ArticlePDF AvailableLiterature Review

Cooperation of Toll-like Receptor Signals in Innate Immune Defence

April 2007
Nature reviews. Immunology 7(3):179-90

DOI:10.1038/nri2038

Source
PubMed

Authors:

National Institutes of Health

The mechanisms by which the recognition of Toll-like receptor (TLR) ligands leads to host immunity remain poorly defined. It is now thought that to induce an effective immune response, microorganisms must stimulate complex sets of pattern-recognition receptors, both within and outside of the TLR family. The combined activation of these different receptors can result in complementary, synergistic or antagonistic effects that modulate innate and adaptive immunity. Therefore, a complete understanding of the role of TLRs in host resistance to infection requires 'decoding' of these multiple receptor interactions. This Review highlights recent advances in the newly emerging field of TLR cooperation and discusses their implications for the development of adjuvants and immunotherapies.

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Mammalian Toll-like receptors (TLRs) are a family

of at least 12 membrane proteins that trigger innate

immune responses through nuclear factor-κB (NF-κB)-

dependent and interferon (IFN)-regulatory factor

(IRF)-dependent signalling pathways

(FIG. 1). TLRs are

evolutionarily conserved molecules and were originally

identified in vertebrates on the basis of their homology

with

Tol l, a molecule that stimulates the production of

antimicrobial proteins in Drosophila melanogaster

2,3

It is of note that Toll was functionally defined on the

basis of its crucial role in determining the resistance of

D. melanogaster to infection with fungi and Gram-positive

bacteria

2,4

. By contrast, mammalian TLRs have been

functionally characterized and distinguished mainly on

the basis of their stimulation by different ligands in vitro

and mice that are deficient in a single TLR rarely show

the extreme susceptibility to pathogenic infection that

was originally observed in D. melanogaster with a func-

tional deletion of Toll

. These considerations have led

to questions concerning whether the role of Toll in host

resistance to infection in D. melanogaster is comparable

to the role of TLRs in mammalian immune defence,

and the functional significance of TLR diversification

in vertebrate hosts.

The TLR-family members are

pattern-recognition

receptors

(PRRs) that collectively recognize lipid, car-

bohydrate, peptide and nucleic-acid structures that

are broadly expressed by different groups of micro-

organisms. Some TLRs are expressed at the cell surface,

whereas others are expressed on the membrane of endo-

cytic vesicles or other intracellular organelles. TLRs are

composed of an ectodomain of leucine-rich repeats

(LRRs), which are involved directly or through acces-

sory molecules in ligand binding, and a cytoplasmic

Toll/interleukin-1 (IL-1) receptor (TIR) domain that

interacts with TIR-domain-containing adaptor mol-

ecules

. The structural basis of ligand binding by TLRs

is poorly understood and several unexpected cross-

specificities (for example, the recognition by

TLR9 of

both CpG-containing oligodeoxynucleotides (ODNs)

and malaria

haemozoin) have now been described

The early hypothesis that individual TLRs might have

evolved to recognize distinct phylogenetic groups of

pathogens has been mainly abandoned because of the

well-documented, broad recognition of microbial prod-

ucts (both pathogenic and non-pathogenic) belonging

to diverse species and phyla by many of the TLRs.

In addition, the recognition of endogenous ligands by

TLRs is now thought to have an important role in the

regulation of inflammation, both in infectious and non-

infectious diseases. However, the precise identification

of these endogenous ligands has remained controversial

because of their possible contamination with microbial

products

. The function of TLRs is further diversified by

the different signalling pathways that can be induced

by ligand interaction

(BOX 1).

Although TLRs are an important system for microbial

sensing, they are not the only PRRs with this function

(BOX 2). At the cell-surface, C-type lectin-like molecules,

such as the mannose receptor and the β-glucan receptors

(for example,

dectin-1), also participate in the binding

and uptake of microbial components

8,9

. Components of

bacteria and viruses that gain entry into the cytoplasm

are recognized by cytosolic receptors, through which

*Cancer and Inflammation

Program, Center for Cancer

Research, National Cancer

Institute, Building 560,

Room 31-93, Frederick,

Maryland 21702-1201, USA.

‡

Immunobiology Section,

Laboratory of Parasitic

Diseases, National Institute

of Allergy and Infectious

Disease, Building 50,

Room 6140, Bethesda,

Maryland 20892-8003, USA.

e-mails: trinchig@mail.nih.gov;

asher@niaid.nih.gov

doi:10.1038/nri2038

Pattern-recognition

receptor

A receptor that recognizes

unique structures that are

shared by different

microorganisms. Signalling

through these receptors

typically leads to the

production of pro-

inflammatory cytokines and

chemokines and to the

expression of co-stimulatory

molecules by antigen-

presenting cells. The

expression of co-stimulatory

molecules, together with the

presentation of antigenic

peptides, by antigen-

presenting cells couples innate

immune recognition of

pathogens with the activation

of adaptive immune responses.

Cooperation of Toll-like receptor

signals in innate immune defence

Giorgio Trinchieri* and Alan Sher

‡

Abstract | The mechanisms by which the recognition of Toll-like receptor (TLR) ligands leads

to host immunity remain poorly defined. It is now thought that to induce an effective immune

response, microorganisms must stimulate complex sets of pattern-recognition receptors,

both within and outside of the TLR family. The combined activation of these different

receptors can result in complementary, synergistic or antagonistic effects that modulate

innate and adaptive immunity. Therefore, a complete understanding of the role of TLRs in

host resistance to infection requires ‘decoding’ of these multiple receptor interactions.

This Review highlights recent advances in the newly emerging field of TLR cooperation

and discusses their implications for the development of adjuvants and immunotherapies.

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REVIEWS

SYK

IRF7

IRF3

IL-10

IL-2

IL-1β

IL-18

Pro-inflammatory cytokines

TLR5 or

TLR11

TLR7,

TLR8

or TLR9

TLR4TLR1 or

TLR6

TLR2

IL-1R or

IL-18R

TIRAP

TRIF

MyD88

TRAM

IκB

NF-

κB

p50 p65

Endosome

TLR3

RICK

dsRNA

ssRNA

or DNA

Caspase-1

ASC

TBK1

RIG-I or MDA5

Proteins LPSLipoproteins

Dectin-1

β-glucan IL-1, IL-18

Various

ligands Peptides

Type I IFN

Cytoplasm

Plasma membrane

NOD or IPAFNALP

TIR domain

Nucleotide-binding

oligomerization

domain

CARD

Pyrin domain

Immunoglobulin-

like domain

C-type lectin

domain

RNA-helicase

domain

Leucine-rich

repeats

ITAM-like domain

Haemozoin

The crystalline product

resulting from digestion of

haemoglobin by the

intraerythrocytic replicative

stage of malaria parasites

(Plasmodium spp.).

C-type lectins

Animal receptor proteins that

bind carbohydrates in a

calcium-dependent manner.

The binding activity of C-type

lectins is based on the

structure of the carbohydrate-

recognition domain (CRD),

which is highly conserved

between members of this

family.

they induce cytokine production and cell activation

1,5

These cytosolic receptors are grouped in two main fami-

lies: the NLR family (nucleotide-binding oligomerization

domain (NOD)-like receptor family), which includes

at least 23 members that are either NOD receptors or

NALPs (NACHT-, LRR- and pyrin-domain-containing

proteins); and a family of receptors that have an RNA-

helicase domain joined to two caspase-recruitment

domains (CARDs), such as retinoic-acid-inducible

gene I (

RIG-I) and melanoma-differentiation-associated

gene 5 (

MDA5). The name RIG-I-like receptor (RLR)

family has been proposed by Creagh and O’Neill

for the

latter family of receptors, for consistency in nomenclature

with the TLR and NLR families.

There is growing evidence that these additional

PRRs can cooperate with TLRs in the innate immune

response to pathogens. The main sentinel cells of

innate immunity (epithelial cells, phagocytic cells and

dendritic-cell (DC) subsets) simultaneously express

overlapping but not identical combinations of TLRs

Figure 1 | Schematic representation of the structure and main signalling pathways of the PRR families. Only the

adaptor molecules and the main signalling pathways that differentiate the different classes of pattern-recognition

receptor (PRR) are shown. In reality, the pathways that are activated by the different receptors are multiple and

complex

(BOXES 1,2). For example, Toll-like receptor (TLR) signalling involves not only nuclear factor-κB (NF-κB)

activation, but also mitogen-activated protein kinases, phosphatidylinositol 3-kinase and several other pathways

that markedly affect the overall biological response to the activation of TLRs. Dectin-1 (a β-glucan receptor) is shown

as an example of various cell-surface PRRs, some belonging to the lectin-like family and some linked with the

immunoreceptor tyrosine-based activation motif (ITAM)-containing adaptor Fc receptor γ-chain (FcRγ), the activation

of which, as described in the text, can markedly affect TLR signalling. ASC, apoptosis-associated speck-like protein

containing a CARD (caspase-recruitment domain); ds, double-stranded; IFN, interferon; IκB, inhibitor of

NF-κB; IL, interleukin; IPAF, ICE-protease-activating factor; IRF, IFN-regulatory factor; LPS, lipopolysaccharide;

MDA5, melanoma-differentiation-associated gene 5; MyD88, myeloid differentiation primary-response gene 88;

NALP, NACHT-, LRR- and pyrin-domain-containing protein; NOD, nucleotide-binding oligomerization domain;

RICK, receptor-interacting serine/threonine kinase; RIG-I, retinoic-acid-inducible gene I; ss, single-stranded;

TBK1, TANK-binding kinase 1; TIRAP, Toll/IL-1R (TIR)-domain-containing adaptor protein; TRAM, TRIF-related

adaptor molecule; TRIF, TIR-domain-containing adaptor protein inducing IFNβ; SYK, spleen tyrosine kinase.

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Cross-tolerance

Cross-tolerance is observed

when the addition of a Toll-like

receptor (TLR) ligand induces

tolerance to subsequent

challenge with the same

stimulus used for priming and

also to subsequent challenges

with other stimuli that signal

through one or more different

TLRs.

Microarray analysis

A technique for measuring

the transcription of genes.

It involves hybridization of

fluorescently labelled cDNA

prepared from a cell or tissue

of interest with glass slides or

other surfaces dotted with

thousands of oligonucleotides

or cDNA, ideally representing

all expressed genes in the

species.

and other PRRs

, and this might result in tissue-

specific responses to microbial stimulation. For example,

CD11c

DCs in the lamina propria of the intestines,

in contrast to DCs at other anatomical locations,

express

TLR5 but not TLR4, a property that might

help the intestinal innate immune system to distinguish

between pathogenic and commensal flora

. Invading

microorganisms are likely to interact with many TLRs

and non-TLR PRRs, and both the magnitude and qual-

ity of the subsequent immune response are likely to

depend on the distribution of receptors on the innate

immune cells encountered by the microorganisms

and the coordinated sum of the signals induced by

these different receptors. Here, we review the emerg-

ing information about how individual TLRs interact

with each other and with members of the non-TLR

PRR families, and how such interactions influence the

generation of host resistance to infection.

TLR cooperation in cellular responses

Initial studies of the cooperation between different

TLRs expressed by haematopoietic cells showed that

the simultaneous activation of

TLR2 and TLR4 resulted

in a synergistic induction of tumour-necrosis fac-

tor (TNF) production

13,14

. However, the induction of

cross-tolerance between the two receptors and the use

of muramyl dipeptide as a putative TLR2 ligand, which

has recently been formally identified as a ligand for

NOD2 (REF. 15), in one of these studies complicates the

interpretation of these early reports

13,14

. A subsequent

study showed that stimulation of mouse macrophages

with both polyI:C (polyinosinic–polycytidylic acid; a

TLR3 ligand) and CpG DNA (a TLR9 ligand) induced

more-than-additive levels of TNF, IL-6 and IL-12 p40

(REF. 16), which confirmed that cooperation between

certain TLRs does exist.

Further studies indicated that gene expression and

protein secretion of TNF, IL-1β, IL-6, IL-10, IL-12,

IL-23 and cyclooxygenase-2 were several-fold higher in

DCs stimulated with combinations of TLR ligands than

in cells stimulated with a single agonist

17,18

. Microarray

analysis

showed that of the genes induced by a single TLR

ligand, the expression of only 1% of these was increased

in a clearly synergistic manner when a combination of

TLR ligands was used

. However, the expression of a few

genes was also downregulated in a synergistic manner

Box 1 | Toll-like receptor signalling pathways

Toll-like receptors (TLRs) (with the exception of TLR3), interleukin-1 receptor (IL-1R)

and IL-18R induce nuclear factor-κB (NF-κB)-dependent cytokine production through

a pathway involving the adaptor molecule myeloid differentiation primary-response

gene 88 (MyD88)

. However, TLR3 (and TLR4) uses a MyD88-independent signalling

pathway that involves the adaptor molecule Toll/IL-1R (TIR)-domain-containing adaptor

protein inducing interferon-β (TRIF; also known as TICAM1)

. Further complexity in the

TLR signalling pathways results from the obligatory use of the adaptor molecule

TIR-domain-containing adaptor protein (TIRAP; also known as MAL) in association

with MyD88 by TLR2 and TLR4, and of the adaptor TRIF-related adaptor molecule

(TRAM; also known as TICAM2) in association with TRIF by TLR4

(REF. 6) (FIG. 1).

Activation of TLR3 or TLR4 induces type I interferon (IFN) production through the

adaptor TRIF, which associates with the kinase TANK-binding kinase 1 (TBK1) and

induces the phosphorylation and nuclear translocation of IFN-regulatory factor 3

(IRF3)

. The production of type I IFNs is further regulated by a positive-feedback

loop. IFNβ (in mice and humans) and IFNα4 (in mice), which are produced in

response to activated IRF3, can induce the transcription of genes encoding other

signalling proteins, such as IRF7 and IRF8

(REFS 90,91). Once phosphorylated, these

transcription factors drive the expression of all type I IFN genes, thereby amplifying

type I IFN production.

The outcome of TLR signalling can also be influenced by the spatio-temporal

regulation of ligand–TLR interactions. For example, in plasmacytoid dendritic cells,

ligation of TLR9 by D-type CpG-containing oligodeoxynucleotides (ODNs) activates

a signalling complex present in early endosomes that is composed of MyD88, IL-1R-

associated kinase 1 (IRAK1) and tumour-necrosis factor receptor-associated factor 6

(TRAF6). This complex physically associates with IRF7 and results in the production

of type I IFNs

92,93

. By contrast, recognition of D-type CpG ODNs by all other cell types

that express TLR9 results in the activation of IRF5 (which forms a complex with

MyD88 and TRAF6 downstream of TLR9 in late endosomes) and the transcription of

pro-inflammatory genes

34,94,95

Box 2 | Signalling pathways of pattern-recognition receptors other than TLRs

The members of the NLR (NOD-like receptor) family of intracellular pattern-recognition receptors (PRRs) include

NALPs (NACHT-, LRR- and pyrin-domain-containing proteins), IPAF (ICE-protease-activating factor; also known as

CARD12 and CLAN) and NOD proteins. NLRs have a tripartite domain structure composed of an amino-terminal

effector domain, which is usually a pyrin domain (PYD) or a caspase-recruitment domain (CARD), followed by a

nucleotide-binding oligomerization domain (NOD), which might be involved in self-oligomerization, and finally a

carboxy-terminal series of leucine-rich repeats (LRRs) that are involved in ligand binding

. Signalling through

members of the NLR family is complex, but in general, activation of several NALPs or IPAF results in the activation

of caspase-1. By contrast, activation of NOD proteins induces nuclear factor-κB (NF-κB) activation through

receptor-interacting serine/threonine kinase (RICK; also known as RIP2 or CARDIAK)

The RLR (RIG-I-like receptor) family is formed by at least two members — retinoic-acid-inducible gene I (RIG-I) and

melanoma-differentiation-associated gene 5 (MDA5) — which are composed of effector CARDs and an RNA-helicase

domain that is involved in the recognition of double-stranded RNA. Their activation results in recruitment of the

kinase TANK-binding kinase 1 (TBK1), activation of NF-κB and interferon (IFN)-regulatory factor 7 (IRF7) and/or IRF3,

and the induction of type I IFN production

Other surface receptors on innate immune cells that are involved directly or indirectly in the recognition of

microbial structures include the mannose and β-glucan receptors

, and other C-type lectins

. The β-glucan receptor

(also known as dectin-1 in mice) has an immunoreceptor tyrosine-based activation motif (ITAM)-like motif in its

intracellular portion that activates the kinase SYK (spleen tyrosine kinase)

. The modulation of innate immune

responses is also mediated by other cell-surface receptors, some with as-yet-undefined ligands, that are associated

with signalling molecules that contain ITAMs, such as Fc receptors (FcRs)

, TREMs (triggering receptors expressed on

myeloid cells)

63,99

and OSCAR (human osteoclast-associated receptor)

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by the combined TLR stimulation

. Cytokine produc-

tion can also be negatively regulated by simultaneous

signalling through certain TLRs

. In particular, the pro-

duction of IL-10 after TLR2 stimulation was shown to

block the expression of IL-12 p35 and CXC-chemokine

ligand 10 (CXCL10; also known as IP10) by human DCs

in response to either TLR3 or TLR4 ligands. Therefore,

the positive or negative regulation of genes by the

combination of TLR ligands is quite selective and it

seems to affect mainly gene products that are involved

in the regulation of the innate and adaptive immune

responses to pathogens, which often express multiple

TLR ligands.

TLR regulation of IL-12 p70 production. The synergis-

tic effect of certain TLR ligands is particularly marked

for the production of the immune-regulating cytokine

IL-12 p70, which is the biologically active, heterodimeric

form of IL-12 composed of the

IL-12 p35 and IL-12 p40

chains

. Stimulation of DCs with single TLR, NLR or

RLR ligands normally induces low levels of the IL-12 p40

homodimer and negligible levels of biologically active

IL-12 p70. By contrast, stimulation of mouse or human

DCs with the

TLR7 and TLR8 ligand R848 (also known

as resiquimod) and either polyI:C or the TLR4 ligand

lipopolysaccharide (LPS) results in amounts of IL-12 p70

that are one or two orders of magnitude higher than the

amounts induced by the individual TLR ligands

17,18,20

A more-than-additive effect on the production of

IL-6 and IL-12 p40 was also observed in these studies,

although the effect was less marked than that shown for

IL-12 p70

(REFS 17,18,20).

It is known that for high levels of IL-12 p70 to be

produced by DCs in response to a single TLR ligand,

co-stimulation by either secreted (for example, IFNγ) or

membrane-bound (for example, CD40 ligand, CD40L)

signals derived from T cells or natural killer (NK) cells

is required

21,22

. Therefore, it could be hypothesized

that IL-12 will be present only at later times during

an immune response when NK cells and/or T cells are

already activated

. This then raises the question of

whether IL-12 p70 can be the initial start-up signal for

the T helper 1 (T

1)-cell response to pathogens. However,

levels of IL-12 as high as or higher than those produced

in response to a single TLR ligand and co-stimulation

with IFNγ and/or CD40L are rapidly produced by simul-

taneous stimulation with two TLR ligands, even in the

absence of NK cells and T cells

17,24

(F. Yarovinsky, A.S.

and G.T., unpublished observations).

In addition to IFNγ, type I IFNs have also been

shown to have a complex role in regulating cytokine

production; type I IFNs upregulate IL-12 p70 produc-

tion while partially inhibiting the production of IL-12

p40. Indeed, DCs derived from mice that are geneti-

cally deficient for the type I IFN receptor (IFNAR) and

human DCs or monocytes treated with a neutralizing

antibody specific for IFNAR have decreased IL-12 p70

production. Specifically, in response to either a single

TLR ligand or a combination of TLR ligands, the pro-

duction of IL-12 p70 and CXCL10, as well as IL-12 p35

mRNA accumulation, but not the production of IL-12

p40 and IL-6, or IL-12 p40 mRNA accumulation, were

dependent on type I IFN signalling

17,24

The promoter of the gene encoding IL-12 p35 is

partially responsive to IFNγ signalling, which indicates

that type I IFN signalling (which partially overlaps with

IFNγ signalling) might also participate in the tran-

scriptional regulation of IL-12 p70 — for example by

inducing the expression of IRF-family members such as

IRF1 and IRF8 that are involved in transcription of the

gene encoding IL-12 p35

(REF. 25). However, it is also

possible that type I IFNs remove negative-feedback

mechanisms for IL-12 production. For example, endo-

genous type I IFNs might inhibit the activation by TLR3

or TLR4 ligands of the phosphatidylinositol 3-kinase

(PI3K)–AKT pathway, which is known to limit IL-12

production

. Although low levels of type I IFN produc-

tion are observed in response to the single TLR ligands

or combinations of TLR ligands that induce IL-12 p70

production, the combination of TLR ligands did not

result in synergistic induction of signal transducer and

activator of transcription 1 (STAT1) phosphory lation

or of CXCL10 production, two responses that are

strictly dependent on endogenous type I IFN produc-

tion in purified DCs

. Furthermore, in the absence

of endogenous type I IFN signalling, the overall level of

IL-12 p70 production by purified DCs is markedly

decreased, but a more-than-additive induction of IL-12

p70 production by a combination of TLR ligands is still

observed

. Therefore, type I IFN is required for optimal

IL-12 p70 production but it is clearly not the response ele-

ment that accounts for TLR synergism. Nevertheless, the

role of endogenous type I IFN in IL-12 p70 production

requires further investigation.

During innate and adaptive immune responses to a

pathogen, the IFNγ and other cytokines produced by

NK cells, natural killer T (NKT) cells and antigen-specific

T cells are important for amplifying IL-12 production by

DCs, which is required for optimal T

1-cell responses.

In addition, activated T cells also increase IL-12 p70 pro-

duction by interacting with DCs through CD40L–CD40

interactions

. Therefore, signals from accessory cells are

important to amplify and maintain IL-12 p70 produc-

tion by DCs. Although signalling through IFNAR is

also required for optimal IL-12 p70 production, type I

IFN, unlike IFNγ, is produced endogenously by DCs in

response to TLR signalling, and combined TLR ligands

can induce high levels of IL-12 p70 in the absence of

accessory cells and their products. Overall, these results

indicate that IL-12 production in response to combined

TLR signalling can be an autonomous DC function.

Because many pathogens express ligands for sev-

eral TLRs

(TABLE 1), it is likely that these ligands can

cooperate to activate multiple TLRs during infection,

resulting in the production of sufficient levels of IL-12

p70 and other pro-inflammatory cytokines early in

infection. These pro-inflammatory cytokines then

initiate and amplify the innate immune response and

adaptive T

1-cell immunity. The possible successive

involvement of different cytokines and cell-surface

receptors in regulating IL-12 p70 production during

the response to infection is shown in

FIG. 2.

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Molecular mechanisms. The synergy between different

TLRs could in part be due to the preferential induction

of IL-12 p40 transcription by myeloid differentiation

primary-response gene 88 (

MyD88)-associated TLRs and

of IL-12 p35 transcription by TIR-domain-containing

adaptor protein inducing IFNβ (

TRIF; also known as

TICAM1)-associated TLRs. The different signalling

pathways activated by receptors that use these two

adaptor molecules (MyD88 and TRIF) have been largely

characterized but the interaction of the signalling path-

ways is not well understood. In both human and mouse

DCs, the production of IL-12 p70 was synergistically

induced when certain MyD88-associated TLR ligands

were combined with TRIF-associated TLR ligands

17,18,20,24

(FIG. 3), which indicates that the two signal-transduction

pathways downstream of MyD88 and TRIF might coop-

erate. Indeed, it has recently been shown that MyD88-

associated TLRs synergize with TRIF-associated TLRs

for the induction of several pro-inflammatory cytokines,

including IFNβ, in part by increasing NF-κB activation

and nuclear translocation. By contrast, sequential stimu-

lation with ligands for either MyD88- or TRIF-associated

TLRs results in cross-tolerance for the responsiveness

of TLRs using the same adaptor

However, when a TLR2 ligand (which uses MyD88

as a signalling adaptor) was combined with any other

TLR ligand (including TRIF-associated TLR ligands),

the synergy for IL-12 p70 production was moderate

or absent. Only a low-level synergy for IL-12 p40,

TNF and IL-6 production was observed when a TLR2

ligand was combined with a TLR3, TLR4

(REFS 17,24)

or TLR9

(REFS 29,30) ligand. In addition, no significant

synergy for IL-12 p70 production was observed when

TLR3 and TLR4 ligands were combined

17,20

. The use

of the co-adaptor TIR-domain-containing adaptor

protein (

TIRAP; also known as MAL) with MyD88

in TLR2 and TLR4 signalling, and of the co-adaptor

TRIF-related adaptor molecule (

TRAM; also known as

TICAM2) with TRIF in TLR4 signalling, might distin-

guish the TLR2 and TLR4 signalling pathways from

the pathways induced by other TLRs that do not use

these co-adaptors.

Although TIRAP has been shown to be required for

the recruitment of MyD88 to TLR4 and probably TLR2,

but not to other MyD88-associated TLRs, no evidence of

a direct role for TIRAP in TLR4 or TLR2 signalling has

been reported

. However, tyrosine phosphorylation of

STAT1, which in purified DCs indicates at least low levels

of type I IFN secretion

, is observed in DCs stimulated

with ligands for all TLRs except TLR2

(REFS 17,32). This

indicates that differences might exist between receptors

that signal through MyD88 alone and those that signal

through MyD88 in association with TIRAP.

The synergy between different TLRs might also be

the result of interactions between the different signal-

ling pathways that affect the production of IL-12 p70 at

both the transcriptional and post-transcriptional stages.

Interestingly, it has been shown that IRF3, which is

activated by TRIF-associated signalling, suppresses the

transcription of IL-12 p35 mRNA, which indicates

the existence of another possible mechanism of IL-12

p70 regulation that might be affected by the interaction

of different TLR signalling pathways

Another important difference between the various

TLRs is their cellular localization and/or the association

of TLRs with other receptors or accessory molecules that

modulate TLR signalling properties and might affect the

ability of TLRs to interact synergistically. An example is

provided by the ability of TLR7 and TLR9 to associate

with different molecular complexes and to activate dif-

ferent signalling pathways when ligands interact with

the receptors in early or late endosomes

. It will also

be important to evaluate how simultaneous triggering

of different receptors can affect the negative-regulatory

mechanisms that modulate TLR responsiveness

. The

molecular basis of some of these negative-feedback

mechanisms is well characterized and includes decoy

receptors, ST2 (an IL-1R-like molecule), the short form

of MyD88, IL-1R-associated kinase M (IRAK-M),

Table 1 | Examples of pathogens expressing ligands for multiple TLRs

Pathogen Toll-like receptor (TLR) TLR ligand

Mycobacterium

tuberculosis

TLR2 Lipoarabinomannan

TLR4 Phosphatidylinositol mannosides

TLR9 DNA

Salmonella

typhimurium

TLR2 Bacterial lipoprotein

TLR4 Lipopolysaccharide

TLR5 Flagellin

Neisseria

meningitidis

TLR2 Porin

TLR4 Lipopolysaccharide

TLR9 DNA

Haemophilus

influenzae

TLR2 Lipoprotein

TLR4 Lipopolysaccharide

Candida

albicans

TLR2 Phospholipomannan

TLR4 Mannan

TLR9 DNA

Murine

cytomegalovirus

TLR2 Viral protein

TLR3 Double-stranded RNA

TLR9 DNA

Herpes

simplex virus

TLR2 Viral protein

TLR3 Double-stranded RNA

TLR9 DNA

Influenza virus TLR7, TLR8 Single-stranded RNA

TLR3 Double-stranded RNA

TLR4 Not determined

Respiratory

syncytial virus

TLR3 Double-stranded RNA

TLR4 Envelope F protein

Trypanosoma

cruzi

TLR2 Glycosylphosphatidylinositol anchor

TLR4 Glycoinositolphospholipid-ceramides

TLR9 DNA

Toxoplasma

gondii

TLR2 Glycosylphosphatidylinositol anchor?

TLR11 Profilin

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Type I IFN

IL-12 p70

Pathogen

a Early innate resistance

TLR

Type I IFN

NLR/RLR

IFNAR

cell

NKT

cell

p40 p35

IFNα

IFNβ

IFNγ

IL-12

IL-18

TNF

b Late innate resistance

c Adaptive immunity

p40 p35

Pro-inflammatory

cytokines

CD40L

IL-12

IL-18

TNF

Pathogen

TLR

p40 p35

Pro-inflammatory cytokines

CD40L

IL-4

IL-13

IL-4

IL-13

TCR

T cell

Pathogen

TLR

IFN

NLR/RLR

IFNGR

IFNγ

IFNGR

NLR/RLR

IL-12 p70

Peptide–MHC class I

Figure 2 | Regulation of IL-12 p70 production by dendritic cells during innate and adaptive immune responses.

a | Early innate resistance. Optimal production of interleukin-12 (IL-12) p70 by dendritic cells (DCs) occurs when ligands

for different pattern-recognition receptors (PRRs) are combined

17,18

. Many pathogens express ligands for several PRRs

and therefore efficiently induce IL-12 p70 production. In addition, high-level production of IL-12 p70 by purified DCs

requires endocrine production of type I interferon (IFN)

. The level of endogenous type I IFN that is required for

optimal IL-12 p70 production is low and sufficient type I IFN can be induced by most single PRR ligands. Therefore, the

induction of type I IFN is not responsible for the synergy of different PRRs that is observed for the activation of IL-12

p70 production

. The production of IL-12 p70 by DCs can be cell autonomous and the cooperation of other cell types

that eventually mediate a marked increase in IL-12 p70 production is not an absolute requirement for the early

production of this cytokine. In an in vivo model of endotoxaemia, this early production of IL-12 p70 is observed within

2–3 hours after the injection of lipopolysaccharide (LPS)

100

. In response to Toll-like receptor 7 (TLR7) and TLR9 ligands,

plasmacytoid DCs produce high levels of type I IFN within a few hours

101

that might replace the need for the autocrine

synthesis of type I IFN by conventional DCs (not shown). However, high levels of type I IFN inhibit IL-12 production

102

which indicates that type I IFN has a complex role in the regulation of IL-12 production. b | Late innate resistance.

The early production of IL-12 and other pro-inflammatory cytokines by DCs rapidly induces natural killer (NK) cells,

natural killer T (NKT) cells and activated/effector T cells (not shown) to produce IFNγ and, in the case of NKT cells, IL-4

and IL-13. IFNγ effectively replaces the need for type I IFN for IL-12 p70 production, probably by using similar molecular

mechanisms

. In addition, activated NK cells and NKT cells increase IL-12 p70 production by DCs through membrane

receptor–ligand interactions

27,103,104

. In an experimental model of endotoxaemia, IL-12-dependent production of IFNγ

is observed 6–7 hours after the injection of LPS

100

. c | Adaptive immunity. When an adaptive immune response to a

pathogen is established at 5 to 7 days after infection, the production of IFNγ by antigen-specific T helper 1 (T

1) cells is

important for amplifying the production of IL-12 p70 by DCs, which is required for optimal T

1-cell responses. In addition,

activated T cells also increase IL-12 p70 production by interacting with DCs through CD40 ligand (CD40L)–CD40

interactions

. IL-4 and IL-13 produced by T

2 cells are also important co-stimulators of IL-12 p70 production by DCs

105

although neither the mechanism nor the biological significance of this interaction is clear. IFNAR, type I IFN receptor;

IFNGR, IFNγ receptor; NLR, nucleotide-binding oligomerization domain (NOD)-like receptor; RLR, retinoic-acid-inducible

gene I (RIG-I)-like receptor; TCR, T-cell receptor; TNF, tumour-necrosis factor.

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Cytoplasm

TLR7, TLR8,

TLR9, TLR11

MyD88

TLR4

TRIF

TLR3

Synergy Synergy

TRIF MyD88

TIRAP

TRAM

Crohn’s disease

A form of chronic inflammatory

bowel disease that can affect

the entire gastrointestinal tract,

but is most common in the

colon and terminal ileum. It is

characterized by transmural

inflammation and granuloma

formation, and it is thought to

result from an abnormal T-cell-

mediated immune response to

commensal bacteria.

Caspases

A family of cytosolic proteases

that contain a cysteine residue

in the active site and that

cleave their substrate after

an aspartic-acid residue.

They can be divided into

pro-inflammatory caspases

(caspase-1, -4, -5 and -11),

which cleave and activate pro-

inflammatory cytokines, and

pro-apoptotic caspases, which

cleave and activate pro-

apoptotic substrates.

Contact hypersensitivity

The initial reaction that occurs

after the first exposure to a

‘sensitizer’ hapten or antigen.

This step requires dendritic-cell

migration to lymph nodes to

prime contact-antigen-specific

T cells.

members of the suppressor of cytokine signalling

(SOCS) family, Toll-interacting protein (TOLLIP) and

IRF4 (a negative regulator of IRF5-mediated TLR sig-

nalling)

. Therefore, these molecules are obvious can-

didates for analysis to determine a possible effect of TLR

cooperation on the negative regulation of TLR signal-

ling. In addition to intracellular regulatory molecules,

secreted cytokines such as type I and type II IFNs and

IL-10 have regulatory effects on TLR-mediated cellular

responses and they might participate in the cooperative

effects of triggering of different PRRs.

TLR cooperation with other PRRs

In addition to the synergy between ligands of differ-

ent TLRs, synergistic induction of cytokine produc-

tion has also been observed for DCs or macrophages

activated by a TLR ligand combined with ligands for

other PRRs. A large amount of evidence supports the

observation that ligands of

NOD1 and NOD2 can

synergize with many TLR ligands, including TLR2

ligands, for the induction of TNF and IL-12 p40 pro-

duction

36–39

. Because bacterial peptidoglycans (TLR2

ligands) are degraded to compounds that can activate

NOD proteins, the synergy between TLR- and NLR-

family receptors can amplify the response not only to

a single pathogen but also to a single component of a

pathogen

However, when the production of IL-12 by human

DCs was analysed, combined stimulation of NOD2 and

TLR2 resulted in only low-level production of IL-12

p70, whereas NOD2 activation effectively increased

IL-12 p70 production in response to the activation of

other TLRs, such as TLR7 and TLR8 (F. Gerosa and

G.T., unpublished observations). Interestingly, the pro-

duction of cytokines induced by TLR2 ligands, but not

by other TLR ligands, was shown to be higher in mice

deficient for NOD2 compared with wild-type mice

But, discordant results were reported in a later study

possibly as a result of the different experimental condi-

tions in the two studies. However, the activation of

NOD2 might involve differential signal-transduction

pathways that result in either amplification or attenuation

of the responses mediated by TLRs (TLR7 and TLR8,

or TLR2, respectively)

. This has important implica-

tions because of the increased frequency of functional

mutations in one or both NOD2 alleles in patients

with

Crohn’s disease

. Therefore, careful dissection of

the molecular mechanisms involved in the interaction

between TLRs and NOD2 and of their role in mucosal

immunity should improve our understanding of the

pathogenesis of inflammatory colitis

A different molecular mechanism of cooperation

between PRRs occurs for the simultaneous activation

of TLRs and NALPs. The stimulation of the latter in

a multiprotein complex containing the adaptor

ASC

(apoptosis-associated speck-like protein containing a

CARD; also known as CARD5), which is known as the

‘inflammasome’

, results in the activation of caspase-1.

Active

caspase-1 cleaves pro-IL-1β and pro-IL-18

to produce the active forms of the two cytokines

46,47

However, TLR stimulation is required for the production

of these pro-cytokines, indicating an essential coopera-

tion between the TLR and NALP families of receptors for

the production of IL-1 and IL-18

(REFS 46,47).

NALP3 is essential for the ATP-driven activation of

caspase-1 in LPS-stimulated macrophages. In addition,

ASC- and NALP3-deficient mice have an impaired

contact hypersensitivity response to the hapten trinitro-

phenylchloride and have decreased secretion of IL-1β

and IL-18 when cultured with the Gram-positive bacte-

ria Staphylococcus aureus and Listeria monocytogenes

48–50

NALP3 is also essential for IL-1β and IL-18 production

in response to small-molecule agonists of TLR7 and

TLR8, to double-stranded RNA, and to viruses such

as Sendai virus and influenza virus

. Interestingly,

endogenous ligands, such as gout-associated uric-acid

crystals, also activate the NALP3-containing inflamma-

some and caspase-1, resulting in the production of IL-1β

and IL-18 in inflammatory and autoimmune diseases,

which indicates that the inflammasome acts as a first line

of defence against cell stress

51,52

. Signals from the cell-

stress-activated inflammasome might cooperate with

signals from TLRs and other PRRs, which are activated

by endogeneous or exogenous ligands, in regulating

inflammatory responses.

Another example of the stimulation of both TLRs

and NLRs by pathogens is the ability of flagellin to

stimulate both TLR5, which is expressed on the cell

membrane, and the NLR ICE-protease-activating fac-

tor (

IPAF; also known as CARD12 and CLAN) in the

cytoplasm of Salmonella-infected macrophages

53,54

IPAF is required for both caspase-1 activation and IL-1β

production in response to infection

53,54

, and the ability

of the CARD-containing IPAF to activate caspase-1,

unlike the NALPs, is partially independent of the adap-

tor ASC

. The recognition of pathogens by different

intracellular receptors, and in some cases the capacity of

the same microbial molecules to activate both TLRs and

cytoplasmic receptors, highlights a new and interesting

perspective in our understanding of the cooperative

effect of the various PRRs.

The ability of RLRs to cooperate with TLR-induced

signalling pathways has not yet been analysed in detail,

Figure 3 | TLR ligand combinations synergistically induce production of IL-12

p70. Ligands for Toll-like receptor 7 (TLR7), TLR8, TLR9 and TLR11 can synergize

with ligands for TLR3 or TLR4 for interleukin-12 (IL-12) p70 production. Although

no or very little IL-12 p70 is synergistically induced by the combination of ligands

for either TLR2 and TLR3 or TLR4, or TLR3 and TLR4, other pro-inflammatory

cytokines are induced more than additively by these receptor combinations.

MyD88, myeloid differentiation primary-response gene 88; TIRAP, Toll/IL-1 receptor

(TIR)-domain-containing adaptor protein; TRAM, TRIF-related adaptor molecule;

TRIF, TIR-domain-containing adaptor protein inducing interferon-β.

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185

owing to the difficulty in studying RLR ligands in isola-

tion without also activating other receptors, particularly

TLRs. For example, polyI:C has been shown to activate

both TLR3 and MDA5, resulting in the production of

different cytokines

55,56

. In particular, activation of TLR3

by polyI:C was mainly responsible for the induction

of pro-inflammatory cytokines other than type I IFNs,

whereas mice genetically deficient for Mda5 were

unable to produce type I IFNs in response to polyI:C.

However, because the signalling pathways downstream

of the TLR3-associated adaptor TRIF and of RLRs are

in part overlapping, it will be of interest to investigate

the possibility that signalling through RLRs might syn-

ergize with ligands for the TLRs that use MyD88.

PRRs other than the TLRs, NLRs and RLRs have

been implicated in the recognition of fungal patho-

gens in particular. Receptors that recognize mannans

and β-glucans have been shown to have an important

role in the recognition and phagocytosis of zymosan

particles

and in the recognition of Mycobacterium

tuberculosis

58,59

. The interaction of these receptors

with TLR signalling, particularly that of TLR2, results

in either synergy or inhibition of pro-inflammatory

cytokine production

58,59

. In particular, the mouse

β-glucan receptor, dectin-1, has been shown in DCs

to cooperate with various TLRs to increase the pro-

duction of pro-inflammatory cytokines, such as TNF

and IL-12 p40

(REF. 60). This study also showed that

dectin-1 can induce synthesis of the anti-inflammatory

cytokine IL-10 and have an anti-inflammatory effect

through a spleen tyrosine kinase (SYK)-dependent,

TLR-independent signalling pathway. By contrast, the

activation of IRAK-M by the polysaccharide antigen

lipoarabinomannan from M. tuberculosis, probably

through the mannose receptor, negatively regulates

TLR-dependent IL-12 p40 production by macrophages

in an IL-10-independent manner

The triggering of immunoreceptor tyrosine-based

activation motif (ITAM)-associated cell-surface recep-

tors has been reported to have a variable effect on TLR

signalling.

TREM1 (triggering receptor expressed on

myeloid cells 1) and

TREM2 activate myeloid cells by

signalling through the adaptor protein DAP12 (DNAX

activation protein 12), which contains an ITAM in its

cytoplasmic tail. TREM1 amplifies TLR- and NLR-

mediated signalling in neutrophils

61,62

, whereas TREM2

inhibits TLR-induced cytokine production by macro-

phages

63,64

. Among the Fc receptor γ-chain-coupled

receptors (which also contain ITAMs), the crosslinking

of Fc receptors specifically inhibits IL-12 production by

monocytes in response to TLR ligands

. By contrast,

signalling through the human osteoclast-associated

receptor (OSCAR) increases the pro-inflammatory

responses of human monocytes and neutrophils to

TLR ligands

. Because the exogenous and/or endo-

genous ligands of these ITAM-associated receptors are

still unknown, with the exception of the Fc receptors,

the biological relevance of the above findings is unclear.

Nevertheless, the published data clearly indicate that

these receptors are closely involved in the regulation of

the inflammatory response.

Evaluating TLR cooperation in vivo

Mice deficient in the TLR and IL-1R adaptor molecule

MyD88 are highly susceptible to a broad range of bacte-

rial, viral, fungal and protozoan pathogens, which indi-

cates that this family of PRRs might have an important

role in host defence in vivo. Although it has been argued

that MyD88 deficiency can lead to TLR-independent

developmental defects in macrophage or DC func-

tion

67,68

, there is as yet no compelling evidence of a role

for such nonspecific defects in the impaired resistance of

Myd88

–/–

mice to infection in vivo. By contrast, knockout

mice lacking a single TLR have, in most cases, either nor-

mal control of infection or more resistance to infection

than Myd88

–/–

mice

1,5

. In such cases, and in particular

when a well-defined TLR–ligand interaction has been

determined in vitro, the absence of an in vivo phenotype

can be explained by redundancy in TLR function or by

the cooperation of multiple TLR and/or IL-1R signals in

host resistance. However, there is no formal way to dis-

tinguish between these two possibilities. It is of interest

that for several bacterial and protozoan pathogens, the

susceptibility of single-TLR-deficient mice to infection

is increased under conditions of high-dose challenge.

For example, whereas Tlr2

–/–

or Tlr9

–/–

mice have nearly

normal resistance to conventional, low-dose aerosol

challenge with M. tuberculosis, at high doses these mice

are clearly more susceptible to infection than are their

wild-type counterparts

29,69

. This indicates that under

high levels of infectious stress, the function of each TLR

involved in pathogen recognition becomes more crucial

for microbial control, an interpretation that is consistent

with TLR cooperation in host defence.

A further consideration in evaluating the function

of TLRs in host resistance to infection in vivo is that

for certain pathogens, the activation of TLRs might

actually promote infection and/or pathology, such

that TLR deficiency leads to increased, rather than

decreased, host resistance. For example, West Nile virus

seems to use its interaction with TLR3 as a mechanism

to induce a local inflammatory response that results

in the disruption of the blood–brain barrier, thereby

allowing the virus to infect the central nervous system

(CNS). Consequently, Tlr3

–/–

mice are less susceptible

to virus-induced CNS pathology than are wild-type

mice

. Similarly, Tlr2

–/–

mice have decreased mortal-

ity when infected with herpes simplex virus 1 (HSV1),

a phenotype that is attributed to the role of TLR2 in

virus-induced encephalitis

Finally, in considering the outcome of TLR deficiency

in vivo, it is important to take into account the possible

role of TLRs in the negative regulation of anti-microbial

effector responses. For example, Tlr2

–/–

mice have been

reported to be deficient in CD4

CD25

regulatory T (T

Reg

)

cells and ligation of TLR2 on adoptively transferred

Reg

cells impairs their suppressive function in vivo, result-

ing in increased resistance to Candida albicans infection

Therefore, the effect (or lack of effect) of TLR deficiency

on the control of pathogenic infection might not neces-

sarily reflect the direct consequences of TLR signalling on

the effector functions driven by antigen-presenting cells,

as was originally thought.

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TLRs in host defence against pathogens

Despite the above complexities, there are many situa-

tions where it has been possible to establish a role for

multiple TLRs in host defence in vivo and in several

cases to investigate TLR cooperation directly using

mice with genetic deletions of these receptors. For

example, both TLR3-deficient and TLR9-deficient

(or mutated) mice have decreased resistance to infec-

tion with murine cytomegalovirus (MCMV). In both

cases, this decreased resistance was associated with

decreased type I IFN production and IL-12 synthesis

In addition, TLR2 is required for resistance to MCMV

infection and to control the production of IL-18 and

other cytokines, as well as NK-cell activation

. These

data indicate that the expression of at least three TLRs

(TLR2, TLR3 and TLR9) is required for the control of

MCMV infection and that deficiency of any of these

three receptors differentially affects distinct effector

mechanisms and patterns of cytokine secretion, result-

ing in decreased resistance to infection. Nevertheless,

mice deficient for the adaptor molecule MyD88 were

much more susceptible to MCMV infection than

were mice deficient for either TLR2, TLR3 or TLR9

(REF. 75). Therefore, in the case of MCMV, differ-

ent TLRs seem to activate complementary defence

mechanisms, as well as to synergize for the induction

of anti-viral cytokines.

LPS-hyporesponsive mice (for example, C3H/HeJ

mice) have been known for many years to be highly

susceptible to infection with Salmonella typhimurium

a phenotype that is now attributed to defective TLR4

function

. S. typhimurium also contains potent TLR2

ligands, which trigger cytokine production during

infection of macrophages. After either intraperitoneal

or oral S. typhimurium infection, whereas Tlr4

–/–

mice

are more susceptible than wild-type mice to infection

(as expected), Tlr2 and Tlr4 double-knockout mice

are more susceptible to infection than either of the

Tlr2

–/–

or Tlr4

–/–

parental strains

. Moreover, in the case

of oral infection, the mortality rate and infection load

in Tlr2

–/–

Tlr4

–/–

mice were similar to those observed in

Myd88

–/–

mice. No increase in susceptibility to infection

over wild-type mice was observed for the two single-

knockout strains or the double-knockout mice when

given a two-log lower challenge dose. This finding was

interpreted by the authors as being the result of the lack

of a requirement in these conditions to respond to the

acute phase of infection normally accompanying high-

dose challenge. On the basis of in vitro studies with

macrophages exposed to bacterial TLR ligands, they pro-

posed a model of sequential TLR interaction and TNF

induction to explain these in vivo data. In this model,

both TLRs can stimulate TNF production in response to

either endotoxin (TLR4) or bacterial lipoprotein (TLR2)

from the pathogen, but TLR4 and TLR2 are triggered at

different times after infection. The expression of TLR2

is regulated as a consequence of the ligation of TLR4,

which, by contrast, is basally expressed on resting macro-

phages; this provides an explanation for the stronger

phenotype observed in TLR4-deficient mice than in

TLR2-deficient mice

Another example of TLR cooperation that involves the

dual activation of TLR2 and TLR9 has been documented

for infection with M. tuberculosis or Trypanosoma cruzi

in mice

29,30

. Both pathogens contain TLR2 ligands that

stimulate the production of TNF by macrophages. In

addition, TLR9 has an important role in regulating the

production of IL-12 p40 by DCs in response to the live

pathogens in vitro (presumably owing to the activa-

tion of this TLR by CpG motifs present in the patho-

gen DNA). DCs from Tlr2 and Tlr9 double-knockout

mice produced even lower levels of IL-12 p40 than did

cells from single TLR9-deficient mice. Importantly,

double-knockout mice had increased susceptibility to

infection and increased pathogen loads compared with

either wild-type or single TLR2- or TLR9-deficient

mice after challenge with either of the microbial agents.

Indeed, during T. cruzi infection, the peripheral-blood

parasitaemia in Tlr2

–/–

Tlr9

–/–

mice was equivalent to that

seen in Myd88

–/–

mice. Nevertheless, in terms of both

mortality and, in the case of M. tuberculosis infection,

bacterial load, the double-knockout mice were clearly

less susceptible to infection than Myd88

–/–

mice, there-

fore indicating that other MyD88-dependent signalling

events, in addition to TLR2 and TLR9 signalling, are

involved in host resistance to these pathogens.

The synergy between TLR2 and TLR9 is also evi-

dent in the induction of IL-12 p40 and other cytokines

by the live pathogens in vitro. These effects were also

observed when the purified glycosylphosphatidy l-

inositol (GPI) anchor and DNA from T. cruzi were used

as TLR ligands. Deficiency of TLR9, but not of TLR2,

selectively affected IFNγ production by CD4

T cells

in vivo (presumably through defective production of

IL-12), whereas TNF secretion by macrophages was

shown to be regulated mainly by TLR2 in both infec-

tion models

29,30

. The explanation for the failure of TLR2

deficiency to affect T

1-cell responses is not clear, but it

might involve the selective activation of TLR9 on DCs

by the pathogens in vivo. Together, the above data indi-

cate that the observed synergy between TLR2 and TLR9

in the control of M. tuberculosis or T. cruzi infection

stems from their regulation of two distinct effector arms

of the immune response (that is, TNF and IFNγ pro-

duction) that are necessary for the control of infection.

This mechanism contrasts with that emerging from the

studies of MCMV

and S. typhimurium

infection, in

which TLRs that interact to activate the host defence

system do so by cooperating for the production of the

same pro-inflammatory cytokines.

Evidence for yet another mechanism of TLR coop-

eration in host defence comes from a recent report

comparing the roles of the MyD88-dependent and

TRIF-dependent signalling pathways in host resistance

to T. cruzi

. Macrophages and DCs from TRIF-deficient

mice had normal control of intracellular infection. By

contrast, cells from Myd88 and Trif double-knockout

mice were highly susceptible to infection, and were

even more susceptible than the same cell populations

from Myd88

–/–

mice. The TRIF-mediated signalling

that is required for optimal in vitro control of infection

was shown to involve the activation of IFNβ. Indeed,

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187

ab c

Cell

Response

Cell X Cell Y

TLR

interactions

for a single

effector

response

Cooperating TLR

signals for multiple

effector responses

Cell

B A B

Response

Paracrine cytokine

enhancing signal

Cell X Cell Y

Response

Cell X Cell Y

Responses Responses

TLR

ligands

TLR

Adjuvant

An agent mixed with an

antigen that increases the

immune response to that

antigen after immunization.

cells from MyD88- and IFNAR-deficient mice had

similar defects in the control of intracellular infection

to Myd88

–/–

Trif

–/–

cells. Importantly, studies of in vivo

infection confirmed these in vitro findings, with both

Myd88

–/–

Trif

–/–

and Myd88

–/–

Ifnar

–/–

mice having higher

levels of peripheral-blood parasitaemia and accelerated

mortality compared with either MyD88

–/–

or Trif

–/–

mice.

Additional studies

indicated that the effects of IFNβ on

parasite growth are due to the induction of IFN-inducible

gene 47 (IRG47), a p47 GTPase that has previously been

implicated in intracellular host defence

. Although the

specific TLR that is involved in TRIF-dependent IFNβ

induction has not been formally identified, TLR4 is a

probable candidate given the presence of known lig-

ands for this PRR in T. cruzi

. Taken together with the

findings of Bafica et al.

, these observations indicate

that TLR2, TLR9 and possibly TLR4 cooperate for the

control of T. cruzi infection, with each TLR triggering a

distinct effector pathway.

Given their crucial role in detecting bacteria and

viruses that gain entry into the cytoplasm and their

important function in caspase activation, NLRs are likely

to work with TLRs to provide innate immune resist-

ance to these pathogens. As already noted, NOD-receptor

and TLR agonists from bacteria can strongly synergize in

the induction of pro-inflammatory cytokine production

and therefore it is expected that in future studies, mice

with combined deficiencies of TLRs and NOD receptors

or other NLRs will have marked susceptibility to several

infectious agents.

Evolutionary and therapeutic implications

The findings discussed in this Review emphasize the

importance of multiple TLR signalling events in the gen-

eration of host resistance to infection and they also show

that TLR cooperation and synergy can arise through dif-

ferent mechanisms. Hypothetically, TLRs might interact

at the level of the same cell or at the level of multiple cell

types

(FIG. 4). The end result of the triggering of multiple

receptors can be either the enhancement of a single effec-

tor function

73,78

or the coordinated induction of distinct

responses

29,30,79

, which together mediate more effective

control of pathogen growth. It is important to remem-

ber that TLRs and other PRRs are widely distributed on

haematopoietic and non-haematopoietic cells, with each

cell type expressing a typical pattern of PRRs. This pat-

tern of expression is further modulated or modified by

the activation, maturation or differentiation of the cells.

Also, the same cell types, for example epithelial cells or

DCs, might express different patterns of PRRs in differ-

ent tissues. Although the distribution and regulation of

PRR expression by different cell types in different loca-

tions is outside the scope of this Review, it is important

to keep this fact in mind when analysing the role of PRR

cooperation in host resistance to infection.

The evolution of such a multi-pronged, integrated

system for sensing pathogens has obvious advantages for

the host in protecting itself against infectious agents that

might delete or mutate crucial ligands for TLRs or other

PRRs. It also has advantages for protecting against muta-

tions in host PRRs or associated downstream signalling

molecules that would cripple the protective immune

response. The quantitatively limited and often pathogen-

selective effects on host resistance of single-nucleotide

polymorphisms in human TLRs

82,83

are consistent with

this concept.

The concept that multiple TLR–ligand inter actions are

required for the induction of effective host resistance to

pathogens has important implications for the design of

improved strategies for vaccination and immunotherapy

against infectious diseases. Individual TLR7, TLR8 and

TLR9 agonists have already been used successfully as

adjuvants to boost CD4

and CD8

T-cell responses to

candidate microbial vaccine antigens. These agonists

seem to be particularly effective when they are cova-

lently conjugated to the immunogens

84–86

. Several stud-

ies have convincingly shown the improved efficacy of

treatment with multiple TLR ligands compared with

single TLR ligands in stimulating cellular immune

responses in vivo. For example, co-administration of

polyI:C and CpG ODNs increases serum cytokine pro-

duction and the expression of nitric-oxide synthase 2

and MHC class I molecules by immune cells, and this

treatment controls pulmonary metastases in a mouse

tumour model when compared with administration

of either of the ligands alone

. Bone-marrow-derived

DCs loaded with antigen and exposed to both polyI:C

Figure 4 | Summary of the basic mechanisms by which TLR signals cooperate

for the generation of host resistance to infection. The signalling pathways that

activate innate resistance to pathogens involve multiple Toll-like receptor (TLR)–

ligand interactions, which function together to generate a single effector response

(for example, interleukin-12 production), as shown in the upper panels, or involve

the cooperation of distinct TLR signals for the generation of multiple effector

responses, as shown in the lower panels. a | Synergy between multiple TLR signals

in one responding cell to generate a single effector response. b | Enhancement of

one TLR-mediated effector response by a paracrine cytokine signal induced by a

different TLR ligand on a separate cell type. c | Additive responses generated by

distinct TLR signals on different cell types. d | Independent activation of different

effector responses by distinct TLR signals in the same cell. e | Triggering of

different effector responses on separate cell types, which together cooperate to

mediate host resistance. Although not considered here, similar schemes might

regulate the interactions between TLRs and other pattern-recognition receptors.

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VOLUME 7 www.nature.com/reviews/immunol

and the synthetic TLR7 ligand R848 induced markedly

increased cytotoxic T-lymphocyte responses compared

with DCs exposed to either TLR ligand alone

. This

study highlights the importance of direct DC target-

ing for the adjuvant effects of such regimens involving

multiple TLR ligands. The relevance of combined TLR

stimulation in the induction of immunity to pathogens

is underscored by the finding that the live, attenuated

yellow fever vaccine, which confers highly efficient

and long-lasting immunity against viral challenge,

simultaneously and potently activates TLR2, TLR7,

TLR8 and TLR9 on different DC subsets

. The stage

is now set for a major research effort to examine the

protective efficacy of multiple TLR- and PRR-ligand

combinations in vaccines against infectious diseases.

The in vitro and in vivo studies discussed here have

provided an important conceptual foundation for this

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Acknowledgements

We gratefully acknowledge the major contributions of S. Akira

and his colleagues to the work summarized in this Review,

which was made possible in a large part by their engineering

of TLR-deficient mouse strains and their generosity in provid-

ing them to the scientific community. G.T. and A.S. are sup-

ported by the intramural research programs of the National

Cancer Institute (USA) and the National Institute of Allergy

and Infectious Disease (USA), respectively.

Competing interests statement

The authors declare no competing financial interests.

DATABASES

The following terms in this article are linked online to:

Entrez Gene: http://www.ncbi.nlm.nih.gov/entrez/query.

fcgi?db=gene

ASC | caspase-1 | dectin-1 | IL-12 p35 | IL-12 p40 | IPAF |

MDA5 | MyD88 | NALP3 | NOD1 | NOD2 | RIG-I | TIRAP | TLR2 |

TLR3 | TLR4 | TLR5 | TLR7 | TLR8 | TLR9 | Toll | TRAM | TREM1 |

TREM2 | TRIF

FURTHER INFORMATION

Alan Sher’s homepage: http://www3.niaid.nih.gov/labs/

aboutlabs/lpd/immunobiologysection/sher.htm

Access to this links box is available online.

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Apr 2023
INT J MOL SCI

Liver transplantation is the only treatment for hepatic insufficiency as a result of acute and chronic liver injuries/pathologies that fail to recover. Unfortunately, there remains an enormous and growing gap between organ supply and demand. Although recipients on the liver transplantation waitlist have significantly higher mortality, livers are often not allocated because they are (i) classified as extended criteria or marginal livers and (ii) subjected to longer cold preservation time (>6 h) with a direct correlation of poor outcomes with longer cold ischemia. Downregulating the recipient’s innate immune response to successfully tolerate a graft having longer cold ischemia times or ischemia-reperfusion injury through induction of immune tolerance in the graft and the host would significantly improve organ utilization and post-transplant outcomes. Broadly, technologies proposed for development aim to extend the life of the transplanted liver through post-transplant or recipient conditioning. In this review, we focus on the potential benefits of nanotechnology to provide unique pre-transplant grafting and recipient conditioning of extended criteria donor livers using immune tolerance induction and hyperthermic pre-conditioning.

Pattern-recognition receptors in endometriosis: A narrative review

Article

Full-text available

Mar 2023

Endometriosis is closely associated with ectopic focal inflammation and immunosuppressive microenvironment. Multiple types of pattern recognition receptors (PRRs) are present in the innate immune system, which are able to detect pathogen-associated molecular patterns (PAMPs) and danger-associated molecular patterns (DAMPs) in both intracellular and external environments. However, the exact role of PRRs in endometriosis and the underlying molecular mechanism are unclear. PRRs are necessary for the innate immune system to identify and destroy invasive foreign infectious agents. Mammals mainly have two types of microbial recognition systems. The first one consists of the membrane-bound receptors, such as toll-like receptors (TLRs), which recognize extracellular microorganisms and activate intracellular signals to stimulate immune responses. The second one consists of the intracellular PRRs, including nod-like receptors (NLRs) and antiviral proteins retinoic acid-inducible gene I (RIG-I) and melanoma differentiation-associated gene 5 (MDA-5) with helix enzyme domain. In this review, we mainly focus on the key role of PRRs in the pathological processes associated with endometriosis. PRRs recognize PAMPs and can distinguish pathogenic microorganisms from self, triggering receptor ligand reaction followed by the stimulation of host immune response. Activated immune response promotes the transmission of microbial infection signals to the cells. As endometriosis is characterized by dysregulated inflammation and immune response, PRRs may potentially be involved in the activation of endometriosis-associated inflammation and immune disorders. Toll-like receptor 2 (TLR2), toll-like receptor 3 (TLR3), toll-like receptor 4 (TLR4), nod-like receptor family caspase activation and recruitment domain (CARD) domain containing 5 (NLRC5), nod-like receptor family pyrin domain containing 3 (NLRP3), and c-type lectin receptors (CLRs) play essential roles in endometriosis development by regulating immune and inflammatory responses. Absent in melanoma 2 (AIM2)-like receptors (ALRs) and retinoic acid-inducible gene I-like receptors (RLRs) may be involved in the activation of endometriosis-associated immune and inflammation disorders. PRRs, especially TLRs, may serve as potential therapeutic targets for alleviating pain in endometriosis patients. PRRs and their ligands interact with the innate immune system to enhance inflammation in the stromal cells during endometriosis. Thus, targeting PRRs and their new synthetic ligands may provide new therapeutic options for treating endometriosis.

Trypanosoma brucei Lipophosphoglycan Activates Host Immune Responses via the TLR-mediated p38 MAP Kinase and NF-κB Pathways

Article

Full-text available

Feb 2023

Objective This study was aimed at investigating the immunoregulatory effects of trypanosomal lipophosphoglycan (LPG) anchored to trypanosome membranes, including the formation of neutrophil extracellular traps (NETs) and neutrophil cytokine release after parasite infection. The interaction of cell surface TLR receptors with LPG, which signals cellular responses during Trypanosma brucei infection, was systematically investigated. Methods The cytokine expression profile in neutrophils after exposure to T. brucei LPG, and the involvement of TLR2, TLR4, p38 MAP kinase, and NF-κB in NET formation were studied with molecular immunological approaches including quantitative PCR, western blotting and immunofluorescence. Results T. brucei -derived LPG induced phosphorylation of p38 MAP kinase and NF-κB, thereby stimulating neutrophil secretion of IL-1β, IL-8, and TNF-α. The blockade of Toll-like receptor 2/4 and specific inhibitors of MyD88, p38 MAP kinase, and NF-κB decreased cytokine release and the phosphorylation of both kinases. Furthermore, the exposure of neutrophils containing LPG to IL-1β and LPG-induced cell supernatants promoted the release of NETs. Conclusion Our findings suggest that T. brucei LPG activates neutrophil IL-1β secretion via the TLR-mediated p38 MAP kinase and NF-κB pathways, thereby promoting the formation of LPG-stimulated NETs.

Potential Regulation of NF-κB by Curcumin in Coronavirus-Induced Cytokine Storm and Lung Injury

Article

Dec 2022
Int J Prev Med

The current pandemic coronavirus disease-19 (COVID-19) is still a global medical and economic emergency with over 244 million confirmed infections and over 4.95 million deaths by October 2021, in less than 2 years. Severe acute respiratory syndrome (SARS), the Middle East respiratory syndrome coronavirus (MERS), and COVID-19 are three recent coronavirus pandemics with major medical and economic implications. Currently, there is no effective treatment for these infections. One major pathological hallmark of these infections is the so-called 'cytokine storm,' which depicts an unregulated production of inflammatory cytokines inducing detrimental inflammation leading to organ injury and multiple organ failure including severe pulmonary, cardiovascular, and kidney failure in COVID-19. Several studies have suggested the potential of curcumin to inhibit the replication of some viruses similar to coronaviruses. Multiple experimental and clinical studies also reported the anti-inflammatory potential of curcumin in multiple infectious and inflammatory disorders. Thus, we hypothesized that curcumin may provide antiviral and anti-inflammatory effects for treating COVID-19. Although these studies suggest that curcumin could serve as an adjuvant treatment for COVID-19, its molecular mechanisms are still debated, especially its potential to modulate the toll-like receptors/TIR-domain-containing adapter-inducing interferon-β/nuclear factor kappa-light-chain-enhancer of activated B cells (TLR/TRIF/NF-κB) pathway. The preliminary results showed that curcumin modulates the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) pathway, a common pathway controlling cytokine production in multiple infectious and inflammatory disorders. Here, we hypothesize and discuss whether curcumin treatment may provide antiviral and anti-inflammatory clinical advantages for treating COVID-19 by modulating the TLR/TRIF/NF-κB pathway. We also review the current data on curcumin and discuss potential experimental and clinical studies that require defining its potential clinical implications in COVID-19.

Immunogenicity of Recombinant Adeno-Associated Virus (AAV) Vectors for Gene Transfer

Article

Full-text available

Mar 2023
BIODRUGS

Recombinant adeno-associated viruses (AAVs) have emerged as promising gene delivery vehicles resulting in three US Food and Drug Administration (FDA) and one European Medicines Agency (EMA)-approved AAV-based gene therapies. Despite being a leading platform for therapeutic gene transfer in several clinical trials, host immune responses against the AAV vector and transgene have hampered their widespread application. Multiple factors, including vector design, dose, and route of administration, contribute to the overall immunogenicity of AAVs. The immune responses against the AAV capsid and transgene involve an initial innate sensing. The innate immune response subsequently triggers an adaptive immune response to elicit a robust and specific response against the AAV vector. AAV gene therapy clinical trials and preclinical studies provide important information about the immune-mediated toxicities associated with AAV, yet studies suggest preclinical models fail to precisely predict the outcome of gene delivery in humans. This review discusses the contribution of the innate and adaptive immune response against AAVs, highlighting the challenges and potential strategies to mitigate these responses, thereby enhancing the therapeutic potential of AAV gene therapy.

Besides TLR2 and TLR4, NLRP3 is also involved in regulating Escherichia coli infection-induced inflammatory responses in mice

Preprint

Full-text available

Feb 2023

The host TLR2 and TLR4 play critical roles in defense against Escherichia coli ( E. coli ) infection. The NLRP3 inflammasome is the most thoroughly characterized and responds to numerous physically and chemically diverse stimuli. However, the underlying molecular mechanism of TLR2, TLR4 and NLRP3 in the host inflammatory response to E. coli infection remains unclear. This study aimed to explore the roles of TLR2, TLR4 and NLRP3 in regulating the inflammatory response in E. coli infection-induced mice. Our result indicated that macrophages from NLRP3-deficient mice showed significantly reduced secretion of TNF-α and IL-1β and cyclooxygenase-2 (COX-2) expression in response to stimulation with lipopolysaccharide (LPS), braun lipoprotein (BLP), or WT E. coli compared with macrophages from wild-type mice. In addition, TNF-α and IL-1β production in mouse serum after stimulation agreed with the macrophage data. Liver damage in TLR2-deficient, TLR4-deficient, and NLRP3-deficient mice significantly decreased compared to wild-type mice after stimulation with LPS, BLP, or WT E. coli . These results indicate that besides TLR2 and TLR4, NLRP3 is also plays a critical role in host inflammatory responses to defense against E. coli infection, and might provide a therapeutic target in combating disease with bacterium infection.

Smac BV6 Has Proapoptotic and Anti-Inflammatory Effects on Rheumatoid Arthritis Fibroblast-Like Synoviocytes Activated by TLR9 Ligand

Preprint

Full-text available

Apr 2021

Rheumatoid arthritis synovial fibroblasts (RASF) are one of the most important cells contributing reumathoid artritis (RA) pathogenesis. The critical role of the Toll-like receptor in inflammation and on autoimmune disease is not fully known. Data rising predominantly from human patients and animal models of autoimmune disease indicate that inappropriate triggering of TLR pathways by exogenous or endogenous ligands may cause the initiation and/or perpetuation of autoimmune reactions and tissue damage. Particularly, the role of TLR9 in RA is still a subject of debate and among TLRs, TLR9 is the only receptor which detects unmethylated CpG motifs in DNA (ODN), and is located intracellularly in endosomes and endoplasmic reticulum. RASFs stimulated by ODN are apoptosis-resistant and contribute to the pathogenesis of RA by producing cytokines and proteolytic enzymes, which degrade the extracellular matrix. Then, we evaluated the proapoptotic and anti-inflammatory activity of the smac BV6 on RASF cultured in synovial fluid (SF), to reproduce the physiopathological environmental characteristics of RA joints. Furthermore, BV6 induces a significant inhibition of the secretion of IL15, TNF alpha, IL6, stimulation of pannus formation, and damage of bone and cartilage in RA. Moreover the secretion of the anti-inflammatory cytokine IL10 and the cleavage of capase 3 and 8 are dramatically increased in the presence of BV6. Furthermore, TLR9 elicited a robust IFN induction and we reported that in RASF treated with ODN and BV6 the expression of IRF7 is enhanced. Our observations demonstrate that BV6 has beneficial regulatory effects on the inflammatory state induced by TLR9 activation.

Identification of Salmonella Pullorum Factors Affecting Immune Reaction in Macrophages from the Avian Host

Article

May 2023

The host-specific Salmonella serovar S. Pullorum (SP) modulates the chicken immune response to a Th2-biased response associated with persistent infection. This is different from the Th1-biased immune response induced by the genetically close serovar, S. Enteritidis (SE). Based on core genome differences between SP and SE, we used three complementary bioinformatics approaches to identify SP genes, which may be important for stimulation of the immune response. Defined mutants were constructed in selected genes, and the infection potential and ability of mutants to stimulate cytokine production in avian derived HD11 macrophages were determined. Deletion of large genomic regions unique to SP did not change infection potential nor immune stimulation significantly. Mutants in genes with conserved single nucleotide polymorphisms (SNPs) between the two serovars in the region 100 bp upstream of the start codon (conserved upstream SNPs [CuSNPs]) such as sseE, osmB, tolQ, a putative immune antigen, and a putative persistent infection factor, exhibited differences in induction of inflammatory cytokines compared to wild-type SP, suggesting a possible role of these CuSNPs in immune regulation. Single nucleotide SP mutants correcting for the CuSNP difference were constructed in the upstream region of sifA and pipA. The SNP corrected pipA mutant expressed pipA at a higher level than the wild-type SP strain, and the mutant differentially caused upregulation of proinflammatory cytokines. It suggests that this CuSNP is important for the suppression of proinflammatory responses. In conclusion, this study has identified putative immune stimulating factors of relevance to the difference in infection dynamics between SP and SE in avian macrophages. IMPORTANCE Salmonella Pullorum is host specific to avian species, where it causes life-threatening infection in young birds. It is unknown why it is host restricted and causes systemic disease, rather than gastroenteritis normally seen with Salmonella. In the present study, we identified genes and single nucleotide polymorphisms (SNPs; relative to the broad-host-range type Salmonella Enteritidis), which affected survival and immune induction in macrophages from hens suggesting a role in development of the host specific infection. Further studies of such genes may enable understanding of which genetic factors determine the development of host specific infection by S. Pullorum. In this study, we developed an in silico approach to predict candidate genes and SNPs for development of the host-specific infection and the specific induction of immunity associated with this infection. This study flow can be used in similar studies in other clades of bacteria.

Synthesis and Evaluation of a Self-Adjuvanting Pseudomonal Vaccine Based on Major Outer Membrane Porin OprF Epitopes Formulated with Low-Toxicity QS-21-Containing Liposomes

Article

Apr 2023
BIOCONJUGATE CHEM

Pseudomonas aeruginosa (PA) is a Gram-negative pathogen that the World Health Organization has ranked as a priority 1 (critical) threat. One potential prophylactic approach to preventing or reducing the incidence of PA would be development of a long sought-after vaccine. Both antibody and CD4+ T-cell responses have been noted as playing key roles in protection against infection. In these studies, we have designed a prototype vaccine consisting of several known linear B-cell epitopes derived from an outer membrane porin F (OprF). The resulting thiol-containing protein was conjugated to a version of the lipopeptide-based Toll-like receptor agonist Pam3CysSK4Mal (10) containing a maleimide moiety and formulated into dipalmitoylphosphatidylcholine (DPPC)/cholesterol (Chol) liposomes. Mice immunized with the resulting vaccine generated antibodies that bound PA14 (serotype O10) in vitro and induced opsonization in the presence of rabbit complement and murine macrophage RAW264.7 cells. The liposome was optimized to contain 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1,2-dimyristoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (DMPG), Chol, Pam3CysSK4-OprF (12) and the Quillaja saponaria-derived saponin adjuvant QS-21. The resulting vaccine formulation produced significantly higher antibody titers, increased the IgG2a antibody isotype, and increased the number of IgG-producing B-cells as well as splenic primed T-cells. In summary, the liposomal vaccine platform was found highly useful for the generation of a robust and balanced TH1/TH2 response.

Synergy and Cross-Tolerance Between Toll-Like Receptor (TLR) 2- and TLR4-Mediated Signaling Pathways

Article

Full-text available

Dec 2000

A family of Toll-like receptor (TLR) mediates the cellular response to bacterial cell wall components; murine TLR2 and TLR4 recognize mycoplasmal lipopeptides (macrophage-activating lipopeptides, 2 kDa (MALP-2)) and LPS, respectively. Costimulation of mouse peritoneal macrophages with MALP-2 and LPS results in a marked increase in TNF-α production, showing the synergy between TLR2- and TLR4-mediated signaling pathways. Macrophages pretreated with LPS show hyporesponsiveness to the second LPS stimulation, termed LPS tolerance. The LPS tolerance has recently been shown to be primarily due to the down-regulation of surface expression of the TLR4-MD2 complex. When macrophages were treated with MALP-2, the cells showed hyporesponsiveness to the second MALP-2 stimulation, like LPS tolerance. Furthermore, macrophages pretreated with MALP-2 showed reduced production of TNF-α in response to LPS. LPS-induced activation of both NF-κB and c-Jun NH2-terminal kinase was severely impaired in MALP-2-pretreated cells. However, MALP-2-pretreated macrophages did not show any reduction in surface expression of the TLR4-MD2 complex. These findings indicate that LPS-induced LPS tolerance mainly occurs through the down-regulation of surface expression of the TLR4-MD2 complex; in contrast, MALP-2-induced LPS tolerance is due to modulation of the downstream cytoplasmic signaling pathways.

Stimulatory and inhibitory effects of interleukin (IL)-4 and IL-13 on the production of cytokines by human peripheral blood mononuclear cells: Priming for IL-12 and tumor necrosis factor ?? production

Article

Full-text available

Feb 1995

The production of cytokines in monocytes/macrophages is regulated by several different cytokines that have activating or inhibitory effects. Interleukin (IL)-10, IL-4, IL-13, and transforming growth factor (TGF)-beta are usually considered to be the most important macrophage-deactivating factors, with inhibitory effects on cytokine production. Unlike IL-10 and TGF-beta, which appear to act as downmodulators of many phagocytic cell functions, the mode of action of IL-4 and IL-13 is more complex. Addition of IL-4 and IL-13 to peripheral blood mononuclear cell (PBMC) cultures inhibited production of IL-12, tumor necrosis factor (TNF)-alpha, IL-10, and IL-1 beta induced by lipopolysaccharide (LPS) or Staphylococcus aureus added simultaneously with the cytokines. However, pretreatment of PBMC with IL-4 or IL-13 for > or = 20 h enhanced the production of IL-12 and TNF-alpha in response to LPS or S. aureus several fold in these cells; this IL-4-induced priming for the two cytokines was inhibited by anti-IL-4 neutralizing antibodies. IL-4 priming also enhanced the accumulation of IL-12 and TNF-alpha mRNA induced by LPS and S. aureus. The enhanced accumulation of transcripts for the IL-12 p35 and p40 chains by IL-4 priming was reflected in enhanced secretion of both the IL-12 free p40 chain and the p70 heterodimer. These results suggest an unexpected complexity in the regulatory role of IL-4 and IL-13 in immune responses.

The interleukin 12 p40 gene promoter is primed by interferon gamma in monocytic cells

Article

Full-text available

Feb 1996

Interleukin (IL) 12 is a proinflammatory cytokine produced by phagocytic cells, B cells, and other antigen-presenting cells that modulates adaptive immune responses by favoring the generation of T helper type 1 cells. IL-12 mediates some of its physiological activities by acting as a potent inducer of interferon (IFN) gamma production by T and natural killer cells. IFN-gamma enhances the ability of the phagocytic cells to produce IL-12 and other proinflammatory cytokines. Thus, IL-12-induced IFN-gamma acts in a positive feedback loop that represents an important amplifying mechanism in the inflammatory response to infections. We show here that IFN-gamma enhances IL-12 production mostly by priming phagocytic cells for lipopolysaccharide (LPS)-induced transcription of the IL-12 p40 gene, which encodes the heavy chain of the IL-12 heterodimer; furthermore, IFN-gamma directly induces transcription of the IL-12 p35 gene, which encodes the light chain of IL-12, and has at least an additive effect with LPS stimulation in inducing its transcription. The priming effect of IFN-gamma on the LPS-induced p40 gene transcription requires preincubation of the cells with IFN-gamma for at least 8 h to obtain a maximal effect. The priming effect of IFN-gamma for IL-12 production is predominantly at the transcriptional level for both the p40 and the p35 gene, and no evidence for a major role of posttranscriptional or translational mechanisms was found. A 3.3-kb human IL-12 p40 promoter construct transfected into cell lines recapitulated the tissue specificity of the endogenous gene, being silent in two human T cell lines, constitutively active in two human Epstein-Barr virus-positive B lymphoblastoid cell lines, and LPS inducible in the human THP-1 and mouse RAW264.7 monocytic cell lines. Because the RAW264.7 cell line is easily transfectable and regulates the endogenous IL-12 p40 gene in response to IFN-gamma or LPS similarly to human monocytes, it was used for analysis of the regulation of the cloned human IL-12 p40 promoter. A requirement for the region between -222 and -204 in both LPS responsiveness and IFN-gamma priming was established. This region contains an ets consensus sequence that was shown to mediate activation of the promoter by IFN-gamma and LPS, as well as by a cotransfected ets-2. The -222 construct was also regulated in a tissue-specific manner. Two other elements, IRF-1 located at -730 to -719, and NF-IL6 at -520 to -512, were also studied by deletion analysis, which did not result in decreased response to IFN-gamma and LPS stimulation.

Role of a transductional-transcriptional processor complex involving MyD88 and IRF-7 in Toll-like receptor signaling

Article

Oct 2004

Toll-like receptor (TLR) activation is central to immunity, wherein the activation of the TLR9 subfamily members TLR9 and TLR7 results in the robust induction of type I IFNs (IFN-alpha/beta) by means of the MyD88 adaptor protein. However, it remains unknown how the TLR signal "input" can be processed through MyD88 to "output" the induction of the IFN genes. Here, we demonstrate that the transcription factor IRF-7 interacts with MyD88 to form a complex in the cytoplasm. We provide evidence that this complex also involves IRAK4 and TRAF6 and provides the foundation for the TLR9-dependent activation of the IFN genes. The complex defined in this study represents an example of how the coupling of the signaling adaptor and effector kinase molecules together with the transcription factor regulate the processing of an extracellular signal to evoke its versatile downstream transcriptional events in a cell. Thus, we propose that this molecular complex may function as a cytoplasmic transductional-transcriptional processor.

Gout-associated uric acid crystals activate the NALP3 inflammasome

Article

Mar 2007

Dectin-1: A signalling non-TLR pattern-recognition receptor

Article

Jan 2006
NAT REV IMMUNOL

G.D. Brown

Stimulatory and inhibitory effects of interleukin (IL)-4 and IL-13 on the production of cytokines by human peripheral blood mononuclear cells: priming for IL-12 and tumor necrosis factor alpha production

Article

Feb 1995

A. D'Andrea

Synergistic stimulation of human monocytes and dendritic cells by Toll‐like receptor 4 and NOD1‐ and NOD2‐activating agonists

Article

Jul 2005
EUR J IMMUNOL

Muropeptides are degradation products of bacterial peptidoglycan (PG) sensed by nucleotide-binding oligomerization domain 1 (NOD1) and NOD2, members of a recently discovered family of pattern recognition molecules (PRM). One of these muropeptides, muramyl dipeptide (MDP) mediates cell signaling by NOD2, exerts adjuvant activity and synergizes with lipopolysaccharide (LPS) to induce pro-inflammatory responses in vitro and in vivo. In contrast, few and contradictory results exist about the stimulatory capacity of NOD1 agonists. Thus, the ability of NOD1 (MurNAc-L-Ala-γ-D-Glu-meso-diaminopimelic acid, MtriDAP) and NOD2 (MurNAc-L-Ala-D-isoGln, MDP; MurNAc-L-Ala-γ-D-Glu-L-Lys, MtriLYS) agonists to activate primary human myeloid cells was examined. We show that both CD14+ monocytes and CD1a+ immature dendritic cells (DC) express NOD1 and NOD2 mRNA. Stimulation of primary human monocytes and DC with highly purified muropeptides (MtriDAP, MDP and MtriLYS) induces release of pro-inflammatory cytokines. We reveal here that NOD1 as well as NOD2 agonists act cooperatively with LPS to stimulate the release of both pro- and anti-inflammatory cytokines in these myeloid cell subsets. Finally, we report that NOD1 as well as NOD2 agonists synergize with sub-active doses of LPS to induce DC maturation, demonstrating that NOD agonists act cooperatively with molecules sensed by Toll-like receptor 4 to instruct the onset of adaptive immune responses.

Genetic Control of Susceptibility to Salmonella Typhimurium in Mice: Role of the LPS Gene

Article

Feb 1980

Interleukin-12 is required for interferon-gamma production and lethality in lipopolysaccharide-induced shock in mice

Article

Mar 1995

Several cytokines, in particular tumor necrosis factor-α (TNF;-α) and interferon-γ (IFN-γ), have been shown to be responsible for pathological reactions which may lead to shock and death observed in infection with Gram-negative bacteria and in response to endotoxins (lipopolysaccharides, LPS). Priming of mice with the avirulent Bacille Calmette Guerin (BCG) vaccine strain of Mycobacterium bovis increases the sensitivity of mice to the lethal effect of LPS and results in an efficient priming for cytokine production. In response to low doses (1 μg/mouse) of LPS, BCG-primed mice produce interleukin-12 (IL-12) which controls IFN-γ production, as demonstrated by the ability of neutralizing anti-IL-12 antibodies to suppress IFN-γ production. However, the concentration of the biologically active IL-12 p70 heterodimer is similar in the serum of both BCG-primed or unprimed mice, reaching levels of 1-3 ng/ml at 3-6 h after LPS injection, whereas IFN-γ production was observed only in BCG-primed mice. The priming effect of BCG on IFN-γ production appears to be mostly due to its ability to increase TNF-α production, which acts as cofactor with LPS-induced IL-12 in inducing IFN-γ production, as shown by the ability of injection of TNF-α and LPS (1 μg/mouse), but not LPS alone, to induce IFN-γ production. However, in addition to TNF-α, other LPS-induced cofactor(s) are required in cooperation with IL-12 to induce optimal IFN-γ production, because co-injection of TNF-α and IL-12, sufficient to induce serum concentrations of both cytokines higher and more persistent than those obtained by injection of LPS, was not sufficient to induce IFN-γ production in vivo. Neutralizing anti-IL-12 antibodies, in addition to inhibiting the in vivo LPS-induced IFN-γ production, also completely protect BCG-primed mice injected with up to 10 μg of LPS from shock-induced death. Thus, IL-12 is required for IFN-γ production and lethality in an endotoxic shock model in mice.

Cooperation of Toll-like Receptor Signals in Innate Immune Defence

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