Lipopolysaccharide from escherichia coli 055 b5

The FH strain of Mp was purchased from the American Type Culture Collection (Manassas, VA, USA). Mp was cultured as described previously (48 (link), 49 (link)). The mice were challenged intranasally with 6 × 10⁷ CFU Mp in 40 μl of PBS under anesthesia. As a control, the mice were treated intranasally with 40 μl of PBS under anesthesia. As a positive control to induce lung injury, the mice were treated intranasally with 40 μl of 100 μg lipopolysaccharide from Escherichia coli 055:B5 (Sigma-Aldrich, St. Louis, MO, USA) under anesthesia. After Mp challenge, the DNA copies of Mp, immune cells, and the level of cytokines in BALF obtained by lavaging the lung with 1.2 ml PBS were analyzed. The DNA copies of Mp in BALF were determined by real-time PCR analysis as described previously (48 (link), 49 (link)).

Wild-type (WT) C57BL/6 mice were originally obtained from School of Veterinary, National University of La Plata. IFN-g^−/− (B6.129S7-Ifngtm1Ts/J strain) mice were obtained from The Jackson Laboratory and IFNAR^−/− (Ifnar1tm1Ag strain) mice were kindly provided by Institut Pasteur. Between 8- and 12-week-old male mice were maintained in the specific pathogen-free barrier facilities at the School of Chemical Sciences animal facility, where all experiments were done in compliance with the procedures outlined in the “Guide for the Care and Use of Laboratory Animals” (NIH Publication No. 86-23, 1985). The experimental protocols were approved by the Institutional Animal Care and Use Committee (IACUC). Our animal facility obtained NIH animal welfare assurance (No. A5802-01, OLAW, NIH, US).
Lipopolysaccharide from Escherichia coli 055:B5 (purified by gel-filtration chromatography) was purchased from Sigma-Aldrich and freshly dissolved in sterile saline prior to intraperitoneal (i.p.) injection. Mice were treated with either vehicle or 40 µg of LPS (1,6 mg/kg) for four consecutive days to induce neuroinflammation, following an injection scheme modified from Cardona et al. (5 (link)).

Lipopolysaccharide (from Escherichia coli, 055:B5) and 2-DG were the products of Sigma (St. Louis, MO, USA). The PKM2 activator ML265, the STAT3 inhibitor stattic, and the myeloperoxidase (MPO) activity assay kit were the products of Cayman Chemical (Ann Arbor, MI, USA). The enzyme-linked immunosorbent assay (ELISA) kits for the determination of mouse tumor necrosis factor alpha (TNF-α) and interleukin 6 (IL-6) were the products of NeoBioscience Technology Company (Shenzhen, China). The nuclear protein extract kit was the product of Genechem Co., Ltd. (Shanghai, China). The rabbit anti-mouse PKM2 (D78A4), STAT3 (79D7), phospho-STAT3 (D3A7), and lamin B (D9V6H) antibodies were the products of Cell Signaling Technology (Danvers, MA, USA). The horseradish peroxidase-conjugated goat anti-rabbit antibody was the product of Proteintech (Wuhan, China). The BCA protein assay kit was the product of Thermoscientific (Rockford, IL, USA). The enhanced chemiluminescence (ECL) reagents were the products of Advansta (Menlo Park, CA, USA).