The parameters of tissue-resident macrophage autonomy

Macrophages are immune cells that reside within the tissues of the body. In normal every-day life these cells play key roles in maintaining healthy tissues, for example guarding against bacteria and clearing away old and dying cells to prevent unwanted inflammation and autoimmunity, and new and unexpected functions are continually being discovered. Macrophages are also of central importance to almost every disease process, and thus are themselves key targets of therapeutic strategies. Therefore, it is important that we understand how macrophages are generated and maintained within the tissues as this will allow us to properly devise ways to intervene in situations where they are damaging or promote their numbers where they are beneficial.

Like most immune cells, it was thought that tissue macrophages were continually replenished from bone marrow-derived cells that circulate in the blood. Recently, we have begun to understand that while some macrophages come from the blood during inflammation, those that reside in normal healthy tissues survive for long periods without recruiting blood cells into their ranks. Even during disease it appears that resident cells self-regenerate in the tissues in order to remain distinct from the recruited blood population. One reason for such independence is that blood and tissue macrophages have highly distinct and possibly opposing functions, a hypothesis that is being borne out by new research. Until now the focus of most research has largely been on blood-derived macrophages, but strategies that target blood macrophages during inflammation may also result in unintended and detrimental effects on the resident macrophage population. Thus, the goals of this proposal are to establish how resident macrophages maintain their independence and determine if this independence deteriorates with age or repeated chronic inflammation, and to identify the functional changes that may occur in the tissue environment should resident macrophages be replaced by blood-derived cells.

The first experimental aim is to determine whether all tissue macrophages have the same ability to regenerate and survive long term, or whether a hierarchy exists whereby a few cells at the top, known as stem cells, are very long lived and give rise to the more numerous cells that carry out most of the important functions in the tissue but have limited regenerative capacity. Our current understanding of stem cells is that they divide relatively infrequently to prevent them acquiring DNA mutations and chromosome changes that result in cells becoming aged, exhausted or cancerous. Discovering if all tissue macrophages have an equal capacity to self-renew could lead to future research to identify those mechanisms within the cell that prevent ageing but at the same time allow regeneration. Alternatively, identification of a stem-like cell would provide a target to expand or contract the resident macrophage pool during disease.

I also plan to explore whether there is a natural limit to the extent that resident macrophages can regenerate, and if so, whether this leads to replacement by blood cells and a change in tissue physiology. This research may help to unravel the processes involved in tissue ageing, but is also pertinent to chronic disease in which repeated cycles of activation and renewal could lead to the accelerated exhaustion of these cells. Ageing and chronic disease are intimately related, with chronic inflammation leading to faster ageing, and a greater incidence of non-resolving inflammation in the elderly. By examining how the origin of tissue macrophage may change with age and chronic disease we may identify a pathway that links these two processes.

A new paradigm is emerging in macrophage biology; the autonomous tissue resident population that exists without replenishment by the hematopoietic system. The limits of this concept are not yet understood, nor have the mechanisms been properly studied that allow the persistence of these cells in the steady state and in the face of inflammation.

In this project, revolutionary tools developed to identify tissue stem cells by permitting spatial and temporal analysis of cell proliferation history and detection of clonal restriction within populations will be applied to the field of murine macrophage biology together with methods that distinguish hematopoietic and tissue-derived macrophages to establish: (1) the contribution of proliferation to steady-state maintenance of tissue macrophages, (2) whether stem-like precursors of resident cells exist and to determine their identity and regenerative capacity, (3) if stable conversion of recruited hematopoietic cells into resident macrophages occurs under physiological conditions, and whether converted cells differ functionally or faithfully reproduce the resident phenotype, and (4) whether conversion of hematopoietic cells is a consequence of exhausting the regenerative capacity of the resident population or their precursors.

These questions will be addressed in several tissues at different stages of neonatal and adult development, during chronic tissue injury, and following treatment with cytokines (IL-4 or CSF-1) that drive elevated self-renewal of resident populations in different inflammatory environments, in order to model changes that may occur with age or under conditions of chronic or recurring inflammation. In addition to fundamentally advancing our basic knowledge of macrophage biology, this work will influence the design of therapeutics that target macrophages during disease and an appraisal of whether stem cells or monocytes can be exploited to manipulate resident cells for our own benefit.

This unashamedly basic research project addresses fundamental principles in macrophage biology. The key role of macrophages in most diseases and in maintaining healthy tissue means that this work will have wide ranging impact, and although this will primarily be the advancement of knowledge affecting a broad section of the academic community (see 'Academic Beneficiaries'), the information gathered will in the long term have far reaching consequences both socially and economically.

Although difficult to predict, a number of outcomes from this project could steer future research towards development of new macrophage-based therapeutic strategies and reassessment of those currently under investigation, and will be of benefit to the pharmaceutical industry, charitable organisations that fund medical research, and ultimately improvement of human health. For example, hematopoietic macrophages have been trialled as vehicles for delivering gene therapy to diseased tissues. A barrier to their effectiveness however, is their limited-life span in the tissue, in contrast to the longevity of resident cells. A major goal of the project is to determine if hematopoietic cells can replace resident macrophages without leading to functional changes in the population, and to reveal the signals that may allow this to happen. Such findings could feasibly be used in combination with gene therapy to introduce long-term expression of transgenes into tissues. Similarly, understanding how hematopoietic cells may be switched to resident phenotype could be harnessed to dampen chronic inflammation.

Should differences be revealed between resident and hematopoietic cells however, then the identification of stem-like precursor cells of tissue macrophages could have numerous similar potential applications. Such cells could also form the basis for adoptive transfer therapies as used for hematopoietic stem cells, for example, isolation from patients prior to high dose radio or chemotherapy allowing transfer and repopulation post treatment, for allogeneic transplantation, or to replenish tissues exhausted of resident cells by chronic inflammation. Here, our aim to determine if resident cells from one tissue can repopulate another becomes important due to the greater accessibility of cells from certain tissues. The alternative, that resident macrophages proliferate randomly over the life of an organism has major implications for ageing and cancer, where accumulation of DNA mutations and telomere loss through repeated rounds of proliferation could lead to transformation or senescence and could identify mechanisms that underlie the link between chronic inflammation and cancer, and changes in tissues during aging. It is also possible that macrophages possess protective mechanisms that allow this stochastic process with limited acquisition of DNA damage and senescence, which could be identified and exploited in other cells/tissues to prolong life, for example, to prolong stem potential in hematopoietic stem cells used to deliver gene therapy.

Thus, while this project is unlikely to directly lead to new treatments, new strategies and approaches for future testing will be defined that would be beneficial to the pharmaceutical industry. Certain elements of the research may also generate exploitable intellectual property that could lead development of spin-out companies to generate commercially exploitable immunological therapies. The timeframe for improvements to human health would be expected in the decades, while increased investment in Research and Development could occur within 3-5 years.

Public understanding of science will benefit via public lectures and attendance at the Edinburgh Science festival by the applicant, in which the data produced during the project will be used to argue for the importance of basic and animal-based research. Younger members of society in particular will be targeted to motivate and recruit the next generation of scientists.