NUCLEIC ACID RECOGNITION BY THE INNATE IMMUNE SYSTEM

Presented by: Ankur Sharma PhD Scholar

CREDIT SEMINARON

Pattern Recognition Receptors (PRRs)

EndolysosomalToll-like receptors

CytosolicRLRs

(RIG-I, MDA5)DNA sensors

NUCLEIC ACID RECOGNIZING PRRs

Severe fungal infection in a fruit fly (color) with a disabling mutation in the signal-transduction pathway required for the synthesis of the antifungal peptide drosomycin.[Electron micrograph adapted from B. Lemaitre et al., 1996, Cell 86: 973; courtesy of J.A. Hoffman, University of Strasbourg.]

Toll-LIKE RECEPTORS (TLRs)

Toll/Interleukin-1 receptor (TIR) domainStructure of a Toll-like receptor (TLR)

• Family of transmembrane PRRs (Human – 11, Mouse – 12)

• Recognize conserved microbial features

•TLR with relevance to Nucleic Acid recognition: TLR-3 dsRNA TLR-7 and 8 ssRNA TLR-9 dsDNA

• Location – Endosome and lysosome

• Expressed in Dendritic cells, B cells macrophages

Contd……..

Kuby Immunology, 6th edition

TLRs AND THEIR LIGANDS

Kuby Immunology, 6th edition

TLR3 recognizes dsRNA derived from viruses or virus-infected cells; dsRNA binds to N- and C-terminal sites on the lateral side of the convex surface of the TLR3 ectodomain, which facilitates the formation of a homodimer via the C-terminal region. TLR3 activates the TRIF-dependent pathway to induce type I interferon and inflammatory cytokines. In pDCs, TLR7 recognizes ssRNA derived from ssRNA viruses in endolysosomes and activates NF-κB and IRF7 via MyD88 to induce inflammatory cytokines and type I interferon, respectively. In addition, autophagy is involved in delivering ssRNA to TLR7-expressing vesicles. TLR9 recognizes DNA derived from both DNA viruses and bacteria. Proteolytic cleavage of TLR9 by cellular proteases is required for downstream signal transduction. TLR9 recruits MyD88 to activate NF-κB and IRF7 in pDCs. TLR3, TLR7 and TLR9 localize mainly to the ER in the steady state and traffic to the endolysosomes, where they engage with their ligands. UNC93B1 interacts with these TLRs in the ER and is essential for this trafficking. Kawai and Akira, 2010

NUCLEIC ACID RECOGNITION BY TLRs

TLR 3First TLR implicated in viral nucleic acid sensing

Binds with dsRNA with very less sequence specificty Minimum 40bp length required for responsiveness and

affinity increased in proportion to dsRNA length (Botos et al., 2009)Also recognize synthetic RNA analogs: Poly inosine – poly cytidylic acid [poly(I:C)] (Alexopoulou et al., 2001)Recognizes the genomic DNA of Reo virus, dsRNA

intermediates of West Nile virus, encephalomyocarditis virus and certain siRNAs

LIGAND SPECIFICITY AND MICROBIAL RECOGNITION

C terminus

(a) TLR3 dimer ( green, only ectodomain shown) in complex with dsRNA (blue). (b) Two regions of the TLR3 ectodomain make contact with dsRNA. Site-directed mutagenesis has revealed an essential role for the residues His39, His60, andHis108 in the N terminus and Asn541 and His539 in the C terminus (residues indicated in magenta).

Liu et al., 2008

Structural motifs mediating interaction between TLR 3 and its cognate ligand

TLR 9Recognizes non methylated CpG motifs in DNAExpressed exclusively by plasmacytoid dendritic cells and B

cells (in human)

Krieg AM., 2006

IFN-alpha secretion requires MyD88 and TLR9 signaling in vitro and in vivo. (A) Bone marrow cells from wild-type or MyD88/ mice were cultured at 5 106 cells/ml for 18 h with either media alone, CpG (5 g/ml) or live HSV-2 (5x106 PFU/ml), UV-HSV-2 (5x106 PFU/ml), or Poly I:C (25 g/ml) and IFN- was measured by ELISA. (B) IFN- levels were measured from bone marrow cells harvested from wild-type (B6 129F2), MyD88/, TLR4/, and TLR9/ mice stimulated for 18 h with media alone, CpG (5 g/ml), or UV-HSV-2 (5x106 PFU/ml) at 5x106

cells/ml. (C) Wild-type, MyD88/, and TLR9/ mice received i.v. injections of 107 PFU of UV-HSV-2. 6 h after injection, serum was collected by cardiac puncture and measured for IFN- by ELISA. Results are representative of three independent experiments.

Lund J et al., 2003

TLR 9 MEDIATED RECOGNITION OF HSV-2 BY pDCs

Synthetic oligodeoxynucleotide used for targeted immune activation.

The two bases to the 5′ and 3′ sides of the CpG dinucleotide comprise a CpG moif (hexamer)

Mice GACGTT Human GTCGTTFactors determining the immune stimulatory activity of ODNs : No. of CpG motifs (2-4) Spacing of the CpG motifs (usually 2 intervening bases) Presence of poly G sequence ODN backboneCpG DNA triggers DNA-PK activation which in turn,

phosphorylates IkB kinase b and leads to NF-kB activation

CpG OLIGODEOXYNUCLEOTIDES

Class A ODN Phosphodiester backbone with nuclease resistant

phosphorothioate 5’ and 3’ ends and poly-G tail Class B ODN• Entire phosphorothioate backbone without poly-G tail

Krieg., 2006

TLR 7

• Specificity to Uridine rich ssRNA and imidazoquinolines

• Type I IFN production by pDC induced by GU-rich sequences (UUGU, GUUC)

• Has been implicated in recognition of RNA viruses pDCs from TLR 7 deficient mice are unable to respond to Influenza ( Heil et al., 2004) HIV-1 ( Diebold et al., 2004)

TLR7 is required for responsiveness to VSV and influenza. (A) pDCs were purified from the bone marrow of WT, TLR7/, TLR9/, or MyD88/ mice by flowcytometric cell sorting and infected with 5x 106 pfu ml VSV for 18 h. IFN alpha and IL-12 p40 levels were measured from culture supernatants by ELISA. (B) Total bone marrow cells were prepared from WT, TLR3/, TLR7/ and MyD88/ mice and cultured with media or 5x106 pfu ml influenza for 18 h. IFN alpha and IL-12 p40 levels were measured from culture supernatants by ELISA.

Lund JM et al., 2004

TLR 7 MEDIATED RECOGNITION OF VSV AND INFLUENZA VIRUS

TLR 8

Recognize viral ssRNA

AU rich sequences (AUUU, UAUC) induce TNF response

Expressed in macrophages and conventional dendritic cells

Kawai and Akira, 2010

TLR7 AND TLR9 SIGNALING IN pDCs : INDUCTION OF TYPE I IFN

RLRs

RIG-I (Retinoic acid inducible gene-I)

Recognizes dsRNA

Consist of : 2 CARD (caspase-recruiting domain)- like domain and Helicase domain (IRF3 activation) Regulatory domain (prevents constitutive expression of protein )

Recognize Poly(I:C) (~70 bp) and and uncapped 5’ triphosphate RNA

CYTOSOLIC SENSORS OF NUCLEIC ACIDS

Upper: RLR structure diagram showing positions of functional domains. Lower: 3-step model of RIG-I activation from a monomeric resting form (left) to RNA-ligand bound, dimeric form (center) and the final active form (right). The positions of the CARDs, helicase domain, and repressor domain (RD) are indicated. Ligand binding by RIG-I facilitates a conformation change that releases it from auto repression by the RD, thus driving downstream signaling of innate antiviral immunity.

Melanoma Differentiation Antigen 5 (MDA 5)

Similar domain structure to RIG-I : N- terminal CARD domain, Central DEAD box helicase/ATPase domain and C- terminal regulatory domainRecognize long poly(I:C) >2Kb

Saito T et al., 2007

RLR Accessory Proteins Mitochondrial Anti Viral Signaling (MAVS )Essential adapter that connects RIG-I/MDA 5 activation with

IRF 3 phosphorylationN- terminal CARD domain associates with RLRsC- terminal Trans membrane domain targets to outer

mitochondrial membrane to activate IRF 3

Recognition of cytoplasmic RNA and DNA. A schematic of the known pathways involved in innate immune recognition of cytosolic RNA and DNA. RIG-I and MDA5 (left) recognize different synthetic ligands. RIG-I recognizes short dsRNAs and RNAs with 5 triphosphate ends, whereas MDA5 recognizes long dsRNA. Both receptors signal through the essential adapter MAVS, which localizes to at least two distinct organelles: mitochondria and peroxisomes.When associated with mitochondria, MAVS leads to type I IFN production; when peroxisome associated, MAVS leads to induction of a distinct set of antiviral genes. Signaling via RIG-I also requires the membrane-bound protein STING. DAI and unidentified cytoplasmic receptor(s) recognize cytoplasmic DNA (middle). After recognition, these DNA sensors require STING to induce type I IFNs.

Barbalat et al.,2011

NUCLEIC ACID RECOGNITION BY CYTOSOLIC PRRs

Innate immune recognition of adenovirus by non-pDCs is TLR independent. (A to C) Splenic cDCs (A), hepatic Kupffer cells (B), and peritoneal macrophages (C) purified from WT, MyD88/, TRIF/, or TLR9/ mice were stimulated with Ad-lacZ at an MOI of 250 for 18 h at 2.5x105 cells/ml and assayed for secretion of IFN-alpha.

Zhu et al.,2007

CYTOSOLIC RECOGNITION OF ADENOVIRUS

Barbalat et al.,2011

MECHANISM OF SELF/NONSELF DISCRIMINATION BY NUCLEIC ACID RECEPTORS

PRRs recognize conserved microbial features and induce both innate and adaptive immune responses.

Non specific recognition of nucleic acids results in autoimmune

diseases.

CpG ODNs have therapeutic applications for cancer, infectious diseases and autoimmune diseases.

Mechanism of ligand binding to TLRs and DNA sensors as well as differential gene induction are yet to be elucidated.

CONCLUSIONS AND FUTURE PROSPECTIVES

Akira S, Takeda K, Kaisho T. 2001. Toll-like receptors: critical proteins linking innate and acquired immunity. Nat. Immunol. 2:675–80

Barbalat R, Ewald SE, Mouchess ML, Barton GM .2011. Nucleic Acid Recognition by the Innate Immune System. Annu. Rev. Immunol. 2011.29:185-214.

Kawai T, Akira S. 2010. The role of pattern-recognition receptors in innate immunity: update on Toll like receptors. Nat. Immunol. 11:373–84

Krieg AM. 2006. Therapeutic potential of Toll-like receptor 9 activation. Nat. Rev. Drug Discov. 5:471–84

Liu L, Botos I, Wang Y, Leonard JN, Shiloach J, et al. 2008. Structural basis of Toll-like receptor 3 signaling with double-stranded RNA. Science 320:379–81

Lund J, Sato A, Akira S, Medzhitov R, Iwasaki A. 2003. Toll-like receptor 9-mediated recognition of Herpes simplex virus-2 by plasmacytoid dendritic cells. J. Exp. Med. 198:513–20

REFRENCES

Lund JM, Alexopoulou L, Sato A, Karow M, Adams NC. 2004. Recognition of single-stranded RNA viruses by Toll-like receptor 7. Proc. Natl. Acad. Sci. USA 101:5598–603

Stetson DB, Medzhitov R. 2006. Type I interferons in host defense. Immunity 25:373–81

Zhu J, Huang X, Yang Y. 2007. Innate immune response to adenoviral vectors is mediated by both Toll-like receptor-dependent and -independent pathways. J. Virol. 81:3170–80

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