Implantation of engineered human microvasculature to study human infectious diseases in mouse models

Summary: Animal models for studying human pathogens are crucially lacking. We describe the implantation in mice of engineered human mature microvasculature consisting of endothelial and perivascular cells embedded in collagen hydrogel that allows investigation of pathogen interactions with the endothelium, including in vivo functional studies. Using Neisseria meningitidis as a paradigm of human-restricted infection, we demonstrated the strength and opportunities associated with the use of this approach

Activation of cGAS/STING pathway upon paramyxovirus infection

International audienc

2D and 3D Human Induced Pluripotent Stem Cell-Based Models to Dissect Primary Cilium Involvement during Neocortical Development

International audiencePrimary cilia (PC) are non-motile dynamic microtubule-based organelles that protrude from the surface of most mammalian cells. They emerge from the older centriole during the G1/G0 phase of the cell cycle, while they disassemble as the cells re-enter the cell cycle at the G2/M phase boundary. They function as signal hubs, by detecting and transducing extracellular signals crucial for many cell processes. Similar to most cell types, all neocortical neural stem and progenitor cells (NSPCs) have been shown harboring a PC allowing them to sense and transduce specific signals required for the normal cerebral cortical development. Here, we provide detailed protocols to generate and characterize two-dimensional (2D) and three-dimensional (3D) cell-based models from human induced pluripotent stem cells (hIPSCs) to further dissect the involvement of PC during neocortical development. In particular, we present protocols to study the PC biogenesis and function in 2D neural rosette-derived NSPCs including the transduction of the Sonic Hedgehog (SHH) pathway. To take advantage of the three-dimensional (3D) organization of cerebral organoids, we describe a simple method for 3D imaging of in toto immunostained cerebral organoids. After optical clearing, rapid acquisition of entire organoids allows detection of both centrosomes and PC on neocortical progenitors and neurons of the whole organoid. Finally, we detail the procedure for immunostaining and clearing of thick free-floating organoid sections preserving a significant degree of 3D spatial information and allowing for the high-resolution acquisition required for the detailed qualitative and quantitative analysis of PC biogenesis and function

DOCK11 deficiency in patients with X-linked actinopathy and autoimmunity

International audienceDedicator of cytokinesis (DOCK) proteins play a central role in actin cytoskeleton regulation. This is highlighted by the DOCK2 and DOCK8 deficiencies leading to actinopathies and immune deficiencies. DOCK8 and DOCK11 activate CDC42, a RHO-GTPase involved in actin cytoskeleton dynamics, among many cellular functions. The role of DOCK11 in human immune disease has been long suspected but has never been described so far. We studied eight male patients, from seven unrelated families, with hemizygous DOCK11 missense variants leading to reduced DOCK11 expression. The patients were presenting with early-onset autoimmunity, including cytopenia, systemic lupus erythematosus, skin, and digestive manifestations. Patients’ platelets exhibited abnormal ultrastructural morphology and spreading as well as impaired CDC42 activity. In vitro activated T cells and B lymphoblastoid cell lines (B-LCL) of patients exhibited aberrant protrusions and abnormal migration speed in confined channels concomitant with altered actin polymerization during migration. A DOCK11 knock-down recapitulated these abnormal cellular phenotypes in monocytes-derived dendritic cells (MDDC) and primary activated T cells from healthy controls. Lastly, in line with the patients’ autoimmune manifestations, we also observed abnormal regulatory T cells (Tregs) phenotype with profoundly reduced FOXP3 and IKZF2 expression. Moreover, we found a reduced T cell proliferation and an impaired STAT5B phosphorylation upon IL2 stimulation of the patients’ lymphocytes. In conclusion, DOCK11 deficiency is a new X-linked immune-related actinopathy leading to impaired CDC42 activity and STAT5 activation, and associated with abnormal actin cytoskeleton remodeling as well as Tregs phenotype culminating in immune dysregulation and severe early-onset autoimmunity