PROJECT SUMMARY Acne vulgaris, or acne, is a disease of the pilosebaceous unit (PSU). Acne is ranked third among chronic skin diseases for causing disability and medical expense and is a major cause of psychological stress in young people. One factor contributing to acne is Cutibacterium acnes, the major bacterial species in the PSU. Using C. acnes as a model to study the interaction between the microbiome and the host immune response, we: 1) previously demonstrated the presence of IL-17 in acne skin lesions and 2) recently discovered that C. acnes phylotypes associated with acne (CA) or healthy skin (CH) differentially regulate the fate of TH17 cells to develop into non-antimicrobial (n-AMTH17) and antimicrobial (AMTH17) subsets. AMTH17 cells release T cell extracellular traps (TETs) and directly kill C. acnes and other bacteria pathogens. To date, the mechanisms by which C. acnes phylotypes induce TETs and their biological impact in acne are unknown. Therefore, our proposed research is innovative because we will define the immune landscape of the acne lesions, provide mechanistic insights into the biological impact of TH17-TET formation in acne, and identify novel immune pathways and potential biomarkers that can be targeted for acne therapy. This is important as future strategies could be developed to modulate TH17 function. Additionally, maintaining the balance among the different phylotypes of C. acnes may represent a strategy for novel probiotic design. The identification of C. acnes ligands will further elucidate the specificity of host receptors involved in microbial surveillance and lead to the development of novel therapeutic approaches skin diseases caused by dysbiosis. to control acne and other human Our central hypothesis is that innate activation of TH17 cells leads to induction of antimicrobial mechanisms, including TETs, which contribute to host defense against C. acnes and other bacteria. To elucidate this, we will determine the antimicrobial mechanisms of AMTH17 cells against C. acnes (Aim 1), investigate the role of AMTH17 cells in acne inflammation (Aim 2), and identify the C. acnes ligands that induce AMTH17 differentiation (Aim 3). Our preliminary findings support the premise that healthy skin commensals are critical to the education of our immune system and our overall defense against pathogens. Our strategy will include: classical immunological techniques involving T cell cloning to dissect the immune effector functions that underlie TH17-mediated antimicrobial host defense; microbiome sequencing of C. acnes phylotypes that inhabit donor biopsies to define how interactions within the skin microbiome and the host immune response influences acne development; high-resolution time-lapse imaging to define mechanisms of TET release and chromatin dynamics that occur during TET formation; scanning electron microscopy to delimit extracellular trap formation in human T cells; and state-of-the-art single cell RNA-seq experiments to...