Kidney transplantation is the only curative treatment for patients suffering from end-stage renal disease but requires the indefinite use of non-specific immunosuppressive drugs to avoid graft rejection. Use of these drugs is associated to increased risks of infections, certain types of cancer and toxicities. Crohn’s disease is an inflammatory bowel disease that is well controlled today by steroids and immunosuppressive drugs although no curative treatment is available. Furthermore, no efficient treatment is developed so far for patients with refractory Crohn’s disease. Cell-based immunotherapy with tolerogenic dendritic cells (DCs) is recognized as an efficient means of promoting antigen specific tolerance since several years. Based on our expertise in rodents, we recently established a GMP-compliant manufacturing process to derive human Autologous Tolerogenic DCs (ATDCs). The safety of ATDC immunotherapy is currently evaluated in a Phase I/II clinical trial in kidney transplant patients. We demonstrated the efficacy of human ATDCs in vitro and in vivo using a model of human-into-mice xenogenic GVHD (Graft-versus-host-Disease). More precisely, our in vitro analyses demonstrated that ATDCs secrete IL-10 in response to TLR ligand stimulations, suppress T cell proliferation and promote Treg expansion. This Treg expansion was confirmed in the humanized mouse model of GVHD. Microarrays analyses highlighted that ATDCs express a very high level of CX3CR1 compared to others populations of monocyte-derived tolerogenic antigen-presenting cells (as Mregs, Rapa-DCs, DC10 and MDSCs). The fractalkine receptor CX3CR1 is widely expressed in the mononuclear phagocyte system. High expression of this chemokine receptor is associated to protective abilities, both in transplantation and in the intestine. Indeed, intestinal CX3CR1+ mononuclear phagocytes are able to promote colitis protection. More precisely, as ATDCs, these cells secrete a high amount of IL-10 and promote Treg expansion. The aim of this proposal is to in depth investigate the correlation between CX3CR1 and ATDCs. My hypothesis is that CX3CR1 expressed by ATDCs plays a crucial role in the functions of these cells as described in mouse intestinal mononuclear phagocytes. Cell therapy using tolerogenic DCs expressing CX3CR1 will be therefore the candidate of choice to treat patients with Crohn’s disease. In this project, the first task will define the molecular role of CX3CR1 in human ATDCs regarding their characteristics, their effects on T cells and their protective effect in vivo using humanized mice. For that, our previous in vitro and in vivo experiments used to define ATDCs will be repeated using ATDCs expressing or not CX3CR1. The knock-down of CX3CR1 in human ATDCs will be possible thanks to the CRISPR/Cas9 technology. The second task will identify whether human ATDCs have an in vivo counterpart. This could reinforce the relevance of ATDC administration in humans, as an enrichment of naturally present protective CX3CR1+ cells. For that, molecular signature of ATDCs will be compared to other public data sets of DCs present in skin, blood, intestine and tumors. We will also investigate whether suppressive cells present in pleural effusions and ascites from cancer patients display similar features to ATDCs. The last task will precise the role of CX3CR1 expressed by ATDCs in protocols of cell therapy performed in mouse models of transplantation and colitis. In that respect, ATDCs will be derived from mouse monocytes (as in humans) from CX3CR1 deficient mice and their WT littermates. Following their in vitro characterization, the efficacy of cell therapy using mouse ATDCs expressing or not CX3CR1 will be evaluated in models of transplantation and colitis. This project will precise the ATDC mechanisms of action and whether CX3CR1 plays a crucial role in their suppressive properties. This study will also pave the way to the expansion of ATDC clinical use in Crohn’s disease.

Azole resistance in Aspergillus is one of the emerging public health concerns, listed as a WHO priority and suited to an integrated One Health approach. Selective pressure due to the use of azole pesticides in agriculture being incriminated, identification of clinical and environmental resistance patterns, and a greater understanding of the factors driving this resistance are urgently needed in order to issue recommendations to the stakeholders. The multidisciplinary AspergillusOne-health project strengthened with model and innovative methodologies (WGS, genotyping, MALDI typing, metabarcoding, AI) aims to identify hotspots as possible sources for selection of azole-resistance in the environment, after the detection of azole-resistant Aspergillus in patients and patiens's home, avian facilities, the environment (farming and sawmills), and detection of the azole fungicides in soil and air. The role of resistance trait on Aspergillus fitness cost will be investigated, using environmental strains and mutants selected after fungicide pressure, to assess its clinical involvement.

The recent rise of high-resolution and depth imaging techniques like photoacoustic microscopy (PA) stimulates novel research areas in biology. In vivo tracking of immune cells, signaling inflammation and severe pathologies thereof, is one of them and attracts great interest. The AZOTICS project thus aims at addressing the current PA microscopy limitations by fabricating innovative biocompatible elastomeric nanolabels relying on azo photochromes. Photostimulated actuation mechanisms will help amplify the PA contrast based on thermal expansion. The photoinduced mechanical deformations of single nano-objects will be assessed at the nanoscale using atomic force microscopy in order to propose a rationale for the performance of photoacoustic probes beyond their sole optical absorption ability. Their PA imaging capability will be validated through an in vitro, in cellulo and in vivo continuum of studies involving macrophage staining, microfluidic systems mimicking microvasculature, and models of acute inflammatory activated in mice. The interdisciplinary AZOTICS consortium gathers experts in chemistry, physics and optics from Nantes and Grenoble, having already tightly worked together and being keen to share their knowledge in order not only to address unexplored fundamental questions but also to propose innovative photoacoustic systems for in vivo imaging.