10 - October 15, 2013.docx

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University of Toronto St. George
Laboratory Medicine and Pathobiology

LMP – October 15, 2013 NOD proteins in mucosal defense against bacteria: implications for Crohn’s disease Epithelial layer line mucosal surfaces; on mucosal side, have macrophages (defense cells in general); major component of lumen of intestine is bacteria (commensals) TLR recognize bacterial and viral products, trigger signalling TLR on luminal side of epithelium? Bad because always active due to presence of commensal bacteria – same products produced by both pathogenic and commensal bacteria, so signalling will always be active Defense mechanism in cytosol – if bacteria found in cytosol, IT IS DEFINITELY PATHOGENIC Location of defense mechanisms is important!!! NOD-like receptors expressed in all sites of the body Recent research has identified key link between NOD receptor and inflammatory disorders of the intestine Mechnikov – father of innate immunity Receptors important for detecting microbes – first were identified in 1995, were TLRs Sensing systems of microorganisms PRMs = pattern recognition molecules Molecules of complement that detect specific microorganisms – all secreted molecules that play specific role in detection and handling of microorganisms OUTSIDE CELLS – key defense organism at level of whole organism At level of cell signalling, have TLRs, NLRs, RLRs, etc. that lead to cell signalling The NOD-like receptor family TLRs are homogenous group; domain organization is conserved VERSUS NLR, all lack transmembrane domain and have ATP/GTP binding domain whose function is unclear and flanked on C-terminus by leucine-rich repeat domain At N-terminus, depending on sub-category of NLR, have different domains, which are responsible for inducing different types of cell signalling Common motifs in domain organization – research in the last year have shown that NLRs have this very interesting ability to detect either microbial motifs and an example is peptidoglycan from bacteria – or danger signals (detection of microbes or danger associated with infection or tissue damage; ex. Host DNA or histones; some molecules released by dying cells, mitochondrial DNA, evolved to be detected by danger signals – important in case of tissue inury where have tissue damage and this dmage can be cured by different means) NOD1 and NOD2 Looking for molecules able to detect intracellular bacteria What are NLR sensing? Luciferase experiment – used for transcription factors; luciferase gene and NFkB promoter, so overexpress luciferase, how much NFkB activity detected, so luciferase dpenedent on NfkB activity Peptidoglycan from different bacteria – NOD1 could only be stimulated by some peptidoglycans, those that could stimulate were gram-positive bacteria Saw difference between types of bacteria – Knock downs can be done down to confirm the results done a decade ago Nod1 – fractionated peptidoglycan in different fractions – analyed by mass spectrometry Only some bacteria were detected by Nod1 – something in peptidoglycan specifically was detected Peptidoglycan is made up of repeating sugars, peptide links – peptides are cross-linking – chains There is heterogeneity in peptidoglycan – some chains made up of 2,3,4, 5, peptides – some cross- linked, creating heterogeneity In mass spec, see different populations – When separate the peaks, repeat the experiments – only 2 peaks activating Nod1 The two molecules that were in fraction were very similar – they had TRIPEPTIDE that were not cross- linked The whole detection system – dGlu and mesoDAP – these are absent from petidostructures of the host; no mesoDAP in mammals Really specific to bacteria It explains why gram-positive bacteria not detected Similar work on NOD2 NDP – worked right away – see difference between NOD1 and 2 NOD2 detects muramic acid; nod1 detecs muramid acids plus mesoDAP Nod 1 detects gram positive bacteria specifically In cytoplasm of cells, have NOD1/2 that were evolved to detected specific bacterial structures i.e. peptidoglycan and different peptidoglycan motifs Once NOD1/2 activated – some variability – nod 1 has cap domain that interatcts with RIp2 which also has CAL domain TLR receptor also TIR domain in cytoplasm – activation of pathway is TIR-TIR hemophilic interaction Here is CAL-CAL hemophilic interaction NOD2 and Chron’s disease Two papers – identified the first gene for Crohn’s disease Most commonly associated locus in CD CD Chronic inflammatory disorder of gastrointestinal system – can affect any part of GI tract Usually have flares of inflammation and periods of remission – there is no cure for Crohn’s disease – but if look at incidence of Crohn’s disease in the world, it is dramatically increasing – reasons unclear Hygiene hypothesis – balance between infectious disease vs inflammatory disorders NLR protein associated with disease of the intestinal mucosa? Pathogens, commensals – the intestine is not any type of mucosal surface because of the nuber of bacteria resident to intestine – there is affinity for immune system in intestine, because have 1) tons of bacteria, so must be silent to microbiota, which si already a challenge to the host; 2) pathogens also present in intestine. Major issue – silent to microbiota, which is made up of bacteria but also aggressive to pathogens that could enter by the intestine; one of the ways the immune system has challenged the problem is by location of immune system sensors – NLRs in cells, where it is supposed to be sterile Expression of functional NOD2 in eptheillial cells Probably CD has something to do with bacteria – simple idea that CD was associated with mishandling of bacteria – only recently due to knowing that NOD2 sensing peptidoglycan, that is known that CD due to mishandling of peptidoglycan NOD2 expressed macropahges – cells in sub-mucosal – not in lumen Begue et al. – when already have inflammation – cytokines released such as TNF; the epithelium changes, and one of the major effects is TLR induces NOD2 expression in epithelial cells ONLY IN INFLAMED STATE Mutations in NOD2 associated with CD – most common one is frame-shift mutation One feature is that the mutated form of NOD2 in CD cannot detect bacteria products – suggesting that there is some misdetection of bacteria in case of CD Other CD associated with NOD2 mutations All the different mutations found in NOD2 associated with CD; most common is SNP13 Took all the different forms of NOD2 and recreate in vitro all the different mutations – even the rare ones Compare the capacity of every mutant to detect peptidoglycan with the wild-type If at 100%, mutant detects as well as wild-type If at 1%, detect 99% less than wild-type Read out is looking at NF kappa B Most of the mutations lead to detrimental function How can you infer causality? What can you conclude of the link between NOD2 and CD?? Maybe NF kappa B is not the only read out; looking from the narrow angle of NF kappa B – maybe the mutants have different functions at different effectiveness Current models to explain the paradoxical link between NOD2 loss of function and CD The mutations associated with NOD2 causes decreased NFkB activity yet inflammation is increased Something is missing… NOD2 is a way to try to dive into those questions 1. Loss of antimicrobial activity – Over expressed NOD2 in Kappa 2 cells – less Salmonella so NOD2 is antimicrobial Overexpress the frameshift mutation – interesting observation even though mechanism was not kn
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