Fig 1: CCN1 binds S. aureus and P. aeruginosa through PGN and LPS, respectively.CCN1 binding to S. aureus (a–c) and P. aeruginosa (d–f) was evaluated using a solid-phase-binding assay. a S. aureus bound to surfaces coated with CCN1-WT or other ECM proteins (FN fibronectin, LN laminin, Col1α1 collagen type I) was quantified using polyclonal anti-S. aureus antibodies. CCN1 and FN bound to S. aureus, whereas LN and Col1α1did not. b Binding assay of S. aureus to recombinant CCN1-WT, CCN1-D125A, and CCN1-DM proteins as above. c Binding of S. aureus to CCN1 deletion mutant proteins as above. d Solid-phase-binding assays for P. aeruginosa were performed as above and detected with polyclonal anti-P. aeruginosa antibodies. e Binding assay showing P. aeruginosa binding to CCN1-WT and CCN1-D125A, but not CCN1-DM. f Solid-phase-binding assays with CCN1 deletion mutants. All assays were done in triplicates and data are expressed as mean ± s.d. Statistical evaluation was performed by one-sided, two-sample with equal variance t-tests. **p < 0.01, n.s. = not significant. g Bacterial molecular patterns (PGN of S. aureus; LTA of S. aureus; LPS of P. aeruginosa; 1 µg each) and BSA as control spotted on nitrocellulose membranes were incubated with CCN1 proteins (WT, D125A, or DM; 2 µg each in PBS) and bound CCN1 was visualized using anti-CCN1 antibodies. Dot blot shown is a representative image. h Senograms of SPR analysis of CCN1 binding to LPS. CCN1 at various concentrations was used as analyte to detect binding to LPS immobilized on HPA hydrophobic sensor chip. Ka, Kd, and KD values were obtained by kinetic analysis.
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: 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 4: Schematic depiction of the synergistical effect of MSNs and PD-1 blockade on tumor immunotherapy. MSNs, mesoporous silica nanoparticles; PD-1, programmed cell death protein 1; TLR4, Toll-like receptor 4.
Fig 5: T cell-recruitment chemokines expression is associated with NF-κB signaling after MSNs treatment. (A) Correlation among the expression of T cell-recruitment chemokines across all B2m-sgRNA B16F10 tumor tissues. (B) Through transcriptomic analysis, all genes with significantly positive correlation with CCL5 were identified. (C) Cell-specific enrichment of CCL5-correlated genes. (D) Transcriptional regulatory enrichment of CCL5-correlated genes. (E) Top 10 signaling pathways by KEGG pathway enrichment of Ccl5-correlated genes. (F) In MSNs-treated tumors, the expression of genes in the top 10 Ccl5-related signaling pathways was presented by heatmap. (G) In tumor tissues, the activation changes of NF-κB, TNF and TLR signaling pathways in αPD-1 plus MSNs groups compared with αPD-1 alone were revealed by GSEA. (H) In tumor tissues after indicated treatments, the levels of proteins in TLR4, TNFR and NF-κB signaling were determined by Western blot. GSEA, Gene Set Enrichment Analysis; MSNs, mesoporous silica nanoparticles; PD-1, programmed cell death protein 1; TLR, Toll-like receptor; TNF, tumor necrosis factor.
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