Integrin CD11b negatively regulates TLR-triggered inflammatory responses by. activating Syk and promoting MyD88 and TRIF degradation via cbl-b
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1 Integrin CD11b negatively regulates TLR-triggered inflammatory responses by activating Syk and promoting MyD88 and TRIF degradation via cbl-b Chaofeng Han, Jing Jin, Sheng Xu, Haibo Liu, Nan Li, and Xuetao Cao
2 Supplementary Fig. 1 FACS assay of expression of CD11b on Itgam +/+ and Itgam +/- peritoneal macrophages. Data are representative of three independent experiments with similar results using pooled macrophages from at least two littermates for each genotype.
3 Supplementary Fig. 2 The intestines of Itgam -/- mice show more severe hyperaemia and hydropsia in response to Poly(I:C) challenge. Hematoxylineosin staining (left panel) ( 100) of intestines from Itgam +/- and Itgam -/- mice i.p. injected with Poly(I:C) (20 mg/kg) 7 hours before dissected (right panel). Data are representative of three independent experiments with similar results.
4 Supplementary Fig. 3 LPS-induced activation of CD11b involves in PI(3)K and RapL pathway. FACS assay of mean fluorescence index (MFI) by staining with CBRM1/5 antibody on /ml human monocyte-derived macrophages pretreated with Wortmannin (100nM) for 1 hour and then stimulated LPS (100ng/ml) as indicated time(a), or transfected with SiRNA specific to RapL (Si-RapL) or negative control (Mock) 72 hours before stimulation of LPS (100ng/ml) (b), or transfected as b and then pretreated as a before stimulation of LPS (100ng/ml, 15 mins) (c). The efficiency of silence was verified by immunobotting with RapL antibody, with p38 as loading control. Data are representative of three independent experiments with similar results and presented as means ± SD. *, P<0.05; **, P<0.01.
5 Supplementary Fig. 4 Increased mrna expression of TNF, IL-6 and IFN-β in TLR-triggered Itgam-deficient macrophages. Quantitative-RT-PCR analysis of TNF, IL-6 and IFN-β in /ml Itgam +/- or Itgam -/- peritoneal macrophages stimulated with LPS (100 ng/ml), Poly(I:C) (10μg/ml) or CpG ODN (0.3 μm), Pam 3 CSK 4 (1μg/ml). TNF and IL-6 were measured 2 h while IFN-β 4 h after TLR ligands stimulation. Data were presented by folds of CpG ODN-induced mrna expression level in Itgam +/- macrophages, presented as mean ± SD of triplicate samples. *, P<0.05; **, P<0.01.
6 Supplementary Fig. 5 Itgam deficiency enhances TLR9 and TLR3 signaling in macrophages. Immunoblot analysis of lysates from Itgam +/- or Itgam -/- macrophages stimulated with CpG ODN (0.3μM) or Poly(I:C) (10μg/ml) for 30 minutes, probed with indicated antibody (p38 as loading control). Data are representative of three independent experiments with similar results using pooled macrophages from at least two littermates for each genotype.
7 Supplementary Fig. 6 Bands intensity quantification of active Erk. Bands intensity of active Erk relative to bands of p38 from Fig. 3d was quantitated by Gene Snap software. Data are presented as means ± SD. *, P<0.05; **P<0.01.
8 Supplementary Fig. 7 Itgam deficiency decreases ROS while increases NO production in macrophages in response to LPS. (a) FACS assay of Itgam +/- or Itgam -/- macrophages stimulated with LPS (100ng/ml) as indicated time by staining with H 2 DCFDA (10μM). (b) Griess reagents assay for NO production in sera of Itgam +/- or Itgam -/- mice i.p. challenged with LPS (15mg/kg body weight). (c) Griess reagents assay for NO production in supernatants of /ml Itgam +/- or Itgam -/- macrophages stimulated with LPS (100ng/ml) as indicated time. Data are representative of three independent experiments with similar results and presented as means ± SD. *, P<0.01.
9 Supplementary Fig. 8 CD11b negatively regulates TLR-triggered inflammatory response by enhancing activation of tyrosine kinase Src/Syk. (a, b) ELISA of cytokines production in supernatants from /ml Itgam +/- or Itgam -/- peritoneal macrophages pretreated with PP1 (10μM) for 1 hour (a), or transfected with constitutively activated Syk (CA-Syk) (b), and then stimulated with (100 ng/ml), Poly(I:C) (10μg/ml) or CpG ODN (0.3 μm). TNF and IL-6 were measured 8 h while IFN-β 12 h after TLR ligands stimulation. Data are representative of three independent experiments with similar results using pooled macrophages from at least two littermates for each genotype and are presented as means ± SD. *, P<0.01, **, P<0.01.
10 Supplementary Fig. 9 Itgam deficiency attenuates TLR9 and TLR3 ligands-induced degradation of MyD88 and TRIF in macrophages. Immunoblot analysis of lysates from Itgam +/- or Itgam -/- macrophages stimulated with 0.3μM CpG-ODN or 10μg/ml Poly(I:C) for 4 hours, probed with anti-myd88 and TRIF antibody (p38 as loading control). Data are representative of three independent experiments with similar results using pooled macrophages from at least two littermates for each genotype.
11 Supplementary Fig. 10 Y275 of MyD88 and Y375 of TRIF are localized on the surface of the modeling crystal structure of MyD88 and TRIF. PDB documents (model NO. 2JS7 and 2Z5V for TIR-domain of MyD88; 1GWA and 1HAY for TIRF) from SWISS-model, green presents tyrosines of 275 of MyD88 and Y375 of TRIF.
12 Supplementary Fig. 11 Overexpression of constitutively active Src prevents enhancement of TLR4 signaling by CD11b blockade. ELISA of cytokines production in supernatants from /ml Itgam +/- macrophages transfected with constitutively active Src and pretreated with IgG (Isotype) or 10μg/ml CD11b blocking antibody (M1/70) for 1 hours before stimulation of LPS (100 ng/ml). TNF were measured 8 h while IFN-β 12 h after TLR ligands stimulation. Data are representative of three independent experiments with similar results using pooled macrophages from at least two littermates for each genotype and are presented as means ± SD.
13 Supplementary Fig.12 CD11b does not affect TNF-induced IKKα/β and Jnk activation. Immunoblot analysis of lysates of splenocytes from Itgam +/- or Itgam -/- mice intravenously injected with TNF (20μg/kg body weight) (a), or Itgam +/- or Itgam -/- macrophages stimulated with TNF (20/ml) as indicated time (b), or Itgam +/- macrophages transfected as indicated and stimulated with TNF (20/ml) as indicated time (c), probed with indicated antibody (p38 or β-actin as loading control). Data are representative of three independent experiments with similar results.
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WT Xid (h) poly(da:dt) Alum. Poly(dA:dT) 0.05
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Supplemental Figure 1 1a 1c PD-1 MFI fold change 6 5 4 3 2 1 IL-1α IL-2 IL-4 IL-6 IL-1 IL-12 IL-13 IL-15 IL-17 IL-18 IL-21 IL-23 IFN-α Mut Human PD-1 promoter SBE-D 5 -GTCTG- -1.2kb SBE-P -CAGAC- -1.kb
3 The abbreviations used are: DC, dendritic cell; TLR, Toll-like receptor; ICAM,
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Spleen. mlns. E Spleen 4.1. mlns. Spleen. mlns. Mock 17. Mock CD8 HIV-1 CD38 HLA-DR. Ki67. Spleen. Spleen. mlns. Cheng et al. Fig.
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