Evolution of recognition of ligands from Gram-positive bacteria: similarities and differences in the TLR2-mediated response between mammalian vertebrates and teleost fish.
ABSTRACT We investigated the role of the TLR2 receptor in the recognition of ligands from Gram-positive bacteria in fish. Comparative sequence analysis showed a highly conserved Toll/IL-1 receptor domain. Although the leucine-rich repeat domain was less conserved, the position of the critical peptidoglycan (PGN)-binding residues in the leucine-rich repeat domain of carp TLR2 were conserved. Transfection of human embryonic kidney 293 cells with TLR2 corroborated the ability of carp TLR2 to bind the prototypical mammalian vertebrate TLR2 ligands lipoteichoic acid (LTA) and PGN from Staphylococcus aureus. The synthethic triacylated lipopeptide N-palmitoyl-S-(2,3-bis(palmitoyloxy)-(2RS)-propyl)-(R)-Cys-(S)-Ser-(S)-Lys(4) trihydrochloride (Pam(3)CSK(4)) but not the diacylated lipopeptide macrophage-activating lipopeptide-2 (MALP-2) also activated TLR2 transfected human cells. We identified clear differences between the mammalian vertebrates and carp TLR2-mediated response. The use of the same ligands on carp macrophages indicated that fish cells require high concentrations of ligands from Gram-positive bacteria (LTA, PGN) for activation and signal transduction, react less strongly (Pam(3)CSK(4)) or do not react at all (MALP-2). Overexpression of TLR2 in carp macrophages confirmed TLR2 reactivity of the response to LTA and PGN, low-responsiveness to Pam(3)CSK(4) and nonresponsiveness to MALP-2. A putative relation with the apparent absence of accessory proteins such as CD14 from the fish TLR2-containing receptor complex is discussed. Moreover, activation of carp macrophages by PGN resulted in increased TLR2 gene expression and enhanced TLR2 mRNA stability, MAPK-p38 phosphorylation and increased radical production. Finally, we could show that NADPH oxidase-derived radicals and MAPK-p38 activation cooperatively determine the level of PGN-induced TLR2 gene expression. We propose that the H(2)O(2)-MAPK-p38-dependent axis is crucial for regulation of TLR2 gene expression in fish macrophages.
- SourceAvailable from: B M Lidy Verburg-van Kemenade[show abstract] [hide abstract]
ABSTRACT: To analyse the functional activity of different leucocyte types, carp pronephros cells were separated on Percoll density gradients and by use of fluorescence-activated cell sorting. Cell populations were characterised by light and electron microscopy and by flow cytometry. Fractions enriched in macrophages and neutrophilic granulocytes were subsequently analysed for phagocytic activity in vitro by quantification of the uptake of Escherichia coli bacteria or yeast cells, and for respiratory burst response by measurement of the production of the reactive oxygen intermediates O2· and H2O2. Both cell types showed very active in vitro phagocytosis and production of both O2· and H2O2. When activated with phorbol myristate acetate or bacteria, it was evident that the neutrophilic granulocytes were significantly more active than the macrophages. Analysis of single-cell respiratory burst activity in fish phagocytes was investigated after preloading of cells with dihydrorhodamine123. Cells were subsequently separated and analysed for fluorescence using flow cytometry. Both the macrophage-enriched fraction and the granulocyte-enriched fraction appeared to consist of active and inactive subpopulations. In comparison with the inactive populations, active populations had characteristic high forward/sideward scatter profiles.Journal of Experimental Biology 02/1994; 187(1):143-58. · 3.24 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: The use of 2',7'-dichlorofluorescein diacetate (DCFH), dihydrorhodamine 123 (DHR) and hydroethidine (HE) has been described for detecting respiratory burst activity by flow cytometry in polymorphonuclear neutrophil (PMN) suspension. However, their specificities for reactive oxygen species are not well defined. We investigated the reactivity of these probes for detecting superoxide anion (O(2)(* -)), hydrogen peroxide (H(2)O(2)) and/or nitric oxide (NO(z.rad;))-dependent mechanisms. PMNs (10(6)/ml) were preincubated for 15 min at 37 degrees C with DCFH (5 micro mol/l), DHR (1 micro mol/l) or HE (10 micro mol/l). Cell suspensions were then split for each probe into five different aliquots containing either no effector or one effector: N-ethylmaleimide (NEM, 150 micro mol/l, NADPH oxidase inhibitor), sodium azide (NaN(3), 50 micro mol/l, peroxidase and catalase inhibitor), N-nitro-L-arginine methyl ester (L-NAME, 1.5 micro mol/l, NO(z.rad;) synthase inhibitor) or H(2)O(2) (30%). At the same time, PMNs were stimulated with phorbol myristate acetate (PMA, 10 micro mol/l) for 10 min at 37 degrees C. Analyses were carried out on a Beckman-Coulter Epics XL equipped with an argon laser (488 nm). Green fluorescences from DCFH and DHR were measured in the FL1 channel and HE fluorescence was analyzed in the FL2 channel. NaN(3) decreased the fluorescence of PMNs incubated with DCFH, indicating that it needs a peroxidase activity to react with H(2)O(2). L-NAME reduced the oxidation of DCFH, showing that it reacts with reactive nitrogen species. DHR was specifically responsive to H(2)O(2) accumulation. HE seemed to be preferentially oxidized by O(2)(* -). Hence the choice of the probe to be used depends on the reactive species of interest.Clinica Chimica Acta 06/2003; 331(1-2):103-10. · 2.85 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: The aim of this study was to investigate the oxidation of two common fluorescent probes, dichlorodihydrofluorescein (DCFH2) and dihydrorhodamine (DHR), and their oxidized forms, dichlorofluorescein and rhodamine, by the radical products of peroxynitrite chemistry, *OH, NO2*, and CO3*-. At pH 8.0-8.2, rate constants for the interaction of carbonate radical with probes were estimated to be 2.6 x 10(8) x M(-1) s(-1) for DCFH2 and 6.7 x 10(8) M(-1) s(-1) for DHR. Nitrogen dioxide interacted more slowly than carbonate radical with these probes: the rate constant for the interaction between NO2* and DCFH2 was estimated as 1.3 x 10(7) M(-1) s(-1). Oxidation of DHR by nitrogen dioxide led to the production of rhodamine, but the kinetics of these reactions were complex. Hydroxyl radical interacted with both probes with rate constants close to the diffusion-controlled limit. We also found that oxidized forms of these fluorescent probes reacted rapidly with carbonate, nitrogen dioxide, and hydroxyl radicals. These data suggest that probe oxidation may often be in competition with reaction of the radicals with cellular antioxidants.Free Radical Biology and Medicine 02/2005; 38(2):262-70. · 5.27 Impact Factor
The Journal of Immunology
Evolution of Recognition of Ligands from Gram-Positive
Bacteria: Similarities and Differences in the TLR2-Mediated
Response between Mammalian Vertebrates and Teleost Fish
Carla M. S. Ribeiro, Trudi Hermsen, Anja J. Taverne-Thiele, Huub F. J. Savelkoul, and
Geert F. Wiegertjes
We investigated the role of the TLR2 receptor in the recognition of ligands from Gram-positive bacteria in fish. Comparative se-
quence analysis showed a highly conserved Toll/IL-1 receptor domain. Although the leucine-rich repeat domain was less conserved,
the position of the critical peptidoglycan (PGN)-binding residues in the leucine-rich repeat domain of carp TLR2 were conserved.
Transfection of human embryonic kidney 293 cells with TLR2 corroborated the ability of carp TLR2 to bind the prototypical
mammalian vertebrate TLR2 ligands lipoteichoic acid (LTA) and PGN from Staphylococcus aureus. The synthethic triacylated
lipopeptide N-palmitoyl-S-(2,3-bis(palmitoyloxy)-(2RS)-propyl)-(R)-Cys-(S)-Ser-(S)-Lys4 trihydrochloride (Pam3CSK4) but not
the diacylated lipopeptide macrophage-activating lipopeptide-2 (MALP-2) also activated TLR2 transfected human cells. We
identified clear differences between the mammalian vertebrates and carp TLR2-mediated response. The use of the same ligands
on carp macrophages indicated that fish cells require high concentrations of ligands from Gram-positive bacteria (LTA, PGN) for
activation and signal transduction, react less strongly (Pam3CSK4) or do not react at all (MALP-2). Overexpression of TLR2 in
carp macrophages confirmed TLR2 reactivity of the response to LTA and PGN, low-responsiveness to Pam3CSK4and non-
responsiveness to MALP-2. A putative relation with the apparent absence of accessory proteins such as CD14 from the fish
TLR2-containing receptor complex is discussed. Moreover, activation of carp macrophages by PGN resulted in increased TLR2
gene expression and enhanced TLR2 mRNA stability, MAPK-p38 phosphorylation and increased radical production. Finally, we
could show that NADPH oxidase-derived radicals and MAPK-p38 activation cooperatively determine the level of PGN-induced
TLR2 gene expression. We propose that the H2O2-MAPK-p38–dependent axis is crucial for regulation of TLR2 gene expression in
fish macrophages.The Journal of Immunology, 2010, 184: 2355–2368.
viruses, fungi, and protozoa), thereby allowing sufficient responses
to limit or eradicate invading microbes. TLRs are type I trans-
he innate immune system of vertebrates uses pattern rec-
ognition receptors (PRRs) to sense invading pathogens.
TLRs act as PRRs and, as such, are able to recognize
membrane proteins with an ectodomain containing interspersed
The cytoplasmic domain is characterized by a Toll/IL-1 receptor
(TIR) motif that is involved in signal transduction (1, 2). In mam-
malian vertebrates, TLR2 senses bacterial lipopeptides (3–5) and
lipoteichoic acids (LTAs) (6, 7) as well as GPI anchors from par-
asites (8, 9). TLR2 recognizes microbial patterns as homodimer
(10) and as heterodimers with TLR1 or TLR6 (11, 12). Expression
of TLR2 in humans is highest in myeloid cells (monocytes, mac-
rophages, dendritic cells, and granulocytes), whereas in mice,
TLR2 expression was also found in T cells (13–15). Investigations
of TLR-mediated immune recognition in nonmammalian verte-
brates, such as teleost (modern bony) fish, can provide new insights
into the evolution of immunity (16). Research on teleost fish TLR2
has been mostly restricted to the sequence identification of the
and catfish) with limited studies on the basal and inducible ex-
pressionlevelsofTLR2transcripts (17–20).Althoughthese studies
demonstrate the presence of a TLR2 ortholog, the role of the TLR2
receptor in the recognition of ligands from Gram-positive bacteria
has not been studied in fish.
Teleost fish possess orthologs of the different mammalian TLR
families and a further two additional fish-specific TLR family
members (17, 21). So far, only for the orthologs of TLR3 in ze-
brafish and rainbow trout (22, 23) and TLR5 in rainbow trout (24)
proof for functional analogy to the mammalian counterparts has
been provided. In fact, clear differences between fish and mam-
malian vertebrates in the recognition and response to certain
PAMPs have been observed. Cells from teleost fish require much
Cell Biology and Immunology Group, Department of Animal Sciences, Wageningen
University, Wageningen, The Netherlands
Received for publication March 26, 2009. Accepted for publication December 31,
This work was supported by the Portuguese foundation Fundac ¸a ˜o para a Cie ˆncia e
Tecnologia under Grant agreement number SFRH/BD/21522/2005 and by the Euro-
pean Community’s Seventh Framework Programme ([FP7/2007-2013] under Grant
agreement number PITN-GA-2008-214505).
The sequence presented in this article has been submitted to www.ncbi.nlm.nih.gov/
Genbank under accession number FJ858800.
Address correspondence and reprint requests to Dr. Geert F. Wiegertjes, P.O. Box 338,
6700 AH, Wageningen, The Netherlands. E-mail address: firstname.lastname@example.org
Abbreviations used in this paper: DHR, dihydrorhodamine; DPI, diphenyleneiodo-
nium chloride; HEK, human embryonic kidney; iNOS, inducible NO synthase;
LRR, leucine-rich repeat; LTA, lipoteichoic acid; MALP-2, macrophage-activating
lipopeptide-2; Nod2, nucleotide-binding oligomerization domain 2; Pam3CSK4,
drochloride;PAMP,pathogen-associated molecular pattern; PDTC,pyrrolidinedithio-
carbamate; PGN, peptidoglycan; PGRP, PGN recognition protein; phospho-p38,
phosphorylated p38; PI, propidium iodide; PI-PLC, phosphatidylinositol-specific
phospholipase C; PRR, pattern recognition receptor; ROS, reactive oxygen species;
RT, room temperature; RT-qPCR, real-time quantitative PCR; SOD, superoxide dis-
mutase; TIR, Toll/IL-1 receptor; WT, wild type.
higher doses (micrograms/milliliter) of LPS from Gram-negative
bacteria than human cells (nanograms/milliliter) for activation (25,
26). In teleost fish, LPS signals via a TLR4-independent manner
(27) and, in fact, it was hypothesized that the TLR4 genes in ze-
thus provides no evidence for a direct relationship between ligand,
the corresponding receptor, and the resulting immune response and
classification of TLR proteins solely based on sequence homology
may lead to false presumptions on functional conservation (28). In
addition, although in mammalian vertebrates cytokine and radical
production have been extensively used as markers of TLR(2) acti-
the ligands for TLR2 and activation of cytokines and nitrogen and
oxygen radicals downstream of TLR2 activation in fish.
from Gram-positive bacteria such as LTA or peptidoglycan (PGN)
results in recruitment of a set of TIR domain-containing adaptor
proteins such as MyD88. These interactions trigger downstream
cascades leading to the activation of NF-kB and MAPKs, which
control induction of inflammatory genes like IL-1b, TNF-a, and
inducible NO synthase (iNOS) and control the activation of anti-
microbial host defense mechanisms such as production of reactive
oxygen species (ROS) and nitrogen species (29, 30, 33, 34). In
general, in teleost fish, the intracellular components acting down-
of genes in mammalian vertebrates and teleosts (35). The extracel-
lular components of receptors such as TLRs seem to be more di-
vergent (36). We analyzed the TLR2 receptor sequence of common
of the position of leucine residues critical for ligand recognition.
Transfection of human embryonic kidney (HEK) 293 cells and carp
macrophages with the carp TLR2 receptor confirmed activation by
Ser-(S)-Lys4trihydrochloride (Pam3CSK4), but not macrophage-
activating lipopeptide-2 (MALP-2). Activation of carp macrophages
indicated that fish cells require high concentrations of ligands from
Gram-positive bacteria (LTA, PGN) for activation and signal trans-
carp TLR2 gene expression and enhanced TLR2 mRNA stability.
Furthermore, our results suggest that H2O2radicals via MAPK-p38
activation play an indispensable role in the regulation of TLR2 gene
observations also apply to other teleost fish species.
Materials and Methods
European common carp (Cyprinus carpio carpio L.) were reared in the
central fish facility at Wageningen University, The Netherlands, at 23˚C in
recirculating UV-treated tap water and fed pelleted dry food (Trouvit,
Nutreco, The Netherlands) daily. R3xR8 carp are the hybrid offspring of
a cross between fish of Hungarian origin (R8 strain) and of Polish origin
(R3 strain) (37). Carp were between 9 and 11 mo old. All studies were
performed with approval from the animal experimental committee of
Stimuli and inhibitors
Purified LTA from S. aureus (tlrl-psLTA), soluble secreted PGN from
S. aureus (tlrl-ssPGN), synthethic tripalmitoylated lipopetide Pam3CSK4
(tlrl-pms) and ultrapure LPS from Porphyromonas gingivalis (tlrl-pgLPS)
were purchased from InvivoGen (Cayla SAS, France). Synthetic MALP-2,
purchased from Alexis Biochemicals (Axxora, Germany). LPS from Es-
cherichia coli (L2880), muramyl dipeptide, and polyinosinic polycytidiylic
acid were purchased from Sigma-Aldrich (St. Louis, MO). NF-kB inhibitor
mitochondrial chain I inhibitor rotenone, NADPH oxidase inhibitor diphe-
nyleneiodonium chloride (DPI), superoxide dismutase (SOD), and catalase
were purchased from Sigma-Aldrich. MAPK-p38 inhibitor SB208530 was
purchased from Calbiochem (Merck Chemicals, Nottingham, U.K.). Phos-
phatidylinositol-specific phospholipase C (PI-PLC) from Bacillus cereus
was purchased from Sigma-Aldrich.
Amplification of carp TLR2 wild type and TLR2DTIR cDNA
Oligonucleotide primers for carp TLR2 were designed based on known
partialfishTLR2 sequences.cDNAfrom macrophages stimulatedwithLTA
for 6 h was used as a template for PCR or nested PCR. The 59and 39ends of
TLR2 were amplified using gene-specific primers (forward TLR2 and re-
verse TLR2, Table I) by 59and 39rapid amplification of cDNA ends (RACE)
(38) using the Gene Racer RACE Ready cDNA kit (Invitrogen, Breda, The
Netherlands) according to the manufacturer´s protocol. Gene-specific
primer (TLR2 Fw) was used in combination with the Gene Racer primers to
amplify first strand of cDNA (Table I). A second round with gene-specific
primers (TLR2Fw, TLR2Rv) was performed to obtain the carp wild type
(WT) TLR2 (TLR2WT, 2367 bp) or TIR-domain truncated TLR2
(TLR2Fw, TLR2Rv nested) (TLR2DTIR, 2100 bp). PCR reactions were
performed in Taq buffer, using 1 U Taq polymerase (Promega, Leiden, The
Netherlands) supplemented with MgCl2(1.5 mM), dNTPs (200 mM), and
primers (400 nM each) in a total vol of 50 ml. PCR and nested PCR were
carried out under the following conditions: one cycle 4 min at 96˚C; fol-
lowedby35 cycles of30 sat 96˚C,30 sat 55˚C,and2 minat 72˚C;andfinal
extension for 7 min at 72˚C, using a GeneAmp PCR system 9700 (PE,
Applied Biosystems, Foster City, CA). Products amplified by PCR, nested
PCR or RACE-PCR were ligated and cloned in JM-109 cells using the
pGEM-Teasykit (Promega)accordingto themanufacturer’s protocol.From
each product, both strands of eight clones were sequenced, using the ABI
prismBigDye Terminator Cycle Sequencing Ready Reaction kit and ana-
lyzed using 3730 DNA analyzer.
The nucleotide sequence was translated using the ExPASy translate tool
(http://us.expasy.org/tools/dna.html) and aligned with Clustal W (www.ebi.
ac.uk/clustalw). The signal peptide cleavage site and the transmembrane
region was predicted by using the SignalP 3.0 (www.cbs.dtu.dk/services/
SignalP/) and the TMHMM 2.0 (www.cbs.dtu.dk/services/TMHMM-2.0/)
servers, respectively. Identification of LRRs within carp TLR2 were pre-
dicted using the method previously described (39). First, LRRs were
predicted using PFAM (http://pfam.sanger.ac.uk/) and SMART (http://
smart.embl-heidelberg.de/) servers. Second, LRRs candidates, that could
not be recognized by PFAM, were identified by multiple sequence align-
ments with TLR2 from other species including human, mouse, and ze-
brafish. Third, protein secondary structure predictions of the LRR
candidate were evaluated by Proteus (http://wks16338.biology.ualberta.ca/
proteus/) server. Posttranslational modifications were predicted using the
NetNGlyc 1.0 (www.cbs.dtu.dk/services/NetNGlyc/) server.
TLR2WT-GFP and TLR2DTIR-GFP expression plasmids
The vivid color pcDNA6.2/C-EmGFP-GW/TOPO (Invitrogen, catalog no.
K359-20) expressionvector combined with TOPO cloning was used to fuse
TLR2WT or TLR2DTIR to EmGFP at the C-terminal end. Isolation of
highly pure plasmid DNA suitable for transfection was performed using
S.N.A.P. Midi Prep Kit (Invitrogen, catalog no. K1910-01) according to the
manufacturer’s protocol. C-terminal fluorescent-tagged protein could be
visualized using confocal microscopy.
Transient transfection of HEK 293 cells
HEK 293 cells were cultured in DMEM supplemented with 10% FBS (Invi-
trogen), 50 U/ml penicillin G (Sigma-Aldrich), and 50 mg/ml streptomycin
sulfate (Sigma-Aldrich). Two days before transfection, HEK 293 cells were
seeded into tissue culture flasks to reach 80–90% confluence at the day of
transfection. For transient transfection, 2.5 mg carp TLR2WT-GFP or
using nucleofactor solution Vand program A-23 (Lonza, Cologne AG, Ger-
many) according to the manufacturer’s instructions. The transfection effi-
ciency, assessed by flow cytometry, was on average 70%. Forty-eight hours
after transfection, cells were trypsinized (0.25% trypsin, Invitrogen), counted
using trypan blue exclusion, and plated overnight at a concentration 6 3 105
2356EVOLUTION OF RECOGNITION OF GRAM-POSITIVE BACTERIA
cells/well ina 24-well tissue cultureplate.The nextday, cellswere stimulated
with (optimized concentrations) 50 mg/ml LTA, 50 mg/ml PGN, 10 mg/ml
Pam3CSK4, 10 mg/ml MALP-2, 50 mg/ml PgLPS, 50 mg/ml pIC, 50 mg/ml
phosphorylated p38 (phospho-p38) activity by Western blot.
Macrophage cell culture
Head kidney-derived macrophages, considered the fish equivalent of bone
Briefly, carp head kidneys were gently passed through a 100-mm sterile nylon
mesh (BD Biosciences, Breda, The Netherlands) and rinsed with homogeni-
zation buffer (incomplete NMGFL-15 medium containing 50 U/ml penicillin
G, 50 mg/ml streptomycin sulfate, and 20 U/ml heparin [Leo Pharmaceutical,
Percoll (Amersham Biosciences, Uppsala, Sweden) and centrifuged at 450g
for 25 min at 4˚C with the brake disengaged. Cells at the interphase were re-
moved and washed twice in incomplete NMGFL-15 medium. Cell cultures
were initiated by seeding 1.75 3 107head kidney leukocytes in a 75 cm2
culture flask containing 20 ml complete NMGFL-15 medium (incomplete
NMGFL-15 medium supplemented with 5% heat-inactivated pooled carp se-
rum and 10% FBS). Head kidney-derived macrophages, named macrophages
throughout the manuscript, were harvested after 6 d of incubation at 27˚C by
placing the flasks on ice for 10 min prior to gentle scraping.
Transient transfection of carp macrophages
For transient transfection, 2.5 mg carp TLR2WT-GFP or TLR2DTIR-GFP
constructs were transfected into carp macrophages by nucleoporation using
nucleofactor Human Macrophage Solution and program Y-001 (Lonza)
a concentration 1 3 106cells/well. The transfection efficiency, assessed by
replaced and macrophages were stimulated for 6 or 9 h with (optimized
concentrations) 50 mg/ml LTA, 50 mg/ml PGN, 10 mg/ml Pam3CSK4,
10 mg/ml MALP-2, or left untreated as control. Cells were lysed for eval-
uation of gene expression by real-time quantitative PCR (RT-qPCR).
Confocal laser scanning microscopy
and 37% formaldehyde (10:1). Sections were embedded in Vectashield
examined with a Zeiss LSM-510 laser scanning microscope. Green fluo-
rescent signal (rhodamine or green-fluorescent protein) was excited with
a 488-nm argon laser and detected using a band-pass filter (505–530 nm)
and detected using a long-pass filter (560 nm).
Macrophage-enriched fractions of head kidney leukocytes were obtained
essentially as previously described (42). Cell suspensions were layered on
a discontinous Percoll gradient (1.020, 1.060, 1.070, and 1.083 g/cm3) and
centrifuged 30 min at 800g with the brake disengaged. Cells at 1.070 and
1.083 g/cm3were collected, pooled, and washed twice with cRPMI (RPMI
the cell membrane. A, All TLR2 molecules have an extracellular, trans-
the N-terminus (LRR1) and is an irregular LRR belonging to the “bacterial”
Regions and amino acid residues shown to be important for ligand recogni-
tion, protein secretion or interaction with MyD88 for the human TLR2
when compared with human TLR2, are depicted in gray and nonconserved
are depicted in black. B, LRR and TIR domain percentage sequence identity
Clustal Walignment. GenBank accession numbers: common carp (Cyprinus
carpio, FJ858800), zebrafish (Danio rerio, NP_997977), human (Homo sa-
piens, NP_003255), and mouse (Mus musculus, NP_036035).
293 cells. HEK 293 cells were transiently transfected with pTOPO con-
taining full-length carp TLR2 (TLR2WT, 2.5 mg) or TIR-domain truncated
TLR2 (TLR2DTIR, 2.5 mg) fused to GFP (green) at the 39-end. PI+cells
are red. Localization of carp TLR2WTand TLR2DTIR was determined by
confocal laser microscopy (original magnification 340).
Localization of carp TLR2 contructs in transfected HEK
The Journal of Immunology2357
were found to be similar as previously described: 1.070 g/cm3interphase
consisted of macrophages (65%) with granulocytes, small macrophages,
and lymphocytes (35%) and 1.083 g/cm3interphase consisted of neutro-
philic granulocytes (85%) with macrophages (15%). The mAb TCL-BE8
(1:50) (43) was used to separate neutrophilic granulocytes from macro-
phages by MACS. After incubation for 30 min with TCL-BE8 on ice, the
leukocyte suspension was washed twice with cRPMI and incubated with
PE-conjugatedgoatanti-mouse(1:50;Dako,Glostrup, Denmark) 30 minon
ice. After washing twice, the total cell number was determined with
a Bu ¨rker countingchamber,and10mlmagneticbeads(anti-PEMicrobeads,
Miltenyi Biotec, GmbH, Gladbach, Germany) was added per 1 3 108cells.
in cRPMI. The magnetic separation was performed on LS-MidiMACS
Columns (Mitenyi Biotec) according to the manufacturer’s instructions.
The purity of the TCL-BE8+(neutrophilic granulocyte-enriched fraction;
.90%) and TCL-BE82(macrophage-enriched fraction; ,10%) fractions
was confirmed by flow cytometricanalysis usinga FACScan flow cytometer
(Becton Dickinson, Mountain View, CA). TCL-BE8+and TCL-BE82were
washed in cRPMI and resuspended in 1 ml cRPMI++(cRPMI medium
supplemented with 1.5% pooled carp serum, 290 mg/ml glutamine [Cam-
brex], 50 U/ml penicillin G, and 50 mg/ml streptomycin sulfate).
Flow cytometric measurement of ROS production
Intracellular ROS levels were evaluated by FACScan flow cytometer using
resuspended in 100 ml rich-NMGFL-15 medium (incomplete NMGFL-15
medium supplemented with 2.5% heat-inactivated pooled carp serum and
5% FBS) and stimulated with PMA (0.05 mg/ml; Sigma-Aldrich) or left
PGN. DHR was added to all samples, and cells incubated for 1 h at 27˚C.
PI (0.1 mg/ml; Sigma-Aldrich) was added to each sample to detect and gate
out PI+cells. For all cytometric measurements the same settings were used
(FS 350 V, gain 2; SS 700 V, gain 10; FL1 600 V; FL2 750 V; FL3 675 V;
FL4 660 V). The baseline offset was on and the FS discriminator was set at
50. Per sample, 104events were measured by flow cytometer.
Macrophages were stimulated with LTA (50 mg/ml), PGN (50 mg/ml), or
left untreated as control, and incubated at 27˚C for 18 h. Nitrite production
was measured essentially as described previously (46). To 75 ml cell
culture supernatant, 100 ml 1% sulfanilamide in 2.5% (v/v) phosphoric
acid, and 100 ml 0.1% (w/v) N-naphthyl-ethylenediamine in 2.5% (v/v)
phosphoric acid were added in a 96-well flat-bottom plate. The absorbance
was read at 540 nm (with 690 nm as a reference) and nitrite concentration
(mM) was calculated by comparison with a sodium nitrite standard curve.
Gene expression profiling of LTA- and PGN-stimulated
seeded in 96-well flat-bottom culture plates and stimulated with LTA, PGN,
Pam3CSK4, or left untreatedas control, and incubated for the indicatedtime
LTA (50 mg/ml), PGN (50 mg/ml), Pam3CSK4(Pam3, 10 mg/ml), MALP-2 (10 mg/ml), PgLPS (50 mg/ml), pIC (50 mg/ml), or EcLPS (50 mg/ml) for 5 min
or left untreated as control. A, MAPK-p38 phosphorylation was analyzed by immunoblotting for phospho-p38, whereas equal loading was confirmed by
immunoblotting for b-tubulin. B, Immunoblot intensity profile (between 0 and 255) of TLR2WT- and TLR2DTIR-transfected HEK 293 cells was de-
termined over a horizontal line on each grayscale image. This is one experiment representative of four experiments.
Activation of carp TLR2 by different PAMPs in HEK 293 cells. TLR2WT and TLR2DTIR transfected HEK293 cells were stimulated with
2358 EVOLUTION OF RECOGNITION OF GRAM-POSITIVE BACTERIA
time point, per treatment, were lysed, and pooled for RNA isolation.
RNA was isolated using the RNeasy Mini Kit (Qiagen, Leusden, The
Netherlands) including the accompanying DNase I treatment on the columns,
ml nuclease-free water. RNA concentrations were measured by spectropho-
performed using DNase I, Amplification Grade (Invitrogen). Briefly, 1 mg
RNAfromeachsamplewascombinedwith103DNase reaction buffer and 1
U DNase I, mixed and incubated at room temperature (RT) for 15 min, fol-
lowed by inactivation of DNase I by adding 1 ml 25 mM EDTA. Synthesis of
cDNAwasperformed with Invitrogen’sSuperScript III First Strand Synthesis
Systems for RT-PCR Systems, according to the manufacturer’s instructions.
Briefly, DNase I-treated RNA samples were mixedwith five times first strand
buffer, 300 ng random primers, 10 mM dNTPs, 0.1 M DTT, 10 U RNase in-
step at 70˚C for 15 min. A nonreverse transcriptase control was included for
each sample. cDNA samples were further diluted 50 times in nuclease-free
water before use as template in real-time PCR experiments.
designed with Primer Express software. IL1-b and TNF-a primer sets were
designed to amplify all known isoforms for each gene. Master mix for each
PCR run wasprepared asfollows:0.32 ml water, 0.84 ml eachprimer (5mM),
and 7 ml Master SYBR Green I mix. To 5 ml diluted cDNA, 9 ml master mix
was added in a 0.1 ml tube. After amplification program was used: one de-
naturation step of 15 min at 95˚C, followed by 40 cycles of RT-qPCR with
three-step amplification (15 s at 95˚C for denaturation, 30 s at 60˚C for an-
specificity. In all cases, the amplifications were specific and no amplification
was observed in negative controls (nontemplate control and nonreverse tran-
scriptase control). Fluorescence data from RT-qPCR experiments were ana-
lyzed using Rotor-Gene version 6.0.21 software and exported to Microsoft
the Rotor-Geneversion6.0.21software.Briefly, the E for eachprimer setwas
set. The relative expression ratio (R) of a target gene was calculated based on
the EAand the Ctdeviation of sample versus control, and expressed in com-
parison with a reference gene (47, 48).
Western blot analysis
Cells were resuspended by pippeting and transferred to precooled eppen-
dorf tubes. Cells were washed twice in ice-cold PBS, lysed on ice with
witha syringe,andincubated 10 minon ice. Cell lysateswere centrifugedat
14,000 rpm for 10 min at 4˚C. Supernatant was collected and total protein
content was determined by the Bradford method. Samples (20–25 mg) were
boiledat 96˚Cfor10min withloadingbuffer containingb-mercaptoethanol
and separated by 10% SDS-PAGE and electrophoretically transferred to
nitrocellulose membranes (Protrans, Schleicher and Schuell, Bioscience
GmbH, Germany). Membranes were blocked in 3% BSA in TBS (10 mM
Tris, 150 mM NaCl, pH 7.5) for 1 h at RTand then incubated with primary
Ab overnight at 4˚C in 3% BSA in TBS. Abs reactive to both humans and
carp were used: rabbit IgG anti–phospho-p38 (1:1000, Thr180/Tyr182,
BioCat GmBh, Heidelberg, Germany) and rabbit IgG anti–b-tubulin
(1:1000, Abcam, Cambridge, U.K.). Membranes were then incubated with
goat anti-rabbit HRP-conjugated (1:1000, Dako) in 10% milk powder in
TBS for 1 h at RT. Between each incubation step, membranes were washed
twice with TBS-Tween/Triton (TBS, 0.05% [v/v] Tween 20, 0.2% [v/v]
Triton X-100) and once with TBS, for 10 min at RT. Signal was detected by
development with a chemoluminescence kit (Amersham Biosciences) ac-
cording to the manufacturer’s protocol and visualized by the use of Lumni-
fil chemiluminescent Detection Film (Roche, Woerden, The Netherlands).
The blots were scanned and the intensity profile (between 0 and 255) of
each lane was determined over a horizontal line on a grayscale image
using the analysis FIVE (Olympus Nederland BV) program.
PI-PLC from B. cereus was resuspended in Tris buffer (10 mM Tris-HCl,
144 mM NaCl, 0.05% BSA, pH 7.4) according to the manufacturers’
protocol. The protozoan carp parasite Trypanoplasma borreli (49) was
incubated for 30 min at 30˚C with 30% Tris-buffer or with 1 U PI-PLC (in
30% Tris-buffer) up to a total of 600 ml in incomplete NMGFL-15 me-
dium. Samples were centrifuged at 800g for 10 min. Supernatants were
collected and filter sterilized (0.22 mm Millex-GV, Millipore, Ireland) to
avoid contamination with parasites. Pellets from PI-PLC–treated were
collected and resuspended in 600 ml incomplete NMGFL-15 medium.
Carp macrophages were stimulated with 25 ml of each fraction.
Transformed values (ln) were used for statistical analysis in SPSS software
(version 15.0). Homogeneity of variance was analyzed using the Levene’s
test. Significant differences (p # 0.05) between a treatment (stimulated
Table I.Primer sequences, gene accession numbers, RT-qPCR melting temperatures, and efficiencies
TLR2 Rv nested
CAA CAC TCT TAA TGT GAG CCA GA
GCT TTC TGC CAC CAC CCT TGG
TCAA CA+C TCT TAA TG+T GAG CCAa
TGT G+CT GGA AA+G GTT CAG AAAa
aThe “+” is before the nucleic acid in which the locked nucleic acid bond was placed.
The Journal of Immunology2359
cells) and the control group (unstimulated cells) were determined by a one-
way ANOVA, followed by a Dunnett t test. Significant differences between
treatments (p # 0.05) were determined by one-way ANOVA, followed by
Bonferroni test. In case of unequal variances between treatments, the one-
way ANOVA was followed by a Games–Howell test.
Carp TLR2 ectodomain contains the critical residues for ligand
Full-length cDNA of the carp TLR2 gene was cloned (GenBank
accession number FJ858800, www.ncbi.nlm.nih.gov/Genbank)
encoding for a TLR2 protein of 788 aa. The carp TLR2 protein is
composed of an extracellular, transmembrane, and intracellular
domain. The ability of carp TLR2 to recognize prototypical TLR2
ligands and to trigger characteristic downstream cascades was
evaluated by comparative sequence analysis. The extracellular do-
main of carp TLR2 has 21 LRRs, whereas human and mouse TLR2
have 20 LRRs (excluding the LRRs at the N-terminal and C-
terminal region) (Fig. 1A). This additional but irregular LRR at the
been found in the zebrafish TLR2 sequence (39). The extracellular
domain of carp TLR2 region containing S40–I64 compassing the
LRR N-terminal and LRR1 motifs, shown to be crucial for the
critical LRR. Furthermore, leucine residues at positions 107, 112,
and 115 (at LRR3) that are critical in the recognition of diacylated
were conserved. The F349 shown to be involved in the recognition
of bacterial lipopeptide was also conserved. N-glycosylation sites
involved in secretion of the N-terminal ectodomain (52) such as
N114 (LLR3) and N442 (LRR16) were conserved (Fig. 1A). Thus,
carp TLR2 LRR domain to mammalian vertebrate LRR domains
(Fig. 1B), the position of the residues critically involved in the
recognition of S. aureus PGN seems well conserved.
The intracellular (TIR) domain of carp TLR2 showed a much
higher sequence identity to the mammalian vertebrate TLR2
sequences than the extracellular LRR domain (Fig. 1B). Proline
P681, involvedintheinteraction withthe MyD88adaptor molecule
(53), was conservedin the carp TLR2 sequence. The high sequence
identity of the carp TIR domain (.67%) suggests that downstream
cascades triggered on TLR2 activation may be conserved in carp.
LTA, PGN, and Pam3CSK4, but not MALP-2, are ligands of
p38 phosphorylation as a measure for responsiveness (15). HEK 293
carp TIR-domain truncated TLR2 (TLR2DTIR). Both constructs
were fused to GFP at the 39-end that enabled us to investigate the
We could confirm the predominant localization of TLR2WT on the
cell surface (TLR2WT; Fig. 2). A more diffuse localization was ob-
served after truncation of the TIR domain (TLR2DTIR; Fig. 2).
MAPK-p38 phosphorylation in transfected HEK 293 cells was
used to study carp TLR2 activation by different TLR ligands, using
gene expression in carp macrophages
PAMPs. RT-qPCR analysis of gene
expression in carp macrophages after
stimulation for 6, 9, 12, and 18 h with
LTA (50 mg/ml), PGN (50 mg/ml),
Pam3CSK4(Pam3, 10 mg/ml), MALP-
2 (10 mg/ml), or left untreated as
control. mRNA levels of IL-1b, iNOS,
IL-11, TNFa, IL-12p35, and IL-12p40
relative to the house keeping 40S ri-
bosomal protein gene level are ex-
pressed as fold change relative to
unstimulated cells at 0 h. Bars show
averages 6 SD of n = 3 fish.
pSignificant (p # 0.05) difference
(control group). Note the differences
in scale of the y-axes.
Kinetics of immune
2360 EVOLUTION OF RECOGNITION OF GRAM-POSITIVE BACTERIA
an Ab specific for phospho-p38. Stimulation of TLR2DTIR
transfected HEK 293 cells with known TLR2 ligands did not in-
crease p38 phosphorylation (TLR2DTIR; Fig. 3A, 3B). This
negative control thereby showed the unresponsiveness of the pa-
rental HEK 293 cells to TLR2 agonists, confirming that HEK 293
cells themselves do not bear receptors for TLR2 ligands. In con-
trast, stimulation with LTA, PGN, and the synthetic agonist
Pam3CSK4[ligand for TLR2/TLR1 (54)] of TLR2WT transfected
HEK 293 resulted in a clear increase of MAPK p38 activity.
Neither MALP-2 [ligand for TLR2/TLR6 (12)], nor PgLPS,
EcLPS, nor polyinosinic polycytidiylic acid were able to modulate
MAPK p38 activation (TLR2WT; Fig. 3A, 3B). The increase of
MAPK-p38 phosphorylation in TLR2WT transfected and un-
responsiveness of TLR2DTIR transfected HEK 293 cells to LTA,
PGN, and Pam3CSK4clearly linked increased MAPK-p38 phos-
phorylation to the presence of carp TLR2.
The above-mentioned conservation of the position of the leucine
residues critical for ligand (PGN) recognition in the carp extra-
cellular LRR domain (Fig. 1) suggested that carp TLR2 should be
able to bind TLR2 ligands. Transfection of HEK 293 cells with
TLR2WT corroborated the ability of carp TLR2 to bind the pro-
totypical mammalian vertebrate TLR2 ligands LTA, PGN, and
Pam3CSK4, but not MALP-2.
Activation of carp macrophages by TLR2 ligands
shown to be most ubiquitous in myeloid-derived cells and highest in
macrophages (data not shown). In mammalian vertebrates, TLR ac-
including several cytokines, chemokines, and radicals such as NO.
immune gene expression profile in carp macrophages (Table I).
Pam3CSK4modulated immune gene expression to a minor extent,
whereas MALP-2 clearly did not modulate immune gene expression
contrast, both LTA and PGN modulated immune gene expression in
two stimulants, with PGN consistently inducing a higher response
than LTA. The kinetics of downstream activation of carp IL-1b,
gene expression at 6 h after PGN-stimulation, whereas LTA stimu-
thattheTLR2ligands LTA,PGN,and Pam3CSK4(toaminorextent)
but not MALP-2, stimulated downstream signaling pathways with
ligand-specific kinetics profiles in carp macrophages.
Higher activation of carp macrophages by LTA, PGN, and
Pam3CSK4, but not MALP-2 after overexpression of TLR2
expression of both TLR2WT and TLR2DTIR constructs after
transfection of carp macrophages were confirmed by Western blot
(data not shown). Stimulation of macrophages overexpressing
TLR2DTIR was used as the negative control (TLR2DTIR; Fig. 5).
macrophages. TLR2WT and TLR2DTIR trans-
fected carp macrophages were stimulated with
LTA (50 mg/ml), PGN (50 mg/ml), Pam3CSK4
(Pam3, 10 mg/ml), MALP-2 (10 mg/ml) for 6
and 9 h, or left untreated as control. mRNA
levels of IL-1b, iNOS, and TNF-a relative to the
house keeping 40S ribosomal protein gene level
are expressed as fold change relative to un-
stimulated cells. mRNA levels of IL-11, IL-
12p35, and IL-12p40 after stimulation were also
determined but no changes were observed (data
not shown). Bars show averages 6 SD of n = 4
fish. pSignificant (p # 0.05) difference com-
pared with TLR2DTIR transfected carp cells.
Note the differences in scale of the y-axes.
Overexpression of TLR2 in carp
The Journal of Immunology 2361
Carp macrophages overexpressing TLR2WT could be further ac-
tivated with PGN and LTA and to a minor extent with Pam3CSK4,
but not with MALP-2 (TLR2WT; Fig. 5). Again, gene expression
(IL-1b, iNOS) was highest at 6 h, after PGN-stimulation, and
highest at 9 h after LTA stimulation.
LTA, PGN, and protozoan GPI-anchors induce expression and
increase mRNA stability of the TLR2 gene in carp macrophages
TLR2 ligands LTA and PGN could activate carp macrophages via
TLR2 as shown by increased MAPK-p38 phosphorylation in HEK
293 cells and enhanced immune gene expression in TLR2WT-
transfected cells. We subsequently tested whether LTA and PGN
could modulate the expression of the carp TLR2 gene itself (Table
I). TLR2 gene expression in carp macrophages was highest at 6 h
and then declined (Fig. 6A). Stimulation with either LTA or PGN
induced a low but significant 1.5–2-fold upregulation of TLR2
To verify the low but consistent fold upregulation of TLR2, two
were examined for TLR2 gene expression. Both macrophage-
and macrophage-enriched MACS-sorted leukocytes (TCL-BE82)
showed a consistent but maximum 2-fold upregulation of expres-
sion of the carp TLR2 gene (data not shown). Second, GPI anchors
from protozoan parasites of carp, T. borreli (Kinetoplastida), were
examined as TLR2 ligands. In mammalian vertebrates, TLR2 rec-
ognizes GPI-anchors from protozoan parasites (8). Macrophages
stimulated with supernatant from PI-PLC–treated parasites as
source of GPI-anchors showed a dose-dependent upregulation of
TLR2 gene expression (Fig. 6B). Macrophages stimulated with
nontreated parasites or the resultant pellet did not show upregula-
tion of TLR2 gene expression (negative controls).
carp TLR2 mRNA (increased mRNA half-life: Fig. 6C). Although
this was not the case for stimulation by PGN. Using an inhibitor of
MAPK-p38, we also studied the posttranscriptional effect of
MAPK-p38 phosphorylation on the mRNA stability of the carp
TLR2 gene, again including IL-1b and iNOS as control genes. In-
hibition of MAPK-p38 destabilized all three gene transcripts. Our
results suggest that the TLR2 ligands LTA and PGN, but also the
the carp TLR2 gene. Increased mRNA stability may lead to an in-
crease in the amount of TLR2 protein induced after ligand binding.
with TLR2 ligands. A, Kinetics of TLR2 gene expression in carp macro-
phages after stimulation for 6, 9, 12, and 18 h with LTA (50 mg/ml), PGN
(50 mg/ml), or left untreated as control. RT-qPCR analysis of gene ex-
pression in carp macrophages. mRNA levels of TLR2 relative to the house
keeping 40S ribosomal protein gene level are expressed as fold change
relative to unstimulated cells at 0 h. Bars show averages 6 SD of n = 3
fish.pSignificant (p # 0.05) difference compared with unstimulated cells
(control group). B, TLR2 gene expression in carp macrophages after
stimulation for 6 h with supernatants from PI-PLC–treated T. borreli
parasites. mRNA levels of TLR2 relative to the house keeping 40S ribo-
TLR2 gene expression and mRNA stability after stimulation
somal protein gene level are expressed as fold change relative to un-
stimulated cells. Bars show averages 6 SD of n = 4 fish.pSignificant (p #
0.05) difference compared with unstimulated cells (control group). C,
TLR2 mRNA stability. TLR2 gene transcription in carp macrophages after
stimulation for 6h with LTA (50 mg/ml) or PGN (50 mg/ml) or pre-
incubated for 30 min with MAPK-p38 inhibitor (SB203530, 25 mM),
followed by stimulation for 6 h with LTA (50 mg/ml). At 6 h, cells were
treated with RNA synthesis inhibitor (actinomycin D, 5 mg/ml) and col-
lected at the time points indicated. mRNA levels of TLR2, IL-1b, and
iNOS, corrected for differences in mRNA levels of the house keeping 40S
ribosomal protein gene, are expressed as % remaining mRNA (ratio
mRNA at indicated time point relative to mRNA of the same gene prior to
the addition of actinomycin D). Graphs do not reflect differences in gene
transcription between treatments before the addition of actinomycin D.
This is one experiment representative of three experiments. Exponential
functions fitting the TLR2 mRNA levels for each treatment were used to
estimate t1/2in hours. Estimated t1/2of TLR2 gene transcripts were t1/2=
1.18 h for unstimulated cells, t1/2= 4.30 h (LTA), t1/2= 1.62 h (PGN), and
t1/2= 0.69 h for MAPK-p38i/LTA–stimulated macrophages.
2362 EVOLUTION OF RECOGNITION OF GRAM-POSITIVE BACTERIA
PGN induces MAPK-p38 activation and radical production in
Stimulation of carp macrophages with LTA and PGN resulted in
higher phosphorylation of MAPK-p38 in carp macrophages within
5 min (Fig. 7A). The signal was stronger in PGN- than in LTA-
stimulated carp macrophages.
The MAPK-p38 pathway is known to be redox-sensitive in
mammalian vertebrates (55). PGN, but not LTA, induced the pro-
duction of nitrogen radicals (NO) in carp macrophages (Fig. 7B).
Similarly, oxygen radical (ROS) production by carp macrophages
could be induced with PGN (141%) (Fig. 7D), but not with LTA
(102%). The ROS production was measured with the DHR 123
probe, which is reduced to the green fluorescent rhodamine 123
Treatment of carp macrophages with SOD (which dismutates O2.2
into H2O2and O2) further increased the ROS production (Fig. 7D)
ROS production modulates TLR2 gene expression
PGN induced MAPK-p38 activation, TLR2 gene expression, NO,
and also ROS production in carp macrophages. To examine the
influence of MAPK-p38 activation and ROS production on carp
TLR2 gene expression, we used inhibitors of signal transduction
and inhibitors of radical production and measured TLR2 gene
expression. Cell viability (assessed by trypan blue exclusion),
TLR2 gene expression, and ROS production were not affected by
the presence of these inhibitors (data not shown).
Signal transduction pathways associated with TLR2 gene reg-
ulation were studied by preincubation of carp macrophages with
inhibitors of NF-kB (PDTC) and MAPK-p38 (SB 208530). TLR2
gene expression, induced by PGN or by LTA (not shown), could
be inhibited by the presence of either inhibitor, although the
strongest by MAPK-p38 inhibition (Fig. 8A). Radical production,
induced by PGN (LTA did not induce radical production, see Fig.
7D), was inhibited only by inhibition of the MAPK-p38 pathway
(Fig. 8B). This suggests that MAPK-p38 activation is involved in
regulating both TLR2 gene expression and radical production.
Enzymatic pathways associated with radical production were
mitochondrial electron transfer chain subunit I (rotenone), NADPH
oxidase (DPI), and xanthine oxidase (allopurinol). TLR2 gene
by the presence of any of the three inhibitors, although to a lesser
extent by the inhibitor of xanthine oxidase (Fig. 8C). Radical
production, induced by PGN, was significantly inhibited only in
the presence of the NADPH oxidase inhibitor (Fig. 8D). This in-
dicates that oxygen radicals, including the NADPH oxidase-de-
rived intracellular ROS (e.g., H2O2), are involved in the regulation
of TLR2 gene expression in carp macrophages.
cells are red (originalmagnification340). D,Radical productionin carp macrophagesby meansof DHRfluorescenceintensity. Macrophageswere incubated
with DHR (0.25 mg/ml) and PMA (0.05 mg/ml), followed by LTA (50 mg/ml) or PGN (50 mg/ml) in the presence or absence of SOD (20 U/ml) for 1 h. Gray
shaded histograms represent a control sample that corresponds to PMA-stimulated cells. This is one experiment representative of four experiments.
Effect of TLR2 ligands on MAPK-p38 activation and ROS production in carp macrophages. A, MAPK-p38 activation in carp macrophages
The Journal of Immunology2363
H2O2is an essential signaling molecule for TLR2 gene
expression and MAPK-p38 activation
ROS, such as H2O2, are able to diffuse across membranes and
in carp macrophages was significantly modulated by H2O2 in
a concentration-dependent manner (Fig. 9A).Consistent with these
could be inhibited by a H2O2scavenger (catalase) (Fig. 9B).
MAPK-p38 activation, carp macrophages were stimulated with in-
considerably upregulated by H2O2, in a concentration-dependent
manner, within 5 min after addition of H2O2(Fig. 9C). MAPK-p38
activation was significantly upregulated by H2O2at concentrations
.1 mM H2O2. The concentration of .1 mM H2O2required to ac-
tivate MAPK-p38 (57) implies that TLR2 gene expression is highly
sensitive to modulation by H2O2(0.1 mM) (Fig. 8A).
Our data indicate that H2O2is able to induce TLR2 gene ex-
pression and activate the MAPK-p38 signaling cascade in carp
macrophages. Futhermore, catalase (H2O2scavenger) but not SOD
(dismutates O2.2into H2O2) inhibited PGN-induced MAPK-p38
phosphorylation (data not shown). These observations suggest that
NADPH oxidase-derived radicals such as H2O2,in particular, are
essential for a complete MAPK-p38 phosphorylation and maximal
expression of the TLR2 gene.
Previous studies have identified TLR2 orthologs in teleost fish
based on sequence homology, but the role of the TLR2 receptor in
the recognition of ligands from Gram-positive bacteria has not been
studied. Transfection of human cells (HEK 293) with the carp TLR2
receptor showed activation of MAPK-p38 by LTA and PGN from S.
aureus, which are prototypical TLR2 ligands from Gram-positive
diacylated lipopeptide MALP-2 also activated TLR2 transfected
human cells. Overexpression of TLR2 in carp macrophages con-
firmed the response to LTA and PGN, low-responsiveness to
Pam3CSK4and nonresponsiveness to MALP-2. Activation of carp
macrophages by LTA and PGN from S. aureus resulted in increased
TLR2 gene expression and enhanced TLR2 mRNA stability. ROS
production and MAPK-p38 activation cooperatively determined the
level of TLR2 gene expression, indicating that the H2O2–MAPK-
in fish cells.
Similar to mammalian vertebrate TLR2, carp TLR2 is primarily
expressed in myeloid cell types as a type I transmembrane protein
TLR2 recognize LTA (6, 7), PGN (58–60), and lipopeptides (5).
Mammalian vertebrate TLR2 contains 20 LRRs (39), whereas the
extracellular domain of carp TLR2 is composed of 21 LRR motifs,
one of which has a “bacterial” motif and may not be important for
ligand recognition. Although, in general, the extracellular LRR do-
of the position of the critical PGN recognition leucine residues (50,
51, 61) in the carp TLR2 extracellular domain. Transfection of hu-
man (HEK 293) cells with carp TLR2 confirmed the ability of pro-
totypical TLR2 ligands (LTA, PGN, and Pam3CSK4) to trigger
MAPK-p38 activation. In contrast, carp TLR2 didnot recognize the
TLR2 ligand MALP-2 in transfected HEK 293 cells.
TLR activation in humans is often measured via quantification of
NF-kB activation or downstream expression of cytokines such as IL-
1b and TNF-a. We characterized activation of fish macrophages by
NO production and MAPK-p38 phosphorylation (62). We identified
NF-kB (PDTC, 5 mM), MAPK-p38 (SB203530, 25 mM), xanthine oxidase (allopurinol, 200 mM), NADPH oxidase (DPI, 20 mM), or mitochondrial electron
transfer chain subunit I (rotenone, 10 mM). A and C, Macrophages were further stimulated for 6 h with PGN (50 mg/ml). TLR2 gene expression was
measured by means of RT-qPCR. 40S ribosomal protein gene was used as a housekeeping gene and results are expressed as mRNA fold change in
stimulated cells relative to untreated cells (fold change = 1). Averages 6 SD of n = 4 fish are given.pSignificant (p # 0.05) difference compared with PGN-
stimulated cells. B and D, Macrophages were further incubated for 1 h with DHR (0.25 mg/ml) and PMA (0.05 mg/ml), followed by PGN (50 mg/ml).
Radical production was measured by means of DHR fluorescence intensity. Results are expressed as mean fluorescence intensity relative to PMA-stimulated
cells (mean fluorescence intensity = 15). Averages 6 SD of n = 4 fish are given.pSignificant (p # 0.05) difference compared with PGN-stimulated cells.
Effect of MAPK-p38 activation and ROS production on TLR2 gene expression. Macrophages were preincubated for 30 min with inhibitors of
2364 EVOLUTION OF RECOGNITION OF GRAM-POSITIVE BACTERIA
similarities but also clear differences between the mammalian verte-
brate and carp TLR2-mediated response. We observed in fish mac-
rophages 1) low responsiveness to Pam3CSK4, 2) nonresponsiveness
to MALP-2, 3) delayed gene expression kinetics and distinct cellular
as compared with PGN, and 4) a requirement for at least five times
higher doses of LTA and PGN than in humans.
The low responsiveness of fish cells to Pam3CSK4is in contrast
with the observation that carp TLR2-transfected HEK 293 cells
recognized by a TLR2-TLR1 heterodimer (54). Apparently,
overexpression of TLR2 homodimers in HEK 293 cells overcomes
for the recognition of Pam3CSK4. Overexpression of TLR2 homo-
dimers in carp macrophages indeed improved recognition of
Pam3CSK4as shown by increased iNOS gene expression. The low
reports in gilthead seabream (63) and rainbow trout (35). In both
studies a minor modulation of cytokine gene expression with
ligands were used.
The unresponsiveness of fish cells to MALP-2, recognized by
TLR2-TLR6 heterodimers in mammalian vertebrates (12), is con-
sistent with the unresponsiveness of TLR2-transfected HEK 293
cells to MALP-2. Apparently, overexpression of TLR2 homo-
dimers in HEK 293 cells does not overcome the requirement for
TLR2-TLR6 heterodimerization. In mammalian vertebrates, it is
clear that distinct TLR2-containing receptor complexes allow for
the accommodation of structurally diverse TLR2 ligands (62) and
the ability of TLR2 to detect a relatively wide array of PAMPs has
been attributed to a functional interaction with a number of other
receptors (64, 65), including TLR1 and TLR6. TLR1, 6 and 10 are
thought to have diverged from a common ancestral gene (66). The
presence of a putative TLR1 homolog in fish (17, 67) is presently
TLR1 heterodimerization and detailed studies into recognition of
teleost fish (17, 21). The apparent absence of a functional TLR6
homolog in carp could contribute to the unresponsiveness to
MALP-2 observed in carp macrophages.
with a delayed kinetic profile when compared with PGN, an obser-
vation alsomade for mammalianvertebrates (62).PGN always more
clearly than LTA induced NO, ROS production, and MAPK-p38
be the result of distinct TLR2 coreceptors usage and signaling after
cellular trafficking of these complexes (62). Although yet to be
characterized, the distinct cellular responses could possibly be also
attributed to the use of distinct TLR2-containing receptor complexes
on fish macrophages. Different recruitment and/or kinetics of TLR
(2)-specific adaptor proteins (68) on activation of TLR2-containing
complex may lead to ligand-specific responses and ultimately have
profound effects on cellular response to LTA and PGN.
Fish cells did respond to both LTA and PGN, albeit at high
to the ligand preparations because we used highly purified TLR2
ligands throughout our study. LTA from S. aureus was obtained by
buthanol extraction that preserves its molecular structure (70, 71).
PGN was a soluble polymeric high molecular-weight preparation
purified from supernatants of S. aureus grown in the presence of
penicillin, devoid of teichoic acids or other proteins (72). As stated
previously, the ability of TLR2 to detect a relatively wide array of
PAMPs has been attributed to a functional interaction with a num-
ber of other receptors (64, 65). These not only include TLR1 and
TLR6, but also the lipid scavenger receptor CD36 and the CD14
protein. A CD36 gene sequence is present in zebrafish but no
functional studies have been reported on the formation of a func-
tional receptor complex of fish TLR2 with the CD36 lipid scav-
anger receptor. LTA and PGN also can interact with CD14 (73–77).
A recent report (78), in fact, suggests that the main function of
CD36 is to bind and transfer diacylglycerol ligands (e.g., lip-
omannan, LTA) onto TLR2/TLR6, in a CD14-dependent manner.
macrophages under influence of hydrogen peroxide. A, TLR2 gene expres-
sion in carp macrophages after stimulation for 6 h with H2O2(0.1, 0.25, 0.5,
and 1 mM). Averages and SD of n = 4 fish are given. mRNA levels of TLR2
are relative to the house keeping 40S ribosomal protein gene level and ex-
pressedasfoldchangein stimulatedcells relativeto unstimulated cells (fold
change= 1).pSignificant (p #0.05)differencecomparedwith unstimulated
cells (control group). B, TLR2 gene expression in carp macrophages after
with PGN (50 mg/ml). Averages 6 SD of n = 4 fish are given.pSignificant
(p # 0.05) difference compared with PGN-stimulated cells. C, MAPK-p38
activation in carp macrophages incubated for 5 min with increasing con-
centrations of H2O2(1, 2.5, and 5 mM). MAPK-p38 phosphorylation was
analyzed by immunoblotting for phospho-p38, whereas equal loading was
confirmed by immunoblotting for b-tubulin.
TLR2 gene expression and MAPK-p38 activation in carp
The Journal of Immunology 2365
fish. If true, the absence of CD14 could not only contribute to the
well-known hyporesponsiveness offish cells to Gram-negative (26,
79) bacteria but also to the hyporesponsiveness of fish cells to li-
gands from Gram-positive bacteria reported in this study.
Of course, carp macrophages undoubtly express innate receptors
additional to TLR2 and it is impossible to unambiguously ascribe
PRRs have been shown to detect PGN from Gram-positive bacteria
(Nod2) and PGN recognition proteins (PGRPs) (80). In zebrafish it
was shown that suppression of PGRP6 decreased significantly the
expression of TLR2 mRNA suggesting that TLR2 and PGRP may
cooperatively recognize PGN (81). Without access to knock-out
phenotypes it remains difficult to exclude the involvement of other
of carp macrophages with muramyl dipeptide; the minimum PGN
fragment recognized by Nod2 (82), did not induce ROS and NO to
the same extent as PGN (unpublished observation). This suggests
that Nod2 did not play the major role in the stimulation by PGN we
observed in carp macrophages. Furthermore, overexpression of
TLR2 in carp macrophages led to a more pronounced induction of
to a minor extent, Pam3CSK4. These results corroborate the ability
of carp TLR2 to bind LTA and PGN from S. aureus and to trigger
TLR2-dependent downstream activation pathways in response to
these ligands in carp macrophages. In conclusion, we provide evi-
positive bacteria that are prototypical activators of mammalian
vertebrate TLR2, but require relatively high concentrations.
We investigated whether TLR2 ligands could modulate the ex-
pression of the carp TLR2 gene itself. Clearly, carp TLR2 gene
expression was regulated in a consistent manner throughout our
studies, although to a maximum of 2-fold upregulation. In mam-
malian vertebrates, expression patterns of the TLR2 gene itself are
divergent. In human monocytes, TLR2 mRNA is upregulated after
In murine monocytes, TLR2 mRNA is low or undetectable in vitro
but can be strongly induced (84). In cell lines, 2- to 3-fold increase
of TLR2 gene expression has been observed in murine (RAW
264.7) and human (HL-60) macrophages (85). In our study, GPI
anchors from protozoan parasites of carp showed a dose-dependent
upregulation of TLR2 gene expression. It is known that the carp
protozoan T. borreli parasite induces radical production in carp
macrophages (86). We are currently defining the involvement of
TLR2 in the recognition of GPI anchors from protozoan parasites.
Further studies will elucidate the contribution of TLR2 to the im-
mune response induced by protozoan parasites in carp.
studied whether TLR2 ligands could induce posttranscriptional
stabilization of carp TLR2 mRNA. Indeed, LTA and PGN specifi-
cally increased TLR2 mRNA stability that could contribute to the
consistent 2-fold inducibility of TLR2 gene expression. Changes in
mRNA stability for TLR2 in mammalian vertebrates are yet to be
reported. Stimulation of carp macrophages with PGN clearly in-
duced MAPK-p38 phosphorylation and increased NO and ROS
production. MAPK-p38 activation was dependent on NADPH ox-
idase-derived radicals, in particular H2O2, suggesting that the
MAPK-p38 activation pathway in fish is redox sensitive. Comple-
mentary, PGN-induced radicals were NADPH oxidase as occurs in
humans (87). Moreover, PGN-induced radicals could be further
occurs after TLR2 engagement. We found that PGN-induced radi-
cals derived from NADPH oxidase were necessary for maximal
expression of carp TLR2 gene expression. Furthermore, catalase
significantly inhibited TLR2 gene expression, wheras H2O2sig-
nificantly induced TLR2 gene expression. In our study, MAPK-p38
was required for an effective PGN-induced TLR2 expression and
posttranscriptional stability, suggesting a clear role for MAPK-p38
in the regulation of TLR2 gene expression in carp. Collectively,
these results suggest that H2O2radicals via MAPK-p38 activation
play an indispensable role in the regulation of TLR2 gene expres-
sion itselfin carp macrophages. In mice, ROS-dependent activation
of the TRAF6-ASK1-p38 pathway is selectively required for TLR4
innate immunity (88). In humans, ASK1-MAPK-p38-p47 phox
activation is essential for inflammatory responses during tubercu-
losis via TLR2-ROS signaling (89). These observations suggest an
important role for ROS as second messengers in TLR-mediated
signaling pathways. We show an important role for ROS, in par-
ticular H2O2, on TLR2 gene expression in carp macrophages. We
demonstrated for the first time, in carp, that bidirectional commu-
nication between ROS and activated MAPK-p38, besides shaping
Biosciences, Tokyo University of Fisheries, Tokyo, Japan) for providing the
mousemAbTCL-BE8. Wegratefullyacknowledge M. Chadzinska,T.Ploe-
gaert, N.I.V. Jimene ´z, J. Wells, I. Konings,H. Schipper, J. van der Veen, and
M. Forlenza for their practical assistance or fruitful discussions.
The authors have no financial conflicts of interest.
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