The Fundamentals of Phagocytosis: Integrating Theoretical Models and Experiments

Bacteria, viruses and other pathogens bombard our bodies every second of every day (and night!). If we didn't respond, this would quickly lead to death. Our immune systems fight back using a whole host of sophisticated methods aimed at destroying the invaders as quickly as possible. One such method is phagocytosis, a word that derives from the Ancient Greek for "eating" and "cell". This is one of the most impressive immune defensive mechanisms and involves immune cells first chasing pathogens and then wrapping themselves around the invader in a process called engulfment. Once inside, the pathogen is then destroyed using special acidic chemicals.

Of course, the pathogen doesn't sit idly by and wait to be destroyed, which leads to an on-going battle between our bodies and foreign invaders. For example, the shape of a bacterium has a huge effect on how easily it can be removed by immune cells. Also the direction that an immune cell attacks the pathogen can make the difference between the invader being destroyed and living to fight another day. This leads to a fascinating question that has never been answered: which pathogen shapes are the easiest for the immune system to eliminate, and which are the hardest?

Answering this question is not easy. Simply looking at phagocytosis under a microscope doesn't help much. Instead, in this project, I plan to answer this question using a combination of mathematics, computing and traditional biology. On the surface this sounds a bit strange. What do mathematics and computers have to do with the immune system!? However, numerous examples have shown how combining mathematics with biology can vastly speed up scientific progress. This is because maths and computing can quickly consider questions that would be very difficult or take a long time with traditional biological techniques. Using this combination of disciplines will allow me, for the first time, to study how phagocytosis is affected by pathogen shape, pathogen size, and the direction of immune cell attack.

One of the most fascinating applications of this is to drug delivery. We normally take drugs either by swallowing a pill or by injecting something into the blood stream. The problem with this is that the drug almost instantly goes everywhere within our body, even to places where it is not required. A much better approach, which people have looked into recently, is to use very (very!) small containers that hold the drug, so called microparticle drug carriers. These drug containers (which are often even smaller than human cells and bacteria) can be injected into the body and directly targeted to where they are needed. Also, since the container takes time to break down, they can be used to slowly release drugs over a period of hours, days or even weeks.

One of the main challenges in developing microparticle drug carriers is that our own immune systems often identify them as foreign bodies and destroy them before they can be useful. What is needed is a way to design these drug carriers so that this cannot happen. And one of the most exciting new avenues for this is to choose the shape of the drug container so that the immune system cannot destroy it. This is exactly what I will do in this project: I will identify a list of shapes that the body finds hard to destroy, which will lead to better design of microparticle drugs in the future.

However, this work is not just about drug design. There are numerous medical conditions that are related to deficiencies in phagocytosis. For example, cancer is the inability of our immune system to identify and destroy our own defective cells. And lupus, which is almost not understood at all, involves our immune systems attacking healthy tissue. In this project, by understanding more about how phagocytosis works, better methods for detecting and treating such diseases will become possible.

Phagocytosis is still poorly understood, despite being first described over 100 years ago. For example, the membrane forces and signalling involved, the precise role of the actin cytoskeleton, and the effect of target shape and size are almost completely unknown. This is surprising since these issues are crucial in multiple issues of human health, including the design of microparticle drug delivery systems and various medical conditions such as Wiskott-Aldrich syndrome.

Here I plan to use a multidisciplinary, integrative approach that combines theoretical studies (mathematical modelling, physical processes and computing) with dual-micropipette experiments that allow the exact shape of the phagocytic cup to be studied with >1Hz time resolution.

In particular, I will, for the first time as far as I am aware, use membrane models (with the membrane represented by a series of points connected by springs) that include signalling (e.g. Meinhardt-like activator-inhibitor pairs) and actin (via a realistic branched network structure). These models will be informed by and compared to real time-lapse movies of phagocytosis, which will be obtained from a dual-micropipette setup. This setup allows precise control over both the immune cell (e.g. mice RAW 264.7 macrophages or neutrophils extracted from whole blood) and the target (e.g. IgG-coated polystyrene beads of various shapes and sizes).

This dual theoretical-experimental approach will allow various questions to be addressed including (i) the role of target shape and size, (ii) the effect of target orientation, (iii) the role of actin and F-actin branching, (iv) the details of the multistage nature of the engulfment rate, (v) the role of different types of signalling molecule, and (vi) the similarities and differences between phagocytosis and EPEC pedestal formation. Perhaps the most exciting outcome will be direct predictions for the optimal shape and size for microparticle drug systems.

I will consider this proposal's impact in three time periods: the short term (1 to 5 years), the medium term (5 to 10 years), and the long term (>10 years).

Short term (1 to 5 years):

(1) My own research team will most immediately benefit from the theoretical and experimental techniques they will learn, and from the group's multidisciplinary philosophy to research. Similarly, my collaborators and their own groups (both at Exeter and elsewhere) will be able to both interact and use the expertise of my own group. This will in turn lead to fresh collaborations and novel research.

(2) My visits to Prof. Volkmar Heinrich's lab at UC Davis in California and Dr Rhoda Hawkins group at Sheffield University will result in the spread of knowledge and techniques amongst those working in similar areas. Further, presentations (by all group members) at both national and international conferences will ensure that impact reaches the broader research community.

(3) As explained in my Communications plan, I will organise a two-day workshop in the third year of this project, which will impact a range of scientists, clinicians and industrial attendees. This workshop will not only focus on my own research, but will involve a range of topics involving mathematics and computing applied to immune processes such as endocytosis and E. coli infections. The aim will be to benefit a diverse group of participants, to initiate new research directions and collaborations, and to highlight the advantages of combining theoretical and experimental approaches in biomedical areas.

(4) Public engagement will lead to impact to the general public and patients. This will involve the forum I will setup with the MAGPIES (an already existent group of non-researchers in Exeter interested in biomedical research), attendance at science festivals, public talks, and visits to local schools. Further, development of my University of Exeter website (www.exeter.ac.uk/davidrichards) and mobile apps will ensure my research benefits as many members of the general public as possible.

Medium term (5 to 10 years):

(1) This project will lead to predictions for the optimal shape for microparticle drugs, which will guide future directions in the design of such drug delivery systems. To ensure benefit to pharmaceutical links, I will work with the University of Exeter's Innovation, Impact and Business (IIB) service to ensure that results are translated into real solutions, with subsequent benefits to health care, as quickly as possible.

(2) The image analysis and mathematical models that will be generated during this project will be of use to range of academics, including image analysts and computer programmers. These will include the analysis of time-lapse movies with tracking of moving beads and cells, using dual micropipettes to control multiple targets, and mathematical models of membrane dynamics.

Long term (>10 years):

(1) The multidisciplinary nature of this project means that the techniques, models and software produced (both during the first five years and in the years after) will be of long term use in various areas within the wider research community. This will include impact to scientists who work in related areas (such as endocytosis and cell migration), technology (via design of non-spherical particles and dual-micropipette time-lapse movies), and computing (via image analysis and particle tracking software).

(2) Potential patient benefits include improved detection and improved treatments for various conditions that include or are related to phagocytosis, such as Wiskott-Aldrich syndrome, systemic lupus erythematosus and E. coli infections.

(3) Long term impact in microparticle drug delivery is also expected. This includes how such systems can be designed to avoid destruction by the immune system. In turn this will also influence health sector policy, guiding the future direction of drug delivery research.