Invertebrate Immunology Bi5615en M 08.00-09.50h; D36-212, Masaryk University https://about.illinoisstate.edu/ntmorti/Documents/whole%20encaps.jpg http://callisto.ggsrv.com/imgsrv/FastFetch/UBER1/ZI-5AMP-2016-JAN00-IDSI-260-1 TODAY HK RECAP Toll, TLR, scavenger receptors Imd pathway, cytokines, memory Schedule, NOTE exams timing Makeup possibility by ZOOM Invertebrate Immunology Bi5615en, tentative schedule, M 08.30-10.00h; D36 212 date lecture topic 17-Feb 1 the mammalian standard, phylogeny 24-Feb 2 absence of invert lymphocytic immunity, red queen 3-Mar 3 lectins, PRRs and PAMPs 10-Mar 4 Immune cascades: clotting, PPO, complement 17-Mar 5 AMPs: discovery, mechanisms, distribution 24-Mar (-) EXAM topics 1-4 31-Mar 6 hemocytes: phagocytosis, cytotoxicity 7-Apr 7 Toll and IMD pathways 14-Apr 8 Cytokines, Diversification of immune factors/memory/novel immune mechanisms 21-Apr 9 velikonoční 28-Apr 10 EXAM 2 lectures 5-8 RECAP NLRs (NOD-like receptors) Scavenger receptors (SR) prophenoloxidase (proPO) PAMPs (LPS, PGN, glucan PRRs (peptidoglycan-binding protein (PGBP), LPS and b-1,3glucan-binding protein (LGBP) and b-1,3-glucan-binding protein (bGBP) This event triggers the activation cascade of several serine proteinase (SPs), leading to a final serine proteinase (clip-SP) designated as a proPO-activating enzyme (PPAE) Inactive proPO zymogen is converted to active phenoloxidase (PO), by PPAE to produce the quinones, which can cross-link neighboring molecules to form melanin around invading microorganisms. Amparyup et al. / Fish & Shellfish Immunology 34 (2013) 990e1001 991 In some cases hemocyanin, an oxygen-carrying pigment related to phenol-oxidase (Type 3 Copper-binding proteins) is modified by to exert phenol-oxidase activity Immune Receptors Toll, Toll-like receptors (TLRs): (in)direct recognition of PAMPs (fungus, yeast, Lys-PGN), AMPs Nod-like receptors (NLRs): intracellular (viruses, bacteria), AMPs, cytokines, apoptosis Scavenger receptors (SC): scavenge LDL, bind apoptotic cells/bacteria, phagocytosis/activation PGRP: PeptidoGlycan Recognition Protein GNBP: Gram Negative (bacteria)-Binding Protein or GlucaN-Binding Protein FRUITFLY GNBP3 BIND GLUCANS (yeast) NOT LPS (FRUITFLY GNBP2 NO IMMUNE FUNCTION) FRUITFLY GNBP1 BIND Peptidoglycan (Lys) NOT LPS Although LPS is a potent immune stimulator in vertebrates and invertebrates, it has no immune stimulating activity in systemic humoral responses in the fruit fly, Drosophila melanogaster, a good model organism for studying the basic principles of innate immunity using genetic and molecular biology techniques (Akira et al., 2006; Lemaitre and Hoffmann, 2007). In contrast to LPS, PGN of Gram-positive and Gramnegative bacteria stimulate multiple immune reactions in Drosophila. Peptidoglycan recognition proteins in Drosophila immunity (2014) Kurata, Dev Comp Immunol. 2014 Jan; 42(1): 10.1016/j.dci.2013.06.006 Remember: (In)direct PAMP detection by fat body (insect) toll receptor. Different from TLR4  LPS, GNBP: Gram Negative (bacteria)-Binding Protein or GlucaN-Binding Protein Bacterial PAMPs: peptidoglycans (PGN) Gram (+) Gram (–) lipoteichoic acids (LTA) LPS Peptidoglycans (PGN) are polymers of β-1,4-linked N-acetylglucosamine (GlcNAc) and Nacetylmuramic acid (MurNAc) cross-linked by short stem peptides, and are categorized into two major types: (a) Lys-type and (b) diaminopimelic acid (DAP)-type, based on the amino acid composition of the stem peptides and the linkage between the stem peptides. http://textbookofbacteriology.net/endotoxin.html Fruitfly: Recognition of Gram (+) and Gram (-) bacteria by PGRPs Peptidoglycan-recognition proteins Similarity to lysozymes from viruses Amidase-activity Short (catalytic) PGRPs bind and cleave PGN Signal peptide (extracellular) Long (receptor) PGRPs (lack a catalytic Cysteine, Bind, BUT NOT cleave PGN) (TM, signaling domains ) PeptidoGlycan (Lys/DAP)-rRecognition Protein(S) PGRPs (Lys Gram +, DAP Gram -) PGRP location PGN response LCa/LCx long DAP PGN monomeric IMD LCx/LCx long DAP PGN polymeric IMD LE long extracellular DAP PGN PO LCx/LCx ->IMD intracellular IMD (autophagy) LF long extracellular (regulates LCa/LCx,LCx/LCx) IMD SC1a/b/s,-LB short extracellular DAP PGN remove (catalytic) antigen regulate IMD SC1a opsonin SB1 short extracellular DAP PGN antibacterial SA, SD LYS PGN Spaetzle, Toll Gram (+) Gram (–) Within the GNBP1/PGRP-SA (-SD) complex, GNBP1 binds, processes Lys-PGN, presents Lys-PGN subunits to PGRP-SA. This complex signals immune activation until Lys-PGN is degraded and GNBP1/PGRP-SA return to inactive state Gram (+) Lys-type PGN PGRP location PGN response LCa/LCx long DAP PGN monomeric IMD LCx/LCx long DAP PGN polymeric IMD LE long extracellular DAP PGN PO LCx/LCx ->IMD intracellular IMD (autophagy) LF long extracellular (regulates LCa/LCx,LCx/LCx) IMD SC1a/b/s,-LB short extracellular DAP PGN remove (catalytic) antigen regulate IMD SC1a opsonin SB1 short extracellular DAP PGN antibacterial SA, SD LYS PGN Spaetzle, Toll Gram (+) Gram (–) PGRP location PGN response LCa/LCx long DAP PGN monomeric IMD LCx/LCx long DAP PGN polymeric IMD LE long extracellular DAP PGN PO LCx/LCx ->IMD intracellular IMD (autophagy) LF long extracellular (regulates LCa/LCx, LCx/LCx) (IMD) SC1a/b/s,-LB short extracellular DAP PGN remove (catalytic) antigen regulate IMD SC1a opsonin SB1 short extracellular DAP PGN antibacterial SA, SD LYS PGN Spaetzle, Toll Gram (+) Gram (–) PGRP location PGN response LCa/LCx long DAP PGN monomeric IMD LCx/LCx long DAP PGN polymeric IMD LE long extracellular DAP PGN PO LCx/LCx ->IMD intracellular IMD (autophagy) LF long extracellular (regulates LCa/LCx,LCx/LCx) IMD SC1a/b/s,-LB short extracellular DAP PGN remove (catalytic) antigen regulate IMD SC1a opsonin SB1 short extracellular DAP PGN antibacterial SA, SD LYS PGN Spaetzle, Toll Gram (+) Gram (–) PGRP location PGN response LCa/LCx long DAP PGN monomeric IMD LCx/LCx long DAP PGN polymeric IMD LE long extracellular DAP PGN PO LCx/LCx ->IMD intracellular IMD (autophagy) LF long extracellular (regulates LCa/LCx and LCx/LCx) SC1a/b/s,-LB short extracellular DAP PGN remove (catalytic) antigen regulate IMD SC1a opsonin SB1 short extracellular DAP PGN antibacterial SA, SD LYS PGN Spaetzle, Toll Gram (+) Gram (–) Regulatory PGRP Down regulation of response Continued response only to pathogenic bacteria, not commensals REMEMBER SHORT PGRPs HAVE BINDING AND ENZYMATIC ACTIVITIES PGRP location PGN response LCa/LCx long DAP PGN monomeric IMD LCx/LCx long DAP PGN polymeric IMD LE long extracellular DAP PGN PO LCx/LCx ->IMD intracellular IMD autophagy LF long extracellular (regulates LCa/LCx and LCx/LCx) SC1a/b/s,-LB short extracellular DAP PGN remove (catalytic) antigen regulate IMD SC1a opsonin SB1 short extracellular DAP PGN antibacterial SA, SD LYS PGN Spaetzle, Toll Gram (+) Gram (–) PGRP function in vertebrates CYTOKINES Oxford dictionary cy·to·kine /ˈsīdəˌkīn/ plural noun: cytokines any of a number of substances, such as interferon, interleukin, and growth factors, which are secreted by certain cells of the immune system and have an effect on other cells. Greek: cyto, "κύτος" kytos "cavity, cell" + kines, "κίνησις" kinēsis "movement". broad category of small proteins (~5–20 kDa: peptides) involved in cell signaling. Cytokines cannot cross the cell membranes, act through receptors as immunomodulating agents cytokines [lower and fluctuations] ≠ hormones. IMMUNOLOGY DEFINITION https://www.thermofisher.com/us/en/home/life-science/cell-analysis/cell-analysis-learning-center/immunology-at- work/proinflammatory-cytokines-overview/_jcr_content/MainParsys/image_4cbd/backgroundimg.img.png/1601907484131.png CYTOKINE COMPLEXITY https://www.thermofisher.com/us/en/home/life-science/cell-analysis/cell-analysis-learning-center/immunology-at- work/proinflammatory-cytokines-overview/_jcr_content/MainParsys/image_4cbd/backgroundimg.img.png/1601907484131.png CYTOKINE COMPLEXITY CYTOKINES OF OTHER ANIMALS Sequence is not (always) reliable identifier, complication for finding cytokines in other animal groups Not characterized at molecular level HETEROLOGOUS REAGENTS, ANTIBODIES AND (RECOMBINANT) CYTOKINES Antibodies against mammalian cytokines: Exposure to mammalian cytokines: Figs 20, 21 Immunocyte cell shape changes in M. galloprovincialis after incubation with IL-8: actin microfilament modifications (21). Control (20). Bar = 10 mm (Modified from Ottaviani et al., 2000). BUT CYTOKINE ACTIVATION OR DETECTION OF NON-SELF? ANTIBODY DETECTION OF CYTOKINE STRUCTURE OR ASPECIFIC BINDING? SPECIFIC BINDING REGION STRUCTURAL FEATURES F(ab) F(c) ANTIBODIES (RAISED AGAINST HETEROLOGOUS CYTOKINES) CAN GIVE FALL POSITIVE, NON-SPECIFIC DETECTION OF CYTOKINES Coelomic fluid of common brandling earthworms Eisenia fetida (Oligochaeta, Annelida) contains numerous molecules with anti-bacterial, hemolytic and cytolytic activities. A cytolytic factor named coelomic cytolytic factor (CCF) (GenBank accession no. AF030028) acts in earthworm defense as a pattern-recognition molecule . Upon binding microbial PAMPs , CCF triggers the prophenoloxidase mechanism . Additionally, CCF has tumor necrosis factor (TNF)-like features: lyses TNF-sensitive tumor cells, CCF expression is up-regulated in macrophage-like coelomocytes upon LPS stimulation Moreover, both TNF and CCF were suggested to interact with and lyse various pathogens. CCF (like TNF), activates macrophages to release other types of cytokines. Interestingly, the functional similarity of CCF and TNF is not based on a structural homology but rather represents a convergence of function based on a similar lectin-like activity . Institute of Microbiology of the Czech Academy of Sciences MIF = Macrophage migration inhibitory factor MIF regulates proliferation and encapsulation dsMIF, shorthand for remove mRNA by RNAinterference WAIT, WHAT???! NO, BAD HYPOTHESIS INVOLVING TIME TRAVEL or COMPREHENSIVE HGT? COMPLEX NOVEL IMMUNE MECHANISMS IN INVERTEBRATES ASSUMED ABSENT, WHY? SIMPLE, NO CELLS FOR CLONAL EXPANSION NO LYMPHOID TISSUES, CELLS, GENES BUT DO THEY HAVE IT? LOOK AND STUDY RNAi: INTRACELLULAR ANTIVIRUS IMMUNE DEFENSES VIRAL dsRNA PAMP Recognition, by Dicer “diced” into ~20 nt siRNA (short interfering RNA, double stranded) One strand (guide strand) loaded in RISC (RNA-Induced Silencing Complex) siRNA binds complementary target RNA (base pairing) SPECIFIC target RNA degraded IMMUNE MEMORY IN INVERTEBRATES ASSUMED ABSENT, WHY? SIMPLE, NO CELLS FOR CLONAL EXPANSION NO LYMPHOID TISSUES, CELLS, GENES BUT DO THEY HAVE IT? LOOK AND STUDY https://www.nature.com/articles/425037a.pdf copepod Macrocyclops albidus infection rates reduced after secondary exposure to (genetic lineages) of tapeworm Schistocephalus solidus Interesting observations but mechanism? PLOS Pathogens | DOI:10.1371/journal.ppat.1005361 Pinaud et al. 2016 FASCINATING BUT MEMORY IN PRESENCE OF INFECTION? vesicles persist for weeks after virus clearance IMMUNE DIVERSIFICATION IN INVERTEBRATES ASSUMED ABSENT, WHY? SIMPLE, NO CELLS FOR CLONAL EXPANSION NO LYMPHOID TISSUES, CELLS, GENES BUT DO THEY HAVE IT? LOOK AND STUDY FREPS, SNAIL PLASMA LECTINS THAT ARE PARASITE ANTIGEN- REACTIVE SPECIFIC MOLECULAR WEIGHT RANGES PROZONE EFFECT, MONOSACCHARIDE INHIBITION, Ca-DEPENDENT nucleotide position 3300 50 100 150 200 250 300 103 90 124 92 122 94 98 12 117 120 112 118 FREP3 variant A FREPs: IgSF CC FReD SOMATIC MUTATION: POINT MUTATIONS AND GENE CONVERSION! Drosophila Dscam (Down’s Syndrome Cell Adhesion Molecule) (original discovery as neuronal wiring /axon guidance factor) 1 gene, 24 exons 4 exons are multiple choice : 12 x 48 x 2 x 33 = 19,008 alternatives IgSF, Fibronectin Dscam with alternative exons occurs throughout insects DIFFERENT MECHANISMS FOR DIVERSIFICATION Final homework question (2 points) How many variant transcripts may results from the DSCAM gene of Lepidoptera? E-mail the number of variants to coenadem@unm,edu CLONES 185/333 UNKNOWN, NOVEL SEQUENCE 185/333 (now called transformer) of different sizes Composed of different units/elements Diversification mechanism remains to be resolved (genome instability?) SUMMARY Group genes mechanism, target Vertebrates antibodies and TcR somatic gene rearrangements (DNA) Molluscs FREPs somatic mutations (DNA) Arthropods Dscam alternative splicing (RNA) Echinoderms 185/333(Transformer) genomic instability (DNA) Different mechanisms, different genes for diversification of immune recognition. ONLY SIMILARITY IS IMMUNE DIVERSITY What and where else? Diversification of immune factors TYPES OF IMMUNITY ACQUIRED IMMUNITY INNATE IMMUNITY DIVERSIFICATION OF IMMUNE FACTORS (SOME) RETENTION OF IMMUNE MEMORY Ig, TcR Sptransformer Dscam, vsRNA, exomes FREPs Darwinian adaptive immunity generates, during the lifespan of each individual, a vast repertoire of ‘random’ receptor specificities, which is then shaped by a series of selection processes. Intricate genetic mechanisms generate variability (MUTATION) in the antigen receptor genes during the development of lymphocyte precursors, and mature lymphocytes typically express a single variant of the receptor gene, which is then transmitted to the daughter cells (CLONAL EXPANSION) Finally, the antigenic specificity of a cell (what molecular patterns it can recognize and react to) affects its chances of survival and proliferation: during maturation, cells are selected (SELECTION) for a functional antigen receptor, while mature lymphocytes are induced to proliferate if their receptors ‘recognize’ non-self antigens (FITTEST). VERTEBRATES Ig, TcR Lamarckian adaptive immunity Adaptive immunity in (archae)bacteria: CRISPR/Cas incorporates short nucleotide sequences (‘spacers’) from bacteriophages or extragenomic DNA to generate complementary ‘guides’ (short RNA molecules) to target foreign DNA or RNA for degradation in a sequence-specific manner. This system generates targeting motifs in response to the invasion of foreign genetic material, using it directly as a template to synthesize and insert a new ‘spacer’ unit into the CRISPR locus of the host chromosome (MUTATION). The integrated spacers are copied as part of the host genome and transmitted as acquired immunity into the next generation(s) (RNA interference is grouped in this category because it takes targeting motifs from pathogens) (archea)bacteria: CRISPR Arthropoda: vsRNA, exomes “All” RNA interference ≈ Lamarck Shotgun immunity Several groups of invertebrates have various mechanisms that generate diversified immune factors. This diversity is generated from germlineencoded templates, using various mechanisms of alternative splicing, RNA editing, post-translational modifications and somatic mutations. BUT, there is no clonal selection and no amplification. A swarm of immune receptors or effectors is generated that does not affect a sharply defined target, but covers a whole region of the possible target range, not unlike pellets fired from a shotgun. Shotgun diversification creates unpredictability in immune targeting, impeding the evasive adaptation of pathogens. The best-studied examples involve Mollusca: FREPs, Arthropoda: Dscam Echinoderms: Sptransformer DIVERSE SYSTEMS OF ADAPTIVE IMMUNITY KEEP LOOKING, WHAT ELSE AND WHERE? Modified from Müller et al, 2018 Invertebrate Immunology BIOL 401 001, CRN 56209 ZOOM, TR 09.30-1045 https://about.illinoisstate.edu/ntmorti/Documents/whole%20encaps.jpg http://callisto.ggsrv.com/imgsrv/FastFetch/UBER1/ZI-5AMP-2016-JAN00-IDSI-260-1