UC Davis

Antibiotic resistant skin infections are an emerging threat to public health. This is driven by the ubiquitous nature of bacteria, their ability to thrive in hostile environments, and their molecular machinery that allows them to quickly develop mechanisms to render antibiotics ineffective. As a result, there is an urgent need to develop therapeutic approaches to treat bacterial infections without relying on antibiotics. Instead, there should be an emphasis on emerging treatments that rapidly and directly enhance immunity against pathogenic infections. Polymorphonuclear leukocytes (PMN) are the most abundant and important effector cells of the innate immune system and should be a primary target for strategies aimed at enhancing innate immunity. Three PMN-centered approaches are presented in this dissertation. The first one revolves around the development and testing of a designed host defense peptide (dHDP) that acts synergistically with PMN to tackle multi-drug resistant S. aureus infection in diabetic mouse wounds. The second approach involves optimizing PMN production and their antibacterial capacity in vitro with the objective of subsequently transferring these cells directly into a site of infection to enhance pathogen clearance. The third strategy aims at investigating signaling pathways that govern PMN functions against S. aureus and P. aeruginosa, with an emphasis on Toll-like receptor signaling and activation of the inflammasome.

Two pathogens of clinical interest are Methicillin-Resistant Staphylococcus aureus (MRSA) and Multi-Drug Resistant Pseudomonas aeruginosa (MDRPA). These are common sources of hospital and community-acquired skin infections in immunocompromised patients, including the elderly and people with diabetes. S. aureus is a gram-positive bacterium capable of forming biofilm and secreting virulence factors that can limit the ability of PMN to be recruited into sites of infection. As a result, therapeutic strategies should amplify PMN quantity and antibacterial functions. Studies presented in Chapter 3 demonstrate that the dHDP RP557 can enhance PMN antibacterial functions in vitro, suppress S. aureus proliferation, and enhance wound healing. The studies presented in Chapter 4 demonstrate that PMN production and antibacterial capacity can be enhanced through encapsulation of HSPCs in a 3D bone-marrow-like environment. These studies are inspired by previous publications demonstrating that local granulopoiesis driven by HSPC recruitment into an infected wound is part of the immune response against S. aureus, and that adoptive transfer of PMN generated in vitro from HSPC cultures can enhance bacterial clearance and survival of immunodeficient mice. This response against S. aureus involves TLR2/MyD88 signaling and IL-1β production via activation of the inflammasome. However, the source of IL-1β during this process, the nature of its effects (paracrine or autocrine to HSPC), and whether activation of other TLRs elicits similar IL-1β-dependent local granulopoiesis, remains elusive. Consequently, the studies presented in Chapter 5 demonstrate the early MyD88 activation is required to contain P. aeruginosa in wounded skin. In the absence of MyD88, P. aeruginosa proliferates and disseminates from the infected wound much faster than S. aureus. Compared to wild-type mice, this phenotype correlates with lower levels of IL-1β in wounds of MyD88-/- mice, as well as impaired PMN movement, and their ability to undergo pyroptosis and NETosis. Thus, suggesting that MyD88 is essential for survival against P. aeruginosa by regulating PMN antibacterial functions via IL-1β signaling. Combined, these studies point to the development of antibacterial strategies that target these key adaptors to amplify the immune response to infection.