PROJECT SUMMARY A craniotomy allows neurosurgeons to access the brain for procedures that include tumor resection, localization and resection of epileptogenic foci, and aneurysm clipping via the removal and intraoperative replacement of a skull fragment (bone flap). Infection following craniotomy occurs at rates of approximately 1-3%, although some reports indicate rates exceeding 10%. Nearly half of craniotomy infections are caused by Staphylococcus aureus (S. aureus) forming a biofilm on the bone flap. As biofilms are inherently difficult to eradicate due to antibiotic tolerance and virulence factors allowing immune evasion, craniotomy infections carry significant morbidity since a second surgery is required for treatment. When craniotomies are complicated by infection, resident immune cells and infiltrating leukocytes throughout the brain, subcutaneous galea, and bone flap tissues are exposed to both damage- and pathogen-associated molecular patterns (DAMPs and PAMPs) originating from the surgical procedure and bacteria, respectively. Triggering receptor expressed on myeloid cells-2 (TREM2) is a major pathology-induced signaling receptor involved in immune remodeling during many neurological conditions. An important feature of TREM2 is its reported ability to bind an array of anionic ligands including DAMPs (DNA, lipoproteins, glycolipids, and phospholipids) and PAMPs (lipoteichoic acid, peptidoglycan, and lipopolysaccharide). This proposal will test the hypothesis that TREM2 plays a critical role in promoting S. aureus craniotomy infection by eliciting an anti-inflammatory response through combined DAMP and PAMP signaling. In support of this hypothesis, my preliminary studies showed increased TREM2 expression and shifts toward an anti-inflammatory metabolic state in microglia after exposure to S. aureus. This proposed work will seek to uncover mechanisms behind TREM2 signaling during craniotomy infection. In Specific Aim 1, I will utilize a mouse model of S. aureus craniotomy infection with TREM2 KO animals to determine the relative contribution of DAMP vs. PAMP signaling, temporal aspects of TREM2 activation post-surgery, the comparative roles of membrane-associated vs. soluble TREM2, and the extent of TREM2 signaling involved in metabolic reprogramming of microglia and individual leukocyte populations. In Specific Aim 2, a novel bacterial scRNA- seq approach will used to identify S. aureus TREM2-targeted virulence factors that are leveraged for immune evasion. I will be the first to use this sequencing technique to study biofilm on a single-cell level and elucidate how bacterial pathogens, specifically biofilm, can hijack host immunity to persist. Results from this work will provide insights into previously unstudied topics on TREM2 signaling in the context of dual DAMP and PAMP exposure during craniotomy infection and elucidate targets for future therapeutic intervention.