Microbial recognition and the immune response Dr. Dana Philpott

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1 Microbial Recognition and the Immune Response Dept. of Immunology University of Toronto 1 Outline of talk Introduction into innate immunity Function of TLRs and NLRs Role of NLRs in microbial detection Link between innate and adaptive immunity 2 Innate immunity Innate vs. adaptive immunity Adaptive immunity Conserved throughout evolution Evolved 500 million yrs ago? In all multicellular organisms Unique to vertebrates Organisms possess a set Infinite number of recognition number of recognition molecules molecules (antibodies) Cells are immediately active Cells require priming Has no memory Provides memory of infection 3 1

2 The discovery of the innate immune system Metchnikoff first described role of phagocytes in invertebrates (star fish) 4 How can our cells tell that something is foreign? Recognition of exogenous microbial products Recognition of endogenous danger signals Dr. Charles Janeway Dr. Polly Matzinger 5 Turns out they are both right! Innate immunity depends upon the recognition of: MAMPs (microbial-associated molecular patterns) Signatures of microbial infection - pieces of microbes DAMPs (danger-associated molecular patterns) Danger signals released from dead or dying cells 6 2

3 Overview of innate immunity Recognition of a: MAMP/DAMP (Danger signal) By a : PRM (Pattern recognition molecule) To induce: Signal transduction (Changes in gene expression) Destruction of microbes 7 What are MAMPs? Also known as PAMPs (pathogen-associated molecular patterns) Represent structural components of microorganisms unable to be modified d Include LPS, peptidoglycan, protein (flagellin) and nucleic acid 8 What are DAMPs? End or by-products of cell death or stress ATP, uric acid Xenogenous compounds Alum, silica, asbestos, amyloid fibers Common denominator of these factors: ROS, K+ efflux, lysosomal damage 9 3

4 MAMPs and DAMPs are the sensees, what are the sensors? PRM - pattern recognition molecules Soluble PRMs Collectins (e.g.: g: surfactant, mannose-binding lectin), Ficolins Cell signaling PRMS Toll-like receptors (TLR), Nod-like receptors (NLR), RIG-like helicase receptors (RLR), Dectin 10 Cell-signaling PRMs Membrane-associated TLR Cytoplasmic NLRs Membrane NLRs : Bruno Lemaitre and Jules Hoffmann: Drosophila use Toll to defend from infection with fungus Toll was first shown to be a protein involved in embryonic development Toll-deficient flies infected with Aspergillus fumigatus succumb to infection Flies use Toll to defend from fungi and also Gram + bacteria Lemaitre et al., Cell

5 Toll-like receptors - outside-in signalling Extracellular stimulation MAMP MAMP TLR3,7-9 TLR1,2,4-6 TLR1: BLP (with TLR2) TLR2: PGN, BLP, LTA TLR3: dsrna TLR4: LPS, taxol TLR5: Flagellin TLR6: BLP (With TLR2) TLR7: Anti-virals, ssrna TLR8: Anti-viral, ssrna TLR9: CpG DNA TLR10:? TLR11 (mouse): Toxoplasma NF-κB JNK Inflammation 13 Two distinct systems to detect microbialassociated molecular patterns? Intracellular Extracellular MAMP TLRs MAMP? NF-κB JNK Inflammation Clearance NF-κB JNK 14 Keeping peace with the intestine LPS Lack or low TLR expression Expression of TLR modulating proteins that interfere with signaling: MyD88s 15 5

6 Keeping peace with the intestine (2) LPS Pathogen NF-κB JNK Inflammation IL-8 16 Shigella flexneri Gram-negative bacteria Induces uptake into epithelial cells lives free in cytoplasm (actin comet tail) Causes intestinal inflammation bacteria Eukaryotic cell Philippe Sansonetti s laboratory 17 NFκB is activated in epithelial cells by invasive Shigella Non-invasive Shigella Shigella NFκB in cytoplasm NFκB in nucleus 18 6

7 Invasive S. flexneri induces NF-κB activation non invasive Shigella Invasive Shigella Electromobility shift assay: nuclear extracts P 32 -labeled DNA probe specific for NF-κB binding p50 p65 P 32 PAGE NF-κB 19 Cytoplasmic exposure of Shigella soluble factors activates NF-κB Supernatant of Shigella + FITC-dextran Medium anti p65 supernatant Immunofluorescence microscopy FITC dextran NF-κB 20 Microflora vs. pathogen Presentation of bacterial products to the intracellular compartment is sufficient to initiate a defensive Intracellular surveillance system of host defence 21 7

8 NLRs are related to plant R proteins involved in disease resistance NOD1 CARD NBS Tobacco N TIR NBS Arabidopsis RPS5 LZ NBS 22 NLR family (Nucleotide-binding domain and leucine-rich repeat) CIITA (NLRA) (CARD) AD NACHT NAD NAIP (NLRB) BIR BIR BIR NACHT NOD1 NOD2 NLRC3 NLRC4 NLRC5 CARD NACHT NAD CARD CARD NACHT NAD (CARD) NACHT NAD CARD NACHT (CARD) NACHT NAD NLRX1 X NACHT NLRP1 PYD NACHT NAD FIIND CARD NLRP2-14 PYD NACHT NAD Modified from Fritz J.H. & Philpott D.J., Nat. Immunol. 2006;7(12): NLRs NOD2 Crohn s disease Blau syndrome NOD1 Asthma / IBD? Vitiligo NLRP1 NLRP3/Pypaf1/CIAS1/ Cryopyrin: NLRP 2-14 Muckle-Wells syndrome Cold stress syndrome CINCA syndrome NLRB/NAIP/Birc1 Susceptibility to Legionella infection (mice) NLRC4 Bare lymphocyte syndrome CIITA Leucine-rich repeats NBS CARD BIR (baculoviral inhibitor of apoptosis) Pyrin DNA interaction domain 24 8

9 NLRs - detectors of MAMPs AND danger signals NOD1 NOD2 NLRP1 NLRP2-14 NLRC4 NAIP CIITA? Nalp3 Peptidoglycan MDP = Nod2 Tri-dap = Nod1 ROS K+ efflux Flagellin ATP Toxins Uric acid Asbestos 25 Nod proteins detect peptidoglycan from the bacterial cell wall Gram-positive Gram-negative From Perry et al., 2002 Microbial Life, Sinauer Associated ed. 26 Nod1 is an intracellular sensor for Gramnegative-specific peptidoglycan AU 205 nm HPLC analysis of peptidoglycan monomers from Neisseria Retention time (min) Only 2 fractions activate Nod1 Stimulation of Nod1 with each fraction NF-κB activation (fold synergy) Girardin et al., Science 2003 HPLC fraction 27 9

10 Nod1 HO O MurNAc O NH O O HN L-Ala O O NH D-Glu HO O Meso DAP HN O O NH 2 OH OH Microbial recognition and the immune Nod proteins detect peptidoglycan from the bacterial cell wall Tri-DAP Nod2 HO O MurNAc HO O NH O O MDP HN L-Ala O O NH D-Glu HO O HO MurNAc-L-Ala-D-Glu Minimal structure detected by Nod2 MurNAc-L-Ala-D-Glu-mesoDAP Minimal structure detected by Nod1 Nod1 recognizes DAP-type peptidoglycan - mostly Gram-negative Nod2 recognizes muramyl dipeptide - both types Inohara et al., J.B.C Girardin et al., J.B.C Girardin et al., Science 2003 Chamaillard et al., Nat. Immunol Proposed mechanism for Nod protein activation by a MAMP (PG) 2. Intracellular bacteria release PG PG 3. Nod1 unfolds and oligomerizes 1. Nod1 is folded in inactive state Nod1 PG 4. Nod1 oligomers provide a focal point for the recruitment of adaptors and kinases (Rip2) Rip2 29 Alerting the cell: Nod1 activates NFκB PG Nod1 IκB Rip2 IKK Nucleus NFκB Defense 30 10

11 Nod1 and Nod2 are intracellular PRMs hpept1 SLC15A4 Shigella flexneri Enteroinvasive E. coli Nod2 PG Nod1 PG H. pylori Rip2 P. aeruginosa IKK Girardin et al., EMBO Reports 2001 Kim et al., IAI 2004 Viala et al., Nat. Immunol Travassos et al., JBC 2005 Pro-inflammatory cytokines 31 Nod1 is recruited to the site of bacterial entry Shigella Actin Nod1 Shigella entry focus Thomas Kufer, University of Cologne 32 Critical role of Nod1 in infected intestinal epithelial cells WT Nod1 -/- NF-κB p65 DAPI Villin NF-κB p65 DAPI Villin Non infected Non Invasive S.flexneri Invasive S.flexneri 33 11

12 NLRs cytoplasmic sensors of MAMPs AND DAMPs Nod proteins and NLRC4 - MAMPs Nod1 and Nod2 detect fragments of peptidoglycan NLRC4 detects flagellin (therefore 2 sensors of flagellin - inside and outside through TLR5) NLRPs - DAMPs K+ and ROS trigger NLRP1 and NLRP3 34 Proposed mechanism for NLRP protein activation by a DAMP Asbestos fibre ROS ROS ROS NLRP3 1. NLRP3 is folded in inactive state 2. Frustrated phagocytosis and generation of ROS; Lysosomal leakage; K+ efflux 3. NLRP3 is triggered (?), unfolds allowing oligomerization 4.NLRP3 oligomers provide a focal point for the recruitment of adaptors and kinases ASC 35 Bacterial-induced inflammasomes Salmonella, Shigella, Legionella Francisella Shigella Salmonella Francisella Legionella NLRC4 ASC Casp-1 ASC Casp

13 Alerting the cell: the inflammasome DAMPs NLRP3 ASC Secretion of IL-1β & IL-18 from the cell Pyroptosis (in some cases) 37 How do innate immune signals translate into adaptive immune s? Detection of microbial constituents Detection of danger Appropriate adaptive (Th1 Th2 Th17) Antigen-specificity 38 Antigen + peptidoglycan Implication of Nod1 and Nod2 in adaptive immune s NLRs Mature DC Immature DC Antigen processing Cytokines/chemokines Co-stimulatory molecules Migration Th1 What kind of pathogen-specific? Naïve T cell Th2 Th

14 s / 10 6 splenocytes Cytokine producing cells No IFNγ a * 200 IL * IL-5 * Nod2 stimulation drives Th2 polarization profile * Microbial recognition and the immune IgG1 (mg/ml) OVA-specific b prime boost * No IgG2c WT Nod2-/- WT Nod2-/- WT Nod2-/- 1 WT Nod2-/- WT Nod2-/- WT Nod2-/- OVA OVA+MDP OVA+FK156 OVA OVA+MDP OVA+FK156 medium control Ovalbumin Magalhaes et al., Journal of Immunology, 2008 MHC class I peptide OVA MHC class II peptide OVA * 40 Overview: TLRS vs. Nods LPS Flagellin Profilin dsrna ssrna Unmethylated CpG DNA Peptidoglycan (Gram + bacteria)? Lipoproteins Lipoarabinomannan (Mycobacteria) LPS (Leptospira) LPS (Porphyromonas) Glycophosphatylinositol - (T. cruzi) Zymosan (Yeast) Tri-DAP MDP Alum CD D14 MD-2 TLR 4 TLR 5 TLR 11 TLR 3 TLR 8 TLR 7 TLR 9 TLR 2 TLR 1 TLR 6 TLR 2 Nod1 Nod2 Nlrp3 High IL-12p70 IFN-a High IL-12p70 IL-12p70 IFN-a IFN-a Low level IL-12p70 Some ligands induce IL-10 or IL-12p35 MCP-1 IL-1b no IL-12p70 IL-18 Th1 Th0/Th2/Treg Th2 41 What happens when TLRs and NLRs are co-activated? 42 14

15 Nod1 is critical for priming adaptive immunity Immunizations with CFA ne producing cells 0 6 splenocytes cytokin per 1 (pg/ml) T cell frequency 600 IFN-γ IL IL-17A /+ -/- OVA +/+ -/- OVA/CFA control CD4 + /CD8 + CD8 + CD4 + globulin (mg/ml) OVA-specific Immunog Antibody titers Fritz et al., Immunity, * IgG1 +/+ -/- +/+ -/- OVA IgG2c * OVA/CFA 43 Nod1 is required for priming adaptive immunity Infections with Helicobacter pylori ri load e Intra-gastric H. pylor Log CFU/g tissue Altered bacterial clearance day 7 day 30 +/+ -/- +/+ -/- H. pylori urease-specific Imm munoglobulin OD value (A 450n m) Lower antigen-specific antibody titers /+ -/- * IgG1 IgG2c Viala et al., Nature Immunology, 2004 Fritz et al., Immunity, What is the compartment that carries Nod1-specific function? Bone marrow Wild-type Nod1-deficient Adaptive immune? 45 15

16 / 10 6 splenocytes Cytokine producing cells / Wild-type hematopoietic cells cannot rescue Nod1-deficent mice for Th2 Therefore, signaling within stromal compartment is required for driving Th2 immunity by Nod proteins Fritz et al., Immunity, Nod-mediated microbial recognition regulates innate and adaptive antimicrobial immunity Summary Nod1 stimulation Injection of the Nod1 agonist Nod1 stimulation + TLRs Infection or Immunization models Nod1 Nod1 Inflammation Inflammation Nod1 expression in radioresistant, non-hematopoietic is essential DC T H1 T H17 T H2 T IL-4 H2 IL-5 Priming of T H 2 Priming of T H 1/T H 17/ T H 2 IFN-γ IL-17A IL-4 IL-17F IL-5 47 Acknowledgements Present lab members: Joao Magalhaes Leonardo Travassos Kaoru Geddes Seamus Hussey Thirumahal Selvanantham Previous lab members: Lionel Le Bourhis Jorg Fritz Thomas Kufer Girardin lab members: Leticia Carneiro Ivan Tattoli Szilvia Benko Kavi Ramjeet Stephen Rubino Fraser Soares Jooeun Lee Arthur Ling 48 16

17 Funding: