The NOX-ATM pathway regulates host microbe interactions, cytokine production and gut epithelial dynamics

About This Grant

PROJECT SUMMARY Infectious stress and damage are important concepts; however, we still lack understanding of how immunity and microbial virulence result in cellular stress, and how this stress in turn influences the immune response. Using a powerful model system, Drosophila melanogaster, we have recently found that phosphorylation of histone H2a (γH2av) is a hallmark of microbial infection (oral infection, septic injury). γH2av is considered a marker of DNA damage response (DDR) downstream of DDR kinases (ATM, ATR, DNA-PKc). We found that pathogens induce host γH2av accumulation in a virulence-, NOX- and ATM kinase-dependent manner, but without DNA damage. We have also found that ATM-mediated signaling promotes inflammatory cytokine production, alters cell dynamics and strongly promotes survival to infection. Based on these results, we hypothesize that ATM- mediated signaling is a central stress response upon infection, altering cytokine production and modulating cell death and survival. To test these hypotheses, we propose the following specific aims: Aim1: We will determine the microbial characteristics (immunogenicity, virulence/tissue damage) that induce γH2av accumulation in midgut enterocytes (ECs). In parallel, we will analyze the role of NADPH oxidases (Duox, NOX) on triggering the ATM pathway. Finally, we will elucidate how cellular age influences the ability of a cell to trigger ATM signaling upon infection. Aim2: We will analyze the impact of ATM signaling in enterocytes where it regulates inflammatory cytokine production, cell dynamics and tissue repair. We will clarify the cell-autonomous requirement of ATM signaling for enterocyte elimination, cytokine induction and tissue repair using a combination of FACS, RNA sequencing, functional genetics, cell tracing and pharmacological treatments. Aim3: We will investigate how ATM signaling decreases host survival upon septic injury. We will first elucidate and characterize spatial-temporal activation of ATM signaling in response to systemic infection and identify key regulators involved. We will determine the mechanisms leading to host lethality downstream of ATM signaling. Finally, we will investigate whether γH2av itself mediates some of the influence of ATM signaling upon infection. This project will demonstrate that NOX-ATM signaling acts as a new central regulator of infectious stress and immunity. Our multi-omics approach coupled with tissue-specific functional genetics and unparalleled Drosophila lineage tracing and clonal systems will help clarify both the upstream regulators and downstream effects of this pathway. Importantly, our work will also show how a stress response can increase or decrease survival to infection in different tissue and infection contexts. We will demonstrate that NOX-ATM is not only central for the control of cytokine production and innate immunity, but also a key regulator of cell death and stem cell-mediated repair. Finally, our research may open new avenues of research on cellular aging and immune stress responsiveness. The key characteristics by which pathogens influence innate immunity are largely conserved, therefore we believe that our studies pave the way to develop strategies aiming to improve the outcome of microbial infections. 1