Journal: Thorax

Article Title: RAGE-induced ILC2 expansion in acute lung injury due to haemorrhagic shock

doi: 10.1136/thoraxjnl-2019-213613

Figure Lengend Snippet: High-mobility group box 1 (HMGB1) induces type 2 innate lymphoid cells (ILC2s) accumulation in the lungs following haemorrhagic shock (HS). (A) ELISA analysis of plasma HMGB1 concentrations and correlation of plasma HMGB1evels with absolute numbers of peripheral blood ILC2s in 30 patients with HS and 29 healthy controls. (B) Western blot analysis of HMGB1 protein expression in lung tissues from wild-type (WT) mice at 0, 6, 12, 24, 36, 48 and 72 hours after HS or sham surgery. Graphs showing quantification of density of Western blot HMGB1 bands normalised to β-actin loading control. (C) ELISA analysis of plasma HMGB1 concentrations in WT mice at time points up to 72 hours after HS. (D) Representative flow cytometry plots, pregated for CD45 + Lin − cells, showing percentages of ILC2s in the lungs of WT mice given intratracheally rHMGB1 (2 mg/kg) or phosphate buffer saline (PBS) for 24 hours. Graphs showing percentages and total numbers of ILC2s in the lungs. (E) Representative flow cytometry plots showing ILC2 percentages and graphs showing ILC2 percentages within the CD45 + Lin − populations and absolute numbers of ILC2s in the lungs at 24 hours after HS or sham surgery in WT mice treated with intraperitoneal neutralising antibody against HMGB1 (anti-HMGB1, 2 mg/kg), or non-specific control antibody (control IgY). (F) Representative flow cytometry plots showing percentages of ILC2s within the CD45 + Lin − populations in the lungs and graphs showing ILC2 percentages and absolute numbers of ILC2s in the lungs of inducible Hmgb1 −/− or control mice at 24 hours after HS. All results are shown as mean±SD. Data were analysed by (A) unpaired two-tailed Student’s t-test and Spearman’s rank correlation, (B, C) Kruskal-Wallis test or (D–F) Mann-Whitney test.

Article Snippet: In some experiments, mice received neutralising antibody against HMGB1 (2 mg/kg, IBL International, Japan) or control non-specific chicken IgY (2 mg/kg, IBL International) by i.p. 30 min before haemorrhage.

Techniques: Enzyme-linked Immunosorbent Assay, Western Blot, Expressing, Flow Cytometry, Two Tailed Test, MANN-WHITNEY

Journal: Thorax

Article Title: RAGE-induced ILC2 expansion in acute lung injury due to haemorrhagic shock

doi: 10.1136/thoraxjnl-2019-213613

Figure Lengend Snippet: High-mobility group box 1 (HMGB1)-receptor for advanced glycation end products (RAGE) is required for type 2 innate lymphoid cell (ILC2)-induced type 2 inflammation. (A–C) Flow cytometry blots and graphs showing percentages of (A) IL-5 + ILC2s, (B) IL-9 + ILC2s and (C) IL-13 + ILC2s in the lungs of wild type (WT), Hmgb1 − /− and Rage −/− mice at 24 hours after haemorrhagic shock (HS) or sham surgery. (D–F) Flow cytometry blots and graphs showing percentages of (D) interleukin (IL)-5 + ILC2s, (E) IL-9 + ILC2s and (F) IL-13 + ILC2s in the lungs at 24 hours after HS or sham surgery in WT mice treatment with HMGB1 neutralising antibody (2 mg/kg) or non-specific control antibody (control IgY). (G) ELISA analysis of plasma IL-5, IL-9 and IL-13 concentration at 24 hours after HS or sham surgery in WT, Hmgb1 − /− and Rage − /− mice. (H) ELISA analysis of plasma IL-5, IL-9 and IL-13 concentration at 24 hours after HS or sham surgery in WT mice treated with HMGB1-neutralising antibody or control IgY. All results are shown as mean±SD. Data were analysed by Mann-Whitney test.

Article Snippet: In some experiments, mice received neutralising antibody against HMGB1 (2 mg/kg, IBL International, Japan) or control non-specific chicken IgY (2 mg/kg, IBL International) by i.p. 30 min before haemorrhage.

Techniques: Flow Cytometry, Enzyme-linked Immunosorbent Assay, Concentration Assay, MANN-WHITNEY

Journal: Thorax

Article Title: RAGE-induced ILC2 expansion in acute lung injury due to haemorrhagic shock

doi: 10.1136/thoraxjnl-2019-213613

Figure Lengend Snippet: High-mobility group box 1 (HMGB1)-receptor for advanced glycation end products (RAGE) activation on innate lymphoid cells (ILC2s) induces eosinophil infiltration into lung. (A) Representative flow cytometric analysis and graphs showing (B) percentages and absolute numbers of eosinophils (CD45 + Gr-1 − Siglec-F + CD11b + ) and (C) percentages of neutrophils (CD45 + Gr-1 + CD11b + Siglec-F − ) in the lungs of wild-type (WT) mice at 24 hours after treatment with intratracheally (i.t.) instilled rHMGB1 (2 mg/kg) or PBS. (D) Graphs showing absolute numbers of eosinophils measured by flow cytometry in the lungs of WT mice at time points up to 72 hours after haemorrhagic shock (HS). (E) Representative flow cytometry blots showing percentages within the CD45 + populations and graphs showing total number of eosinophils at 24 hours after HS in the lungs of WT mice treated with HMGB1-neutralising antibody or control IgY. (F) Representative flow cytometry plots showing percentages of eosinophils and graphs showing absolute numbers of ILC2s in the lungs of WT, Hmgb1 − /− and Rage − /− mice at 24 hours after HS or sham surgery. (G) Flow cytometry plots showing percentages and graphs showing absolute numbers of lung eosinophils from isotype and αThy1.2-treated Rag1 −/− mice 24 hours after given i.t. rHMGB1 gated on CD45 + populations. (H, I) Flow cytometry blots showing percentages and graphs showing absolute numbers of eosinophils in the lungs at 24 hours after HS in (H) Hmgb1 −/− and (I) Rage −/− mice transferred with ILC2s (5×10 4 cells/mouse) isolated from WT mice or saline at 30 min post-HS. All results are shown as mean±SD. Data were analysed by Mann-Whitney test.

Article Snippet: In some experiments, mice received neutralising antibody against HMGB1 (2 mg/kg, IBL International, Japan) or control non-specific chicken IgY (2 mg/kg, IBL International) by i.p. 30 min before haemorrhage.

Techniques: Activation Assay, Flow Cytometry, Isolation, MANN-WHITNEY

Journal: Thorax

Article Title: RAGE-induced ILC2 expansion in acute lung injury due to haemorrhagic shock

doi: 10.1136/thoraxjnl-2019-213613

Figure Lengend Snippet: High-mobility group box 1 (HMGB1)-receptor for advanced glycation end products (RAGE)-dependent type 2 innate lymphoid cells (ILC2s) expansion promotes lung injury after haemorrhagic shock (HS). (A) H&E stained sections and histopathological lung injury scores at 24 hours after HS of wild-type (WT), Hmgb1 − /− and Rage − /− mice. (B) H&E stained sections and histopathological lung injury scores at 24 hours after HS from WT mice treated with HMGB1-neutralising antibody or control IgY. (C, D) H&E stained sections and histopathological lung injury scores at 24 hours after HS from (C) Hmgb1 − /− and (D) Rage −/− mice transferred with ILC2s (5×10 4 cells/mouse) isolated from WT mice or saline at 30 min post-HS. Magnification is ×200. All results are shown as mean±SD. Data were analysed by Mann-Whitney test.

Article Snippet: In some experiments, mice received neutralising antibody against HMGB1 (2 mg/kg, IBL International, Japan) or control non-specific chicken IgY (2 mg/kg, IBL International) by i.p. 30 min before haemorrhage.

Techniques: Staining, Isolation, MANN-WHITNEY