Fig 1: Schematic of CCN1 functions in bacterial clearance and activation of TLR signaling.CCN1 functions as a PRR and opsonizes Gram-positive and Gram-negative bacteria through binding PGN and LPS, respectively. CCN1 activates phagocytosis by engagement of integrin αvβ3 in phagocytes, thereby promoting the engulfment of bacteria. In macrophages, CCN1 also stimulates ROS production through activation of Rac1 and NOX2, thus enhancing bacterial killing. Independent of the presence of bacteria, CCN1 functions as a DAMP and activates TLR2 and TLR4 by direct binding to these receptors, leading to MyD88-dependent expression of inflammatory cytokines and chemokines.
Fig 2: Specific interaction of HP-NAP with recombinant human TLR2 protein. (A) Binding of TLR2 to HP-NAP as determined by an ELISA-based solid-phase binding assay. The indicated amounts of recombinant human TLR2-10xHis and TLR4-10xHis proteins were incubated with 0.05 µg of HP-NAP coated on an ELISA plate. The binding of TLR2 and TLR4 to HP-NAP was detected by anti-His tag antibody or its isotype control antibody using the ELISA-based solid-phase binding assay as described in Materials and Methods. The results are presented as absorbance at OD450 nm. Data are expressed as mean ± SD of two independent experiments. (B) Specific binding of TLR2 to HP-NAP as determined by competitive binding with Pam3CSK4. The indicated amounts of Pam3CSK4 (Pam3) and BSA, as a negative control, were incubated with 0.625 µg of the recombinant human TLR2-10xHis protein at 4 °C overnight and then incubated with 0.05 µg of HP-NAP coated on an ELISA plate. The binding of TLR2 to HP-NAP was detected by the ELISA-based solid-phase binding assay as described in A. The results were presented as absorbance at OD450 nm. Data are expressed as mean ± SD of two independent experiments.
Fig 3: CCN1 binds and activates TLR2 and TLR4.a Solid-phase-binding assays between CCN1 and TLR2/4. Recombinant TLR2 or TLR4 proteins (200 ng per well) were added to 96-well plates pre-coated with serially diluted CCN1 protein at indicated amounts. Specific interaction was detected and quantified using polyclonal anti-hTLR2 or anti-hTLR4 antibodies. Recombinant CD14 (200 ng per well) was used as a control. b SPR analyses of CCN1 binding to TLR2. TLR2 was immobilized on CM5 chip and various concentrations of CCN1 was injected as analyte. c Sensorgrams of CCN1 binding to TLR4 analyzed by SPR as in b. d Dot blot analyses of CCN1 and mutant proteins binding to TLR2/4. CCN1-WT, CCN1-D125A, or CCN1-DM proteins (1 µg each) were spotted onto nitrocellulose membrane and incubated with TLR2 or TLR4 proteins (2 µg each in PBS) for 4 h. Binding was detected using polyclonal anti-hTLR2 or anti-hTLR4 antibodies. A representative image is shown. e Tnfa and Il6 mRNAs were quantified in BMDMs from either B6 or MyD88−/− mice treated with CCN1-WT, CCN1-D125A, and CCN1-DM mutant proteins using qRT-PCR analysis. f Tnfa and Il6 mRNAs from BMDMs of B6, Tlr2−/−, or Tlr4−/− mice treated with CCN1 proteins (CCN1-WT, -D125A, -DM, 2 μg per ml each) were quantified using qRT-PCR analyses. All data are represented as mean ± s.d. acquired in triplicate determinations. Statistical evaluation was performed by one-sided, two-sample with equal variance t-tests. *p < 0.05, **p < 0.01, and n.s. = not significant. Source data are provided as a Source Data file.
Fig 4: Involvement of TLR2 and PTX-sensitive heterotrimeric G proteins in HP-NAP-induced secretion of IL-8 by ATRA-induced differentiated HL-60 cells and neutrophils. (A) TLR2-dependent secretion of IL-8 from ATRA-induced differentiated HL-60 cells induced by HP-NAP. ATRA-induced differentiated HL-60 cells at a density of 2 × 106 cells/mL were pretreated with 10 µg/mL of anti-TLR2, anti-TLR4, or IgG2a isotype antibodies at 37 °C for 30 min and followed by the stimulation with 1 µM HP-NAP, 10 ng/mL Pam3CSK4, 10 µg/mL LPS or D-PBS, pH 7.2, as the unstimulated control at 37 °C for 16 h. The amount of IL-8 release was determined as described in Figure 6. Data are expressed as mean ± SD of four independent experiments. (B) TLR2-dependent secretion of IL-8 from neutrophils induced by HP-NAP. Neutrophils at a density of 5 × 106 cells/mL were pretreated with 10 µg/mL of anti-TLR2, anti-TLR4, or IgG2a isotype antibodies at 37 °C for 30 min and followed by the stimulation with 1 µM HP-NAP, 1 µg/mL Pam3CSK4, 10 µg/mL LPS or D-PBS, pH 7.2, as the unstimulated control at 37 °C for the indicated time. The amount of IL-8 release was determined as described in A. Data from neutrophils stimulated with HP-NAP for 3 h are shown as the bar graph and expressed as mean ± SD of four independent experiments. (C) PTX-sensitive secretion of IL-8 from ATRA-induced differentiated HL-60 cells induced by HP-NAP and Pam3CSK4. ATRA-induced differentiated HL-60 cells at a density of 2 × 106 cells/mL were pretreated with 100 ng/mL PTX or the vehicle control at 37 °C for 16 h and followed by the stimulation with 1 µM HP-NAP, 10 ng/mL Pam3CSK4, or D-PBS as unstimulated control at 37 °C for 16 h. The amount of IL-8 release was determined as described in A. Data are expressed as mean ± SD of three independent experiments. (D) PTX-sensitive secretion of IL-8 from neutrophils induced by HP-NAP. Neutrophils at a density of 5 × 106 cells/mL were pretreated with 100 ng/mL PTX or the vehicle control at 37 °C for 4 h and followed by the stimulation with 1 µM HP-NAP or D-PBS, pH 7.2, as the unstimulated control at 37 °C for the indicated time. The amount of IL-8 release was determined as described in A. Data from neutrophils stimulated with HP-NAP for 3 h are shown as the bar graph and expressed as mean ± SD of four independent experiments. *: p value < 0.05, **: p value < 0.01 as compared with unstimulated cells. #: p-value < 0.05, ##: p value < 0.01 as compared with stimulated cells in isotype control group or vehicle control group.
Fig 5: The effect of TLR2-neutralizing antibodies on HP-NAP-induced ROS production by ATRA-induced differentiated HL-60 cells and neutrophils. (A) TLR2-independent ROS production in ATRA-induced differentiated HL-60 cells induced by HP-NAP. ATRA-induced differentiated HL-60 cells at a density of 4 × 106 cell/mL were pretreated with 10 µg/mL of anti-TLR2, anti-TLR4, or IgG2a isotype antibodies at 37 °C for 30 min and followed by the stimulation with 1 µM HP-NAP, 1 µg/mL Pam3CSK4, 10 µg/mL LPS, or D-PBS, pH 7.2, as the unstimulated control at 37 °C for 30 min. ROS production was measured as DHE-derived fluorescence by flow cytometry as described in Figure 2C. Representative histograms are shown in the left panel. Data in the right panel are expressed as the fold change as described in Figure 2C and as mean ± SD of three independent experiments. (B) TLR2-independent ROS production in neutrophils induced by HP-NAP. Neutrophils at a density of 2 × 106 cell/mL were pretreated with 10 µg/mL of anti-TLR2, anti-TLR4 or IgG2a isotype antibodies at 37 °C for 30 min and followed by the stimulation with 1 µM HP-NAP, 1 µg/mL Pam3CSK4, 10 µg/mL LPS or D-PBS, pH 7.2, as the unstimulated control for the indicated time. ROS production by neutrophils was determined by H2DCF-DA-derived derived fluorescence assay as described in Figure 2A. Data from neutrophils stimulated with HP-NAP for 2.5 h are shown as bar graph and expressed as mean ± SD of four independent experiments in duplicate. *: p value < 0.05, **: p value < 0.01, ***: p value < 0.001 as compared with the unstimulated cells. ##: p value < 0.01, ###: p value < 0.001 as compared with HP-NAP-stimulated cells in the isotype control group. n.s.: non-significant.
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