The chitinase family: Does sibling rivalry shape lung homeostasis, inflammation, and response to helminth-induced damage?

Chitin is a sugar molecule found abundantly in nature and the human lung is repeatedly exposed to chitin due to inhalation of fungal spores and other debris such as insect faeces. Mammals do not make chitin but secrete extremely large amounts of 'chitinases', proteins that can bind and breakdown chitin. The genes that code for these chitinases are very ancient and they exist in all animals, presumably to fight infection with organisms, like yeast, that contain chitin. Additionally, mammals may need to break down the large volume of chitin inhaled everyday as there is evidence that chitin itself triggers inflammation in the lung. However, this role for breaking down chitin in the lung is not sufficient to explain all the different places in the body in which these proteins are found. Also, all mammals have two different chitinase proteins but also contain several more proteins, called chitinase-like proteins (CLPs) that are very similar but can no longer break down chitin. The function of these CLPs is completely unknown but they are secreted by cells of the immune system during allergic responses and infection with worms. They are also associated with some forms of cancer and seemed to be turned on in response to injury. The purpose of this application is try to understand the difference between the active chitinases and these CLPs. We know that both are expressed at very high levels in the lung during an allergic response and we are going to use a mouse model to manipulate these proteins to reveal their function. Our preliminary work suggests that the chitinases and CLPs have opposing roles in the lung and may have evolved to regulate each other. To test this hypothesis, we propose to use blockers to the chitinases and the CLPs both alone and in combination and in mice that are deficient in each of these genes. We can also transfer DNA into mice that will lead to production of these proteins in the lungs of normal mice. We can then compare the effects they have on lung tissue. This work is important because these proteins are found in a vast array of diseases of both humans and animals and we are remarkably ignorant of what they do. Importantly, our collaborators are developing specific inhibitors that can be used to block either the chitinases or the CLPs that could be used as drugs in the future. This work, therefore, has the potential to be translated into therapies in situations where these proteins may be contributing to disease.

Glycoside hydrolase family 18 (GH18) members, acidic mammalian chitinase (AMCase) and chitinase-like proteins (CLPs), are dramatically increased in Th2-driven lung pathologies, but their functions remain largely unknown. We have strong evidence for cross-regulation between GH18 members and thus aim to delineate the specific functions of AMCase/CLPs in the context of the entire family. Whilst AMCase is highly conserved across species, CLPs differ due to the rapid evolutionary divergence of the CLP family in mammals. By investigating the dynamics of AMCase-CLP family interactions, we can shed light on both the evolutionary and proximal purpose of GH18 proteins, with relevance to human disease.
For this study we have a range of unique tools, including a potent AMCase inhibitor, a neutralizing monoclonal antibody against 2 CLPs and mice genetically deficient in each of the family members.
Our 1st aim is to assess and compare the impact of each of the Th2-induced family members on the naïve lung by gene transfection. A microarray analysis will provide an unbiased view of the genes altered by exposure to different GH18 family members. Co-transfection, blocking reagents, and gene KO mice will highlight inter-family dynamics. In vitro studies, that assess the effects of AMCase and CLPs on cell lines, will elucidate common patterns in function between human and mouse family members, an important step toward translation.
Our 2nd and 3rd aims are to apply our blocking tools and KO mice to two distinct models of lung pathology in which the family 18 members are known to be major players. Using the OVA allergy model, with a focus on eosinophils and neutrophils, as we have evidence these are differentially regulated by AMCase and CLPs, we will assess the downstream impact of AMCase/CLP manipulation on airway inflammation. Using a nematode migration model we will investigate AMCase/CLPs alters the level of physical damage to the lung that leads to long-term structural changes.

This proposal aims to understand the relationship between the family 18 chitinase (-like) proteins, which include both active chitinolytic enzymes and related family members who have lost the enzymatic activity but whose functions remain unknown. The beneficiaries of this research will include any system in which these proteins are found. As such, the impact of this work is extra-ordinarily wide ranging. In terms of mammalian biology, these proteins have been associated with cancer, wound healing, helminth infection, allergy, development and normal immune system homeostasis (reviewed by Sutherland, Maizels & Allen in Clin Exp Allergy 2009). The finding that chitinase gene duplication and loss-of-function has occurred independently in other non-mammalian lineages including plants and invertebrates (Bussink et al. Genetics 2007) mean the findings here will also be of interest to evolutionary biologists. Indeed, in collaboration with Tom Little, we are exploring chitinase/CLP family as invertebrate innate immune genes (Decaestecker et al. 2011 Dev Comp Immunol). Finally, because we are looking at the possible co-regulation of these proteins, it is relevant for the structural biologists interested in the relationship between chitinases and CLPs.

Acidic mammalian chitinases (AMCase) has been strongly implicated in asthma (Zhu et al. Science 2004), and thus drug inhibitors are under development. Our recently published data suggests that targeting AMCase specifically may be counter productive for asthma, but chitinases and CLPs are involved in so many pathophysiological conditions, that there are likely to be many applications for these drugs. In particular, we are collaborating with academic groups at the University of Dundee and the University of Bath, who are developing highly specific inhibitors of different chitinase family members. Unlike the inhibitor allosamidin used previously in the asthma studies, these molecules can be used as drugs, and we have shown they can function effectively in vivo (Sutherland et al. Chem Biol 2011). Our studies should provide critical information on whether targeting individual members of the family 18 chitinases has potential therapeutic value, and what the potential implications of such intervention will be. Thus, the outcome of our studies should provide critical direction to our collaborators and drug companies.

Keeping a focus on the broader evolutionary implications of this gene family should help everyone who has started to study these molecules in a diverse range of projects. Understanding the relationship of these proteins to one another will help interpretation of already published data. Additionally, our specific findings on granulocyte recruitment should be relevant to any inflammatory studies. The micro-array analysis will be a valuable resource to the whole 'chitinase' and lung allergy community. Although most of the impact will initially be on basic research, the efforts by numerous drug companies to develop chitinase inhibitors mean that our findings have important implications for the drug development community.

We ourselves have not set up specific partnerships to exploit this data, but the extensive collaborative base we have established (see pathways to impact) will lead to the dissemination of our results to other groups with more 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. Additionally, Dr Sutherland will be the lead investigator and communicator on this project and has direct experience working with the pharmaceutical industry as a part time researcher during her PhD and thus is well placed to identify when our work has relevance beyond academia.