Innate ImmunityReview

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TOLL-LIKE RECEPTORS (TLRS)

TLRs were the first PRRs to be identified and remain the best char-

acterized. Signaling by TLRs initiates key inflammatory responses

and also shapes adaptive immunity.

All TLRs (10 in humans and 12 in mice) are type I transmembrane

proteins characterized by an extracellular leucine-rich domain

and a cytoplasmic tail that contains a conserved Toll/IL-1 receptor

(TIR) domain. TLRs recognize various PAMPs, including lipid-based

bacterial cell wall components such as lipopolysaccharide (LPS) and

lipopeptides; microbial protein components such as flagellin; and

viral or microbial nucleic acids such as single-stranded or dou-

ble-stranded RNA, and CpG DNA. TLR ligands also include DAMPs

liberated from damaged or dying host cells, including nucleic acids

and proteins.

TLRs initiate shared and distinct signaling pathways by recruiting

different combinations of four TIR-domain-containing adaptor

molecules: MyD88, TIRAP (Mal), TRIF and/or TRAM. These signal-

ing pathways activate the transcription factors NF-kB and AP-1,

leading to production of inflammatory cytokines and chemokines.

They also activate interferon regulatory factors (IRFs) such as IRF3

and IRF7, which lead to production of type I interferons (IFNs) and

upregulation of interferon-stimulated genes (ISGs).

TLRs can be organized by cellular localization: TLRs 1, 2, 4, 5 and

6 are cell-surface receptors, whereas TLRs 3, 7, 8, 9 and 13 are

endosomal receptors. This localization mirrors the chronology

of PAMP contact with host cells: the surface TLRs detect surface

components from incoming microbes, whereas the endosomal

TLRs detect nucleic acids released by pathogens into the host cell

cytoplasm. Intriguingly, upon activation, TLR2 and TLR4 move from

the surface into endosomes.

An exceptional case among TLRs is TLR10, a surface TLR that pro-

vides anti-inflammatory, rather than pro-inflammatory, responses.

It does this by negatively regulating other TLR pathways.

The innate immune system is an evolutionarily conserved system acting as a first-line of defense against exogenous and endogenous

threats to the host, such as pathogenic infection, tissue damage or cancer. It includes diverse cells such as macrophages, dendritic cells

(DCs), neutrophils, natural killer (NK) cells and innate lymphoid cells (ILCs). The destruction and clearance of invading pathogens, and the

resolution of other threats to the host, requires complex coordination of multiple innate immune pathways.

Importantly, the innate immune system not only precedes the highly specialized adaptive immune system chronologically, but also enables

the long-lasting immunological memory characteristic of adaptive immunity. This is done partly through the work of innate antigen-pre-

senting cells (APCs), which interact with adaptive immune components such as B cells and T cells.

In order to detect and gauge threats, innate immunity employs an arsenal of specialized receptors. Chief among these are the numerous

pattern-recognition receptors (PRRs), which are found at varying levels in immune and non-immune cells alike. The cognate ligands of

PRRs comprise pathogen-associated molecular patterns (PAMPs), which are found exclusively on or in viruses, bacteria, fungi and other

microbes, and danger-associated molecular patterns (DAMPs), which are released by damaged or dying host cells.

The recognition of PAMPs and DAMPs by PRRs generates an acute inflammatory response. This involves secretion of cytokines and

chemokines, production of antimicrobial peptides, triggering of apoptotic or pyroptotic cell death, induction of autophagyand recruitment

of phagocytic cells.

The main PRR families are the Toll-Like receptors (TLRs), the NOD-Like receptors (NLRs), the RIG-I-Like receptors (RLRs), cytosolic DNA

sensors (CDSs), the C-type lectin receptors (CLRs) and inflammasomes. Many of these processes induce, or are regulated by, autophagy.

2017

TLR PRINCIPAL LOCALIZATION

ADAPTOR PROTEIN

PRINCIPALLIGANDS

TLR1/TLR2 (dimer)

Surface

MyD88,TI-RAP and TRAM

Bacterial cell-wall components

TLR1/TLR6 (dimer)

TLR4 LPS

TLR5MyD88

Flagellin

TRL10 Unknow

TLR3

Endosome

TRIF dsRNA

TLR7 ssRNA

TLR8

TLR9 CpG DNA

TLR13 23S rRNA

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TLR2

Canonicalinflammasomes

Non-canonicalinflammasome

MyD88

TLR2

TRAM

pro-IL-1β

pro-IL-18

IL-1β

IL-18

IL-1β

IL-18

pro-IL-1β

pro-IL-18

TLR1/6

Bacterial cell wall components

IL-18

IL-1β

IL-18

Pyroptosis

Pro-Casp-11 Caspase-11

CytosolicLPS

NLRP3Pro-Casp-1

ASC

Caspase-1

Danger signals (e.g. ATP) Crystalline substances (e.g. alum, uric acid) Microbial toxins (e.g. nigericin)

Pyroptosis

TLR13

23SrRNA

IRF3

IRF3IRF3

ISRE3

IFN-β Pro-inflammatory cytokinesPro-inflammatory cytokines

MyD88

AP-1

MKKs

ERK JNK p38

Junc-fos

IKKεTBK1DDX3

NLRP3Pro-Casp-1

ASC AIM2Pro-Casp-1

ASC

dsDNA

Caspase-1

IL-1β

TLR & NLR Signaling Pathways

NLRP1Pro-Casp-1

ASCNLRC4

Pro-Casp-1ASC NAIPs

Flagellin

MDP, Anthrax toxin

MyD88

TLR

PAMPs

IKKβ

IκB

p50 p65

p50 p65

NEMO

RIP2

NOD1NOD2

iE-DAPMDP

NF-кB

Pro-inflammatory cytokines

AP-1

MKKs

ERK JNK p38

Junc-fos

CARD9TAB2 TAB3

TAK1

IRAK1 IRAK2IRAK4

Bcl-10

MALT1

Pellino

TIRAPMyD88

TLR6TLR2TLR1

MyD88

TRAM

TLR8TLR3

TLR7TLR9

TLR4ssRNA

dsRNAssRNA

CpGDNA

Endosome

TRAF6

TIRAPMyD88

LBP

TLR4

MD2

CD14

LPS

IKKβ

IκB

p50 p65

p50 p65

NEMO

NF-кB

TRIF

MyD88

TLR5

Flagellin

IRAK1 IRAK2

TRAF6

IRAK4

TRAF6

TRAF3TANK NAP1

RIP1TRADD

IRAK4

IKKβ

IκB

p50 p65

p50 p65

NEMO

NF-кB

IKKα IKKα IKKα IKKα

IRF7

IRF7IRF7

ISRE7

IFN-α

Bacterial cell wall components

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Dec�n-1

P P

P

P

P

P

Apoptose

DHX29PKR

NEMO

IKKβIKKα

IкBp50 p65

Ub

ZDHHC1

LSP1

IKKε

CYLD

Bcl3

Bcl3

An�-inflammatory cytokines

NF-кB

Fucose

DC-SIGN

CNK

KSR1

Raf-1

LSP1

Raf-1

p65

STING

cGAMPc-di-GMPc-di-AMPcGAMP

p65

IKKβIKKα

ISRE7

Type I IFNs IFN-l1 IFN-βIL-2

NF-кB

Pro-inflammatory cytokines

Caspase 8

FADD

DAI

DDX41

TRAF6

TBK1

Pro-Casp-1ASC

Caspase-1

AIM2

pro-IL-1β IL-1β

IFI16cGAS

MRE11

MyD88

IRF1IRF3/7 IRF3/7

ISRE1/7

p50 p65

DHX15

DDX3

IKKεTBK1

IRF3/7

IRF7 IRF7 IRF7

IFN-α

ISRE3/7

Pro-inflammatory cytokines

CLR, RLR & CDS Signaling Pathways

IкBp50 p65

p50 p65 Junc-fos

Pro-inflammatory cytokines

NF-кBNFAT

FcRγ

SykFcRγ

Syk

TBK1

IRF3

IRF3 IRF3

ISRE3

c-di-GMPc-di-AMP

AP1

dsRNA

IPS1

MDA-5 RIG-I

LRRFIP1

Ku70

DHX36

IFIX

dsDNA

β-catenin

Pathogen dsDNA Host dsDNA

LGP2

Mitochondrion

Endoplasmic re�culum

DHX9

High mannose

Dec�n-2

NEMO

IKKβIKKα

ERK JNK p38

PLCγ

IP3DAG

RNA Virus

Calcineurin

NFAT

CARD9

MALT1Bcl-10

Ca2+PKCδ

β-Glucan

Syk

DC-SIGN

MAPKs

Mincle

α-Mannose, cord factor

High mannose, α-mannan

NF-кB

RNAPol III

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An�bodies & InhibitorsReporter plasmidsLigand screening service

TLR-NLR PATHWAYS

CLR-RLR-CDS PATHWAYS

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NOD-LIKE RECEPTORS (NLRS)

NLRs constitute a family of intracellular pattern recognition

receptors (PRRs) that contains more than 20 members in mammals.

Although the ligands and functions of many of these receptors are

not known, their primary role is to recognize cytoplasmic pathogen-

associated molecular patterns (PAMPs) and/or DAMPs, inducing

immune responses.

NLRs are characterized by a tripartite-domain organization with

a conserved nucleotide binding oligomerization domain (NACHT/

NOD), leucine-rich repeats (LRRs) involved in microbial sensing and

one of four N-terminal effector domains.

NLRs are classified in to four sub-families, which are named

according to the N-terminal effector domain: NLRA (Acidic

transactivation domain), NLRB (Baculovirus inhibitor repeat

domain), NLRC (Caspase recruitment domain) and NLRP (Pyrin

domain). There is also a sub-family known as NLRX, whose

members do not contain an effector domain analogous to those

mentioned above.

RIG-I-LIKE RECEPTORS (RLRS) AND CYTOSOLIC DNA SENSORS (CDS)

RLRs are a family of cytoplasmic RNA helicases that are critical for

host anti-viral responses. The RLRs include the RNA sensors RIG-I

and MDA-5, which, upon activation, drive transcription factors that

control the transcription of genes encoding interferons and other

cytokines.

RIG-I and MDA-5 sense double-stranded RNA, a replication inter-

mediate for RNA viruses. Upon binding of specific types of dsRNA,

each of these sensors activates the mitochondrial-bound adaptor

protein MAVS, which leads to production of type I interferons. The

cytosolic protein LGP2, which contains a RNA-binding domain, acts

as a negative feedback regulator of both RIG-I and MDA-5.

Recent advances in the recognition of nucleic acids have identified

several CDSs, which detect double-stranded DNA (dsDNA) of

pathogen, self or tumor origin, leading to the induction of inter-

ferons and/or the processing of pro-inflammatory cytokines. The

best-known CDSs are cGAS, AIM2 and IFI16.

Upon detection of dsDNA and DNA/RNA hybrids, cGAS produces

the cyclic dinucleotide 2’3’-cGAMP, which is the endogenous ligand

of the adaptor protein STING. Once activated, STING induces type

I IFNs and pro-inflammatory cytokines through the IRF3 and NF-

kB pathways, respectively.

AIM2 is unique among CDSs in that it forms an inflammasome (see

“Inflammasomes” section, below), which, like other inflammasomes,

contains the accessory proteins ASC and Pro-Casp-1.

Unlike other CDSs, IFI16 shuttles between the cytoplasm and the

nucleus.

Other noteworthy CDSs include DAI, DDX41, IFIX and LRRFIP1.

C-TYPE LECTIN RECEPTORS (CLRS)

CLRs, also called the C-type lectin receptors encompass a large

family of phagocytic receptor proteins that bind to carbohydrate

moieties of various pathogens. The importance of CLRs in shaping

the adaptive immune response is becoming increasingly apparent.

The lectin activity of CLRs is mediated by conserved carbohy-

drate-recognition domains (CRDs). These receptors are involved in

fungal recognition and in modulation of innate immune mechanisms

for pathogen clearance or for antigen presentation to T lympho-

cytes.

The principal human CLRs are the surface receptors DC-SIGN,

Dectin-1, Dectin-2 and Mincle, and the soluble receptor man-

nose-binding lectin (MBL).

PRODUCT DESCRIPTION UNIT SIZE

NLRAAcidic transactivation

(A)CIITA

NLRBBaculovirus inhibitor repeat

(BIR)NAIP

NLRCCARD

(Caspase recruitment domain)NOD1/2, NLRC 3/4

NLRP Pyrin NLRP1, NLRP3

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INFLAMMASOMES

Inflammasomes are caspase-1-activating protein complexes

assembled by certain NLRs. Caspase-1 is activated by

inflammasomes through autoproteolytic maturation, leading to

processing and secretion of the pro-inflammatory cytokines IL-1b

and IL-18.

Four inflammasomes have been identified to date. These include

three inflammasomes defined by their constituent NLR protein:

the NLRP1 (or NALP1b) inflammasome, the NLRC4 (or IPAF)

inflammasome and the NLRP3 (or NALP3) inflammasome. There

is also an inflammasome built around the DNA sensor AIM2, a

member of the IFI16 family.

Inflammasomes fulfill a central role in innate immunity by detecting

and responding to specific DAMPS and PAMPS, see table.

AUTOPHAGY AND INNATE IMMUNITY

Autophagy is a mechanism that cells use to sequester, remove and

recycle waste. In autophagy, macromolecules in the cytosol are

engulfed in a newly formed phagocytic body and subsequently

digested in a lysosome that releases the resultant metabolites back

into the cytosol.

Autophagy, often referred to as macroautophagy, serves to recycle

large chunks of cytoplasm as a source of nutrients, which enables

cells to maintain macromolecular synthesis and energy homeosta-

sis during starvation and other stressful conditions.

Moreover, cells use autophagy to regulate the activity of specific

signaling proteins, to prevent accumulation of damaged organelles

or long-lived, aggregate-prone proteins, and to remove incom-

ing threats such as intracellular pathogens. Thus, autophagy has

emerged as a critical component of innate immunity.

The interplay between autophagy, PRRs and inflammation is highly

complex and encompasses several regulatory mechanisms that

ensure balanced innate immune responses.

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PRODUCT DESCRIPTION

AIM2 dsDNA

NLRC4 Flagellin

NLRP1 MPD, anthrax, toxin

NLRP3ATP, uric, acid, alum, nigericin,

oxidized mtDNA, bacterial RNA

PRR & INNATE IMMUNITY - INVIVOGEN.COM

PRRS FAMILIES

Toll-Like Receptors - TLRs Best characterized receptors involved in early innate immune response to invading pathogens

NOD-Like Receptors - NLRs Intracellular pattern recognition receptors that recognize cytoplasmic pathogen-associated molecu-lar patterns

RIG-I-Like Receptors - RLRs Cytoplasmic RNA helicases that are critical for host antiviral responses

C-type Lectin Receptors - CLRs Receptors involved in fungal recognition and modulation of the innate immune response

Cytosolic DNA Sensors & STING Receptors to diverse molecules of microbial origin (PAMPs), or released from damaged or dying cells (DAMPs)

Inflammasome Large intracellular multiprotein complexes that play a central role in innate immunity

TOOLS FOR PRRS STUDY

PRR & PAMP Detection Rapid, convenient and reliable detection of pattern recognition receptors and pathogen-associated molecular patterns