Periodic Reporting for period 1 - ActinSensor (Identification and characterization of a novel damage sensor for cytoskeletal proteins in Drosophila)
Período documentado: 2019-01-01 hasta 2020-12-31
Cell death-associated sterile inflammation plays a critical role in a range of human diseases from cancer to autoimmunity. Damaged tissues are thought to elicit their inflammatory effects through the sudden release from cells of endogenous damage-associated molecular patterns (DAMPs) that serve to recruit and modulate the function of immune cells. What provided the impetuousness for this project was the discovery that purified extracellular actin elicited a JAK-STAT-dependent inflammatory response in the fruit fly (Drosophila melanogaster. The JAK-STAT pathway in fruit fly is activated by a broad range of cellular stresses including mechanical pressure, infection, and septic wounds. A unifying feature of all these forms of stress is cell death and it has been speculated that STAT activation might occur in response to the release of DAMPs during the disparate cellular insults, however, the nature and identity of these DAMPs remain obscure. The major objective of this project was to discover the actin sensor in Drosophila that senses actin to promotes a JAK-STAT response. Corrective action was taken during the early phases of this project to refocused on how a mammalian cytoskeletal sensor (DNGR-1, expressed on dendritic cells, signals to promote the presentation of exogenous antigens to cytotoxic T lymphocytes (CTLs), through a process called 'cross-presentation' (XP). Following cell death and plasma membrane rupture in mammals, Filamentous-actin (F-actin) is recognised as a DAMP by the C-type lectin receptor DNGR1, expressed on Type 1 conventional dendritic cells (cDC1), that signals to favour the cross-presentation of dead-cell-associated antigens to CTLs. The function of DNGR1 requires the presence of an immunoreceptor tyrosine-based activation motif (ITAM)-like domain in its intracellular tail that allows the recruitment and activation of the spleen tyrosine kinase (SYK). Mice that are deficient in DNGR-1 or SYK lack protective CTL responses to viral and tumor challenges. Therefore, an understanding of how cDC1, and DNGR-1 more specifically mediates XP is crucial to better understand immune control of cancer and viruses. Understanding how cDC1 promote presentation of exogenous antigens (e.g. from tumor cells) would constitute a big step forward in developing a new, and potentially more effective, category of immunotherapies.Therefore we sought to address what mechanism(s) does DNGR-1 utilize to mediate XP of dead-cell associated antigen? During this project, we demonstrated that DNGR-1 is a dedicated XP receptor that signals upon ligand engagement to promote phagosomal rupture. These rupturing events allow for the escape of phagosomal contents into the cytosol where they access the endogenous MHC class I antigen processing pathway. The activity of DNGR-1 maps to its signalling domain, which activates SYK and NADPH oxidase to cause phagosomal damage. These findings reveal the existence of innate immune receptors that couple ligand binding to endocytic vesicle damage to permit MHC class I antigen presentation of exogenous antigens and regulate adaptive immunity.
Previous data from our lab have demonstrated that DNGR-1 and its adaptor kinase SYK contributed to the ability of cDC1 to translate dead cell recognition into adaptive immunity via XP. However, it was unclear at the outset of this project whether this reflected a function of DNGR-1-SYK in routing dead cell cargo into specialised endocytic compartments that are permissive for XP (i.e. poorly degradative) or a more active role of DNGR-1-SYK signalling in the process leading to XP. We first demonstrated through flow cytometry and microscopy experiments that DNGR-1 accumulates on phagosomes selectively regulates XP via a cytosolic pathway. During these experiments, we noticed that a small proportion of these DNGR-1+ phagosomes accumulated markers of endomembrane damage (Galectins and Lysenin). We generated SYK deficient cell lines (via CRISPR-Cas9) to demonstrate by microscopy that SYK was required for the observed endomembrane damage. Consistent with a role for SYK in phagosomal damage, the accumulation of endomembrane damage markers was also dependent on a functional DNGR-1 hemITAM motif which is located on its intracellular tail and critical for SYK recruitment and signalling. SYK has previously been implicated in the activation of the NADPH oxidase, which is the primary source of reactive oxygen species (ROS) in phagocytic cells. ROS has recently been linked to damaging endomembranes via a poorly described lipid peroxidation mechanism. We sought to test if NADPH oxidase was responsible for the observed endomembrane on a subset of our DNGR-1 phagosomes. By inhibiting NADPH oxidase activity we blocked the accumulation of damage markers on phagosomes was attenuated. Furthermore, inhibiting the NADPH oxidase or by scavenging ROS we attenuated XP of exogenous antigen in in vitro assays of XP. We further refined these experiments and demonstrated a role for the NADPH oxidase in phagosomal damage and XP through the use of primary Flt3L-derived cDC1 deficient in a crucial subunit of the NADPH oxidase, namely NOX2. Primary cDC1s were exposed to DNGR-1 ligand (F-actin/myosin-II) beads. Notably, in cells grown from WT but not NOX2-deficient bone marrow, many of these phagosomes were positive for the endomembrane damage marker (Galectin-3). We further extended these data in vivo through the use of mixed bone marrow chimeras, confirming that cDC1 deficient for NOX2 had an attenuated capacity to XP dead cell-associated antigen. Most of the results of this project have been published in a peer-reviewed manuscript (See uploaded publication). Furthermore, results from this project have been disseminated to the scientific community by oral and poster presentations at national and international meetings. This project has already led to the initiation of a number of follow up projects on the cell biology of cross-presentation with collaborators in the UK and the USA.
Work from this project proposal allowed me to acquire new skills and expertise in Drosophila genetics, dendritic cell biology and adaptive immunity. The skills and expertise I gained during the last two years of work as a postdoc within the scientific environment at the Francis Crick Institute are reflected in an additional project published where I investigated the role of the cytoskeletal protein a-actinin as an activator of the JAK-STAT pathway in Drosophila (Gordon, Henry et al., eLife 2018). Secondly, my postdoctoral work has allowed me to initiate a follow up project in which I am investigating the pathway that promotes phagosomal rupture in dendritic cells. Thirdly and most importantly, the funding of my postdoctoral work allowed me to develop my own area of research interest within the field of dendric cell biology. The unique combination of expertise and techniques I acquired during my research as a Marie Sklodowska-Curie fellow has ideally positioned me to reach the next level of professional maturity as an independent group leader. Beyond basic research, these findings are highly relevant for biotechnology and biomedicine. Exploiting a better understanding as to how cDC1 promote presentation of extracellular derived antigens (e.g. from tumor cells), by devising ways to enhance cross-presentation for instance, would constitute a big step forward in developing a new, and potentially more effective, category of immunotherapies.