Mouse ril 33

Bone marrow (BM) cells from the femur and tibia were isolated and red blood cells were lysed. BM cells were cultured in DMEM-F12 media supplemented with 10%FBS and 20% supernatant derived from CMG14–12 (M-CSF producing) cell line for 5–7 days prior to experiments. BMDM polarization was assessed by cell morphology and later confirmed by cell surface expression of CD64, F4/80, CD11b, and CD11c. For some experiments, BMDMs were exposed to neuron-derived supernatants overnight. On the day of experiment, cells were exposed to vehicle, 50 or 250ng/mL of LPS (Sigma-Aldrich) for 6–8hours. BD GolgiPlug™ Protein Transport Inhibitor (containing Brefeldin A) and BD GolgiStop^™ Protein Transport Inhibitor (containing Monensin) were added in the last 5 hours of stimulation. Cells and supernatant were recovered to be analyzed for cytokine expression by flow cytometry and cytokine concentration by ELISA, respectively. For IL-1β release, BMDMs were treated with 250ng/mL of LPS (Sigma-Aldrich) for 4 hours followed by exposure to vehicle, 0.5 or 2.5mM of ATP(Sigma-Aldrich). Supernatants were assessed for cytokine concentration by ELISA. For some experiments, control BMDMs were treated with 500ng/mL of neutralizing anti-ST2 antibody (R&D systems) or CD11c-IL-33KO BMDMs were exposed to 500ng/mL of mouse rIL-33 (R&D systems) 24hours prior to LPS stimulation.

Groups of six WT, IL-33-KO, and ST2-KO mice were anesthetized by isoflurane inhalation and then injected subcutaneously with 1 mL of air to form an air pouch one day before the infection. One day later, 0.1 mL of bacterial suspension containing 3 × 10⁸ S. pyogenes NZ131 cells was inoculated into the air pouch [48 (link)]. The animals were observed every day for a total of 13 days. Survival curves were then determined. At 48 h post-infection, the degrees of skin lesion were quantitated by an exudates-absorbed method, which is based on the premise that the more severe the skin damage, the more that the exudates will effuse. A Kimwipes (Kimberly-Clark Global Sales, Inc., Roswell, GA, USA) paper was attached to the air pouch area, and the wet area of the paper was then gauged [49 (link)]. The average lesion area in each group was generated by examination of skin lesions from six mice. After that, the skin lesion tissue around the air pouch was excised, fixed in 3.7% formaldehyde, and embedded in paraffin. The 5-µm-thick tissues were sliced and stained with hematoxylin and eosin. In some experiments, 0.1 mL of mouse rIL-33 (R&D Systems) (1 µg per mouse) and sST2 protein (R&D Systems) (1 µg per mouse) were injected into the air pouches of IL-33-KO mice and WT mice, respectively, at 30 min before GAS infection or 24 h post-infection.