Lps o111 b4

The LPS and LTA binding assay was performed to investigate the interaction between PN5 and E. coli O111:B4 LPS and S. aureus LTA (Sigma-Aldrich) by CD spectroscopy. E. coli LPS and S. aureus LTA were diluted in 10 mM sodium phosphate buffer (pH 7.4) to 1 mg mL⁻¹. The peptide (50 μM) was added to E. coli LPS and S. aureus LTA. The temperature was regulated by a PTC-423S controller and was set to 20°C. The spectra were measured from 190 to 250 nm using a CD spectropolarimeter (Jasco) operating at room temperature (25°C) (61 (link)).

EV isolation from MSCs at the flask-scale was performed essentially as described previously [10 (link), 26 (link)]. Low passage number [3 (link)–6 (link)] of non-senescent frozen stocks of MSCs from the six human isolates (F_1 to F_6) were expanded in T-75 cm² plastic flasks and grown to near confluence. The cells were washed with PBS (Hyclone) and replaced with MSC serum-free media (SFM) (StemPro A103332-01, ThermoFisher Scientific, Waltham, MA, USA) at 10 mL/flask. To produce LPS-EVs, MSCs from 3 isolates (F_1-F_3) were primed with LPS as described [10 (link)]. Briefly, MSCs were either unprimed (EVs) or primed with 1.0 ug/mL of E. coli LPS O111:B4 (L4391 Sigma, St Louis, MO, USA) (LPS-EVs) in SFM for 18–24 h. The conditioned media was collected, then centrifuged at a low-speed spin (2000 × g at 4 °C for 20 min) to remove any cell debris, followed by an ultra-centrifugation (UC) step (100,000 g _avg at 4 °C for 2 h) of the supernatant using Optima™ L-80XP Ultracentrifuge and SW28 rotor (Beckman Coulter Inc. Indianapolis, IN, USA). EVs or LPS-EVs were re-suspended in 1 ml PBS (Hyclone) per 300 mL of conditioned media, aliquoted, then stored at − 80 °C.

LPS (O111:B4, #L2630) was procured from Sigma-Aldrich Ltd. (St. Louis, USA). APExBIO (Houston, TX, USA) supplied Ang-(1–7) (#A1041; ≥ 99% purity) and A-779 (#B6056; ≥ 98% purity).

ADSCs were isolated from the epididymal adipose tissue of four-week-old male SD rats [23 (link)]. Cells were cultured in low-glucose DMEM (Gibco, USA) supplemented with 10% FBS at 37 °C with 5% CO₂. For LPS preconditioning, the culture medium was replaced by a fresh medium containing 1 µg/mL LPS (O111:B4, Sigma-Aldrich, USA) for 48 h when the ADSCs reached 80% confluence. CD11b, CD29, CD44, CD45 and CD90 antibodies (eBioscience Inc., USA) were used to verify the surface markers of L-ADSCs. The multipotency of L-ADSCs was demonstrated based on osteogenic, adipogenic and chondrogenic differentiation, as described previously [24 (link)]. All cells were used at passages 3–7.
Corpus cavernosum smooth muscle cells (CCSMCs) were extracted as described in a previous study [25 (link)]. The cavernosal tissues were cut into 1-2 mm pieces and placed in a T25 flask with DMEM supplemented with 10% FBS. The cells migrating from the explants were passaged and purified. CCSMCs were identified by immunofluorescence of an anti-SMA antibody (19245T, 1:500, CST). All cells were used at passages 2–3.

All cell culture reagents were purchased from Gibco-Invitrogen (Carlsbard, CA, USA). Cell culture dishes and other plasticware were obtained from Nalgene Nunc (Rochester, NY, USA). Salts and buffers were purchased Sigma (St. Louis, MO, USA). Leptomycin B (LMB), α-tocopherol, LPS O111:B4 and (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) tetrazolium (MTT) were obtained from Sigma-Aldrich. Primary antibodies used in protein detection of CALM, α-adaptin, β-adaptin, eps 15, c-Myc, c-Jun, c-Abl and SR-B1 as well as secondary antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA, USA) and Abcam (Cambridge, UK). Fluorescent conjugates of LPS BODIPY FL, and 6-carboxy-2′,7′-dichlorodihydrofluorescein diacetate for ROS detection were obtained from Thermo Fisher Scientific (Waltham, MA, USA).

Adult male 3-month-old C57BL/6 J mice (27–30 g) were obtained from Envigo (Barcelona, Spain), while Tg (TcraTcrb)425Cbn (OT-II) mice were obtained from the Jackson Laboratory (Bar Harbor, ME, USA). The mice were housed at 21 °C in a humidity controlled environment on a 12-h light/dark cycle, fed ad libitum with standard rodent pellet diet (Envigo, Barcelona, Spain) and free access to water. Wild type animals received one intraperitoneal administration of LPS (O111:B4; 5 mg/kg in saline; Sigma-Aldrich, St. Louis, MO, USA) or saline alone, and they were sacrificed 48 h later. All procedures involving animals were carried out in accordance with the Spanish National Research Council’s guide for the care and use of laboratory animals, and the experimental design was approved by the Ethical Committee for Animal Testing at the University of Navarra (ref. 109-18).