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Cutting Edge |


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Epithelial Pathobiology Division, Department of Pathology and Laboratory Medicine, Emory University, Atlanta, GA 30322; and
Department of Molecular Biology, Genentech, Inc., South San Francisco, CA 94080
| Abstract |
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B gene expression in response to
flagellin. The response depended on both extracellular leucine-rich
repeats and intracellular Toll/IL-1R homology region of TLR5 as well as
the adaptor protein MyD88. Furthermore, immunolocalization and cell
surface-selective biotinylation revealed that TLR5 is expressed
exclusively on the basolateral surface of intestinal epithelia, thus
providing a molecular basis for the polarity of this innate immune
response. Thus, detection of flagellin by basolateral TLR5 mediates
epithelial-driven inflammatory responses to
Salmonella. | Introduction |
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Toll-like receptors
(TLR)4 are an
evolutionarily conserved family of receptors that function in innate
immunity via recognition of conserved patterns in bacterial molecules
(for review, see Ref. 7). Given that the ligands for the
majority of cloned TLRs have yet to be identified, we explored the
possibility that a TLR would be a good candidate to mediate
inflammatory responses to flagellin. Indeed, Hayashi et al.
(8) recently screened bacterial products for ability to
activate TLR5 and found that flagellin could activate NF-
B-mediated
gene expression in TLR5-transfected cells. We report here that this
function is specific to TLR5 in that of all known TLRs
(1, 2, 3, 4, 5, 6, 7, 8, 9, 10), only TLR5 could activate proinflammatory gene
expression in response to flagellin. Further, TLR5 is expressed on the
basolateral, but not apical, surface of model epithelia, thus providing
a mechanism by which microbes that invade or translocate flagellin, but
not commensal bacteria, induce intestinal epithelia to orchestrate an
inflammatory response.
| Materials and Methods |
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Model human intestinal epithelia were prepared by culturing the
colonic cell line T84 on collagen-coated permeable supports as
previously described (9). Model epithelia were used 614
days after plating and after verification that they had achieved a
transepithelial electrical resistance of at least 1000
·cm2, thus indicating a high degree of
surface polarity. COS-7, 293, and HeLa cell lines were cultured
in DMEM with 10% FBS, 2 µM L-glutamine, 100 U/ml
penicillin, and 100 µg/ml streptomycin. Transient transfections were
conducted using Superfect (Qiagen, Chatsworth, CA) for COS and 293
cells and Effectene (Qiagen) for HeLa cells, according to the
manufacturers instructions. MG-262 was obtained from Affiniti
(Exeter, U.K.). Purified flagellin was prepared as previously described
(3).
Construction of TLR expression plasmids
Full length human TLR cDNAs (TLRs 110) were cloned from a
human fetal lung library into the pRKN. N-terminal epitope tag versions
(gD.TLR) linking TLRs to the first 53 aa of HSV-1 glycoprotein D (gD)
were constructed as previously described (10). The
predicted amino acid sequence for TLR5 matched that previously
published (11) except for the following substitutions:
V233L, C352L and L616F. TLR5 deletion plasmids were constructed as
follows: gD.TLR5 in pRKN were restricted with XhoI (deletion
of aa (
) 1191), EcoRV and MfeI
(
408591), and BsrGI (
695858). The resulting cDNAs
were gel purified and religated. Protein expression was confirmed by
immunoprecipitation and immunoblotting using gD mAb (Genentech, South
San Francisco, CA).
Luciferase reporter and other proinflammatory assays
293, COS, or HeLa cells were plated (in 12-well plates; 1
x 105 cells/well) and transfected 18 h
later with expression plasmids and 0.5 µg luciferase reporter plasmid
pGL3-ELAM.tk and 0.1 µg Renilla luciferase reporter vector
as an internal control. After 24 h, cells were treated with
flagellin (100 ng/ml) or TNF-
(60 ng/ml) for 6 h, and
luciferase was assayed via the Dual-Luciferase system (Promega,
Madison, WI). Data are expressed as relative luciferase activity
representing the mean ± SE of duplicate experiments and were
obtained by calculating the ratio of firefly luciferase activity and
Renilla luciferase activity. IL-8 secretion, I
B
degradation, and I
B
phosphorylation were measured as previously
described (12).
Fluorescence microscopy and FACS analysis
Confocal microscopy was performed on paraformaldehyde model
epithelia as previously described (13). FACS analysis was
performed on confluent T84 cells, disassociated with EGTA as previously
described (13) using a goat polyclonal Ab to the
N-terminal region of TLR5 and competing immunogen peptide from Santa
Cruz Biotechnology (Santa Cruz, CA). Isolation of apically or
basolaterally biotinylated proteins was performed as previously
described (14). Western blotting of such lysates was
performed with a 1/800 dilution of TLR5 rabbit polyclonal Ab directed
at aa 154280 purchased from Santa Cruz Biotechnology. Abs to
E-cadherin and IFN-
-inducible protein 82 were a gift from C. Parkos
(Emory University, Atlanta, GA).
| Results |
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B activation with the proteasome inhibitor MG-262
(15) blocked this IL-8 secretion by
80% (data not
shown). Further, basolateral flagellin induced NF-
B nuclear
translocation (Fig. 1
B
(Fig. 1
,
confirming that flagellin induces epithelial chemokine expression via
the activation of NF-
B.
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B activation is not specific to intestinal
epithelial cells. Rather, NF-
B-mediated responses also occur in
monocytic cells (16) as well as in such well-characterized
cell lines as COS cells and 293 cells (Fig. 2
B activation in
response to flagellin. We observed that transfection of 293 cells with
a dominant-negative TLR effector protein MyD88 (
152296) that
blocks most TLR responses (17, 18) also prevented NF-
B
activation in response to flagellin, but not TNF-
(negative
control), suggesting that a TLR was mediating NF-
B activation in
response to flagellin (Fig. 2
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B in response to flagellin and found HeLa cells fit this
criteria (Fig. 2
B reporter gene in
response to flagellin was measured. HeLa cells transfected with TLR5
exhibited
10-fold activation of NF-
B-mediated gene expression,
whereas no activation was observed for HeLa transfected with any of the
other TLRs (Fig. 3
B
activation in TLR5-expressing HeLa was prevented by coexpressing
dominant-negative MyD88 (Fig. 3
695858), but not
from analogous mutants of TLR4 (Fig. 3
|
100 kDa (Fig. 5C) likely due to differential
glycosylation. As controls (not shown), we verified that
IFN-
-inducible protein 82 and E-cadherin were preferentially
expressed on the apical and basolateral surfaces, respectively, as
previously described (19, 20). Thus, the polarity of TLR5
expression matches the polarity of the response to flagellin, further
supporting the notion that flagellin activation of intestinal epithelia
is mediated by TLR5.
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| Discussion |
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B pathway, is an essential component of
innate immunity (21). TLRs play a key role in such innate
immune pathways via recognition of conserved microbial molecular
patterns, thus activating immune inflammatory responses. However,
because components of both pathogenic and commensal bacteria can
activate TLRs, host tissues that are normally densely colonized by
commensal bacteria must be able to distinguish commensal from
pathogenic organisms to avoid being in a constant state of
inflammation. For example, bacterial flagellins that are secreted by
both pathogenic and commensal bacteria have the potential to activate
epithelial chemokine secretion but can only do so upon translocation
from the luminal domain (where commensal strains are located) to the
basolateral membrane domain (3). Such translocation of
flagellin can be mediated by pathogens such as Salmonella
typhimurium, but not by commensal Escherichia coli
strains. This report identifies the molecular mechanism by which
intestinal epithelia distinguish between basolateral and apical
flagellin, namely, the basolateral expression of TLR5 that serves as
the flagellin receptor. In addition to its expression on colonic
epithelium, TLR5 is expressed in internal tissues including heart,
brain, spleen, kidney, and testis, suggesting a wide role for TLR5 in
host defense (11). Supporting this notion, mutations in
TLR5 result in an increased susceptibility of mice to
Salmonella infection (11). TLR5 expressed on the basolateral surface of intestinal epithelia ought to be able to detect the invasion of a large variety of microbes and is positioned to do so at a very early step in the invasive process. Importantly, this innate immune mechanism could be activated not only by invasive pathogens but also by commensal organisms that had somehow, perhaps opportunistically, breached the epithelium. Recruitment of inflammatory cells in response to TLR5-mediated chemokine secretion could thus prevent systemic penetration of such locally invading microbes, hence preventing the dire consequences that occur when pathogenic or commensal bacteria achieve systemic infection in humans. Interestingly, S. typhimurium may translocate flagellin independent of bacterial invasion (3) and thus may use TLR5 as a mechanism to induce intestinal inflammation and the associated diarrhea that aids in their dissemination to new hosts. Additionally, ligation of basolateral TLR5 by flagellin secreted from commensal organisms may occur in states of epithelial barrier dysfunction such as inflammatory bowel disease and thus may play a role in inducing or exacerbating the inflammation that characterizes these disorders.
| Acknowledgments |
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| Footnotes |
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2 Address correspondence and reprint requests to Dr. Andrew T. Gewirtz, Epithelial Pathobiology Division, Department of Pathology and Laboratory Medicine, Emory University, Atlanta, GA 30322. E-mail address: agewirt{at}emory.edu; or Dr. Paul J. Godowski, Genentech Inc., South San Francisco, CA 94080. E-mail address: ski{at}gene.com ![]()
3 A.T.G. and T.A.N. contributed equally to this work. ![]()
4 Abbreviations used in this paper: TLR, Toll-like receptor; gD, glycoprotein D;
, deletion of amino acids. ![]()
Received for publication May 30, 2001.
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