L3012

LysM-Rictor^WT or LysM-Rictor^KO mice (6–8 months old) were injected ip with a solution of LPS (2.5 mg/kg) (Sigma Aldrich L3012). Body temperature was monitored over the course of the experimentation using a rectal temperature sensor. Blood was collected before and after LPS injection. Preliminary experiments carried in our laboratory showed that mice having a core body temperature dropping down to 33+/−0.2°C or below, following LPS injection, have a poor recovery rate and a high probability of death and this was set as a criteria for an ethical sacrifice: mice reaching this limit were humanely sacrificed by CO² asphyxiation.

BMDMs from WT mice or deficient mice (Asc^−/−, Nlrc4^−/−, Aim2^−/−, Nlrp3^−/−, Casp-1/11^−/−, and Gsdmd^−/−) were infected with the SS2 WT or ∆SLY strain ex vivo at a multiplicity of infection (MOI) of 1 for 16 h without lipopolysaccharide (LPS,100 ng/ml, L3012, Sigma) priming. BMDMs treated with phosphate buffer saline (PBS) or LPS plus adenosine triphosphate (ATP; 500 μM, A2383, Sigma) were used as the negative and positive control, respectively.
In parallel, BMDMs from the WT mice or deficient mice (Asc^−/−, Nlrc4^−/−, Aim2^−/−, Nlrp3^−/−, Casp-1/11^−/−, and Gsdmd^−/−) were pretreated with LPS priming (100 ng/ml) for 3 h and washed off with PBS. Then, the cell-free concentrated supernatant proteins SS2-S/N (containing SLY) or ∆SLY-S/N (without SLY) were incubated with the primed BMDMs in a 24-well plates at a concentration of 200 ng/ml for 16 h, respectively.
Both the unprimed BMDMs and LPS-primed BMDMs were pre-incubated with various inhibitors, including KCl (50 mM, PB0440), oxidized ATP (oATP, 500 μM, A6779), N-acetyl-L-cysteine (NAC; 20 mM, A7250), Nlrp3 inhibitor MCC950 (10 μM, S7809), and Caspase-1 inhibitor Z-YVAD-FMK (10 μM, A3707) at the indicated concentrations for 1 h. Then, these unprimed BMDMs were infected with the SS2 WT or ∆SLY strain, and LPS-primed BMDMs were treated with the concentrated supernatant proteins SS2-S/N or ∆SLY-S/N for 16 h ex vivo, as described above.

After deep anaesthesia via an intra-peritoneal injection of ketamine (100 mg/kg body weight: BW) and xylazine (10 mg/kg BW), mice were placed in a stereotactic frame. For injection into the cerebellum, the skin covering the occipital bone was cut, and a burr hole was made 5 mm caudal from the bregma. The tip of a Hamilton syringe (33 gauge) with an attached micropump (UltraMicroPump II; World Precision Instrument (WPI) Sarasota, FL, USA) was inserted 1.8 mm below the pia mater of the cerebellar vermis. Ten microliters of viral solution (titre; 5.0 × 10¹² vector genomes (vg)/ml) was injected at a rate of 400 nl/min using a microprocessor-based controller (Micro4; WPI). Two micrograms of LPS extracted from Escherichia coli O111:B4 and purified via gel filtration chromatography (L3012; Sigma-Aldrich, St. Louis, MO, USA) was dissolved in 10 μl of PBS containing AAV9 vectors (final LPS concentration; 0.2 μg/μl, AAV9 titre; 5.0 × 10¹² vg/ml). This dosage of LPS was shown to induce the microglial response in the mouse brain upon intracerebroventricular injection46 (link).

BMDMs were seeded in a six-well plate at an initial density of 5×10⁵ cells per well and induced with 100 ng·mL⁻¹ LPS (L3012, Sigma-Aldrich), 100 ng·mL⁻¹ IFN-γ (285-IF, R&D System), LPS+IFN-γ, and RANKL (462-TEC, R&D Systems, Minneapolis, MN, USA) at different concentrations (1, 10, and 100 ng·mL⁻¹) for 12 h. Polarized cells were harvested for mRNA analysis by reverse reverse transcription-quantitative polymerase chain reaction (RT-qPCR).

A total of 1 mg/mL of Hp1404 peptide solution was mixed with an equal volume of 5 mg/mL of the lipoteichoic acid (LTA, sigma-aldrich: L2515) or lipopolysaccharide (LPS, sigma-aldrich: L3012) solution. Then, the MICs of Hp1404 treated with LTA or LPS against S. aureus AB94004 were measured. The experiment was verified by three independent trials.