Lecture 3 - Nod-like Receptors and the Complement System

LBP (LPS recognition by TLR4)

when LPS is shed it will bind first in the ECM to initiate the complex

CD14 (LPS recognition by TLR4)

moves through membrane to molecule of TLR4 so it comes together

MyD88 (LPS recognition by TLR4)

adaptor transmitting signal inside the cell, signal transmission occurs through TIR domain

NOD1

it is made of one CARD domain which allows receptor to interact with adaptor containing domain so it dimerizes, one NOD and LRRs, it has many cell types

E-DAP

ligand for NOD1 receptor, mostly in gram-negative bacteria

MDP

ligand for NOD2, found in gram + and gram - bacteria

NOD2

it is made of 2 card domains so it creates 2 dimers, one nod and LRRs, more restricted as its only found in phagocytes and paneth cells

NLRP3

instead of a card domain it has a pyrin domain, allows adaptor interaction and it is related to card but its different, one nod and LRRs

DAMPs

danger associated molecular patterns, it is the ligand for NLRP3 and it could be extracellular ATP or K+ efflux for example, its an inflammasome recognizing molecules produced by the host ligand, post recognition it forms the inflammasome

DAMP function

they are endogenous molecules released because of cellular damage, inducing the formation of NLRP3 inflammasome to produce active forms of IL-1B and IL-18

NOD receptors

intracellular sensors of bacterial infection that recognize fragments of peptidoglycan, they reside in the cytoplasm in an inactive form, binding of bacterial ligands to these proteins induces the recruitment of RIP2

RIP2

activates TAK1 leading to the activation of NFkB, it has the same card domain and they interact causing a kinase cascade

paneth cells

in the gut they use NOD2 signaling to constantly produce antimicrobial peptides like a-defensins and REG3, AMPs and mucins are part of low grade protective inflammatory response that maintains a healthy microbiota and avoids microbial encroachment/overgrowth, it recognizes peptidoglycan from microbiota and producing AMP in response to it

NOD2 loss of function mutations

disrupts paneth cell and AMP function, this can lead to impaired barrier function and changes in gut microbiota composition, the consequent dysbiosis and chronic inflammation can contribute to the development of crohn’s disease

IL-1B and IL-18

proinflammatory cytokines that are similar in terms of function, it needs to be cleaved so it can be activated

caspase 1

starts oligomerization, produces active form of NLRP3

sterile activators

activates NLRP3, they can be self derived (ATP, cholesterol crystals, urate crystals, glucose, amyloid B and hyaluronan) or environment derived (alum, asbestos, silica, alloy particles, UV radiation and skin irritants), they reduce intracellular K+, Ros generation and lysosomal disruption

how do inflammasomes form?

it requires the concomitant activation of other innate receptors that activate NFkB to produce cytokine precursors and NLRP3 itself, there is a priming and activation step

how do inflammasomes form? (priming)

first there is the priming for pro-IL-1B, pro-IL-18 and NLRP3 production, these pro-inflammatory cytokines are not produced all the time so they need some kind of trigger, priming is mediated by IL-1B, PAMPs, DAMPs and TNF

how do inflammasomes form? (activation)

this step is mediated by inflammation triggers recognized by the inflammasome, these include ATP, K+ efflux, crystals and ROS, first DAMP recognition induces NLRP3 oligomerization in a structure that contains ASC and capsase 1, caspase 1 will cleave pro-IL-1B, pro-IL-18 and the pore formation protein gasdermin which causes IL-1 and IL-18 release

cryopyrin associated periodic syndromes (CAPS)

these are autoinflammatory diseases that are caused by autosomal gain of function NLRP3 mutations, this mutation causes constant inflammation independent of triggers due to excess IL-1B production, symptoms include recurrent attacks of fever and localized inflammation in the absence of infection due to inappropriate triggering of the inflammasome, it can be treated with IL-1ra which blocks IL-1B receptor

muckle wells syndrome

causes periodic fever, urticarial rash, joint pains, conjunctivitis and progressive deafness

familial cold autoinflammatory syndrome (FCAS)

causes cold induced fever, urticarial rash, joint pains and conjunctivitis

chronic infantile neurologic cutaneous and articular syndrome (CINCA)

causes neonatal onset recurrent fever, urticarial rash, chronic arthropathy, facial dysmorphia and neurological involvement

the complement system

a collection of proteins that respond to pathogens through 3 effector arms that produce inflammation, enhance phagocytosis and lyse microbial membranes

what are the 3 arms of the complement system

recruitment of inflammatory cells, opsonization of pathogens facilitating uptake and killing by phagocytes and lastly perforation of pathogen cell membranes all resulting in death of the pathogen

inactive complement proteins

these are known as components (C) which are in the common pathway or factors which are found in the alternative pathway

activation by proteolysis

this is when proteins are cleaved into active fragments that each have a particular function, for instance C3 is cleaved by C3 convertase into C3a which recruits phagocytes and C3b which tags the bacterium for destruction, several complement proteins are proteases synthesized in a zymogen tat is activated by a complement protein and acts on another complement protein (cascade)

naming convention (complement system)

suffix a indicates the smaller fragment and suffix b indicates the larger one, there are exceptions where a is used to indicate larger fragment of C2

C1 complex

suffix C1q, C1r and C1s form it and these proteins are not proteolytic fragments,

sensors

these are proteins that act as complement initiators recognizing the pathogen, C1q binds to the antigen antibody complexes and pathogen surfaces while MBL, ficolins and properdin/factor p bind to carb structures such as mannose or GlcNAc on microbial surfaces

membrane attack proteins

these come together to form a complex and kill bacteria, these include C5b, C6, C7, 8 and 9

opsonins

includes C4b and C3b, surface binding proteins facilitating phagocytosis

complement activation pathways

alternative, lectin and classical pathways

how does the complement system work

first some kind of pathogen binding occurs through one of the pathways causing the generation of C3 convertase, this will cleave C3 into C3a and C3b where the first is a small fragment for cytokine recruitment and the second is a bigger fragment that tags the pathogen, C3b binds covalently to pathogens and is the main effector molecule of the complement system, C4b does the same thing

what allows C3 and C4 covalent binding?

the presence of a highly reactive thioester bond that gets exposed upon activation, it can be attacked by anything with an amino group/any protein, cleavage of C3 or C4 exposes the bond so it can be attacked by water hydrolyzing it so it gets inactivated (nucleophilic attack), or by a hydroxyl or amino group

C3 structure

the newly synthesized C3 protein is cleaved to generate a B chain and an a chain held together by disulfide bonds, before cleavage the thioester bond within TED is protected from reacting, the cleavage releases C3a and a change in conformation of C3b allows the thioester bond to react with a chemical group on the pathogen surface, the reactive thioester group of C3b in TED is cleaved so C3b is either bound to the pathogen surface or its inactivated by hydrolysis if the pathogen is not present

lectin pathway of complement activation

Mannose binding lectin and ficolins act as soluble PRRs recognizing repetitive carbs on microbial surfaces, they are oligomeric proteins, 3 monomers form trimers which combine to create4-6 clusters with carb binding domains, each cluster has 12-18 carb binding sites, enabling high avidity binding to sugars on microbial surfaces, host glycans are protected by sialic acid preventing recognition, MBL and ficolines bind MASP1 and MASP2 (zymogens, inactive form of the proteins), the ligand binding activates them

how is C3 convertase generated through the lectin pathway?

activated MASP-2 associated with MBL or ficolin cleaves C4 to C4a and C4b which binds to the microbial surface, C4b then binds C2 which is cleaved by MASP-2 to C2a and C2b forming C4b2a complex/C3 convertase, it cleaves C3 to C3a and C3b which binds to the surface or convertase itself, multiple C3b molecules will bind to the microbial surface, those C4 fragments who don’t bind are rapidly inactivated by water, the activation of complement is confined to the surface its initiated on

classical pathway of complement activation

this is highly homologous to the lectin pathway and its initiated by C1q which acts like MBL in the lectin one, it associates with C1r2s2 protease complex acting like MASP1 and MASP2 in the lectin pathway

C1r2s2 complex (classical pathway)

it is a protease complex made of 2 C1r and 2 C1s

C1q activation (classical pathway)

it either binds to the FC fragment of Ig molecules bound to pathogens and there are at least two molecules of antibody bound to antigen, binds to C-reactive protein attached to phosphorylcholine on pathogens or directly to some bacteria like lipoteichoic acid on gram pos bacteria

globular heads

interacts with C1q ligand and binds Fc of antibodies

classical pathway of activation

C1q binds antigen bound antibody and induces a conformational change in one C1r molecule, activating it, this then activates the second C1r and the two C1s molecules, C1s cleaves C4 and C2, C4 is cleaved first and C4b binds to the membrane close to C1, C4b binds C2 and exposes it to the action of C1s, it cleaves C2 creating the C3 convertase (C4b2a), this will then cleave C3 and many molecules of C3b and C4b deposit on the pathogen, this boosts phagocytosis cause of the multiple opsonins/fragments on the microbial surface

alternative pathway of complement activation

this is activated by spontaneous activation of C3, this is done by hydrolysis to C3(H2O) which binds to factor B allowing it to be cleaved by factor D which is a protease into Ba and Bb, this creates the C3 convertase (C3(H2O)Bb complex) cleaving more C3 into C3a and C3b, C3b is rapidly inactivated unless it binds to a cell surface, factor B will bind noncovalently to C3b on a cell surface and is cleaved to Bb by factor D

properdin

stabilizes the alternative C3 convertase on pathogen surfaces, it is necessary cause the convertase alone has a short half life if its not stabilized, it acts as a positive regulator extending the ½ life so there is more C3 cleavage and C3b binding amplifying it, this causes more opsonization and activation of terminal complement components

lectin pathway effector functions

C3a and C5a recruit phagocytic cells to the site of infection and promote inflammation

classical pathway effector functions

phagocytes with receptors for C3b engulf and destroy the pathogen

alternative pathway effector functions

completion of the complement cascade leads to the formation of a membrane attack complex (MAC) which disrupts cell membrane and causes cell lysis

membrane attack complex (MAC) assembly

this requires the generation of C5 convertase, it cleaves C5 to form C5a and C5b and the steps downstream are the same regardless of activation, C5a is a potent mediator of inflammation

terminal complement proteins function

they polymerize to form pores in the lipid bilayer membrane of certain pathogens, C5b will bind C6 and C7, this forms a complex that binds to the membrane via C7 which starts insertion into the membrane, C8 will bind to the complex and inserts into the cell membrane, C9 binds to the complex and polymerizes and 10-16 molecules of it bind to form a pore in the membrane

anaphyltoxins

by products of enzymatic cleaved steps of complement activation that have pro-inflammatory and chemoattractant effects, C5a, C3a and C4a act on endothelial cells to induce further recruitment of cellular mediators and humoral mediators to the site of infection, it also triggers degranulation of mast cells and basophils, overall increasing inflammation

cellular mediators

neutrophils, macrophages, and lymphocytes

humoral mediators

complement and antibodies

how do anaphylatoxins induce local inflammatory responses?

when C5a being the most potent, C3a and C4a are produced in large amounts, and they are found in the blood they can cause systemic circulatory collapse similar to anaphylactic allergic reactions involving IgE antibodies, the small complement cleavage products act on blood vessels to increase vascular permeability and cell adhesion molecules, the increased permeability allows increased fluid leakage from blood vessels and extravasation/diapedesis of Ig and complement molecules, migration of monocytes polymorphonuclear leukocytes and lymphocytes is increased, microbicidal activity of macrophages and PMNs is also increased

C3a and C5a receptors

those expressed on endothelial cells (leukocyte recruitment) and mast cells allow the release of histamine and TNF as well as amplification of inflammation

what is the most important function of complement?

opsonization of microbial cells with complement components, phagocytosis can be mediated by many different complement receptors on the surface of macrophages and neutrophils including CR1, CR3, CR4 and CRIg, complement receptors bind C4b and C4b and/or C4b/C3b cleavage products, using their Fc receptors phagocytes also bind to antigens opsonized with antibodies (decorates pathogens with opsonins

cleavage of pathogen bound C3b by factor I

this produces small fragments recognize by different complement receptors, C3b is bound to pathogen surface, its cleaved by factor I and MCP cofactor released the C3f fragment and leaves iC3b on the surface, cleavage of iC3b by factor I and CR1 releases C3c and leaves C3dg bound to the surface, C3b degradation products cannot form a C3 convertase so it stops complement activation

CR1 (CD35)

recognizes C3b and C4b, it promotes their decay, stimulates phagocytosis when working with C5a, responsible for erythrocyte transport of immune complexes, found in erythrocytes, macrophages, monocytes, polymorphonuclear leukocytes, B cells and FDC

CR2 (CD21)

recognizes C3d, iC3b, C3dg and epstein-barr virus, it is part of the B cell co receptor and the epstein-barr virus receptor, it is found in B cells and FDC

CR3 (mac-1, CD11b/CD18)

it recognizes iC3b and stimulates phagocytosis, it is found in macrophages, monocytes, polymorphonuclear leukocytes and FDC

complement receptor 1 (CR1, CD35)

it binds to C3b and C4b, it is a cofactor for factor I so it displaces Bb from C3b and C2a from C4b

decay accelerating factor (DAF, CD55)

it binds to C3 convertase which is blocked when C3b is cleaved from the small fragment, it displaces Bb and C2a from C3b and C4b respectively

membrane cofactor of proteolysis (MCP,CD46)

it binds to C3b and C4b, it is a cofactor for factor I

protectin (CD59)

it binds to C8 and inhibits MAC (membrane attack complex) formation

regulatory mechanisms for C3 convertases

displacement so proteins remove components to stop convertase function or inactivation so factor I and cofactors cleave active fragments into inactive ones to stop further activation preventing host cell damage

C1 inhibitor (C1INH)

it binds to C1r, C1s (C1q) and MASP-2 (MBL), it displaces C1r/s and MASP-2 inhibiting the activation of C1q and MBL (stops activation of lectin and classical pathways) , if defective it causes hereditary angiodema

Factor H

it binds to C3b and displaces Bb, the cofactor for factor I, it is is defective it causes age-related macular degeneration and atypical hemolytic uremic syndrome

factor I

it binds to C3band C4b, its a serine protease cleaving C3b and C4b, if defective it causes low levels of C3 and hemolytic uremic syndrome

regulation of complement activation of C1

C1q binding to antigen:antibody complexes activates C1r and C1s, the C1 inhibitor (C1INH) dissociates C1r and C1s from the active complex, removes the enzymatic part of the complex so complement cant start, this is common to the lectin and classical pathways

regulation of complement inhibition of MAC formation

the terminal components of complement form a membrane pore (MAC), CD59 prevents final assembly of the MAC at the C8 to C9 stage, it blocks the assembly of C5 and C9 interfering with binding so the pore is not formed