Project summary Oral mucositis is one of the most common comorbidities of cancer chemotherapy. Due to its symptoms and associated complications, oral mucositis can impact the delivery of optimal cancer treatment. Oral mucositis primarily results from the cytotoxic effect of chemotherapeutics on the rapidly dividing oral epithelium. However, the specific cellular events involved in mucosal injury remain incompletely understood, thereby hampering the development of effective treatments. Cell death and inflammation are hallmarks of mucositis. Although DNA damage and apoptosis have been considered the main mechanisms that lead to oral mucositis, lytic forms of cell death triggering the release of host-derived damage-associated molecular patterns (DAMPS) and activation of inflammatory responses by resident microbial commensals could also be involved. Preliminary observations in a mouse model of 5-FU-induced oral mucositis suggest apoptosis of basal epithelial cells is an early response, but non-apoptotic forms of cell death are likely to mediate late tissue injury. There is therefore a need to evaluate the temporal progression of cell death pathways and mediators activated during chemotherapy-induced oral mucositis. Here we will explore the hypothesis that apoptosis mediates early responses, while non-apoptotic forms of cell death and activation of inflammation by sensing of DAMPs or a dysbiotic microbiome contribute to late stages of tissue injury. We will specifically focus on evaluating the role of the pro-apoptotic mediator PMAIP1, which was found as upregulated in human oral mucosa during chemotherapy. We will also test if signaling through tumor necrosis factor (TNF)-α and/or Toll-like receptor 4 (TLR4) leads to oral mucosal injury. Levels of TNF-α increase during oral mucositis and are associated with its severity. TLR4, which senses DAMPs, is expressed in the oral mucosa and has been shown to mediate lower gastrointestinal mucositis, but its role in oral mucositis has not been evaluated. Furthermore, oral mucositis is associated with microbiome dysbiosis, with an enrichment of pathobionts, which could contribute to amplify tissue injury in a TLR-dependent or independent manner. Accordingly, in this proposal we will use our mouse model of 5-FU-induced oral mucositis to identify mediators of tissue injury. In Aim 1, we will longitudinally characterize pathways leading to cell death and inflammatory damage during 5-FU-induced mucositis and mechanistically evaluate via knockout strains and pharmacological inhibitors the contribution of PMAIP1, TNF-α and TLR4 as mediators of pathophysiology. In Aim 2 we will characterize dysbiotic changes in the oral microbiome and microbial tissue invasion during 5-FU- induced oral mucositis. We will then test the effect of antibiotic regimens, shown to selectively or totally deplete oral commensals, on the course of mucositis and will evaluate if transfer of a dysbiotic community modulates oral mucositis severity...