Results obtained in in vivo studies in PTX3-deficient mice agree with our hypotheses and indicate that PTX3 is involved in tissue repair and in modulating complement-dependent cancer-related inflammation.
Activities performed showed that PTX3 plays a non-redundant role in tissue repair in a skin wound healing model (Figure 1) as well as in different models of tissue damage (i.e. chemically-induced sterile liver and lung injury). Under these conditions, macrophages and mesenchymal cells produced PTX3 in response to TLR activation and amplification by IL-1, localizing to the pericellular matrix of macrophages and mesenchymal-remodeling cells. PTX3-deficiency was associated with increased clot formation, fibrin deposition and persistence, followed by increased collagen deposition (Figure 2). We found that PTX3, and in particular its N-terminal domain, interacts with fibrin and plasminogen, with higher affinity observed at acidic pH, a condition which occurs in damaged tissues (Figure 3). In vitro and in vivo studies demonstrated that by these interactions, PTX3 promoted remodelling of the fibrin-rich inflammatory matrix ensuring a normal tissue repair, thus providing a novel link between innate immunity, haemostasis and tissue repair.
Concerning PTX3 in cancer, activities performed showed that PTX3-deficiency in mice caused increased susceptibility to mesenchymal and epithelial carcinogenesis (Figure 4). PTX3-deficiency was associated with exacerbated cancer-related inflammation as revealed by enhanced macrophage infiltration in tumours, pro-inflammatory cytokine production, and complement activation. PTX3 regulated C3-deposition on sarcoma cells by interacting with and recruiting the negative regulator Factor H (Figure 5). Indeed, genetic inactivation of C3 reverted the increased susceptibility to 3-MCA-induced carcinogenesis and macrophage recruitment. These results indicate that unleashed complement activation and increased C5a production associated to PTX3-decifiency are responsible of exacerbated production of chemokines, increased recruitment of tumor promoting macrophages.
In the context of tissue remodeling we investigated the role of PTX3 in physiological skeletal remodeling and bone healing. Micro-computed tomography (μCT) and bone histomorphometry of distal femur showed that PTX3 gene-targeted mice had lower trabecular bone volume than their wild-type littermates (Figure 6). Using a tibial fracture model, we found that PTX3-deficient mice formed significantly less mineralized callus during the anabolic phase following fracture injury compared to wild type mice. We speculate that PTX3 could support maintenance of the bone mass, possibly by inhibiting FGF2 and its negative impact on bone formation. Our results indicate that PTX3 plays an important role in bone homeostasis and in proper matrix mineralization during fracture repair.
Efforts to obtain high resolution structural models of the PTX3 protein were based on a combination of X-ray crystallography, Cryo Electron Microscopy (Cryo-EM), and Small-Angle X-ray Scattering (SAXS) techniques. An X-ray model of the isolated monomeric PTX3 C-terminal domain (C_PTX3) was generated that was superimposed onto a Cryo-EM structure of the C_PTX3 octamer. The resulting structure was refined to a final resolution of 2.7Å (Figure 7).
We addressed the role of tumor-associated neutrophils (TANs) and described a novel antitumor pathway in which TANs promote IL-12 production by macrophages, leading to type 1 polarization of a subset of unconventional T cell (UTCalpha/beta). Type 1 UTCalpha/beta possess an innate-like phenotype and antitumor potential in vivo. In selected human tumors, neutrophil infiltration is associated with type 1 immunity and better clinical outcome (Figure 8).
Finally, we analyzed the role of Complement as a component of tumor promoting inflammation. We observed that C3-/-, MBL1/2-/-, C4-/- and C3aR-/-mice showed reduced susceptibility to 3-MCA sarcomagenesis and transplanted sarcomas (Figure 9). C3 and C3aR deficiency were associated with reduced accumulation and functional skewing of TAMs, increased T cell activation and response to anti-PD-1 therapy. Transcriptional profiling of sarcoma infiltrating macrophages and monocytes revealed the enrichment of MHC II-dependent antigen presentation pathway in C3-deficient cells. In patients, C3aR expression correlated with a macrophage population signature and C3 deficiency-associated signatures predicted better clinical outcome. These results suggest that the lectin pathway and C3a/C3aR axis are key components of complement and macrophage-mediated sarcoma promotion and immunosuppression.
