Identification Of Age-associated Factors That Alter Immune Function During Tissue Regeneration In A Combined In Vivo And In Vitro Pipeline

Ageing involves an altered function of the immune system arising from changes to immune cell populations, chronic inflammation and altered cell behaviour. Immune cells are critical for effective tissue homeostasis and repair, and aberrant immune cell function in the context of ageing likely underpin a variety of age-related frailties. Specifically, a loss of muscle strength in ageing can be causally linked to ineffective resident muscle stem cell (muSC) function. These cells act to repair or regenerate muscle after injury, to maintain the neuromuscular junction and signal to the myofiber to regulate extracellular matrix composition. During regeneration muSC behaviour is regulated by interactions with immune cells, particularly macrophages. In ageing muscle there is an increased presence of immune cells coinciding with sporadic generation of new myofibres, indicating continuous activity of muSCs. How macrophage-muSC interactions change during ageing and whether these can be manipulated to enhance muscle health is a key question that forms the basis of this proposal. Macrophages secrete a number of factors that have been shown to alter resident stem cell function in the context of regeneration and disease. As altered macrophage dynamics and inflammatory profiles are associated with ageing and disease they are an attractive target cell population for designing therapeutics to promote more effective stem cell function. One major barrier to identifying relevant macrophage modifiers to enhance tissue homeostasis and repair is that it is not clear how these might act to regulate cell behaviour in vivo. Furthermore, heterogeneity between macrophages in tissue makes it difficult to know which macrophage sub-populations interact with stem cells to promote regeneration. To develop interventions that can alter macrophage function to promote healthy ageing this proposal aims to identify candidate molecules that alter muSC behaviour in vivo. A zebrafish telomerase mutant model of ageing will be used to visualise behaviour of macrophages and muSCs during tissue repair and relate cell interactions to gene function. Specifically we will test how manipulating age-associated molecules in macrophages affect muSC responses to injury in zebrafish using advanced microscopy and artificial intelligence-based classification tools. Candidate molecules will be identified from transcriptomic datasets generated from zebrafish, mouse and human to establish a core molecular signature of ageing. Combined with spatial expression profiling of zebrafish tissues using the powerful CosMx system from Nanostring this will pinpoint specific signatures of interacting macrophages and muSCs in ageing tissues. By mapping cell transcriptomes to spatial gene expression we will determine which genes are active in muSCs and mø and whether these show age-related changes. Functional evaluation of gene function in zebrafish will be performed by CRISPR knockout, small molecule manipulation and macrophage specific over-expression and cell behaviour measured by live cell imaging and immunolabelling. Genes shown to affect cell behaviour in vivo will then be tested for their effects on muSCs isolated from young and old people in vitro. Human muSCs previously isolated from a cohort of volunteers are available in a biobank and will be used to investigate the effects of manipulating candidate gene function using over-expression systems or small molecule modifiers. A co-culture system will be used to determine how manipulation of candidate molecules in macrophages affects macrophage-muSC interactions and muSC biology. This will be further explored using small molecule modifiers of candidate genes with a preference for clinically approved compounds. Outcomes will be to establish which genes are important for macrophage regulation of muSC and how these are altered by ageing. A key focus of this proposal is to identify molecules that can be targeted for improving muscle function in ageing. An important consideration for investigating gene function is to therefore select those molecules that can be manipulated using small molecule compounds or dietary supplements to change muSC function in vivo. The value of our proposed approach is that we will be able to clearly identify how macrophage cell behaviour and muSC function are affected by manipulating specific molecules with direct relevance to their interactions in vivo. This will therefore provide a fast route for testing candidate palliative drugs that can modify immune cell function in elderly people suffering from muscle weakness and promote increased strength and endurance.