Design and validation of a human brain endothelial microvessel-on-a-chip open microfluidic model enabling advanced optical imaging

We describe here the design and implementation of an in vitro microvascular open model system using human brain microvascular endothelial cells. The design has several advantages over other traditional closed microfluidic platforms: (1) it enables controlled unidirectional flow of media at physiological rates to support vascular function, (2) it allows for very small volumes which makes the device ideal for studies involving biotherapeutics, (3) it is amenable for multiple high resolution imaging modalities such as transmission electron microscopy (TEM), 3D live fluorescence imaging using traditional spinning disk confocal microscopy, and advanced lattice light sheet microscopy (LLSM). Importantly, we miniaturized the design, so it can fit within the physical constraints of LLSM, with the objective to study physiology in live cells at subcellular level. We validated barrier function of our brain microvessel-on-a-chip by measuring permeability of fluorescent dextran and a human monoclonal antibody. One potential application is to investigate mechanisms of transcytosis across the brain microvessel-like barrier of fluorescently-tagged biologics, viruses or nanoparticles

The Role of c-di-AMP in Listeria monocytogenes

L. monocytogenes is a Gram-positive intracellular pathogen that can cause serious disease, like septicemia and meningitis. It is detected in the cytosol by the innate immune system by sensing pathogen ligands. c-di-AMP has recently been shown to be one of them, inducing an interferon response. L. monocytogenes produces this molecule with the DacA protein and secretes it in the host cell cytosol upon infection. A dacA mutant shows a growth defect indicating an important role for c-di-AMP. To get insights into the role of c-di-AMP this study proposes a forward genetic screen on a dacA mutant to find transposon insertions rescuing growth. The screening of 10 thousands mutants led us to find two insertions. A transposon insertion in a gene involved in DNA metabolism partially rescues growth in broth and intracellular culture. A second transposon insertion in a gene involved in energy metabolism was found to partially rescue intracellular growth only

Quantitative Single-Cell Analysis of Host-Pathogen Interactions

During infection, microbial pathogens encounter phagocytic cells of the host innate immune system, such as macrophages and neutrophils. These encounters typically lead to uptake and killing of the bacteria by the host cell or, conversely, parasitization of the host cell by the bacteria. Microscopic analysis of these host-microbe interactions is complicated by the fact that phagocytic cells are motile and tend to stray out of the field of view. Most host-pathogen studies have therefore been undertaken at the population level. However, the success of many pathogens likely lies in their ability to generate heterogeneity within the population, facilitating adaptation to every host micro-environment and to environmental perturbations (such as application of an antibiotic). We believe that studying host-pathogen interactions at the single-cell level can reveal not only new mechanisms of pathogenesis but also why current treatments are not always successful. This thesis presents a novel microfluidic device for the long-term co-culturing of phagocytic cells and bacteria within spatially confining microchambers. Each device is patterned with thousands of microchambers, permitting high-throughput fluorescence timelapse microscopy of host-microbe interactions at single-cell resolution. We demonstrate the utility of this approach by co-culturing an established host-cell model, Dictyostelium discoideum, with the extracellular pathogen Klebsiella pneumoniae. We observed that K. pneumoniae is readily taken up and killed by D. discoideum but some events can take ten times as long as others. The capsule, a polysaccharide layer present at the surface at the bacterial cell wall, reduces the phagocytosis efficiency by 1.5 fold, but does not influence the killing time. The opportunistic human pathogen Mycobacterium marinum was then used to study intracellular bacterial virulence. As expected, bacteria could infect host cells, replicate inside, and eventually lyse them. However, surprisingly, in the majority of the infections, the bacteria were actually released from the host, with no apparent harm to either the host or the bacteria. Less frequently, bacteria were even seen being killed. The timing of these events was highly heterogeneous, ranging from a couple of hours to several days. In summary, the tools presented in this thesis provide a new and simple approach for following individual host-microbe interactions at high spatiotemporal resolution from the onset to the end of infections. This work unveiled that the timing and outcome of host-microbe interactions are unexpectedly heterogeneous, which may have important consequences for the bacterial pathogenesis and ultimately for the development of future treatment of microbial infections

The developmental cycle of Dictyostelium discoideum ensures curing of a mycobacterial infection at both cell-autonomous level and by collaborative exclusion

During its life cycle, the social amoeba alternates between a predatory amoeba and a facultative multicellular form. The single-celled amoeba is a well-established model system to study cell-autonomous mechanisms of phagocytosis and defence against intracellular bacterial pathogens, whereas the multicellular forms are arising as models to study the emergence of innate immune defence strategies. Importantly, during evolution, prokaryotes have also evolved their own strategies to resist predation. Considering these complex ecological relationships, we wondered whether cells infected with intracellular pathogenic mycobacteria would be able to undergo their developmental cycle and what would be the fate of the infection. We show that the combination of cell-autonomous mechanisms and the organisation into a multicellular organism leads to the efficient multistep-curing of a mycobacteria-infected population, thereby ensuring germ-free spores and progeny. Specifically, using a microfluidic device to trap single infected cells, we revealed that in the first curing phase, individual cells rely on three mechanisms to release intracellular bacteria: exocytic release, ejection and lytic release. The second phase occurs at the collective level, when remaining infected cells are excluded from the forming cell aggregates

A microfluidic cell-trapping device for single-cell tracking of host-microbe interactions

The impact of cellular individuality on host-microbe interactions is increasingly appreciated but studying the temporal dynamics of single-cell behavior in this context remains technically challenging. Here we present a microfluidic platform, InfectChip, to trap motile infected cells for high-resolution time-lapse microscopy. This approach allows the direct visualization of all stages of infection, from bacterial uptake to death of the bacterium or host cell, over extended periods of time. We demonstrate the utility of this approach by co-culturing an established host-cell model, Dictyostelium discoideum, with the extracellular pathogen Klebsiella pneumoniae or the intracellular pathogen Mycobacterium marinum. We show that the outcome of such infections is surprisingly heterogeneous, ranging from abortive infection to death of the bacterium or host cell. InfectChip thus provides a simple method to dissect the time-course of host-microbe interactions at the single-cell level, yielding new insights that could not be gleaned from conventional population-based measurements

High-throughput single-cell activity-based screening and sequencing of antibodies using droplet microfluidics

International audienceMining the antibody repertoire of plasma cells and plasmablasts could enable the discovery of useful antibodies for therapeutic or research purposes1. We present a method for high-throughput, single-cell screening of IgG-secreting primary cells to characterize antibody binding to soluble and membrane-bound antigens. CelliGO is a droplet microfluidics system that combines high-throughput screening for IgG activity, using fluorescence-based in-droplet single-cell bioassays2, with sequencing of paired antibody V genes, using in-droplet single-cell barcoded reverse transcription. We analyzed IgG repertoire diversity, clonal expansion and somatic hypermutation in cells from mice immunized with a vaccine target, a multifunctional enzyme or a membrane-bound cancer target. Immunization with these antigens yielded 100–1,000 IgG sequences per mouse. We generated 77 recombinant antibodies from the identified sequences and found that 93% recognized the soluble antigen and 14% the membrane antigen. The platform also allowed recovery of ~450–900 IgG sequences from ~2,200 IgG-secreting activated human memory B cells, activated ex vivo, demonstrating its versatility