Our work demonstrates that peptidoglycan (PGN), a major component of the cell wall of all Gram-positive bacteria, promotes inflammation and coagulation, which are major features of severe infections with Bacillus anthracis or other Gram positive pathogens. We found robust cytokine production in human innate immune cells and prothrombinase activity in human platelets exposed to highly purified polymeric PGN derived from 6. anthracis or Staphylococcus aureus. Innate immune cell responses required PGN phagocytosis, digestion in lysosomes, and stimulation of cytoplasmic NOD sensors; these processes were all dependent on anti- PGN antibodies. In addition, PGN-stimulated human monocytes expressed tissue factor (TF), an initiator of coagulation, in a process that required anti-PGN antibodies but not phagocytosis. We recently showed that anti-PGN IgG also promoted activation of the classical complement pathway, resulting in stimulation of platelet prothrombinase activity by the C5b-9 complex. These results suggest that, unlike responses to all other pathogen-associated molecular patterns, the PGN- stimulated inflammation and coagulation in human immune cells requires pre-existing anti-PGN IgG and antibody opsonization. We recently discovered that baboons, like humans, carry anti-PGN IgG, while mice lack such antibodies. Thus, it is not surprising that mouse macrophages in vitro and challenges of mice in vivo showed minimal responses to PGN. However, baboons responded to in vivo PGN challenge with features of systemic inflammation and disseminated intravascular coagulopathy (DIC) similar to those seen in human cells challenged with PGN in vitro and in patients with inhalation anthrax. This project will provide mechanistic insight into PGN-stimulated, anti-PGN-dependent pathologies. First, we will identify the pathways that regulate PGN-induced TF expression by monocytes. We will also test the relationship between the pre-existing anti-PGN titer in humans and the extent of PGN-stimulated inflammation and complement activation. Lastly, we will generate a panel of recombinant human monoclonal anti-PGN antibodies as activating IgGI or blocking lgG4 isotypes. These antibodies will be tested in vitro and in vivo for their ability to support or block PGN-induced inflammation. The lgG4 blocking antibodies could be developed into an effective therapeutic for all Gram-positive pathogens.
Primates but not mice respond to peptidoglycan of Gram-positive bacteria like the bioterrorist agent Bacillus anthracis by inflammation and blood coagulation in a process that requires human antibodies. This project will test ways in which blood coagulation is triggered by S. anthracis, and test the contribution of the antibodies in the pathology caused by 6. anthracis using human and non-human primate models.
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