Phagocytic cells and Streptococcus pyogenes in invasive infections : an inflammatory relationship

Abstract: Severe Streptococcus pyogenes infections, such as streptococcal toxic shock syndrome and necrotizing soft tissue infections, are rare but life threatening conditions. They are characterized by high bacterial load and a hyper-inflammatory state. The aim of this thesis was to investigate interactions between S. pyogenes and the phagocytic cells neutrophils and macrophages and how this correlates to cell activation and inflammation. First, we investigated high mobility group box 1 (HMGB1), a marker of inflammation and necrosis, as a potential biomarker and mediator of tissue pathology in S. pyogenes tissue infections. Analysis of tissue biopsies collected from patients with streptococcal soft tissue infections of varying severity showed that HMGB1 was present in the tissue, and that the amount correlates with severity. Further investigations showed that HMGB1 co-localized with IL-1β suggesting the potential for immunostimulatory complexes to form at the site of infection. HMGB1 was also demonstrated to act as a chemoattractant for neutrophils. Next, we assessed neutrophil activation and degranulation in response to different bacterial species, focusing on the release of the sepsis-associated factors heparin-binding protein (HBP) and resistin. Stimulations of neutrophils showed that streptococcal strains were potent inducers of neutrophil activation and degranulation. The results also showed a difference in signaling requirements for the release of HBP and resistin, respectively. While HBP release was mainly dependent on a previously described mechanism involving dual ligation of integrins and Fc-receptors, the release of resistin appeared to be multifactorial and involve multiple bacterial structures and host signaling pathways. Finally, we set out to define the macrophage phenotype present at the site of infection. Using a multi-parameter imaging workflow, we were able to assess the phenotype of macrophages present at the site of infection, in tissue from patients with severe S. pyogenes soft tissue infections as well as infected organotypic skin tissue models. These investigations showed that macrophages in S. pyogenes infected tissue displayed a shift towards a more anti- inflammatory M2-like phenotype, in spite of the hyper-inflammatory environment in the tissue. Gene expression analysis of infected patient tissue, skin tissue models as well as a murine model of severe streptococcal soft tissue infection showed an overrepresentation of signaling pathways associated with anti-inflammatory macrophage polarization. Taken together these findings highlight the complex pathophysiology of severe S. pyogenes infections, where on the one hand the bacteria and mediators present in the infected tissue potently activates neutrophils. While macrophages on the other hand, display a more anti- inflammatory phenotype upon infection, potentially promoting intracellular survival and persistence of S. pyogenes. Emphasizing the importance of careful patient characterization with regards to immune status, to ensure optimal treatment.