Lps from escherichia coli 0111 b4

Twenty-four hours after the last doses of B. breve or PBS control, mice received an IP injection of 1.25 mg kg⁻¹ LPS from Escherichia coli 0111:B4 (Sigma) or sterile saline (control) and mice were sacrificed 1.5 h post-challenge with LPS. Proximal small intestine was collected in 10% neutral buffered formalin saline (Sigma) and fixed for 24 h followed by paraffin embedding. Samples of proximal small intestine were also collected into RNA Later (Qiagen) for transcriptome analysis or frozen on dry ice for subsequent ELISA analysis. In some cases, proximal small intestine was also collected into Hanks buffered saline solution (HBSS) for isolation of IECs.

DHI was purchased from Shandong Buchang Pharmaceutical Co., Ltd (Jinan, China) (drug approval number: Z20026866). LPS from Escherichia coli 0111:B4 was purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). D-galactosamine hydrochloride (GalN, 98%) and N-acetyl-L-cysteine (NAC, 98%) were purchased from Alfa AesarCo. (Tianjin, China). Detection kits for alanine aminotransferase (ALT), aspartate aminotransferase (AST), glutathione S-transferase (GST), and total bilirubin (TBil) were purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). The lipid peroxidation malondialdehyde (MDA) assay kit was purchased from Beyotime Institute of Biotechnology (China). Tumor necrosis factor (TNF)-α Mouse ELISA Kit was obtained from e-Bioscience Co. (USA). The DeadEnd Colorimetric TUNEL System and ImProm-II Reverse Transcription System were purchased from Promega Co. (USA). The Mammalian Cell Lysis Kit and UNIQ-10 column Trizol total RNA extraction kit were bought from Sangon Biological Engineering Technology & Services Co., Ltd. (Shanghai, China). The FastStart Universal SYBR Green Master (ROX) kit was purchased from Roche (Mannheim, Germany).

BALB/c and C3H/HeJ (TLR4 defective) mice received at days 0 and 7 subcutaneous (s.c.) injection of S1P (10 ng; Enzo Life Science, Italy) dissolved in sterile saline containing bovine serum albumin (0.001%) according to the manufacturer’s instructions. Subcutaneous administration of S1P induces in BALB/c mice asthma-like disease (17 (link)). As previously shown, these effects are sustained by a Th2 response as confirmed by the fact that the adoptive transfer of CD4⁺ T cells obtained from mice injected with S1P increases bronchial reactivity and induces pulmonary inflammation of recipient (non-treated) mice (17 (link)). In another set of experiments, BALB/c or C3H/HeJ (TLR4 defective) mice received lipopolysaccharide (0.1 µg; intranasal instillation LPS from Escherichia coli 0111:B4; Sigma Aldrich, Italy) or S1P (10 ng) or the association LPS + S1P at day 0 and 7. Another group of S1P-treated mice received the purified rabbit anti-TLR4 (10 µg; i.p. H-80, sc-10741; Santa Cruz) 30 min prior to S1P administration. Mice were sacrificed at days 10 and 21 and bronchi and lungs harvested and used to perform functional and molecular studies.

All experimental animal protocols were performed in accordance with European Community policies, and approved by local committees (AZ: 35-9185.81/G-86/16). To analyze BMP-9/LPS effects in vivo C57/Bl6 mice were used (n = 5 per group). Mice received one intraperitoneal injection of either PBS (control), lipopolysaccharide (LPS, from Escherichia coli 0111:B4; Sigma Aldrich) (25 µg/mouse), BMP-9 (5566-BP-10/CF; R&D Systems) (100 ng/mouse) or a mixture of both. Mice were sacrificed at 2 h and 12 h after injections and livers were processed for RT-qPCR.