Download - Differential inductions of TNF-alpha and IGTP, IIGP by structurally diverse classic and non-classic lipopolysaccharides

Cellular Microbiology (2006)

8

(3), 401–413 doi:10.1111/j.1462-5822.2005.00629.xFirst published online 20 October 2005

© 2005 The AuthorsJournal compilation © 2005 Blackwell Publishing Ltd

Blackwell Science, Ltd

Oxford, UK

CMICellular Microbiology 1462-5814© 2005 The Authors; Journal compilation © 2005 Blackwell Publishing Ltd8

3401413

Original Article

LPS activation patternsN. Lapaque et al.

Received 24 June, 2005; revised 17 August, 2005; accepted 23August, 2005. *For correspondence. E-mail [email protected];Tel. (

+

33) 4 91 26 93 15; Fax (

+

33) 4 91 26 94 30.

Differential inductions of TNF-

αααα

and IGTP, IIGP by structurally diverse classic and non-classic lipopolysaccharides

Nicolas Lapaque,

1

Osamu Takeuchi,

2

Fernando Corrales,

3

Shizuo Akira,

2

Ignacio Moriyon,

4

Jonathan C. Howard

5

and Jean-Pierre Gorvel

1

*

1

Centre d’Immunologie INSERM-CNRS-Université Méditerranée, case 906, 13288 Marseille, Cedex 9, France.

2

Department of Host Defense, Research Institute for Microbial Diseases, Osaka University, 3-1 Yamada-oka, Suita Osaka 565-0871, Japan.

3

Centro de Investigación Médica Aplicada, University of Navarra, Avda. Pío XII, 5531008 Pamplona, Spain.

4

Department of Microbiology, University of Navarra, c/Irunlarrea 1, 31008 Pamplona, Spain.

5

Institute for Genetics, University of Cologne, Zülpicher Strasse 47, D-50674 Cologne, Germany.

Summary

The innate immune system recognizes microbesby characteristic molecules like the Gram-negativelipopolysaccharide (LPS). Lipid A (the LPS bioactivemoiety) signals through toll-like receptors (TLRs)to induce pro-inflammatory molecules and smallGTPases of the p47 family involved in intracellularpathogen control. We tested TNF-

αααα

and p47-GTPaseinduction in macrophages using classical LPSs [lipidAs with glucosamine backbones, ester- and amide-linked C14:0(3-OH) and C12 to C16 in acyloxyacylgroups] of wild type and mutant

Escherichia coli

and

Yersinia

species and non-classical LPSs [lipid As withdiaminoglucose, ester-linked 3-OH-fatty acids andC28:0(27-OH) and C23:0(29-OH) in acyloxyacylgroups] of plant endosymbionts (

Rhizobium

), intrac-ellular pathogens (

Brucella

and

Legionella

) andphylogenetically related opportunistic bacteria(

Ochrobactrum

). Classical but not non-classical LPSsefficiently induced TNF-

αααα

, IIGP and IGTP p47-GTPaseexpression. Remarkably, the acyloxyacyl groups inclassical LPSs necessary to efficiently induce TNF-

αααα

were not necessary to induce p47-GTPases, suggest-ing that different aspects of lipid A are involved in thisdifferential induction. This was confirmed by usingPPDM2, a non-endotoxic lipid A-structurally relatedsynthetic glycolipid. Despite their different bioactivity,all types of LPSs signalled through TLR-4 and notthrough TLR-2. However, whereas TNF-

αααα

inductionwas myeloid differentiation factor 88 (MyD88)-depen-dent, that of p47-GTPases occurred via a MyD88-independent pathway. These observations show thatdifferent aspects of the LPS pathogen-associatedmolecular pattern may be triggering different signal-ling pathways linked to the same TLR. They also rein-force the hypothesis that non-classical lipid As act asvirulence factors by favouring the escape from theinnate immune system.

Introduction

Innate immunity was formerly thought to be non-specific,characterized mostly by digestion of pathogens and for-eign components by professional phagocytes. However,innate immunity has considerable specificity and discrim-inates between pathogen components and self so that aresponse is triggered well before effectors of adaptiveimmunity are available. Initial detection of microbial invad-ers is achieved through the recognition of pathogen-asso-ciated molecular patterns (PAMPs) by specific peptides,proteins and cell receptors, among which the toll-likereceptors (TLRs) are of paramount importance. More than10 members of the TLR family have been reported inhuman and mouse databases, and TLRs 1, 2, 3, 4, 5, 6and 9 are critical signal transducers for molecules bearingPAMPs and as well as for some host stress proteins.Whereas recognition by TLRs 1, 3, 5, and 9 is morerestricted [some lipopeptides, double stranded viral RNA,bacterial flagellin protein, and unmethylated (bacterial)CpG DNA, respectively], TLRs 2 and 4 recognize a largervariety of microbial products, mostly amphiphilic lipopro-teins and glycolipids of which the most significant is thelipopolysaccharide (LPS) of Gram-negative bacteria(reviewed by Takeda

et al

., 2003; Akira and Takeda,2004). LPS (or endotoxin) is a major envelope componentand may be massively released at the onset of infection

402

N. Lapaque

et al.

© 2005 The AuthorsJournal compilation © 2005 Blackwell Publishing Ltd,

Cellular Microbiology

,

8

, 401–413

by the action of bactericidal peptides and proteins, com-plement and other innate immunity effectors targeted tothe bacterial envelope but also in lesser amounts duringbacterial division. The majority of host cells recognizeminute amounts of the released LPS through the incom-pletely characterized CD14-MD2-TLR4 system (Akashi

et al

., 2000a,b; Jiang

et al

., 2000; da Silva Correia

et al

.,2001) in which the last molecule plays a critical role asillustrated by the fact that TLR4 genetically deficient indi-viduals are essentially unresponsive to LPS (Poltorak

et al

., 1998; Takeda

et al

., 2003). Although some LPSswere reported to be recognized by TLR2 and not by TLR4,the consensus is that, when free of contaminants, allLPSs and synthetic lipid As are initially recognized byTLR4 (Hirschfeld

et al

., 2000; Tapping

et al

., 2000). Theoutcome of LPS recognition by TLR4 is the expression ofpro-inflammatory molecules, including TNF-

α

, IL-6 and IL-12, all critical for an effective immune response, which inturn may also lead to endotoxic shock (reviewed by Alex-ander and Rietschel, 2001; Erridge

et al

., 2002).It has been observed that LRG-47, IGTP and IIGP, three

members of the p47 GTPase family, are induced uponexposure of mouse cells to

Escherichia coli

LPS (Sorace

et al

., 1995; Taylor

et al

., 1996; Zerrahn

et al

., 2002), sug-gesting a role for these GTPases in defence againstGram-negative bacteria. The p47 GTPase family (TGTP,IIGP, IRG-47, GTPI, IGTP and LRG-47), previously shownto be induced by interferon-

γ

(IFN-

γ

) (Boehm

et al

., 1998)has no sequence homology to other GTPases outside theGTP-binding region. IGTP (Taylor

et al

., 1997) and IIGP-1 (Martens

et al

., 2004) localize predominantly to theendoplasmic reticulum while LRG-47 is predominantlylocalized in the Golgi (Martens

et al

., 2004). Although themechanism of action of these GTPases is still unknown,some of them have been implicated in host resistanceagainst intracellular pathogens (reviewed in Taylor, 2004).Thus, IGTP- and IRG-47-knockout mice are susceptibleto

Toxoplasma gondii

but not to

Listeria monocytogenes

whereas LRG-47-knock-out mice are highly susceptible toboth these organisms. LRG-47 has also been involved inhost resistance against the Gram-negative bacteria

Sal-monella typhimurium

(Taylor, 2004). Although it has beenreported that TGTP-1 expression in fibroblasts can induceresistance to vesicular stomatitis infection

in vitro

, no viralresistance due to p47 GTPases has yet been demon-strated

in vivo

(Carlow

et al

., 1998).The specificity ofthese p47-mediated resistances contrasts with IFN-

γ

knockout mice, which are susceptible to most infections.The LPS molecule is composed of three parts: the O-

polysaccharide, the core oligosaccharide and the lipid A.The latter moiety carries the PAMP directly responsible forendotoxicity. Most common Gram-negative pathogenicbacteria express lipid As (henceforth called classical lipidAs) formed by a bisphosphorylated glucosamine disac-

charide carrying two amide and two ester-linked 3-OH-hydroxymiristate [C14:0(3-OH)]. In turn, additional C12:0to C16:0 fatty acids may be ester linked to those 3-OHgroups in the tetraacyl form, thus creating one or twoacyloxyacyl groups and penta or hexaacyl lipid As respec-tively. In most wild-type bacteria hexaacyl lipid A is themost abundant one, but variations are found in pathogenssuch as

Yersinia

(Rebeil

et al

., 2004; Knirel

et al

., 2005).In addition, some bacteria express non-classical lipid As.For instance, lipid As of

Brucella

,

Ochrobactrum

and

Legionella

(members of the

α

2- and

γ

-

Proteobacteria

,respectively) contain mono- or bisphosphorylated diami-noglucose disaccharides and, accordingly, only possess3-OH acyl groups in amide linkages. Moreover, the acy-loxyacyl groups carry much longer chains, includingC28:0(27-OH) and C30:0(29-OH).

In this study, we have investigated the effects of lipid Astructure on the induction of IIGP and IGTP expression inmacrophages by using classical and non-classical lipidAs, related synthetic glycolipids as well as phospholipids.Classical lipid As strongly induced TNF-

α

and the expres-sion of both p47-GTPases, regardless of their particularstructural variations. However, the structural modificationsof this PAMP present in non-classical LPSs led to ineffi-cient TNF-

α

induction. In addition, although they stillretained the ability to induce IIGP and IGTP expression,we show that non-classical lipid As were capable of induc-ing markedly lower levels of IIGP and IGTP expression.We report that these non-classical LPSs signal through aTLR-4-positive and myeloid differentiation factor 88(MyD88)-negative pathway.

Results

Lipopolysaccharide structural requirement for inducing IGTP and IIGP expression and TNF-

α

expression

We used an array of LPSs, including O-polysaccharide-bearing and O-polysaccharide -defective forms, differingin lipid A structure to test whether they would induce IIGPand IGTP expression in mouse peritoneal macrophages.Classical S-LPSs from

E. coli

and

Yersinia enterocolitica

serotypes O:3 and O:9 (containing mostly hexaacyl lipidA; Fig. 1 and Table 1), induced IGTP and IIGP expressionat concentrations equivalent to 0.5–5 nM of lipid A(Fig. 2A and data not shown). Similar results wereobtained with

S. typhimurium

and

Shigella flexneri

LPSs (data not shown). Moreover, LPSs from

E. coli

mutants that produce mostly pentaacyl or tetraacyl lipid A(Fig. 1 and Table 1) (Clementz

et al

., 1996; Clementz

et al

., 1997) or from

Yersinia pestis

KIM (with tri- andtetraacyl lipid A, Fig. 1, Table 1) were also able to inducethe expression of IGTP and IIGP under equivalent condi-tions (Fig. 2A). As some of these LPSs lack the O-

LPS activation patterns

403



,

8

, 401–413

polysaccharide, this first set of observations demonstratenot only that hexaacyl as well as penta and tetraacylenterobacterial-type lipid As induce similar levels of IIGPand IGTP, but also that O-polysaccharides are not neededfor the activation process. In contrast, the ability of theseclassical LPSs to induce TNF-

α

decreased in parallel totheir lipid A acylation defects (Fig. 2B). This is consistentwith previous evidence that showed the reduced endotox-icity of these non-fully acylated compounds (Somerville

et al

., 1996; Schromm

et al

., 2000; Seydel

et al

., 2003).To assay whether the above conclusions could beextended to non-classical LPSs, we tested those from the

α

-2

Proteobacteria

intracellular animal pathogens

Bru-cella abortus

and

Brucella melitensis

, the soil living oppor-tunistic pathogen

Ochrobactrum anthropi

and the plantendosymbiont

Rhizobium etli

(Fig. 1 and Table 1). As illus-trated in Fig. 2B for

B. abortus

LPS, these LPSs were lessefficient in inducing TNF-

α

secretion, an observation con-sistent with their reduced endotoxicity (Zahringer

et al

.,1995; Neumeister

et al

., 1998; Vandenplas

et al

., 2002;Moriyon, 2003). Moreover, when tested at the same con-centrations as the classical LPS, they failed to elicit detect-

able IGTP expression (not shown) and only a weakinduction was observed at tenfold higher concentrations(Fig. 2C). Interestingly, this result was not restricted toLPSs

α

-2

Proteobacteria

, because the LPSs of threestrains of

Legionella pneumophila

(

γ

-

Proteobacteria

mem-bers like

Salmonella

and

E. coli

) also induced a weakIGTP expression at higher concentrations (Fig. 2C). Com-pared with wild-type

L. pneumophila

(RC1) LPS, strain5215 possesses a different O-chain (Kooistra

et al

., 2001)and 2–4 CH2 units shorter fatty acids are found in thereducing diaminoglucose of the lipid A of strain 811 (Koo-istra

et al

., 2002) (Fig. 1 and Table 1). These slight modi-fications seemed not to alter the ability to induce IGTPexpression (Fig. 2C). Likewise, comparison of the sugarbackbones of the non-classical lipid As shows differenceswith regard to the phosphate groups and, in the case

ofR. etli

, presence of galacturonic and aminoglucuronic acid(Fig. 1). On the other hand, and regardless of the taxo-nomical or biological group (e.g. intracellular pathogens,environmental or endosymbiont) of the correspondingbacteria, amide-linkages at the backbone disaccharideand long-chain fatty acids in acyloxyacyl linkages are fea-

Table 1.

Lipopolysaccharide structural characteristics.

Type of LPS/Strain(growth temperature) Description monoisotopic mass of the dominant lipid A forms)

a

References

Classical

E. coli

0127 (37

°

C)(EC)

Smooth-LPS;

>

90% hexaacyl 1823.3 Da); penta and tetraacyl traces This work

E. coli

MLK53;

htrB-

(37

°

C) (EC003)Rough-LPS; pentaacyl lipid A deficient in C12 oxyacyl of 3-OH-C14 acyl at GlcN

C2

′

1615.1 Da)Clementz

et al

. (1996)

E. coli

MLK 1067;

msbB-

(37

°

C)(EC004)

Rough-LPS;

>

90% pentaacyl 1587.0 Da); tetraacyl traces Clementz

et al

. (1997) andthis work

E. coli

MLK986;

msbB

-,

htrB

-(26

°

C) (EC005)

Rough-LPS; 29% pentaacyl 1643.0 Da); 54% tetraacyl 1404.8 Da); and 17%triacyl 1178.6 Da)

Clementz

et al

. (1997);Clementz

et al


Y. enterocolitica

O:3WE245/92 (26

°

C)(YE)

Smooth-LPS; 9% heptaacyl; 54% hexaacyl 1795.0 Da); 11% arabinosamine-hexaacyl 1928.3); 18% tetraacyl 1404.8 Da)

This work

Y. enterocolitica

O:9MY79 (26

°

C)Smooth-LPS bearing a

B. abortus

-type N-formylperosamineO-polysaccharide; hexaacyl lipid A is dominant

Aussel

et al

. (2000);Perry and Bundle (1990)

Y. pestis

KIM (26

°

C)(YP)

Rough-LPS, 9% hexaacyl 1797.2 Da); 10% pentaacyl; 40% tetraacyl 1404.8 Da); 7% arabinosamine-tetraacyl 1535.9 Da); 30% triacyl 1178.6 Da)

This work

Non-classical

B. abortus

2308(37

°

C) (BA)Smooth-LPS; hexaacyl lipid A several high mass forms; 2101. 6 and 2079.7 Da

are representative)Ferguson

et al

. (2004);Velasco

et al


B. melitensis

16M(37

°

C)Smooth-LPS; lipid A similar to that of

B. abortus

by HPTLC This work

O. anthropi

LMG3331 (37

°

C) (OA)Smooth-LPS; hexaacyl lipid 2101.6 Da) Velasco

et al

. (2000)

L. pneumophila

RC1 Smooth -LPS; hexaacyl lipid A 2229.7 Da) Zahringer

et al

. (1995) andU. Zähringer (pers. comm.)

L. pneumophila

811(37

°

C)Smooth-LPS; mutant of RC1, 3-OH fatty acids at C2 and C2

′

are 2–4 CH2 unitsshorter

Luneberg

et al

. (1998)

L. pneumophila

5215(37

°

C)Smooth-LPS; isogenic mutant of RC1 differing in O-chain Luneberg

et al

. (1998)

R. etli

CE3 (26

°

C) Smooth-LPS; pentaacyl lipid A 1985.5 and 2001.5 Da) Que

et al

. (2000)

a.

For structures see Fig. 1.

404

N. Lapaque

et al.



,

8

, 401–413

Fig. 1.

Lipid As and analogue structures. The proposed structures correspond to the lipid A dominant forms observed (mass is in parenthesis) here and in previous works (see also Table 1).A. The hexaacyl, pentaacyl, tetraacyl and triacyl lipid A forms present in the

E. coli

and

Yersinia

LPSs (less abundant forms of the same degree of acylation were also observed). See Table 1 for the proportion of acyl chains in

E. coli

or

Yersinia

LPS species. These LPSs possess bisphosphorylated glucosamine disaccharide backbones carrying amide and ester-linked C14:O(3-OH), in some cases decorated with arabi-nosamine at 4

′

.B. In some lipid As (

Brucella-Ochrobactrum

and

Legionella

), the most abundant forms have diaminoglucose backbones that carry very long-chain fatty acids such as 27OHC28 in acyloxyacyl groups. In others (

Rhizobium etli

CE3), the most abundant forms have backbones of glucosamine disaccharides with galacturonate at 4

′

, or of aminogluconate, glucosamine and galacturonate. The latter lipid As are pentaacylated, with very long-chain fatty acids in acyloxyacyl groups decorated with

β

-hydroxybutyrate at the subterminal hydroxyl group. PPDM2 is a synthetic compound that possesses two 3-hydroxymyristic acyl groups amide-linked to a bisphosphorylated diaminoglucose.

LPS activation patterns

405



,

8

, 401–413

Fig. 1.

cont

.

406

N. Lapaque

et al.



,

8

, 401–413

Fig. 3.

PPDM2 induce p-47 GTPases expression. Peritoneal macrophages stimulated with different

E. coli

and

B. abortus

LPS concentrations equivalent to 0.5 (a), 5 (b) and 50 (c) nM of lipid A, or with the synthetic compound PPDM2. Macrophages lysates were collected 24 h after stimulation, and equal protein amounts of each lysate were analysed for IGTP and IIGP expression by western blotting.

Contro

l

PPD

M2 c

E. col

i aB. a

bortu

s a

E. col

i b

B. abo

rtus b

B. abo

rtus c

PPD

M2 a

PPD

M2 b

IGTP

IIGP

Fig. 2.

p47-GTPases and TNF-

α

induction do not require similar lipid A structures.A. IGTP and IIGTP expression by peritoneal macrophages from wild-type mice stimulated with IFN-

γ

(20 Units ml

−

1

) or buffer (control) or LPSs from

E. coli

0127 (

E. coli

),

E. coli

lipid A mutants MLK53

htrB

- (EC003), MLK1067

msbB

- (EC004) and MLK986

htrB

-/msbB- (EC005),

Y. pestis, Y. enterocolitica

O:3 or

Y. enterocolitica

O:9, all adjusted to a concentration equivalent to 5 nM lipid A. Macrophage lysates were collected 24 h after stimulation, and equal protein amounts of each lysate were analysed for IGTP and IIGP expression by Western blotting.B. TNF-

α

expression by peritoneal macrophages from wild-type mice stimulated with LPSs from

Y. enterocolitica

O:3 (YE),

Y. pestis

(YP),

E. coli

0127 (EC),

E. coli

lipid A mutants MLK53

htrB

- (EC003), MLK1067

msbB

- (EC004), MLK986

htrB

-/msbB- (EC005),

O. anthropi

(OA) and

B. abortus

(BA) adjusted at concentrations equiv-alent to 0.05; 0.5 and 5 nM lipid A. Supernatants were collected 24 h after stimulation and TNF-

α

determined by ELISA.C. IGTP expression by peritoneal macrophages from wild-type mice incubated with LPSs from

Y. pestis

,

E. coli

and

O. anthropi

at a concentration equivalent to 5 nM lipid A, or with LPSs from

B. abortus, B. melitensis, L. pneumophila

WT 5215, 811 and 55215, and

R. etli

at a concentration equivalent to 50 nM lipid A. Macrophage lysates were collected 24 h after stimulation, and equal protein amounts of each lysate were analysed for IGTP expression by Western blotting.

IFN-g

20 U

Control

Y. ente

rocoli

tica 0

:3

Y. pest

is

EC003

EC004

EC005

E. coli

Y. ente

rocoli

tica 0

:9

IGTP

IIGP

A

50 nM

B. meli

tens

is O. a

ntro

pi

E. coli

B. abo

rtus

L. pne

umop

hila

RC1

L. pne

umop

hila 8

11

L. pne

umop

hila 5

312

R. etli

Y. pes

tis

Contro

l

IGTP

C

B

tures shared by these less active non-classical LPSs(Fig. 1). Therefore, induction of IGTP and IIGP as well asTNF-

α

expression seem to be negatively correlated withone or both of these structural traits. Moreover, for classi-cal lipid As (

E. coli wild type vs. E. coli mutants), IGTPand IIGP expression is independent from the degree oflipid A acylation, contrasting with the requirement of theseacyl chains for TNF-α expression (Fig. 2).

Synthetic lipid A-structurally related monosaccharide glycolipid induces IGTP and IIGP expression

The above results showed that variations in endotoxicitymediated by structural changes in classical LPSs did notcorrelate with IGTP and IIGP expression. To strengthenthese findings and to establish the role of the lipid Asugars in IGTP and IIGP expression, we tested PPDM2,a synthetic lipid A-related structure (Fig. 1) that behavesas an antagonist of endotoxic shock and does not induceTNF-α at nanomolar concentration (Chaby et al., 1993;Pedron et al., 1994; Charon et al., 1998). Despite being amonosaccharide glycolipid, PPDM2 displayed substan-tially more p47-GTPase inducing activity than non-classi-cal LPSs, although less than the classical LPSs (Fig. 3).This confirms that endotoxicity and IGTP/IIGP expressionare unrelated mechanisms. Furthermore, it seems thatester linkages (missing in PPDM2) are not critical in theinduction of these GTPases.

To study whether by themselves the very long-chainfatty acids in non-classical LPS correlated with a poorinduction of IGTP and IIGP, we used phosphatidylcholineand phosphatidylethanolamine carrying C12 to C24 acylchains. None of these compounds was able to inducesignificant expression of IGTP and IIGP (data not shown).Thus, acyl chains linked to a C3 backbone linked to phos-

LPS activation patterns 407

© 2005 The AuthorsJournal compilation © 2005 Blackwell Publishing Ltd, Cellular Microbiology, 8, 401–413

phoethanolamine (altogether 5 C units) or phophorylcho-line (altogether 8 C units) are not sufficient for theinduction of p47-GTPases. Therefore, the bisphosphory-lated pyranose moiety seems to be essential, probablyproviding the required conformation and spacing of theacyl chains.

Lipopolysaccharide does not induce the expression of TNF-α, IGTP and IIGP in TLR-4 deficient macrophages

To investigate the pathway of LPS-dependent induction ofp47-GTPase expression, we exploited peritoneal mac-rophages from C3H/HeJ mice, which are resistant toclassical LPS due to a spontaneous mutation on thecytoplasmic tail of the TLR4 (Poltorak et al., 1998; Pol-torak et al., 2000). As controls, were used macrophagesfrom C3H/HeN mice, which do not carry this mutation. Thepattern of strong induction of IGTP and IIGP expressionby E. coli LPS (at 0.5 and 5 nM lipid A) and weak inductionby B. abortus LPS (at 50 nM lipid A) was maintained inC3H/HeN but not in C3H/HeJ murine macrophages whichfailed to response to either LPS (Fig. 4A). In contrast,GTPase induction by IFN-γ was not affected by the muta-tion in C3H/HeJ (Fig. 4A). Thus, TLR4 is involved in induc-tion of the p47-GTPases by classical and non-classicalLPSs. In order to confirm and extend this, we performedsimilar experiments using wild type and TLR4 knockoutmice and investigated a potential role for TLR2 in p47-GTPase induction using TLR2 knock-out mice. Consistentwith the above-described results, neither IGTP nor IIGPwas detected in macrophages from TLR4 knockout micestimulated with either E. coli or B. abortus LPS (Fig. 4B).However, in both wild type and TLR2 knockout macroph-ages, E. coli LPS and B. abortus LPS induced a strongand a weak induction of the p47-GTPases respectively(Fig. 4B). As expected, the levels of TNF-α were muchhigher with classical than with non-classical LPSs in wildtype and TLR2 knockout macrophages. No TNF-α wasproduced by TLR-4 knockout macrophages (Fig. 4D). Thisis consistent with the observation that LPSs are depen-dent on TLR-4 but not on TLR-2. In addition, the slightinduction of TNF-α and p47-GTPases by non-classicalLPSs correlates with their low endotoxicity.

Induction of IIGP and IGTP is MyD88-independent

It has been proposed that the signal translocated by TLR-4 occurs via two different main pathways, one dependenton MyD88-IRAK and the other bypassing these mole-cules, usually called MyD88-independent pathway(reviewed by McGettrick and O’Neill, 2004). In order toidentify which of these two pathways is used to inducep47-GTPases expression by LPSs, we treated macroph-ages from MyD88 knockout and wild-type mice with dif-

Fig. 4. Induction of p47 GTPases by LPS is dependent on TLR4 but not on TLR2 or MyD88. Peritoneal macrophages from (A), C3H/HeN and TLR4-mutants (C3H/HeJ) or (B), wild type (WT), and TLR2 and TLR4 knockout mice were stimulated for 24 h with IFN-γ at 1 and 5 U ml−1 or with E. coli and B. abortus LPSs concentrations equivalent to 0.5 (a), 5 (b) and 50 (c) nM lipid A. In (C), peritoneal macrophages from wild type C57/Bl6 (WT) and MyD88-deficient mice were stimu-lated for 24 h with IFN-γ at 1 and 5 U ml−1 or with E. coli and B. abortus LPSs concentrations equivalent to 0.5 (a), 5 (b) and 50 (c) nM lipid A. In (D), peritoneal macrophages from wild type (WT), TLR2, TLR4 and MyD88 deficient mice were stimulated for 24 h with LPSs from Y. enterolitica (YE), E. coli (EC), O. antropi (OA) or B. abortus (BA) adjusted at a concentration equivalent to 0.5 nM lipid A. Mac-rophage lysates were collected 24 h after stimulation, and equal protein amounts of each lysate were analysed for IGTP and IIGTP expression by Western blotting (A–C), or TNF-α in culture superna-tants measured by ELISA (D).

B

A Control

IFN-γ

1U

B. abo

rtus a

B. abo

rtus b

B. abo

rtus c

IFN-γ

5U

E. coli

a

E. coli

b

WTTLR2-/-TLR4-/-

WTTLR2-/-TLR4-/-

C3H/HeNC3H/HeJ

C3H/HeNC3H/HeJ

IGTP

IIGP

IGTP

IIGP

CWT

Control

IFN-γ

1U

B. abo

rtus a

B. abo

rtus b

B. abo

rtus c

IFN-γ

5U

E. coli

a

E. coli

b

MyD88-/-

WT

MyD88-/-

IGTP

IIGP

D

408 N. Lapaque et al.


ferent LPSs. No differences were observed between theinduction of the p47 GTPases in MyD88-deficient andwild-type macrophages (Fig. 4C). Therefore, IGTP andIIGP induction by LPS is MyD88-independent.

Lipopolysaccharide induces the expression of IIGP and IGTP through type-I IFN

In time-course experiments, we observed that p47-GTPase induction by LPS was delayed by several hoursrelative to IFN-γ-mediated induction (data not shown).This result suggested that LPS might be acting throughan indirect pathway, in contrast to IFN-γ. As it has beendescribed that IIGP induction by E. coli LPS dependscompletely on the type-I IFN receptor (Zerrahn et al.,2002), we tested whether this would be also the case forlow endotoxic non-classical LPSs. To this end, wild-typemurine macrophages were stimulated with B. abortusLPS, E. coli LPS, IFN-β or IFN-γ, with or without neutral-izing antibody specific for type I IFNs and assayed forinduction of IGTP and IIGP expression. The induction ofIGTP and IIGP by LPS was clearly reduced in the pres-ence of anti-type I interferon antibody and, on the otherhand, this antibody had no effect on the induction of theGTPases by IFN-γ (Fig. 5). Therefore, p47-GTPaseexpression is induced by LPS through a type-I IFN-depen-dent indirect pathway.

Discussion

The molecular characterization of PAMPs, particularly thatof LPSs, is of a great interest because the innate immunesystem responds to minute amounts of these microbialcompounds by releasing pro-inflammatory and inflamma-tory mediators that act as earlywarning messengers. Adramatic pathophysiological consequence of this system

is endotoxic shock, which occurs when microbial media-tors, mainly LPSs, induce a massive release of cytokines(reviewed by Alexander and Rietschel, 2001; Erridgeet al., 2002). However, some Gram-negative bacteria thatreplicate extensively to reach high numbers in tissues andcells do not induce endotoxic shock. For instance, Bru-cella and other intracellular pathogens specialized inchronic disease outcome and not acute disease outcomesuch as salmonellosis must hide into host cells and lowerthe host responses in order to survive and replicate. Bru-cella and Legionella LPSs are built in this way and it isnot surprising that they behave as poor inducers. Thus,infections caused by the intracellular parasites Brucella,Legionella as well as by several rickettsiae do not causetypical manifestations of endotoxicity, unlike systemicinfections mediated by Escherichia, Salmonella,Pseudomonas and many other Gram-negative bacteria(Moreno and Moriyón, 2003). This divergence is explainedin part by the different structures of classical and non-classical lipid As. Classical endotoxic lipid As bear PAMPmoieties efficiently recognized by the CD14-MD2-TLR-4system in cooperation with LBP (Akashi et al., 2000b;Tapping et al., 2000). It is also known that the degree andtype of lipid A acylation modulates endotoxicity (Seydelet al., 1999), and that tetraacyl forms of lipid A (lackingthe acyloxyacyl linked fatty acids) such as the biosyntheticprecursor lipid IVa are not endotoxic (Rossignol et al.,1999). Indeed, it has been shown that pure tetraacyl formsdo not signal efficiently through human TLR-4 and thathexa- and pentaacyl lipid A structures are distinguishedby human TLR4 (Hajjar et al., 2002; Janusch et al., 2002;Backhed et al., 2003). Moreover, there are host peculiar-ities in the recognition of lipid A forms differing in thedegree of acylation that are linked to TLR-4 species-spe-cific features (Golenbock et al., 1991; Akashi et al., 2001;Hajjar et al., 2002). Recently, it has been shown thataggregates, rather than single molecules, are the biolog-ically active forms of LPSs and that the activity of hexaacyllipid A is strongly potentiated by the simultaneous pres-ence in the aggregate of underacylated forms, includingtetraacyl lipid A (Mueller et al., 2004). This is relevantbecause wild-type LPSs are, in fact, aggregates carryingseveral lipid A structures differing in the degree of acyla-tion. As illustrated here, small proportions of penta andtetraacyl forms are present along hexaacyl lipid A in wild-type E. coli LPS, and penta and tetraacyl forms increasesignificantly or even dominate in the LPS of Yersinia spe-cies depending upon the growth temperature (Rebeilet al., 2004; Knirel et al., 2005). By using LPSs represen-tative of the above-summarized lipid A structure andaggregate possibilities, we compared their influence onthe stimulation of mouse macrophages for TNF-α or p47-GTPase expression. For the classical LPS, and withregard to their ability to induce TNF-α, we confirmed the

Fig. 5. LPSs induce p47-GTPase via type-I IFN. Peritoneal macroph-ages from wild-type mice were activated with IFN-γ 10 U ml−1, IFN-α (1000 and 100 U ml−1), or with E. coli and B. abortus LPSs adjusted at a concentration equivalent to 5 and 50 nM of lipid A respectively. Anti-type I IFN neutralizing antibodies were added where indicated by a (+) sign. Macrophage lysates were collected 24 h after stimula-tion, and equal protein amounts of each lysate were analysed for IGTP and IIGTP expression by Western blotting.

a n t i -I F Nα / β - + - + - + - + - +

IGTP

IIGP

Control

IFN-γ

10U

IFN-α

1000

UE. c

oli b

B. abo

rtus c

IFN-α

100U



above-summarized generalizations. We also extendedprevious observations (Moreno et al., 1981; Rasool et al.,1992; Neumeister et al., 1998; Vandenplas et al., 2002;Moriyon, 2003; Zahringer et al., 2004) on the low endot-oxicity of LPS carrying the non-classical lipid A structuresused in this study, and show that these failed to inducehigh levels of TNF-α even at high concentrations. More-over, we characterized structural requirements that markdifferences between TNF-α induction and the expressionof two members of the p47 GTPase family. Although themode of action of these intracellular small GTPases is stillbeing clarified (MacMicking et al., 2003; Martens et al.,2004), it becomes apparent that they are part of powerfulmechanisms of resistance against intracellular pathogens(reviewed in Taylor, 2004). As shown above, expressionof these proteins by LPS seems to follow a type I IFN-dependent indirect pathway. In our experiments, bothIGTP and IIGP were induced by classical LPSs, no matterthe degree of acylation. This contrasts with the markedlyreduced ability of pentaacyl and tetraacyl dominant lipidAs to induce TNF-α. Especially striking was the p47-GTPase stimulatory ability of the synthetic LPS analogue,PPDM2, which has only a diacyl monosaccharide struc-ture and which does not display a TNF-α-mediated induc-ing activity (Chaby et al., 1993; Pedron et al., 1994). Non-classical LPSs were very weak inducers of the p47-GTPases, just as they were weak inducers of TNF-α, aset of traits useful for an intracellular parasite.

Induction of p47-GTPases by all classical LPS variantsor the non-classical LPS was completely inhibited by lossof TLR4 but not TLR-2 showing that both classical LPSand non-classical LPS signal through TLR4. A similarresult has recently been described for the LPS of Bar-tonella, another member of the α2-Proteobacteria (Zahr-inger et al., 2004).

In addition, in contrast to TNF-α induction, whichrequires the adaptor molecule MyD88, induction ofexpression of the p47 GTPases by wild type, underacy-lated classical and non-classical LPS was unaffected byloss of MyD88. Because induction of the p47 GTpases byLPS seems to be indirect and dependent on type I IFNs(Zerrahn et al., 2002 and the present study), this is con-sistent with a growing body of evidence that shows thatinduction of type I IFNs in macrophages by LPS is MyD88-independent. One possibility is therefore that the signal-ling uses the TRAM/TRIF pathway (Yamamoto et al.,2003a; Yamamoto et al., 2003b), as it has been shown fordendritic cells after lipid A stimulation (Weighardt et al.,2004). Additional experiments are necessary to determi-nate if this pathway is involved in p47-GTPase induction.

Here, we show for the first time that structural modifica-tions of LPS target preferentially the MyD88-independentpathway. Hence, our results suggest that the alternativeor simultaneous triggering of the MyD88-dependent and

-independent pathways relies on fine conformationalaspects of the ligand and receptor complexes.

An obvious question that remains is what kind of lipidA configurations and determinants are the most relevantPAMPs. Lipid As with long fatty acids chain in acyloxya-cyl groups linked to a disaccharide or trisaccharide (forR. etli) backbone with or without phosphate groups,dephosphorylated classical lipid A as well as some bio-synthetic precursors and analogues lacking the acyloxy-acyl substitutions have considerably less endotoxicityand some behave as antagonists of classical lipid As(Rossignol et al., 1999; Seydel et al., 1999). In addition,classical lipid A variants and analogues lacking the acy-loxyacyl substitutions are still able to induce strongexpression of GTPases whereas non-classical LPSssuch as those of Brucella and Legionella are weakinducers. Furthermore long fatty acid chains linked to aglycerol backbone decorated with choline or ethanola-mine do not perform the role of lipid A because phos-phatidylcholine and phosphatidylethanolamine carryingshort or long aliphatic chains were not biological active.Therefore, it is feasible that bisphosphorylated amino-sugars substituted by relatively short (from 10 to 16 Catoms) acyloxyacyl residues in ester or/and amide link-ages constitute the major PAMPs determinants of LPS,and that long fatty acid chains linked to aminosugarbackbones weaken recognition and signalling. Both fromthe physicochemical and stereochemical perspectives, itis important that the non-classical lipid As display alonger and more hydrophobic section. First these fea-tures result in a stronger aggregate state, more difficultto break up by innate immunity agents like cationic pro-teins and peptides (Moriyon, 2004), so that it is likelythat delivery of these LPSs by LBP to the CD14-TLR4-MD2 system is hampered. Moreover, their larger hydro-phobic section and relatively low negative charge(Velasco et al., 2000) may produce unsuitable ligands forthe anionic sites and hydrophobic pocket of MD-2 thatare essential for the activity of this TLR4 adapter (Gruberet al., 2004). This is compatible with the view that lipidAs vary in their three-dimensional shape and that thisparameter relates to their biological activity. Dependingon the degree of acylation and the length and asymme-try of acyl groups, lipid A shapes ranges from conical tocylindrical, and at least the cytokine induction propertiesare characteristic of the former and lost in the latter, asdemonstrated by studies with both natural lipid As andsynthetic analogues (Schromm et al., 2000; Seydelet al., 2003). So far, no X-ray diffraction analyses thatwould allow the inference of lipid A shapes are availablefor the non-classical LPSs. Such studies would help tounderstand the lipid A peculiarities that result in analtered PAMP and, by contrast, to the definition of theclassical LPS PAMP (Moriyon, 2004).



Lipopolysaccharides require TLR-4 but not TLR-2 forearly cell activation and, whereas the induction of TNF-αoccurs via TLR-4-dependent/MyD-88-dependent pathway,the generation of p47-GTPases occurs through a TLR-4-dependent/MyD-88-independent pathway. Within this per-spective, it seems reasonable to propose that the lowactivation of TNF-α and p47-GTPases by the LPS of theintracellular parasite Brucella, which has coevolved withits mammalian host, may constitute an evolutionaryadvantage for adapting to intracellular life. The fact that avery similar LPS is present in Ochrobactrum, the Brucellaclosest know phylogenetic relative but not an overtlypathogen, and in a plant endosymbiont does not contra-dict this hypothesis. Indeed, a LPS apt to withstand soilpeptide antibiotics of action akin to that of the innateimmunity peptides may represent a scaffold to developstrategies useful within animal and plant cells (Moriyon,2004). Low activation of cellular killing mechanisms maybe essential during the intracellular trafficking and replica-tion of these bacteria in host cells. Certainly, macroph-ages activated through the action of IFN-γ and othermediators are better adapted to destroy intracellular Bru-cella than naïve cells (Jiang and Baldwin, 1993; Gebranet al., 1994). Likewise, members of the p47-GTPases areimplicated in host resistance against intracellular patho-gens and LRG-47 has been implicated in resistance to S.typhimurium (quoted in Taylor, 2004) as well as Gram-positive intracellular bacteria and Protozoa (reviewed inTaylor, 2004).

Experimental procedures

Mice

Eight-week-old female C57Bl/6 and C3H/HeN mice were pur-chased from Jackson ImmunoResearch (West Grove, PA). C3H/HeJ (LPS-resistant mice) mice were purchased from Harlan(UK). The generation and breeding of all knockout mice used inthis study have been reported previously (Hoshino et al., 1999;Takeuchi et al., 1999). Background-matched control wild-typemice were bred at Centre d’Immunologie de Marseille-Luminy,and TLR2-, MyD88- and TLR4-deficient mice were bred at OsakaUniversity (Osaka, Japan).

Lipopolysaccharides

O-polysaccharide-bearing (smooth) wild-type classical LPSswere obtained by the phenol-water method from representativewild-type enterobacterial strains E. coli 0127 (grown at 37°), Y.enterocolitica O:9 MY79 and O:3 WE245/92 (both grown at26°C). The same method was used to obtain O-polysaccharidedeficient classical LPSs with partially acylated lipid As from theavirulent Y. pestis KIM6 strain (grown 26°C) and lipid A E. colimutants MLK53 htrB– (lauroyl-transferase), MLK 1067 msbB–

(miristoyl-transferase) (both grown at 37°C) and MLK 986 htrB–/msbB– (grown at 22°C) (Clementz et al., 1996; Clementz et al.,

1997). B. abortus LPS from the virulent 2308 strain and O.anthropi LPS from strain LMG3331 were obtained by the phenol-water method with modifications (Velasco et al., 2000). All theabove-described LPSs were extensively purified to removetraces of contaminant lipids and lipoproteins following the phenol-deoxycholate protocol (Hirschfeld et al., 2000) or, in the case ofthe phenol-soluble LPSs (Y. enterocolitica O:9 and B. abortus),by repeated nuclease and proteinase K digestions followed byseveral cycles of chloroform-methanol extraction, and absenceof such contaminants was then determined by SDS-PAGE, GLCand amino acid and amino sugar analyses as described before(Velasco et al., 2000). Purified LPSs from L. pneumophila RC1and its spontaneous 811 (3-OH fatty acids at C2 and C2′ 2–4CH2 units shorter than in RC1) and isogenic 5215 (bearing adifferent O-chain but same lipid A as RC1) mutants (Luneberget al., 1998) were a generous gift of U. Zähringer (BorstelResearch Center, Division of Immunochemistry, Borstel, Ger-many) whom also provided the structurally defined R. etli CE3LPS (originally extracted and purified by R.W. Carlsson, CCRC,Athens, GA, USA) (Que et al., 2000). PPDM2, a synthetic lipidA agonist, was kindly provided by Girard R. (URA-1961, Paris,France) (Chaby et al., 1993; Pedron et al., 1994; Charon et al.,1998). To verify the degree of acylation in the lipid A structuresof classical LPSs preparations, they were subjected to acidhydrolysis in the presence of SDS and the lipid A fractions exam-ined by high-performance thin layer chromatography (Bengo-echea et al., 2003) and nano-electrospray ionization time-of-flightmass spectrometry (ESI-TOF-MS) with a Q-TOF-MS instrument(Q-TOF Micro, Waters, Milford, Massachusetts). Lipid As werediluted in chloroform-methanol (2:1, v/v in 0.02% formic acid) andintroduced into the PicoTip nanospray ionization source by infu-sion through a 100 µl syringe with the assistance of the infusionpump provided with the instrument, at a flow rate of 0.5 µl min−1.Spectra were collected in negative-ion mode over 10 min at2.4 s scan−1 with an interscan delay of 0.1 using a cone voltageenergy of 1600 V. Background subtraction and smoothing wasperformed with the MassLynx V4.0 software. Phosphoric acidwas used to calibrate the instrument.

For testing, lyophilized LPSs were dissolved by sonication indistilled water and autoclaved before used. All LPSs were usedat concentrations such that equal amounts of lipid A were tested,regardless of the presence or absence of the O-polysaccharide.To this end, the amount of the LPS core sugar 3-deoxy-D-manno-2-octulosonic acid (Kdo) was measured (Velasco et al., 2000)and its reported proportion in the particular LPS core (Holst,1999) (depending upon the LPS, 2 or 3 nanomols of Kdo areequivalent to 1 nanomol of lipid A) used to calculate the nano-mols of lipid A in the LPS solution (from approximately 20 nano-mols of lipid A/mg for S E. coli LPS to 80 nanomols lipid A/mgfor R E. coli LPS).

Antibodies and reagents

Rabbit anti-IIGP antibody n°165 was raised against IIGP proteinpurified from an E. coli expression system (Uthaiah et al., 2003).IGTP (clone 7) monoclonal antibody was from Transduction lab-oratories (Lexington, KY, USA). TNF-α enzyme-linked immun-osorbent assay (ELISA) kit, IFN-γ and anti-type I IFN were fromR and D Systems (Minneapolis, MN). Secondary antibodies,rabbit IgG-peroxidase and anti-mouse IgG-peroxidase anti-rabbitIg conjugates were from Sigma (St Louis, MO).



Macrophage preparation

Four days after intraperitoneal injection of 2 ml mouse−1 of 4%sterile fluid thioglycollate, peritoneal exudate macrophages wereextracted by washing with 10 ml of Dulbecco’s modified Eagle’smedium (DMEM) (Gibco Biological Research Laboratories,Gaithersburg, MD) at 4°C, sedimented and resuspended inDMEM supplemented with 10% FCS, 10 mM HEPES, 10 mMsodium pyruvate, 10 mM non-essential amino acids, 2 mMglutamine, 100 U ml−1 penicillin and 100 µg ml−1 streptomycin (allfrom Life Technologies). For all experiments, peritoneal cells wereplated and incubated for 2–4 h at 37°C in a 7% CO2 atmosphere.Non-adherent cells were removed from wells or dishes by aspi-ration, and the adherent macrophages were rinsed and incu-bated in fresh medium. For stimulation, macrophages wereincubated with LPSs (0.5; 5; 50 mM), IFN-β (100–1000 U ml−1)or IFN-γ (5–10 U ml−1) during 24 h. Macrophages were then lysedwith 1% Nonidet 40 in PBS, and the protein concentration of eachlysate was evaluated with a BCA protein assay kit (Pierce, Rock-ford, USA).

Western blotting

Equal protein amounts of each sample were run in 12% SDS-PAGE and transferred onto Immobilon-P membranes (Millipore,Bedford, MA). Membranes were blocked in TBS/5% skim milk/0.1% Tween-20 (Sigma) for 2 h. Primary and secondary Abswere successively added in this buffer, each being left for 2 hbefore developing with the enhanced chemoluminescence sys-tem (ECL; Amersham, Courtaboeuf, France).

Acknowledgements

We are grateful to Drs S. Salcedo, S. Garvis and E. Moreno forcritically reading the manuscript. We thank Dr F. Forquet fortechnical assistance, Dr U. Zärhinger for providing L. pneumo-phila LPSs and Pr. Girard R for the PPDM2. This work wassupported by institutional grants from the CNRS, INSERM, andARC (Grant number 7541), by grants from the European Union(QLK2-CT-2002-00918 and MRTN-CT-2003-504227), SpanishCAICYT (AGL2004-01162/GAN) and Red Temática de Investi-gación en Brucellosis (G03/204), and a grant in the Schwerpunk-tprogramm SP1110 of the Deutsche Forschungsgemeinschaft‘Innate Immunity’ to J.C.H.

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