From expression to signaling: Roles of TREM-1 and TREM-2 in innate immunity and bacterial infection

ARTICLE IN PRESS

Immunobiology 213 (2008) 701–713

0171-2985/$ - se

doi:10.1016/j.im

Abbreviations

derived macrop

cyclooxygenase

growth factor r

kappa light cha

based activation

differentiation p

linositol 3-kinas

NTAL, non-T

transcription po

trem-like transc

TREM-1.�Correspond

Guertel 18-20,

E-mail addr

www.elsevier.de/imbio

REVIEW

From expression to signaling: Roles of TREM-1 and TREM-2 in innate

immunity and bacterial infection

Omar Sharifa,b,�, Sylvia Knappa,b

aCenter for Molecular Medicine (CeMM) of the Austrian Academy of Sciences, Medical University Vienna,

Waehringer Guertel 18-20, 1090 Vienna, AustriabDepartment of Medicine I, Div. of Infectious Diseases and Tropical Medicine, Medical University Vienna,

Waehringer Guertel 18-20, 1090 Vienna, Austria

Received 23 July 2008; accepted 23 July 2008

Abstract

Triggering receptor expressed on myeloid cells (TREM) proteins play important roles in innate and adaptiveimmunity. Since the discovery of TREM-1 and TREM-2 in 2000, the field has exploded over the past 8 years andcurrently TREM-1 is thought of as an amplifier of the immune response, while TREM-2 is believed to be a negativeregulator. Here, we discuss the current state of the literature on TREMs, with a focus on TREM-1 and TREM-2 andtheir expression, signaling in mononuclear phagocytes and roles in innate immunity and bacterial infection.r 2008 Elsevier GmbH. All rights reserved.

Keywords: Bacterial infection; Innate immunity; TREM-1; TREM-2

Contents

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 702

The TREM gene family and their expression . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 702

TREM-1. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 702

TREM-1 signaling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 702

e front matter r 2008 Elsevier GmbH. All rights reserved.

bio.2008.07.008

: AP-1, activator protein 1; Bcl-10, B cell lymphoma 10; BMDCs, bone marrow-derived dendritic cells; BMDM, bone marrow-

hages; CARD, caspase recruitment domain; CD14, monocyte differentiation antigen CD14; CLP, cecal ligation and puncture; COX,

; DAP-12, DNAX activation protein 12; ERK, extracellular signal-related kinase; FACS, fluorescence activated cell sorting; Grb2,

eceptor-bound protein 2; HSP70, heat shock protein 70; HMGB1, high-mobility group box 1; IL, interleukin; IkB-a, inhibitor ofin gene enhancer in B cells; IKK, inhibitor of kappaB kinase; iNOS, inducible nitric oxide synthase; ITAM, immunoreceptor tyrosine-

motif; LPS, lipolysaccharide; MCP-1, monocyte chemotactic protein 1; MMP, matrix metalloproteinase; MyD88, myeloid

rimary response gene 88; NCL, necrotic cell lysate; PAMPs, pathogen-associated molecular patterns; PI3-kinase, phosphatidy-

e; PKA, protein kinase A; NF-kB, nuclear factor kappa B; NLR, nucleotide-binding oliogomerization domain (Nod)-like receptor;

cell activation linker; RelA and RelB, V-REL avian reticuloendotheliosis viral oncogene homolog A and B; RT-PCR, reverse

lymerase chain reaction; ShRNAi, short hairpin RNA interference; sTREM-1, soluble TREM-1; TLR, Toll-like receptor; TLT,

ript; TNF, tumor necrosis factor; TREM, triggering receptor expressed on myeloid cells; TREM-1sv, alternatively spliced form of

ing author at: Center for Molecular Medicine (CeMM) of the Austrian Academy of Sciences, Medical University Vienna, Waehringer

1090 Vienna, Austria. Tel.: +43 1 40400 5140; fax: +43 1 40400 5167.

ess: [email protected] (O. Sharif).

www.elsevier.de/imbio

dx.doi.org/10.1016/j.imbio.2008.07.008

mailto:[email protected]

ARTICLE IN PRESSO. Sharif, S. Knapp / Immunobiology 213 (2008) 701–713702

Regulation of TREM-1 expression . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 705

TREM-1: a positive regulator of inflammatory responses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 706

TREM-1 and bacterial infection: in vivo studies using agents which either activate or block TREM-1

signaling show it is an important player in bacterial infection and sepsis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 706

Soluble TREM-1 (sTREM-1): from enigma to diagnostic marker . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 707

The mystery of the TREM-1 ligand . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 708

TREM-2. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 708

TREM-2 signaling: similarities and differences between TREM-1 signaling . . . . . . . . . . . . . . . . . . . . . . . . . . . 708

Regulation of TREM-2 expression . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 709

TREM2: a negative regulator of inflammatory responses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 709

TREM-2 and bacterial infection: an unexplored area . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 709

The mystery of the TREM-2 ligand . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 710

Concluding remarks. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 710

Conflict of interest . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 710

Acknowledgment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 710

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 711

Introduction

Mononuclear phagocytes (monocytes and macro-phages) play a key role in the first line of defenceagainst invading microbes, and molecules secreted bythese cells upon recognition of microbial danger signalsshape the vertebrate adaptive immune response.A plethora of cell surface receptors (members of theToll-like receptor (TLR), Siglec, C-type lectin familyand intracellular sensing mechanisms such as nucleo-tide-binding oliogomerization domain (Nod)-like recep-tors (NLR) exist which allow mononuclear phagocytesto fulfill this function. These are areas of intenseresearch and the reader is directed to excellent recentreviews on these subjects (Robinson et al., 2006; Wertset al., 2006; Banerjee and Gerondakis, 2007; Crocker etal., 2007; Kanneganti et al., 2007; Krishnan et al., 2007).However, the purpose of this review is to discussanother set of cell surface receptors known as thetriggering receptor expressed on myeloid cells (TREM)family, with particular emphasis given to TREM-1 andTREM-2, their expression, signaling in mononuclearphagocytes and their roles in innate immunity andbacterial infection.

The TREM gene family and their expression

The TREM-1 and TREM-2 genes are present onhuman chromosome 6p21 and mouse chromosome17C3. In addition, there are homologous genes calledTREM-1 like (trem-like transcript (TLT)-1) andTREM-2 like (TLT-2) transcript, which like TREM-1and TREM-2 encode a single variable-type immunoglo-bulin domain (Allcock et al., 2003). Additionally presentin mice is functional TREM-3, which is a pseudogene inhumans (Allcock et al., 2003). TREM proteins arehighly conserved in evolution as indicated by their

presence not only in mice and humans, but also inchicken, pig and cow (Ramanathan et al., 2004, 2005;Viertlboeck et al., 2006). Phylogenetic analysis indicatesthat bovine and pig TREM-1 are more closely related toeach other than the murine and human TREM-1, whichcluster together (Ramanathan et al., 2004). With theexception of TLT-1, expression of all other TREMfamily members has been detected in either macrophagecell lines or a variety of primary macrophages usingvarious techniques (Table 1). It has recently beendemonstrated that expression of TREM family membersis not restricted to myeloid cells, as hepatic endothelialcells also exhibit constitutive expression of TREMs1–3,although the authors of this study did not demonstratecell surface expression (Table 1; Chen et al., 2007).Importantly, not all macrophages express TREM-1.While lymph node, peritoneal and alveolar macrophages(our unpublished observations and Wiersinga et al.,2007), exhibit surface expression of TREM-1, intestinalmacrophages do not and also fail to induce TREM-1surface expression in response to tumor necrosis factor(TNF; Table 1; Schenk et al., 2005). Reverse transcrip-tion polymerase chain reaction (RT-PCR) analysis ofbovine TREM-1 indicates expression is high in the bonemarrow, thymus and spleen, while a similar analysis ofpig TREM-1 indicates high expression in bone marrow,lung and colon (Ramanathan et al., 2004, 2005).

TREM-1

TREM-1 signaling

TREM-1 consists of an extracellular domain, atransmembrane region which contains a conservedlysine residue (Ramanathan et al., 2005), and a shortcytoplasmic domain, which lacks any signaling motif(Bouchon et al., 2000; Radaev et al., 2003; Kelker et al.,

ARTICLE IN PRESS

Table 1. Expression of the TREM family members

Cell type Species Technique References

TREM-1

Peripheral blood CD14+ monocytes Human FACS, RT-PCR Bouchon et al. (2000), Bleharski et al. (2003), Knapp

et al. (2004), Gomez-Pina et al. (2007), Gingras et al.

(2002)

Neutrophils FACS; IF Bouchon et al. (2000, 2001a), Knapp et al. (2004),

Fortin et al. (2007)

Lymph node macrophages FACS Schenk et al. (2005)

Alveolar macrophages IHC, FACS Colonna and Facchetti (2003), Knapp (unpublished)

Differentiated U937 (pre-monocytic) RT-PCR Gingras et al. (2002)

HT1080 (fibrosarcoma cell line) RT-PCR Allcock et al. (2003)

Blood monocytes and neutrophils Mouse FACS Wiersinga et al. (2007)

Alveolar macrophages FACS Wiersinga et al. (2007)

MH-S cells (alveolar macrophage) RT-PCR Knapp (unpublished)

Hepatic macrophages and endothelial

cells

RT-PCR Chen et al. (2007)

TREM-2

Immature monocyte derived DC Human FACS Bouchon et al. (2001b)

THP-1 (monocyte) and U937 (pre-

monocyte) cells

RT-PCR Allcock et al. (2003)

Microglia CM Sessa et al. (2004)

CHME-5 microglial cell line T98G

neuroblastoma cell line

WB, FACS, CM Sessa et al. (2004)

Newly recruited peritoneal and alveolar

macrophages

Mouse FACS Turnbull et al. (2006)

Bone marrow-derived macrophages FACS Hamerman et al. (2006), Turnbull et al. (2006)

Hepatic macrophages and endothelial

cells

RT-PCR Chen et al. (2007)

Microglia RT-PCR, CM,

FACS

Schmid et al. (2002), Sessa et al. (2004), Takahashi et

al. (2005)

J774.2 macrophage cell line WB, FACS Sessa et al. (2004), Piccio et al. (2007)

N2A neuroblastoma cell line WB Sessa et al. (2004)

N9 microglial cell line WB, FACS Sessa et al. (2004), Piccio et al. (2007)

RAW 264 NB, FACS Daws et al. (2001), Turnbull et al. (2006)

IC21 and MT2 macrophage cell lines NB Daws et al. (2001)

TLT-1

Platelets Human NB, WB Washington et al. (2004)

U937 cells RT-PCR Allcock et al. (2003)

Platelets Mouse NB, WB, FACS Washington et al. (2004)

Megakaryocytes CM, IHC Washington et al. (2004)

TLT-2

Jurkat T cell line Human RT-PCR Allcock et al. (2003)

Raji B cell line RT-PCR Allcock et al. (2003)

U937 cell line RT-PCR Allcock et al. (2003)

Resident peritoneal and alveolar

macrophages

Mouse FACS King et al. (2006)

Peritoneal B1b cells FACS King et al. (2006)

Neutrophils FACS King et al. (2006)

TREM-3

RAW 264 & MT2 macrophage cell lines Mouse NB Chung et al. (2002)

RBL-5T cell line NB Chung et al. (2002)

Hepatic macrophages RT-PCR Chen et al. (2007)

TREM family members have been identified in a variety of primary cells as well as cell lines using different techniques. The species (only human and

murine shown), the technique used for identification in the corresponding cells and the reference are indicated. CM, confocal microscopy; FACS,

fluorescence activated cell sorting; IHC, immunohistochemistry; IF, immunofluoresence; NB, Northern blotting; RT-PCR, reverse transcriptase

polymerase chain reaction; WB, Western blotting.

O. Sharif, S. Knapp / Immunobiology 213 (2008) 701–713 703


2004). The ligand for TREM-1 is unknown and manystudies on TREM-1 have made use of an agonisticantibody, which induces receptor cross-linking. Uponcross-linking the conserved lysine residue in the trans-membrane domain of TREM-1 associates with anaspartate residue in the immunoreceptor tyrosine-basedactivation motif (ITAM) of the adaptor protein DNAXactivation protein 12 (DAP-12), leading to its tyrosinephosphorylation (Bouchon et al., 2000). This thenresults in downstream signal transduction events whichinclude the phosphorylation of phospholipase (PL)Cgand extracellular signal-related kinase (ERK) 1/2 and anincrease in intracellular calcium and pro-inflammatorycytokine secretion (Bouchon et al., 2000; Fig. 1A).When U937 cells which ectopically express TREM-1and DAP12, are treated with an short hairpin RNAinterference (shRNAi) against non-T cell activationlinker (NTAL), it was shown that NTAL knockdownresults in an increase in TREM-1 induced ERKphosphorylation, a reduced but prolonged calcium flux

Rel

A

p50

IκBα

PP

UbUb

UbUb

IκBα

IκBα

Rel

A

p50

CARD9

Bcl-10

IKKγ

IKKγ

IKKβIKKα

P

?

TREM-1

R D

D

IRAK-1

P

JAK AktP

P

FPSTAT5

?IκBα

UbUb

UbUb

IκBα

IκBα

IκBα

Rel

A

p50

?

?

TREM-1

R D

?

IRAK-1

P

AktP

Akt

E

P

?

?

P

Fig. 1. TREM-1 signaling. Reported signaling pathways associated w

letters (A–F). Unknown aspects of TREM-1 signaling in mononucl

and increased levels of the inflammatory cytokines TNFand interleukin-8 (IL-8), demonstrating that NTALnegatively regulates TREM-1/DAP12 signaling (Tessarzet al., 2007; Fig. 1B). In addition, NTAL was shown tobe phosphorylated upon TREM-1 ligation and phos-phorylated NTAL associates with the growth factorreceptor-bound protein 2 (Grb2) (Tessarz et al., 2007;Fig. 1C). TREM-1 has also been reported to result inincreased nuclear levels of the nuclear factor kappa B(NF-kB) transcription factor (Gibot et al., 2004), whichplays a crucial role in the transcription of inflammatoryresponse genes such as TNF-a, IL-2, IL-12p40 andIL-1b (Sharif et al., 2007). Recent data in caspaserecruitment domain 9 (CARD9) knockout murine bonemarrow-derived dendritic cells (BMDCs) has shed somelight on how NF-kB activation in response to TREM-1occurs (Hara et al., 2007). Briefly, the caspase recruit-ment domain (CARD) is a protein-binding modulewhich mediates the assembly of CARD-containingproteins (such as members of the Nod family) and

GGGACTTTCC

mRNA

Extracellular

Intracellular

Nucleus

TREM-1 ligand ?

DAP12

PLC-γP

Ca2+

Rel

A

p50

P

ERK1/2

NTAL

PPP

Grb2

P

B

TNFIL-8

mRNA

TREM-1 ligand

PLC-γP

Ca2+

?

ERK1/2

PPP

Grb2

P

A

C

ith TREM-1 (details are explained in the text and indicated by

ear phagocytes are highlighted by question marks.

ARTICLE IN PRESSO. Sharif, S. Knapp / Immunobiology 213 (2008) 701–713 705

modulates apoptosis, inflammasome or NF-kB activa-tion (Werts et al., 2006; Kanneganti et al., 2007).CARD-9 associates with another CARD-containingprotein called B cell lymphoma 10 (Bcl-10) and mediatesactivation of NF-kB (Bertin et al., 2000). Importantly,in response to TREM-1 agonistic antibody, CARD9�/�BMDCs produce significantly less TNF, IL-2 and IL-12p40 compared to wild-type BMDCs. Bcl10�/�BMDCs also produce significantly less IL-2 in responseto TREM-1 receptor ligation compared to wild-typeBMDCs (Hara et al., 2007). This suggests that down-stream of TREM-1 signaling, NF-kB activation mayoccur through a CARD9/Bcl10 complex. The identity ofboth the upstream events which lead to the activation ofthis complex following TREM-1 cross-linking and thedownstream events, which link this to NF-kB signaltransduction remain to be determined. The downstreammechanism is likely to involve activation of the inhibitorof kappaB kinase (IKK) complex; a central regulator ofNF-kB activity (Srinivasula et al., 1999; Perkins, 2007;Fig. 1D). The mammalian NF-kB family is composed ofa variety of homo and heterodimers made from fivesubunits: V-REL avian reticuloendotheliosis viral onco-gene homolog A (RelA) (p65), p100/p50 (NFkB1), c-rel,V-REL avian reticuloendotheliosis viral oncogenehomolog B (RelB) and p100/p52 (NFkB2) (Perkins,2007). TREM-1 ligation results in increased nuclearlevels of RelA/p50, but it is as yet unknown whetherother combinations of subunits are involved.

All of the aforementioned studies have investigatedTREM-1 signaling in mononuclear phagocytes, but it isimportant to mention that some aspects of TREM-1signaling are shared between mononuclear phagocytesand neutrophils, which also express TREM-1 (Table 1).For example, in response to agonistic TREM-1 anti-body in monocytes, PLCg is phosphorylated within1min and maximum ERK1/2 phosphorylation occurs at2min, with levels decreasing 10min post-receptorligation (Bouchon et al., 2000). PLCg and ERK1/2 arealso phosphorylated in neutrophils, albeit with ERKphosphorylation being sustained till 30min (Fortinet al., 2007). Significantly, although this indicatescommonalities in TREM-1 signaling between these celltypes, it also indicates subtle differences. In addition toPLCg and ERK1/2 phosphorylation, the kinases AKT,Lyn, Jak2, IRAK-1 and the transcription factorsSTAT5 and RelA are phosphorylated in neutrophils inresponse to TREM-1 cross-linking (Fortin et al., 2007).Whether similar signaling events occur in mononuclearphagocytes is unclear (Fig. 1E). Phosphorylation ofRelA at S536, increases the association of RelA withcomponents of the basal transcriptional machinery andthis may lead to enhanced signal transduction (Buss etal., 2004). RelA can be phosphorylated by a variety ofkinases, such as AKT and IKKb (Viatour et al., 2005),but the kinase responsible for the phosphorylation in

response to TREM-1 ligation awaits further study(Fig. 1F). TREM-1 ligation alone can induce cytokinesecretion, as well as synergistically enhance cytokinesecretion in response to TLR or NLR ligands (Bouchonet al., 2000, 2001a; Bleharski et al., 2003; Gibot et al.,2004; Netea et al., 2006). The transcription factors andpromoter elements involved in this TREM-1-mediatedresponse are still elusive, and molecular studies usinggelshift, reporter gene analysis and ChIP are required toidentify the relevant cis- and trans-elements and theirinteraction.

A study conducted by Ornatowaska and colleagues,utilizing a combination of PCR-based arrays andshRNAi to TREM-1 in RAW 264.7 macrophagesshowed that TREM-1 silencing results in decreasedtranscription of key proteins involved in the TLR4signaling pathway, including myeloid differentiationfactor 88 (MyD88), monocyte differentiation antigenCD14 (CD14) as well as downstream molecules in theNF-kB pathway (inhibitor of kappa light chain geneenhancer in B cells (IkB-a) and p100), followinglipolysaccharide (LPS) treatment compared to controlcells (Ornatowska et al., 2007). This suggests thatTREM-1 may impact inflammatory responses byincreasing the availability of key proteins involved inTLR signaling. It remains to be seen whether TREM-1silencing will affect similar downstream effectorproteins following ligation of other TLR receptors, (suchas TLR2) or NLR receptors, which have been pre-viously shown to amplify TREM-1-mediated responses(Bleharski et al., 2003; Netea et al., 2006).

Regulation of TREM-1 expression

Treatment of pigs with the Salmonella enterica

serovar typhimurium results in increases in TREM-1expression 8 and 24 h post-infection, indicating a role ofthis orthologue in bacterial infection (Ramanathanet al., 2005). It is possible that LPS present on the cellwall of this bacterium induces TREM-1 expressionbecause treatment of pig bone marrow-derived macro-phages (BMDM) with purified LPS induces up-regula-tion of TREM-1 expression (Ramanathan et al., 2005),similar to that described for both human and mouseTREM-1 (Bouchon et al., 2000, 2001a; Bleharski et al.,2003; Knapp et al., 2004). LPS activates a plethora ofgenes including the pro-inflammatory mediator, TNFand the stress response gene cyclooxygenase 2 (COX-2),which have been reported to increase TREM-1 surfaceexpression (Bleharski et al., 2003; Murakami et al.,2007). LPS also activates the anti-inflammatory cyto-kine IL-10, which has been shown to abrogate TREM-1up-regulation in response to TLR stimulation (Bleharskiet al., 2003; Schenk et al., 2005). This indicates thatboth pro-inflammatory and anti-inflammatory cytokines


produced by TREM-1 and TLR ligation can inturn modulate TREM-1 expression in an autocrinemanner.

Following treatment of RAW 264.7 macrophageswith LPS, the transcription factors PU.1, activatorprotein 1 (AP-1) and RelA were shown to bind to theTREM-1 promoter using ChIP assay (Zeng et al., 2007).LPS and Pseudomonas aeroginosa-mediated expressionof TREM-1 was shown to be impaired in RAW264.7 macrophages pre-treated with NF-kB inhibitors,whereas macrophages which had been silenced forPU.1 exhibited increased expression of TREM-1 RNAcompared to control cells (Zeng et al., 2007). Thisstudy indicates that expression of TREM-1 in responseto Gram-negative bacteria or LPS in macrophagesis dependent on activation of NF-kB, whereasPU.1 negatively regulates TREM-1 expression. It hasbeen described that PGE2-mediated synthesis of TREM-1 in peritoneal macrophages is at least partiallydependent on protein kinase A (PKA), p38 andphosphatidylinositol 3-kinase (PI3-kinase), but indepen-dent of MAPKK since a PKA inhibitor (H89), ap38 MAPK inhibitor (SB203580), and a PI3K inhibitor(LY294002) significantly suppressed PGE2-inducedTREM-1 expression, whereas a MAPKK inhibitor(PD98059) did not (Murakami et al., 2007). In termsof regulation of TREM-1 expression by the PI3-kinasepathway, these observations correlate well with ourprevious studies showing that LPS-induced surfaceexpression of TREM-1 in human monocytes isdependent on the PI3-kinase pathway (Knapp et al.,2004). Given that the TREM-1 promoter is regulatedby NF-kB and the NF-kB pathway does not existin isolation but cross-talks with other kinase path-ways such as the PI3-kinase pathway (Perkins, 2007), itwould be of interest to examine how transcriptionfactor recruitment to the TREM-1 promoter is influ-enced by these pathways in response to pathogen-associated molecular patterns (PAMPs). Furthermore, itremains to be defined whether similar transcriptionalmechanisms exist that regulate other TREM familymembers.

TREM-1: a positive regulator of inflammatory

responses

Administration of mononuclear phagocytes with anagonistic TREM-1 antibody followed by activation ofpattern recognition receptors such as those of the TLRor NLR family, results in a greater increase in cytokineand chemokine secretion, compared to either stimulusalone (Bouchon et al., 2000, 2001a; Bleharski et al.,2003; Gibot et al., 2004; Netea et al., 2006). Treatmentof monocytes with an agonistic TREM-1 antibody wasalso shown to increase expression of molecules related to

antigen presentation and T cell activation such as CD86and MHC class II (HLA-DR), which correlated with agreater ability of these cells to activate T cells in vitro

(Bleharski et al., 2003). These studies show thatTREM-1 is an amplifier of the immune response toinfectious stimuli and suggest that TREM-1 signalingmay modulate both the innate and adaptive immuneresponses during infectious disease.

TREM-1 and bacterial infection: in vivo studies

using agents which either activate or block TREM-1

signaling show it is an important player in bacterial

infection and sepsis

Severe sepsis and septic shock are major causes ofmorbidity and mortality in humans (Hunter, 2006).Sepsis can be described as a systemic immune responseto TLR activation that has gone awry, resulting inactivation of various immunological mechanisms andcoagulation cascades that are detrimental to the host(Riedemann et al., 2003; Hunter, 2006).

Though a TREM-1 knockout mouse does not exist asyet, several in vivo studies have corroborated a roleof TREM-1 in both bacterial infection and sepsis.TREM-1 is highly expressed in inflammatory lesionscaused by infectious agents such as bacteria and fungi,but not in lesions caused by non-infectious agents(Bouchon et al., 2001a). An indirect role for TREM-1in the control of bacterial infection has been suggestedby studies using DAP12�/� mice which exhibitdecreased levels of plasma IL-6, TNF and monocytechemotactic protein 1 (MCP-1) at 24 h post-cecalligation and puncture (CLP)-induced septic shock, andconsequently have a survival advantage over wild-typemice (Turnbull et al., 2005). It is difficult to interpretthese results in the context of TREM-1 since DAP-12 israther promiscuous and associates with many otherreceptors (Turnbull and Colonna, 2007). Direct evidenceon the role of TREM-1 in vivo in bacterial infection hasbeen obtained using agents which either interfere oraugment TREM-1 signaling. Blocking experimentsusing a fusion protein containing the murine TREM-1extracellular domain fused to the Fc portion of humanimmunoglobulin-g (mTREM-1/IgG1), demonstrated areduced inflammation resulting in improved survival inmodels of murine endotoxemia and septic peritonitis(Bouchon et al., 2001a). In models of LPS-inducedseptic shock, activation of TREM-1 signaling with anagonistic TREM-1 antibody has been shown to doublethe mortality rate (Gibot et al., 2004). Additional insightinto the in vivo role of TREM-1 in bacterial infection hasbeen obtained using a peptide called LP17, which hasbeen designed to the complementary determining region3 (CDR3) and the ‘‘F’’ b strand of the extracellulardomain of TREM-1, which has been proposed to


mediate dimerization of the TREM-1 receptor (Radaevet al., 2003; Gibot et al., 2004, 2006b). It has beensuggested that this compound either impairs TREM-1dimerization and/or competes with the natural ligand ofTREM-1 (Gibot et al., 2004, 2006b). In vitro, LP17 hasbeen shown to reduce TNF and IL-1b production frommonocytes cultured with LPS or with LPS and aTREM-1 agonistic antibody (Gibot et al., 2004). In vivo

either pre-treatment of mice with a single dose of LP17before a lethal dose of LPS, or treatment of mice 4 hafter LPS treatment, conferred a survival advantagecompared to control mice (Gibot et al., 2004). LP17 isalso protective in a CLP model. Interestingly, in thismodel of infection, although LP17 was shown todecrease serum levels of TNF and IL-1b by 30%,bacterial counts in the peritoneal lavage and blood weresimilar in animals treated with LP17 to controls, 24 hpost-infection (Gibot et al., 2004). It was later shownthat LP17 decreased levels of plasma nitrates/nitrites inthis model of infection, maintained arterial bloodpressure and again conferred a survival advantage,ratifying previous results (Gibot et al., 2006b). Theprotective effects of modulating TREM-1 signalingusing LP17 are also evident in other models of infectionsuch as P. aeroginosa pneumonia and Burkholderia

pseudomallei pneumonia (Gibot et al., 2006a; Wiersingaet al., 2007). In both of these studies, although LP17 wasshown to confer a survival advantage, it had no impacton bacterial clearance in the BAL compartment.However, in B. pseudomallei pneumonia, LP17 causeda decrease in bacterial burden in the spleen (Gibot et al.,2006a; Wiersinga et al., 2007). In P. aeroginosa

pneumonia, LP17 prevented cellular infiltration, histo-logical damage and decreased levels of both BAL andplasma TNF, IL-1b and IL-6 at 24 h post-infectioncompared to control mice (Gibot et al., 2006a). Thisdecrease in cytokine levels correlates with previousstudies utilizing the CLP model (Gibot et al., 2004,2006b), but not with the model of B. pseudomallei

pneumonia (Wiersinga et al., 2007). These studies areimportant to mention because they demonstrate howmodulation of the TREM-1 pathway can have differenteffects depending on the infectious model. The degree bywhich the TREM-1 pathway is modulated could haveprofound effects on a septic patient and may actuallyeven be detrimental. Experiments in a septic mousemodel of fecal peritonitis, demonstrate that the extent towhich TREM-1 is silenced using in vivo siRNA dictateseither survival or lethality (Gibot et al., 2007). The exactrole of TREM-1 in vivo in response to various bacterialinfectious disease models will undoubtedly be answeredby conditional knockout approaches and the discoveryof the ligand(s). Nonetheless, it is clear that TREM-1plays a very important role in bacterial infection andsepsis through its ability to amplify the immuneresponse.

Soluble TREM-1 (sTREM-1): from enigma to

diagnostic marker

A soluble form of TREM-1 (sTREM-1) has beendetected in cultured mononuclear phagocyte super-natants treated with LPS, in plasma of mice which havebeen treated with endotoxin, in human volunteersinjected with LPS, in the plasma of septic patients andin the BAL of patients with pneumonia (Gibot andCravoisy, 2004; Gibot et al., 2004; Knapp et al., 2004).How this form of TREM-1 is generated was an enigmafor many years and subject to some debate. It wasinitially proposed that the presence of sTREM-1 wasdue to an alternatively spliced form of TREM-1(TREM-1sv) in monocytes and CD34+ bone marrowcells, which produces a protein devoid of both thetransmembrane and cytoplasmic domains with a pre-dicted molecular mass of 17.5 kDa (Gingras et al., 2002).However, western blotting of supernatant of monocytestreated with LPS for sTREM-1 with an antibody specificfor the extracellular domain, showed that sTREM-1 is27 kDa, suggesting it is unlikely that TREM-1sv is thesource of sTREM-1 (Gibot et al., 2004). A later studyshowed that pre-treatment of monocytes with a generalinhibitor of the matrix metalloproteinases (MMPs)called GM6001, followed by LPS treatment results ina decrease in sTREM-1 production and maintenance ofTREM-1 surface expression, compared to monocytespre-treated with a protease inhibitor cocktail whichblocks the activity of a broad range of serine andcysteine proteinases but not MMPs (Gomez-Pina et al.,2007). These findings correlate well with an earlier studyconducted by Gibot and colleagues who also found nodecrease in the appearance of sTREM-1 in LPSchallenged monocytes which had been pre-treated witha protease inhibitor cocktail (Gibot et al., 2004).Southern blotting of monocytes treated with LPS usinga biotin-labeled probe designed to hybridize to both fulllength TREM-1 and TREM-1sv, showed only thepresence of a 900 bp band corresponding to the fulllength receptor, excluding the possibility of alternativesplicing as a mechanism for the generation of sTREM-1(Gomez-Pina et al., 2007). Therefore, despite the exactfunction of TREM-1sv remaining elusive, it is likely thatsTREM-1 is generated through proteolytic cleavage ofmembrane anchored TREM-1 by MMPs. Interestingly,it has been shown that treatment of monocytes withLP17 and LPS results in an even greater increase insTREM-1 compared to those monocytes treated withLPS alone (Gibot et al., 2004). How LP17 leads toenhanced production of sTREM-1 is currently un-known.

The role of sTREM-1 is unclear but since sTREM-1 hasthe same extracellular domain as TREM-1, it is possiblethat when released into the serum, it may compete withTREM-1 for ligand(s) and act to down-regulate the


TREM-1 pathway. Indeed, as discussed earlier, a solubleform of TREM-1 generated artificially has previously beenshown to be protective in models of murine endotoxemiaand septic peritonitis (Bouchon et al., 2001a). Significantly,sTREM-1 is an important predictor and diagnostic markerfor sepsis, bacterial and fungal pneumonia (Gibot andCravoisy, 2004).

The mystery of the TREM-1 ligand

To date, the nature of the TREM-1 ligand remains amystery. There is evidence which suggests the ligandfor TREM-1 may be expressed on human platelets(Haselmayer et al., 2007). Although the authors of thisstudy did not show whether TREM-1 was expressed onhuman platelets (to our knowledge this has not beendemonstrated before), they did show that in their systemsTREM-1 bound to human platelets and this bindingcould be blocked by LP17 (Haselmayer et al., 2007).Because sTREM-1 contains the same extracellulardomain as the TREM-1 receptor, it could bind to anunidentified ligand present on human platelets. Insupport of this hypothesis, Haselmayer and colleaguesshowed that co-incubation of platelets (which had beenfixed with paraformaldehyde, excluding influences ofsoluble factors released by platelets) enhanced LPS-induced activation of neutrophils. This could besuppressed by sTREM-1, but the association of plateletswith neutrophils could not, suggesting that platelet-mediated neutrophil activation is dependent on aTREM-1 ligand interaction with neutrophils, but thephysical association of platelets with neutrophils in-volves other interactions (Haselmayer et al., 2007).TLT-1 has been shown to be expressed in platelets(Table 1; Washington et al., 2004) and this member ofthe TREM family also has activatory functions (Barrowet al., 2004). Therefore, although there is evidencesuggesting that the TREM-1 ligand is expressed onplatelets and enhances neutrophil activation, the exactcontribution of TLT-1 to platelet-mediated neutrophilactivation requires further study.

Interestingly, there is recent evidence to suggest thathigh-mobility group box 1 (HMGB1) and heat shockprotein 70 (HSP70) could be ligands for TREM-1(El Mezayen et al., 2007). Treatment of THP-1 cells,with necrotic cell lysate (NCL) from THP-1 cells (whichhad been treated with LPS to induce secretion ofHMGB1 and HSP70), showed that NCL could augmentLPS-induced secretion of IL-6, TNF and IL-8. Thisaugmentation was dependent on HMGB1 and HSP70,since neutralization of these proteins with antibodies,prevented it (El Mezayen et al., 2007). Importantly,blocking TREM-1 using a recombinant TREM-1 fusionchimera did not prevent pro-inflammatory cytokinesecretion in response to LPS, but decreased the

augmentation in response to LPS and NCL. Moreover,blocking TREM-1 simultaneously with blockingHMGB1 and HSP70 did not have any additional effectsto blocking TREM-1 alone (El Mezayen et al., 2007).Although the authors of this study did not show surfaceexpression of TREM-1 in THP-1 cells, this worksuggests that HSP70 and HMGB1 released by necroticcells augments LPS-induced cytokine secretion in aTREM-1-dependent manner. HMGB1 is believed to bea late mediator of sepsis and neutralization of HMGB1protects mice against LPS-induced shock and septicperitonitis (Riedemann et al., 2003; Hunter, 2006;Bianchi and Manfredi, 2007). Given that signalingthrough the TREM-1 receptor plays such an importantrole in bacterial infections and sepsis via its capacity topromote inflammation, results that suggest an associa-tion with a ligand like HMGB1, which is involved in thesame syndrome (and is also capable of promotinginflammation (Bianchi and Manfredi, 2007)) are ex-tremely intriguing and merit further investigation.

TREM-2

TREM-2 signaling: similarities and differences

between TREM-1 signaling

Similar to TREM-1, the ligand for TREM-2 isunknown and studies on TREM-2 signaling haveutilized an agonistic TREM-2 antibody, which revealsthat TREM-2 also signals through DAP-12, leading toan increase in intracellular calcium and phosphorylationof ERK1/2 (Bouchon et al., 2001b). Importantly,TREM-2 receptor ligation does not induce the degrada-tion of IkB-a and the subsequent nuclear translocationof NF-kB, showing clear differences between TREM-2and TREM-1 signaling (Bouchon et al., 2001b).Receptor cross-linking of TREM-2 on immature den-dritic cells triggers up-regulation of molecules involvedin T cell co-stimulation such as CD86, CD40 and MHCclass II, as well as up-regulation of the chemokinereceptor CCR7 (Bouchon et al., 2001b). TREM-2 is alsoexpressed on microglia, where receptor cross-linkingresults in an increase in ERK1/2 phosphorylation andCCR7 but not CD86 or MHC class II expression,suggesting possible cell type-specific differences inTREM-2 signaling (Takahashi et al., 2005). Addition-ally, activation of TREM-2 signaling in microglia resultsin an increase in phagocytosis, which is accompanied bya polarization and re-organization of F-actin in anERK-dependent manner (Takahashi et al., 2005).Whether actin is similarly modified in other cellswhich express TREM-2 remains to be determined.Interestingly, treatment of monocytes with an agonisticTREM-1 antibody leads to increased expression of


CD86 and MHC class II (HLA-DR) (Bleharski et al.,2003), indicating that despite differences betweenTREM-1 and TREM-2 signaling, some of the down-stream effector genes activated are similar.

Regulation of TREM-2 expression

Contrary to TREM-1, TREM-2 is not expressed onperipheral blood monocytes, but is expressed onimmature dendritic cells, macrophages which have beennewly recruited into the peritoneum or air spaces andBMDM, as well as microglia and a variety of macro-phage cell lines (Table 1; Bouchon et al., 2001b;Turnbull et al., 2006; Piccio et al., 2007). While in thecase of TREM-1 cell surface expression is induced byLPS, cell surface expression of TREM-2 decreasesfollowing LPS treatment of RAW 264.7, J774.2 macro-phages, BMDM and microglia (Bouchon et al., 2001b;Turnbull et al., 2006; Piccio et al., 2007). TREM-2 cellsurface expression is induced in resident peritonealmacrophages (which do not exhibit constitutive cellsurface expression of TREM-2) following treatmentwith IL-4 for 48 h (Turnbull et al., 2006). Interestingly,in microglial cell lines, it has been reported that TREM-2 expression is mainly intracellular, localizing in part tothe golgi complex (Sessa et al., 2004). Treatment ofmicroglia with ionomycin causes rapid TREM-2 cellsurface expression but upon wash-out, there is aprogressive decline to basal levels, suggesting thatintracellular pools of TREM-2 can be rapidly translo-cated to the cell surface (Sessa et al., 2004). WhetherTREM-2 is stored intracellularly in other types ofmacrophages is as yet uncertain. Furthermore, theidentity of the transcription factors that regulate theexpression of the TREM-2 gene is currently unknown.

TREM2: a negative regulator of inflammatory

responses

BMDM which have been silenced for TREM-2 usingshRNAi display increased secretion of TNF in responseto the TLR2/6 ligand zymosan and the TLR9 ligandCpG, compared to those cells which have been treatedwith a non-specific shRNAi (Hamerman et al., 2006),indicating that TREM-2 negatively regulates cytokinesynthesis in macrophages. These results have beenconfirmed using BMDM from TREM-2 knockout mice(Turnbull et al., 2006). The authors of this studyadditionally extended these findings to show that levelsof TNF and IL-6 were also higher in TREM2�/�BMDMs in response to LPS, compared to wild-typecontrols (Turnbull et al., 2006). TREM-2 overexpressionin microglia has been demonstrated to lead to less TNFand inducible nitric oxide (iNOS) mRNA after cultureof these cells with apoptotic neurons, whereas TREM-2

knockdown resulted in a modest increase in TNF andiNOS (Takahashi et al., 2005). These studies indicate incontrast to TREM-1, which is a positive regulator ofcytokine synthesis, TREM-2 is a negative regulator.This effect of TREM-2 on inflammation appears to beindependent of the type of macrophage as it occurs inboth microglia and BMDMs.

TREM-2 and bacterial infection: an unexplored area

Although a TREM-2 knockout mouse exists(Turnbull et al., 2006), there are no in vivo studies onthe role of TREM-2 in bacterial infection, which utilizethese mice. Also no studies have been reported onactivation or inhibition of TREM-2 signaling in thecontext of bacterial infectious disease models. TREM-2and DAP-12 mutations occur in patients with a diseasecalled Nasu–Hakola syndrome, which manifests itselfwith bone cysts and dementia (Klunemann et al., 2005).Therefore, many studies on TREM-2 have focused onthe role of TREM-2 in microglia and brain function(Sessa et al., 2004; Takahashi et al., 2005; Piccio et al.,2007). Moreover, there are no reports on increasedsusceptibility to bacterial infection or increased severityof sepsis in patients with Nasu–Hakola syndrome. Thesepatients also exhibit mutations in DAP-12, which haspreviously been demonstrated to be important inbacterial infection (Turnbull et al., 2005), suggestingthat investigations into the in vivo role of TREM-2 inbacterial infection are warranted. The in vitro studiesmentioned earlier indicate that TREM-2 is a negativeregulator of inflammatory responses. Negative regula-tion of inflammation is important as it acts to ensurethat production of pro-inflammatory cytokines is keptin check, as this can be detrimental to the host.However, at the onset of infection, low expression levelsof negative regulators are required to ensure a properimmune response. Many negative regulators, includingthose which modulate TLR responses, have been shownto be important in bacterial infection (Krishnan et al.,2007; Lang and Mansell, 2007). A study by Aoki andcolleagues hints to a possible role of TREM-2 in lunginfections because TREM-2 expression was shown toincrease in the lung following Mycobacterium bovis

infection of mice and this correlates with an increase inDAP-12 mRNA and protein expression (Aoki et al.,2004). However, this increase does not occur in vitro inisolated alveolar macrophages which have been treatedwith mycobacteria, indicating that the increase could beeither on another cell type present in lung tissue orrelated to an increase in alveolar macrophage influx intothe lung (Aoki et al., 2004). Regarding the formerpossibility, it is of interest that recently hepaticendothelial cells have been shown to express TREM-2transcripts (Table 1; Chen et al., 2007), so it is possible


that the increase of TREM-2 mRNA observed by theseauthors in the lung, could be on lung endothelium.Regarding the latter possibility, it has been shown thatTREM-2 is expressed on the cell surface of newlyrecruited alveolar macrophages (Table 1; Turnbull et al.,2006). Importantly, there is evidence to suggest thatTREM-2 is capable of binding to bacteria (discussedbelow and Daws et al., 2003). Therefore, it is temptingto speculate that like TREM-1, TREM-2 also plays animportant but as yet unknown role in bacterialinfection.

The mystery of the TREM-2 ligand

Like TREM-1, the nature of the TREM-2 ligandremains elusive. There is some evidence to suggest thatthe TREM-2 ligand could be polyanionic microbialproducts (Daws et al., 2003). Cells transfected withTREM-2 are able to bind to fluorescently labeledStaphylococcus aureus and Escherichia coli as deter-mined by fluorescence activated cell sorting (FACS) andconfocal microscopy. The binding of bacteria to over-expressed TREM-2 can be blocked by sTREM-2(a TREM-2 consisting of the extracellular domain ofTREM-2), dextran sulfate and polyinosinic, suggestingthat TREM-2 binds to bacteria through the recognitionof anionic bacterial surface structures, such as carbohy-drate structures (Daws et al., 2003). However, thespecific molecules present on bacteria responsible forbinding to TREM-2 are unknown. Additionally, im-munoprecipitation experiments indicate that TREM-2can associate with another receptor on the cellmembrane called Plexin-A1. The association withTREM-2 is needed for plexin-A1 to associate withDAP-12 (Takegahara et al., 2006). Functionally,TREM-2 and DAP-12 are required for plexin-A1signaling (Takegahara et al., 2006), and this is interest-ing considering that plexin-A1�/� mice have a defect inosteoclast development, similar to that previouslyattributed to TREM-2 and DAP-12 (Humphrey et al.,2006). The results of this study show that TREM-2 ispart of a higher order receptor complex and suggest thatidentification of TREM-2 ligands may require ap-proaches which encompass co-receptors (Klesney-Taitet al., 2006). Whether, TREM-1 like TREM-2 is capableof forming higher order receptor complex remains to bedetermined.

Concluding remarks

TREM-1 and TREM-2 play central roles in mono-nuclear phagocyte biology and innate immunity. How-ever, a proper understanding of these receptors has beenrestrained because of the elusive nature of their ligands.

Studies indicate that while TREM-1 and TREM-2 sharesome similarities in signaling, there are also differences.Importantly, the outcome of these signaling pathwaysupon ligand recognition is that TREM-1 acts as apositive regulator of the immune response followingrecognition of danger signals from microbes, whileTREM-2 acts a negative regulator.

The distinct roles of TREM-1 and TREM-2 in innateimmunity could be related to differential proteinexpression of these receptors in different resident tissuemacrophages. TREM-1 is constitutively expressed onresident alveolar macrophages, while TREM-2 isexpressed on microglia. Resident tissue macrophagesdisplay functional heterogeneity (Morrissette et al.,1999). For example, alveolar macrophages are impor-tant for airway defense, while microglia play a role inneuro-endocrine homeostasis and are refractory to awide variety of inflammatory signals, which serves toprotect the surrounding neurons in the brain. (Morris-sette et al., 1999). Given the role of TREM-1 as apositive regulator of inflammation and TREM-2 as anegative regulator, this suggests that different popula-tions of macrophages in the body may use differentialexpression of TREMs as a layer of control in the innateimmune system, which, on the one hand, protectsagainst infection and on the other hand, preventsexcessive tissue damage. The situation is likely to bemore complex though because newly recruited alveolarmacrophages have been shown to display surfaceexpression of TREM-2, but the exact role of TREM-2in these circumstances remains to be determined.

By its capacity to promote inflammation, TREM-1 isan important player in bacterial infection and sepsis.This is evidenced by sTREM-1, which acts to counteractthe pro-inflammatory signaling pathways which areactivated following recognition of the TREM-1 receptorby its ligand. Considering TREM-2 has been shown tobind to bacteria, it is tempting to speculate that similarto TREM-1, TREM-2 has an undiscovered role inbacterial infection. The exact role of both TREM-1 and2 in bacterial infection will ultimately be answeredthrough in vivo studies which utilize knockout mice forthese receptors, in combination with their ligands, in thecontext of various bacterial infectious disease models.

Conflict of interest

The authors declare there is no conflict of interest.

Acknowledgment

We would like to take this opportunity to thankUlrich Matt and Tanja Furtner for critically reading thismanuscript.


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Documents

From expression to signaling: Roles of TREM-1 and TREM-2 in innate immunity and bacterial infection