Fig 2: CCN1 induces Myd88-dependent inflammatory response.a–d C57BL/6J (B6) and Myd88−/− mice (n = 6 each genotype) were i.p. injected with CCN1 protein (5 µg in 300 µl PBS), and peritoneal exudates were analyzed for TNFα (30 min) and IL6, KC, and MCP1 (2 h) using ELISA. e Complete blood count (CBC) analysis was performed, and neutrophils and lymphocytes contents are shown. f Gene expression induced by CCN1. BMDMs from B6 or Myd88−/− mice were treated with CCN1 protein (2 µg per ml), LPS (50 ng per ml), or PGN (5 µg per ml) for 6 h. Tnfa and Il6 mRNAs were quantified by 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 < 0.01, and n.s. = not significant. Source data are provided as a Source Data file.

Fig 3: CCN1 enhances bacterial killing after phagocytosis through ROS production.a Lysostaphin protection assay for assessing S. aureus killing after phagocytosis. After BMDMs phagocytosed S. aureus and extracellular bacteria were eliminated by lysostaphin, cells were treated with CCN1 or BSA (2 μg per ml each) and viable bacteria inside BMDMs were enumerated at indicated chase periods. b Superoxide (O2−) was measured by dihydroethidium (DHE, 5 µM) staining in BMDMs treated with recombinant CCN1 proteins (WT, D125A, or DM; 2 µg per ml each) for 30 min. Representative images shown were acquired from fluorescence microscopy. Bar = 40 µm. c High-magnification images were taken from at least ten random fields and mean fluorescence intensity (MFI) was calculated using Image J software. d ROS production was quantified as MFI using DHE fluorescence in BMDMs pretreated with cilengitide (1 μM), followed by CCN1 or BSA (2 µg per ml each). e S. aureus killing assays were performed as in a with cilengitide pretreatment. f The effects of various inhibitors on CCN1-induced ROS production. All inhibitors were added 30 min prior to CCN1 treatment. Rac1 inhibitor NSC23766 (10 µM); NOX inhibitor Apocynin (10 µM); NOX2 inhibitor GSK2795039 (10 µM); NOX1 inhibitor ML-171 (10 µM). g Effects of various inhibitors on bacterial killing using lysostaphin protection assays as above. All data were acquired from at least three independent assays and are expressed as mean ± s.d. in triplicate determinations. Statistical evaluation was performed by one-sided, two-sample with equal variance t-tests. **p < 0.01, n.s. = not significant. Source data are provided as a Source Data file.

Fig 4: 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 5: 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.