MDP stimulation downregulates PGN mediated IL1β transcription by downregulating NF-κB and AP1 activation.

<p>(<b>A-E</b>), (<b>A</b>) Normal or Nod2 knockdown macrophages were treated as indicated. Culture supernatants were collected after 12 hours and the level of secreted IL1β was measured by ELISA. Each well contained 1×10⁶ macrophages. Statistical significance was checked by one way ANOVA. P value was found to be significant (<0.0001). (<b>B</b>) Real time RT-PCR analysis of IL1β mRNA. Normal or Nod2 knockdown Macrophages were treated as indicated. Total RNA was isolated used for checking IL1β transcripts. Each bar represents fold expression relative to untreated macrophages. Statistical significance was checked by one way ANOVA. P value was found to be significant (<0.0001). (<b>C</b>) Western blot analysis of pre-IL1β. Peritoneal macrophages were treated as indicated. Cell extracts were collected after 4 hours and analysed for the presence of pre-IL1β. ATP stimulation (1 mM) was given for 20 minutes after primary stimulation (MDP or PGN) was over. Lane1: untreated macrophages (123); lane2: MDP treated macrophages (127); lane3: PGN treated macrophages (140); lane4: ATP stimulated macrophages (124); lane5: MDP+PGN treated macrophages (132); lane6: Macrophages treated with MDP followed by ATP stimulation (108). Values in brackets indicate average integrated density values of spot densitometric analysis using software Alpha Imager (<b>D</b>) Electrophoretic mobility shift Assay for NF-κB. Normal or TLR2 knockdown macrophages or macrophages pre-treated (for 20 minutes) with TLR2 specific blocking antibody (1 µg/ml) were stimulated as indicated. Nuclear extracts were prepared and incubated with 100 fmol of biotinylated NF-κB binding sequence. EMSA was performed as described. Lane1: untreated macrophages; lane2: MDP treated macrophages; lane3: PGN treated macrophages; lane4: MDP+PGN treated macrophages; lane5: PGN treated TLR2 knockdown macrophages; lane6: Macrophages treated with TLR2 blocking antibody for 20 min and then stimulated with PGN; lane7: Nuclear extract of PGN stimulated macrophages is incubated with 100 fmol biotinylated NF-κB binding DNA and 100 pmol of unlabelled NF-κB binding DNA. Macrophages were treated for 40 minutes. (<b>E</b>) Electrophoretic mobility shift Assay for AP1. Macrophages were treated for 40 minutes as indicated. Nuclear extracts were prepared and EMSA was performed as described. Lane1: untreated macrophages (25); lane2: MDP treated macrophages (45); lane3: PGN treated macrophages (106); lane4: MDP+PGN treated macrophages (87); lane5: Pam3CSK4 treated macrophages (82); lane6: Pam3CSK4+MDP treated macrophages (69); lane7: Nuclear extract of PGN stimulated macrophages is incubated with 100 fmol biotinylated NF-κB binding DNA and 100 pmol of unlabelled AP1 binding DNA (12). Values in brackets indicate average integrated density values of spot densitometric analysis using software Alpha Imager. Western blot and EMSA correspond to one representative experiment of three and two independent experiments respectively.