Innate Immunity II - Receptors and Cytokines (PDF)

Innate Immunity II Randal Gregg, PhD Receptors and Cytokines harry potter symbol - Bing images defense against the dark arts - Bing images 1 Learning Objectives 1) Define PAMP and PRR and identify the cellular localization, ligands, and actions of TLR-3, -4, -7, and 9. 2) Identify the recepto...

Innate Immunity II Randal Gregg, PhD Receptors and Cytokines harry potter symbol - Bing images defense against the dark arts - Bing images 1 Learning Objectives 1) Define PAMP and PRR and identify the cellular localization, ligands, and actions of TLR-3, -4, -7, and 9. 2) Identify the receptor components, signaling molecules, and major cytokines produced upon TLR ligation. 3) Identify the cellular localization, ligands, and actions of NLRs and RLRs. 4) Recall the effector functions of TNF-α, IL-1β, IL-6, IL-8, and type I interferons. 5) Distinguish between the physiological outcomes associated with local and systemic release of TNF-α. 6) Recognize the role of the inflammasome in cytokine responses and associated diseases related to defects in this process. 7) Identify the major functions of the acute phase response (C-reactive protein, serum amyloid A, and mannose binding lectin). 8) Recall the molecules and steps associated with lymphocyte trafficking into tissues as part of the inflammatory process. 2 The next line of defense involves receptors for detecting pathogens and production of chemicals to mediate inflammation harry potter using petronis charm background - Search Images (bing.com) 3 Detection and response • If the cellular and molecular defenses are successful in repelling the pathogen within a few hours – the pathogen encounter may not be perceived by the host • This occurs on a daily basis! • We next consider what happens when a pathogen expands at a greater rate than the immediate defenses can eliminate the invader • The next wave of innate immunity can take up to 4 days to develop and involves soluble chemicals and cellular receptors • Inflammation operates to permit entry of innate cells from the blood into the site of infection and the local temperature rises (can develop systemically) Material and energy resources normally used for work, thought and other activities are diverted to the innate immune response producing symptoms (not usu. due to the pathogen) – most infections are resolved at this stage 4 Detection of pathogens • Cellular barriers (epithelial cells, keratinocytes and endothelial cells) and immune cells (macrophages, dendritic cells and lymphocytes) express innate receptors • These receptors recognize structures “conserved” among classes of microbes • Most of these innate receptors trigger phagocytosis without cell signaling • The portion of the “conserved” structure that is bound by an innate receptor is the pathogen associated molecular pattern (PAMP) • Innate receptors are known as pattern recognition receptors (PRR) K. Murphy. 2015. Janeway’s Immunobiology. Garland Science. 5 Nonsignaling innate receptors trigger receptor-mediated phagocytosis K. Murphy. 2015. Janeway’s Immunobiology. Garland Science. RTLD = ricin-like lectin domain; CTLD = C-type lectin domain; SR-A, SR-B = scavenger receptor-A and -B; MARCO = macrophage receptor with collagenous structure 6 TLRs are signaling innate receptors • Toll-like receptors (TLR) are a family of 13 PRR that have an extracellular domain for PAMP engagement (leucinerich repeat region, LRR) and a cytoplasmic signaling domain (Toll Interleukin-1 receptor, TIR) • Each TLR consists of either a homodimer or heterodimer • Specific TLR are expressed on the cell surface while others are found in the membranes of endosomes K. Murphy. 2015. Janeway’s Immunobiology. Garland Science. 7 Toll-like receptor distribution Detect bacteria and other extracellular microbes Detect viruses and other intracellular microbes 8 Toll-like receptor pathogen recognition K. Murphy. 2015. Janeway’s Immunobiology. Garland Science. 9 Recognition of bacterial lipopolysaccharide (LPS) by TLR4 • A tissue infected with a Gram-negative bacteria will have some death due to receptor-mediated phagocytosis of macrophages • A component of the cell wall, LPS (lipopolysaccharide) is released and binds to CD14 on the tissue macrophage • CD14 acts as a co-receptor to TLR4; LPS can also be captured by LBP (LPS binding protein) and delivered to CD14 • MD2, associated with TLR4, binds LPS-CD14 • Binding alters the conformation of the TIR (Toll Interleukin-1 Receptor) domain to bind a signaling molecule known as MyD88 K. Murphy. 2015. Janeway’s Immunobiology. Garland Science. 10 TLR signaling leads to cytokine production K. Murphy. 2015. Janeway’s Immunobiology. Garland Science. IRAK-4 = interleukin-1 receptor-associated kinase 4; TRAF6 = tumor necrosis factor receptor-associated factor 6; IKK = inhibitor of kB kinase; IkB = Inhibitory kB; NFkB = nuclear factor kB 11 IL-1β and TNF-α production by macrophages NF-κβ IL-1β TNF-α Capillary Endothelial Cells Tissue Fibroblasts Chemokines Adhesion Molecules Vascular Permeability Matrix Metalloproteinases LEUKOCYTE ATTACHMENT CELL MIGRATION INTO TISSUE CELL MIGRATION THROUGH TISSUE ECM Neurons Prostaglandin E2 cAMP MYALGIA and ARTHRALGIA FEVER (IL-1) IL-1β = interleukin-1 beta; TNF-α = tumor necrosis factor alpha. Both are primarily involved in vasodilation, increased vascular permeability and cell adhesion to endothelium. K. Murphy. 2015. Janeway’s Immunobiology. Garland Science. 12 IL-1 (IL-6 and TNF-α) trigger fever • Cytokines bind to receptors on hypothalamic endothelium • Increases levels of prostaglandin E2 (PGE2) • PGE2 increase in the periphery is responsible for nonspecific myalgias and arthralgias often accompanying fever • PGE2 binds to EP-3 (PGE2 receptor or PGE2R) on glial cells • Glial cells release cAMP • cAMP activates neuron terminals that extend into the thermoregulatory center of the brain • Some microbial toxins induce fever by binding to TLR on hypothalamic endothelium 13 Local effects of TNF-α • TNF-α can also stimulate endothelial cell expression of receptors for platelets which promote platelet adhesion and blood clotting • This prevents pathogen entry into the bloodstream and disseminating to other sites K. Murphy. 2015. Janeway’s Immunobiology. Garland Science. 14 Systemic effects of TNF-α • Presence of an infection in the bloodstream (sepsis) is accompanied by a massive release of TNF-α by macrophages in the liver, spleen and other sites throughout the body • Systemic vasodilation and increased vascular permeability → loss of blood pressure → loss of plasma volume → septic shock • Systemic presence of TNF-α → blood clotting in small vessels throughout the body → disseminated intravascular coagulation (with depleted clotting factors) → organ failure and patient blood cannot clot appropriately K. Murphy. 2015. Janeway’s Immunobiology. Garland Science. 15 IL-1β and TNF-α production by macrophages NF-κβ IL-1β TNF-α Liver Natural Killer (NK) cells IL-1β and IL-12 are effective at activating natural killer cells. IFNγ Cytotoxins CYTOTOXICITY K. Murphy. 2015. Janeway’s Immunobiology. Garland Science. Acute Phase Proteins [CRP, SAP (serum amyloid protein), fibrinogen, MBL (mannose binding lectin)] PHAGOCYTOSIS COMPLEMENT ACTIVATION IL-6 is the primary cytokine for the acute phase response; TNFα also a strong inducer of acute phase protein production by hepatocytes 16 Amplification of IL-1β production Inflammasome • Secreted IL-1β binds to the IL-1 receptors on activated macrophages • Feedback to increase the amount of pro-IL-1β (inactive) gene expression • Uptake of ATP and release of K+ induces assembly of the complex: o Procaspase-1 o NLRP3 (NLR family pyrin domain 3) o Adaptor protein • Procaspase-1 autocleaves to active caspase-1 • Caspase-1 cleaves pro-IL-1β into IL1β which is then secreted K. Murphy. 2015. Janeway’s Immunobiology. Garland Science. Without the inflammasome, IL-1 levels are nearly too low to be physiologically effective. 17 Cryopyrin-associated periodic fever syndrome (CAPS) CAPS     Fever Hives (urticaria) Red eyes Joint pain (arthralgia) • Autosomal dominant inflammatory disorder associated with mutations of NLRP3 gene (overactivation) • Affects 1-3 per 1 million; onset at 8 mo; diagnosis generally at 15 y • Leads to excessive production of IL-1β o  Neurological:  Headache (chronic or migraine)  Hearing loss  Aseptic meningitis  Muscle aches (myalgia)  Optic nerve damage Promotes fever, vasodilation and induction of other inflammatory cytokine production • ~50% have recurrent course; most bouts of symptoms resolve within 24 h • Anti-IL-1β antibody (Canakinumab) administered 1 x 8 wks or daily dose of IL-1 receptor antagonist (Anakinra) 18 IL-6 production by macrophages NF-κβ IL-6 Hypothalamus Bone Marrow Prostaglandin E2 cAMP MYALGIA and ARTHRALGIA FEVER K. Murphy. 2015. Janeway’s Immunobiology. Garland Science. INCREASED OUTPUT OF NEUTROPHILS Liver Acute Phase Proteins [CRP, SAP (serum amyloid protein), fibrinogen, MBL (mannose binding lectin)] PHAGOCYTOSIS COMPLEMENT ACTIVATION Adipocytes Myocytes INCREASED GLUCOSE METABOLISM LOCAL HEAT 19 Acute phase proteins (APP) • Induced primarily by IL-6 (lesser IL-1β and TNF-α) • Soluble proteins produced and secreted by the hepatocytes of the liver • About 30 proteins impacted (some increased, some decrease in concentration usually within 48 h) o o C-reactive protein (CRP) Albumin Serum amyloid A protein Transferrin Mannose-binding lectin Transthyretin Retinol-binding protein Binds phosphorylcholine of bacterial LPS and that of fungi, yeasts and some parasites Haptoglobulin  Can trigger classical pathway of complement activation Ceruloplasmin Serum amyloid A protein Interacts with bacterial lipoprotein and triggers various TLR signals (cytokines) as well as CD36 scavenger receptor Mannose-binding lectin (MBL)  C-type lectin that binds mannose-containing carbohydrates of bacteria, fungi, some parasites and viruses  Triggers lectin pathway of complement activation Decreasing APP C-reactive protein   o Increasing APP α2-macroglobulin α1-acid glycoprotein Fibrinogen Complement C3, C4 20 Acute phase response operates to detect and destroy pathogens Coagulation activation yields proteases that not only interact with coagulation protein zymogens but also with specific cell receptors to induce signaling pathways that mediate inflammatory responses K. Murphy. 2015. Janeway’s Immunobiology. Garland Science. 21 Other modes of complement activation than the alternative pathway can respond hogwarts castle in the distance - Bing images 22 Lectin pathway of complement activation • MBL complexed with MASP (MBL-associated serine proteases) in plasma • When MBL binds to mannose on the pathogen surface → autocleavage of MASP-2 then cuts the second MASP-2 (active) • Unclear the role of MASP-1 • Active MASP-2 cleaves C4 and C2 o C4 resembles C3; C2 is similar to Factor B o C4 → C4a (released as an anaphylatoxin) + C4b (fixes to the pathogen surface) o C2 → C2a (interacts with C4b) + C2b (released) o C4bC2a = classical C3 convertase K. Murphy. 2015. Janeway’s Immunobiology. Garland Science. 23 Lectin pathway of complement activation • Classical C3 convertase (C4bC2a) binds and cleaves C3 to produce C3b fragments which attach to the pathogen surface • C3b will combine with cleaved Factor B (Bb) to produce alternative C3 convertase (C3bBb) • Complement activities follow as for alternative pathway • Alleles of nonfunctional MBL occur in about 10% of the population leading to the following: o Patients are highly susceptible to meningitis caused by Neisseria meningitidis o N. meningitidis can be a nasopharyngeal commensal in a small portion of the population o Deficiency of C5-C9 can also lead to increased susceptibility o Complement-mediated killing is important to prevent disease in healthy carriers K. Murphy. 2015. Janeway’s Immunobiology. Garland Science. 24 CRP and classical pathway of complement activation • CRP binds to the surface of pathogens • It can interact with C1 (complement component 1) of the classical pathway of complement activation • C1 component consists of C1q + C1r / C1s serine proteases • C1q binds to CRP → C1r autocleaves and then cuts the other C1r and the two C1s proteases • Cleaved C1s protease is active and cuts C4 and C2 leading to the formation of classical C3 convertase (C4bC2a) • Complement activation and function proceeds as for the alternate pathway • The classical pathway can also be activated by C1 interaction with antibody that is bound to the pathogen surface (more on this later) K. Murphy. 2015. Janeway’s Immunobiology. Garland Science. 25 Temporal contribution of complement activation in the immune response K. Murphy. 2015. Janeway’s Immunobiology. Garland Science. 26 Result of initial response = inflammation “The pillars of inflammation” 27 Other signaling pathogen recognition receptors hogwarts castle in the distance - Bing images 28 Additional signaling PRR – NLR NOD-like receptors (NLR) • Receptors that detect products from degraded phagocytosed pathogens • Recognize components of bacterial cell walls • Best studied are the intracellular NLR, NOD1 and NOD2 o NOD1 recognizes γ-glutamyl diaminopimelic acid (degraded peptidoglycan of Gram-negative bacteria) o NOD2 recognizes muramyl dipeptide (degraded peptidoglycan of many bacteria) • The terminal end of the NOD molecules express a CARD (caspase-recruitment domain) • Same CARD found on the ends of RIPK2 (receptor interacting serine/threonine kinase 2) – allows binding to the NOD receptors K. Murphy. 2015. Janeway’s Immunobiology. Garland Science. CARD CARD 29 Additional signaling PRR – NLR NOD-like receptors (NLR) • When NOD binds the ligand → NOD dimerizes and RIPK2 binds via the CARD • RIPK2 then phosphorylates TAK1 (transforming growth factor beta-activated kinase 1) • pTAK1 phosphorylates and activates IKK • pIKK degrades Iκβ, bound to NF-κB • NF-κB translocates to the nucleus and induces cytokine production, phagocytosis and defensin synthesis K. Murphy. 2015. Janeway’s Immunobiology. Garland Science. 30 Additional signaling PRR – RLR RIG-I-like receptors (RLR) • Most cells express RIG-I (retinoic acid inducible gene 1) and MDA-5 (melanoma differentiation associated protein 5) which are cytoplasmic detection proteins for viral RNA • Viral RNA binds to RIG-I and MDA-5 and through CARDs interact with MAVS (mitochondrial antiviral signaling proteins) • Results in phosphorylation of IRF3 (interferon response factor 3) → dimerizes → transcription factor for type I interferons (type I IFNs) • Another site aids in the activation of NF-κβ to promote other cytokine production K. Murphy. 2015. Janeway’s Immunobiology. Garland Science. 31 Interferon response (antiviral state) RIG-I-like receptors (RLR) • IRF3 promotes expression of the family of type I IFNs: o IFN-α o IFN-β o IFN-γ, IFN-κ, IFN-λ, IFN-θ, IFN-ω • Autocrine (infected cell) and paracrine (uninfected cells) type I IFN signaling induces the interferon response • Acts to prevent viral protein synthesis and production of new virions 32 Mechanism of the interferon response Type I IFNs 1) Serine/threonine protein kinase (PKR)  Phosphorylates (inactivates) EIF2α (elongation initiation factor 2 alpha)  Protein synthesis inhibited 2) 2’,5’-oligoadenylate synthetase  Oligo A activates RNase L  RNase L degrades RNA molecules 3) Mx GTPases  Interferes with nuclear transport of viral RNA into nucleus  Blocks viral assembly 33 Early responder cells infiltrate the site of inflammation but how? hogwarts castle in the distance - Bing images 34 Vascular adhesion to promote cell rolling selectin ligand IL-1 and TNF-α induce the expression of Selectins on the surface of vascular endothelial cells K. Murphy. 2015. Janeway’s Immunobiology. Garland Science. Selectin ligands constitutively expressed on the surface of immune cells and can bind transiently to selectins = cells roll along the vascular surface 35 Selectins and ligands = cell rolling Endothelium Immune cells (leukocytes) Sialylated Lewis X antigen (SLex) 36 Integrins and ligands = cell arrest • • Integrin Neutrophils interact with vascular endothelia using selectinselectin ligand interactions to slow the neutrophils at the site of inflammation Integrin (LFA-1) – integrin receptor (ICAM-1) are used to firmly adhere the neutrophils to the blood vessel surface Neutrophil PSGL-1 E-selectin Chemokine receptor Chemokine Integrin receptor Proteoglycan Endothelial cell (Platelet/endothelial cell adhesion molecule-1 or PECAM-1) K. Murphy. 2015. Janeway’s Immunobiology. Garland Science. 37 CXCL8 production by macrophages NF-κβ CXCL8 (IL-8) Capillary Endothelial Cells Chemokines Adhesion Molecules NEUTROPHIL ATTACHMENT and MIGRATION INTO TISSUE K. Murphy. 2015. Janeway’s Immunobiology. Garland Science. Neutrophil Chemokine receptor (CXCR1, CXCR2) NEUTROPHIL MIGRATION IN THE TISSUE 38 Chemokines increase integrin binding • Selectin interaction slows cells to allow for interactions between chemokines and integrins with receptors/ligands • Integrin ligand expression induced on the surface of endothelial cells by TNF-α • ICAM-1 and ICAM-2 are common ligands of CR3 and LFA-1 on neutrophils 2 1 1 • Initial engagement transiently stops the neutrophil • Chemokines diffuse into the vascular lumen and are captured by sticky proteoglycan molecules on the surface of the endothelial cells 2 • Chemokine (CXCL8) binds to the chemokine receptor (CXCL8R) on the neutrophil surface → induces cell signaling to increase the binding affinity of the integrin • Neutrophil arrests on the surface of the endothelia K. Murphy. 2015. Janeway’s Immunobiology. Garland Science. 39 Tissue infiltration via chemokine gradient • As more integrins bind firmly → cell begins to flatten along the vascular surface • TNF-α weakens tight junctions and neutrophil proteases loosen PECAM-1 interactions • Concentration gradient of chemokine induces cytoskeletal rearrangements and migration to the source (mediated by G protein-coupled receptors) • Cells slide through endothelial cell layer (extravasation) Diapedesis = penetration of cells into and through the ECM K. Murphy. 2015. Janeway’s Immunobiology. Garland Science. A.K. Abbas, A.H. Lichtman, S. Pillai. 2015. Cellular and Molecular Immunology 8th ed. Elsevier Saunders. 40 Cytokine IL-1β Cell releasing Macrophages Monocytes Dendritic cells Cell targeted Endothelium Hypothalamic endothelium Natural killer cells TNF-α Macrophages Endothelium Hypothalamic endothelium IL-6 Macrophages Hepatocytes Neutrophils Adipocytes Myocytes Hypothalamic endothelium Type I IFN IFN-α IFN-β IL-8 Primary Effect(s) ↑ Adhesion, chemokines, MMP, vascular permeability (promote inflammation) Other functions Also ↑ acute phase proteins (+IL-6) by liver ↑ PGE2 (induce fever) ↑ Cytotoxicity ↑ Adhesion, chemokines, MMP, vascular permeability (promote inflammation) ↑ PGE2 (induce fever) Also ↑ acute phase proteins (+IL-6) by liver ↑ Acute phase proteins (complement activation and phagocytosis) ↑ Bone marrow output of neutrophils ↑ Glucose metabolism (heat) ↑ PGE2 (induce fever) Plasmacytoid dendritic cells Most cells (lower level) All cells ↑ PKR, 2’,5’-oligoA synthetase, Mx GTPases (antiviral state) Macrophages Monocytes Neutrophils Capillary endothelial cells Activation and migration Attachment of neutrophils in circulation 41 Questions? hogwarts dining hall wallpaper - Bing images 42 Next… Innate immunity III Induced Cellular Response Hogwarts Express Wallpaper (65+ images) (getwallpapers.com) 43