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Figure S1. Phenotypic characterization of Itgb2-/- macrophages. (A) The expression of integrin alpha subunits, CD11a, CD11b, CD11c and F4/80 was determined on bone marrow-derived macrophages by flow cytometry. (B) Macrophages were stimulated with the indicated concentrations of IFNγ for 48 hours and MHC II expression was assessed by flow cytometry. (C) Macrophage surface expression of TLR4, TLR2 and Dectin-1 was determined by flow cytometry. (D) TLR9 mRNA expression was determined by qPCR, with levels normalized to GAPDH. The data are shown as mean +/- SD of triplicate wells and representative of 3 experiments.!

Figure S2. Itgb2-/- macrophages are hypersensitive to TLR stimulation. (A) Representative data of the results shown in Fig. 1A. Macrophages were stimulated with the indicated TLR agonists and supernatant cytokine concentrations were determined by ELISA 24 hours later. Results are displayed as mean +/- SD of independently stimulated wells from one experiment. (B) Expression of IL-23 p19 and IL-12 p35 was determined by qPCR, with values normalized to GAPDH. Results are representative of 2 experiments and shown as mean +/- SD of triplicate wells. (C) Representative data of the results shown in Fig. 1B. Kinetics of cytokine secretion as assessed by ELISA. Results are shown as mean +/- SD independently stimulated triplicate wells from one experiment. * p < 0.05, ** p < 0.01, *** p < 0.001!

Figure S3. Isolation of thioglycollate-elicited macrophages (A) Mice were injected i.p. with 3% thioglycollate broth and peritoneal exudates were examined for F4/80highGr-1low macrophages and F4/80negGr-1high neutrophils by flow cytometry up to 5 days post injection. (B) Representative plots for F4/80highGr-1low peritoneal macrophages after magnetic bead enrichment of D5 post-injected peritoneal exudates. !

Figure S4. Itgb2-/- dendritic cells are hypersensitive to TLR stimulation. (A) and (B) Bone marrow-derived dendritic cells were isolated by magnetic bead separation for MHC II+ cells after GM-CSF culture. DCs were stimulated with TLR agonists overnight and cytokine concentrations in the supernatant were determined by ELISA. The data are representative of 3 experiments and shown as mean +/- SD of independently stimulated triplicate wells. * p < 0.05.

Figure S5. CD11a, CD11b, and Cbl-b deficiency does not induce macrophage TLR hypersensitivity or disturb MyD88 degradation. (A) Representative data of the results shown in Fig. 4A. WT, Itgal-/- (CD11a KO), Itgam-/- (CD11b KO) and Itgb2-/- macrophages were stimulated with 1 ng/mL LPS, 100 nM CpG DNA or 100 μg/mL zymosan particles for 24 hours and supernatant IL-12 p40 concentrations were determined by ELISA. Data are shown as mean +/- SD of independently stimulated triplicate wells from one experiment. (B) Representative data of the results shown in Fig. 4C. Macrophages were stimulated as in (A) and cytokine concentrations were determined by ELISA. The results are displayed as mean +/- SD of independently stimulated wells from one experiment. (C) Macrophages were stimulated with 10 ng/mL LPS and cytoplasmic lysates were assessed for MyD88 by Western blot, with β actin used as a loading control. Results are representative of 2 independent experiments. !

Figure S6. β2 integrin deficiency enhances NF-κB pathway activation downstream of TLR activation. (A) and (C) Western blot analysis for macrophages stimulated with 1 ng/mL LPS for phospho- IκBα, with β actin used as a loading control. In (A) and (C), macrophages were pre-treated with 10 μM MG-132 for 30 min. prior to LPS treatment. (B) and (D) Relative densitometry ratios (phospho-IκBα/β actin) for the data represented in (A) and (C) respectively. The results in (B) and (D) are set at WT time 0 set to 1 and shown as mean +/- SD of 2 separate experiments. (E) Macrophages were stimulated with 20 ng/mL TNF and expression of NF-kB-dependent genes was determined by qPCR, with results normalized to GAPDH expression and set relative to WT at time 4 hours. The results are shown as mean +/- SD of 2 independent experiments. !

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