Educating Macrophages in vivo

Macrophages are white blood cells which can exist in many different activation states, some of which drive tissue damage and some of which promote repair. The balance of macrophage subpopulations that exists in vivo is therefore central to controlling long term damaging responses and, as such, getting the balance right can prevent disease. Our understanding of the macrophage has grown over the last decade and it is now recognised that macrophages exist in different activation states which have different functions: the so called "M1" cell drives tissue damage whilst "M2" cells are thought to be involved in healing. Much of our understanding, and the definition of M1 and M2, however, derives from refined in vitro cell cultures and it is not known how well these ideas translate to a complex in vivo tissue environment. Thus, in vivo the macrophage may receive multiple activation signals from the tissue and may transit through different activation states. As the outcome of damage to tissues depends on the balance of M2-like cells and M1-like cells in favour of M2 it is clear that defining the driving signals in vivo is paramount. There thus remains an unmet need to enrich our understanding of the biology of the tissue macrophage in vivo. Armed with the knowledge of what important signals are, which support the M2-like cell in vivo, we can then begin developing therapies to prevent disease by enriching for this macrophage subpopulation.

We have established a mouse model of gut inflammation as a robust system for defining intestinal macrophage populations and have shown that subpopulations of macrophages, firstly M1, followed by M2-like macrophages, emerge in a robust and predictable fashion.

Using our model we ask whether removing specific cell signals in vivo, in the context of a complex tissue environment, prevents the emergence of the M2-like cell. In addition we address function of the tissue macrophages in the context of tissue repair, protection from damage and protection from infection.

Our central hypothesis is that the balance of macrophage cell types that exist during tissue damage determines the potential for the tissue to repair. This is important for health and improved disease outcomes as it is important to prevent tissues being exposed to inflammation for prolonged periods of time.

We will identify the signals driving RELMa+ Arginase-1+ Ym1+ M2-like macrophage populations in vivo in a complex tissue environment, and to address the function of the intestinal macrophage. This can only be addressed in vivo where an inflamed physiologically relevant environment can be provided. We use Trichuris muris in the mouse, as an in vivo model of resolving gut inflammation. We have established this system as a robust tool for defining intestinal macrophage populations, including the emergence of waves of M1, and then RELMa+ Arginase-1+ Ym1+ M2-like macrophages. We combine this tissue inflammation model with cell-type specific gene-targeting mouse methodology. We use the CX3CR1Cre.IL4Rflfl mouse and the CX3CR1Cre.IL10Rflfl mouse to identify whether RELMa+ Arginase-1+ Ym1+ macrophages fail to emerge if the macrophage, in an environment where multiple stimuli are present, cannot receive a signal from IL-4/IL-13 or IL-10. We use an iNOS legacy mouse to identify in vivo whether RELMa+ Arginase-1+ Ym1+ macrophages transit through an M1 activation state, and whether this is essential for their emergence. We use multi-colour flow cytometry, and immunohistochemical approaches to identify macrophage subpopulations and other leukocytes in the gut (Little et al 2014 Journal of Immunology). We will identify the in vivo function of the intestinal macrophage by analysing (1) the reparative processes in the gut post T. muris infection, (2) the response to second infection and (3) the response to an inflammatory driver (DSS), in the presence of absence of the intestinal macrophage. We believe that there is an unmet scientific need to improve our understanding of macrophage biology in vivo; that our proposal will allow testing of in vitro paradigms in in vivo settings and that understanding macrophage behaviour in vivo is an essential prerequisite for the management of tissue inflammation, preluding the design of new immune-therapies to regulate/prevent chronic inflammation

Chronic inflammation underlies a spectrum of debilitating diseases many of which occur at mucosal surfaces such as the lung and gut. In order to determine how our immune system maintains health it is essential that the underlying biological mechanisms that regulate immune responses are understood. The macrophage is widely recognised as being central in tissue homeostasis, resolving inflammation and non-resolving inflammation. This paradox is explained by the different activation states that a macrophage can exist in, and thus the macrophage's role as a driver or regulator of inflammation. Both roles are important therapeutic targets in chronic inflammation. Perhaps surprisingly therefore, the drivers of macrophage activation in the complex inflamed tissue setting are not well understood and function in vivo is often inferred rather than defined.
The aim of this project is to develop a fundamental understanding of how sub populations of tissue macrophages acquire their different activation states in the complex in vivo inflamed tissue setting, and to define their function. Our project will therefore have important impact in the medical field, given that chronic inflammation is detrimental to a healthy life. Our work will benefit a wide range of people interested in the the regulation of immune responses.
There will thus be a wide range of impacts as detailed below:

1. Basic scientists: Our data will be of interest to a wide cross-section of immunologists involved in inflammation biology and researching the underlying mechanisms that regulate immunity during health and disease. Our work will be of particular interest to macrophage biologists, addressing unanswered questions around the activating signals and function of tissue macrophages in inflammation (Time frame 1-3 years)

2. General public: Our data will be used as a basis for a range of ongoing public engagement events achieving educational benefits to the general public. This will increase awareness of how the immune system works, the importance of immune cell populations in the maintenance of health, and the ways scientists carry out basic research with a view to better understanding complex systems (Time frame 1-3 years)

3. Clinicians and clinical scientists: These data will feed in to clinical understanding of biologic therapies designed to regulate tissue inflammation (Time frame 2-3 years)

4. Drug discovery industry: This research will inform the development of second generation biologic therapies designed to manipulate macrophage balance during chronic inflammation, countering pro-inflammatory and supporting anti-inflammatory immune responses (Time frame 2-5 years)