TLRs and Innate immune response Prof. Dr. Mohammad Al-Sweify 2010 Q. After bypassing the first line of defense, How the other defense lines work as antibacterial/antiviral protective systems ? A. Here is the whole story, and the sequence of events: Response is initiated by activation of innate immune response, on local basis. Then, it progresses to acute-phase and antigen- specific responses, on a systemic scale.. – This includes an inflammatory process, and then an adaptive humoral and cell-mediated immune responses. First; How the innate response becomes activated by bacterial cells ? Bacteria have potent activators of innate response, such as: 1) Direct activators through TLRs of dendritic cells (DC) and Mθ and other cells: LPS, Lipotechoic acid, lipoarabinomannan, Glycolipids and glycopeptides, ployanions, N-formyl peptides, and PGs fragments. 2) Indirect activators by chemotaxis: C3a and C5a are chemotactic to innate system effector cells. The complement was activated beforehand by bacterial PGs fragments and contact to cell surface (alternative and lectin pathways). P.S.: Lectin pathway is activated by the binding of serum mannose-binding protein to bacterial cell surface and in turn produce C3 convertase How foreign invaders are recognized by the innate immune cells as Enemy? 1. Two systems of microbial detection exist in our body called: pattern recognition receptors (PRRs). 2. One system comprises a family of membrane-bound receptors called the Toll-like receptors or TLRs. 3. TLR4, detects LPS present in the extracellular milieu. 4. The other family of detection proteins is the nucleotide- binding site/leucine-rich repeat (NBS/LRR) family; so called Nod receptors (Nod1 and Nod2). 5. These proteins are located in the cytoplasm and are involved in the detection of bacterial PAMPs that enter into the cell either with an invasive microbe or by translocation by certain pathogenic bacteria through specialized transfer apparatuses. TLRs 1. TLRs are essential for the recognition of microbial pathogens. 2. A subset of TLRs, TLR3, TLR7/8, and TLR9, is involved in antiviral responses by triggering the production of antiviral cytokines such as type I interferons (IFNs). 3. TLR3 responds to double stranded RNA, a replication intermediary for many viruses. 4. TLR7/8 recognize viral single-stranded RNAs, whereas TLR9 recognizes certain motifs within viral DNA. 5. Interestingly, the contact between a virus and a TLR- expressing cell is often sufficient to induce type I IFN production without need for infection, allowing uninfected cells to participate in the antiviral responses. Toll-like receptor (TLR) leukocyte expression patterns and PAMP specificities. Each of the TLRs is expressed on a different subset of leukocytes and each of the TLRs detects different subsets of pathogens allowing vigilant surveillance by the immune system. Q: Why do we need an intracellular means of pathogen detection in addition to the TLRs? 1. This cytoplasmic detection system (Nod) plays a key role in host defense in the tissues where TLRs are absent or expressed at low levels (in epithelial cells that line mucosal surfaces, e.g.; in colon)… but why ? 2. Nod 2 is more or less restricted to monocytes/macrophages 3. Activation of Nod occurs when a bacterial ligand is sensed by the LRR domain. 4. Nod1 and Nod2 can then activate the expression of a number of pro-inflammatory genes. 5. Nods detect unique PGN fragments: 1. Nod1 binds to N-acetyl glucosamine-N-acetyl muramic acid linked to a tripeptide, in which terminal amino acid is D- glutamine and meso-diaminopilimic acid. 2. This minimal motif is a signature of bacterial infection since neither of these 2 amino acids exist in mammals. Gram –ve peptidoglycan structure Inflammasomes NLR family: The nucleotide-binding oligomerization domain-like receptor. Certain NLR sense PAMPs in cytosol induce assembly of large caspase-1-activating complexes, called inflammasomes. Activation of caspase-1 (autoproteolytic maturation) processing and secretion of the pro-inflammatory cytokines IL-1β and IL-18. Several inflammasomes have been identified and defined by NLR protein that they contain. 1) NALP3/cryopyrin inflammasome assembles in response to a variety of PAMPs and „danger‟ signals, such as uric acid crystals. 2) AIM2 inflammasome (absent in melanoma 2) is new, recognizes cytoplasmic double-stranded DNA. 3) IPAF (ICE protease-activating factor) inflammasome: is triggered by bacterial flagellin 4) NALP1b (NACHT domain-, leucine-rich repeat-, and PYD-containing protein 1b) inflammasome is induced by anthrax lethal toxin IL-1β and IL-18 are related cytokines that cause a wide variety of biological efffects associated with infection, inflammation and autoimmune processes. IL-1β participates in the generation of systemic and local responses to infection and injury by generating fever, activating lymphocytes and by promoting leukocyte infiltration at sites of infection or injury. IL-1β initiates cascades secretion of inflammatory cytokines. IL-18 induces IFN-γ production and contributes to T-helper 1 (Th1) cell polarization. Both cytokines share a common maturation mechanism that requires Caspase-1. Caspase-1 is activated within the inflammasome. It is now generally accepted that activation and release of IL-1β requires two distinct signals. In vitro studies indicate that the first signal can be triggered by various PAMPs following Toll-like receptor (TLR) activation which induces the synthesis of pro-IL-1β. The second signal is provided by the activation of the inflammasome and caspase-1 leading to IL-1β processing. The requirement for a second signal for IL-1β maturation might constitute a fail-safe mechanism to ensure that induction of potent inflammatory responses occurs only in the presence of a bona fide stimulus, such as pathogen infection and/or tissue injury. 1) NALP3 Inflammasome Its activation occurs in macrophages by PAMPs, provided the cells are exposed to ATP. Indeed, in the absence of ATP, macrophages stimulated with LPS produce large quantities of pro-IL-1β, but release little mature cytokine to the medium. ATP and certain bacterial toxins, such asnigericin and maitotoxin, cause a change in the intracellular ion composition leading to the activation of the NALP3 inflammasome. ATP rapid potassium efflux from the cytosol A recent study monosodium urate and calcium phosphate dihydrate crystals activate caspase-1 in a NALP3-dependent manner (in joints: gout and pseudogout, respectively). Thus, the NALP3 inflammasome was suggested to participate in the etiology of these auto-inflammatory diseases. In addition to its role in gout, uric acid is a major component released into the extracellular milieu by necrotic cells, suggesting that an important role of NALP3 in the detection of endogenous ‘danger’ signal. Crystalline silica and asbestos were also shown to induce the activation of the NALP3 inflammasome, suggesting that the NALP3 inflammasome participates in the pathogenesis of silicosis and asbestosis. AIM2 Inflammasome Recently, AIM2 is identified as a new receptor for cytoplasmic DNA, which activate caspase-1 AIM2 is an interferon-inducible. AIM2 senses cytoplasmic dsDNA ++ caspase-1. Clearly, inflammasomes fulfill a central role in innate immunity. They detect and respond to bacterial components, „danger signals‟ and potentially dangerous cytoplasmic DNA. Further understanding on how they are activated should provide new insights into the mechanism of host defense and the pathogenesis of autoimmune diseases. The Innate cellular responses Early, these innate cells respond to bacterial invasion: 1. Immature dendritic cells 2. Langerhans cells 3. NK cells 4. γδ T lymphocyte residing in tissue. In addition to the acute inflammatory cells: 1. Neutrophils (PMNL) 2. Monocytes 3. ? Eosinophils ** Reminding you that Acute inflammation is an early defense mechanism to contain an infection, prevent its spread from the initial focus, and to signal subsequent specific immune responses. ** The three major events in acute inflammation are: 1. expansion of capillaries to increase blood flow 2. increase in permeability of the microvasculature structure to allow escape of fluid, plasma proteins, and leukocytes from the circulation 3. exit of leukocytes from the capillaries, and their accumulation and response to infection at the site of injury (pus). (see next figure) TNF-α and chemokines activate the expression of selectins and intercellular adhesion molecules (ICAM-1) on the endothelium near the inflammation. ligands on the neutrophil: integrins, L-selectin, and LFA-1 (leukocyte function-associated antigen). The neutrophil binds progressively tighter to the endothelium until it finds its way through the endothelium Neutrophil diapedesis in response to inflammatory signals. The progression of protective responses to a bacterial challenge. Protection is initiated by activation of innate responses on a local basis and progresses to acute-phase and antigen-specific responses on a systemic scale. Mechanisms for escaping the immune system ** Facts 1. the longer a bacterial infection remains in a host, the more time the bacteria have to grow and also cause damage. 2. bacteria that can evade or incapacitate the host defenses have a greater potential for causing disease. Pathogens, in general, can evade the body immune response by: 1. Encapsulation: – CHO are poorly immunogenic; – Capsule is slimy and hard to grasp – Capsule tears away when grabbed by a phagocyte. – Capsule protects a bacterium from destruction within the phagolysosome. 2. Antigenic mimicry – hyaluronic acid capsule of S. pyogenes is similar to CT of human body. 3. Antigenic masking: capsule, biofilm, S. aureus protein A binds IgG Fc portion. 4. Antigenic shift: influenza virus, N. gonorrheae 5. Production of anti-immunoglobulin proteases: Gonococci (IgA) …..Pathogens, in general, can evade the host immune response by (continued) 6. Destruction of phagocyte: TB, Streptolysin and ∂-toxins. 7. Block activation of phagocyte by γ-interferon: TB 8. Inhibition of chemotaxis: C5a is degraded by S. pyogenes. 9. Inhibition of phagocytosis: capsule, M-protein,coagulase, granuloma of TB. 10. Inhibition of phagolysosome fusion: e.g., Legionella sp., Mycobacterium tuberculosis, Chlamydia sp. 11. Resistance to lysosomal enzymes: Salmonella typhimurium, Coxiella sp., Ehrlichia sp., Mycobacterium leprae, Leishmania sp. 12. Intracellular replication: Salmonella, TB, Brucella, Chlamydia, Listeria, Francisella, and Rickettsia spp. They need TH1 T-helper avtivation of phagocytes to be killed or isolated by a wall around it as in TB!! 13. The long “O” Ag of LPS prevents complement from reaching the cell membrane of bacteria. List of encapsulated bacteria : Staphylococcus aureus Escherichia coli Streptococcus Klebsiella pneumoniae pneumoniae Salmonella sp. Streptococcus pyogenes Yersinia pestis (group A) Campylobacter fetus Streptococcus agalactiae Pseudomonas aeruginosa (group B) Bacteroides fragilis Bacillus anthracis Cryptococcus neoformans Bacillus subtilis (yeast) Neisseria gonorrhoeae Neisseria meningitidis Haemophilus influenzae The response to a viral infection is quite different from that seen in a bacterial infection. Viral infections are intracellular, this has two implications. – much of the damage is going to occur inside infected cells. – elimination of the infection is going to require destruction of host cells. Therefore, the immune system needs a method of discriminating virally infected cells from healthy cells. (MHC I molecules). Cell-mediated immunity is much more important for clearing a viral infection. The Innate host responses are often sufficient to limit viral spread The interferon response: – First line of defense, induced early after virus infection, before any other defense mechanisms. – Types: IFN-α by leukocytes and epithelium, IFN-β by fibroblasts IFN-γ by lymphocytes. – Normal cells do not contain preformed IFN nor do they secret interferon constitutively; because IFN genes are not transcribed in normal cells. Inducers of IFN-α and IFN-β production: – ds RNA, produced as the replicative intermediates of RNA virus, or from the interaction of sense/antisense mRNAs for some DNA viruses. (strongest inducer; one copy is enough) – Interaction of enveloped v. (herpesvirus and HIV) with iDC ++ IFN-α Non viral inducers are: – I.C. pathogenic organisms. – Certain TLRs activators – mitogens and antigen – ds-Polynucleotides – Synthetic polyanion polymers – Antibiotics (kanamycin, cyclohexamide) In general, IFN inducers may act either by: – Preventing synthesis of the gene repressor protein – Increasing the levels of the gene activator proteins Effect of IFN on the infected cell and adjacent cell: I. IFN- α/β are released within hours after infection. ++ Antiviral state proteins production in the adjacent cells. Antiviral state lasts for 2-3 days. Antiviral state proteins (enzymes) are only activated on viral infection and production of dsRNA, which activate the enzymes. These Enzymes are: 1) Protein kinase R (PKR): it phosphorylates ribosomal eIF-2α (euk. Initiation factor) inhibition of protein synthesis in the cell. 2) 2`,5`oligoadenylate synthetase (unusual polymerase) forms the oligonucleotide 2`,5`oligoadenosine, which activates cellular Ribonuclease L degradation of mRNA (preferentially the viral mRNA). II. IFN enhance APC antigen presentation by increasing the expression of MHC antigens III. IFN initiate the clearance of infected cells by activating NK cells and antigen-specific responses. Mode of IFN action (in general).. Role of each IFN type: IFN α, β, and γ Molecular basis of antiviral state, initiated by IFN: -Protein kinase R - 2`5`oligoadenylate synthetase -Ribonuclease L.