Characterization of the Platelet Inflammatory Response in NAFL and NASH

Background NonAlcoholic Fatty Liver Disease (NAFLD) is one of the most commonly encountered liver disorders worldwide. NAFLD is a spectrum of liver disease including (i) simple hepatic steatosis also called Non-Alcoholic Fatty Liver (NAFL) and (ii) Non-alcoholic SteatoHepatitis (NASH).

When more than 5% hepatic steatosis is present, patients are considered to have NAFL. If steatosis is present along with hepatocyte ballooning degeneration and lobular inflammation, patients are considered to have NASH. About 20% of patients with NAFLD have NASH.

Over time, NAFLD may progress to cirrhosis and ultimately to hepatocellular carcinoma (HCC) with a greater proportion of NASH patients (20%).

Platelets are specialized blood cells that continuously monitor and preserve the integrity of the cardiovascular system. Beyond their well-established role in hemostasis, platelets have been recently shown to actively participate in the inflammatory response and in tissue remodeling, by releasing bioactive molecules and by interacting with leukocytes.

The liver-platelet relationship is very complex. In physiological conditions the liver regulates platelet number by producing thrombopoietin and platelet lifespan by clearing aged platelets. In pathological conditions, increasing evidence demonstrate that platelets have an important role in regulating liver inflammation and chronic disease. Platelet-derived cytokines, such as TGF-β, platelet-derived growth factor-β (PDGF-β), and CXCL4, promote hepatic fibrosis, and platelet count has been used for many years as a surrogate marker of liver fibrosis (FIB-4 index). Recent studies demonstrate that platelet number and platelet aggregation are increased in liver sinusoids of NASH patients. Mechanistic insights provided by mouse models suggest that the role of platelets in NAFLD progression is mediated through the interaction with immune cells. Platelet colonization of the liver is mediated through the interaction with specialized macrophages lining the liver sinusoids (Kupffer cells) and it is a critical step for the recruitment of CD8+ T cells and NKT cells, which drive NASH progression through the release of cytokines and the metabolic reprogramming of hepatocytes. The importance of platelets in the development of NASH and subsequent progression to cirrhosis and HCC has been confirmed in humans by inhibiting platelets with a combination of aspirin and clopidogrel in a small pilot case study.

There is mounting evidence suggesting that distinct signaling pathways regulate the hemostatic and the inflammatory function of platelets. For instance, the platelet ITAM- (GPVI, CLEC2) and ITIM- (PECAM-1, TLT-1) coupled receptors regulate the platelet inflammatory response but have a minor role in hemostasis. Thus, one could envisage targeting the platelet inflammatory response as a strategy to limit the progression of NAFLD, without undermining hemostasis.

Hypotheses The overarching hypothesis of the proposed project is that platelets amplify the inflammatory state that drives the progression from NAFL to NASH.

Platelets participate in the inflammatory response by releasing bioactive compounds and establishing heterotypic interactions with leukocytes. However, these mechanisms in the context of chronic liver disease are poorly understood and have been studied mainly in mice.

Our working model is that platelets docked in the liver of NAFLD patients amplify the inflammatory state by releasing pro-inflammatory cytokines, which in turn recruit and activate leukocytes in the liver sinusoids. Combined stimuli from leukocytes and platelets would then lead to metabolic reprogramming of hepatocytes and progression to NASH. In this context the investigators expect to identify an important role of GPVI and CLEC-2 and their inhibitory counterparts PECAM-1 and TLT-1, which are critically implicated in the regulation of platelet activation at sites of inflammation.

The investigators anticipate that features of the platelet inflammatory response could be both sensitive and sex-specific biomarkers for NASH progression and novel therapeutic targets.