 mg/ml), neuregulin (10 nM) and forskolin (2 μM). Cytosine arabinoside (Ara-C, 10 mM) was added three days later, to remove fibroblasts. The culture medium was replaced every 3 days. Cells were cultured at 37 °C in 5% CO2 and 95% O2 for 15 days before experiments.
Human Schwann cells (HSCs). HSCs (#P10351; Innoprot) were grown in Schwann cell medium (#P60123, Innoprot) at 37◦C with 5 % CO2 and 95 % O2 and discarded and replaced after 12 passages 55.
The murine macrophage cell lineage, RAW 264.7 (#TIB-71™; ATCC, RRID: CVCL_0493), was cultured and maintained in DMEM supplemented with FBS (10%), penicillin (100 U/mL), streptomycin (100 mg/mL) and L-glutamine (2 mM) at 37 ◦C in 5% CO2 and 95% O2.
The human monocytic cell line, U937 (#CRL-1593.2™; ATCC, RRID:CVCL_0007) was maintained at 37 ◦C, 5% CO2 and 95% O2 in Roswell Park Memorial Institute (RPMI-1640) medium supplemented with heat-inactivated FBS (10 %), L-glutamine (2 mM), 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES; 10 mM), and sodium pyruvate (1 mM). The differentiation of U937 cells (1 × 105 cells/mL) towards macrophage-like lineage was promoted by the exposure to 200 ng/mL of phorbol 12-myristate 13- acetate (PMA) for 2 days before experiments 56.
AAVpro293T cell line. AAVpro 293T cells (#632273, Takara, RRID:CVCL_B0XW), were maintained in DMEM high glucose supplemented with 10% heat inactivated FBS, 4 mM L-glutamine, 1 mM penicillin/streptomycin and 1 mM sodium pyruvate at 37 °C in 5% CO2 and 95% O2. The day before transfection, cells were plated in DMEM supplemented with 2% FBS.
Induction of endometriosis
Endometriosis was induced in mice as previously reported 57. Briefly, donor mice, received a subcutaneous injection of estradiol benzoate (3 μg/100 μL) to stimulate the endometrium growth. Seven days later, the uteri of the donor mice were dissected and divided longitudinally. Uterine horns from each donor mouse were minced in tissue fragment smaller than 1 mm in Hank’s balanced salt solution (HBSS). Each dissociated uterine horn was then injected intraperitoneally (50 mg/500 μL i.p.) in recipient mice. One donor mouse was used for every 2 endometriosis mice. Sham mice received an intraperitoneal injection of 500 μL of HBSS. Behavioral experiments were performed during a 28-day period. After 28 days from the injection of the endometrium suspension, mice were sacrificed, and tissues collected. Lesion implantation was also quantified in a time-course manner by counting the number of lesions in endometriotic mice at day 7, 14, 21, and 28.
Treatment protocols
If not otherwise indicated, all reagents were obtained from Merck Life Science SRL. Mice received systemic administration of C5a complement antagonist (DF2593A, 1 mg/kg, intragastric, i.g.) or vehicle (0.5% carboxymethyl cellulose, CMC) twice a day, by two different time schedules: from one day before the endometriotic tissue injection to day 6 or from day 12 to day 16.
Anti-IL1β (#BE02463, clone B122, Bio X Cell, RRID:AB_2687727) monoclonal antibody (mAb) or IgG isotype control were administered (50 µg/200 μL, i.p.) at day 8 and day 14 after endometriosis/sham induction. MaFIA mice were treated with B/B homodimerizer (AP20187, 2 mg/kg, i.p., once a day) or vehicle (10% PEG-400, 1.7% tween 80 in 0.9% NaCl) by three different time schedules: from day -2 to day 2; from day 2 to day 6, and from day 21 to day 25. Plp-Cre+ and control mice were infected with an intravenous (1 mL/kg, 1x1012 v/g, i.v.) injection of different AAVs. Adv-Cre+ and control mice were infected with an intrathecal (1x1012 v/g, i.th., 5 μL) injection of AAV. Animals were used 3 weeks after AAVs infection. Sciatic and trigeminal nerve tissues were harvested for evaluating AAVs infection.
Behavioral assays
Abdominal Mechanical allodynia (AMA). The mechanical pain sensitivity of the abdominal region was evaluated with von Frey filaments by methods described previously 58 . Mice were habituated in small compartments on a perforated grid for 1 h before the test. Subsequently von Frey filaments were applied to the abdomen (between diaphragm and genitals). The test was performed by a trained blinded observer. The individual filaments were tested in an ascending order covering 0.008, 0.04, 0.16, and 0.4 g forces. Each force was applied 10 times to the abdominal surface. The maximal duration of each force application was 2 s, and the inter-stimulus interval was 2–3 min. Following each challenge, the withdrawal response was quantified either as 1 (withdrawal of abdominal wall, licking or retraction of animal) or 0 (no response). All counts in response to an individual filament were averaged. Withdrawal responses to low forces reflect high mechanical pain sensitivity.
Periorbital mechanical allodynia (PMA). Periorbital mechanical allodynia (PMA) was assessed using the up-down paradigm 59, 60. Briefly, mice were placed in a restraint apparatus designed for the evaluation of periorbital mechanical thresholds 61. PMA was evaluated in the periorbital region over the rostral portion of the eye (i.e., the area of the periorbital region facing the sphenoidal rostrum) 62 before (basal threshold) and after treatments. On the day of the experiment, after 20 min of adaptation inside the chamber, a series of 7 von Frey filaments in logarithmic increments of force (0.02, 0.04, 0.07, 0.16, 0.4, 0.6 and 1.0 g) were applied to the periorbital area perpendicular to the skin, with sufficient force to cause slight buckling, and held for approximately 5 s to elicit a positive response. The response was considered positive by the following criteria: mouse vigorously stroked its face with the forepaw, head withdrawal from the stimulus, or head shaking. Mechanical stimulation started with the 0.16 g filament. Absence of response after 5 s led to the use of a filament with increased force, whereas a positive response led to the use of a weaker (i.e., lighter) filament. Six measurements were collected for each mouse or until four consecutive positive or negative responses occurred. The 50% mechanical withdrawal threshold (expressed in g) was then calculated from these scores by using a δ value of 0.205, previously determined 62.
Hindpaw Mechanical allodynia (HMA). Paw mechanical allodynia was evaluated by measuring the paw withdrawal threshold by using the up-down paradigm 59, 63. Mice were acclimatized (1 h) in individual clear plexiglass boxes on an elevated wire mesh platform, to allow for access to the plantar surfaces of the hind paws. von Frey filaments of increasing stiffness (0.07, 0.16, 0.4, 0.6, and 1.0, 1.4 and 2 g) were applied to the hind paw plantar surfaces of mice with enough pressure to bend the filament. The absence of a paw being lifted after 5 s led to the use of the next filament with an increased force, whereas a lifted paw indicated a positive response, leading to the use of a subsequently weaker filament. Six measurements were collected for each mouse or until four consecutive positive or negative responses occurred. The 50% mechanical withdrawal threshold (expressed in g) was then calculated.
Plasmid construction
All short hairpin RNAs were designed according to Vector builder and RNAi Consortium of Broad Institute libraries. pAAV[FLEXon]-CMV>EGFP:LL:rev(mCherry:miR30-mC5ar1[shRNA#1]):rev(LL):WPRE and pAAV[FLEXon]-CMV>LL:rev(EGFP:5' miR-30E:{shRNA-NLRP1a}:3' miR-30E):EGFP:5' miR-30E:{shRNA-NLRP1b}:3' miR-30E:rev(LL):WPRE were used. pAAV[FLEXon]-CMV> EGFP-LL-rev(mCherry)-mNLRP3[mir30-shRNA]-rev(LL)-WPRE was generated in two steps. In the first step two overlapping fragments, were obtained by amplifying regions from pAAV[FLEXon]-CMV>EGFP-LL-rev(mCherry)-mC5ar1 [mir30-shRNA]-rev(LL)-WPRE with P1/P4 and P2/P3 primer couples. P1 and P2 were designed to include partial shNlrp3 overlapping regions. A third PCR, using external primers P3/P4 generated the extended fragment, that was cloned (AgeI/SpeI) into pAAV[Exp]-CMV>EGFP-LL-rev(mCherry)- mC5ar1 [mir30-shRNA]-rev(LL)-WPRE. To obtain pAAV[Exp]-CMV>EGFP-LL-rev(mCherry)-mIL-1β[mir30-shRNA]-rev(LL)-WPRE two oligonucleotides (P5 and P6) containing half short hairpin sequence were used to amplify a fragment from the pAAV[Exp]-CMV>EGFP:LL:rev(mCherry:miR30-mC5ar1[shRNA#1]):rev(LL):WPRE. Purified product was phosphorylated with T4 PNK (NEB, #M0201S) according to the manufacturer instructions and finally subjected to circular ligation with a T4 ligase (Thermo Fisher #K1423). All the new-generated plasmids were validated by Sanger sequencing. Primer sequences are reported in Supplementary table S1.
AAV Generation
Recombinant AAV particles (rAAVs) were produced by using polyethylenimine (#23966, PEI, Polyscience) triple transfection strategy as previously described 55. In brief, AAVpro 293T cells (#632273, Takara, RRID:CVCL_B0XW), were transfected with a 1:3 ratio of DNA:PEI 64. To obtain rAAVs, AAVpro 293T cells were transiently transfected with 2.5 mg total DNA (plasmid expressing genes of interest, pAdDeltaF6; (#112867, Addgene) and Rep/Cap, 1:1:1 molar ratio). To infect with high efficiency Schwann cells and primary sensor neurons, Rep/Cap 2/rh10 or 2/9n were used (pAAV2/rh10 #112866 or pAAV 2/9n #112865, respectively Addgene). rAAVs virions were extracted 72 h post-transfection, purified by iodixanol gradient ultracentrifugation, concentrated, and titrated using a RT-qPCR assay (#6233 AAVpro Titration Kit, Takara) according to the manufacturer instructions.
AAV production and cell lysis
Detailed method is reported in Supplementary information.
Iodixanol-based purification protocol
Detailed method is reported in Supplementary information.
Immunofluorescence
Detailed method is reported in Supplementary information.
Protein extraction and western immunoblot assay
Detailed method is reported in Supplementary information.
Proteome profiler array
Mouse plasma samples were collected at different days after endometriosis induction for a cytokine array analysis using the Proteome Profiler Mouse Cytokine Array Kit, Panel A (#ARY006. R&D Systems) according to manufacturer’s instructions. Signal was developed using an imaging system (ChemiDoc; Bio-Rad). The density of specific cytokine dots was measured using HLImage++ (v. PCM.25.5.1.a).
Caspase-Glo® 1 Inflammasome Assay
The caspase-1 activity was evaluated using the bioluminescent based assay Caspase-Glo® 1 inflammasome assay (#TM456, Promega) according to manufacturer’s instructions. Briefly, HSCs were grown on in 96-well poly-L-lysine-coated (8.3 μM) plates and incubated with C5a (100 ng/ml) in the presence of DF2593A (1 µM), Ac-YVAD-CHO (1µM) or veh (0.001 % DMSO) at 37 °C in 5% CO2 and 95% O2. After 16 h, the cell culture medium was collected and transferred to a 96-white, opaque-walled multiwell plates (with a clear bottom) (#6005181, PerkinElmer) and incubated with Caspase-Glo® 1 Reagent for 60 min. The luminescence signal was measured by FlexStation3 Multi-Mode Microplate Reader (Molecular Devices;) using SoftMax® Pro7 software (Molecular Devices). Results were expressed as relative light units (RLU).
Caspase-1 in vitro imaging
The genetically encoded fluorescent sensor Single-Cell Imaging of Caspase-1 (SCAT1) based on fluorescence resonance energy transfer (FRET) was used to detect the inflammasome-mediated caspase-1 activation in HSCs. Briefly, HSCs were plated on 96-well poly-L-lysine-coated (8.3 μM) black clear bottom (5 × 105 cells/well; #6055302 PerkinElmer) and transfected with cDNA of SCAT1 (130-300 ng) using jetOPTIMUS® DNA transfection reagent (#55-250, Polyplus) for 16-24 h at 37 °C in 5% CO2 and 95% O2. On the day of experiments, HSCs were washed and added with HBSS at pH 7.4 at 37 °C and transferred to a chamber on the stage of a fluorescent microscope for recording (Axio Observer 7; with a fast filter wheel and Digi-4 lens to record excitations and Ultra-fast Sutter Lambda DG4 Xenon excitation source-range 300-700 nM) (Zeiss) with 20x objective. Cells were exposed to C5a (10 µg/ml) in the presence of DF2593A (1µM) or Ac-YVAD-CHO (1 µM) vehicle (0.001% DMSO). FRET changes were measured as a ratio of the acceptor fluorophore emission (530 nm) to donor emission (435 nm). The ΔF/F0 ratio was calculated for each experiment and the results were expressed as the AUC after inverting the curve.
C5a and IL-1β assays
C5a and IL-1β content was assayed in mouse sciatic and trigeminal nerve tissue homogenates and in mouse plasma samples using a single-analyte enzyme-linked immunosorbent assay (ELISA) kit (#ab193718 and #ab197742, Abcam, Cambridge, UK) according to the manufacturer’s protocol. Data are expressed as pg/mg of protein and pg/mL. C5a and IL-1β content was assayed in human plasma samples and HSCs cultured medium by ELISA kit (#ab193695 and #ab214025, Abcam) according to the manufacturer’s protocol. Data are expressed as pg/mL
Reverse transcription-quantitative real-time PCR (RT-qPCR)
Detailed method and the sets of primers for HSCs and MSCs are reported in Supplementary information and Supplementary Table S2.
Trans-well migration assay
Noncontact coculture transwell cell culture system between macrophages and Schwann cells was obtained using the Boyden migration assay 56. Briefly, the noncontact cocultured cells were prepared as follows: macrophage-like U937 cells or RAW 264.7 were seeded at a density of 2.5 × 104 / chamber into the upper of a 24-well transwell cell culture system (6.5 mm in diameter, with 8-μm pores, (#CLS3458 ™; Corning) by using the complete media described above and allowed to grow 2 days after PMA treatment (U937 cells) or overnight (RAW 264.7) before migration assay 65. In the meantime, HSCs and MSCs were plated on a 24-well plate. On the day of the experiment, all the cells were replaced with serum free media, and the macrophage-like U937 cells and RAW 264.7 cells cultured on the membrane of 24-well transwell insert were placed into the 24-well plate cultures containing the HSCs and MSCs to initiate the experiment. HSCs and MSCs were treated with C5a (100 ng/ml) also in the presence of DF2593A (1 µM) or veh (0.001 % DMSO) or in the presence mAb-IL-1β (1 µg/ml) or IgG control (1 µg/ml). After 16 h of incubation, migrated cells on the lower surface of the filter were fixed and stained with Diffquick staining set following the manufacturer’s directions (#726443™), and nonmigratory cells on the upper surface of the filter were wiped with a cotton swab. Random fields were counted under a light microscope using a 20x objective (1.5 x 105 µm2).
Statistical analysis
Data are presented as mean ± SEM. For multiple comparisons, a one-way ANOVA followed by a post-hoc Bonferroni’s test was used. The two groups were compared using Student’s t-test. For behavioral experiments with repeated measures, a two-way mixed-model ANOVA followed by a post-hoc Bonferroni’s test was used. Statistical analyses were performed on raw data using GraphPad Prism 8 (GraphPad Software Inc.). P-values less than 0.05 (P < 0.05) were considered significant. EC50 values were determined from non-linear regression models using GraphPad Prism 8. The statistical tests used and sample size for each analysis are shown in the Fig. legends.