Supplemental Figure 1

Transcription

1 Supplemental Figure 1 A IL-12p7 (pg/ml) Medium then TLR ligands MDP then TLR ligands Medium then TLR ligands + MDP MDP then TLR ligands + MDP B IL-12p4 (ng/ml) Medium MDP Medium CD4L IL-6 (ng/ml) Medium CD4L 4 CXCL8 (ng/ml) C PI Medium MDP Annexin V 2 IL-1 (ng/ml) 1 1 CD83 CD8 CD86

2 Supplemental Figure 2 18 Medium then E.coli stimulation MDP then E.coli stimulation IL-12p4 (ng/ml) E.coli (cfu/ml)

3 Supplemental Figure 3 A NOD2 +/+ Medium B NOD2 +/+ MDP NOD2 -/- Medium I L-12p7 (pg/ml) 4 3 NOD2 -/- MDP 2 1 Medium MDP PGN Pam LPS Flagellin CpG FL2-H MHC Class II 3 TNF (ng/ml) FL2 -H CD8 Medium MDP PGN Pam LPS Flagellin CpG F L2-H CD86

4 Supplemental Figure 4 A IRF4 Actin h 12h 24h 36h MDP stimulation h 12h 24h 36h LPS stimulation B Control sirna -Medium Control sirna -MDP pre-treatment IL-12p4 (ng/ml) PGN LPS IRF4 sirna #1-MDP pre-treatment IRF4 sirna #2-MDP pre-treatment

5 Supplemental Figure Control IRF4 PBS MDP PBS MDP IRF4 Actin I L-12p4 (ng/ml) 1 1 Control vector-medium Control vector-mdp IRF4 vector-medium IRF4 vector-mdp Medium PGN Pam 3 CSK4 LPS

6 A Supplemental Figure 6 Medium MDP! DGDAP IRF4 IRF Actin B IL-12p4 (ng/ml) Medium then TLR ligands! DGDAP then TLR ligands Medium then TLR ligands + MDP! DGDAP then TLR ligands + MDP 4 TNF (ng/ml) CXCL1 (ng/ml) 3 2 1

7 Supplemental Figure 7 Control FLAG-IRF4.2 1 µg FLAG-IRF4 Actin IL-12p4 (ng/ml) Control µg.2 µg 1 µg FLAG-IRF4 µg Medium PGN LPS IL-6 (ng/ml) 2 1 Medium PGN LPS

8 Supplemental Figure Legends Supplemental Figure 1. NOD 2 pre-stimulation does not induce cell exhaustion or cell death. (A) IL-12p7, CXCL8, and IL-1 production by human DCs pre-incubated with MDP or medium and then stimulated with TLR ligands in combination with MDP as described in Figure 6A. P<., P <.1 when compared with the concentrations of cytokines produced by DCs pre-incubated with medium and stimulated with TLR ligands (light blue bar). (B) Production of IL-12p4 and IL-6 in human DCs pre-incubated with MDP or medium and then stimulated with CD4 ligands (1µ g/ml). (C) Human DCs stimulated with MDP or medium for 24 hrs were stained with FITC-conjugated Annexin V and propidium iodide (PI). The number in each quadrant shows the percentage of cells (top). PI-negative DCs were stained with FITC-conjugated anti-cd83, CD8, or CD86 Ab. Red and green colors show the expression in cells with MDP and medium, respectively. Supplemental Figure 2. MD P pre-treatment suppresses the IL-12p4 production of human D C s in response to bacterial stimulation. Human DCs were pre-incubated with MDP or medium for 24hrs and then stimulated with various concentrations of E.coli. Culture supernatants were collected at 24hrs and analyzed for IL-12p4 production by ELISA. P <.1 when compared with the concentrations of IL-12p4 produced by DCs pre-incubated with medium and stimulated with E.coli. Supplemental Figure 3. MD P pre-stimulation suppresses TLR-induced cytokine secretion in mouse B MD C, but MD P stimulation has no effect on B MD C maturation markers. (A) CD11c + DCs (1x1 6 /ml) derived from bone marrow cells from NOD2-intact (NOD2 +/+ ) and NOD2-deficient (NOD2 -/- ) mice were pre-incubated with MDP (µ g/ml) or medium alone for 24hrs and stimulated with broad range of TLR ligands. Culture supernatants were collected at 24hrs and analyzed for cytokine production by ELISA. P<., P <.1 when sups are compared to NOD2-intact DCs pre-incubated with medium and stimulated with TLR ligands (light blue bar). (B) CD11c + DCs - 1 -

9 derived from NOD2-intact mice were pre-incubated with MDP (µ g/ml) or medium alone for 24hrs and then their co-stimulatory molecules were stained and analyzed by flow cytometry. Red and green colors show the expression in cells with MDP and medium, respectively. Supplemental Figure 4. Silencing of IRF4 with IRF4-siRNA reverses MD P- mediated suppression. (A) Human DCs were stimulated with either MDP (1µ g/ml) or LPS (1µ g/ml). Cell lysates were collected at the indicated time points and IRF4 expression was assessed by immunoblot. (B) Human DCs were transfected with either control scrambled sirna or two different IRF4- specific sirna (2µ g). After transfection, cells were washed and either left alone or pre-stimulated with MDP for 24hrs, and then stimulated with either PGN (1µ g/ml) or LPS (1µ g/ml) after washing. Culture supernatants were collected at 24hrs and analyzed for IL-12p4 production by ELISA. P <.1 when compared with the concentrations of cytokines by DCs transfected with control sirna and pre-incubated with medium (white bar). Supplemental Figure. Susceptibility to MD P suppression is restored by IRF4 reconstitution. THP1 cells were transfected with a vector expressing FLAG-tagged human IRF4 or a control vector (1µ g) by the Amaxa nucleofection method; the cells were then pre-incubated with medium or MDP for 24hrs and then stimulated with PGN, Pam 3 CSK4, and LPS. Expression of IRF4 was analyzed 48hrs after the transfection by immuno-blotting with anti-flag Ab (top); culture supernatants were collected at 24hrs and analyzed for cytokine production by ELISA (bottom). P <., P <.1 when compared with the concentrations of cytokines by cells transfected with a control vector and preincubated with medium (white bar). Supplemental Figure 6. Pre-activation of NOD 1 does not lead to reduced cytokine production upon re-stimulation with TLR ligands. Human DCs were pre-incubated with!dgdap or medium for 24hrs and then stimulated with broad range of TLR ligands in combination with MDP for 24hrs. (A) Expression - 2 -

10 of IRF4 and IRF in human DCs stimulated with MDP,!DGDAP, or medium for 24hrs. (B) Production of cytokines and chemokines measured by ELISA. P<., P<.1 when compared with the concentrations of cytokines by DCs pre-incubated with medium and stimulated with TLR ligands (light blue bar). Supplemental Figure 7. PGN-signaling is more sensitive to IRF4-mediated suppression than LPS-signaling. Expression of transfected IRF4 in human DCs (top); DCs prepared from three healthy donors were transfected with various doses of FLAG-tagged IRF4 cdna; whole cell extract were prepared 24hrs after transfection followed by immuno-blotting. IL-12p4 (middle) and IL- 6 (bottom) production by IRF4 transfected DCs; after transfection, cells were stimulated with PGN or LPS for 24hrs. Culture supernatants were analyzed for cytokine production by ELISA. P<., P <.1, compared to control vector-transfected cells