Supplemental Information. By Capturing Inflammatory Lipids Released. from Dying Cells, the Receptor CD14 Induces

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1 Immunity, Volume 47 Supplemental Information By Capturing Inflammatory Lipids Released from Dying Cells, the Receptor CD14 Induces Inflammasome-Dependent Phagocyte Hyperactivation Ivan Zanoni, Yunhao Tan, Marco Di Gioia, James R. Springstead, and Jonathan C. Kagan

2 Figure S1. oxpapc is an agonist for CD14 but not for TLR4. Related to Figure 1. 1

3 (A) DCs were treated with oxpapc at the indicated concentrations. Surface levels of CD14 were measured by flow cytometry. Line graphs represent means and standard deviations of two independent experiments. (B-D) DCs were treated with LPS (1μg/ml) or oxpapc (120μM) for the indicated times in the presence or absence of cycloheximide (100μg/ml). Surface levels of CD14 (B), TLR4 (C) and TLR4 dimerization (D) were measured by flow cytometry. Line graphs represent means and standard deviations of two independent experiments. 2

4 Figure S2. oxpapc-induced CD14 deficiency selectively blocks TLR4 signaling from endosomes. Related to Figure 2. (A-C) Immortal macrophages were treated with LPS (indicated concentrations), or pretreated with oxpapc (120μM) for 30 min and then with LPS. (A) STAT-1 phosphorylation was measured 4 hours after LPS treatment by western analysis. (B) Type I IFN secretion was measured 18 hours after LPS stimulation by using the ISRE-luciferase reporter cell assay. (C) TNFα secretion was measured 18 hours after LPS stimulation by ELISA. nt: cells not treated with LPS;; LPS: cells primed with LPS. Bar graphs represent the average and error bars represent the standard deviation of triplicate readings from one representative experiment of three. Statistical significance of LPS + oxpapc treated cells compared to only LPS treated cells is shown. 3

5 (D) oxpapc binding to MD-2 was determined by biotinylated oxpapc pull down assay. Lysates of 293T cells expressing MD-2 were incubated with biotinylated oxpapc. MD-2-oxPAPC complexes was captured using neutravidin beads. The amount MD-2 retained by oxpapc was determined by western analysis. 4

6 Figure S3. CD14-deficiency does not alter splenic DC activation. Related to Figure 3. (A, B) WT or CD14-deficient splenic DCs were treated, or not, for 18 hours with LPS (1μg/ml) or oxpapc (120μM) or both. CD86 expression was measured by cytofluorimetry gating on CD11c+ cells. 5

7 Figure S4. CD14 is not required for the priming phase of inflammasome activation. Related to Figure 4. (A) WT DCs and CD14-deficient DCs were treated with LPS or Pam3CSK (P3C). Gene expression relative to TBP was analyzed by qpcr at times indicated. Results are shown as gene expression compared to untreated cells. Line graphs represent the average of triplicate readings from one representative experiment of three. (B) WT DC and CD14-deficient DC were treated with LPS (1μg/ml), oxpapc (120μM) or were primed with LPS for 3 hours and then treated with oxpapc in the presence or absence of rifnβ (100U/ml). 18 hours after LPS administration, IL-1β and TNFα secretion was measured by ELISA. Means and standard deviations of two independent experiments are shown. 6

8 (C) WT DC and CD14-deficient DC were treated with LPS or P3C (1μg/ml). Gene expression relative to Tbp was analyzed by qpcr at times indicated. Untreated cells were used as a negative control in all experiments. Line graphs represent the average and error bars represent the standard deviation of triplicate readings from one representative experiment of three. 7

9 Figure S5. oxpapc is a mixture of phospholipids. Related to Figure 5. (A) Mass spectrometry analysis of oxpapc. (B) oxpapc component chemical structures. 8

10 Figure S6. POVPC and PGPC do not induce pyroptosis. Related to Figure 6. A, B) Primary WT and CD14- deficient macrophages were treated for 3 hours with LPS (1μg/ml) and stimulated with: oxpapc (120μM), POVPC (120μM), PGPC (120μM), and ATP (5mM). (A) Images of cell morphology were taken 24 hours post- stimulation. (B) To remove dead, non- adherent cells, macrophages were subjected to one gentle PBS wash and warmed media was then added. Images of adherent macrophages were then captured. For each treatment, at least three randomly chosen fields were documented. Representative images of one experiment out of two independent experiments are shown. 9

11 (C) WT and CD14-deficient macrophages were treated (or not) for 3 hours with LPS and stimulated, or not, with: oxpapc (120μM), POVPC (120μM), PGPC (120μM), ATP (5mM). LDH release was assessed 24 hours later. Means and standard deviations of two replicates of one representative experiment out of three are shown. 10

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13 Figure S7. CD14 is required for macrophage hyperactivation. Related to Figure 7. (A, B) Primary WT and CD14-deficient macrophages were primed (or not) for 3 hours with P3C (1μg/ml) plus rifnβ (50 U/ml) and stimulated, or not, with oxpapc (100 µm), POVPC (120 µm), and PGPC (120 µm), IL-1β secretion was measured at indicated time points post stimulation. Means and standard deviations of two replicates of one representative experiment out of three are shown. (C, D) Primary WT and CD14-deficient macrophages were primed (or not) for 3 hours with P3C (1μg/ml) plus rifnβ (50 U/ml) and stimulated, or not, with ATP (5 mm), IL-1β secretion was measured at indicated time points post stimulation. Means and standard deviations of two replicates of one representative experiment of three are shown. (E) Primary WT and CD14-deficient macrophages were primed (or not) for 3 hours with P3C (1μg/ml) plus rifnβ (50 U/ml) and stimulated, or not, with: oxpapc (120μM), POVPC (120μM), PGPC (120μM), and ATP (5 mm) or lysed using triton (0.1%). LDH release was measured at 18 hrs post stimulation. (F) Primary WT and CD14-deficient macrophages were primed for 3 hours with P3C (1μg/ml) plus rifnβ (50 U/ml) and stimulate with: oxpapc (120μM), POVPC (120μM), PGPC (120μM), and ATP (5 mm). Images of cell morphology were captured at 24 hours post stimulation. For each treatment, at least three randomly chosen fields were documented. Representative images of one experiment out of two independent experiments are shown. 12