MDP co-stimulation facilitates export of nuclear cRel subunits in PGN stimulated macrophages.

<p>(<b>A-E</b>), (<b>A</b>) Nuclear translocation of cRel. Normal or cycloheximide (5 µg/ml) pre-treated (20 minutes prior to PGN or MDP+PGN stimulus) macrophages were stimulated for 40 minutes as indicated. Nuclear extracts were prepared and run on 10% SDS-PAGE. 30 µg of nuclear extract was loaded per well. Results correspond to one representative experiment of three independent experiments. (<b>B</b>) Phosphorylation of IκB. Normal or cycloheximide (5 µg/ml) pre-treated (20 minutes prior to PGN or MDP+PGN stimulus) macrophages were stimulated for 30 minutes as indicated. Whole cell lysates were checked for the presence of phosphorylated form of IκB. Results correspond to one representative experiment of three independent experiments. (<b>C</b>) Electrophoretic mobility shift Assay for NF-κB. Macrophages were stimulated for 40 minutes as indicated. Nuclear extracts were prepared and incubated with 100 fmol of biotinylated NF-κB binding sequence. EMSA was performed as described. Cycloheximide treatment was for 20 minutes prior to PGN or MDP+PGN stimulation. Results correspond to one representative experiment of two independent experiments. (<b>D</b>) Phosphorylation of IκB. Macrophages were stimulated for 30 minutes as indicated. Whole cell lysates were checked for the presence of phosphorylated form of IκB. Results correspond to one representative experiment of three independent experiments (<b>E</b>) Activation of MAPKs. Macrophages were stimulated as indicated for 30 minutes. Cell extracts were analysed for the presence of phosphorylated (activated) forms of p38, ERK and JNK. Results shown correspond to one representative experiment of three independent experiments.</p

Stimulation of peritoneal macrophages with PGN, but not with MDP, triggers IL1

<p>β <b>release.</b> (<b>A-F</b>), (<b>A</b>) Mouse peritoneal macrophages (1×10⁶/well/ml) were left untreated or treated with MDP (10 µg/ml) or PGN (8 µg/ml) for indicated time intervals. Culture supernatant was checked for the presence of IL1β by ELISA. Statistical significance was checked by two way ANOVA. P value was found to be significant (<0.0001). (<b>B</b>) Macrophages were left untreated or treated with MDP or PGN for 4 hours and cell extracts were analysed for the presence of IL1β using western blotting. (<b>C</b>) Mouse peritoneal macrophages were left untreated or treated with MDP (10 µg/ml) or PGN (8 µg/ml) for indicated time intervals. Total RNA was isolated using TRI reagent and was checked for the presence of IL1β transcripts using real time RT-PCR. The bars show fold increase compared to control macrophages. Statistical significance was checked by two way ANOVA. P value was found to be significant (<0.0001). (<b>D</b>) Macrophages were left untreated or treated as indicated (time in minutes) and cell extracts were analysed for the presence of phosphorylated forms of JNK, ERK and p38 using western blotting. (<b>E</b>) Macrophages were pre-treated or left untreated with ERK inhibitor PD98059 (50 µM) for 30 minutes and then stimulated with MDP for 2 hours. Total RNA was isolated and checked for the presence of IL1β transcripts. The graph shows fold increase in IL1β transcripts relative to untreated macrophages. Statistical significance was checked by one way ANOVA. P value was found to be significant (<0.0001). (<b>F</b>) Macrophages were left untreated or treated as indicated (time in minutes) and cell extracts were analysed for the presence of phosphorylated forms of IκB using western blotting. Blots correspond to one representative experiment of three independent experiments. Results in graph are presented as the mean of triplicate wells ± SD.</p

Differential modulation of PGN mediated gene expression by MDP co-stimulation.

<p>(<b>A</b>) ELISA for IL10, TNFα and RANTES. Normal of Nod2 knockout or scrambled sequence siRNA treated macrophages were stimulated for 12 hours with various ligands as indicated. Culture supernatant was checked for the presence of indicated cytokines. Results are presented as the mean of triplicate wells ± SD. Data is plotted on split semi-log graph. Statistical significance was checked by two way ANOVA. P value was found to be significant (<0.0001). (<b>B</b>) Western blot analysis of iNOS and COX2. Macrophages were stimulated for 6 hours as indicated and whole cell lysates were checked for the presence of iNOS and COX2 proteins. Results correspond to one representative experiment of three independent experiments.</p

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.</p

PGN mediated IL1β production is through TLR2 and involves NF-κB and JNK pathways.

<p>(<b>A-E</b>)<b>,</b> Blocking antibody (1 µg/ml) was added 20 minutes prior to stimulation, transfection with gene specific siRNA or scrambled sequence siRNA was done for 72 hours. MAPK inhibitors, SP600125 (20 µM) or PD98059 (50 µM) or SB202190 (5 µM) were added 20 minutes prior to stimulation (<b>A</b>) Normal macrophages or TLR2 knockdown macrophages or cRel knockdown macrophages or macrophages pre-treated with TLR2 specific blocking antibody were treated as indicated. After 12 hours culture supernatant was collected 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 macrophages or TLR2 knockdown macrophages or macrophages pre-treated with TLR2 specific blocking antibody were treated as indicated. Total RNA was isolated after 2 hours and 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>) Macrophages were left untreated or pre-treated MAPK inhibitors (JNK inhibitor SP600125 (20 µM) or ERK inhibitor PD98059 (50 µM) or p38 inhibitor SB202190 (5 µM)) for 30 minutes and then stimulated with MDP or PGN as indicated. Culture supernatants were collected after 12 hours and checked for the presence of secreted IL1β using 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>D</b>) Real time RT-PCR analysis of IL1β mRNA. Macrophages were pre-treated with inhibitors or left untreated as described and stimulated with PGN or left unstimulated and checked for the presence of IL1β transcripts after 2 hours of stimulation. 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>E</b>) Western blot analysis of phospho-cJun. TLR2 knockdown macrophages or Nod2 knockdown macrophages or macrophages pre-treated with TLR2 specific blocking antibody were stimulated as indicated. Cell extracts were collected after 40 minutes of stimulation and checked for the presence of phosphorylated form of cJun. Results in graph are presented as the mean of triplicate wells ± SD. Blots correspond to one representative experiment of three independent experiments.</p

Cartoon diagram showing the effect of Nod2 co-stimulation on gene expression induced by different TLRs.

<p>Gene 1 and gene 2 are independently induced by different TLRs and Nod2. Different induction level upon stimulation of different receptors indicates involvement of receptor specific TF profile. Co stimulation of macrophages with MDP and TLR ligands alters the profile of TFs bound to some gene promoters resulting in modulation of expression in various ways.</p

MDP mediated downregulation is specific for TLR2/1 ligands.

<p>(<b>A-E</b>), (<b>A</b>) ELISA for IL1β; macrophages were stimulated as indicated. Culture supernatants were collected after 12 hours and checked for the presence of IL1β. 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: Macrophages were stimulated as indicated for 2 hours. Total RNA was isolated and checked for the presence of IL1β transcripts. Statistical significance was checked by one way ANOVA. P value was found to be significant (<0.0001). (<b>C and D</b>) Western blot analyses of nuclear translocation of cRel subunit of NF-κB. Macrophages were stimulated for 40 minutes as indicated. Nuclear and cytosolic extracts were prepared and run on 10% SDS-PAGE. 30 µg of nuclear protein was loaded in each well corresponding to nuclear fraction. (<b>E</b>) Electrophoretic mobility shift Assay for NF-κB. Macrophages were stimulated for 40 minutes 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 (43); lane2: MDP+Pam3CSK4 treated macrophages (59); lane3: Pam3CSK4 treated macrophages (75); lane4: MDP+zymosan A treated macrophages (94); lane5: zymosan A treated macrophages (91); lane6: MDP+LPS treated macrophages (83); lane7: LPS treated macrophages (87); lane8: Nuclear extract of Pam3CSK4 stimulated macrophages is incubated with 100 fmol biotinylated NF-κB binding DNA and 100 pmol of unlabelled NF-κB binding DNA (43). Values in brackets indicate average integrated density values of spot densitometric analysis using software Alpha Imager. Results in graph are presented as the mean of triplicate wells ± SD. Western blot and EMSA correspond to one representative experiment of three and two independent experiments respectively.</p

Involvement of PKC in MIP supernatant induced cell death of macrophages.

<p>(a) Macrophage monolayers were pretreated with H7 or Rottlerin for 45 min, incubated with MIP supernatant for 8 hr and % cytotoxicity was determined by MTT assay. Untreated macrophages were taken as control with 0% cytotoxicity. (b) Macrophages monolayers were pretreated with H7, incubated with different doses of MIP supernatant & activated caspase 3 was quantified using caspase3 FL detection kit. Inhibiting PKC also resulted in decreased caspase 3 activities. RFU: Relative Fluorescence Units. (c) MIP supernatant induced nuclear translocation of PKC δ. Macrophage monolayers were treated with MIP supernatant for 1, 2 or 3 hr. Cytoplasmic and nuclear fractions were harvested and immunoblotted for PKC δ. Lanes 1–4 (Cyto): control, 1 hr, 2 hr, 3 hr; lanes 5–8 (Nuc): control, 1 hr, 2 hr, 3 hr. Cyto-cytoplasmic fraction, Nuc-nuclear fraction.</p

MIP supernatant induces apoptosis in mouse peritoneal macrophages.

<p>(a) MIP supernatant induced cell death of macrophages was not due to LPS contamination. Macrophage monolayers were treated with MIP supernatant (1 µg/ml) for 8 hr and % cytotoxicity was determined by MTT assay. Before incubation of macrophages with MIP supernatant, the supernatant was also treated either with Polymyxin B (PB). Untreated cells were taken as control with 0% cytotoxicity. (b) Murine peritoneal macrophages after treatment with MIP supernatant for 4 hr were dual stained with Sigma APOAC kit. Annexin V stained early apoptotic cells (annexin V positive, 6-CFDA positive) show red fluorescence on cell surface. Untreated viable cells (annexin V negative, 6-CFDA positive) fluoresce green with no signal for Annexin V. (c) Macrophage monolayers were treated with MIP supernatant for 6 hr, fixed & stained for DAPI. Arrows show nuclear condensation & fragmentation. Scale bar: 10 µm. (d) Macrophage monolayers were treated with MIP supernatant for 6 hr, lysed and the DNA fragmentation was detected by quantitative Nucleosome ELISA with anti-histone antibody. Untreated cells were taken as control. (e) MIP cell-free supernatant was lyophilized and re-suspended in sterile PBS & run on SDS-PAGE. The gel was stained with AgNo₃ and observed. Lane1: Low molecular weight SDS marker, lane2: MIP supernatant. The indicated molecular weights are in kDa.</p

MIP Supernatant resulted into downregulation of LPS induced proinflammatory responses.

<p>Macrophage monolayers were treated with LPS without or in the presence of 40 ul of MIP supernatant for 12 hrs, supernatant was collected & ELISA was performed for IL-1β & IL-10. Supernatant led to enhanced expression of IL-10.</p