Project Summary Patients with both types of diabetes mellitus have greater thrombotic risk than non-diabetic subjects. The exact reasons are unclear, but is partly due to increased number of reticulated platelets (RPs) and pro-inflammatory leukocytes. We recently reported that elevated blood glucose in type 1 diabetes (T1D) increases the number of RPs and platelet-leukocyte aggregates (PLAs). Mechanistically, we found that enhanced glycolytic stress in neutrophils promote secretion of certain alarmins that in turn bind to their receptor (RAGE) on Kupffer cells (KCs) to produce thrombopoietin (TPO) from hepatocytes. The released TPO interact with its receptor, cMPL on hematopoietic stem cells and platlelet progenitors to enhance the formation of RPs from the bone marrow (BM). Surprisingly, unlike in T1D, the TPO levels in models of obesity/ type 2 diabetes (T2D) and humans were unaltered, yet, the number of KCs in the liver and RPs in the blood were elevated. Normalizing blood glucose did not reverse these changes implying that pathways that regulate thrombopoiesis in T2D are different. Further interrogations led to the identification of enhanced NLRP3 inflammasome signaling in macrophages (fat and liver) as a source of thrombopoietic cytokines (i.e., IL-1β, IL-1α and CCL5). These cytokines may interact with megakaryocytes (MK) in a TPO-independent manner to promote thrombopoiesis. Based on these findings, we hypothesize that, “the NLRP3 inflammasome signaling in macrophages of fat and/or liver drives thrombopoiesis via TPO-independent mechanisms in obesity/ T2D”. We will test the hypothesis using 2 specific aims in a mouse model of diet-induced obesity (DIO)/ T2D. Aim 1 will define the integral role of NLRP3 inflammasome-Gasdermin D signaling in promoting thrombopoiesis. In this Aim, first, we will identify the major cellular source of inflammasome signaling using flow cytometry and scRNA seq. Second, we will study the functional role of inflammasome-GSDMD signaling in driving thrombopoiesis by employing pharmacological and, various global, conditional and cell-specific Nlrp3 or Gasdermin D (Gsdmd) deletion strategies. For each intervention, the effect of Nlrp3/ Gsdmd inhibition on downstream signaling mediators and its overall impact on thrombopoiesis will be studied. Aim 2 will define the TPO-independent pathways of thrombopoiesis downstream of inflammasome. In this aim, first, we will study the role of a MK-biased vWF+ HSC subset in driving thrombopoiesis mediated by IL- 1β, IL-1α and/ or CCL5. Strategies include global or MK-specific deletion of Il1r1, Ccr5 and cMpl. These studies will reveal if the products of inflammasome are capable of driving thrombopoiesis independent of TPO-cMPL by directly interacting with MKs.