pattern recognition receptors

gram +ve/-ve. intestinal recognition. acute inflammation. TLRs. PAMPs. MyD88 (dependent/independent). NLRs. RLRs. CLRs. vaccine adjuvants. drugs/compounds. sepsis.

gram positive bacteria

surrounded by single thick peptidoglycan cell wall.

termed monoderms.

gram negative bacteria

thinner peptidoglycan cell wall.

outer membrane containing lipopolysaccharides.

termed diderms.

pathogenic surface molecules and intracellular components

lipoteichoic acid.

peptidoglycan.

lipoproteins.

DNA.

flagellin - on cell surface.

lipopolysaccharide - on cell surface.

innate immune system

driven by neutrophils, dendritic cells, macrophages, and epithelial cells.

occurs within minutes of pathogen detection.

pattern recognition receptors recognise the cellular wall components and other RNA/DNA of invading pathogen.

leads to activation of adaptive immune response and confers level of memory.

very broad.

adaptive immune response

driven by lymphocytes and natural killer cells.

highly specific, individual pathogen directed.

intestinal recognition of bacteria

invading pathogens entering/crossing epithelial barrier.

detected to some extent by epithelial cells and receptors on epithelial cells.

this detection/recognition elicits production of inflammatory mediators, mainly cytokines, chemokines, and lipid mediators.

early responders recruit innate cells (neutrophils - polymorphonuclear leukocytes).

these invade interstitial space and primes to destroy pathogen.

also recruit second responders (monocytic cells) which join with tissue resident macrophages.

monocytes infiltrate and become differentiated.

they release cytokines and other inflammatory mediators designed to combat infection.

feed into adaptive immune response.

lipid mediators

products of cyclooxygenase and activity of cytooxygenase and lipoxygenase.

leads to production of prostaglandins, thromboxane, and resolving lipids (e.g. resolvins).

initiation phase

pro-inflammatory lipid mediators including cysteinyl leukotrienes and prostaglandins.

kicked off by vasodilation and build up oedema in tissue.

accumulation neutrophils (polymorphonuclear).

resolution phase

anti-inflammatory lipid mediators (e.g. resolvins) signal for clearance of apoptotic cells by macrophages (Mo efferocytosis).

halt polymorphonuclear recruitment.

stimulate pro-resolving macrophage phenotype (M2).

prolonged.

accumulation monocyte macrophages.

driven by changes in profile of pro-inflammatory lipid mediators (e.g. leukotrienes and prostaglandins) giving way to resolving inflammatory mediators (e.g. resolvins).

acute inflammation

2 phases: initiation and resolution.

ideal outcome is complete resolution.

temporal switch in lipid mediators biosynthesised by leukocytes.

uncontrolled inflammation leads to sepsis and diseases associated with chronic inflammation.

pattern recognition receptor families

toll-like receptors - prototypical member.

nucleotide binding and oligomerisation domain (NOD)-like receptor.

retinoic acid inducible gene 1 (RIG)-like receptors.

c-type lectin receptors.

PAMPs

pathogen associated molecular patterns - barcodes on pathogen surface.

innate immune system recognises conserved motifs in pathogens (e.g. PAMPs).

pattern recognition receptors play important role in recognising PAMPs.

TLR structure

leucine rich regions - LRR.

immunoglobulin (Ig) domain.

toll-IL1 receptor (TIR) domain.

TLRs and PAMPs

TLRs can form dimers - homodimers (TLR4) or heterodimers (TLR2/1 or TLR2/6).

TLR4 homodimer interacts LPS.

TLR2 bound to TLR6 interacts lipoproteins (found in bacteria).

heterodimers highly specific for specific pathogens or pathogenic components.

plasticity/heterogeneity gives high level diversity in specificity for individual pathogenic products.

TLR location

some not all expressed on surface immune cells where interact with pathogen products.

TLR4 has co-receptor called MD2 (key component allowing dimerise with another TLR4 molecule).

on endosome, intarcellular organelles responsible for internalisation of pathogen products (dsRNA and ssRNA).

TLR expressed on interior side endosome.

binding sites on inner side endosome where interact with and endocytose pathogen components.

signalling TIR domains located on outside endosome.

TIR domains facilitate TLR signalling pathways leading to activation transcription factors (NFkB).

direct recognition PAMPs (TLR2/1)

the convex surfaces of TLR1 and TLR2 have binding sites for lipid side chains of triacyl lipopeptides.

binding of each TLR to same lipopetide induces dimerisation, bringing their cytoplasmic TIR domains into close proximity.

TLR2 forms ‘m’ shaped heterodimers with either TLR6 or TLR1, with diacyl moiety bound by TLR2.

TLR1 has hydrophobic channel that binds third diacyl moiety.

TLR2/1 heterodimers bind to triacylated lipoproteins.

hydrophobic channel closed off in TLR6 by presence phenylalanine at positions 343 and 365.

TLR2/1 heterodimers bind diacylated lioproteins.

direct recognition of PAMPs (TLR4)

LPS has multiple fatty acyl chains linked to glycan head.

five acyl chains can bind to pocket within MD2, but one acyl chain is free.

free acyl chain of LPS molecule binds to outer convex surface of another TLR4, inducing dimer.

LPS molecule bound to second TLR4/MD2 stabilises dimer.

MD2 cofactor for TLR4.

MyD88 and TLRs

MyD88 key molecule co-associates with intracellular region of TLRs, except TLR3.

apart TLR4, MyD88 directly coupled to TIR domain of TLR.

for TLR4, intermediate molecule (MAL or TIRAP - interchangeable).

MAL acts as intermediate/bridging molecule that allows MyD88 to interact TLR4 (and TLR2 complex with 6 or 1).

endosome molecules, also signal by MyD88 but don’t have MAL.

TLR3 doesn’t use MyD88 or MAL, associates witrh TRIF (adapter protein that TRL3 signals through).

TRIF associates with TLR4.

MyD88 coupled pathways culminate in activation of NFkB (transcription factor driving (crucial to) production pro-inflammatory cytokines such as IL1, IL6, and TNF).

TLR3, via TRIF, activates another cascade of signalling events that culminate in activation of another transcription factor (IRF3 - interferon response factor 3).

IRF3 leads to production of interferon a and b.

only activated by TLR3, and one arm TLR4.

TLR7, 8, and 9, and other endosome TLRs, also activate NFkB by same pathway.

MyD88 knockout mouse unable fight pathogens - key to innate immune response.

MyD88 dependent signalling

dimerised TLRs recruit IRAK1 and IRAK4 (both kinases), activating E3 ubiquitin ligase TRAF6.

TRAF6 polyubiquitinated (phosphorylated and ubiquitinated), creating scaffold for activation TAK1 (phosphorylated by IRAK).

TAK1 associates IKK and phosphorylates IKKb, which phosphoryates IkB (cytoplasmic inhibitor NFkB).

IkB phosphorylated, dissociates and activates NFkB (p50/p65 components).

activated NFkB translocates to nucleus and drives gene transcription and translation.

IKKy = NEMO.

mitogen activates kinases (MAPKs), p38, and JNK leading to activation transcription factor (AP1).

NFkB and AP1 induce release of classic inflammatory response - two pronged attack (NFkB and AP1).

MyD88 independent signalling

required for signalling by TLR4 and TLR3.

requires adapter protein TRAM and TRIF.

TRAM associates with only TLR4.

activate 2 signalling arms.

(1) RIP1 (kinase) leading to phosphorylation and activation IKKa/b/y leading to activation NFkB (p50/p65).

(2) TRAF3 leading to TBK1/IKKi and activation (phosphorylation) interferon response factor (IRF3; induced anti viral response include release of IFNa/b/y).

type 1 interferons

resistance to viral replication.

induction MHC class 1 and antigen presentation.

activate NK which selectively kill virus infected cells.

IL1b - local and systemic effects

local effects:

• activates vascular endothelium.

• activates lymphocytes.

• local tissue destruction.

• increases access of effector cells.

systemic effects:

• fever.

• production IL6.

TNF-a - local and systemic effects

local effects:

• activates vascular endothelium and increases vascular permeability, leads to increased entry IgG, complement, and cells to tissues and increased fluid drainage to lymph nodes.

systemic effects:

• fever.

• mobilisation metabolites.

• shock.

IL6 - local and systemic effects

local effects:

• lymphocyte activation.

• increased antibody production.

systemic effects:

• fever.

• induces acute phase protein production.

CXCL8 - local effects

chemotactic factor recruits neutrophils, basophils, and T cells to site infection.

IL12 - local effects

activates NK cells.

induces differentiation CD4 T cells into Th1 cells.

NLR family

predominately detect PAMPs in bacteria.

localised in cytoplasm - not membrane associated proteins.

contain multiple protein domains:

• leucine rich region.

• caspase activation and recruitment domain.

• pyrin domain - allows interaction with other PYDs.

• NACHT domain - contains nucleotide triphosphatase.

initiate downstream signalling cascade that leads activation NFkB or inflammasome (caspase 1 that c;eaves and activates IL1b).

NLR sub families

NLRA (e.g. CIITA).

NLRB (e.g. NAIPs).

NLRC (e.g. NOD1, NLRC4, NOD2, NLRC3, NLRC5, NLRX1).

NLRP (e.g. NLRP1, NLRP2-9, NLRP11-14, NLRP10).

RLR and cytosolic DNA sensors

cytoplasmic replication of virus produces uncapped RNA with 5’triphosphate.

retinoic acid inducible gene 1 (RIG1) - single stranded RNA containing 5’-triphosphate.

melanoma differentiation associated gene (MDA5) - dsRNA.

localised in cytoplasm.

contain multiple protein domains:

• caspase activation and recruitment domain.

• DEAD helicase - interacts with dsRNA.

• CTD domain.

initiate downstream signalling cascades lead to activation interferon response factor (IRF), NFkB, or caspase 1 (inflammasome).

they’re cytosolic DNA sensors.

CLR sub family

type 1 (e.g. DEC205, MMR).

type 2 (e.g. dectin1, dectin2, mincle, DC-SIGN, DNGR1).

soluble (e.g. MBL).

CLRs

detect carbohydrate motifs (PAMPs) in fungi membrane.

predominantly localised in plasma membrane.

contain multiple protein domains - carbohydrate recognition domain.

initiate downstream signalling cascade leads to activation NFkB, AP1, and NFAT (nuclear factor activated T cells).

dectin 1 - CLRs

receptor for b-glucans.

components of fungal membranes.

ligand binding triggers - CLRs

phagocytosis.

activation src and syk kinases.

syk induces CARD9-Bcl10-Malt1 complex.

activation NFkB and subsequent secretion pro-inflammatory cytokines.

ROS production - CLRs

affect IL1b secretion by activating NLRP3 inflammasome.

NFAT - CLRs

modulation cytokine expression by inducing NFAT through Ca2+-calcineurin-NFAT pathway.

TLR agonists as adjuvants during vaccination

modern vaccine contain DNA or recombinant proteins - poorly immunogenic.

needs adjuvant to boost immunological response by T and B cells.

agonists TLR receptors often employed to boost immune response (adjuvants).

imiquimode (aldara)

TLR7 agonists.

stimulates release interferons and activation immune cells.

treatment of early superficial basal cell carcinoma (skin).

applied as 5% cream on skin.

monophosphoryl lipid A (MPL)

adjuvant in Cervarix.

LPS derivative from Salmonella minnesota.

TLR2/4 agonist.

vaccine against human papillomavirus 16 and 18.

HEPLISAV-BM

FDA approved 2017.

hepatitis B surface antigen combined with TLR9 agonists to induce maximal immune response - 2 doses in one month.

more effective and improved on limitations of current vaccines:

• slow onset of protection - 3 doses over 6 months.

• poor protection in hypo-responders (obese, smokers, diabetics).

• poor compliance - needs 3 doses.

current therapies: Engerix-B (GSK) and Recombivax-HB (Merck’s).

eritoran

investigational drug against severe sepsis.

TLR4 antagonist - synthetic LPS analogue.

treatment severe sepsis - blood poisoning.

phase III failed to reach end-point - increase in 28-day survival.

indication to limit cytokine storm.

MN-166 (Ibudilast)

progressive MS, amyotrophic lateral sclerosis.

designed as PDE inhibitor and anti-inflammatory drug which:

• suppresses pro-inflammatory cytokines IL1b, TNFa, and IL6.

• TLR4 antagonist activity - may attenuate neuroinflammation.

• currently phase II drug US.

tumour progression

TLR2, 4, 7, 8.

production of immunosuppressive cytokines.

increased cell proliferation.

resistance to apoptosis.

tumour inhibition

TLR2, 3, 4, 5, 7, 8, 9.

Th1 biased immune response.

long-term anti-tumour immunity.

overcoming PDL1 resistance/enhancing efficacy of immune checkpoint inhibitors.

metastasis suppression.

induction of apoptosis.

sepsis

systemic inflammatory response to infection.

sever sepsis - sepsis complicated by organ dysfunction progressing to life-threatening septic shock.

few effective treatments except antibiotics, fluid resuscitation, and organ support (intensive care).

sepsis causes and treatment

early death caused by overwhelming inflammation:

• block pro-inflammatory response.

• promote anti-inflammatory response.

late death due to immunosuppression and failure to clear infection:

• promote adaptive immune response (similar to cancer).

late stage of disease associated with immunosuppression - sepsis

immune cell depletion - T cells most affected.

compromised T cell effector functions.

T cell exhaustion.

impaired antigen presentation.

increased susceptibility to opportunistic hospital infections.

dysregulated cytokine secretion.

reactivation of latent viruses.

sepsis symptoms - adult

fast breathing.

skin rash or clammy, sweaty skin.

fast heartbeat.

weakness or aching muscles.

not passing much (any) urine.

feeling confused disorientated, or slurring speech.

feeling very hot or cold, chills or shivering.

feeling very unwell, extreme pain or the ‘worst ever’.