Our defense against pathogens relies on the complex and sophisticated orchestration of immune cells recruitment by external cues. Our main research goal is to analyze the key steps of the leukocyte recruitment sequence by using adapted in vitro tools to quantitatively characterize and decipher the phenotypes of interest.
When circulating in the blood stream and patrol the body, leukocytes travel across the narrow and intricate network of the blood microvasculature associated to organs. Leukocytes experience there extreme conditions of hydrodynamic shear stress, mechanical deformation, and intimate friction with capillary walls. Specific physiopathological situations develop in this specific microenvironment. We have developed microfluidic approaches to study single cell passage in constrictions and then applied our knowledge and tools to samples of patient suffering from the acute respiratory distress syndrom.
In order to leave the blood stream and eventually travel to lymph nodes or sites of inflammation, leukocytes are captured by the endothelium of blood vessels, where they migrate along the to permissive sites of transmigration. These migration and transmigration processes are supposedly orchestrated by chemical signals and take place under the influence of a strong hemodynamic shear stress. Although the capacity of cells to orient versus chemical gradients (chemotaxis) has long been identified, existing information on actual chemokines distribution in vivo is still very sparse. Moreover, mechanical cues as efficient players in immune cell orientation (mechanotaxis) have been acknowledged only recently, and their function and mechanisms remain largely unexplained. In this context, we proposed to investigate quantitatively the guidance mechanisms of immune cells by both mechanical and chemical cues.
Leukocyte arrest in the microcirculation related to the Acute Respiratory Distress Syndrome (ARDS).
M Biarnes-Pelicot, P Bongrand, P Robert, O Theodoly. Coll J Bico, MC Julien ESPCI; JM Forel, L Papazian AP-HM.
Microfluidic tools were developed since 2007 to explore the passage of circulating leukocytes through narrow constrictions mimicking lung blood capillaries (Biophys J 2009, Lab Chip 2010). , Novel microfluidic devices and methodologies were then created to achieve the first microfluidic rheometer yielding quantitative absolute measurements of cells viscosity (Biomicrofluidics 2013). A microfluidic diagnostic tool was also built and tested at hospital to assess rigidification of leukocytes in whole blood samples (lab chip 2013). Finally, an in depth medical study of adhesiveness and stiffness of leukocytes using serum samples of ARDS patients allowed identification of cytokines triggering a massive arrest of leukocytes in the lung upon early ARDS (Critical Care 2016).
Friction of model objects in confined environments. (O Theodoly, MP Valignat with MC Jullien from Institut PG de Gennes, Paris). The physical analysis of cells friction in microchannels developed for quantitative rheometry in microfluidics has triggered a fruitful collaboration with MC Julien to perform similar experiments on model systems of droplets/bubbles in microfluidics Hele-Shaw chambers (ANR TRAM 2013, PRL 2015, Lab Chip 2016).
Integrin mediated migration and guiding of T cells.
L Aoun, M Biarnes-Pelicot, Y Koo, A Lellouch, C Mionnet, , X Luo, P Negre, T Sbarrato, O Theodoly, MP Valignat. Collab. M Bajenof, JP Gorvel, CIML; C Hivroz, Inst. Curie-Paris; V Studer, IIN-Bordeaux; Alveole company.
A model system of primary effector human T lymphocyte migrating on glass substrates functionalized with adhesion molecules ICAM-1 (ligand of integrin LFA-1) allowed us to show that cells were sensitive to flow and oriented against the flow (Biophys J 2013). This surprising and unique phenotype was then deciphered as a passive guiding mechanism requiring no mechano-tranduction signaling. The lymphocyte tail, called uropod, acts as a wind vane that detects flow direction and biases cell direction (Nat Comm 2014). We are now engineering various substrates in order to step by step decipher the behavior of crawling lymphocytes in vivo: substrates with fixed ligands on glass or diffusing ligands on lipid bilayers, substrates with varying amounts of integrin-ligands ICAM-1 and V-CAM, substrates covered with endothelial cells. Alterations of leukocytes migration and guiding properties during inflammation are also being investigated using our in vitro tools via collaborations with biologists performing similar investigations in vivo (ANR Recrute 2015).
Optical nanodetection without labelling
O Theodoly, MP Valignat. Coll. T Mignot, LCB; Nanolane Company
Ongoing collaboration with the company Nanolane yielded to the adaptation of the wet-SEEC technique to biological applications, i.e. in immerse conditions and on transparent substrates. This technique is instrumental for characterization of all substrate coating in the lab. Unprecedented imaging and quantification of molecular tracks in the wake of bacteria M. Xanthus were published in PNAS (2012).