Flagellin/TLR5 responses in epithelia reveal intertwined ...

Flagellin/TLR5 responses in epithelia revealintertwined activation of inflammatory andapoptotic pathwaysHui Zeng, Emory UniversityHuixia Wu, Emory UniversityValerie Sloane, Emory UniversityRheinallt Jones, Emory UniversityYimin Yu, Emory UniversityPatricia Denning, Emory UniversityAndrew Gewirtz, Emory UniversityAndrew Neish, Emory University

Journal Title: AJP - Gastrointestinal and Liver PhysiologyVolume: Volume 290, Number 1Publisher: American Physiological Society | 2006-01-01, Pages G96-G108Type of Work: Article | Post-print: After Peer ReviewPublisher DOI: 10.1152/ajpgi.00273.2005Permanent URL: https://pid.emory.edu/ark:/25593/rwxjj

Final published version: http://dx.doi.org/10.1152/ajpgi.00273.2005

Copyright information:© 2006 the American Physiological Society

Accessed October 16, 2021 9:08 PM EDT

http://open.library.emory.edu/profiles/hwu2/

http://open.library.emory.edu/profiles/rjones5/

http://open.library.emory.edu/profiles/pllin/

http://open.library.emory.edu/profiles/aneish/

https://pid.emory.edu/ark:/25593/rwxjj

http://dx.doi.org/10.1152/ajpgi.00273.2005

Flagellin/TLR5 responses in epithelia reveal intertwined activation of inflammatory and apoptotic pathways

Hui Zeng1,*, Huixia Wu1,*, Valerie Sloane1, Rheinallt Jones1, Yimin Yu1, Patricia Lin2, Andrew T. Gewirtz1, and Andrew S. Neish1

1Epithelial Pathobiology Unit, Department of Pathology and Laboratory Medicine, Emory University School of Medicine, Atlanta, Georgia

2Division of Neonatal-Perinatal Medicine, Department of Pediatrics, Emory University School of Medicine, Atlanta, Georgia

Abstract

Flagellin, the primary structural component of bacterial flagella, is recognized by Toll-like

receptor 5 (TLR5) present on the basolateral surface of intestinal epithelial cells. Utilizing

biochemical assays of proinflammatory signaling pathways and mRNA expression profiling, we

found that purified flagellin could recapitulate the human epithelial cell proinflammatory

responses activated by flagellated pathogenic bacteria. Flagellin-induced proinflammatory

activation showed similar kinetics and gene specificity as that induced by the classical endogenous

proinflammatory cytokine TNF-α, although both responses were more rapid than that elicited by

viable flagellated bacteria. Flagellin, like TNF-α, activated a number of antiapoptotic mediators,

and pretreatment of epithelial cells with this bacterial protein could protect cells from subsequent

bacterially mediated apoptotic challenge. However, when NF-κB-mediated or phosphatidylinositol

3-kinase/Akt proinflammatory signaling was blocked, flagellin could induce programmed cell

death. Consistently, we demonstrate that flagellin and viable flagellate Salmonella induces both

the extrinsic and intrinsic caspase activation pathways, with the extrinsic pathway (caspase 8)

activated by purified flagellin in a TLR5-dependant fashion. We conclude that interaction of

flagellin with epithelial cells induces caspase activation in parallel with proinflammatory

responses. Such intertwining of proinflammatory and apoptotic signaling mediated by bacterial

products suggests roles for host programmed cell death in the pathogenesis of enteric infections.

Keywords

Salmonella; inflammation; Toll-like receptor; cDNA microarray

GRAM-NEGATIVE BACTERIA of the Salmonella enteritidis group are common human

pathogens often isolated from cases of acute food-borne gastroenteritis in the United States

and developing countries (49). S. enteritidis interaction with the intestinal epithelia provokes

Address for reprint requests and other correspondence: A. S. Neish, Epithelial Pathobiology Unit, Dept. of Pathology and Laboratory Medicine, Emory Univ. School of Medicine, 105-F Whitehead Bldg., 615 Michaels St., Atlanta, GA 30322 ([email protected]).*H. Zeng and H. Wu contributed equally to this work.Present address of H. Zeng: Influenza Branch, Centers for Disease Control and Prevention, Atlanta, GA 30333.

HHS Public AccessAuthor manuscriptAm J Physiol Gastrointest Liver Physiol. Author manuscript; available in PMC 2017 February 28.

Published in final edited form as:Am J Physiol Gastrointest Liver Physiol. 2006 January ; 290(1): G96–G108. doi:10.1152/ajpgi.00273.2005.

Author M

anuscriptA

uthor Manuscript

Author M

anuscriptA

uthor Manuscript

secretion of chemokines and subsequent luminal translocation of neutrophils (51). The result

of this acute inflammatory response is the disruption of barrier integrity permitting

transepithelial fluxes of ions and water as well as neutrophils. These events correlate

clinically with an acute inflammatory diarrhea. Other enteric bacterial infections, such as

those caused by Shigella, enteropathogenic Escherichia coli, and Yersinia, elicit similar

responses (10).

Many of the epithelial responses to intestinal infections involve the transcriptional activation

of inflammatory mediators such as chemokines, adhesion molecules, and antibacterial

peptides. These gene products are controlled by the action of proinflammatory signaling

cascades such as the NF-κB (Rel) and MAPK pathways that culminate in the activation of

nuclear transcription factors and the initiation of de novo mRNA transcription. Indeed, work

in our and other laboratories has identified NF-κB activation as a central event in the

proinflammatory gene expression that mediates infectious enterocolitis (13, 16). Enterocytes

(and most other cells) can perceive bacterial threats and activate the NF-κB pathway via a

battery of transmembrane Toll-like receptors (TLRs) (35). These sentinel pattern recognition

receptors are potently activated by pathogen-associated molecular patterns (PAMPs),

complex molecules with a macromolecular structure limited to and characteristic of

prokaryotic life, permitting stable and efficient recognition by eukaryotic TLRs (48).

Flagellin is a bacterial product that is generally considered a PAMP, with TLR5 as its

physiological receptor in vertebrates (22). This pattern recognition receptor is present on the

basolateral aspect of intact intestinal epithelial cells (15) and binds a 13-amino acid motif

present in the flagellin protofilament but not accessible in polymerized flagella (46). In S. typhimurium, the flagellar protofilaments are 55-kDa monomers encoded by the two similar

but not identical genes FliC and FljB (34). Approximately 20,000 subunits of flagellin

assemble to form the extracellular filament structure that is necessary for bacterial motility.

Flagellin is found on a wide variety of bacteria, and it is assumed that structural constraints

necessary for motor function permit TLR5 recognition of flagellin encoded in a variety of

organisms (47).

Purified flagellin can activate transcription and secretion of the proinflammatory chemokine

IL-8 in in vitro cell culture systems (45). Flagellin is also a potent activator of systemic

inflammation in murine models (12), and, in humans, serum levels of the protein correlate

with clinical severity of bacteremic shock syndromes (31). Interestingly, studies of

circulating antibodies in the serum of human Crohn’s disease patients and in murine colitis

models identified flagellin as a dominant antigen, suggesting a role for this bacterial protein

in the immunopathogenesis of inflammatory bowel disease (32).

In our laboratory, we have shown that infection of model epithelia with live S. typhimurium elicited a classic proinflammatory transcriptional response involving NF-κB (16). Strikingly,

mutation of both Salmonella genes encoding flagellin (FliC and FljB) could totally abolish

this response (17, 53), indicating that this PAMP is necessary for the proinflammatory

responses to this pathogen. Because of the vital role flagellin plays in Salmonella pathogenesis (and probably other bacterial infections) as well as the ability of this protein to

activate known proinflammatory genes, we sought to characterize the signaling pathways

Zeng et al. Page 2

Am J Physiol Gastrointest Liver Physiol. Author manuscript; available in PMC 2017 February 28.

Author M

anuscriptA

uthor Manuscript

Author M

anuscriptA

uthor Manuscript

and transcriptional responses elicited by this exogenous prokaryotic protein. We report

herein that flagellin is sufficient to recapitulate epithelial proinflammatory responses to S. typhimurium. Furthermore, we demonstrate that flagellin also activates apoptotic signaling

pathways. We also observe transcriptional upregulation of antiapoptotic factors that serve to

arrest caspase activation in a NF-κB-dependant manner; however, when the NF-κB pathway

is blocked, caspase activation proceeds to cell death. We speculate that this parallel

activation may be a general feature of activators of innate immunity and that flagellin may

play a previously under appreciated role in host monitoring of, and response to, microbes.

MATERIALS AND METHODS

Reagents and constructs

Flagellin (FliC) from wild-type S. typhimurium (SL3201) was purified through sequential

cation and anion-exchange chromatography and used at the concentrations indicated in the

figures (16). Purity was verified as previously described (15). Cycloheximide and

wortmannin were obtained from Calbiochem (La Jolla, CA), MG-262 was from BioMol

(Plymouth Meeting, PA), staurosporine was from Sigma (St. Louis, MO), 4′,6-diamidino-2-

phenylindole dihydrochloride was from Molecular Probes (Eugene, OR), and TNF-α was

from R&D Systems (Minneapolis, MN). Antibodies used included caspase 3, cleaved

caspase 3, caspase 8, caspase 9, phospho-p38, phospho-Akt, phospho-IκBα, phospho-JNK,

phospho-ERK, p38 (all from Cell Signaling; Beverly, MA), poly(ADP) ribose polymerase

(PARP; BD Pharmingen; San Diego, CA), IκBα (Santa Cruz Biotechnology; Santa Cruz,

CA), cIAP-2 (R&D Systems), and β-actin (Sigma). Epitope-tagged antibodies included T7

(EMD Biosciences; Madison, WI) and V5 (Invitrogen; Carlsbad, CA). The pDsRed2

construct was obtained from BD Biosciences. The mutant IκBα pCMV4 T7-IκB-α S32/36A construct, containing Ser→Ala replacements at Ser32 and Ser36, was kindly

provided by Dr. Dean W. Ballard (Vanderbilt University School of Medicine). The cellular

inhibitor of apoptosis-2 (cIAP-2) expression vector pcDNA4.1-cIAP2 was constructed by

cloning RT-PCR products into the pcDNA4.1A vector (Invitrogen).

Cell culture and bacterial infection

Model human intestinal epithelial cells (T84) were prepared on 0.33- or 5-cm2 permeable

filters and used 9–14 days after they had been plated and achieved a stable transepithelial

resistance of >1,000 Ω · cm2. Monolayers were washed twice with Hanks’ balanced salt

solution at 37°C and equilibrated for 15 min before treatment. Primary rat intestinal IEC-6

epithelial cells were maintained in DMEM, 10% fetal bovine serum, and 4 μg/ml insulin

(Invitrogen). For bacterial treatments, prepared bacterial cultures were washed, concentrated,

and applied to the apical aspects of cells (both T84 and IEC-6 cells) at a multiplicity of

infection of 30 organisms/cell as described (53). HeLa/TLR5 cells were generated by stable

transfection of HeLa cells with a vector encoding a V5-TLR5 and were maintained in

DMEM supplemented with 10% fetal bovine serum and 5 μg/ml blasticidin (52).

Zeng et al. Page 3


Author M

anuscriptA

uthor Manuscript

Author M

anuscriptA

uthor Manuscript

Transfection

IEC-6 cells were transiently transfected by FuGENE 6 reagent (Roche Diagnostics;

Indianapolis, IN) according to the manufacturer’s instruction. HeLa and 293T cells were

transfected with Lipofectamine 2000 (Invitrogen).

SDS-PAGE immunoblot analysis

Differently treated cells, as described in the figures, were collected and lysed in ice-cold

buffer containing 50 mM Tris·HCl (pH 7.5), 150 mM NaCl, 1% Nonidet P-40, 0.5% sodium

deoxycholate, 0.1% SDS, and protease inhibitor cocktail (Sigma). Protein concentration was

determined with the Bradford reagent (Bio-Rad; Hercules, CA). Equal amounts of proteins

were loaded on SDS-PAGE and blotted with different antibodies as indicated, following

standard protocols.

Microarray analysis

Details of cDNA microarray fabrication, hybridization, scanning, and labeling of RNA

samples are described in Ref. 53. Briefly, total RNA from treated T84 cells (sample) and

untreated T84 cells (reference) was prepared using TRIzol reagent (Invitrogen). The

integrity of RNA transcripts was verified by gel electrophoresis. With the use of an Oligo-dT

primer and Superscript II Reverse Transcriptase (Invitrogen), labeled cDNA was synthesized

from 40 μg of total RNA. Reference and experimental RNA samples were labeled with Cy3-

and Cy5-coupled dCTP (Amersham Biosciences; Piscataway, NJ), respectively, hybridized

to microarrays, washed, and scanned. Three repeats for each experimental condition were

performed. All experiments were compared with the same reference to allow the relative

expression level of each gene to be compared across all experiments. After normalization,

genes with intensity over four times of background mean were selected. To minimize

variability, the mean of three independent experiments for each gene was calculated and

used for final data clustering. Only genes that showed significant change (over 2-fold

difference) were selected for further characterization. Cluster/Tree view (Michael Eisen,

Stanford University; Stanford, CA) analytic software packages were used for hierarchical

clustering.

Real-time quantitative PCR

Total RNA (1.0 μg) from sample and reference T84 cells was reverse transcribed with a

Taqman RT kit (Applied Biosystems; Foster City, CA). One microliter of the product was

subjected to SYBR green Real-Time PCR assay (Applied Bio-systems). Reactions were

performed in triplicate and normalized to 18s rRNA. The level of expression for a given

gene was first normalized by subtracting the mean value of the cycle threshold (Ct) with that

of 18s rRNA (ΔCt). Relative levels of gene expression were then determined by subtracting

the individual ΔCt values of samples with those of reference (ΔΔCt) and expressing the final

quantification value as 2−ΔΔCt. Primers for the genes of interest were designed with

PrimerExpress (Applied Biosystems), and their sequences are available upon request.

Zeng et al. Page 4


Author M

anuscriptA

uthor Manuscript

Author M

anuscriptA

uthor Manuscript

Caspase staining and quantification

T84 cells on permeable supports or IEC-6 cells grown on glass coverslips to 95% confluence

were stimulated with purified flagellin. After experimental incubation, caspase 8 and

caspase 9 activation was detected using APO LOGIX carboxyfluorescein Caspase Detection

Kits (Cell Technology; Minneapolis MN) through irreversible binding of substrate to

enzymatically active caspases in living cells. Cells were visualized by confocal microscopy

(Zeiss LSM 510) at 505 nm. The numbers of caspase positive cells from 5 representative

viewing fields including at least 500 cells were counted. Data are presented as one of three

representative experiments. Caspase 8 and caspase 9 activities were also determined with

luminescent substrates, using Caspase-Glo 8 and 9 Assays (Promega; Madison, WI)

according to the manufacturer’s protocol, and quantified by a TD-20/20 luminometer

(Turner Designs; Sunnyvale, CA).

Programmed cell death assay

Two different assays were applied to IEC-6 cells (both floating and attached cells) to detect

apoptotic cells after treatment at various times. In the propidium iodide staining assay, cells

were fixed in 70% ethanol and analyzed for DNA content by flow cytometry. With this

method, the percentage of apoptotic cells was determined by quantification of the sub-G1

fraction of cells. For TdT-mediated dUTP nick-end labeling (TUNEL) staining, the In Situ

Cell Death Detection Kit (Roche) was used according to the manufacturer’s instructions.

Briefly, IEC-6 cells were harvested, fixed in 2% paraformaldehyde, permeabilized in 0.1%

Triton X-100–0.1% sodium citrate, and labeled with TUNEL reaction mixture. In both

assays, 10,000 fluorescent events were measured by a FACScalibur flow cytometer (Becton

Dickinson Immunocytometry Systems; San Jose, CA) for each sample. Flow cytometric data

were analyzed using FlowJo software.

RESULTS

Flagellin elicits proinflammatory signaling in epithelial cells

We have previously utilized genetic “loss of function” experiments using Salmonella flagellin mutants to show that this protein was necessary for proinflammatory cellular

responses (53). In a complementary “gain of function” approach, we sought to determine

whether purified flagellin was sufficient to mediate the proinflammatory effects of the viable

pathogen. We evaluated kinetics of established proinflammatory signal response pathways in

T84 model epithelia activated by flagellin (applied basolaterally) and directly compared

responses with those elicited by the canonical endogenous proinflammatory agonist TNF-α (also applied basolaterally). When fully polarized T84 model epithelia were stimulated with

purified flagellin (100 ng/ml, a concentration comparable with that seen during coculture

with live S. typhimurium), we observed rapid (within 5 min) activation of the NF-κB

pathway, as measured by the appearance of phosphorylated IκBα and subsequent

degradation of the unmodified form (Fig. 1). We also noted robust activation of p38 and JNK

kinases of the MAPK signaling group as well as ERK and Akt kinase activation within 5

min of treatment with both flagellin and TNF-α. Notably, flagellin elicited a more prolonged

phosphorylation of these effectors relative to TNF-α, particularly JNK and p38, but overall

cellular responses to these proinflammatory agonists appeared highly similar. These results

Zeng et al. Page 5


Author M

anuscriptA

uthor Manuscript

Author M

anuscriptA

uthor Manuscript

demonstrated the ability of purified flagellin, a prokaryotic protein, to activate cellular

proinflammatory response pathways highly similar to those activated by an endogenous

proinflammatory agonist.

Flagellin elicits a proinflammatory transcriptional response in epithelial cells

To study the transcriptional program elicited by bacterial flagellin, we utilized the same

epithelial model stimulated with proinflammatory agonists over 1–6 h and measured mRNA

abundance by cDNA microarray. This time frame was chosen to evaluate primary, largely

transcriptional changes in epithelial gene expression. The microarray system and data

management methodology have been reported previously (53). Model epithelia were treated

with 1) purified flagellin, applied basolaterally; 2) TNF-α, also applied basolaterally; and 3)

the wild-type pathogen S. typhimurium (strain SL3201, from which we purified flagellin),

cocultured apically with the monolayer.

Model epithelia were stimulated for 1, 2, 3, 4, 5, and 6 h. After experimental manipulation,

RNA was prepared and subjected to microarray analysis. From the collected data, 263 genes

reproducibly exhibited a greater than doubling differential expression; for display purposes,

103 genes showing significantly greater than twofold change relative to control are shown.

The genes comprising the data set are represented in matrix form with the expression

patterns of individual genes organized by hierarchical clustering (Fig. 2), and also listed as

supplementary Table 1. This clustering method calculates the mathematical degree of

relatedness between the expression patterns of each gene in an iterative fashion and arranges

similar expression patterns adjacent to each other along the y-axis. A striking aspect of this

series of experiments was the degree to which cellular responses to S. typhimurium could be

recapitulated by endogenous (TNF-α) or exogenous (flagellin) proinflammatory protein

agonists. Most of these genes have recognized roles in innate immune and inflammatory

reactions, and many of them are regulated by the NF-κB transcription factor pathway (37).

The characteristic differences between early induced genes [often acute inflammatory

mediators such as IL-8 and chemokine, CXC motif, ligand 3 (CXCL3)] and later induced

genes [typically chronic mediators such as inter-feron regulatory factor 1 (IRF-1) and IFN-γ receptors 1 and 2] were clearly apparent. Overall, TNF-α- and flagellin-mediated

upregulation was more rapid than induction elicited by S. typhimurium, consistent with the

kinetics of bacteria-mediated activation of NF-κB reported earlier (16).

Flagellin mediates antiapoptotic effects in epithelial cells

In addition to transcriptional activation of genes with well-known roles in cellular

inflammation, flagellin also activated a panel of antiapoptotic mediator genes including

cIAP-1, cIAP-2, and A20 (indicated in red in Fig. 2). These genes are rapidly induced in a

NF-κB-dependent manner (7) and can inhibit caspase activation and subsequent

proapoptotic signaling (9, 27). For example, the IAPs are ubiquitin ligases that can bind to

and induce degradation of activated caspases (9), whereas A20 is a zinc finger protein that

disrupts assembly of proapoptotic adaptor molecules to the TNF receptor (23). The

unexpected appearance of antiapoptotic mediator genes induced by an exogenous stimulus

led us to confirm this regulation by real-time PCR. cIAP-1, cIAP-2, and A20 antiapoptotic

proteins are well known TNF-α-responsive genes; as shown in Fig. 3A, flagellin is also a

Zeng et al. Page 6


Author M

anuscriptA

uthor Manuscript

Author M

anuscriptA

uthor Manuscript

potent inducer. Given the very high levels of cIAP-2 transcript induced by flagellin, we also

verified expression of this antiapoptotic protein by Western blot analysis (Fig. 3B). Purified

flagellin (FliC) induced this antiapoptotic protein with kinetics consistent with the

microarray analysis.

To test whether flagellin-induced gene expression could protect cells from subsequent

apoptotic stimuli, we used a nontransformed epithelial model, rat IEC-6 cells. These cells

are commonly used in studies of epithelial cell apoptosis (6). IEC-6 epithelial cells were

pretreated for 4 h with flagellin (this duration elicited the maximum induction of

antiapoptotic genes; Fig. 2) and subsequently challenged them by incubation with the

intestinal commensal bacteria E. coli for 6 h. Non-pathogenic E. coli was used to stimulate

multiple TLR/PAMP receptor ligand pairs simultaneously without the presence of type III

secretion system (TTSS) effectors that might be present in pathogens. Under conditions

where cells were not treated with flagellin, this coculture with E. coli induced potent caspase

8 activation within 6 h, as measured quantitatively with luminescent substrates and

morphologically with fluorescent caspases substrates (Fig. 3, C and D). However, cells

pretreated with flagellin before identical E. coli coincubation showed a marked reduction in

caspase-positive cells in both assays. Flagellin treatment alone for a total of 10 h showed no

activation. To determine flagellin-mediated cytoprotective effects against agents that

stimulate the intrinsic, mitochondrial pathway, we performed similar experiments utilizing

the apoptotic activator staurosporine (11). Pretreatment of IEC-6 cells with flagellin reduced

caspase 9 stimulated by staurosporine (as expected, staurosporine had no effect on caspase 8

activity; data not shown), although the effects were not statistically significant (Fig. 3E).

Collectively, these data indicate that the flagellin-induced epithelial transcriptional program

shown in Fig. 2 involves an antiapoptotic component.

Flagellin can induce caspase activation and apoptosis in cells under blockade of NF-κB

Given the similarity of proinflammatory signaling between TNF and flagellin, we attempted

to determine whether flagellin could also positively influence apoptotic processes in the

manner of the TNF receptor. TNF-α signaling has a dualistic nature: although it is potently

proinflammatory and strongly induces antiapoptotic genes, TNF-α is also an activator of the

extrinsic pathway of apoptotic activation through caspase 8 activation (50). Usually, the

concurrent activation of proinflammatory pathways, especially NF-κB, results in

upregulation of antiapoptotic effector genes, which aborts caspase action and resultant cell

death (27). Activation of apoptotic pathways by ligands/TLRs is poorly defined, although

many studies have evaluated the TNF/TNF receptor (death receptor) interaction (50).

To investigate a potential role of flagellin in proapoptotic signaling, we examined T84 cells

stimulated with basolateral flagellin over a time course for evidence of caspase activation.

We utilized specific antibodies to evaluate activation of initiator caspases of the extrinsic

pathway (caspase 8) and intrinsic pathway (caspase 9) as well as executioner caspases

(caspase 3) and substrates (PARP) in Western blots. These antibodies react with the

processed forms of the specific caspases, a proteolytic event necessary for activation of

enzymatic function, whereas PARP cleavage is another marker of the execution phase of

apoptosis. With treatment with flagellin alone, trace appearances of the cleaved/activated

Zeng et al. Page 7


Author M

anuscriptA

uthor Manuscript

Author M

anuscriptA

uthor Manuscript

forms of caspases were seen over 4–8 h (Fig. 4A). In cells under blockade of the NF-κB

pathway with proteasomal inhibitors (MG-262), activation of caspases (capase 8, caspase 9,

and caspase 3) was markedly more intense than that with flagellin or MG-262 alone, most

noticeable with caspase 8. In cells treated with wortmannin (an inhibitor of P13K/Akt

kinase) flagellin potently induced caspase-3 cleavage, suggesting a role for this pathway in

flagellin-mediated caspase activation (Fig. 4B).

To further study caspase activation, carboxyfluorescein (FAM) caspase substrates were

employed. Polarized T84 cells were stimulated with flagellin and/or MG-262 as above,

treated with the fluorescent substrates, and evaluated by fluorescence microscopy.

Occasional fluorescent cells were seen within 6 h of flagellin treatment alone with substrates

specific for both caspase 8 and caspase 9 (Fig. 4, C and D). Similar activation was observed

in cells treated with MG-262. However, the combination of flagellin stimulation and NF-κB

blockade revealed a marked quantitative upregulation of caspase 8 and caspase 9 activation,

a pattern highly consistent to the data obtained with anti-active caspase antibodies.

To demonstrate that viable Salmonella could activate apoptotic signaling in cultured model

epithelia, we cocultured live wild-type Salmonella with IEC-6 cells and evaluated caspase 8

and caspase 9 activation with fluorescent substrates (Fig. 4E). High levels of caspase 8

activation was seen within 12 h postinoculation with the wild-type strain, whereas activation

of caspase 9 was less significant. These data are consistent with prior evaluation of

Salmonella-mediated apoptotic activation (28) and suggests that Salmonella-mediated

apoptotic stimulation is predominately via the extrinsic pathway.

Collectively, these data indicate that flagellin and flagellated bacteria can initiate caspase

activation, a process that is markedly enhanced when proinflammatory (survival) pathways

are blocked. Further experiments were undertaken to determine whether this caspase

activation proceeded to cell death. We observed that at 6 h of flagellin stimulation, no cell

death occurred in IEC-6 epithelial cells, as measured by an increase in the sub-G1 DNA

fraction (Fig. 5A), as would be expected with the simultaneous and unimpeded activation of

the NF-κB pathway and secondary arrest of activated caspases. However, during flagellin

stimulation under conditions of NF-κB blockade (with the proteasome inhibitor MG-262),

IEC-6 cells showed evidence of programmed cell death. The sub-G1 fraction was

comparable with that induced by staurosporine. Proteasome inhibitors are commonly used to

augment apoptosis experimentally (21). MG-262 alone did show some apoptotic activation,

presumably due to inhibition of cyclin turnover.

To support the notion that specific inhibition of the NF-κB pathway would allow flagellin-

induced caspase activation to proceed to irreversible cell death, we transfected cells with an

IκBα mutant bearing Ser to Ala transitions (S32/36A) on the phosphorylation motif. This

mutant cannot be degraded by proinflammatory stimuli and also acts as a dominant negative

substrate for inhibitor of κB, kinase (IKK), effectively and selectively inhibiting the NF-κB

pathway. IEC-6 cells were transfected (with expression confirmed by Western blot) and

assayed for apoptosis by TUNEL staining or immunoblot for activated caspase 3 with and

without stimulation of the cells with flagellin (Fig. 5, B and C). Whereas the mutant IκBα construct alone had relatively little affect on TUNEL positively, transfected cells exposed to

Zeng et al. Page 8


Author M

anuscriptA

uthor Manuscript

Author M

anuscriptA

uthor Manuscript

flagellin for 12–24 h showed a more than doubling of the numbers of apoptotic cells. TNF-α was also tested as a positive control; this cytokine was equally effective in activating

apoptosis under conditions of NF-κB blockade. Collectively, these data indicate that

flagellin-induced signals can augment apoptosis during specific inhibition of the

proinflammatory/antiapoptotic NF-κB signaling pathway.

Flagellin activates the extrinsic pathway in a TLR5-dependant fashion

Proinflammatory signaling elicited by flagellin requires TLR5. To investigate whether

flagellin-elicited apoptotic responses are also TLR5 dependent, we utilized a cell line stably

transfected with TLR5 that can confer the ability to secrete IL-8 in response to flagellin (the

parent cell line does not react to flagellin) (52). These cells were stimulated with flagellin

with and without cycloheximide (a protein synthesis inhibitor) for 4–6 h and evaluated with

immunoblots for activated caspases. As expected, strong caspase 3 and caspase 8 activation

was only seen in the presence of both flagellin and cycloheximide, at 4–6 h (Fig. 6, A–C).

Caspase 9 activation was not consistently observed with these reagents. The control HeLa

cell line, without the transfected TLR5, did not show any caspase activation under identical

conditions. Taken together, these results indicate that flagellin can activate the extrinsic

pathway of caspase processing through its physiological TLR5 receptor.

We have shown that flagellin induces antiapoptotic proteins as part of its transcriptional

program, with cIAP-2 being the most highly induced example. We have also shown that

when survival pathways are inhibited, TLR5-mediated signaling can activate caspases. We

hypothesize that in most flagellin-TLR5 interactions, these antiapoptotic proteins inhibit

caspase activation and permit inflammation. To obtain mechanistic data, we overexpressed

cIAP-2 under conditions where flagellin induces apoptotic activation, by blockade of the

NF-κB pathway with proteasome inhibitors, as demonstrated in Fig. 5C. As shown in Fig. 7,

cIAP-2 reduced executioner caspase 3 cleavage by ~50% under these conditions.

DISCUSSION

Flagellin is increasingly recognized as a bacterial inducer of innate and adaptive immunity

in mammals. It also serves to stimulate cellular defensive responses in plants [acting through

a specific pattern recognition receptors (FLS2)] (18, 19, 55) and invertebrates (43),

suggesting a wide range of eukaryotic life has evolved mechanisms to perceive this protein

and react to it as a threat. In our experiments, flagellin largely recapitulated the

transcriptional response induced by viable S. typhimurium. The responses are also highly

similar to those responses induced by TNF-α, a classical endogenous promoter of innate

immunity/inflammation. The differences that were observed, such as the preferential

activation of inducible nitric oxide synthase and more prolonged MAPK (both JNK and p38)

phosphorylation by flagellin, presumably stems from different receptor utilization (TNF

receptor vs. TLR5) and distinct receptor specific secondary signaling intermediates (i.e.,

MyD88) that ultimately control downstream transcriptional responses (35). Our current data

showing the essentially similar transcriptional responses of flagellin and TNF-α underscore

the importance of acute neutrophilic inflammation in control of infection by flagellated

organisms.

Zeng et al. Page 9


Author M

anuscriptA

uthor Manuscript

Author M

anuscriptA

uthor Manuscript

Although signaling cascades such as NF-κB are generally understood as components of the

proinflammatory response mediating upregulation of cytokines, chemokines, adhesion

molecules, etc., it is becoming increasingly apparent that integral components of this

response are protein modifiers of cellular apoptotic pathways such as the IAP family and

A20. This observation is highly supportive of the notion that apoptotic activation proceeds in

parallel with proinflammatory activation (7, 27). Certain inducers of inflammation, such as

TNF-α, can initiate apoptotic signaling via the extrinsic (caspase 8) pathway (2). The TNF

receptor proteins employ differential utilization of adaptor proteins to control the bifurcation

of proapoptotic and proinflammatory signaling. Receptor-bound TNF receptor-associated

death domain protein (TRADD) may interact with TNF receptor-associated factor (TRAF2)

to activate downstream IKK and NF-κB, or it may bind the death effector domain-containing

adaptor FAS-associated death domain protein (FADD) to initiate caspase 8 processing (50).

Evidence is beginning to emerge that indicates that the TLRs can also mediate both

proinflammatory and proapoptotic signaling in a similar fashion. TLR4/LPS interactions

have been demonstrated to induce apoptotic pathways in macrophages under conditions of

proteasomal blockade, and TLR4-deficient macrophages were shown to be resistant to

Yersinia-induced apoptosis (21). Aliprantis et al. (3) reported that TLR2/bacterial lipoprotein

interactions activated the extrinsic pathway (caspase 8) via signaling involving MyD88 and

subsequent recruitment of FADD, indicating that TLRs can act as “death receptors.”

Proapoptotic signaling via TLR4 has been described in macrophages (25, 41), involving the

TLR interacting adaptor protein inducing IFN-β (TRIF). TRIF-null macrophages show

reduced TLR4-dependant apoptosis, and overexpressed TRIF itself is proapoptotic (42). Our

data indicate that flagellin-induced signaling can also mediate activation of the proapoptotic

cascades. The potential roles of MyD88, FADD, and TRIF in transducing caspase activation

via TLR5 are under investigation.

It is evident that epithelial cells exposed to a microbial challenge can eventuate in cellular

proinflammatory responses or in programmed cell death. Natural infection can vary in

numbers of microbes, duration of interaction, associated organisms, concurrent state of

innate or adaptive immune activation, and undoubtedly many other variables. It is probably

accurate to say that proinflammatory and proapoptotic activation both occur rapidly and

reliably during the initial interactions of bacterial organisms with a potential host. The

cellular end point of these biochemical events depends whether proinflammatory or

proapoptotic signaling “gains the upper hand.” We propose that epithelial cells, once they

perceive bacterial flagellin in an inappropriate location (i.e., intracellular or basolateral) via

the TLR5 receptor, initiate in parallel tandem pathways, both proinflammatory, conducted

along sequential transfer of covalent modifications, and apoptotic, conducted along

proteolytic cascades (Fig. 8). In most cases, proinflammatory upregulation of antiapoptotic

effectors, such as the IAP molecules, serves to arrest apoptotic pathways before activation of

executioner caspases and irreversible cell damage occurs. In the event of inhibition of

proinflammatory signaling and consequent transcription, caspase activation could occur

unimpeded and terminate in the dismantling of the cell. Such inhibition is now recognized to

play an important role in the pathogenesis of certain bacterial infections. Yersinia possess a

translocated effector protein, YopJ, with demonstrated effects against NF-κB and MAPK

survival pathways, which allows them to induce macrophage apoptosis in a TLR4-dependent

Zeng et al. Page 10


Author M

anuscriptA

uthor Manuscript

Author M

anuscriptA

uthor Manuscript

manner, efficiently eliminating an immunomodulatory cell (54). Salmonella are well known

inducers of apoptosis in macrophages (28, 29) and can induce apoptosis in HT-29 epithelial

cell cultures after at least 24 h of coculture with wild-type Salmonella (36). These bacteria

also possess a YopJ homolog, AvrA, which has been demonstrated to inhibit NF-κB and

promote apoptosis in epithelial cells (8). While it is possible that such bacterial effectors

may be directly activating caspases, prokaryotic proteins and small molecules that

specifically inhibit proinflammatory pathways would not be proapoptotic unless

accompanied by a concurrent signal that set caspase processing in motion. Because flagellin

and other PAMP induced signaling through the TLRs fulfill this requirement, it will be

interesting to determine whether the presence of these molecules is required for bacteria-

mediated apoptosis.

Flagellin is emerging as a key regulator of innate and adaptive immunity against flagellated

pathogens (40). Why are cells wired to respond to flagellin (and probably most

proinflammatory agonists) in part by activation of the apoptotic program? It has been

suggested that apoptosis itself may represent the earliest form of “immunity,” whereas the

responses we now consider “innate immunity (i.e., inflammation)” are a later evolutionary

development (4, 26). Colonial, multicellular eukaryotes may have utilized a form of

programmed cell death to eliminate infected cells without lethal effects to the whole

organism. Indeed, such a strategy is common in plant immunity and in lower metazoans

such as nematodes (1), and it is well known that PAMPs including flagellin, peptidoglycan

(PGN), and LPS are potent inducers of apoptosis in these organisms (30, 44). Pattern

recognition receptors may have evolved to altruistically eliminate a threatened cell via

activation of apoptotic proteolytic cascades or, if such drastic steps are unwarranted, induce

a parallel biochemical signaling pathway the results in synthesis and release of soluble

mediators including chemotactic chemokines and antimicrobial peptides.

How flagellin (an extracellular agonist) stimulates the intrinsic/mitochondrial pathway is not

clear, although potential mechanisms can be envisioned. Bid is a proapoptotic member of the

Bcl-2 family of apoptotic regulators (33). These proteins, defined by the “BH3” domain, can

heterodimerize with each other; a relative excess of pro- over antiapoptotic members is

thought to permit formation of a mitochondrial “pore,” allowing the escape of cytochrome c (20) and subsequent initiation of the intrinsic pathway. Bid is regulated by cleavage and

activation by caspase 8, thus providing an amplifying link from extrinsic proapoptotic

signals to intrinsic pathways. Transient Bid cleavage was observed maximally in T84 cells at

3 h of flagellin treatment, but, again, the exposure of cells to flagellin under conditions

where survival genes could not be upregulated (MG-262) resulted in a strong appearance of

the cleaved, 15-kDa active form, tBid (data not shown). Further experiments to characterize

this process are underway.

Our data demonstrating that flagellin pretreatment and cIAP-2 overexpression can reduce

caspase activation (in normal cells with intact proinflammatory pathways) elicited by a

separate potentially injurious stimulus are consistent with our current understanding of the

role of antiapoptotic effectors (27). Recent work has also suggested that constitutive TLR

stimulation by PAMPs normally present in the intestinal lumen is cytoprotective to

immunochemical epithelial injury (39). Although flagellin per se was not identified as a

Zeng et al. Page 11


Author M

anuscriptA

uthor Manuscript

Author M

anuscriptA

uthor Manuscript

cytoprotective PAMP in that study, our results suggest that abundant lumenal flagellin may

mediate a similar effect. Consistently, other studies have shown flagellin-mediated

proinflammatory signaling in vivo induced by flagellated commensals or purified flagellin

(5, 24).

Acknowledgments

GRANTS

This study was supported by National Institutes of Health Grants RO1 AI-51282 and AI-49741 (to A. S. Neish) and DK-062851 (to P. Lin), Emory Digestive Disease Research Center Grant R24 DK-064399, and the Woodruff Research Foundation.

References

1. Aballay A, Ausubel FM. Programmed cell death mediated by ced-3 and ced-4 protects Caenorhabditis elegans from Salmonella typhimurium-mediated killing. Proc Natl Acad Sci USA. 2001; 98:2735–2739. [PubMed: 11226309]

2. Adams JM. Ways of dying: multiple pathways to apoptosis. Genes Dev. 2003; 17:2481–2495. [PubMed: 14561771]

3. Aliprantis AO, Yang RB, Weiss DS, Godowski P, Zychlinsky A. The apoptotic signaling pathway activated by Toll-like receptor-2. EMBO J. 2000; 19:3325–3336. [PubMed: 10880445]

4. Ameisen JC. On the origin, evolution, and nature of programmed cell death: a timeline of four billion years. Cell Death Differ. 2002; 9:367–393. [PubMed: 11965491]

5. Bambou JC, Giraud A, Menard S, Begue B, Rakotobe S, Heyman M, Taddei F, Cerf-Bensussan N, Gaboriau-Routhiau V. In vitro and ex vivo activation of the TLR5 signaling pathway in intestinal epithelial cells by a commensal Escherichia coli strain. J Biol Chem. 2004; 279:42984–42992. [PubMed: 15302888]

6. Bhattacharya S, Ray RM, Viar MJ, Johnson LR. Polyamines are required for activation of c-Jun NH2-terminal kinase and apoptosis in response to TNF-α in IEC-6 cells. Am J Physiol Gastrointest Liver Physiol. 2003; 285:G980–G991. [PubMed: 12869386]

7. Burstein E, Duckett CS. Dying for NF-kappaB? Control of cell death by transcriptional regulation of the apoptotic machinery. Curr Opin Cell Biol. 2003; 15:732–737. [PubMed: 14644198]

8. Collier-Hyams LS, Zeng H, Sun J, Tomlinson AD, Bao ZQ, Chen H, Madara JL, Orth K, Neish AS. Cutting edge: Salmonella AvrA effector inhibits the key proinflammatory, anti-apoptotic NF-kappa B pathway. J Immunol. 2002; 169:2846–2850. [PubMed: 12218096]

9. Deveraux QL, Reed JC. IAP family proteins–suppressors of apoptosis. Genes Dev. 1999; 13:239–252. [PubMed: 9990849]

10. Donnenberg MS. Pathogenic strategies of enteric bacteria. Nature. 2000; 406:768–774. [PubMed: 10963606]

11. Duan S, Hajek P, Lin C, Shin SK, Attardi G, Chomyn A. Mitochondrial outer membrane permeability change and hypersensitivity to digitonin early in staurosporine-induced apoptosis. J Biol Chem. 2003; 278:1346–1353. [PubMed: 12403774]

12. Eaves-Pyles T, Murthy K, Liaudet L, Virag L, Ross G, Soriano FG, Szabo C, Salzman AL. Flagellin, a novel mediator of Salmonella-induced epithelial activation and systemic inflammation: I kappa B alpha degradation, induction of nitric oxide synthase, induction of proinflammatory mediators, and cardiovascular dysfunction. J Immunol. 2001; 166:1248–1260. [PubMed: 11145708]

13. Elewaut D, DiDonato JA, Kim JM, Truong F, Eckmann L, Kagnoff MF. NF-kappa B is a central regulator of the intestinal epithelial cell innate immune response induced by infection with enteroinvasive bacteria. J Immunol. 1999; 163:1457–1466. [PubMed: 10415047]

14. Franke TF, Hornik CP, Segev L, Shostak GA, Sugimoto C. PI3K/Akt and apoptosis: size matters. Oncogene. 2003; 22:8983–8998. [PubMed: 14663477]

Zeng et al. Page 12


Author M

anuscriptA

uthor Manuscript

Author M

anuscriptA

uthor Manuscript

15. Gewirtz AT, Navas TA, Lyons S, Godowski PJ, Madara JL. Cutting edge: bacterial flagellin activates basolaterally expressed TLR5 to induce epithelial proinflammatory gene expression. J Immunol. 2001; 167:1882–1885. [PubMed: 11489966]

16. Gewirtz AT, Rao AS, Simon PO Jr, Merlin D, Carnes D, Madara JL, Neish AS. Salmonella typhimurium induces epithelial IL-8 expression via Ca2+-mediated activation of the NF-kappaB pathway. J Clin Invest. 2000; 105:79–92. [PubMed: 10619864]

17. Gewirtz AT, Simon PO Jr, Schmitt CK, Taylor LJ, Hagedorn CH, O’Brien AD, Neish AS, Madara JL. Salmonella typhimurium translocates flagellin across intestinal epithelia, inducing a proinflammatory response. J Clin Invest. 2001; 107:99–109. [PubMed: 11134185]

18. Gomez-Gomez L, Boller T. Flagellin perception: a paradigm for innate immunity. Trends Plant Sci. 2002; 7:251–256. [PubMed: 12049921]

19. Gomez-Gomez L, Boller T. FLS2: an LRR receptor-like kinase involved in the perception of the bacterial elicitor flagellin in Arabidopsis. Mol Cell. 2000; 5:1003–1011. [PubMed: 10911994]

20. Green DR, Kroemer G. The pathophysiology of mitochondrial cell death. Science. 2004; 305:626–629. [PubMed: 15286356]

21. Haase R, Kirschning CJ, Sing A, Schrottner P, Fukase K, Kusumoto S, Wagner H, Heesemann J, Ruckdeschel K. A dominant role of Toll-like receptor 4 in the signaling of apoptosis in bacteria-faced macrophages. J Immunol. 2003; 171:4294–4303. [PubMed: 14530354]

22. Hayashi F, Smith KD, Ozinsky A, Hawn TR, Yi EC, Goodlett DR, Eng JK, Akira S, Underhill DM, Aderem A. The innate immune response to bacterial flagellin is mediated by Toll-like receptor 5. Nature. 2001; 410:1099–1103. [PubMed: 11323673]

23. He KL, Ting AT. A20 inhibits tumor necrosis factor (TNF) alpha-induced apoptosis by disrupting recruitment of TRADD and RIP to the TNF receptor 1 complex in Jurkat T cells. Mol Cell Biol. 2002; 22:6034–6045. [PubMed: 12167698]

24. Honko AN, Mizel SB. Mucosal administration of flagellin induces innate immunity in the mouse lung. Infect Immun. 2004; 72:6676–6679. [PubMed: 15501801]

25. Hsu LC, Park JM, Zhang K, Luo JL, Maeda S, Kaufman RJ, Eckmann L, Guiney DG, Karin M. The protein kinase PKR is required for macrophage apoptosis after activation of Toll-like receptor 4. Nature. 2004; 428:341–345. [PubMed: 15029200]

26. James ER, Green DR. Infection and the origins of apoptosis. Cell Death Differ. 2002; 9:355–357. [PubMed: 11965487]

27. Karin M, Lin A. NF-kappaB at the crossroads of life and death. Nat Immun. 2002; 3:221–227.

28. Kim JM, Eckmann L, Savidge TC, Lowe DC, Witthoft T, Kagnoff MF. Apoptosis of human intestinal epithelial cells after bacterial invasion. J Clin Invest. 1998; 102:1815–1823. [PubMed: 9819367]

29. Knodler LA, Finlay BB. Salmonella and apoptosis: to live or let die? Microbes Infect. 2001; 3:1321–1326. [PubMed: 11755421]

30. Koropatnick TA, Engle JT, Apicella MA, Stabb EV, Goldman WE, McFall-Ngai MJ. Microbial factor-mediated development in a host-bacterial mutualism. Science. 2004; 306:1186–1188. [PubMed: 15539604]

31. Liaudet L, Szabo C, Evgenov OV, Murthy KG, Pacher P, Virag L, Mabley JG, Marton A, Soriano FG, Kirov MY, Bjertnaes LJ, Salzman AL. Flagellin from gram-negative bacteria is a potent mediator of acute pulmonary inflammation in sepsis. Shock. 2003; 19:131–137. [PubMed: 12578121]

32. Lodes MJ, Cong Y, Elson CO, Mohamath R, Landers CJ, Targan SR, Fort M, Hershberg RM. Bacterial flagellin is a dominant antigen in Crohn disease. J Clin Invest. 2004; 113:1296–1306. [PubMed: 15124021]

33. Luo X, Budihardjo I, Zou H, Slaughter C, Wang X. Bid, a Bcl2 interacting protein, mediates cytochrome c release from mitochondria in response to activation of cell surface death receptors. Cell. 1998; 94:481–490. [PubMed: 9727491]

34. Macnab, RM. Escherichia coli and Salmonella typhimurium. Washington, DC: American Society for Microbiology; 1999. Flagella; p. 123-145.

35. O’Neill LA. Signal transduction pathways activated by the IL-1 receptor/toll-like receptor superfamily. Curr Top Microbiol Immunol. 2002; 270:47–61. [PubMed: 12467243]

Zeng et al. Page 13


Author M

anuscriptA

uthor Manuscript

Author M

anuscriptA

uthor Manuscript

36. Paesold G, Guiney DG, Eckmann L, Kagnoff MF. Genes in the Salmonella pathogenicity island 2 and the Salmonella virulence plasmid are essential for Salmonella-induced apoptosis in intestinal epithelial cells. Cell Microbiol. 2002; 4:771–781. [PubMed: 12427099]

37. Pahl HL. Activators and target genes of Rel/NF-kappaB transcription factors. Oncogene. 1999; 18:6853–6866. [PubMed: 10602461]

38. Park JM, Greten FR, Li ZW, Karin M. Macrophage apoptosis by anthrax lethal factor through p38 MAP kinase inhibition. Science. 2002; 297:2048–2051. [PubMed: 12202685]

39. Rakoff-Nahoum S, Paglino J, Eslami-Varzaneh F, Edberg S, Medzhitov R. Recognition of commensal microflora by toll-like receptors is required for intestinal homeostasis. Cell. 2004; 118:229–241. [PubMed: 15260992]

40. Ramos HC, Rumbo M, Sirard JC. Bacterial flagellins: mediators of pathogenicity and host immune responses in mucosa. Trends Microbiol. 2004; 12:509–517. [PubMed: 15488392]

41. Ruckdeschel K, Mannel O, Schrottner P. Divergence of apoptosis-inducing and preventing signals in bacteria-faced macrophages through myeloid differentiation factor 88 and IL-1 receptor-associated kinase members. J Immunol. 2002; 168:4601–4611. [PubMed: 11971008]

42. Ruckdeschel K, Pfaffinger G, Haase R, Sing A, Weighardt H, Hacker G, Holzmann B, Heesemann J. Signaling of apoptosis through TLRs critically involves toll/IL-1 receptor domain-containing adapter inducing IFN-beta, but not MyD88, in bacteria-infected murine macrophages. J Immunol. 2004; 173:3320–3328. [PubMed: 15322195]

43. Samakovlis C, Asling B, Boman HG, Gateff E, Hultmark D. In vitro induction of cecropin genes–an immune response in a Drosophila blood cell line. Biochem Biophys Res Commun. 1992; 188:1169–1175. [PubMed: 1445351]

44. Shimizu R, Taguchi F, Marutani M, Mukaihara T, Inagaki Y, Toyoda K, Shiraishi T, Ichinose Y. The DeltafliD mutant of Pseudomonas syringae pv. tabaci, which secretes flagellin monomers, induces a strong hypersensitive reaction (HR) in non-host tomato cells. Mol Genet Genomics. 2003; 269:21–30. [PubMed: 12715150]

45. Sierro F, Dubois B, Coste A, Kaiserlian D, Kraehenbuhl JP, Sirard JC. Flagellin stimulation of intestinal epithelial cells triggers CCL20-mediated migration of dendritic cells. Proc Natl Acad Sci USA. 2001; 98:13722–13727. [PubMed: 11717433]

46. Smith KD, Andersen-Nissen E, Hayashi F, Strobe K, Bergman MA, Barrett SL, Cookson BT, Aderem A. Toll-like receptor 5 recognizes a conserved site on flagellin required for protofilament formation and bacterial motility. Nat Immun. 2003; 4:1247–1253.

47. Smith KD, Ozinsky A. Toll-like receptor-5 and the innate immune response to bacterial flagellin. Curr Top Microbiol Immunol. 2002; 270:93–108. [PubMed: 12467246]

48. Takeda K, Kaisho T, Akira S. Toll-like receptors. Annu Rev Immunol. 2003; 21:335–376. [PubMed: 12524386]

49. Tauxe, R., Pavia, A. Bacterial infections in humans: epidemiology and control. New York: Plenum; 1998. Salmonellosis: nontyphoidal; p. 613-630.

50. Wajant H, Pfizenmaier K, Scheurich P. Tumor necrosis factor signaling. Cell Death Differ. 2003; 10:45–65. [PubMed: 12655295]

51. Wick MJ. Living in the danger zone: innate immunity to Salmonella. Curr Opin Microbiol. 2004; 7:51–57. [PubMed: 15036140]

52. Yu Y, Zeng H, Lyons S, Carlson A, Merlin D, Neish AS, Gewirtz AT. TLR5-mediated activation of p38 MAPK regulates epithelial IL-8 expression via posttranscriptional mechanism. Am J Physiol Gastrointest Liver Physiol. 2003; 285:G282–G290. [PubMed: 12702497]

53. Zeng H, Carlson AQ, Guo Y, Yu Y, Collier-Hyams LS, Madara JL, Gewirtz AT, Neish AS. Flagellin is the major proinflammatory determinant of enteropathogenic Salmonella. J Immunol. 2003; 171:3668–3674. [PubMed: 14500664]

54. Zhang Y, Bliska JB. Role of Toll-like receptor signaling in the apoptotic response of macrophages to Yersinia infection. Infect Immun. 2003; 71:1513–1519. [PubMed: 12595470]

55. Zipfel C, Robatzek S, Navarro L, Oakeley EJ, Jones JD, Felix G, Boller T. Bacterial disease resistance in Arabidopsis through flagellin perception. Nature. 2004; 428:764–767. [PubMed: 15085136]

Zeng et al. Page 14


Author M

anuscriptA

uthor Manuscript

Author M

anuscriptA

uthor Manuscript

Fig. 1. Flagellin (FliC) and TNF-α induce similar patterns of proinflammatory signaling activation.

Polarized T84 cells were stimulated with TNF-α (10 ng/ml) or purified flagellin (100

ng/ml) applied basolaterally for the indicated time points. Whole cell lysates were subjected

to immunoblots with the indicated antibodies. β-Actin is shown as a loading control. Data

shown are derived from a single set of cell lysates and are representative of 3 independent

experiments.

Zeng et al. Page 15


Author M

anuscriptA

uthor Manuscript

Author M

anuscriptA

uthor Manuscript

Fig. 2. Transcriptional response of polarized model epithelia to proinflammatory stimuli. Cells were

treated with TNF-α (3 ng/ml, applied basolaterally), wild-type (WT) Salmonella typhimurium (SL3201, applied apically), and purified flagellin (5 ng/ml, applied

basolaterally) for 1, 2, 3, 4, 5, and 6 h. Flagellin (FliC) was purified from the SL3201 strain.

Data are a time course of expression profiles of 103 genes differentially regulated by

proinflammatory stimuli with >2-fold induction. The expression patterns of individual genes

were organized by uncentered correlation complete linkage hierarchical clustering. Data are

presented as a matrix; each row represents an individual array element (gene), and each

column represents an experimental condition. Red designates relative upregulation, whereas

green designates downregulation, in a semicontinuous fashion (see scale, bottom left). The

intensity of the color corresponds to the mean ratio of transcript abundance of the

Zeng et al. Page 16


Author M

anuscriptA

uthor Manuscript

Author M

anuscriptA

uthor Manuscript

experimental condition relative to the transcript abundance of the untreated cell line (T84).

The green vertical bar delineates the “bacterially enhanced” group of genes, whereas the red

bar denotes “flagellin-enhanced” genes. Antiapoptotic effector genes are shown in red.

Zeng et al. Page 17


Author M

anuscriptA

uthor Manuscript

Author M

anuscriptA

uthor Manuscript

Fig. 3. Flagellin mediates antiapoptotic effects in epithelial cells. A: real-time PCR quantification of

3 selected antiapoptotic effector genes: cellular inhibitor of apoptosis (cIAP)1, cIAP2, and

A20. Cells were stimulated with flagellin as described in Fig. 2 for the indicated time points.

Data are shown as fold upregulation relative to basal (unstimulated) levels. B: immunoblot

for cIAP-2. T84 cells were stimulated with flagellin for the times indicated. Actin is

included as a loading control. C: IEC-6 cells were stimulated with flagellin (100 ng/ml) for 4

h before challenge by the addition of viable Escherichia coli for an additional 6 h at a

multiplicity of infection (MOI) of 30. Caspase activation was detected and quantitated with a

luminescent caspase 8 substrate. Data are from a single experiment and representative of 3

experiments that showed an identical pattern of results.D: IEC-6 cells were treated as in C.

Zeng et al. Page 18


Author M

anuscriptA

uthor Manuscript

Author M

anuscriptA

uthor Manuscript

Caspase 8 activation in viable cells was detected with an APO LOGIX carboxyfluorescein

(FAM) caspase detection kit, and nuclei were counterstained with 4′,6-diamidino-2-

phenylindole dihydrochloride (DAPI). Data are from a single experiment and representative

of 3 experiments that showed an identical pattern of results. E: IEC-6 cells were treated with

flagellin as in C and then stimulated with 1 μM staurosporine (STS) for 4 h. Caspase

activation was detected and quantitated with a luminescent caspase 9 substrate. Data are

from a single experiment and representative of 3 experiments that showed an identical

pattern of results.

Zeng et al. Page 19


Author M

anuscriptA

uthor Manuscript

Author M

anuscriptA

uthor Manuscript

Zeng et al. Page 20


Author M

anuscriptA

uthor Manuscript

Author M

anuscriptA

uthor Manuscript

Fig. 4. Flagellin induces caspase activation. A: polarized T84 cells were stimulated with flagellin

(100 ng/ml), MG-262 (1 μM), or a combination for the indicated durations. Whole cell

lysates were subjected to immunoblots with the indicated antibodies. Cleaved fragments are

designated with arrows. β-Actin is shown as a loading control. Data shown are derived from

a single set of cell lysates and are representative of 5 independent experiments. B: T84 cells

were pretreated with 100 nM wortmannin (WTN) for 30 min and then stimulated with 100

ng/ml flagellin for 3 h. Cells were lysed and immunoblotted with antibody to cleaved

caspase 3. β-Actin is shown as a loading control. C: polarized T84 cells were stimulated

with flagellin (100 ng/ml), MG-262 (250 ng/ml), or a combination for 6 h. Monolayers were

treated with fluorescent caspase substrates (caspase 8: FAM-LETD-FMK and caspase 9:

FAM-LEHD-FMK) and visualized by confocal microscopy. D: quantification of images

described in C. Results are displayed as fold increase of caspase positive cells over the

Zeng et al. Page 21


Author M

anuscriptA

uthor Manuscript

Author M

anuscriptA

uthor Manuscript

unstimulated baseline. E: IEC-6 cells were colonized with flagellated viable S. typhimurium for the indicated times, and cells were treated with fluorescent caspase substrates as in C.

Zeng et al. Page 22


Author M

anuscriptA

uthor Manuscript

Author M

anuscriptA

uthor Manuscript

Fig. 5. Flagellin can elicit apoptosis under conditions of NF-κB blockade. A: IEC-6 cells were

treated with or without 100 ng/ml of flagellin and/or 1 μM MG-262 or 2 μM STS for 6 h.

Cells were fixed, stained with propidium iodide, and analyzed by flow cytometry to assay

DNA cleavage (sub-G1 fraction); 10,000 fluorescent events were measured for each sample.

The percentages of positive cells are graphed on the y-axis. B: IEC-6 cells were transiently

cotransfected with expression vector pDsRed2, encoding red fluorescent protein; mutant

IκBα, pCMV4 T7-IκBα S32/36A; or empty control vector by FuGENE 6 reagent according

to the manufacturer’s instruction. Twenty hours after transfection, cultures were treated with

flagellin (100 ng/ml) or TNF-α (10 ng/ml). Cells were harvested 24 h later, a TdT-mediated

dUTP nick-end labeling (TUNEL) assay was performed, and cells were analyzed by flow

Zeng et al. Page 23


Author M

anuscriptA

uthor Manuscript

Author M

anuscriptA

uthor Manuscript

cytometry; 10,000 fluorescent events were measured for each sample. C: 293T cells were

treated as in B. Lysates were prepared and probed for indicated antibodies.

Zeng et al. Page 24


Author M

anuscriptA

uthor Manuscript

Author M

anuscriptA

uthor Manuscript

Fig. 6. Flagellin activates caspase 8 (extrinsic pathway) in a Toll-like receptor (TLR)5-dependant

manner. A: HeLa cells with and without a stably transfected TLR5 expression plasmid were

stimulated with flagellin (100 ng/ml), cycloheximide (CHX; 5 μg/ml), or a combination for

the indicated durations. Whole cell lysates were subjected to immunoblots with the indicated

activated caspase antibodies. V5 is an epitope tag of the transfected TLR5. B and C:

densitometric analysis of caspase 8 (B) and caspase 3 (C) immunoreactivity. Cleaved

caspase 8 and caspase 3 bands were scanned with Bio-Rad Imaging Densitometer GS-700

and quantitated with Bio-Rad Quantity One acquisition software.

Zeng et al. Page 25


Author M

anuscriptA

uthor Manuscript

Author M

anuscriptA

uthor Manuscript

Fig. 7. cIAP-2 can inhibit flagellin-mediated caspase activation. A: cells were transfected with

vector for cIAP-2 and subsequently treated with flagellin and MG-262 as in Fig. 4C. B:

densitometric analysis of caspase 3 immunoreactivity was performed as in Fig. 6C.

Zeng et al. Page 26


Author M

anuscriptA

uthor Manuscript

Author M

anuscriptA

uthor Manuscript

Fig. 8. Model of pro- and antiapoptotic signaling from flagellin/TLR5. Flagellin activates the

proinflammatory NF-κB pathway in parallel with caspase cascades. Usually, transcriptional

activation of antiapoptotic effectors arrests caspase activation, and reversible inflammation

occurs. If proinflammatory pathways are blocked, caspase activation can proceed to

irreversible cell death.

Zeng et al. Page 27


Author M

anuscriptA

uthor Manuscript

Author M

anuscriptA

uthor Manuscript

Flagellin/TLR5 responses in epithelia reveal intertwined ...

Documents

Transcript of Flagellin/TLR5 responses in epithelia reveal intertwined ...