The applicant seeks a VA Career Development Award-2 (CDA-2) to further our understanding of the pathogenesis of ?-cell loss in type 2 diabetes (T2D) and support his career development at the Veteran?s Affairs Puget Sound Health Care System (VAPSHCS). His expertise in cell and molecular biology, islet inflammation, and islet amyloid deposition, along with his strong mentors (Steven Kahn and Jane Buckner), collaborators (Andrew Oberst and Matthias von Herrath), Advisory Committee (Rebecca Hull, Jerry Palmer, Alvin Powers), and location at the VAPSHCS, make him uniquely qualified to execute the proposed studies. With the support of this award, he will investigate novel and underappreciated aspects of ?-cell death in T2D, interact with internationally-recognized diabetologists and immunologists, and undertake didactic and laboratory-based immunology training. These activities will allow the candidate to author additional high impact publications, gain research independence, and position himself to successfully compete for a VA Merit Review Award. The scientific goals of this proposal are to define the roles of necroptosis (a novel form of cell death) and T cells in islet amyloid-induced ?-cell loss in order to develop new treatments for individuals afflicted by T2D. Islet amyloid is a pathologic hallmark of T2D, where it elicits islet inflammation and ?-cell death. Investigation of the mechanisms of amyloid-induced ?-cell death have focused largely on apoptosis signaling. Preliminary data in this application show for the first time that a recently described alternative form of cell death signaling called necroptosis is activated by islet amyloid formation. These data also show that components of this cell death pathway, including receptor interacting protein kinase 3 (RIPK3) and mixed lineage kinase domain-like pseudokinase (MLKL), are present in ? cells. A central characteristic of necroptosis is its ability to elicit immune responses to cellular antigens that are released by this form of lytic cell death. Consistent with this process occurring in response to islet amyloid, preliminary data show that amyloid-laden islets exhibit significantly higher T cell abundance and mRNA expression. Based on these findings and the literature, I hypothesize that islet amyloid-induced TNF? production by macrophages leads to RIPK3-mediated ?-cell necroptosis, which results in T lymphocyte recruitment to amyloid-laden islets and exacerbation of amyloid-induced ?-cell dysfunction and death. To test this hypothesis, the following specific aims are proposed:
Specific Aim 1 : Determine the role of RIPK3 signaling in amyloid-induced ?-cell death, ?-cell dysfunction, and islet immune responses. The contribution of RIPK3 signaling to islet amyloid-induced ?-cell death will be determined in vitro using 1) a RIPK3 inhibitor, and 2) islets from Ripk3-/- mice crossed with amyloid-prone human IAPP (hIAPP) transgenic mice. These Ripk3-/- x hIAPP mice will also be used to determine whether RIPK3 loss ameliorates amyloid-induced islet immune responses and ?-cell death in vivo.
Specific Aim 2 : Determine the role of T lymphocytes in amyloid-induced ?-cell death and dysfunction. Islet amyloid-prone mice lacking lymphocytes (Rag1-/- x hIAPP) will be used to determine the role of T cells in amyloid-induced ?-cell death and dysfunction, and in amyloid deposition itself. The relationship between islet lymphocytes, amyloid deposition, inflammation, and ?-cell death will be evaluated 1) in vitro using isolated islets, and 2) in vivo using this novel amyloid-prone, lymphocyte deficient mouse model. The relationship between islet amyloid deposition, immune cell abundance, and ?-cell death will also be evaluated in human pancreas sections.
Type 2 diabetes disproportionally affects the veteran population, with recent studies reporting that ~20% of Veteran?s Affairs (VA) patients have this disease compared to ~10% of the general population. Since diabetes contributes to multiple morbidities including heart disease, stroke, kidney failure, and blindness, new therapeutic strategies to treat or prevent type 2 diabetes are likely to provide substantial health benefits to Veterans. The process of pancreatic islet amyloid formation leads to ?-cell death and loss of insulin production in most individuals with type 2 diabetes. In this application, the relationship between islet amyloid deposition, islet immune responses, and ?-cell death signaling will be examined. By gaining a greater understanding of the contribution of immune responses to amyloid-induced ?-cell death, this research aims to develop new approaches to preserve ? cells and prevent or treat type 2 diabetes. It will also enable the PI to develop scientific independence and further his career within the VA Puget Sound Health Care System.
