New Interplay Between Interstitial and Alveolar Macrophages in Pulmonary Alveolar Proteinosis

New interplay between interstitial and alveolar macrophages in pulmonary alveolar proteinosis (PAP)induced by indium-tin oxide particles

François Huaux*,Valentin De Gussem, Astrid Lebrun, YousofYakoub, MihalyPalmai-Pallag, Saloua Ibouraadaten, Francine Uwambayinema and Dominique Lison.

Supplementary figures

Supplementary Figure 1: ITO particles induce pulmonary alveolar proteinosis (PAP) in mice.(A) PAS-stained lung sections (first line) and BAL pellets (second line) of control mice (Ctl) and ITO-treated animals after 3, 15 and 30 days. PAS-stained BAL pellets of Ctl and corresponding silica-treated mice (3, 15 or 30 days as indicated in the third line) (Magnification: 200x and 400x for the inserts). Kinetics (3 to 30 days) of protein (B) and LDH activity (C) levels in BAL supernatants after ITO or silica aspiration. Each group comprised at least 4 mice, and 2 mg of ITO or SiO2 were administered by pharyngeal aspiration. Data are given as mean ± SEM. These results were statistically analyzed using a one-way ANOVA and Dunett’s comparison post-test. *p<0.05, **p<0.01 and ***p<0.001 indicate a statistically significant difference between treated and corresponding control mice.

Supplementary Figure 2: Inflammatory phagocyte accumulation and cytokine expression in response to ITO or SiO2in mouse lungs. Number of Ly6G+neutrophils (A) and Ly6C+monocytes (B),IL-6 (C) and CCL-2 (D) cytokine levels in BAL after ITO (2mg) or silica (2mg) treatment. Data are given as mean ± SEM (n = 4 or 8). These results were statistically analyzed using a one-way ANOVA and Dunett’s comparison post-test. *p<0.05, **p<0.01 and ***p<0.001 indicate a statistically significant difference between treated and control mice.

Supplementary Figure 3: FACS strategy and analysis of lung neutrophils, monocytes and IL-1α-treated bone marrow-derived macrophages.(A) The flow cytometry dot plots show the gating strategy used to identify lung neutrophils (CD45+ Ly6G+ GR1+) and monocytes (CD45+ F4/80+ CD11b+ Ly6C+) and the proportion of these cells in lung tissue cell suspensions obtained from ITO-treated mice at day 3 (FSC; forward scatter). (B) Phenotypic analysis by flow cytometry of macrophages (F4/80, CD11c, CD64 TfR and Siglec-F positive cells) derived from bone marrow macrophage progenitors cultured with IL-1α.

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