Volume 18, Issue 5, Pages 1298-1311 (January 2017) SR-B1 Is a Silica Receptor that Mediates Canonical Inflammasome Activation  Misato Tsugita, Nobuyuki Morimoto, Manabu Tashiro, Kengo Kinoshita, Masafumi Nakayama  Cell Reports  Volume 18, Issue 5, Pages 1298-1311 (January 2017) DOI: 10.1016/j.celrep.2017.01.004 Copyright © 2017 The Author(s) Terms and Conditions

Cell Reports 2017 18, 1298-1311DOI: (10.1016/j.celrep.2017.01.004) Copyright © 2017 The Author(s) Terms and Conditions

Figure 1 SR-B1 Recognizes Silica through K151 and K156 (A) The indicated BW5147 cells were cultured with (shaded histograms) or without (open histograms) FITC-labeled amorphous nano-silica (3 μg/mL) for 30 min. Numbers indicate the mean fluorescence intensity (MFI). (B) The indicated BW5147 cells were cultured with FITC-amorphous silica for 30 min. Delta MFI was calculated by subtracting MFI of silica-treated cells from MFI of untreated cells. (C and D) The molecular surface of mSR-B1 was built and the electrostatic surface of the molecule is shown in (C). The boxed apex area in (C) was focused and is shown as a ribbon model in (D) where basic and acidic amino acid residues are shown using a ball-and-stick model colored by blue and red, respectively. (E) HEK293T cells transiently expressing wild-type (WT) or mutant mSR-B1-eGFP were cultured with rhodamine-labeled amorphous nano-silica (30 μg/mL) for 30 min. Numbers indicate the MFI. Delta MFI was calculated as in (B). Data are shown as mean + SD. ∗∗p < 0.01 (Student’s t test). See also Figure S1. Cell Reports 2017 18, 1298-1311DOI: (10.1016/j.celrep.2017.01.004) Copyright © 2017 The Author(s) Terms and Conditions

Figure 2 SR-B1 Discriminates between Particulate Substances (A) Hydrodynamic diameter and zeta-potential of particles in PBS (pH7.4) were measured by dynamic light scattering. (B) The indicated NIH 3T3 cells were cultured with particles for 30 min. Delta MFI was calculated as in Figure 1B. Delta mean side scatter intensity (MSI) was calculated by subtracting MSI of crystal-treated cells from MSI of untreated cells. (C) Cells were cultured with FITC-amorphous silica (1 μg/mL) or crystalline silica (30 μg/mL) for 30 min and analyzed by confocal microscopy. White bars, 20 μm. Number of particles per cell (n = 5–7 per group) was quantified by NIH ImageJ software. Data are shown as mean + SD. ∗∗p < 0.01 ∗p < 0.05 (Student’s t test). See also Figure S1. Cell Reports 2017 18, 1298-1311DOI: (10.1016/j.celrep.2017.01.004) Copyright © 2017 The Author(s) Terms and Conditions

Figure 3 SR-B1 Contributes to Macrophage Recognition of Amorphous and Crystalline Silica, but Not to TiO2 or MSU Crystals (A) BMDMs were treated with FITC-amorphous silica (3 μg/mL), FITC-TiO2 (10 μg/mL), crystalline silica (30 μg/mL), or MSU (100 μg/mL) for 30 min. Numbers indicate the MFI or MSI. (B) BMDMs were cultured with the indicated dose of particles for 30 min. Delta MFI and delta MSI were calculated as in Figure 2B. (C) BMDMs were treated as in (A) and analyzed by confocal microscopy. Crystal particles are shown by DIC intensity. Yellow bars, 5 μm. Number of particles per cell (n = 8–16 per group) was quantified by NIH ImageJ software. N.D., not detected. Data are shown as mean + SD. ∗p < 0.05, ∗∗p < 0.01 (Student’s t test). Cell Reports 2017 18, 1298-1311DOI: (10.1016/j.celrep.2017.01.004) Copyright © 2017 The Author(s) Terms and Conditions

Figure 4 SR-B1 Contributes to Inflammatory Responses to Amorphous and Crystalline Silica, but Not to TiO2 or MSU Crystals (A and B) LPS-primed BMDMs were stimulated with particles for 4 hr and the secretion of cytokines was measured by ELISA in (A). Percent cell death was quantified by LDH release assay in (B). (C) LPS-primed BMDMs were stimulated with particles (100 μg/mL) or ATP (1 mM) for 2 hr. Maturation of caspase-1, IL-1β, and caspase-11 and SR-B1 expression in pooled sup. and cell extracts were analyzed by immunoblot. (D) LPS-primed BMDMs were stimulated as in (C) for 4 hr. Protein in sup. was cross-linked with DSS, and ASC oligomerization was analyzed by immunoblot. Data are shown as mean + SD. ∗∗p < 0.01 (Student’s t test). See also Figure S2. Cell Reports 2017 18, 1298-1311DOI: (10.1016/j.celrep.2017.01.004) Copyright © 2017 The Author(s) Terms and Conditions

Figure 5 Anti-Mouse SR-B1 Blocking mAb Inhibits BMDM Responses to Silica (A) The indicated Jurkat.EcoR cells were stained with biotinylated control rat IgG2a or anti-mSR-B1 mAb AOB-1 followed by PE-streptavidin. (B and C) The indicated Jurkat.EcoR cells pretreated with 50 μg/mL (B) or the indicated dose (C) of mAb were cultured with or without FITC-amorphous silica (10 μg/mL) as in Figure 1A. Delta MFI was calculated as in Figure 1B. (D and E) B6 mouse BMDMs were pretreated with mAb (50 μg/mL) and were then cultured with or without 100 μg/mL (D) or the indicated dose (E) of crystalline silica for 30 min. Numbers indicate the MSI. Delta MSI was calculated as in Figure 2B. (F) LPS-primed B6 mouse BMDMs were pretreated with mAb (50 μg/mL) or YVAD-CHO (20 μM) and were then cultured with particles or ATP for 4 hr, and the secretion of cytokines was measured by ELISA. Data are shown as mean + SD. ∗p < 0.05, ∗∗p < 0.01 (Student’s t test). See also Figures S3–S5. Cell Reports 2017 18, 1298-1311DOI: (10.1016/j.celrep.2017.01.004) Copyright © 2017 The Author(s) Terms and Conditions

Figure 6 Anti-Mouse SR-B1 Blocking mAb Inhibits Silica-Induced Lung Inflammation and Fibrosis (A and B) B6 mice pretreated with control rat IgG or AOB-1 (300 μg/head, N = 4 each) were injected i.t. with crystals (1 mg/head). As a negative control, mice (N = 3) were injected i.t. with PBS. Twenty-four hours later, lung inflammation was analyzed by micro-computed tomography (A) and H&E staining (B). Red arrowheads indicate areas of inflammation in (A). Black bars, 100 μm in (B). (C and D) B6 mice were treated as in (A), and BALF was harvested. Cells in BALF were stained with anti-Gr-1 mAb and were counted and analyzed by flow cytometry in (C). Cytokines in BALF were measured by ELISA in (D). (E) Six weeks after i.t. injection with the indicated dose of silica (N = 5 each) or with PBS (N = 3), mouse lung fibrosis was analyzed. The boxed areas in top panels were shown at a higher magnification in middle panels and then were processed by NIH ImageJ software in bottom panels. Bars, 100 μm. Arrowheads indicate picrosirius red-positive fibrotic area. The percentage of fibrotic area on four fields per mouse (n = 12 per PBS-treated group, n = 20 per each silica-treated group) was quantified by NIH ImageJ software. Data are shown as mean + SD. ∗p < 0.05. See also Figure S4. Cell Reports 2017 18, 1298-1311DOI: (10.1016/j.celrep.2017.01.004) Copyright © 2017 The Author(s) Terms and Conditions

Figure 7 Human SR-B1 Recognizes Silica and Is Associated with the Inflammatory Response (A) The recognition of FITC-amorphous silica was analyzed as in Figure 1B. (B) The indicated BW5147 cells were stained with biotinylated control mouse IgG2a or anti-hSR-B1 mAb FRI-1 followed by PE-streptavidin. (C) The indicated BW5147 cells pretreated with mAb (10 μg/mL) were cultured with FITC-amorphous silica (3 μg/mL) and the delta MFI was calculated as in Figure 1B. (D) Human PBMCs were stained with the indicated cell markers plus control mIgG2a or FRI-1. (E and F) Human PBMCs pretreated with mAb (10 μg/mL) were treated with crystalline silica (50 μg/mL) for 30 min. Then cells were stained with anti-CD14 mAb. Numbers indicate MSI (E). Delta MSI was calculated as in Figure 2B (F). (G) LPS-primed human PBMCs were pretreated with mAb (10 μg/mL) or YVAD-CHO (20 μM) and were then treated with crystals or ATP for 2 hr. IL-1β in sup. was measured by ELISA. (H) LPS-primed human PBMCs were pretreated with mAb (20 μg/mL) and were then treated with silica (30 μg/mL), MSU (90 μg/mL), or ATP (1 mM) for 2 hr. Maturation of caspase-1 and IL-1β in pooled sup. and cell extracts was analyzed by immunoblot. Data are shown as mean + SD; ∗∗p < 0.01 (Student’s t test). Cell Reports 2017 18, 1298-1311DOI: (10.1016/j.celrep.2017.01.004) Copyright © 2017 The Author(s) Terms and Conditions