Cardiovascular diseases (CVD) are the leading cause of death worldwide with 17 millions deaths every year and represent a major public health challenge. Atherosclerosis is the main cause of CVD characterized by the accumulation of lipids and leukocytes in the lumen of arteries and causing their narrowing and dysfunction. Despite current therapeutic interventions, the incidence of CVD is expected to markedly progress imposing an enormous burden on the health care systems. It is now well recognized that the state of chronic low-grade inflammation favor the onset and progression of CVD but the underlying mechanism is still poorly understood. Leukocyte counts, and monocytes in particular, have been shown to independently predict risk for CVD. Indeed, recruitment of inflammatory Ly6Chi monocytes in early vascular injury give rise to plaque macrophages and supply therefore the growth of the plaque. In line with these observations, circulating monocyte levels correlate with the degree of disease severity in humans and mice, and targeting C-C motif chemokine receptor 2 (CCR2)-dependent monocyte plaque infiltration prevents the development of atherosclerosis. Although extensive research has focused on elucidating the role of cytokines and the microenvironment in the migration and infiltration of monocytes, the cellular metabolic pathways that regulate these processes are not well understood. Therefore, there is a considerable therapeutic interest in better understanding the mechanisms linking monocyte metabolism to CV risks.

Failure in glycemic control by hepatocytes is a pivotal event in the development of metabolic diseases such as type2-diabetes, NAFLD and NASH. However, although the complex network of transcription factors and enzymes allowing hepatocytes to reorganize their metabolic pathways for glucose homeostasis is relatively well established, it remains incompletely understood how hepatocytes integrate these extracellular changes and translate them into the non-genomic and the genomic programs for the glucose metabolic adaptation. Therefore, filling this gap in knowledge is an important challenge for fundamental knowledge and future clinical translation. I previously contributed to demonstrate that the endocytic system is involved in the regulation of glucose metabolism in hepatocytes, raising a novel concept of a role for regulators of endosomal trafficking in hepatocytes metabolic adaptation to nutritional cues. However, the identity of these specific endocytic regulators remains unknown and how they control the hepatocyte metabolism is far to be understood. Recently, I identified an endocytic component, the small GTPase Rab4b, which is controlled at the mRNA level by the nutritional cues, underlining its potential role in the regulation of glucose metabolism during the fasting to feeding transition. Taking advantage of a well-controlled culture system of primary hepatocytes I developed, that recapitulates fasted and fed conditions, as well as a mice model depleted for Rab4b specifically in hepatocytes, I will explore the role of this endocytic regulator on the regulation of glucose metabolism in vitro and in vivo. For this I will 1) determine whether Rab4b-dependent endocytosis is involved in the switch in hepatocyte glucose metabolism during the fasting-feeding transition, 2) investigate whether Rab4-dependent endocytosis is modulated between the fasting and feeding state, and 3) investigate the implication of liver Rab4b in the metabolic complications of obesity. Highlighting the feasibility of this proposal, I already obtained key preliminary results validating the important role of Rab4b in the response of hepatocyte to insulin and glucose, which are essential for the metabolic adaptation of hepatocytes to the fasting/feeding transition. In addition, the methodologies required for the success of this proposal are already established and work in my hands. Moreover, my expertise in both endocytosis and liver metabolism well positions my emerging team for the success of this proposal. This “young investigator” project will be a great opportunity for the emergence of my team on this new field at the interface between endocytosis and glucose metabolism.

Activation of innate immune cells by Pathogen-associated molecular patterns (PAMPs) and Damaged-associated molecular patterns (DAMPs) via Toll like receptors (TLRs) signaling is a key mechanism in the pathogenesis of chronic inflammatory diseases. Spleen tyrosine kinase (SYK) emerges as a novel regulator of TLR pathway. In this proposal, the SYK pathway will be investigated in two chronic inflammatory diseases: Cherubism, a form of genetically defined autoinflammatory bone disease and the hepatitis associated with obesity, the progressive form of the Non-Alcoholic Fatty Liver Disease (NAFLD), which is associated with chronic low-grade inflammation. NAFLD is one of the main causes of cirrhosis and increases the risk of liver-related death and hepatocellular carcinoma. Despite this major public health concern, clinical management of autoinflammatory diseases such as Cherubism and NAFLD are still elusive as there is a lack of efficacious pharmacological treatment. These chronic inflammatory diseases are associated with 1) pathogenic myeloid cells that are key players in disease development and severity and 2) endogenous substances (DAMPs) released from damaged cells (Cherubism and NAFLD) and increased microflora-derived bacterial products (PAMPs) (NAFLD), which activate innate cells via TLRs, leading to amplified danger signaling and exacerbated inflammatory response. The consortium shows that SYK deficiency in myeloid cells corrected systemic and hepatic inflammation in a mouse model of autoinflammatory genetic disease Cherubism. In obesity-related liver complications, hepatic SYK expression correlates with steatohepatitis in human and mouse and deficiency of the SYK effector 3BP2 prevents hepatic inflammation, liver injury and, in turn, fibrogenesis. The aims of INFLAMMASYK consortium are: -to better understand how SYK-pathways regulate innate cell responses to TLR engagement in our cherubic and NAFLD mouse models and transgenic animals (3BP2KI, 3BP2KO and myeloid SYK deficient mice). We will focus on systemic, bone, gut and liver inflammation; -to better decipher SYK-dependent signaling pathways regulating macrophage functions in our models of chronic inflammation. -to evaluate pre-clinically how pharmacological targeting of SYK-dependent pathways improve the pathogenic chronic inflammation in both diseases and -to evaluate the relevance of SYK signaling in our NAFLD patients (cohort of 1006 obese patients)

How the innate immune system recognizes virulent microbes? Knowing that pathogenic bacteria express specifically virulence factors, we have worked-out to identify defense mechanisms that respond to microbial virulence factors. We have unraveled the host monitoring of the activity of virulence factors. We found that the CNF1 toxin-induced activation of the RhoGTPase Rac2 triggers an innate immune Anti-Virulence pathway. We have demonstrated the importance of that Anti-Virulence Immunity (AVI) in the context of the infection in mammals using a mice model of Escherichia coli bacteremia. The aim of this proposal is to identify the molecular players involved from virulence sensing to bacterial clearing in mice and the conservation of this pathway in humans. The accomplishment of this project will decipher the innate immune signaling that confers immunity to the host, with the long-term goal of proposing new strategies to stimulate host defense to cure virulent microorganisms from infected patients or to detect AVI immunodeficient patients. On top of that, this work will improve our understanding of the host interaction with virulent microbes and be of broad relevance to many infectious diseases.

Obesity is a major risk factor for numerous metabolic diseases including insulin resistance (IR) and Type 2 Diabetes (T2D). During obesity, the excessive expansion of white adipose tissue (WAT) is accompanied by the accumulation of immune cells, mostly macrophages. Metabolic stress in obese adipose tissue (AT) modify the function and fate of AT macrophages (ATMs), resulting in a low-grade inflammation within the AT and in the circulation. Among others, my work has contributed to demonstrate that inflammation could impair AT functions and participate to the development of IR. However, there are major gaps in our understanding of the molecular mechanisms underlying ATMs reprogramming in response to metabolic stress during obesity. Therefore, identification of new actors and pathways modulating these responses would be of great interest for the treatment of metabolic diseases associated to obesity. My preliminary findings indicate that the transcription factor p53, which is an important cellular stress integrator, is upregulated in ATMs in obese mice. Interestingly, the upregulation of p53 is an early event, since the upregulation is detected after only three days upon high fat diet (HFD). Importantly, we found that silencing p53 specifically in ATMs improved the glucose tolerance of obese mice. Furthermore, exploration of published microarray data performed on human showed an upregulation of p53 pathway in ATMs of obese and diabetic patients, compared to obese non-diabetic patients. Based on these unpublished data, I hypothesize that p53 upregulation in ATMs during obesity could participate in the early step of the development of obesity-induced metabolic diseases such as IR. Therefore, the global objective of the MacP53 project is to explore the functional role of p53 in macrophages and to decipher the mode of action of p53 by identifying key actors of the transcriptional program induced by p53 in ATMs, and to investigate their role in obesity-associated metabolic complications. Thus, I will 1) investigate the role of p53 in ATMs in AT dysfunctions and IR in obesity. I will take advantage of a mouse model allowing a macrophage-specific invalidation of p53, to determine whether the invalidation of p53 in macrophages could prevent the development of diet-induced obesity and IR. I will also take advantage of a technology allowing to silence p53 specifically in ATMs in obese adult mice to determine whether silencing p53 in ATMs could improve the IR of obese mice; 2) identify and investigate the role of key actors of p53 transcriptomic program. I will use a sophisticated and unique methods that I have acquired during my training, and which are not accessible in many laboratories such as ChIP-seq and GRO-seq to identify the direct targets of p53 in ATMs. I will focus on micro-RNAs (miRNAs) and on a new class of non-coding RNAs (ncRNAs), the enhancer-derived RNAs (eRNAs). Indeed, miRNAs and eRNAs have been shown as crucial component of p53 mode of action in response to cellular stress by targeting multiple pathways. These ncRNAs could be very pertinent targets since they can modulate the expression of several genes at once and in a cell-type specific manner. This project will improve our understanding of the connection between p53 activation, AT dysfunctions and IR, and may identify new targets against IR.