Proteomic and genomic analysis of inflammatory resolution in mouse and man

Many chronic debilitating diseases including bronchitis, psoriatic arthritis, systemic lupus erythematosus and Crohn?s disease have dysregulated inflammation as their main driving force. However, such diseases fail to resolve and become recurrent in nature. In contrast, there are other inflammatory diseases such as streptococcal-induced pneumonia, which typically resolve without causing lasting tissue injury. Therefore, it?s reasonable to suggest that some inflammatory responses are under the control of endogenous factors that signal their cessation, which may be dysregulated in others. The idea behind this research proposal comes from the simple observation that acute inflammation elicited, for example, by a finger prick or bee-sting injury has a natural progression from start (swelling, pain and redness) to finish (reduction in swelling and pain and restoration of the tissue to its prior form, collectively called resolution). However, it has remained unappreciated for many years that resolution of inflammation is under the control of factors manufactured by the injured tissue. Our thinking is that we could use these factors or develop drugs that mimic their action in order to force chronic inflammatory diseases into resolving or switching off. This approach would allow the inflammatory response to progress as normal and neutralise the injurious agent, which is the basis of any inflammatory response, but bring about resolution of the event in a timely manner with minimal tissue injury. However, unlike the multitude of signals known to drive inflammation very little is known about the pre-resolution factors that switch it off. In order to address this we wish to apply powerful state-of-the-art technology to identify the genes and proteins that are present as inflammation resolves. The idea being that the expression of such factors during resolution acts as ?stop switches? for inflammation. One of the unique aspects of this project is that of human samples derived from simple but well-understood models of self-limiting inflammation to complement the experiments that will be done in mouse. By using samples obtained from human resolving inflammation we can get a direct correlation with our animal studies and add greater strength to the overall objectives of this project and data outcome. The overall philosophy behind this body of research is to generate an information bank of soluble factors and genes involved in resolution with the intention of developing drugs based on their mode of action i.e. to help drive ongoing/chronic inflammation down a pro-resolution pathway.

Inflammation is characterised by the sequential release of pro-inflammatory mediators that cause cell accumulation and edema formation to sites of infection and injury. There are also breaking signals that simultaneously temper the severity of this early onset phase to prevent the response from becoming over exuberant and causing collateral tissue damage. In our attempts to understand the aetiology of chronic inflammatory diseases, we are beginning to look beyond this early phase of cell recruitment and realise that, in addition to the above endogenous checkpoints that regulate PMN trafficking, there are also signals that control the transition of PMNs to phagocytosing macrophages, scavenge cytokine/chemokine from the inflammatory environment as well as clear monocyte-derived macrophages to draining lymph nodes leading to resolution. Advancing this paradigm further, our group have recently identified the post-resolution influx of innate-type lymphocytes to sites of inflammation. These repopulating lymphocytes have no role in switching inflammation off (i.e. clearing apoptotic PMNs/macrophages), but are critical for controlling responses to superinfection and secondary inflammation. Additionally, we have identified phenotypically-unique resolution-phase macrophages (rM) that are functionally distinct from classically-activated M1 cells and that we suspect signal post-resolution lymphocyte repopulation and tissue homeostasis. Thus, resolution of acute inflammation is increasingly regarded as an active event where temporally-released pro-resolution factors lead to inflammation switching off and tissues returning to normal physiological function. However, while a great deal is known about the early-release signals that drive inflammation, we are still trying to understand more about the resolution-phase mediators that help switch it off and restore homeostasis. Therefore, the aim of this proposal is to carry out a detailed proteomic and genomic analysis of resolution-phase exudates and cells, respectively, obtained from mouse and human models of self-limiting inflammation. To validate the pro-resolving potential of candidate mediators identified, we will use an in-house in vitro screen based on the ability of identified factors to switch the phenotype of M1 cells to that of rM. This will be complemented with an in vivo screen that will not only define resolution agents by their ability to clear PMNs from inflammatory sites but also signal lymphocyte repopulation and tissue restitution i.e. recruitment of rM cells. With this strategy, we will generate, for the first time, a compendium of soluble factors and genes involved in resolution with the intention of developing drugs based on their mode of action i.e. to help drive ongoing/chronic inflammation down a pro-resolution pathway.