Description of scientific achievement of the LipSTIC LabEx

As major public health concerns, cancer and inflammatory diseases are pathologies with urgent medical requirements. A novel concept has recently emerged and relies on the ability of lipoproteins to distribute essential bioactive compounds and drugs throughout the body, and to neutralize and eliminate bacterial proinflammatory mediators. The LipSTIC LabEx is a multidisciplinary research program forming an innovative cluster at the crossroads between lipoprotein metabolism, inflammation, and cancer. The main goal of the LipSTIC LabEx project are :

  • 1) to study the role of lipoproteins in the absorption, transport and delivery of natural bioactive molecules,
  • 2) to determine the implication of lipids and lipoproteins in the initiation and progression of cancer and inflammatory diseases,
  • 3) to take advantage of lipoprotein carriers in a new way to prevent, diagnose, treat and monitor inflammatory diseases and cancer,
  • 4) to develop a novel approach in the prevention and treatment of the endotoxin-mediated inflammatory response and its harmful consequences,
  • 5) to set up and test a technical system of communication aiming at improving patient care and at linking researchers/teaching-researchers of the LipSTIC project with medical doctors, with patients and their relatives, and with the general public.

As outlined below, the LipSTIC LabEx was highly effective in developing new scientific strategies, gathering groups from INSERM/University laboratories, Clinical Investigations Centers, clinical departments of University Hospitals, CGFL and Etablissement Français du Sang (French blood bank), as well as private companies. It contributed to the initiation of new transversal axes (for instance in fields devoted to “lipids, nutrition and inflammation”, “lipids and immunity”, “lipoproteins and sepsis”, …). Furthermore, creation of the LabEx lipidomic plaform led to propose a broad spectrum of analyses (fatty acids, phospholipids, lipoprotein subclasses, lipopolysaccharides,…). They are made accessible to all LabEx members. In addition, sustained efforts have been done to systematically constitute biobanks associated with clinical cohorts.

Thanks to the Ferdinand Cabanne Biological Resources Center, it is largely opened to other academic researchers and is proposed to private companies for economic valorization. Financial support from the LipSTIC LabEx has been essential for upgrading platform equipment, for recruitment of research technicians and post-doctoral fellows (noticeably at the interface of clinical departments and biological laboratories), and for operating costs (including project support and storage of biological samples). Importantly, repeated interactions of LipSTIC LabEx partners had incentive effects for initiating new scientific projects in yet unexplored areas, and sometimes through partnerships with other research projects (some of them are also supported through the PIA program).

Point-by-point description of main LipSTIC advances in individual workpackages :

WP1 – Role of lipoproteins in the absorption, transport and delivery of natural bioactive molecules
Beyond cholesterol, lipoproteins carry and distribute a wide variety of hydrophobic biomolecules, being thus likely to modulate numerous pathophysiological processes. In this context, creation of the LipSTIC Labex has provided an unique opportunity to identify new biological functions of lipoproteins and lipid transfer proteins through the determination of their role in the detection and transport of fatty acids (in particular essential polyunsaturated fatty acids – PUFAs), lipophilic vitamins (in particular antioxidant vitamin E), and organic pesticides (such as a natural A6 anilinopyrimidine compound from marigold). Among the main LabEx achievements in this area, demonstration of the impact of the lipoprotein-bound phospholipid transfer protein (PLTP) on vitamin E transport into the brain in a non-transgenic model of Alzheimer’s disease (consisting in the intracerebroventricular injection of an oligomeric preparation of the amyloid-β [25–35] peptide fragment (Aβ25–35), inducing within one week learning and memory deficits, cholinergic cell loss, oxidative stress, neuroinflammation, Aβ1-42 seeding and Tau hyperphosphorylation). We demonstrated that partial brain vitamin E depletion associated with PLTP deficiency results in an increased memory decline in response to the toxic effect of amyloid beta oligomers. This could be counteracted through dietary vitamin E supplementation. Thus, lipoproteins plus PLTP appear as a relevant and previously unrecognized combination in vitamin E transport and endogenous neuroprotection (Desrumaux et al. Neuropsychopharmacology 2013). Although earlier studies from LabEx teams demonstrated that PLTP can modify macrophage activation, the implication of PLTP in the modulation of T cell-mediated immune responses had never been investigated. In the context of the LipSTIC LabEx project, we showed that PLTP deficiency in mice has a profound effect on CD4+ Th0 cell polarization with a shift towards the anti-inflammatory Th2 phenotype, both under normal and pathological conditions. In a model of contact hypersensitivity, a significantly impaired response to skin sensitization with the hapten 2,4-dinitrofluorobenzene (DNFB) was observed in PLTP-deficient compared to wild-type mice. Interestingly, PLTP-deficiency was accompanied by a significant decrease in the production of the pro-Th1 cytokine interleukin (IL)-18 by accessory cells. We concluded that PLTP exerts a physiological role in the polarization of CD4+ T cells towards the pro-inflammatory Th1 phenotype (submitted manuscript).

As far as the role of lipoproteins in the biodistribution of hydrophobic pesticides is concerned, recent studies reported the presence of lipophilic pesticides in human biological fluids. Moreover, and several epidemiological and toxicological studies in populations exposed chronically or to high doses of these compounds have underlined the role of pesticides in the occurrence of cancers as well as Parkinson’s disease. Investigation of the role of lipoproteins in the transport of pesticides is under way in the LipSTIC Labex. Thanks to the LabEx lipidomic platform and the ultra-high sensitivity 6490 Triple Quadrupole LC/MS2 system with iFunnel technology (Agilent), pesticides are quantified in lipoprotein and lipoprotein-free plasma fractions, urine, bile, and tissue homogenates. Preliminary results are very encouraging and show a high sensitivity of the assay which is compatible with very low values of pesticide concentrations that are expected in biological samples of the present project.

Finally, the LipSTIC LabEx project has led to investigate in a more specific way the impact of dietary fat intake on inflammation and oro-intestinal sensing of lipids. Recent mouse studies from the LipSTIC LabEx teams revealed that feeding a high fat/high sucrose diet reduces the oral sensitivity of the fatty taste, and is associated with the appearance of pro-inflammatory markers in the gustatory papillae. In support of a direct link between high fat intake and inflammation in the context of a leaky gut, we observed an inverse relationship between plasma endotoxin levels and the lipid preference. Interestingly, observed changes were reversed when animals were subjected to caloric restriction. From a mechanistic point of view, the signaling cascade initiated by the fatty acid activation of the CD36 lipid receptor was found to be disturbed in taste bud cells of mice fed the high fat/high sucrose diet, resulting in lower fatty acid-mediated neurotransmitter release (ongoing studies). Through the HumanFATaste1 observational study, obese patients overconsuming fatty foods were found to detect lipids poorly. An additional translational study (HumanFATaste2) has been recently set up in collaboration of several LipSTIC LabEx Teams and food companies (SIA and TEREOS). A main goal is now to explore the effect of a drastic weight loss after bariatric surgery on lipid receptor expression in taste bud cells, on inflammatory parameters, and on plasma lipoprotein profile. In addition, the contribution of postprandial hypertriglyceridemia to endotoxemia and low grade inflammation will be investigated further.

WP2 – Implication of lipids and lipoproteins in the initiation and progression of cancer and inflammatory diseases
Several teams of the LipSTIC LabEx have joined their expertise to investigate the role of fatty acids, lipoproteins and phospholipids in the initiation and progression of cancer, for instance hepatocellular carcinoma (HCC) among patients with cirrhosis. Such an exploration had never been done in large-scale studies. The CiRCE study of the LipSTIC LabEx is a multicenter case-control study in cirrhotic patients (431 with HCC and 739 matched controls without HCC) with a dedicated biobank. Recently, CiRCE data have been included in a European meta-analysis that showed a marked influence of a variant in the PNPLA3 gene encoding an enzyme highly expressed in the liver but with unclear function (Limagne et al. Hepatology 2014). In addition, collaborative studies through the LipSTIC LabEx indicated that 40 % of patients with cirrhosis suffer from diabetes, and in patients with diabetes and cirrhosis a poor prognosis was more strongly related to the liver disease than to diabetes or cardiovascular diseases (Petit et al. Acta Diabetol. 2014). Biological analyses and technician recruitments were covered by the PIA grant to the LipSTIC LabEx. Using the LipSTIC lipidomic platform, ongoing projects are focused on the exploration of fatty acid and phospholipid composition of lipoproteins and erythrocyte membranes in cirrhosis complications and risk of HCC in cirrhotic patients, on the prognosis value of lipid parameters, and on the role of lipoproteins and lipopolysaccharides in cancerous and non-cancerous cirrhosis complications.

A multicenter case-control study was designed to examine the role of fatty acids in the risk of ColoRectal Cancer (CRC) and the role of biomarkers of adipose tissue metabolism and inflammation (AGARIC study). By using fatty acid composition of subcutaneous adipose tissue as a biomarker of fatty acid intake, both dietary patterns and altered fatty acid metabolism could be related to colorectal carcinogenesis (Cottet et al. AJCN 2015). Collaborations of LabEx teams led to demonstrate that serum neutrophil gelatinase-associated lipocalin (NGAL), soluble tumor necrosis factor receptor 1 and 2 (sTNFR-1, sTNFR-2), and C reactive protein (CRP) were independent determinants of CRC risk, although not suitable as biomarkers for the early diagnosis of CRC (Duvillard L et al. BMC Cancer. 2014). Analysis of AGARIC data is pursued with the investigation of the inter-relationships of fatty acid composition in adipose tissue and erythrocyte membranes, of serum phospholipids as potential biomarkers of CRC, and of fatty acid composition of adipose tissue and erythrocyte membranes as determinants of CRC mortality.

From the DIADE study, we highlighted that the detection process of colorectal adenoma, but not of CRC, is highly dependent on socioeconomic disparities and access to general practitioners throughout a well-defined territory (Fournel et al. submitted manuscript).
Collaborative studies of the anesthesiology department of the University Hospital of Dijon and of LipSTIC LabEx teams demonstrated in a cohort of 217 patients undergoing cardiac surgery with cardiopulmonary bypass that a low preoperative cholesterol level is associated with an increased risk of sepsis. In addition, elevated post-operative IL8 and procalcitonin levels were found to predict poor clinical outcome when combined with low plasma lipoprotein levels (Lagrost, Crit Care Med 2014 and patent EP13306433.7).
Additional multicenter cohort studies are still in progress in the LipSTIC LabEx. Biological analyses of TOXIB study are schedule, the 5-year follow-up of the CiRCE study is ongoing, patient recruitments in IDEAL study (291/864), IVOIRE study (1005/1500), VERROU-REA study (258/300) and CALIPSO study (151/650) are in progress.

Whereas earlier studies reported that n-3 PUFAs such as docosahexaenoic acid (DHA) can dampen the production of proinflammatory cytokines from innate immune cells (including natural killer cells, macrophages and dendritic cells), direct effect of DHA on CD4+ T cells remained elusive. The LipSTIC LabEx consortium has enabled the development of novel activities in this field, including the exploration of the effects of fatty acids on CD4+ T cell differentiation. Thus, DHA was demonstrated to inhibit Th17 cell differentiation and to prevent tumor growth of B16F10 melanoma and 4T1 mammary adenocarcinoma murine tumors in an IL-17-dependent manner (Berger et al. Cancer Research 2013). In additional studies, 5-Fluorouracil (5-FU) (i.e. a drug widely used in the treatment of breast and colon cancer) was found to deplete selectively the immunosuppressive myeloid derived suppressor cells (MDSC) in vivo, thus restoring CD8+ T cell anticancer immune responses and improving its therapeutic effect. However, further research uncovered an ambivalent effect of 5-FU on anticancer immunity. Indeed, an additional molecular pathway through which the 5-FU-mediated cell death of MDSC can limit 5-FU efficacy and contributes to immune evasion was deciphered. Specifically, we have shown that 5-FU triggers activation of the Nlrp3 inflammasome in MDSC (Bruchard et al. Nat Med 2013). It leads to the release of IL-1, which favors IL-17 secretion by intratumoral CD4+ T cells. Since IL-17 secretion from CD4+ T-cells could favor tumor progression not only through cell-intrinsic effects on tumor cells but also by enhancing the secretion of proangiogenic mediators within the tumor microenvironment, we hypothesized that interrupting this series of molecular events would enhance the anticancer efficacy of 5-FU. Accordingly, 5-FU administration along with the IL-1 receptor antagonist Anakinra into tumor-bearing mice magnified the anticancer effect of 5-FU. In addition, 5-FU was found to exhibit a stronger therapeutic action in Nlrp3-, Casp1-, IL-1R1- and IL-17-deficient mice compared to wild-type mice, indicating that activation of the Nlrp3 inflammasome in MDSC is detrimental to 5-FU anticancer efficacy. The aforementioned findings may suggest that future anticancer therapies would most likely benefit from combinatorial treatments associating chemotherapy with immunomodulation. Ongoing studies in the LipSTIC LabEx are now addressing the impact of complex lipid composition on tumor progression and on the chemotherapeutic treatment responses. This project aims at establishing a putative relationship between lipid metabolism dysregulation and tumor resistance. A translational research program has been set up in partnership with the Division of Gastroenterology of the University Hospital of Dijon and the Anticancer GF Leclerc Center (CGFL). Recently, a phase II clinical trial has been initiated at CGFL to investigate the relevance and efficacy of a combinatory treatment made of conventional 5-Fluorouracil plus the IL-1 receptor antagonist Anakinra known to block inflammation. This project is carried out in partnership with the SOBI Pharma Company. Whether manipulation of lipid metabolism may contribute to the development of new therapeutic approaches with the aim to increase the sensitivity to conventional anticancer drugs will be also addressed.

Macrophages are other key cells involved in the inflammatory response and innate immunity. In collaboration with the Gustave Roussy Institute, specific attention has been paid through the LipSTIC LabEx to macrophage polarization and to the role of caspases, which are key effectors of apoptosis. Importantly, a postdoctoral fellow from the LipSTIC LabEx has demonstrated non-apoptotic functions of caspases 3, 7 and 8 in monocyte-derived macrophages. Interestingly, caspase activation was found to be restricted to mitochondria and responsible for the generation of cytosolic Reactive Oxygen Species (ROS). Observations suggest that caspase inhibition could be used to modulate macrophage polarization. In support of this hypothesis, caspase inhibitors prevent the development of lung fibrosis in bleomycin-treated mice, which is associated with a change in macrophage polarization. Thus, caspase inhibition could find therapeutic applications in fibrosis, cancer, and other diseases as atherosclerosis.

In the context of myeloproliferative/myelodysplastic diseases, the LipSTIC LabEx teams identified TIF1 (tripartite motif family 33/transcription intermediary factor 1) as a crucial regulator of transcription during hematopoiesis, modulating the balance between lymphoid- and myeloid-derived Hematopoietic Stem Cells. Thanks to the LabEx lipidomic platform, Tif1γ deficiency was found to be associated with dysregulation of lipid metabolism. Gene clusters involved in synthesis and transport of cholesterol are overexpressed whereas those implicated in the lipid catabolism or lipid storage and transport exhibit a lower expression. In bone marrow, the levels of cholesterol, sphingomyelin, and phosphatidylcholine, as well as lipid raft domains were found to be increased (Quéré et al. PNAS 2014).

Through a partnership of the LipSTIC LabEx with the Curie Institute, a family of five pyridopyrazine (PyPy) compounds inhibiting the Granulocyte Monocyte Colony Stimulating Factor (GM-CSF) pathway and controlling leukemic cell proliferation has been identified. A European patent was registered, describing a PyPy compound as a potential chemotherapeutic and anti-leukemic agent in humans (Quéré & Nguyen. 2013). Through the LabEx lipidomics platform, phosphatidylcholine (PC) was identified as the target of the PyPy compounds. PC is the most abundant phospholipid and an integral component of the lipoproteins, especially HDL. Characterization of the role of PyPy on PC and of the associated alterations of lipid metabolism in leukemic stem cells of acute leukemia is under way in the LipSTIC LabEx. Therapeutic valorization of the project was initiated in 2014 in collaboration with the Society for Acceleration of Technology Transfer (SATT Grand Est) which provided a specific grant dedicated to the project in order to foster pre-clinical investigations, pharmacokinetic studies and large-scale production of the PyPy compound. Project is conducted in collaboration with the Department of Clinical Hematology of the Dijon university hospital and the hospital network organized through the Cancéropôle Grand-Est which will help in the development of PyPy compounds through phase I trials in Acute Myeloid Leukemia.

WP3 – Lipoprotein carriers for drug delivery in the treatment of inflammatory diseases and cancer
One potential application of lipoproteins is their use for drug targeting. After loading the drug into the lipid core of the lipoprotein structure it appears to be promising to deliver its cargo selectively to the site of action.

Design of drug delivery systems for more selective approaches in anti-inflammatory and anti-cancer therapy is a major challenge. The LipSTIC LabEx gathers distinct groups specialized in prevention and treatment of cancer and inflammatory diseases, as well as in drug targeting. Over the former LabEx period, they have joined their expertise to investigate lipoprotein-inspired or lipoprotein-based drug delivery systems. In a first step, they have created polymeric carrier systems similar to lipoprotein structure to entrap drugs for a more selective deposition in the inflamed tissue (Lamprecht et al. 2015). In order to increase selectivity, the polymeric nanocarriers have been decorated with plant lectins, thus increasing selective adhesion to the inflamed tissue and therapeutic effect (Moulari et al. 2014). In a second step, a more ‘bioinspired’ approach has taken advantage from natural lipoproteins by entrapping pharmacological compounds (cyclosporine, paclitaxel, etoposide,…) into high density lipoproteins (HDL), low density lipoproteins (LDL), and very low density lipoproteins (VLDL). In a murine experimental colitis model, cyclosporine-loaded LDL lowered significantly the inflammatory response, thus emerging as a very promising therapeutic tool. Ongoing steps are related to pharmacokinetics and delivery pathways of drug-loaded lipoproteins.

Another important goal of the LipSTIC LabEx is to develop inhibitors of Heat Shock Proteins (HSP70 and HSP110) to be used as chemosensitizing agents in cancer therapy. Tumor cells overexpress HSP70 and/or its co-chaperone HSP110 to overcome their increased proteotoxic stress when compared to healthy cells. In experimental models, HSP70 and HSP110 depletion reduced cancer cells tumorigenicity and increased their sensitivity to therapeutic drugs. Further, clinical retrospective studies have demonstrated, notably in colon cancer, an association between HSP levels and poor prognosis. A LipSTIC LabEx team has already screened a chemical library for HSP70 inhibitors and selected a hit. Unfortunately, this potent HSP70 inhibitor is poorly soluble in water. Thanks to joint expertise and know-how of LipSTIC teams, the idea to use lipoproteins to transport and target the HSP70 inhibitors has emerged. Results recently obtained have been quite spectacular at least concerning the solubility issues (patent pending). From hit to lead, one objective of the work is to study how the lipoprotein-dependent vectorization affects stability and bio-distribution of the molecule as well as its anti-cancer properties (both in vitro in cultured cancer cells treated with anticancer agents and in vivo in mouse models of colon cancer and melanoma). The same experimental strategy is currently applied to HSP110 inhibitors in the LipSTIC LabEx. With the best-selected conditions (molecule/vector), the drug approval process for clinical use will be initiated to determine pharmacokinetics (with sub-therapeutic doses – Phase 0), safety, dosage range and side effects of the best drug candidate (phase I). This will be done in collaboration with the anticancer Georges François Leclerc Center (CGFL, Dijon), one partner of the LabEx consortium. Patent registration and publication are in progress, and a spin off about cancer detection using HSPs has been recently created (Nano-Diag). This start up will be in charge of the clinical development of drug-candidates for HSP70/HSP110 inhibition (with Inserm Transfert and SATT Grand-Est).

WP4 – Novel approaches in the prevention and treatment of the inflammatory response and its harmful consequences
Both non-septic/sterile and septic/microbial-induced inflammation are characterized by the recruitment of inflammatory cells such as neutrophils and macrophages. The inflammatory response triggered by these cells involves the production of pro-inflammatory cytokines, PUFA-derived eicosanoids, and ROS. The research activities of the LipSTIC consortium has focused on three main goals :

  • 1) the development of new biotherapies based on the immune-modulatory properties of apoptotic cells or monocyte-derived suppressive cells,
  • 2) the exploration of the metabolism of lipids and lipoproteins in inflammatory diseases (in particular with specific interests in glucose homeostasis, mitochondrial ROS production and PUFA-derived lipid mediators),
  • 3) the deciphering of the “Reverse Lipopolysaccharide Transport” pathway and its pathophysiological relevance.

The LipSTIC LabEx has focused on several inflammatory diseases, including graft-versus-host disease (GvHD) after allogeneic hematopoietic cell transplantation, chronic kidney diseases/kidney transplantation, and autoimmune diseases. A main objective here is to develop new treatments taking into account individual profile of each patient by using relevant biomarkers. New therapeutic approaches are based on the development of biotherapies (Angelot-Delettre et al. Hematologica 2015) which take advantage of the immune-modulatory properties of cell-derived products. We reported that IL-22, which contributes to homeostasis of the gastro-intestinal tract, can affect the severity of experimental GvHD (Couturier et al. Leukemia 2013). Interestingly, an innovative treatment with human monocyte-derived suppressing cells (HuMoSC) has been tested successfully by LipSTIC LabEx teams in a xenogenic model of GvHD with the induction of CD8+ regulatory T cells. The impact of IL-22 deficiency in donor T cells on intestine permeability and microbiota during GvHD is currently investigated. Moreover, biologic agents used to collect peripheral hematopoietic grafts, such as G-CSF and an inhibitor of CXCR4 may affect the severity of experimental GvHD (Arbez et al. Cytotherapy 2015). We also reported that chronic antigenic stimulation of T cells during kidney transplantation is associated with immune senescence and chronic inflammation which may impact on clinical complications (Crepin et al. Am J Transplant 2015). We identified CMV as a major factor responsible for chronic antigenic stimulation and here we are currently studying the impact of bacterial translocation due to T cell depletion after anti-thymocyte globulins (ATG). To reach the goals, two patient cohorts were constituted : the ORLY-Est involving 856 kidney transplant recipients (partnership with the INCREASE FHU), and the GABII cohort including 24 patients. Moreover, the relationship between circulating lipid profile, abdominal adiposity as assessed by dual-energy X-ray absorptiometry, and systemic inflammation is addressed in several autoimmune diseases, including rheumatic diseases before (Toussirot et al Front Immunol 2013) and after anti-TNF treatment (Toussirot et al. J Rheumatol 2014; Toussirot et al. Eur J Nutr 2014), as well as psoriasis (Toussirot et al. Front Immunol 2014). In immune thrombocytopenia, the autoimmune mechanisms involved have been deciphered (Audia et al Med Sci 2014) and the effect of anti-CD20 mAb therapy (rituximab) has been evaluated. The latter was conducted in collaboration with the INFLAMEX LabEx. While we previously reported that the alternative transcript of CD20 may be associated with resistance of malignant B cells to rituximab (Henry C et al. Blood, 2011), access to the INFLAMEX cohort of rheumatoid arthritis patients resistant to rituximab led to demonstrate that the alternative transcript of CD20 is not associated with rituximab resistance in these patients (Gamonet et al Rheumatlogy 2015).

The above studies relied on the tight cooperation involving LabEx research laboratories, Clinical Investigation Centers, and University Hospitals. Importantly, two patents relating to the novel anti-inflammatory treatments were registered. The first patent is dealing with an anti-inflammatory product derived from the supernatants of macrophages eliminating apoptotic cells (SuperMApo – patent # WO2014106666-A1). As Co-Inventor, Sylvain Perruche was awarded a national prize for the creation of a startup (15ème concours national d’aide à la création d’entreprises de technologies innovantes, BPI France, 2013). This award offers a unique opportunity to foster the creation of the SuperMApo startup which should occur by the end of september 2015. The second patent is dealing with human monocyte-derived suppressive cells (HuMoSC – patent # FR1452286) and their use in a novel cellular therapy against GvHD which remains a major complication of allogeneic hematopoietic cell transplantation (Janikashvili N et al., J Allergy Clin Immunol. 2015).

In its acute form, GvHD has also been shown to produce gastrointestinal tract injury, with subsequent bacterial translocation and endotoxemia, thus worsening further the systemic inflammatory response. Endotoxins or lipopolysaccharides (LPS) are located at the surface of Gram negative bacteria. They are constituted of an antigenic polysaccharidic chain and a lipid A moiety which can activate toll-like receptor 4 (TLR4) to trigger the inflammatory response. As amphipathic molecules, LPS can bind readily to lipoproteins, resulting in alterations of their detoxification and proinflammatory properties, i.e. a specific pathway that we named Reverse LPS Transport (RLT) in analogy with the well-known Reverse Cholesterol Transport (RCT) pathway. Because LPS cannot be metabolized in the body, RLT actually constitutes the only way to get rid of LPS, to resolve endotoxemia and to prevent its harmful effects. Whereas it is of major importance to distinguish between non-infectious and septic inflammatory response, to date none of microbial identification tests has proven to be reliable enough to ascertain the occurrence and extent of infection. In particular, previously described methods for LPS detection have shown some limitations. As a major outcome of the LipSTIC LabEx, a new LCMS2 method for the sensitive, accurate, and direct quantitation of total amounts of LPS in biological media was set up, thanks to new equipment acquisition (co-funding through LipSTIC LabEx and INSERM/AVIESAN grants). It led to the novel, so-called EndoQuant method, combining the LCMS2 quantitative method with the classical Limulus Amaebocyte Lysate bioassay. These developments led to the recent creation of the EndoQuant business unit. As part of the LipSTIC LabEx, it was set up in close partnership with SATT-Grand Est and today has secured several customers in France and abroad.

Among other technological breakthroughs of the LipSTIC LabEx, a new, dual-labelling technique for LPS and HDL has been developed. It was set up through a collaboration of LipSTIC LabEx teams with the ICMUB laboratory, the 3MIM CNRS project, and the IMAPPI EquipEx, all of them combining their know-how and efforts to promote pharmaco-imaging studies at the University of Bourgogne. Capacity of this consortium to achieve a dual labelling of LPS was reported in a recent article (Duhéron et al. ACS Chemical Biology 2014). This strategy allows the real-time monitoring of LPS localization in mice through the detection of radioactivity by SPECT Scan as well as the assessment of tissue distribution by fluorescence microscopy. The ongoing validation of the same labelling strategy for HDL will allow to determining further LPS and HDL plasma kinetics, body distribution and organ uptake under inflammatory conditions.
Through the LipSTIC LabEx program, and in collaboration with the BioProtein Technologies company as a founder company in the LipSTIC consortium, we took advantage of a newly created line of PLTP transgenic rabbits (with the human PLTP transgene placed under the control of the Whey Acidic Protein promoter) to produce large amounts of purified and active human PLTP from the milk of transgenic females. PLTP, as a member of the lipid transfer/lipopolysaccharide binding protein gene family, was found to play a driving role in RLT, thus modulating inflammation and innate immunity.

LipSTIC LabEx teams demonstrated that endotoxemia, as initiated by a single acute injection or by a continuous infusion of LPS, is associated with an increased glucose-stimulated secretion of insulin through a glucagon-like peptide 1 (GLP-1)-dependent pathway (Nguyen et al. Diabetes 2014). These advances from the LipSTIC LabEx strongly suggest that GLP-1, a key component of enteroendocrine cells involved in insulin secretion stimulated by glucose, may constitute an unrecognized link between endotoxemia and glucose metabolism. The expression of genes involved in the synthesis and secretion of GLP-1 in response to LPS is currently studied in GLUtag cells (entero-endocrine lineage capable of secreting GLP-1) that have been made accessible to LipSTIC LabEx teams through collaboration with Pr. Daniel Drucker (Toronto, Canada).

Importantly, the LPS-mediated inflammatory response is tightly dependent of the n-6/n-3 fatty acid balance in immune cells which release active fatty acid derivatives. Many genes involved in lipid metabolism and production of oxygenated lipid derivatives are regulated by the liver X receptors (LXR) which are expressed at high levels in monocyte-derived macrophages, thus modulating cholesterol metabolism, innate immune response and inflammation. Among important determinant of the availability of eicosanoid precursors in membrane phospholipids, the lysophosphatidylcholine acyltransferase 3 (LPCAT3) was identified by LipSTIC LabEx teams as a master regulator of arachidonate incorporation at the sn-2 position of glycerophospholipids. Thus, LPCAT3 was found to prime human macrophages for subsequent eicosanoid secretion (Ishibashi et al ATVB 2013; Varin et al ATVB 2015). A new line of LPCAT3-KO mice was recently generated by LipSTIC LabEx teams in collaboration with ICS Institute in Strasbourg. The impact of LPCAT3 in vivo on phospholipid metabolism and LPS-triggered inflammation is currently under investigation.

In another way to modulate the PUFA content of immune cells, transgenic fat-1 (fat1Tg) mice expressing a C. Elegans gene encoding a n-3 fatty acid desaturase were available for LipSTIC LabEx teams through a collaboration with Pr JX Kang (Harvard Medical School). Fat1Tg mice were found to display a high pancreatic and intestinal content of n-3 fatty acids, to be protected against streptozotocin-induced diabetes and breast cancer, and to display decreased levels of proinflammatory arachidonic acid-derived mediators. It illustrates the relevance of the fat-1Tg mouse model over conventional dietary supplementation. Whether high tissue content of n-3 fatty acids can alleviate metabolic endotoxemia is under way in the LipSTIC LabEx.

As outlined above, translational research, clinical investigation and applied biotechnology to the benefit of patients are the results of a major leverage effect of the LipSTIC LabEx. In the context of endotoxin neutralization and detoxification, this strategy is currently strengthened and extended in a new and relevant approach to improve clinical outcome. To this end, several observational and interventional studies have been initiated in collaboration with Clinical Investigation Centers, Center for Biological Resources and the INCREASE FHU (hospital-university federation). It includes an observational study in 300 patients with septic shock (collaboration with Pr Meziani – Strasbourg), the NUTRIVOIRE cohorts (patients admitted in the Intensive Care Unit of the University Hospital of Dijon), and the IDEAL-ICU study (860 hemodialyzed patients from Intensive Care Unit-ICU). Main goals here are to investigate lipid profiles, cytokine levels and endotoxemia in patients from the ICU, and to work out the influence of renal replacement therapy and mechanical ventilation. Finally, ongoing interactions with several companies (Baxter, Fresenius, Alteco-Medical) aim at investigating the impact of novel hemofiltration devices on endotoxin detoxification and clinical outcome.

WP5 – Health economics and communication.
Because cancer and inflammatory diseases have both non-monetary and monetary consequences, a major concern is to integrate medico-economic evaluations of patient care. The production of global utility scores associated with innovative strategies in specific diseases (especially in cancer) is a major challenge. As an example, through the APPACH study, the LipSTIC LabEx is currently developing and validating a specific questionnaire to estimate the objective burden associated with informal care given by spouses of patients treated by adjuvant or palliative chemotherapy for a colon cancer. In collaboration with a Social sciences team and the national platform “Quality of life and cancer” (partner in the LipSTIC LabEx), the aim is to identify demographic, clinical, psychological and socio-economic factors characterizing patients and spouses associated with the burden. We expect the questionnaire will be a routine tool to identify the spouses who are experiencing difficulties. Then, they can be directed towards existing support options. Use of this questionnaire may be further valorized in future trials evaluating the economic impact of new chemotherapy techniques on the daily life of relatives.

In the context of multi-professional group practice and the diffusion of innovations into primary care, LipSTIC LabEx teams conducted an analysis of the coordination between different health professionals (GPs, nurses, physiotherapist…). The efficiency of primary care centers was compared to that in the context of single practice, and analysis of the remuneration schemes in this context was conducted. Analyses of the two surveys set up for this study with the support of the Bourgogne Health Regional Agency (ARS) are completed, and the final results were presented in July 2014 for phase 1 (assessment of the impact of multi-professional groups for patients), and in February 2015 for Phase 2 (analysis of the coordination within healthcare networks or multi-professional groups). Assessment of the efficiency of primary care centers compared to that in the context of single practice is conducted in collaboration with the Institute for Research and Information in Health Economics (IRDES), which supplies the national database. The authorization of the CNIL has been requested in 2013 and is expected soon and the methodology to assess the efficiency is under progress. For analysis of remuneration schemes, the remuneration of medical activity in the context of multi-professional group practice in France is compared to other countries (USA, Canada …). A major goal here is to address whether the multi-professional group practice is more efficient that the traditional practice and how to compensate health care professional who are practicing in group in the French context.

Another aim is dealing with the medico-economic evaluation of a regional (Burgundy) program of telemedicine. This program gathers several telemedicine services connecting primary care, hospital care (cardiology, dermatology, geriatrics) and a telestroke organization. The assessment of the telestroke program is largely completed. The assessment of the other telemedicine services will be completed in the fall of 2016. The cost-effectiveness evaluation of telestroke network required the retrospective collection of data on 780 patient records. The aim of the study was to assess the contribution of telemedicine in terms of mortality and disability, with former results obtained in patients with stroke. Expected results will provide evaluation of the financial impact of telemedicine experiments for hospitals. For example, a LipSTIC LabEx team has recently evaluated the actual cost of cerebral fibrinolysis and assessed the balance hospital cost-income for all patients, this innovation being currently not reimbursed in French hospitals. Still in the context of the telestroke assessment, differential rates of mortality and disability were demonstrated with or without telemedicine, depending on the distance from the stroke center.

Since the real beginning of the LipSTIC LabEx, it was decided to develop a strong policy concerning social issues of the scientific fields to be explored by researchers. This policy comes as a permanent support for LipSTIC researchers to participate in any outreach activities (radio and TV programs, public talks and lectures, newspapers interviews…) thanks to their regular promotion on the website or in any occasion (scientific council,…).

Two main communication programs were initiated in the LipSTIC LabEx :

  • Face-to-face communication program.
    This program is implemented by services of University of Bourgogne and University of Franche-Comte. It offers a specific training to researchers who would like to be involved in Face-to-Face activities. Dedicated to the general public, it aims at establishing fruitful relationships between this public and researchers in the fields covered by the LipSTIC LabEx.
  • Terre de Louis Pasteur program

Part of the Investment for the Future program (PIA), the Terre de Louis Pasteur project is implemented under the auspices of the French Academy of Sciences. It benefits from a long-lasting and large set of activities dedicated to Louis Pasteur. Both Louis Pasteur’s birthplace in Dole and family home in Arbois turned into museums. Recently, a third house, belonging to the Vercel family (Pasteur’s friends), has been purchased through the Public Institution for Cultural Cooperation. In the general framework, the Vercel house will be turned towards a Centre of Interpretation of Contemporary Science. As highlighted in the signed consortium agreement, the LipSTIC Labex has been appointed by The French Academy of Sciences and local authorities as a major partner for the promotion of biomedical sciences and research in the Terre de Louis Pasteur network. It will reinforce the presence and visibility of LipSTIC Labex in the public sphere. LipSTIC LabEx researchers have already contributed to several meetings dedicated to science and communication, as well as a science and art (Public lecture in Academy of Arts and Literature in Mâcon in 2013, Symposium on Innovation in Health Communication in Dole Town Hall in 2013, three-day Face-to-Face Communication workshop ‘La Main à la Pâte” in 2014). In 2014, one steering committee of the LipSTIC LabEx has been held in Pasteur’s House of Arbois and introduced by the Mayor and the director of the Public Institution for Cultural Cooperation “Terre de Louis Pasteur”. Thus, LipSTIC LabEx researchers were given a good opportunity to get involved deeply in the project which aims at transforming the Pasteur and Vercel houses into an interpretation center dedicated to contemporary research.

A PhD fellowship is funded 50% through the Bourgogne Regional Council grant to the LipSTIC LabEx, and 50% by the city of Dole. Main goals are to study pasteurian scientific outcome issues, to understand what can be transposed from laboratory life to school curriculum, and to develop the tremendous added value of Pasteur achievements in setting up scientific activities with children at school. In addition, most relevant knowledge and practices are determined through interviews with LipSTIC researchers, thus opening to what could be the communication to the general public of main LabEx advances. A specific attention is currently paid to an educational package dedicated to cancer and its unmentionable aspects.