Pattern Recognition Receptors in the innate immune response

host: discerns its surroundings & responds to changes

pathogen: decrease or increase in virulence

environment: friendly changes (temperature, nutrients) or detrimental changes (cold, lack of food, irradiation)

—>coexistance or disease

—>neuronal circuits detect pain & temperature alterations

—>immune pathway detect microbial encounters and tissue injury

macrophages

epithelial cells

neutrophils

innate lymphoid cells

starts after minutes and ends after days

B- & T-lymphocytes

starts after 12-24 hours and lasts for weeks

bridged to innate immune response by dendritic cells

receptors of the innate immune response

limited number (10 TLR, 22 NLR)

germline-encoded

ligand present of non-self (conserved and invariant microbial motifs) MAMP, PAMP & non-pathogen associated molecular patterns but also on seld (alarmins) & DAMP

expressed on DC, neutrophils, macrophages, B-cells & endothelial & epithelial cells (exposed to microbes in mucus membrane)

free receptor

membrane-bound phagocytic receptors

membrane-bound signaling receptors

cytosolic signaling receptors

in serum and extracellular fluids

C1q molecule of the classical pathway of the complement system

collectins: Mannose-Binding Lectins (MBL) & Surfactant Protein A&D (SP-A & SP-D)

ficolins

pentraxins

involved in phagocytosis of extracellular pathogens —> initiation of phagocytosis

opsonin receptors: CR, Fc receptors

mannose receptor (mannosylated ligands present on virus, bacteria & fungi)

dectin: glucans—>polymers of glucose on fungi

scavenger receptors: SR-AI/II, MARCO & CD36 (lipids, lipoproteins of bacteria)

expressed by DC, macrophages, neutrophil & B-cell

detect extracellular and endosomal pathogen—>initiation of inflammatory chemokine & cytokine synthesis

Toll-like receptors (with LRR domain)

expressed by DC, neutrophil, macrophage & epithelial cell

PAMPs detected: extracellular & endosomal microbial compounds

detect intracellular pathogens —> initiation of inflammatory chemokine & cytokine synthesis & pyroptosis (a particular type of cell death)

NLR: nucleotide-binding domain NBD and Leucine-rich repeat LRR receptor or NOD-like receptor

RLR: RIG1 helicase (retinoicacid inducible gene)-like receptor)

ALR: absent in melanoma 2-like receptors

expressed by nearly all cell types

PAMPs detected: cytoplasmic microbial compounds & alarmins

all compartments are equipped with several PRR

cell surface: TLR recognizes extracellular pathogens (bacteria, fungi, or viruses)

endosome: TLR recognizes endocytosed invaders

cytoplasm: NLR/RLR/ALR—>cytosolic microbes necessarily virulent pathogens

no receptor described in the nucleus

cell membrane & peptidoglycan

cell membrane, thin peptidoglycan & outer membrane with lipopolysaccharide

endotoxin

very important part for the inflammatory activity of LPS

anchors LPS in the membrane

has 6 acy-chains for anchoring in outer leaflet

extracellular part with polysaccharides of various öength

highly sensitive to fungi (Gram+) infections—>Drosophila shows dramatic reduction in survival after infection

mice resistant to LPS due to punctual mutation in the TIR domain of TLR4—>no TNFα produced after stimulation with LPGS (Gram-)

endosomal TLRs detect DNA/RNA

all detect different ligands of microbes

TLR6/2: parasite (GPI-anchor), yeast (zymonsan). bacteria (Diacyllipopeptides)

TLR1/2: bacteria (Triacyllipopeptides)

TLR4: LPS

TLR5: flagellin

on cell surface

TLR3,7/8,9: in endosome detecting dsRNA, ssRNA (virus) and DNA without CpG (bacteria)

—>limited number of TLR can detect presence of many different microorganisms by recognizing common features that are typical of different groups of pathogens

different PAMPs (LPS, Taxol, fusion protein, Env proteins)

DAMPs (oxidized LDL,…)

endogenous ligands

alarmins

extracellular: Leucine-rich repeat LRR containing proteins or Ig domain

membrane anchor: helical transmembrane domain or GPI anchor (CD14)

intracellular: Toll/interleukin-1 TIR superfamily —> high homology

20-27 LRR modules constitute the main extracellular part of TLR

each LRR is composed of 20-30 amino acids rich in the hydrophobic amino acid leucine

hydrophobic core (ß-strand), asparagine network & variable region (α-helix or α-helix like)

α-helix convex

ß-sheet concave

C-terminal & N-terminal with Asn ladder

central part more flexible without Asn

MD2 (associated with both monomers) binds 5 acyl-chains from LPS

LPS (glycan chains & 1 acyl-chain) binds TLR4

MD2 induces the formation of dimerization of TLR4 due to the binding of the lipopeptide

induced by binding of the ligand (LPS, dsRNA etc)

1. dimerization induced by the ligand binding

2. adaptor protein: Myd88 (universal adaptor) or TRIF

3. serine threonine kiases

4. transcription factor translocation in the nuclues

induced by ligand binding

LPS bound by LPS binding protein LBP (soluble in the serum)

transfer of LPS to GPI-anchor protein (CD14)

transfer of LPS to MD2 bound to TLR4

homodimerization TLR4

2 TIR domains interact with TIR domain of Myd88—>oligomerization of 6 Myd88 (Myddosome)

death domain of Myd88 interacts with IRAK (kinase)

TRAF-6 (E3 ligase) binds Myddosome & self-ubiquitinylation

polyubiquitin allows interaction with TAK1, IKKγ (NEMO) & IKKα/ß

TAK1 gets phosphorylated—> phosphorylation MAP kinase-kinase 4 —> JNK —> AP-1 (activates gene expression)

IKKγ phosphorylation —> IKKß phosphorylation —> proteolysis & release of NFκB —> translocation in nucleus & expression of pro-inflammatory cytokines

also internalization of TLR4 homodimer into endosome —> interacts with TRIF (instead of Myd88)—> other signaling pathway —> TRAF3 phosphorylated —> IRF3 phosphorylation & translocation into IRF3 —> expression of IFN type 1 (α&ß)

helical structure & compact structure

all TLR use the same adaptor: Myd88 except endosomal TLR4 & TLR3 which use TRIF adaptor

TLR3/4/7/8/9 leads to secretion of IL-12 & IFNα

engages differentiation of T helper into Th1

secretion of Il-12, TNFα & IFNγ (against intracellular/bacterial infections)

cell-mediated immunity: B cells produce IgG & IgA

TLR5,2/1,2/6 leads to secretion of Il-10 &-4 by DC—>differentation into Th2

secretion of Il-4, Il-5, Il-10 (parasitic infections, also involved in allergy)

B cells secrete IgE, IgG & IgA

• MHC/peptide/TCR

• costimulator: CD80/CD28 or CD86/CD28

• cytokines: secreted by DC binding receptors on T-cell

increased expression of HLA-DR & CD80 on all

CD86 not by all ligands increased

if TLR4 KO: no production of TNFα & RANTES after induction with LPS or Lipid A (ligand TLR4), no impact if Pam2 (ligand of TLR2)

impact on gene regulation: 700-800 genes differently regulated —>huge amount of genes regulated regulated by TLR4 regulation

internalized peptides (ligands of TLR) can bind MHCI/ MHCII —>expressed at the surface for interaction with TCR/BCR

also induction of costimulatory molecules & cytokine production & other genes

stimulation via the TLR initiates & modulates the adaptive cellular & humoral immune response

recurrent pneumococcal meningitis

stroke, seizure, middle-ear infections, intestinal intussusception, perforation, boils

develops low-grade fevers late in the course of illnesses

—>normal complete blood count, complement function & immunoglobulin titers

—>ability to make a protective response against protein antigens but failed to mount a response against polysaccharide antigen

—>significant decrease in TNFα secretion after TLR stimulation

WB for phospho-p38 & phospho-IκBα —>both not present in the patient

—> Sequencing revealed a homozygous nonsense mutation in the kinase domain of IRAK4

—>no downstream signaling

high number of infections in first 40 month (meningitis, septicemia & arthritis)

50% dead before 40 months of life

after 5 years no more problems—> in first period of life innate system with a great role to protect organism, after 5 years adaptive immune system completely functional

MAL encoded by TIRAP

impairs cellular responses to TLR2 & TLR4 stimulation

causes staphylococcal disease

Anti-LTA antibodies rescue TLR2-dependent responses to LTA in TIRAP-deficient cells

—>compensates absence of TIRAP

—>adaptive immune response can compensate for deficiency in the TLR pathway

TLR encoding genes are extremely polymorphic

10% of the European population has a mutated allele of TLR5 (homozygous mutation not characterized by severe immunodeficiency —>redundant role)

adaptive evolution (infections) & nonadaptive evolution (genetic drift, population bottlenecks & migration routes) contribute to polymorphisms in various populations

give resistance to infections via sensing of PAMPS but also greater risk of autoimmunity (endogenous ligands acting via PRRs)

common single nucleotide polymorphism

Asp299Gly protects from mortality due to cerebral malaria but also increased risk of septic shock from infection by Gram-negative bacteria

15% in Africa (malaria) lost during migration to Europe, not prevalent in South America

CTL: granzyme & perforin activate apoptotic cell death

NK

phagocytic cells

type I Interferon (α & ß): inhibit viral replication

Il-1ß & Il-18 —> key mediator of inflammatory response—>cleaved from pro-peptide by activation of caspase 1 (also induces pyroptosis)

cell death mechanism

characterized by cell swelling, lysis & release of cytoplasmic content

Multi-protein complexes that activate caspase 1 to induce processing of pro-interleukin-1ß & pro-interleukin 18 and to drive pyroptosis

—>cell autonomous immunity

central signaling hub regulating innate immunity

speck in fluorescence & confocal microscopy

PAMP or DAMP (self: ATP, MSU, CPPD, amyloid-ß; or non-self: asbestos, silica, alum)

• classical signaling pathway (NOD1, NOD2)—>NFκB IRF —>TNF, Il-6, IFN type 1 —-> regulation of transcription

• inflammasome signaling pathway (NLRP1, NLRC4, AIM2)—> cytosolic platform (oligomerization of the sensor) —>caspase 1 activation —>Il-1 & Il-18 (from pro-peptides) & pyroptosis —> post-transcriptional effects

NLRC4

NLRP1, NLRP3

NAIP1, NAIP2, NAIP5/6

AIM2

ancestral nucleotide-binding domains (NBDs)

NLR present in animals & plants

similar number in mouse (30) & human (22)

no TLR in Drosophila (use other PRR)

around 1000 AA

N-terminal domain, functional domain, or homotypic interaction domain (150 AA), differ

NACHT or NOD oligomerization domain (500 AA)

LRR ligand binding domain (300 AA), TLR common structure

NLRP1B: activated by Bacillus anthracis (secretes Anthrax proteins into phagolysosome) & T. gondii (also secretes protease) —> protease cleaves NLRP1B —>activates oligomerization

NLRP3: activated by diverse stimuli that might damage cellular membranes (pore formation) —>e.g. ATP (only extracellular after cell lysis) —> unknown endogenous signal induces the formation of the inflammasome by oligomerization of NLRP3

NLRC4: activated by flagellin & different proteins of the T3SS of bacteria (e.g. Salmonella) —> sensed by NAIP protein —> interacts with ligand & NLRC4 —> oligomerization

all lead to activation of caspase 1 cleaving pro-Il1ß / 18 & gasdermin D (N-terminal fragment induces pyroptosis)

first signal required to induce their production

not constitutively expressed

need TLR4 signal for their expression

latent factors

trigger: T3SS proteins (in NAIP-NLRC4)

sensor: self-proteins detecting the sensor having a NACHT & BIR domain (NIAP)

nucleator: oligomerizes to form inflammasome, has NACHT & CARD domain (NLRC4)

adaptor: links nucleator & effector (ASC)

effector: function to proteolyse gasdermin & pro-cytokines (caspase 1)

ligand binding (flagellin) leads to the opening of the molecule—>LRR released from the central NACHT domain

opening of NAIP exposes nucleating surface (basic)

interacts with the acidic surface of NLRC & its opening (exposure of LRR) & newly formed nucleating surface (basic)

self-propagating oligomerization

fully formed wheel-like structure consists of 11 NLRC4 molecules & 1 NAIP2

CARD domain of NLRC allows interaction with the adaptor protein ASC

ASC polymerization by homotypic interaction of their PYD domains & CARD domains of fiber protrude outside (high number of fibers leads to speck=

followed by caspase-1 recruition (via CARD) —>pro-caspase closer —> auto-activation to heterotetramer (2 p20 & 2 p10 domains)

active caspase 1 leads to release of Il-1ß & Il-18 from pores formed by gasdermin N-terminal fragment (also cleaved by caspase 1) & induces also pyroptosis

doesn’t always lead to cell death

membrane repair process can eliminate pore —> activation of inflammasome not automatically death

inflammasome-mediated cleavage of cytokines

inhibition Il-33 production —> no Th2 polarization

IFNγ production, Th17 differentiation, T-cell proliferation & Il-17 production

don’t use NLR and caspase-1

Il-1ß has a major role in inflammation

in some pathologies, inflammasome is constantly activated in the absence of a ligand

—>cyropryinopathies, familial Mediterranean fever, macrophage-activating syndrome (life-threatening, recurrent fever, hepatobiliary dysfunction, coagulopathy, low peripherical cell counts)

inflammatory symptoms affect CNS, muscles, eyes & pulmonary skeletal muscles

causes syndrome of enterocolitis & autoinflammation

mutation in NACHT domain (normally interacting in inactive state with LRR) but constantly open due to mutation

increased cell lysis & cytokine production

serious pathologies

inflammasome activated by alum (present in 50% of vaccines)

respiratory infectious disease due to coronavirus-2

common human coronaviruses (common cold)

dangerous human coronaviruses (SARS-CoV & MERS-CoV)

three viruses in bats with 95% shared overall genome

direct passage to human or through pangolines

S protein binds ACE2 (Angiotensin Conversion Enzyme 2) which is present in many different cells (lungs, heart, kidney, liver —>many different syndroms)

cleaved by TMPRSS2 (Transmembrane Protease Serine 2)

30-40% asymptomatic

60-70% symptomatic

—>80% mild (runny nose & dry cough, low grade fever)

—>15% severe: fluid in lungs, decreased blood oxygen (oxygenation with mask)

—>5% critical: organ failure due to lack of oxygen, inflammation & septic shock (oxygenation by intubation & anesthesia)

obesity

diabetes

heart dieases

hypertension

aging (90% of death over 65 years old)

vaccines

protease inhibitor

nucleoside analogs

anti-inflammatory molecule JAK1/2 inhibitor

cytokine inhibition with anti-Il-6 antibodies

inappropriate hyperinflammatory responses important

co-morbidities associated with high basal inflammation

PRR involved in sensing

MDA & RIG-1 are RLR

TLR2 senses E protein (secretion proinflammatory cytokines)

TLR4 probably senses S protein (sequence analysis)

elevated serum LDH (lactate dehydrogenase) concentration strongest single predictor of severe covid

Il-1ß & Il-18 also highly predictive biomarker of death in Covid patients

Il-1ß controls production of Il-6/TNF

molecules to escape detection by signaling proteins

inhibition TLR2&4

inhibit translocation —>no antiviral response

cytokine storm —>hyperactivity