Aspergillosis Patients Support Meeting August 2011


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  • Editorial notes (Graham Atherton): Nicola worked on Chronic and allergic forms of aspergillosis, NOT invasive aspergillosis. This is partly because those are the patients groups we mainly treat here at the National Aspergillosis Centre and partly because we think that the genes that make us susceptible to invasive infection are probably quite different to those that may be making us susceptible to chronic and allergic disease.
  • Editorial notes (Graham Atherton): There are 3 cases reported of ABPA occuring in a parent and child or in siblings – it is very rare but does weakly suggest that changes in genes may be involved There are also some small studies already done that suggest some gene changes are associated with aspergillosis Consequently we looked for any evidence of changes in how genes are switched on or off, and for changes in genes (mutations) that occur in ABPA & CPA patients
  • Everything in our body is made up of lots of different proteins working together Different hair colour, eye colour depends on what genes you inherit from your parents
  • Editorial notes (Graham Atherton): This is where patients’ donations are vitally important. Nicola was able to take blood samples from all volunteers (approx. 460 in all) and in each case isolate a particular group of white blood cells – the macrophages A macrophage literally eats germs such as fungal spores that you may have just breathed in. The surface of our lungs are covered in these cells, and the same cells are also present in our blood.   In order to eat germs a macrophage has to perform quite specialized tasks so it must switch particular genes on. It is normally very successful and cleans our lungs very efficiently but what about the macrophages in the lungs of ABPA patients? Are they less efficient? If so is this our first clue as to why ABPA patients cannot clean all of the fungi out of their lungs?
  • Editorial notes (Graham Atherton): To find out Nicola performed some very sophisticated experiments were she mixed macrophages with Aspergillus spores and then looked whether dozens of genes in the macrophage were switched on or off in response to the fungus (i.e. gene expression) – replicating what happens inside our lungs. She carried this out using macrophages taken from ABPA & CPA patients and compared them with macrophages taken from uninfected people.
  • Editorial notes (Graham Atherton): Nicola also followed the expression of the same genes before adding the Aspergillus spores and while the spores grew over the next 9 hours
  • Editorial notes (Graham Atherton): 3 different stages of growth of the Aspergillus spores were followed: ungerminated spores at the beginning, then when they sent out their first growing tube (germtube) and then some time later (9hrs) when nearly all of the culture was taken up by the fungal mass of hyphae. At each time point different genes in the Macrophages may be turned on – it is a lot easier to eat a tiny spore (conidia) compared with a huge fungal thread (hyphae)!
  • Editorial notes (Graham Atherton): The isolation of Macrophages from your blood is the start point of these experiments. We purify the macrophages using special tubes that are spun very fast until the different types of cells in your blood are separated out. Your cells can be used straight away or frozen to use later.
  • Editorial notes (Graham Atherton): 84 genes were looked at. Here we see a horizontal line with columns representing genes, going upwards to indicate a gene being switched on, downwards to indicate a gene being switched off. The different colours show the levels of expression of each gene at each time it was looked at (0 to 9 hrs).
  • Editorial notes (Graham Atherton): Red = switched on. Green = switched off. This is how the macrophage genes look before Aspergillus is added to the macrophages. These macrophages were taken from ABPA patients. 2 genes appear to be on and 15 off, though 3-4 stand out in particular.
  • Editorial notes (Graham Atherton): Red = switched on. Green = switched off. This is how the macrophage genes look before Aspergillus is added to the macrophages. Macrophages taken from CCPA (now called CPA) patients
  • Editorial notes (Graham Atherton): Once Aspergillus was added to the macrophages Nicola found 2 groups of genes that are switched off in ABPA & CPA patients compared with macrophages from non-ABPA/CPA people. Some genes belong to one of the major groups of genes that fight infection and others belong to a group of genes responsible for detecting infection.
  • Editorial notes (Graham Atherton): Perhaps this is a clue or a ‘signpost’ that is pointing us towards looking at these specific groups of genes in more detail. Perhaps we have an indication here that ABPA & CCPA sufferers cannot fight off infection or cannot detect the infection as well as they should because they cannot switch on these important genes?
  • Editorial notes (Graham Atherton): ABPA: Once the genes involved are off they stay off throughout the course of growth of Aspergillus.
  • Editorial notes (Graham Atherton): CCPA: In one gene – TNF – the gene isn’t switched on at first but does come on more slowly than it should
  • Editorial notes (Graham Atherton): It isn’t possible (yet) to look at all genes for every patient so we have to try to look at those for which we have some reason to suspect that they are important in the immune response to Aspergillosis. The work Nicola had already carried out has, as we have already explained, identified several groups of genes that are switched off in ABPA & CPA patients. These are therefore a good place to start so Nicola has been looking at the DNA sequence of the genes highlighted by that research.
  • Editorial notes (Graham Atherton): Several genes do show changes in CCPA, ABPA and SAFS patients. In all cases those small changes could have one or several effects including changes in how the protein coded for by the gene does its job and changes in whether the gene is switched on or off. In several cases here we can see genes associated with CCPA and ABPA that are switched off or are on only at very low levels – so perhaps here is a clue that the small change in DNA sequence in those genes might also be causing the gene to be switched off?
  • Editorial notes (Graham Atherton):
  • Editorial notes (Graham Atherton):
  • Editorial notes (Graham Atherton): CONCLUSIONS   Nicola’s work has shown us that we can detect differences between the way genes are switched on & off in response to Aspergillus ‘infection’ in the laboratory and in the DNA sequences of genes when comparing macrophages taken from ABPA & CPA/CCPA patients with those taken from people who do not suffer from those infections. Those changes fall into groups of genes known to be involved in our immune systems and in how we detect infections. This suggests that the genes we have detected could be directly involved in fighting off infection. Given that one of the things chronic aspergillosis patients cannot do is fight off infection by Aspergillus we are encouraged that we are working along the right lines and are going about this research using the right methods to get meaningful results.   As yet all we had demonstrated is that some genes are controlled differently in chronic aspergillosis patients and some mutations are more frequent in the same patient group compared with non-infected people. This ISN’T the same as showing that these genes and mutations are responsible for ABPA & CPA as there are still several other possible explanations – for example we could as easily argue that the changes in the levels of gene expression in ABPA patients is caused BY ABPA rather than causing ABPA! An illustrative example might be that we have done the equivalent of demonstrating that your nose runs when you have a cold – we cannot conclude that a runny nose causes a cold BUT it does point us in the right direction to finding out if it does.   Consequently we cannot yet say that susceptibility to aspergillosis is an inherited trait. There have been 3 cases reported but in all cases you could just as easily state that as they will have been brought up in the same house & environment and that may have been the cause of their ABPA rather than them having similar genes. No clarifying work has been done on the cases mentioned (the work was carried out in India) so we do not know if there are common DNA changes etc.   Even if we do show at some point in the future that some gene differences can make a person susceptible to infection, it is very likely that these gene differences will be rare and usually present with other genes that compensate for them in some way so as to reduce their effect. Only on rare occasions will enough ‘susceptibility genes’ come together in an individual to make them vulnerable to ABPA or CPA, and even then they would need the right conditions to actually become infected e.g. CPA normally needs a prior infection such as TB. If this were not the case then ABPA & CPA would be much more common.  
  • Editorial notes (Graham Atherton): For the future we now have several candidate genes on which to do further research thanks to Nicola’s work and the amazing generosity of every patient who donated a sample of blood.   Graham Atherton 5/8/2011
  • Aspergillosis Patients Support Meeting August 2011

    1. 1. NICOLA SMITH NATIONAL ASPERGILLOSIS CENTRE UHSM MANCHESTER Support Meeting for Aspergillosis Patients Fungal Research Trust
    2. 2. Nicola Smith The University of Manchester, Manchester Academic Health Science Centre, University Hospital of South Manchester, UK Genetic Susceptibility to Aspergillosis
    3. 3. Introduction: Aspergillosis (1) <ul><li>Disease caused by fungus </li></ul><ul><ul><li>Usually A. fumigatus </li></ul></ul><ul><li>Causes multiple types of lung disease </li></ul><ul><ul><li>Allergic (ABPA, SAFS) </li></ul></ul><ul><ul><li>Chronic (CCPA, CNPA, CFPA) </li></ul></ul><ul><ul><li>Invasive (IPA) </li></ul></ul><ul><li>Project focuses on chronic and allergic diseases </li></ul>
    4. 4. Genetic Susceptibility in Aspergillosis <ul><li>ABPA occurs in families – S hah et al ., 1990; Shah et al ., 2008 </li></ul><ul><li>Genetic associations found in literature </li></ul><ul><li>My Project </li></ul><ul><li>Investigating possible genetic associations </li></ul><ul><li>Find genes underlying susceptibility to aspergillosis </li></ul>
    5. 5. DNA, Genes & Proteins <ul><li>Genes are made of DNA </li></ul><ul><li>Genes are the blueprint for proteins </li></ul><ul><li>Proteins are the building blocks of our bodies </li></ul>Fig: <ul><li>Genes (and proteins) vary between people </li></ul><ul><ul><li>We look different (eye, hair colour etc) </li></ul></ul><ul><li>Differences in genes can also affect susceptibility to disease </li></ul>
    6. 6. Gene Expression (1) <ul><li>When cells in the body make proteins we say that the gene is being expressed </li></ul><ul><li>Some genes are expressed all the time </li></ul><ul><li>Others are turned on and off as required </li></ul><ul><li>Gene expression can be turned on or off by various factors </li></ul>
    7. 7. Gene Expression (2) <ul><li>Example: Person gets an infection </li></ul><ul><li>Cells see germs – gene expression of anti-germ genes is turned on </li></ul><ul><li>Cells produce proteins to fight of the germ </li></ul><ul><li>Germ is killed by proteins </li></ul><ul><li>Cells no longer see germs – gene expression is turned off </li></ul><ul><li>Cells stop making anti-germ protein </li></ul>Immune cell Protein Germ
    8. 8. Gene Expression: Do the cells of aspergillosis patients respond differently to those from healthy volunteers?
    9. 9. Gene Expression and Aspergillosis (1) <ul><li>Which genes are expressed in aspergillosis? </li></ul><ul><li>Macrophages: cells in blood, fight germs </li></ul><ul><li>Aspergillus : fungus (germ) causing aspergillosis </li></ul><ul><li>Grow macrophages and Aspergillus together and look at gene expression </li></ul>Aspergillus Macrophage
    10. 10. Gene Expression and Aspergillosis (2) <ul><li>Changes in expression after exposure to fungus = genes are important in fighting fungus </li></ul><ul><li>Look at cells from healthy volunteers and patients </li></ul><ul><li>Differences in expression between the groups = may be important in disease susceptibility </li></ul><ul><li>Differences may be at before or after exposure to fungus </li></ul>
    11. 11. Timepoints for gene expression (1) Gene expression is measured at 30min, 1hr, 3hr, 6hr and 9hr – how does this relate to the fungus? Fungal growth: resting conidia (RC)  germinating conidia (GC)  germtube (GT)  hyphae (H) Counted the amount of fungus at each stage over time
    12. 12. Timepoints for gene expression (2) Gene expression is measured at 30min, 1hr, 3hr, 6hr and 9hr – how does this relate to the fungus? 3hr – mostly resting conidia 6hr – mostly germtubes 9hr – mostly hyphae
    13. 13. Method Ficoll prep PBMCs stored in liquid nitrogen Cultured for 12-15 days to differentiate to macrophages (~2x10 5 ) 24well plate – one plate per timepoint 0hr timepoint – investigate gene expression Aspergillus spores added (4x10 5 ) 30min, 1hr, 3hr, 6hr, 9hr timepoints - Investigate gene expression Macrophages and Aspergillus cultured together Heparinised Blood Macrophages PBMCs thawed and plated out (2x10 6 ) PBMCs Ficoll prepped blood Plasma Ficoll Granulocytes
    14. 14. Gene Expression Results I studied the expression of 84 genes!
    15. 15. RED: more expression in ABPA (2) GREEN: less expression in ABPA (12) Expression before Exposure: ABPA ABPA 0hr vs Asthma 0hr >2-fold higher expression in ABPA >2-fold lower expression in ABPA
    16. 16. RED: more expression in CCPA (4) GREEN: less expression in CCPA (5) CCPA 0hr vs Healthy 0hr >2-fold higher expression in CCPA >2-fold lower expression in CCPA Expression before Exposure: CCPA
    17. 17. <ul><li>Less expression in ABPA: </li></ul><ul><li>9 genes involved in IL1 pathway </li></ul><ul><li>TREM1: receptor </li></ul><ul><li>Less expression in CCPA: </li></ul><ul><li>TLR10, TLR3, TLR1, TLR8: receptors </li></ul>Expression before Exposure: ABPA and CCPA
    18. 18. <ul><li>Less expression in CCPA than controls: </li></ul><ul><li>TLR10, TLR3, TLR1, TLR8 </li></ul><ul><li>TLRs: </li></ul><ul><ul><li>are receptors on the cell surface </li></ul></ul><ul><ul><li>are important in recognising germs </li></ul></ul><ul><ul><li>tell the cell to respond to the germ (to fight it off) </li></ul></ul><ul><li>Less expression  fewer receptors  harder for cell to recognise fungus  fungus grows  get disease </li></ul>Expression before Exposure: TLRs
    19. 19. <ul><li>Less expression in ABPA than in controls: </li></ul><ul><li>9 genes involved in IL1 pathway </li></ul><ul><li>IL1 pathway: </li></ul><ul><ul><li>Proinflammatory – leads to inflammation </li></ul></ul><ul><ul><li>Inflammation is often useful in fighting off fungus </li></ul></ul><ul><ul><li>Uncontrolled inflammation can be a problem </li></ul></ul><ul><li>What happens in this pathway over time? </li></ul>Expression before Exposure: IL1 pathway
    20. 20. <ul><li>Asthma (control) - expression alters in response to fungus </li></ul><ul><li>ABPA - expression remains low </li></ul><ul><li>ABPA cells don’t respond to presence of fungus  less inflammation  less fungal death  disease </li></ul>Expression over time: IL1 pathway IL6 IL1A IL1B
    21. 21. <ul><li>TNF expression increases over time in all groups (CCPA, ABPA and controls) </li></ul><ul><li>TNF is proinflammatory – is important in fighting fungus </li></ul>Expression over time
    22. 22. Genetic Variation: Are there similar differences in the genes (DNA)?
    23. 23. Genetic Variation <ul><li>DNA is like a string of letters (A, T, G, C) </li></ul><ul><li>The letters in the string tell the body how and when to make protein </li></ul><ul><li>Small changes in the letters (SNPs) can change: </li></ul><ul><ul><li>When a protein is made </li></ul></ul><ul><ul><li>How much of a protein is made </li></ul></ul><ul><ul><li>Whether a protein works or not (or how well) </li></ul></ul>ATGGGTAATGCCATGTCAC = normal protein ATGGGTAA C GCCATGTCAC = more protein ATGG A TAATGCCATGTCAC = non-functional protein
    24. 24. Example: STAT6
    25. 25. Example: MBL <ul><li>Protein Function </li></ul><ul><li>Binds to germs, helps cells see them </li></ul><ul><li>SNPs cause: </li></ul><ul><li>Low/High expression levels </li></ul><ul><ul><li>Low expression = less protein to fight infection </li></ul></ul><ul><li>Functional/Non-functional protein </li></ul><ul><ul><li>Non-functional protein = can’t fight infection </li></ul></ul><ul><li>Associated with CPA (Vaid et al ., 2007; Crosdale et al ., 2001) </li></ul>
    26. 26. Genetic Variation and Disease <ul><li>Most SNPs have 2 possible letters (e.g. C and G) </li></ul><ul><li>Count how many patients and how many controls and each letter: </li></ul><ul><ul><li>More C in patients than in controls </li></ul></ul><ul><ul><li>Statistics prove it’s not due to chance </li></ul></ul><ul><ul><li>SNP is associated with disease (C in particular) </li></ul></ul>
    27. 27. Genetic Variation Results (1) CCPA 0hr vs Healthy 0hr >2-fold higher expression in ABPA >2-fold lower expression in CCPA <ul><li>TLR1: SNP associated with CCPA </li></ul><ul><li>TLR10: SNP associated with ABPA+CPA </li></ul><ul><li>Both show low expression in CCPA </li></ul><ul><li>Do the SNPs cause this? </li></ul>
    28. 28. Genetic Variation Results (2) <ul><li>IL6: 2 SNPs associated with ABPA </li></ul><ul><li>Differences are seen in IL6 expression by ABPA and control cells </li></ul><ul><li>Do the SNPs cause this difference? </li></ul>IL6
    29. 29. Genetic Variation Results (3) <ul><li>TREM1: 1 SNP associated with SAFS </li></ul><ul><li>Lower expression by ABPA than control cells – before and after exposure to fungus </li></ul><ul><li>Does the SNP cause this difference? </li></ul>ABPA 0hr vs Asthma 0hr >2-fold higher expression in ABPA >2-fold lower expression in ABPA
    30. 30. Genetic Variation Results (4) <ul><li>IL1RN: </li></ul><ul><li>1 SNP associated with CCPA </li></ul><ul><li>2 SNPs associated with ABPA+CPA </li></ul><ul><li>1 SNP associated with ABPA </li></ul><ul><li>Expression increases over time: must be important in response – do the SNPs affect the function? </li></ul>
    31. 31. Genetic Variation Results (5) <ul><li>Dectin-1: receptor that recognises A. fumigatus </li></ul><ul><li>2 SNPs associated with CCPA </li></ul><ul><li>1 SNP associated with ABPA+CPA </li></ul><ul><li>1 SNP associated with ABPA </li></ul><ul><li>1 SNP associated with SAFS </li></ul><ul><li>SNPs may affect the ability of the cells to recognise and respond to the fungus </li></ul><ul><li>Expression of Dectin-1 was not investigated </li></ul>
    32. 32. So what? <ul><li>We can find differences between patients and controls – but so what? </li></ul><ul><li>Differences in expression over time: tells us which genes are important in fighting the fungus (and preventing the disease) </li></ul><ul><li>Differences between patients and controls: suggests genes that may be involved in disease susceptibilty </li></ul><ul><li> Knowledge of these helps us know which proteins may be useful drug targets/therapies… </li></ul>
    33. 33. How? <ul><li>Knowledge of these helps us know which proteins may be useful drug targets/therapies… how? </li></ul><ul><li>This is a long way in the future, but… </li></ul><ul><li>If a protein is shown to be important and is reduced in a patient, it may be possible to replace this protein as a therapy </li></ul><ul><li>If a receptor is shown to be important in recognition of the fungus, it may be possible to boost the response of this receptor with drugs </li></ul>
    34. 34. Summary <ul><li>DNA  Genes  Proteins </li></ul><ul><li>Small changes in DNA (SNPs) can affect protein expression or function </li></ul><ul><li>Differences in expression can be seen between ABPA and CCPA patients and controls </li></ul><ul><li>SNPs can be found in these and other genes </li></ul><ul><li>Future work: </li></ul><ul><li>Discover the function of these SNPs </li></ul><ul><li>Use this information to further understanding aspergillosis </li></ul>
    35. 35. Acknowlegements <ul><li>Patients </li></ul><ul><li>Supervisors: </li></ul><ul><li>Dr Paul Bowyer </li></ul><ul><li>Dr Angela Simpson </li></ul><ul><li>Dr David Denning </li></ul><ul><li>Advisor: </li></ul><ul><li>John Curtin </li></ul><ul><li>Blood Processing: </li></ul><ul><li>Liz Hadley </li></ul><ul><li>Lucy Yates </li></ul><ul><li>Recruitment / clinics: </li></ul><ul><li>Dr David Denning </li></ul><ul><li>Dr Robin Gore </li></ul><ul><li>Dr Rob Niven </li></ul><ul><li>NWLC nurses </li></ul><ul><li>NAC nurses (Georgina & Debbie) </li></ul><ul><li>Marie Kirwan </li></ul><ul><li>Marian Denson </li></ul><ul><li>Funding / Resources: </li></ul><ul><li>MRC </li></ul><ul><li>NHS </li></ul>