How particle shape determines phagocytic uptake: a systems-biology analysis

Phagocytosis is the fundamental cellular process by which our immune cells bind, engulf, and destroy unwanted bacteria and particles that enter our bodies. This process is complex, relying on an estimated 1000 different signalling molecules inside the cell, making it difficult to build a mathematical or computer model that includes all of these regulatory components. However, despite the signalling complexity, phagocytosis also depends on simple biophysical parameters; the process is sensitive to particle shape and stiffness. A simple analogy would be the difficulty in wrapping a soft toy compared to a book. It costs the cell different amounts of energy to bend and stretch its membrane around particles of regular shapes compared to irregular soft particles. The influence of shape on phagocytosis is clearly important for our immune response as bacteria exist in various shapes, ranging from round spheres, to rods, and even spiral forms. Particle shape is also important for efficient drug delivery, in determining whether or not drugs remain outside cells, or are taken up by cell in order to reach their targets. For instance, tuberculosis starts with an infection of cells in the lung and could be targeted efficiently by aerosolised drugs, transmitted in the form of micro-sized particles of optimal shape for phagocytosis by cells in our lungs. Recently, phagocytosis-like engulfment processes were also found in bacteria themselves, such as during the formation of spores. This points to the universal importance of cells being able to engulf particles. Despite the tremendous importance of engulfment in biology, little is known about the biophysical aspects of uptake. Recent work by us and others described some of the early events in phagocytic engulfment using simple mathematical equations to describe the forces for engulfment. However, these studies did not address the sensing of rod- or spiral-shaped bacteria, nor the stiffness of the particles. Furthermore, these models do not sufficiently include signalling and force generation in the cell interior, and hence are unable to explain the dynamics of cellular proteins and mechanosensitivity. In particular, motor proteins are important to contract the cell membrane around the particle to complete internalisation. Here, integrated into the rich research activities at Imperial College London and its interdisciplinary centres, we aim to develop a quantitative biophysical model for phagocytosic engulfment focussing on explaining the importance of particle-shape on uptake. Our proposed simulations will be more realistic and specifically include aspects of signalling and contraction by motor proteins. In order to determine if our model describes phagocytosis correctly, we will conduct high-resolution imaging experiments to see where key proteins localize in the cell, and what the cell shape and dynamics of uptake look like. Additional collaborations and contacts with industry are being initiated to ensure relevance to improving health, and the economy.

Phagocytosis allows a cell to engulf another cell (or a particle) for feeding purposes or as part of our immune response. It is a complex spatio-temporal, multi-step process, involving recognition of opsonised and unopsonised particles by cell-surface receptors, which, when ligand-bound, signal to the actin cytoskeleton and myosin motor proteins. As a consequence, remodelling of the actin cytoskeleton and contraction by motor proteins lead to zipper-like engulfment and finally enclosure of the particle by the cell membrane. Despite the signalling complexity, the engulfment process also depends on simple biophysical parameters of the cell and particle. Most importantly, engulfment depends on the shape and stiffness of the particle, important for both host-pathogen interactions and efficient drug delivery. Here, we aim to develop such a model based on biochemical reaction diffusion equations and physical conservation laws, which will be implemented and solved in 3D using a computational finite-element scheme. To address the mechanosensitivity during uptake and cup closure, the model will include the cell's porooelastic, wave-like actin cytoskeleton, its contractility by myosin motor proteins, and their regulation by Rho family GTPases. The cell membrane constitutes a moving boundary for such solutions. Model parameters will be estimated by comparison with experiments imaging uptake, using 4D fluorescence resonance confocal and scanning electron microscopy. Experiments will be used to validate the model and to test its predictions. Phagocytic cell morphology will be imaged, key myosins will be knocked down by RNAi, and the resulting phenotypes compared with simulation results. The validated model and experimental assays will then be used to determine particle shape and size for best/worse uptake. To demonstrate industrial applicability, together with collaborators we will design optimally shaped, biodegradable PLGA particles for efficient drug delivery.

(1) Who will benefit from this research? The proposed work constitutes high-impact research in systems biology, one of the strategic aims of BBSRC. More specifically, our proposal fits into the BBSRC Principal Strategic Priorities of (i) 'Build national capability in integrative mammalian biology, and drive the use of modelling and simulation to complement in vivo approaches', (ii) 'Develop model organisms and systems that provide insight into physiological processes that are key for maintaining health in humans', and (iii) 'Develop new tools in areas such as chemical biology, high-resolution structural analysis, lipidomics, proteomics, biomarkers and bioimaging, high-throughput and comparative genomics and modelling'. Our Impact Plan has been designed to ensure that the research in this proposal will impact directly or indirectly the academia sector, industry, policy makers, as well as the general public. (a) Academia: Our proposed interdisciplinary research will promote gain in experimental and theoretical techniques and assays, as well as employment for two individuals. As our research is broad and touches on many areas, it will impact researchers working in cell biology, molecular biology, host-pathogen interactions, immunology, biological physics, computational and theoretical biology. As such, our research will strengthen the systems-biology community in the United Kingdom. (b) Industry: Our proposed research on the particle-shape dependence of phagocytosis and on the design of particles for optimal uptake by cells will have potential applications in the nano-technological and pharmaceutical industry. As such we will contribute to the economy and wealth of the UK. (c) Policy makers and general public: As our research will determine key parameters which influence our innate immune response, it will ultimately affect policy makers and the wider public, promoting health and well-being in the UK. (2) How will they benefit? (a) Academia: Due to similarity in signalling pathways, our research will contribute knowledge, skills, and methodology to the research conducted in eukaryotic chemotaxis, cell migration, and cell adhesion. The proposed research will also contribute to our understanding of the innate immune system. Furthermore, we will develop new computational tools for simulating cell signalling, cell morphological changes, and mechanosensitivity. This is not only important for phagocytosis and above mentioned areas, but also for bacterial sporulation, endocytosis, viral uptake, and embryonic development. The project will afford opportunities for employment, career development and training of individuals. In addition to project-specific training, Imperial College offers a variety of transferable skills courses, which the employed researchers for this project would be encouraged to attend. After finishing the project, the researchers will be highly skilled persons, and increase creative output of the UK (see also Academic Beneficiaries section). (b) Industry: Our project will provide predictive computational and experimental assays to design particles for best/worst uptake by cells. This can be exploited for delivering drugs to specific areas or cells in our body. From our discussions with such companies, there is real need for an predictive approach, directing experiments and ultimately saving resources. (c) Policy makers and general public: In the long term, a better understanding of our phagocytic immune response, host-pathogen interactions, and development of novel interventions will be beneficial to health policy-makers in government agencies as well as the general public.