PATHOGEN RECOGNITION AND INFLAMMATOY SIGNALING IN INNATE IMMUNE RESPONSE
What is the Innate Immune Response? A universal and evolutionarily conserved mechanism of host defense against infection First line of Defense Predates the adaptive immune response Found in all multicellular organisms Adaptive only in vertebrates Uses receptors and effectors that are ancient in their lineage Must provide protection against a wide variety of pathogens Distinguishes self from non-self perfectly Defects in innate immunity are very rare and almost always lethal
PAMPs: P athogen A ssociated M olecularP atternsPRRs: P attern R ecognition R eceptors
The Innate Immune Response: Common Misconceptions The innate immune system is an evolutionary rudiment whose only function is to contain the infection until the “real” immune response can kick in. Adaptive immunity developed because of the inflexibility of the nonclonal receptors used by the innate immune response. The innate system cannot cope with the high mutational rate and heterogeneity of pathogenic organisms.
The Innate immune system instructs the adaptiveimmune response to respond to microbial infection The major decision to respond or not respond to a particular ligand is decided by the genome-encoded receptors of the innate immune system
PRRs common characteristics1.Recognize microbial components, known as pathogen associated molecular patterns (PAMPs), that are essential for the survival of the microorganism and are therefore difficult for the microorganism to alter.2.Expressed constitutively in the host and detect the pathogens regardless of their life- cycle stage.3.Germline encoded, nonclonal, expressed on all cells of a given type, and independent of immunologic memory.
Adapted from Medzhitov and Janeway PAMP Cur. Opin. Immunol. 1997 9:4-9 Phagocytosis Y PRR APCComplement Endosome Direct Bactericidal Activity MHC B7 PhagocytosisPathogen-specific Antibody Y Oxygen burst Anti-microbial peptides Naive Inflammatory T Cell and effector cytokines Activated CD40L, FasL, CD30L, CD27L T Cell B Cell
Machineries underlying innate immune recognition are highly conserved among species, from plants and fruit flies to mammals.
Recognition mechanisms of innate immunity (concepts) What mediates the recognition of PAMPs? Diverse recognition elements; 4 key families of “Pattern Recognition Receptors”: Toll-like receptors (TLRs; transmembrane receptors) RigI-like receptors (RLRs; cytoplasmic RNA helicases) NOD-like receptors (NLRs; cytoplasmic sensors) C-type lectin receptors (CLRs; transmembrane receptors) Also, recognition of molecules released from necrotic cells, tissue damage (“damage associated molecular patterns” DAMPs or “danger”; recognized by some NLRs, TLRs, CLRs) New hypothesis: recognition of perturbations induced by pathogens (“patterns of pathogenicity”) such as bacterial pore- forming toxins, perturbations of the cytoskeleton, various types of cell stress etc.)--recognition mechanisms less well understood (inflammasome, etc.)(Lectin: a protein that binds to carbohydrates)
TLRS-DISCOVERY Toll, the founding member of the TLR family, was initially identified as a gene product essential for the development of embryonic dorsoventral polarity in Drosophila. Later, it was also shown to play a critical role in the antifungal response of flies (Lemaitre et al., 1996). To date, 12 members of the TLR family have been identified in mammals.
Discovery of the mammalian Toll-like receptors (TLR):1997: Janeway and Medzhitov discovered a human proteinwith structural similarity to drosophila Toll that couldactivate immune response genes human cells (TLR4). 1998: Beutler discovered that a mouse strain with analtered response to bacterial lipopolysaccharide (called LPSor endotoxin) was due to a mutation in the TLR4 gene.There are 11 TLR family members in human and 12 inmice. Each responds to a distinct set of microbialproducts.
STRUCTURE TLRs are type I integral membrane glycoproteins characterized -extracellular domains containing varying numbers of leucine-rich-repeat (LRR) motifs a cytoplasmic signaling domain homologous to that of the interleukin 1 receptor (IL-1R), termed the Toll/IL-1R homology (TIR) domain (Bowie and O’Neill, 2000).
The LRR domains are composed of 19–25 tandem LRR motifs, each of which is 24– 29 amino acids in length, containing the motif XLXXLXLXX as well as other conserved amino acid residues. Each LRR consists of a β strand and an α helix connected by loops.
SUBFAMILIES Based on their primary sequences, subfamily of TLR1, TLR2, and TLR6- recognizes lipids, TLR7,TLR8, and TLR9 -recognize nucleic acids . However, the TLRs are unusual in that some can recognize several structurally unrelated ligands.
Expressed on Various immune cells- macrophages, dendritic cells (DCs), B cells, specific types of T cells Nonimmune cells - fibroblasts and epithelial cells. Expression is not static but rather is modulated rapidly in response to pathogens, a variety of cytokines, and environmental stresses.
TLRs may be expressed - Extracellularly on the cell surface -TLRs 1, 2, 4, 5, and 6 are Intracellularly-TLRs 3, 7, 8, and 9 almost exclusively in intracellular compartments such as endosomes, and their ligands, mainly nucleic acids, require internalization to the endosome before signaling is possible.
Different mammalian Toll-like receptors (TLRs) are specific for different classes of microbial products Insert Fig 3-11
Toll-like receptors and recognition of pathogensssRNA ViralLRR extracellular domainTIR domain inside K. Takeda & S. Akira, Cell. Microbiol. 5: 143-53, 2003
TLR1, TLR2 and TLR6 TLR2 recognizes- lipoproteins/lipopeptides from various pathogens, peptidoglycan and lipoteichoic acid from Gram-positive bacteria, lipoarabinomannan from mycobacteria, glycosylphosphatidylinositol anchors from Trypanosoma cruzi, a phenol- soluble modulin from Staphylococcus epidermis, zymosan from fungi and glycolipids from Treponema maltophilum .
Also recognises- LPS preparations from non-enterobacteria such as Leptospira interrogans, Porphyromonas gingivalis and Helicobacter pylori . TLR2 recognizes a wide range of microbial products through functional cooperation with several proteins that are either structurally related or unrelated.
TLR3 TLR3 is implicated in the recognition of dsRNA and viruses. However, TLR3-independent mechanisms of dsRNA recognition exist.
TLR4 TLR4 is an essential receptor for LPS recognition , recognition of taxol, a diterpene purified from the bark of the western yew (Taxus brevifolia) ,endogenous ligands, such as heat shock proteins (HSP60 and HSP70), the extra domain A of fibronectins, oligosaccharides of hyaluronic acid, heparan sulfate and fibrinogen.
TLR5 TLR5 recognize an evolutionarily conserved domain of flagellin. TLR5 expression is also observed in the intestinal endothelial cells of the subepithelial compartment. In addition, flagellin activates lung epithelial cells to induce inflammatory cytokine production. These findings indicate the important role of TLR5 in microbial recognition at the mucosal surface.
TLR9 Its stimulatory effect is due to the presence of unmethylated CpG dinucleotides in a particular base context designated CpG-DNA. Although the CpG motif is abundant in bacterial genomes, its frequency is suppressed and it is highly methylated in mammalian genomes. The methylated CpG motif does not activate mammalian immune cells.
Toll-like receptors (TLRs)link microbial products (PAMPs) to transcription factor activation in a signalingpathway that is conserved betweenmammals and insects
Adaptor molecules MyD88 TIR-associated protein (TIRAP)/MyD88-adaptor-like (MAL) TIR-domain-containing adaptor protein- inducing IFN-β (TRIF)/TIR-domain-containing molecule 1 (TICAM1) (Oshiumi et al., 2003; Yamamoto et al., 2002b) TRIF-related adaptor molecule (TRAM). The differential responses mediated by distinct TLR ligands can be explained in part by the selective usage of these adaptor molecules.
Toll-like receptor signaling pathways•Ligand induceddimerization of TLR-->induced assembly with TIR-domain containing adaptors•MyD88 pathway andTRIF pathway;•Activate Transcriptionfactors and MAP kinases
A more detailed look at the signaling pathway down-stream of Toll-like Receptors (TLRs)
A more detailed look at the signaling pathway down-stream of Toll-like Receptors (TLRs)
Type I IFN Production via TLRs TRIF-Dependent Pathway- Stimulation with TLR3, TLR4, TLR7, and TLR9 ligands, but not the TLR2 ligand, induces type I IFN production in addition to proinflammatory signals.
Triggering of PRRs on macrophage or dendritic cells caninduce a LARGE variety of events including: Increased phagocytosis Production of cytokines and inflammatory mediators: Interferons to induce anti-viral state Chemokines to attract migrating cells Etc, etc. Increased cell migration Changes in expression of molecules involved in T cellantigen presenting cell function.
TLR signaling within phagosomes determines fate of that phagosome (destruction vs antigen presentation).Pathogen Dying infected cell(non-self, TLR signaling) (self, no TLR signaling) Material in Material in phagosome phagosome disposed enters antigen of inside cell-no presentation pathway- presentation to T cells presentation to T cells Blander and Medzhitov 2006 Nature v440 p808
Common and Distinct Themes in TLR Signaling TLR4/4 TLR2 TLR1/6 CD14 MD-2Rac PI3K TRIF PI3K TIRAP MyD88 IRF3 MyD88 TIRAP IFN-β IRAK IRAK TRAF6 TAK1/AKT NIK Death domain MAP kinases IKK Complex TIR domain IkB p65 p50
MyD88 is used by all TLRs except TLR3; TIRAP is used by TLR2 and TLR4; TRIF is used by TLR3 and TLR4; and TRAM is used only by TLR4 .
Pathways of NF-κ B activation NF-κB is a family of transcription factors: p50, p52, p65 (Rel-A), c-Rel, Rel-B; plus inhibitors (I-κB)Canonical Non-pathway canonical Pathway (activated by some TNF receptor family members)
The NF-kB Family of Transcription Factors Eukaryotic transcription factor found in essentially all cell types First described in 1986 as a nuclear factor required for the transcription of the immunoglobulin kappa light chain in B cells. Binds to a 10-bp sequence GGGGYNNCCY Important component in the inducible expression of many proteins: cytokines, acute phase proteins, adhesion molecules The NF-kB signaling system is evolutionarily conserved Three NF-kB molecules in Drosophila dorsal controls dorsal/ventral polarity during development Regulates antifungal gene expression dif and relish: regulate expression of antifungal and antibacterial genes
NF-κB exists in the cytoplasm as an inactive heterotrimer composed of 2 Rel family proteins and an inhibitory IkB moleculeStress, infection, or cytokine IKK P P (Ub)n IkB p65 p50 26S proteosome Nuclear Translocation Activation of NF-KB Responsive genes
Luke A.J. O’Neil www.stke.org/cgi/content/full/sigtrans;2003/171/re3
Cytoplasmic Pathogen Recognition System A large family of cytoplasmic PRRs has been cloned to date. Currently, they are roughly subclassified into the NOD-LRR proteins and the CARD helicase proteins. These protein families are implicated in the recognition of bacterial and viral components, respectively.
NOD-LRR Proteins and Their Functions Proteins in this family possess LRRs that mediate ligand sensing; a nucleotide binding oligomerization domain (NOD); and a domain for the initiation of signaling, such as CARDs, PYRIN, or baculovirus inhibitor of apoptosis repeat (BIR) domains (Inohara et al., 2005; Martinon and Tschopp, 2005).
NOD-Like Receptors - NLRs The cytosolic NOD-Like Receptors (NLRs, also known as CATERPILLERs, NODs or NALP/PAN/PYPAFs) are nucleotide- binding oligomerization domain containing receptors. 22 NLRs have been identified in humans and constitute a major class of intracellular pattern recognition receptors (PRRs).
The designated subfamilies are (based on the initial of the domain name): NLRC (formely known as NODs), NLRP (formerly known as NALPs), NLRB (formely known as NAIP or Birc) and NLRA.
These proteins include NOD1 and NOD2, which both contain N-terminal CARD domains. NOD1 and NOD2 detect g-D-glutamyl- meso-diaminopimelic acid (iE-DAP) and muramyl dipeptide (MDP), found in bacterial PG, respectively (Chamaillard et al., 2003; Girardin et al., 2003).
Common alleles of NOD2 are a genetic risk factor for Crohn’s disease•Several moderately common alleles of the NOD2 gene (7% of totalalleles) increase susceptibility to Crohn’s disease (a form ofinflammatory bowel disease)•Two copies of these alleles increase susceptibility by 40X•Pretty strong evidence that these alleles of are “loss of function”alleles•NOD1/2 have been shown to have 4 immune functions: activation ofinflammatory cytokine gene expression; induction of anti-microbialpeptide synthesis by Paneth cells in intestines; activation ofinflammasome; autophagy of bacteria in cytoplasm
Processing of IL-1 and related cytokines: an important regulatory step•Some “NLRs” assemble to form the “inflammasome” whichproteolytically processes IL-1 and related cytokines to their active,secreted forms.•Inflammasome is activated by cellular stress or recognition ofmicrobial components in the cytoplasm•Genetic periodic fever syndromes are due to activating mutationsin inflammasome•Activated by small crystals, important role in Gout•Suggestive evidence that inflammasome may be activated bycholesterol crystals (atherosclerotic lesions?); possible role in type2 diabetes? Possible role in alzheimer’s disease?
CYTOPLASMIC BACTERIAL DETECTERS AND SIGNALLING
RNA Helicases and Double- Stranded RNA Fibroblasts and cDCs lacking MyD88 and TRIF are still capable of inducing type I IFNs after viral infection, indicating that the TLR system is not required for viral detection in at least several cell types (Kato et al., 2005). Retinoic-acid-inducible protein I (RIG-I) is an IFN inducible protein containing CARDs and a DExD/H box helicase domain and has been identified as a cytoplasmic dsRNA detector (Yoneyama et al., 2004).
RIG-I and MDA-5 RIG-I (retinoic-acid-inducible protein 1, also known as Ddx58) and MDA-5 (melanoma- differentiation-associated gene 5, also known as Ifih1 or Helicard) sense double-stranded RNA (dsRNA), a replication intermediate for RNA viruses, leading to production of type I interferons (IFNs) in infected cells. In cDCs, macrophages and fibroblasts, RLRs are the major sensors for viral infection, while in pDCs, TLRs play a more important role.
RIG-I participates in the recognition of Paramyxoviruses (Newcastle disease virus (NDV), Sendai virus (SeV)), Rhabdoviruses (vesicular stomatitis virus (VSV)), Flaviviruses (hepatitis C (HCV)) and Orthomyxoviruses (Influenza), whereas MDA-5 is essential for the recognition of Picornaviruses (encephalo-myocarditis virus (EMCV)) and poly(I:C), a synthetic analog of viral
Downstream of RIG-I-IPS-1, TBK1 and IKK-i are activated to phosphorylate IRF-3 and IRF-7, indicating that the signaling pathways triggered by TLR stimulation and RIG-I converge at the level of TBK1/IKK-i.
These suggest that RNA viruses actively replicating in the cytoplasm are recognized by RIG-I, but not TLR3, irrespective of their route of entry. On the other hand, TLR3 has been suggested to be responsible for the recognition of dsRNA contained in the apoptotic bodies of virus-infected cells taken up by DCs.
C-Type Lectin Receptors large family of receptors that bind to carbohydrates in a calcium-dependent manner. The lectin activity of these receptors is mediated by conserved carbohydrate- recognition domains (CRDs). On the basis of their molecular structure, two groups of membrane- bound CLRs can be distinguished and a group of soluble CLRs.
Type I transmembrane proteins Containing several CRDs or CRD-like domains DEC-205 and the macrophage mannose receptor (MMR)
Type II transmembrane CLRs Carry a single CRD domain Dectin-1, Dectin-2, macrophage-inducible C- type lectin (Mincle), the dendritic cell- specific ICAM3-grabbing nonintegrin (DC-SIGN), and DC NK lectin group receptor-1 (DNGR-1).
Soluble CLRs MBL, an oligermeric protein that binds an array of carbohydrate patterns on pathogen surfaces. CLRs expressed by most cell types including macrophages and dendritic cells (DCs), which phagocytoze various glycoproteins and microbes for the purposes of clearance and antigen presentation to T lymphocytes
MBL MBL (Mannose-binding lectin) is a soluble C- type lectin. MBL plays a crucial role in innate immunity against yeast by enhanced complement activation and enhanced uptake of polymorphonuclear cells. MBL binds to repetitive mannose and/or N- acetylglucosamine residues on microorganisms, leading to opsonization and activation of the lectin complement pathway.
MBL also interacts with carbohydrates on the glycoprotein (gp)120 of HIV-1. MBL may inhibit DC-SIGN-mediated uptake and spread of HIV.
Dectin-1 Antifungal innate immunity. Dectin-1 is a specific receptor for β-glucans . Dectin-1 signaling has been shown to collaborate with TLR2 signaling to enhance the responses triggered by each receptor
Mincle Is a member of the Dectin-2 family . Variety of exogenous and endogenous stimuli, such as mycobacteria, certain fungi and necrotic cells
DC-SIGN The recognition of several viruses (HIV-1, HCV, dengue virus, CMV, ebola virus) and other microbes of the Leishmania and Candida species. This type II transmembrane protein has a single C-type lectin domain and is expressed on immature monocyte-derived DCs. DC-SIGN modulates TLR signaling at the level of the transcription factor NF-κB, however, prior TLR activation of NF-κB is required.
DNGR-1 DNGR-1 binds damaged or dead cells via exposed actin filaments . DNGR-1 is therefore considered to be DAMPs receptor since no microbial ligand has yet been identified.
Plasmacytoid dendritic cells Many cell types produce small amounts of type 1 interferons upon infection There is a dendritic cell subtype (“plasmacytoid dendritic cell”; “natural interferon-producing cell”) that produces 100-1000x more interferon upon contact with viruses, does not need a productive infection. Also produces a large amount of TNF Recognition mechanism: probably TLR7, TLR9
Inflammasome Inflammasomes are large intracellular multiprotein complexes that play a central role in innate immunity. Inflammasomes comprise a member of the NOD-like receptor (NLR) family, such as NLRP3 and IPAF, and are defined by the NLR protein that they contain. The NLR protein recruits the inflammasome- adaptor protein ASC, which in turn interacts with caspase-1 leading to its activation.
Once activated, caspase-1 promotes the maturation of the proinflammatory cytokines interleukin (IL)-1β and IL-18.