Macrophage provenance, proliferation and plasticity in nematode infection

Macrophages are white blood cells (WBC) involved in killing microbes but also have important roles in repairing damaged tissue. Macrophages that are activated by the immune system to kill bacteria are called 'classically activated macrophages'. However, during infection with large migrating worms, macrophages take on an 'alternative' activation state. Alternatively activated macrophages (AAM) are induced by interleukin-4, an immune molecule that is induced by worm infection and other conditions such as allergy. AAM have important roles in repairing damaged tissue, but how they effect worms that live in the tissues is not known. We are particularly interested in filarial worms that cause debilitating diseases like elephantiasis and river blindness.

During infection with either microbes or worms our bodies mount an inflammatory response, in which large numbers of macrophages accumulate at the site of infection. It has always been presumed that the increase in macrophage numbers during an inflammatory response was caused by recruitment of WBC that are made in bone marrow and circulate in the blood. While investigating the killing of filarial worms in the body tissues, we made a major discovery. We found that the big increase in macrophage numbers at the site of worm infection can occur without recruitment of cells from the blood because locally resident macrophages rapidly divide within the tissues. We have shown that that the main factor leading to macrophage cell division is interleukin-4, the same factor that induces AAM. We believe this new form of inflammation may be less damaging to the tissues, and more suited to repairing damage. However, we don't know whether it contributes to worm killing or helps worms to survive.

We were recently able to demonstrate that macrophages are important in killing worms but we don't know how. We want to investigate whether macrophages that come in from the blood or macrophages that divide locally are better at killing worms. However, our discovery that macrophages can be generated by local cell division is so new that many other fundamental questions need to be addressed as well.

So the major aims of this proposal are:

What controls macrophage division during worm infection?
Do macrophages that come in from the blood have different functions than macrophages which divide locally?
What are the consequences for worm infection of blocking macrophage division or blocking their ability to become alternatively activated?
How does cell division vs. recruitment from the blood effect the ability of macrophages to switch between alternative and classical activation states and how does this effect situations in which an individual is infected with both worms and bacteria?

The results of these studies are important for many reasons. First, we need to know how worms are killed so we can develop better treatment strategies and vaccines. Secondly, understanding more about the function of locally dividing macrophages vs. macrophages that come in from the blood, is relevant to almost all inflammatory conditions. Currently, treatment for most inflammatory diseases is targeted at preventing recruitment of macrophages, or enhancing their clearance. The contribution (good or bad) of the locally dividing population has not been investigated. Finally, many diseases such as cancer, are treated with drugs that block cell division but we don't know what effect that might have on local macrophage function. We believe the interleukin-4-driven immune response that involves AAM and local marcrophage division is an evolutionary ancient means to cope with large and damaging parasites without minimal self damage. By understanding these evolutionarily ancient pathways we can shed light on modern inflammatory diseases as well as important infectious diseases of the tropics.

Proliferation of resident macrophages in response to the Th2 cytokine, IL-4, is a newly discovered form of inflammation that can occur without the need for recruitment of monocytes from the blood. However, when a pro-inflammatory stimulus leads to blood monocyte recruitment, IL-4 will also induce proliferation of the recruited population. In both settings, IL-4 induces an alternative macrophage activation programme characterised by anti-inflammatory and tissue reparative genes. Both IL-4 and macrophages are required for the control of nematode infection but the specific factors required are not known. Our primary aim is to understand the functional consequences of alternative macrophage activation and proliferation in the context of nematode infection and to investigate functional differences between recruited and resident macrophages.

We have developed an elegant tool box to look at global vs. cell intrinsic aspects of macrophage activation/proliferation that include bone marrow chimeras to track cell origins and restrict mutations to specific cell compartments as well as macrophage transfer procedures. We will use serous cavity models (both reductionist IL-4 injection and nematode infection) for the ease with which cells can be enumerated, manipulated and assessed. The tractability of these models means that we can address key questions about the basic requirements for proliferation and alternative activation that can be applied more broadly in different tissues. For readouts, we will use our well- developed and quantitative assessments of alternative activation but additionally assess macrophage genes that are IL-4-dependent in both and human and mouse and determine whether elements that regulate cell cycle do so independently of these genes or not.

By unravelling pathways that the mammalian host has evolved to resist or tolerate nematode infection, this programme will expand our basic understanding of inflammatory processes.

First and foremost, this research will advance our understanding of immunity to filariasis, a family of diseases that currently infects over 120 million people, with about 40 million disfigured and incapacitated by the disease. Additionally, this research will impact a very wide range of diseases where IL-4 and macrophages are implicated in either control or progression. In addition to the research areas listed below, our serous cavity models will be directly relevant to peritonitis, peritoneal fibrosis and peritoneal adhesions, which are frequent complications of abdominal surgery and peritoneal dialysis. The studies with M-CSF will provide a critical link to human translation, not least because the CSF-1R axis is a key pharmaceutical target. CCR2 is also a therapeutic target and we will define its role in several models.

Beyond immunity to filariasis and peritoneal/pleural cavity inflammation our research will provide insight and potentially new leads in the following areas:

Helminth infections: Our collaborators observe proliferation of IL-4 activated Kuppfer cells in the liver of schistosome infected mice and see evidence of involvement of both resident and recruited macrophages in granuloma formation. Our findings on macrophage origins will help guide this research area. The impact of GI nematode infection on the peritoneal cavity is relatively unexplored area to which we will directly contribute.

Co-infection: Co-infection with helminths and microbial pathogens is the norm in developing countries and macrophages are the host cell for a wide range of important pathogens including TB, Leishmania, Toxoplasma, Salmonella and many more. Understanding the true in vivo mechanisms of macrophage plasticity and the consequences for secondary infection are relevant to all of these diseases.

Wound Healing, Fibrosis, and Liver disease: IL-13 activated macrophages have been directly implicated in liver fibrosis. Further, increasing evidence suggests macrophagse are central mediators of acute liver injury in which macrophage proliferation is observed. IL-4Ra macrophage activation is also part of the normal wound healing process, and we are pursuing this area of research through collaboration.

Asthma: Macrophages in the lung have a very high level of steady state proliferation as well as alternative activation. We find that macrophages in the lung increase their proliferation when IL-4 is administered i.p. As well as the strong links with IL-4 and IL-13, CSF1R gene polymorphisms are linked to asthma. Thus our studies on the relationship between IL-4 and M-CSF will have relevance to allergic lung disease and asthma.

Cardiovascular and Metabolic Diseases: There is increasing evidence that IL-4 activated macrophages are beneficial in regulating host metabolism. We have collaborations to further develop the connection between IL-4 inducing helminth infections and host metabolism.

Cancer: A heterogenous population of myeloid cells support the tumor microenvironment and a number of studies have implicated alternative activation of macrophages as a major contributing factor to the immunosuppressive, and tumor promoting environment. It is particularly relevant to understand macrophage proliferation as many chemotherapeutic approaches involve blocking cell division.

Alzhiemer's research - Recent studies indicate that microglial proliferation may play a role in limiting plaque formation during Alzheimer's and we observe microglial proliferation in the brain following IL-4C injection.

In addition to partnerships discussed in the pathways to impact, our extensive collaborative base will lead to the dissemination of our results to other groups with direct translational impact. We believe our current practice of frequently speaking at international conferences, and publication in many open access and/or high-profile journals ensures that the data reaches its target audience.