Lipopolysaccharides (lps)

LPS-induced ALI was performed to establish an ALI mouse model. Briefly, 1 mg/mL of LPS (Escherichia coli, serotype 055:B5; Sigma-Aldrich, St. Louis, MO, USA) was injected into mice through intratracheal instillation, and the control group was injected with the same volume of sterile phosphate-buffered saline (PBS), as in Wang et al. [27 (link)]. Mice were then sacrificed under ether narcotization at 24 h after challenge with LPS or PBS to collect 1% heparin-anticoagulated peripheral whole blood, spleen, bronchoalveolar lavage fluid (BALF), and lung tissues. The WT+LPS+PGRN and PGRN^−/−+LPS+PGRN groups were treated with 2 μg of recombinant-mouse PGRN protein (CF, R&D Systems, Minneapolis, MN, USA) 30 min after LPS challenge through intratracheal instillation [19 (link)]. Blood, spleen, BALF, and lung tissues were collected as indicated for further analysis.

A murine model of neutrophilic asthma that was characterized by Th17 cell responses was generated as described previously [18 ]. In brief, mice were intranasally sensitized with 75 μg LPS-depleted OVA (grade V; Sigma-Aldrich, St. Louis, MO, USA) plus 10 μg LPS (E. coli serotype 026 : B6; Sigma-Aldrich) on days 0, 1, 2, and 7 and then challenged with 50 μg OVA alone on days 14, 15, 21, and 22. One day after the final challenge, the mice were euthanized for further analyses.
In this study, the mice were divided randomly into four groups (n = 6 mice) as follows: (i) mice sensitized with phosphate-buffered saline (PBS) and challenged with OVA (control group); (ii) mice sensitized with OVA plus LPS and challenged with OVA (asthma group); (iii) mice treated with control IgG (R&D Systems) for half an hour before sensitization to OVA plus LPS and the same challenge with OVA later (control IgG group); (iv) mice treated with anti-HMGB1 IgG (R&D Systems) half an hour before sensitization to OVA plus LPS and the same challenge with OVA later (anti-HMGB1 group). Anti-HMGB1 IgG or control IgG was administered intranasally (200 μg/kg) on days 14, 15, 21, and 22 a half an hour before OVA challenge (Figure 1(a)). The dose of anti-HMGB1 IgG was predetermined using staining analysis of airway inflammation in mice receiving 100–400 μg/kg of anti-HMGB1 IgG.

The matured BMDMs obtained above were divided into four groups with 1 × 10⁶ cells for each group: (i) BMDMs incubated with Ts-AES (4 μg/ml) (AES + Mφ); (ii) BMDMs incubated with LPS (100 ng/ml) (Solarbio, China) and IFN-γ (10 ng/ml) (R&D Systems, USA) (LPS + IFN-γ + Mφ); (iii) BMDMs incubated with LPS (100 ng/ml) and IFN-γ (10 ng/ml) in the presence of Ts-AES (4 μg/ml) (AES + LPS + IFN-γ + Mφ); (iv) BMDMs incubated with PBS as the control group (PBS + Mφ). After being incubated for 24 h, cells were collected, and M1 macrophage-associated marker CD86 and M2 macrophage-associated marker CD206 expressed on the surface of the cells were measured by flow cytometry.