Elucidating novel roles of selective autophagy in inflammation during ageing

Lead Research Organisation: University of Oxford
Department Name: Biochemistry

Abstract

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Technical Summary

Ageing is associated with the lifelong, gradual accumulation of molecular and cellular damage and this has been observed in species ranging from yeast to humans. One of the phenotypic hallmarks of ageing tissues is chronic inflammation. We found that a protein called Kenny, which participates in the control of inflammation in Drosophila, is selectively degraded by the cellular catabolic process of autophagy. Autophagy is an essential process that involves the degradation of cytoplasmic material through the lysosomal pathway. Accumulating evidence indicates that autophagic degradation declines with age and this gradual reduction of autophagy might have a causative role in the functional impairment of biological systems during ageing. Our proposal aims to elucidate how selective autophagic degradation of Kenny regulates innate immunity and inflammation during ageing.
We will 1) examine how selective autophagic degradation of Kenny controls the production of antimicrobial peptides (AMPs), 2) examine the role of Kenny in the selective degradation of invading bacteria as a xenophagy receptor , 3) examine the role of Kenny in the selective degradation of mitochondria and how this is related to inflammation and ageing, 4) elucidate the physiological significance of elevated AMPs in autophagy mutants and how this is related to ageing.
We will address our objectives using a combination of cell and molecular biology, genetics, biochemistry, and advanced imaging. Using this approach we expect to identify novel mechanisms that regulate inflammation during ageing.

Planned Impact

INTRODUCTION: One of the phenotypic hallmarks of ageing tissues is chronic inflammation. Age-related inflammation is the low-grade, systemic inflammation in the absence of any apparent infection, and is a significant risk factor for mortality in the elderly. However the molecular and cellular mechanisms of age-related inflammation remain elusive. We recently found that autophagy, a cellular degradation process, selectively degrades Kenny, the fruit fly homologue of IKKgamma/NEMO, in order to terminate the innate immune response. Our proposal aims to understand at molecular level how autophagy, a cellular catabolic process, regulates innate immunity and chronic inflammation during ageing.

BASIC SCIENCE: This is a basic science project; it addresses fundamental, yet unresolved issues that will allow us to identify the cellular and molecular pathways involved in inflammation during ageing and potential new targets for therapeutic intervention. The project is most likely to have long term impact in the biomedical and health science areas.

IMPACT ON HUMAN HEALTH, PHARMA AND BIOTECH: Our research project will elucidate the role of autophagy in chronic inflammation during ageing. This will have a major impact on understanding how treatments that regulate autophagy can modulate inflammation during ageing and may be beneficial for healthy ageing during the life course. In other words, our research proposal is likely to have a potential long-term impact on human ageing. This impact will be realised through dissemination of our research findings to industry, healthcare professionals, government and public sector bodies and charities. Dr Nezis is currently establishing relationships with industrial partners (GlaxoSmithKline and Unilever) and will continue to do so.

IMPACT ON WIDER PUBLIC: The project will also provide scope for public engagement having impact on better understanding and appreciation of basic science of chronic inflammation during ageing among the local community. The general public will benefit from the proposed project not only because of the potential therapeutic applications developed from our discoveries, but also because of our efforts to enhance public understanding of our research by engaging school students and participating in University activities directed to a general audience such as Open Days and Research Showcases.

IMPACT ON GENERATION OF SCIENTIFICALLY LITERATE WORK FORCE: One of the more immediate outcomes of the project will be the professional training of the post-doctoral researcher employed. The post-doc scientist will have an opportunity to learn and improve a wide range of techniques in genetics, molecular and cell biology as well as in vivo techniques. This will equip him/her well for a career as scientist in academia or in a private sector. The highly skilled post-doc that we produce will most certainly lead ultimately to wealth creation through the applications of this transferable skills base.

BBSRC STRATEGIC PRIORITIES: The proposed project fulfils a number of BBSRC strategic aims such as: "maintaining world-class UK Bioscience by supporting the best people and best ideas" and "providing skilled researchers needed for academic research". It also has particular relevance to BBSRC's strategic priorities in "Ageing research: lifelong health and wellbeing" and "The replacement, refinement and reduction (3Rs) in research using animals".

CONCLUSION: This study will directly and indirectly contribute to both improved health and economic wealth.
 
Description We are finding that autophagy is important for intestinal stem cell division and coordination of this division with basal-level immune activity following infection as well as following infection.
Exploitation Route Take forward potential drug targets
Sectors Healthcare,Pharmaceuticals and Medical Biotechnology
 
Title Ex vivo phagocytosis assay using Drosophila primary larval macrophages 
Description To visualize phagocytosis by primary macrophages, we devised an ex vivo system of hemocyte imaging. Briefly, we bleed Drosophila larvae on a slide to isolate macrophages, which were then kept in a humid chamber for live imaging. Bacteria were stained with DAPI and added to the macrophage preparation. The protocol is as follows: 1. Macrophage preparation. Three third instar larvae were washed in 1 mL of ddH2O followed by 50% (v/v) bleaching and vortex. The larvae were quickly washed three times with autoclaved MilliQ H2O and left swimming whilst the slides were prepared. Into previously washed metal slides (100% (v/v) Ethanol) with a 1.0 mm coverslip it was put Schneider medium supplemented with 5% Fetal Calf Serum. The larvae were bled into the medium and the macrophages were let settle for 1 h at RT in a humid chamber. 2. Preparation of the bacteria. 200 µL of an O/N culture were harvested at RT and washed with of Schneider. The cells were resuspended in 200 µL of Schneider with 0.5 µL of a DAPI solution (at 1 mg/mL in MilliQ H2O) and incubated for 5 min at RT, without shaking. The cells washed with 200 µL of Schneider and resuspended in 100 µL of Schneider. Finally, they were dilute 1/10 in Schneider in a 200 µL final volume. 3. Imaging of the macrophages with the bacteria. To image the interactions between bacteria and macrophages, 200 µL of bacterial suspension was added on macrophages cultures in a "well slide" as previously described (Parton et al, 2010). Briefly, well slides were prepared by gluing a coverslip with SYLGARD 184 Elastomer (Dow Corning) to an aluminium metal slide with a central hole of 1.5cm diameter. Approximately 200µl of medoum was added to the well, macrophages isolated into this and allowed to settle onto the coverslip in a humidifier chamber. Before mounting on the microscope, an YSI 5775 Standard Membrane (YSI Incorporated, Japan) was placed over the medium to reduce dehydration and improve imaging. This was covered with a humidifier chamber for long-term imaging. Live imaging of UAS-GFP-PGRP-LCx haemocytes was performed at 25ºC in a humid chamber for 12 h at 15 min intervals. With the same conditions, live imaging of moesin-GFP hemocytes was performed for 18h at 3min intervals. In all experiments we used a GE Healthcare DeltaVision Elite integrated imaging system and an Olympus MPLAPON-Oil immersion objective (100X 1.40 NA). For high-sensitivity imaging and to allow lower intensities of excitation light, we used the Photometrics Evolve back-thinned EMCCD camera. Time-lapse images were denoised post acquisition using an implementation of the Patch-Based Denoising algorithm (Kervann and Boulanger, 2008). Default parameters were used, except: number of iterations (=3), adaptability (=10) and patch radius (=3). 
Type Of Material Technology assay or reagent 
Year Produced 2019 
Provided To Others? Yes  
Impact The ability to screen for the genetic basis of phagocytosis in a tractable model organism using primary macrophages and any bacterial pathogen.