Tackling bacterial infections of the bone: PTX3 as new diagnostic tool and therapeutic target

Osteomyelitis (OM) is a severe musculoskeletal infection mainly caused by the opportunistic bacterium S. aureus (SA) that occurs after fracture, surgery, or secondary to vascular spread of the pathogen to skeletal sites. Several factors, including presence of foreign bodies (implants) and dysmetabolic diseases (diabetes), make the bone susceptible to infections. OM manifests with extensive tissue remodeling and inflammation. Host immune reactions and SA survival strategies coordinately affect the bone microenvironment (BME), which becomes an elusive niche for systemic drugs, thus favoring the emergence of antimicrobial resistance. Hard to prevent, diagnose, and cure, OM poses a social and sanitary burden on healthcare systems. A consensus on OM pathogenesis, with major regard to the SA/skeletal cells crosstalk, is lacking. With the general aim of filling this gap, my PhD project was designed to investigate the host/pathogen interface in OM with a focus on the long pentraxin 3 (PTX3), a soluble pattern recognition molecule (PRM) of innate immunity. This PRM is made both by immune and non-immune cells in response to microbial moieties and inflammatory cytokines (IL-1β, TNF-α), and is involved in resistance to opportunistic pathogens, regulation of inflammation and tissue remodelling. Established evidence points to this protein as an independent prognostic marker of opportunistic infections, including those sustained by SA. Furthermore, PTX3 is increasingly acknowledged as a key player of bone pathophysiology and recently emerged as a diagnostic marker of periprosthetic joint infections, which makes it an ideal target for original research on OM. Using a mouse model that recapitulates aspects of surgery-related OM of the lower limbs in humans, we found that infection with SA (but not with S. epidermidis) induced over-expression of the Ptx3 gene and production of the PTX3 protein mostly in haematopoietic and osteogenic cells. Consistent with this, we have observed an increase in PTX3 production in an in vitro 3D fluid-dynamic model of the human bone niche following stimulation with TNF-α. Over-expression of PTX3 in SA-injected mice was confined to the acute phase of the experimental infection (6 to 14 days from challenge). PTX3 deficiency in genetically modified mice (Ptx3-/-) was accompanied by reduced bacterial burden in the infected limbs and decreased levels of cellular and soluble markers of systemic inflammation. Importantly, this phenotype was dose- and sex-independent and SA-specific. Also, targeting of the endogenous protein in vivo with a blocking antibody reduced the bacterial load in PTX3-competent animals and administration of the recombinant protein exacerbated the infection in PTX3-deficient animals, pointing to a non-redundant pathogenic role of this pentraxin in SA-OM. Moreover, we observed that the PTX3-deficient BME had a stronger antimicrobial response with elevated concentrations of inflammatory cytokines and chemokines in the bone tissue (IL-1, IL-6, IFN-γ, CXCL1) and enhanced expression of cell-borne and soluble PRMs in bone cells (TLR2 and 4, NOD1 and 2, β-defensin-1). Proteomics and animal experimentation suggest that this anti-inflammatory activity of PTX3 in the bone is likely dependent on the interplay with the complement system. Our study provides evidence that PTX3 is a major host player in the acute phase of OM pathogenesis and might be exploited as a potential molecular target for the development of new strategies of prophylaxis and therapy of bone infections. This is of significant translational importance in an era of increasing antibiotic resistance.