BACKGROUND
intestinal epithelial cells serve as a barrier between the luminal microflora and the body and as such are perfectly positioned to monitor the approach/invasion of pathogens.
these intestinal epithelial cells  serve as innate immune sentinels and monitor their environment and constantly give out innate host defense instruction to local immune effector cells  <cit> .
pathogens such as salmonella and other enteroinvasive pathogenic bacteria such as enteroinvasive e. coli, shigella and yersinia upon infection of iecs leads to the up-regulation of the expression of host genes, the products of which activate mucosal inflammatory and immune responses and alter epithelial cell functions  <cit> .
previously we and others demonstrated that ikk via nf-κb and the sapk signaling pathways via jun n-terminal kinase  and p <dig> kinase were key regulators of the up-regulation of the proinflammatory gene program  <cit> , with nf-κb appearing to be the most critical  <cit> .
typically salmonella infects thirty-forty percent of iecs in culture models of infection  <cit> , however, we and others have found that salmonella infection activates nf-κb dna binding activity to levels equivalent to that of tnfα which activates nf-κb in all of the cells  <cit> .
previous studies examining nf-κb activation by salmonella in ht <dig> colonic intestinal epithelial cells, which serve as model colonic epithelial cells in culture, indicated that delivery of salmonella proteins into the host cell via its type iii secretion system , such as sope and sope <dig>  the bacterially encoded exchange factors for the rho-family members rac <dig> and cdc <dig>  result in exchange factor activation, cytoskeletal rearrangements and activation of the mapk, sapk and nf-κb signaling pathways  <cit> .
recent observations that utilized salmonella strains that were defective in invasion and delivery of invasion proteins by the ttss but not attachment indicated that additional factors other than those delivered by the ttss could lead to nf-κb activation  <cit> .
presently it is not clear what protein dictate the activation of key signaling pathways that lead to the temporal expression of the proinflammatory gene program, although the sope proteins have been given extreme attention recently  <cit> .
in searching for additional salmonella proteins that could activate the proinflammatory gene expression program, bacterial flagellin was recently found to be such a protein  <cit>  and had been shown previously to activate il- <dig> expression in monocytes  <cit> .
flagellin was found to activate nf-κb in polarized epithelial cells only when flagellin was present on their basolateral surface  <cit>  consistent with the idea that a cell surface receptor was present there and could recognize it.
the toll-like receptors  have been found to recognize pathogen associated molecular patterns  reviewed in  <cit> .
tlr <dig> interacts with tlr <dig> and tlr <dig> to recognize bacterial lipopeptides and zymosan respectively  <cit> .
tlr <dig> recognizes lps only when associated with its co-receptor md <dig> and cd <dig>  <cit> .
recently, flagellin was demonstrated to be recognized by tlr <dig> and activate an innate host response  <cit> .
however, little was known or demonstrated about the endogenous levels of tlr <dig> in cells used in those studies and why those cells failed to respond to flagellin.
here we have identified flagellin as the primary initiator and temporal regulator of not only the major signaling pathways activated during salmonella infection but also of key target genes of the proinflammatory gene program too.
we have also found flagellin expression to be required for salmonella bacterial invasion.
independently we found tlr <dig> recognizes flagellin but its signaling activity toward this pamp is consistent with either the aid of another flagellin-recognizing co-receptor  or the use of another adapter protein, perhaps similar to myd <dig>  that is absent or present at low levels in flagellin non-or low-responding cells.
RESULTS
salmonella infection leads to a minority of cells invaded but activates nf-κb in nearly all cells
previously, we have noted that pathogenic salmonella sp.
infection leads to potent ikk and nf-κb activation and activation of the proinflammatory gene program  <cit> .
previous studies suggest that about thirty-forty percent of the intestinal epithelial cells are infected during a typical salmonella infection in cultured intestinal epithelial cells  <cit> .
we wished to address the question of how bacterial infection of about thirty percent of the host cells could give rise to nf-κb dna binding activity equivalent to activation of nf-κb in nearly all of the host cells as tnfα treatment of the cells does.
to examine this phenomenon in detail ht <dig> cells either mock-infected or infected at a moi of fifty for one-hour with wild-type s. typhimurium that had been transformed with the plasmid pfm <dig> , that encodes green fluorescent protein  under the control of the salmonella ssah promoter and only functions once the bacteria has invaded the host cell  <cit> .
as can be seen in fig.
1a, gfp expression occurs in about thirty to forty percent of the cells.
we next examined the localization of the nf-κb subunit p <dig>  in non-treated , salmonella infected or tnfα  stimulated cells and found that p <dig>  was localized to the cytoplasm in non-treated cells whereas, in salmonella infected cells or in tnfα treated cells p <dig>  had localized to the nucleus .
these results demonstrate that salmonella infection activates nf-κb in virtually all of the cells even though only a minority of them become infected and is consistent with and aids in explanation of our previous results examining salmonella infection and nf-κb activation  <cit> .
soluble bacterial product identified as flagellin can activate nf-κb in intestinal epithelial cells
since salmonella sp.
infection of intestinal epithelial cells in culture led to only roughly thirty percent infection but activation of nf-κb in nearly all of the cells, we anticipated that nf-κb activation was in response to host cell recognition of bacteria structural components or products produced by the bacteria and not by the invasion process.
invasion itself has been demonstrated not to be required for activation of the proinflamatory gene program as had previously been thought  <cit> .
to investigate this possibility sterile-filtered s. dublin culture broth left either untreated or boiled for twenty minutes was used to challenge ht <dig> intestinal epithelial cells and nf-κb dna binding activity was monitored by electromobility shift assays  of whole cell extracts  prepared forty-five minutes after exposure  <cit> .
potent activation of nf-κb in response to the broth under both conditions was observed indicating the activating factor was heat-stable  and is not lps since ht <dig> cells are not responsive to lps  <cit> .
the native sterile-filtered concentrated broth was subsequently treated with dnase, rnase, proteinase k or crudely size fractionated on  <dig> kda centricon filters.
the variously treated broths were then used to challenge ht <dig> intestinal epithelial cells and wces were prepared after forty-five minutes and nf-κb dna binding activity was analyzed by emsa .
direct infection of ht <dig> cells by s. typhimurium  <dig> or exposure to the culture broths , as indicated, induced nf-κb dna binding activity, while the activity-inducing factor was found to be sensitive to protease digestion and was retained by a  <dig> kda filter .
to further determine the identity of the nf-κb inducing activity, sterile-filtered concentrated broth culture was fractionated by superose  <dig> gel permeation chromatography  and by anion exchange chromatography .
aliquots of chromatography fractions were assayed for their ability to activate nf-κb in ht <dig> cells and analyzed by emsa.
as can be seen from the coomassie blue stained gel  increased nf-κb dna binding activity  corresponded to the increased abundance of an approximately  <dig> kda protein.
anion exchange chromatography on poros hq matrix and elution of bound proteins with an increasing salt gradient as indicated  demonstrated that nf-κb dna binding-inducing activity corresponded to chromatographic fractions containing an increased abundance of the  <dig> kda protein .
eluted fractions observed in fig.
2c were concentrated and fractionated on preparative 12% sds-page gels and bands corresponding to b1-b <dig> were cut from the gels and the proteins eluted, precipitated and renatured as described in experimental procedures and used to stimulate ht <dig> cells.
whole cell extracts from these cells were assayed for nf-κb dna binding-inducing activity by emsa and only band  <dig>  corresponding to the  <dig> kda protein  was able to elicit nf-κb dna binding activity while buffer from the beginning or end of the salt gradient failed to activate nf-κb dna binding activity.
proteins corresponding to protein bands b1-b <dig> and blank areas of the gel were further processed for peptide sequencing as described in experimental procedures.
trypsin digestion of the protein corresponding to b <dig> and analysis by electrospray ion trap lc/ms identified the amino acid sequence of twenty-one peptides.
flagellin  was unambiguously identified as the protein consistent with inducing nf-κb dna binding activity .
flagellin is required to activate nf-κb in intestinal epithelial cells
to determine if flagellin was indeed the factor that was responsible for triggering activation of nf-κb after exposure of intestinal epithelial cells to direct bacterial infection or to filtered culture broths of pathogenic salmonella sp.
we prepared infectious bacteria and boiled and filtered culture broths from the non-flagellated e. coli dh5α, pathogenic s. dublin strain  <dig>  an isogenic s. dublin  <dig> sope- mutant, isogenic s. dublin  <dig> sopb- mutant, isogenic s. dublin  <dig> double sope-/sopb- mutant , s. typhimurium strain  <dig>  and isogenic s. typhimurium flic::tn <dig> insertion mutant  and a s. typhimurium  <dig> isogenic double mutant flic-/fljb- and were used to challenge ht <dig> cells.
bacteria and culture broths were used to challenge ht <dig> intestinal epithelial cells and wce extracts were prepared after forty-five minutes and analyzed for nf-κb dna binding activity by emsa.
salmonella strains could activate nf-κb, while salmonella strains failing to produce flagellin  also failed to activate nf-κb .
e. coli dh5α is non-flagellated and does not produce flagellin failed to activate nf-κb.
we also noticed through numerous experiments that s. dublin direct infections always activated nf-κb to a greater extent than s. typhimurium as observed in fig.
4a while culture broths from both species activated nf-κb almost equally well .
we believe this difference is due perhaps to s. dublin releasing more flagellin into the cell culture media than s. typhimurium during infection since purification of flagellin from both s. dublin and s. typhimurium and addition of equivalent amounts of chromatographically purified flagellin gave similar nf-κb activation profiles .
of note is the total failure of the double flagellin gene mutants to activate nf-κb as compared to the very minor activation observed in the single phase i flagellin flic::tn <dig> insertion mutant  which likely is due to the extremely limited expression of the phase ii flagellin , although the strains of salmonella used here genetically are unable or rarely shift phases of flagellin production.
these results are consistent with previous reports identifying flagellin as a potent inducer of the proinflammatory response and il- <dig> production  <cit> .
since flagellin appears required for activation of the nf-κb pathway upon direct infection of intestinal epithelial cells it appeared possible that flagellin may also be the major determinant of other major mitogenic and stress activated signaling pathways activated upon pathogenic salmonella infection of intestinal epithelial cells.
previously others and we have demonstrated that direct salmonella infection of intestinal epithelial cells results in jnk activation  <cit>  and also the activation of nf-κb via ikk  <cit> .
the identification of flagellin as a potent nf-κb activator is significant since sope had previously been shown to be a pathogenic salmonella bacteriophage encoded protein that is injected into the host cell and acts as an exchange factor for the small rho gtpases rac <dig> and cdc <dig> initiating cytoskeleton rearrangements and eventual activation of the mapk, sapk and nf-κb pathways  <cit> , while sopb is a salmonella protein that functions as an inositol phosphate phosphatase and participates in cytoskeletal rearrangements and stimulates host cell chloride secretion  <cit> .
flagellin triggers activation of the mitogen activated protein kinase, stress activated protein kinase and ikk signaling pathways
intestinal epithelial cells act as sentinels for invasion of luminal surfaces and orchestrate the attraction of effector immune cells to the area by production of chemokine genes like il- <dig> and macrophage chemoattractant protein  <dig>  proinflammatory cytokine genes such as tnfα, il- <dig> and il- <dig>  <cit> .
expression of these genes primarily depends upon the action of transcription factors that are activated in response to the transmission of signals via the mapk, sapk and ikk signaling pathways.
since nf-κb is considered a central regulator/activator of the proinflammatory gene program we decided to examine the effect that non-flagellin producing mutant strains of salmonella had on activation of the mapk, sapk and ikk signaling pathways compared to infection of intestinal epithelial cells with wild-type salmonella or by exposure of the intestinal epithelial cells to purified flagellin.
infection of ht <dig> cells with wild-type s. typhimurium resulted in activation of mapks erk1& <dig>  the sapks p <dig>  jnk and ikk  as determined by use of activation-indicating phospho-specific antibodies in immunoblot  analysis or antibody-specific immuno-kinase assays  for jnk and ikk using their respective substrates gst-cjun 1– <dig> and gst-iκbα1– <dig>  <cit> .
interestingly, mapk stimulation is transient in nature as activation declines beginning at forty-five minutes while p <dig>  jnk and ikk activity increases with time through one hour.
as seen in fig.
<dig>  the flic-/fljb- double mutant salmonella also failed to induce ikk and nf-κb activity .
surprisingly, the flic-/fljb- double mutant salmonella failed to induce the sapks p <dig> and jnk and only briefly  activated mapk.
this result is puzzling since other salmonella proteins such as sope and sope <dig> can activate the small gtpases rac and cdc <dig>  and these rho family gtpases have been linked to jnk and p <dig> activation  <cit>  yet appear not to function in the flagellin minus strain.
the flic-/fljb- double mutant salmonella failed to invade ht <dig> cells compared to the wild-type salmonella strain as determined by gentamycin protection/invasion assay .
the flagellin flic-/fljb- double mutant displayed a four orders of magnitude difference in its ability to invade ht <dig> cells .
to demonstrate this point further, we infected ht <dig> cells with either wild-type salmonella or the flic-/fljb- double mutant salmonella , both strains were transformed with the plasmid pfm <dig>  that encodes gfp under the control of the salmonella ssah promoter and only functions once the bacteria has invaded the host cell  <cit> .
the wild-type salmonella clearly was able to infect ht <dig> cells  while the flagellin mutant bacteria failed to invade ht <dig> cells as evidenced by the lack of gfp expression .
to determine if flagellin is sufficient or that other bacterially produced proteins are required for invasion, we added either purified flagellin or sterile-filtered culture broths or a combination of both to ht <dig> cells that were challenged with the salmonella flic-/fljb- double mutant and assayed for invasion.
intestinal epithelial cells failed to be invaded using all tested combinations of purified flagellin and/or culture broths with the flic-/fljb- double mutant strain .
to our knowledge there is no known direct connection between expression of flagellin genes and the effectiveness of the type iii secretion system to deliver bacterially produced proteins such as sope, sope <dig> and sipa or other sip or sop proteins  <cit>  that play important roles in initiating bacterial internalization.
furthermore, to evaluate the effectiveness of flagellin to stimulate p <dig>  nuclear localization in intestinal epithelial cells we challenged ht <dig> cells with purified flagellin and examined p <dig>  localization using indirect immunofluorescence and found p <dig>  nuclear localization in nearly every cell .
purified flagellin  was added to the culture media of ht <dig> cells and wce were prepared at various times as indicated after exposure and assayed for nf-κb dna binding activity in emsas .
flagellin potently activated nf-κb in a time dependent manner similar to that observed for tnf  treatment of ht <dig> cells .
analysis of the mapk, sapk and ikk signaling pathways  at various times after flagellin treatment of ht <dig> cells using activation-specific phospho-antibodies to monitor mapk and p <dig> kinase activation or antibody-specific immunoprecipitation kinase assays for jnk and ikk activities demonstrated that jnk and ikk activity increased through time to one-hour while p <dig> and mapk  activity peaked at thirty minutes and began to decline to noticeably lower levels by one-hour .
the activation profile of the mapk, sapk and ikk signaling molecules erk1& <dig>  p <dig>  jnk and ikk in intestinal epithelial cells in response to purified flagellin exposure remarkably resembled that of intestinal epithelial cells infected with wild-type salmonella .
from these observations we conclude that the temporal activation of the signaling pathways examined here , which reflect early events in salmonella infection, are determined almost exclusively by recognition and response of intestinal epithelial cells to flagellin.
we wished to further examine the effect of purified flagellin and flagellin present on salmonella on the temporal pattern of proinflammatory cytokine gene expression in intestinal epithelial cells in order to differentiate the effects of flagellin alone vs. flagellated salmonella or non-flagellated salmonella infection.
ht <dig> cells were left untreated, stimulated with tnfα , or stimulated with flagellin , or infected with wild-type salmonella typhimurium or the salmonella flic/fljb double mutant .
after the indicated times after treatment or infection, ht <dig> cells were harvested in ice-cold pbs and the cell pellets lysed in trizol and rna was purified and used to prepare first-strand cdna .
aliquots of the cdna were used in semi-quantitative rt-pcr reactions using il1α, il-1β, il- <dig>  tnfα, mcp <dig> and β-actin gene specific primers  and the products were fractionated on ethidium bromide containing  <dig> % agarose gels.
expression of the known nf-κb target genes il-1β, il- <dig>  tnfα and mcp <dig> was increased in response to tnfα or purified flagellin exposure .
wild-type salmonella infection also led to activation of these same genes although the expression of tnfα and mcp <dig> was transient in comparison and occurred immediately after infection.
the salmonella flic-/fljb- double mutant failed to induce il-1β, il- <dig> and tnfα expression, however mcp <dig> expression was induced, although at lower levels than that induced by wild-type salmonella, and also, the expression of mcp <dig> was not transient in nature and continued throughout the time course  .
the expression level of β-actin served as an internal standard for comparison.
interestingly, il-1α, which is not an nf-κb target gene was stimulated in response to ht <dig> cell challenge by all of the treatments.
obviously, the salmonella flic-/fljb- double mutant can activate other signaling pathways leading to il-1α expression.
we presently do not know what these signaling pathways are.
flagellin activates nf-κb dna binding in a myd88-dependent manner
flagellin was capable of activating the requisite signaling pathways consistent with proinflammatory gene activation similar to that of a cytokine like tnfα that activates all cells on which a functional cell surface receptor for it is present  we decided to examine the potential of the toll-like receptors, to activate the nf-κb pathway in response to flagellin exposure.
to quickly test this hypothesis we examined the effect that a dominant-negative myd <dig>   <cit>  expressing adenovirus had on flagellin-mediated nf-κb activation in ht <dig> cells.
myd <dig> is an adapter protein utilized by the il- <dig> receptor and all of the known tlrs, which share homology to il- <dig> through their cytoplasmic signaling domain and is required for immediate activation of the nf-κb pathway  <cit> .
we found that expression of dn-myd <dig> in ht <dig> cells blocked the activation of nf-κb dna binding activity assayed by emsa analysis in response to il- <dig> or flagellin exposure, consistent with the action of a tlr-mediated activation of nf-κb .
to examine this possibility further we initially used wild-type, myd88-/- and tlr2-/-/tlr4-/- mefs  to verify the role of myd <dig> and to examine the potential role of two of the tlrs to respond to flagellin or to direct wild-type salmonella infection and lead to nf-κb activation .
wild-type salmonella infection activates nf-κb potently in both the wild-type and tlr deficient mefs  but this activation is somewhat defective in the myd <dig> deficient mefs .
challenge of all three types of cells with concentrated sterile-filtered wild-type s. dublin or the double sope-/sopb- isogenic mutant s. dublin strain se1sb <dig> culture broths activated nf-κb in wild-type mefs and tlr2/ <dig> double deficient cells but failed to activate nf-κb in myd <dig> deficient cells .
nf-κb was potently activated in wild-type mefs by exposure to purified flagellin  and therefore eliminated the possibility that lps played a role in nf-κb activation in these experiments.
the exclusion of lps as a major contributor to nf-κb activation is also provided by the potent activation of the tlr2/ <dig> double deficient mefs .
tlrs  <dig> and  <dig> respond to bacterial lipopeptides, peptidoglycans, certain lpss and gram negative lps respectively  <cit> .
il- <dig> stimulation verified the functional requirement of myd <dig> in transmission of il- <dig> and flagellin-mediated signals.
to further define a possible role for the tlrs in flagellin recognition we assayed for the ability of overexpressed tlrs to activate nf-κb in cells that normally respond poorly to flagellin exposure.
choosing cells that responded slightly to purified flagellin ensured that the signaling components and adapters that flagellin uses were present and functional and that the limiting factor was likely only to be the receptor that responds to flagellin.
we found that hela cells and hek293t cells activated nf-κb dna binding activity in response to il- <dig> stimulation but poorly to flagellin exposure   and we chose hek293t cells to use further because of their greater transfection efficiency.
amino-terminus flag epitope-tagged tlrs 1– <dig>   <cit>  were overexpressed in hek 293t cells in transient transfections along with the 2×-nf-κb-dependent promoter driven luciferase reporter gene  <cit>  and the expression of luciferase in response to no treatment, flagellin  or tnfα  was determined.
tlr <dig> was the only tlr whose expression resulted in a noticeable response to flagellin challenge of the cells .
293t cells were transfected with empty vector  or the individual listed wild-type tlr alleles in triplicate in 6-well dishes.
cells were left untreated , tnfα  or flagellin .
nf-κb reporter activity was adjusted by normalizing expression to control renilla luciferase activity and fold induction was calculated as reporter gene activity in treated cells/reporter gene activity in non-stimulated cells.
nd is not determined.
to further determine the likelihood of tlr <dig> being the tlr through which flagellin activated nf-κb, we constructed dominant-negative signaling mutations by deletion of the carboxyl portion of each tlr to a conserved tryptophan in the tir domain .
a similar mutation in the il- <dig> receptor abrogates its ability to lead to nf-κb activation  <cit> .
each dn-tlr along with a reverse cloned tlr <dig>  were cloned into the mammalian expression vector pcdna <dig>  .
all mutant proteins were expressed well .
each dn-tlr mammalian expression vector and empty expression vector along with 2× nf-κb luc was transfected as previously described  <cit>  into ht <dig> cells which respond very well to flagellin.
the transfected cells were left untreated, stimulated with tnfα  or with purified flagellin .
reporter gene expression was observed not to be affected by dn-tlr expression in response to tnfα stimulation of transfected cells  however, only expression of either the dn-tlr <dig> or an antisense tlr <dig> construct resulted in a nearly fifty percent and twenty-five percent inhibition of flagellin-mediated reporter gene activation respectively , while dn-tlr <dig> also was found to mildly inhibit flagellin-mediated reporter expression.
these results imply that tlr <dig> takes part in cell surface recognition of flagellin and initiates the signaling pathway leading to nf-κb activation.
the effect of dn-tlr <dig> on nf-κb-dependent reporter gene activation may be non-specific since its expression also inhibited tnfα-mediated reporter activation as compared to the other dn-tlrs.
dn-tlr <dig> may also compete for an unknown adapter protein that both tlr <dig> and tlr <dig> might share.
in any event, tlr <dig> and tlr <dig> were shown by the results presented in fig.
<dig> not to be required for flagellin-mediated activation of nf-κb.
flagellin-mediated activation of nf-κb in intestinal epithelial cells leads to increased and decreased expression of a subset of tlrs
stimulation of intestinal epithelial cells with s. typhimurium or with purified flagellin led to activation of the proinflammatory gene program .
we wished to examine whether or not expression of tlr genes would also be altered in flagellin stimulated cells.
ht <dig> cells were treated or not with purified flagellin  or with tnfα  and total rna was isolated from non-treated and treated cells three hours after stimulation and used to make first-strand cdna.
real-time rt-pcr using gene-specific primers for each of the tlrs  and first-strand cdna prepared from non-stimulated or flagellin stimulated cells was used to generate sybr-green  labeled dna products that were detected in an icycler™ .
interestingly, flagellin only mildly activated the expression of tlr <dig>  while expression levels of tlrs  <dig>   <dig>   <dig> and  <dig> were decreased by 2-fold .
contrastingly, tnf stimulation led to increased expression of tlrs  <dig> and  <dig> , while tlrs  <dig>   <dig> and  <dig> were decreased by approximately 2-, 5- and 3-fold respectively.
gapdh expression served as comparative standard.
nd none detected by rt2pcr
m none detected above level of minus rt control.
reverse transcription and real time pcr -rna was prepared from cells left untreated, stimulated with tnfα  or flagellin  for  <dig> h. rt2pcr was performed with an icycler  to quantify sybr-green labeled products generated from pcr products of 1st strand cdna prepared from tlr <dig> through tlr <dig> mrna, 18s rrna, and gapdh mrna.
rt2pcr  was performed with the appropriate primers  in triplicate with hotstart taq dna polymerase  at 95°c for  <dig> min to activate taq and amplified for  <dig> cycles .
rt2pcr was performed on the minus rt controls with tlr <dig> primers to detect dna contamination.
fold change in mrna expression was expressed as 2ΔΔct.
Δct is the difference in threshold cycles for the tlr mrnas and 18s rrna.
ΔΔct is the difference between Δct non-simulated control and Δct stimulated sample.
tlr <dig> is expressed in cells that don't respond well to flagellin
this study and others  <cit>  have identified tlr <dig> as the likely tlr through which flagellin activates nf-κb.
previous reports made no determination on the presence or abundance of tlr <dig> in the cells that they used to ascertain its function  <cit> .
we wished to determine if tlr <dig> protein abundance was absent or greatly decreased in cells that failed to respond or responded poorly to challenge by flagellin.
tlr <dig> abundance in a number of cell lines was examined by immunoblot analysis using a tlr5-specific antibody and compared with the ability of purified flagellin to induce nf-κb dna binding activity of wces prepared from them.
intestinal epithelial cell lines t <dig> and ht <dig> were used as was the lung adenocarcinoma cell line a <dig>  the human cervical adenocarcinoma cell line hela, the human embryonic kidney cell line expressing large t antigen hek293t, and the glioblastoma cell line t98g.
tlr <dig> protein was detected in all cell lines examined by immunoblot with tlr5-specific antibody .
t <dig> cells exhibited the highest abundance while expression levels of the other cell lines were similar and appeared not to differ by more than two-fold .
nf-κb dna binding activity in non-stimulated, tnfα and flagellin stimulated cells was analyzed by emsa assays of wces prepared from each cell type .
ht <dig> and a <dig> cells responded strongly to flagellin and to tnfα stimulation while hela, 293t and t98g cells responded poorly  or not at all  to flagellin stimulation.
the authenticity of the nf-κb dna binding complex was determined using p65-specific antibody to supershift the nf-κb dna:protein complex.
it is of interest that some cells that express tlr <dig> either do not respond at all or do so very poorly.
this may be due to either lack of receptor presence at the plasma membrane and intracellular localization, inactivating or detrimental mutations in the tlr <dig> gene in these cell lines or lack of or low abundance of a required co-receptor or adapter protein .
il- <dig> can activate nf-κb dna binding activity in all of the examined cell lines so it appears that the signaling apparatus downstream of myd <dig> to nf-κb is intact.
recently muc <dig> a secreted and membrane bound mucin protein was shown to serve as a receptor that bound pseudomonas aeruginosa and its flagellin, leading to activation of the mapk pathway  <cit>  although nf-κb activity was not examined.
we examined the muc <dig> abundance levels in ht <dig> , a <dig> , hela, 293t  and t98g  to determine if its expression correlated with the activation profile of nf-κb and mapk in these cells in response to flagellin  <cit> .
should this be the case then muc <dig> might serve as a viable co-receptor for tlr <dig> in propagating activation signals leading to nf-κb activation.
we observed that only ht <dig> cellular proteins gave a strong signal by immunoblot analysis using an muc1-specific antibody while muc <dig> was barely detectable in the other cell lines .
these results suggest that muc <dig> does not serve the role of a tlr <dig> co-receptor that leads to nf-κb activation and likely plays little to no role activating mapk pathways in a <dig> cells where we have observed similar temporal mapk activation in response to flagellin exposure as we do in ht <dig> cells .
further examination of muc1's role in ht <dig> cells in regards to nf-κb and mapk signaling using sirna is warranted.
discussion
intestinal epithelial cells at mucosal surfaces serve as innate immune sentinels controlling the innate host defense instruction to the immune effector cells inside the body in response to the external environment  <cit> .
previous studies examining pathogenic salmonella invasion of intestinal epithelial cells demonstrated activation of the proinflammatory gene program and invasion of only a minor portion of the cells  <cit> .
we previously demonstrated that nf-κb is as potently induced in pathogenic salmonella .sp infected cells similar to those treated with the proinflammatory cytokines that are potent nf-κb activators such as tnfα and il-1β and that this activity was ikk-mediated  <cit> .
here we examined how bacterial invasion of only a third of the cells could give rise to nf-κb activity profiles consistent with activation of nf-κb in every cell such as the profile tnfα stimulation provides.
we found that bacterial infection activates nuclear translocation of p <dig>  in nearly all of the intestinal epithelial cells consistent with the hypothesis that a cell surface receptor was recognizing either a soluble product that bacteria were producing, or a bacterial product on the bacteria, or both.
we examined bacterial culture broths and found a bacterial product that was protein in composition and when used to challenge intestinal epithelial cells it potently activated nf-κb dna binding activity .
we further purified this protein by gel permeation and anion exchange chromatography and found the protein to be flagellin by electrospray ion trap mass spectroscopy .
while our studies were in progress, flagellin was identified as being a potent proinflammatory mediator leading to il- <dig> production and secretion  <cit> .
we demonstrate in this study that flagellin appears to be exclusively responsible for activating nf-κb in intestinal epithelial cells since flagellin mutant strains do not activate nf-κb  nor lead to their internalization .
furthermore, flagellin challenge of intestinal epithelial cells leads to p <dig>  nuclear localization in nearly all of the treated cells .
transcription factors like activator protein  <dig>  and nf-κb, which are key regulators/activators of the proinflammatory gene program  <cit>  are activated by engagement of the mapk, sapk and ikk signaling pathways.
we demonstrate that the mapk, sapk and ikk signaling pathways activation fails to occur in host cells by infection/exposure to salmonella strains devoid of flagellin or products in the culture broths derived from those mutant salmonella strains .
we also demonstrate here that combined mutants of both flic and fljb exhibit a severe lack of invasion  and failure to activate stress response signaling, which has not been revealed previously.
it is likely that the lack of flagellin production interferes with the functioning of the type iii secretion system  although flagellin is not known to effect expression of ttss-required gene products.
this hypothesis seems credible since supply of flagellin or bacterial culture components from wild-type salmonella cultures in trans to the double flic-/fljb- mutant bacteria fails to compliment their lack of infectivity in gentamycin invsion assays .
in fact, we found the abundance of a subset of sip and sop proteins  released into the bacterial culture media to be drastically reduced in the flagellin mutant strains used here .
these two proteins have not previously been identified as activators of nf-κb nor are they considered as such here.
the ttss translocates the salmonella invasion proteins  and the sope proteins into the host cell initiating cytoskeletal rearrangements that ultimately lead to bacterial internalization,  <cit> .
in any event, it is clear that purified flagellin activates a similar cadre of proinflammatory genes as does infection of intestinal epithelial cells with wild-type flagellated salmonella.
the temporal expression pattern of these genes was found to be remarkably similar  indicating that flagellin-mediated temporal activation of the mapk, sapk and ikk signaling pathways can suffice for signaling pathways activated by sips or sope and sope <dig> and largely recapitulates the temporal activation of key proinflammatory genes as does infection of intestinal epithelial cells with wild-type flagellated salmonella.
the rapid, and potent activation of the mapk, sapk and ikk signaling pathways by flagellin was consistent with and indicative of the activation of a cell surface receptor.
in this study and in other studies tlr <dig> has been demonstrated to play an integral role in the recognition of flagellin leading to activation of nf-κb and expression of the il- <dig> gene   <cit> .
identification of tlr <dig> utilized transfection of tlrs  <dig> –  <dig> into cell lines which responded poorly to flagellin  or not at all  <cit>  and challenging the transfectants with flagellin to identify which tlr responded to this pamp.
previous studies that identified tlr <dig> as the receptor for flagellin did not examine the abundance of tlr <dig> in these cells or account for the lack of tlr5-mediated signaling in response to flagellin  <cit> .
we demonstrate here that cells which respond poorly  or not at all  contain tlr <dig> in at least equivalent abundance as ht <dig> cells which are highly responsive to flagellin.
we propose at least three possibilities to account for this discrepancy, first, this may be due to either lack of tlr <dig> receptor presence at the plasma membrane and intracellular localization; second, inactivating or detrimental mutations in the tlr <dig> gene in these cell lines; and lastly, lack of or low abundance of a required co-receptor or adapter protein required for either efficient ligand recognition and/or signaling.
these possibilities are currently being investigated.
we favor the last possibility since surface biotinylation experiments indicate that tlr <dig> is present on the cell surface in both flagellin responding cells and in non-responders mentioned above .
invocation of the second hypothesis would require inactivating mutations be present in three different cell lines, a highly improbable outcome.
how do the findings presented here correlate with events during a "normal" salmonella infection?
we have indicated in this study that defective type iii secretion system functioning leads to loss of host cell infectivity and underscores the importance of this system in the normal course of infection.
in the in vivo setting, polarized epithelial cells express tlr <dig> on the basolateral surface  <cit>  and flagellin can only reach the receptor either after either breaching the tight junction barrier by physical damage or by loosening of the junctions in response to sips and sops delivered into the intestinal epithelial cells by the ttss or by delivery of flagellin across the intestinal epithelial cell by the bacteria itself  <cit> .
this scenario would imply the main function of the type iii secretion system would be to trigger stress response signaling facilitating invasion and lead to loosening the tight junctions and result in flagellin/ flagellated bacteria to passing through the junctions and infected cells allowing access the basolateral surface and then systemic dispersion.
tlr <dig> on the basolateral surface of the intestinal epithelial cells, in response to flagellin, could then lead to activation of nf-κb and the proinflammatory gene program and host protection.
this model is consistent with activation of the proinflammatory gene program observed in response to flagellated salmonella sp.
infection in many reports too numerous to cite here and would allow the innate host defense system a fail-safe way to recognize pathogen exposure.
in instances where infection of intestinal epithelial cells by naturally occurring non-flagellated salmonella occurs, a strong proinflammatory response would not initially be presented but the salmonella would instead lead to systemic infection as is the case in chickens with s. galinarum and s. pollorum and result in typhoid-like disease  <cit> .
infection of chicken epithelial cells does not lead to proinflammatory gene expression by these non-flagellated pathogens but does when infected with s. typhimurium or s. dublin  <cit> .
argument for the existence of an additional tlr <dig> co-receptor/adapter being in limited abundance or absent might be in evidence from the transfection results presented in table  <dig> which demonstrated that overexpression of cell surface localized flag-tagged tlr <dig> only resulted in slightly over a two-fold increase in nf-κb reporter gene expression in response to flagellin.
if only tlr <dig> was required for activation of the signaling pathway should not a much more robust response been observed?
we have also used dn-tlr <dig> transfections and nf-κb-dependent reporter gene assays or overexpresssion of dn-tlr <dig> using recombinant adenoviruses and analysis of resulting nf-κb dna binding activity in response to flagellin to examine its effectiveness to completely inhibit tlr5-mediated flagellin activation of nf-κb.
we have found it difficult to gain more than a fifty-percent reduction in either reporter gene activation or nf-κb dna binding activity in ht <dig> cells .
these results suggest that the resting tlr <dig> signaling complex may be quite stable as has recently been suggested  <cit> .
should the endogenous tlr <dig> signaling complex be extremely stable it would therefore be expected that titration of a required pre-stimulus bound adapter or co-receptor away would be inefficient and this is what we have observed.
expression of a dn-mal , a myd88-related tlr adapter  <cit>  had little to no effect on flagellin-mediated nf-κb activity in transient transfection nf-κb reporter gene assays .
recently, tlr <dig> has been shown to bind flagellin  <cit>  and that this is likely a direct interaction due to failure of the human tlr <dig> to respond to a purified flagellin derived from a mouse-specific salmonella strain  <cit> .
these observations still do not preclude the existence of a co-receptor or adapter that is critical for signal transmission.
detailed biochemical characterization of the tlr <dig> signaling complex will resolve this issue.
muc <dig>  a recently described flagellin interacting membrane protein by virtue of its ability to trigger activation of the mapk pathway in response to flagellin exposure  <cit>  was considered a viable candidate for such a co-receptor but our observations suggest that it can not serve as the putative tlr <dig> co-receptor as it is expressed at similar levels in flagellin non-responding cell lines examined here as it is in a <dig> cells which respond strongly to flagellin and both cell line types express similar levels of tlr <dig> .
CONCLUSIONS
in conclusion, our data clearly demonstrates that flagellin can act as the major determinant in activating key stress response signaling pathways and proinflammatory gene program expression in a temporal and qualitative fashion as observed during infection of intestinal epithelial cells by wild-type salmonella sp.
that express flagellin, a point that was not well established until this study.
in addition, expression of the fli c gene appears to play an important role in the proper functioning of the ttss since mutants that fail to express fli c are defective in expressing a subset of sip proteins and fail to invade host cells.
flagellin added in trans cannot restore the ability of the fli c mutant bacteria to invade intestinal epithelial cells.
flagellin is "sensed" by tlr <dig> and in response propagates signaling pathways culminating in potent proinflammatory gene expression.
interestingly we found that tlr <dig> is expressed in weakly responding and also in some flagellin non-responding cells, 293t, hela and t98g cells respectively at levels similar to cells such as ht <dig> and a <dig> cells that respond strongly to flagellin and can be found on the cell surface, raising a strong possibility that productive tlr <dig> signaling may require an additional factor/adaptor other than those already known to be key in the il- <dig> signaling pathway, which shares extensive similarities to the tlrs signaling pathways.

 intestinal epithelial cells serve as a barrier between the luminal microflora and the body and as such are perfectly positioned to monitor the approach/invasion of pathogens. these intestinal epithelial cells serve as innate immune sentinels and monitor their environment and constantly give out innate host defense instruction to local immune effector cells. pathogens such as salmonella and other enteroinvasive pathogenic bacteria such as enteroinvasive e. coli, shigella and yersinia upon infection of iecs leads to the up-regulation of the expression of host genes, the products of which activate mucosal inflammatory and immune responses and alter epithelial cell functions. previously we and others demonstrated that ikk via nf-κb and the sapk signaling pathways via jun n-terminal kinase and p kinase were key regulators of the up-regulation of the proinflammatory gene program, with nf-κb appearing to be the most critical. typically salmonella infects thirty-forty percent of iecs in culture models of infection, however, we and others have found that salmonella infection activates nf-κb dna binding activity to levels equivalent to that of tnfα which activates nf-κb in all of the cells. previous studies examining nf-κb activation by salmonella in ht colonic intestinal epithelial cells, which serve as model colonic epithelial cells in culture, indicated that delivery of salmonella proteins into the host cell via its type iii secretion system, such as sope and sope the bacterially encoded exchange factors for the rho-family members rac and cdc result in exchange factor activation, cytoskeletal rearrangements and activation of the mapk, sapk and nf-κb signaling pathways. recent observations that utilized salmonella strains that were defective in invasion and delivery of invasion proteins by the ttss but not attachment indicated that additional factors other than those delivered by the ttss could lead to nf-κb activation. presently it is not clear what protein dictate the activation of key signaling pathways that lead to the temporal expression of the proinflammatory gene program, although the sope proteins have been given extreme attention recently. in searching for additional salmonella proteins that could activate the proinflammatory gene expression program, bacterial flagellin was recently found to be such a protein and had been shown previously to activate il- expression in monocytes. flagellin was found to activate nf-κb in polarized epithelial cells only when flagellin was present on their basolateral surface consistent with the idea that a cell surface receptor was present there and could recognize it. the toll-like receptors have been found to recognize pathogen associated molecular patterns reviewed in. tlr interacts with tlr and tlr to recognize bacterial lipopeptides and zymosan respectively. tlr recognizes lps only when associated with its co-receptor md and cd. recently, flagellin was demonstrated to be recognized by tlr and activate an innate host response. however, little was known or demonstrated about the endogenous levels of tlr in cells used in those studies and why those cells failed to respond to flagellin. here we have identified flagellin as the primary initiator and temporal regulator of not only the major signaling pathways activated during salmonella infection but also of key target genes of the proinflammatory gene program too. we have also found flagellin expression to be required for salmonella bacterial invasion. independently we found tlr recognizes flagellin but its signaling activity toward this pamp is consistent with either the aid of another flagellin-recognizing co-receptor or the use of another adapter protein, perhaps similar to myd that is absent or present at low levels in flagellin non-or low-responding cells.  salmonella infection leads to a minority of cells invaded but activates nf-κb in nearly all cells previously, we have noted that pathogenic salmonella sp. infection leads to potent ikk and nf-κb activation and activation of the proinflammatory gene program. previous studies suggest that about thirty-forty percent of the intestinal epithelial cells are infected during a typical salmonella infection in cultured intestinal epithelial cells. we wished to address the question of how bacterial infection of about thirty percent of the host cells could give rise to nf-κb dna binding activity equivalent to activation of nf-κb in nearly all of the host cells as tnfα treatment of the cells does. to examine this phenomenon in detail ht cells either mock-infected or infected at a moi of fifty for one-hour with wild-type s. typhimurium that had been transformed with the plasmid pfm, that encodes green fluorescent protein under the control of the salmonella ssah promoter and only functions once the bacteria has invaded the host cell. as can be seen in fig. 1a, gfp expression occurs in about thirty to forty percent of the cells. we next examined the localization of the nf-κb subunit p in non-treated, salmonella infected or tnfα stimulated cells and found that p was localized to the cytoplasm in non-treated cells whereas, in salmonella infected cells or in tnfα treated cells p had localized to the nucleus. these results demonstrate that salmonella infection activates nf-κb in virtually all of the cells even though only a minority of them become infected and is consistent with and aids in explanation of our previous results examining salmonella infection and nf-κb activation. soluble bacterial product identified as flagellin can activate nf-κb in intestinal epithelial cells since salmonella sp. infection of intestinal epithelial cells in culture led to only roughly thirty percent infection but activation of nf-κb in nearly all of the cells, we anticipated that nf-κb activation was in response to host cell recognition of bacteria structural components or products produced by the bacteria and not by the invasion process. invasion itself has been demonstrated not to be required for activation of the proinflamatory gene program as had previously been thought. to investigate this possibility sterile-filtered s. dublin culture broth left either untreated or boiled for twenty minutes was used to challenge ht intestinal epithelial cells and nf-κb dna binding activity was monitored by electromobility shift assays of whole cell extracts prepared forty-five minutes after exposure. potent activation of nf-κb in response to the broth under both conditions was observed indicating the activating factor was heat-stable and is not lps since ht cells are not responsive to lps. the native sterile-filtered concentrated broth was subsequently treated with dnase, rnase, proteinase k or crudely size fractionated on kda centricon filters. the variously treated broths were then used to challenge ht intestinal epithelial cells and wces were prepared after forty-five minutes and nf-κb dna binding activity was analyzed by emsa. direct infection of ht cells by s. typhimurium or exposure to the culture broths, as indicated, induced nf-κb dna binding activity, while the activity-inducing factor was found to be sensitive to protease digestion and was retained by a kda filter. to further determine the identity of the nf-κb inducing activity, sterile-filtered concentrated broth culture was fractionated by superose gel permeation chromatography and by anion exchange chromatography. aliquots of chromatography fractions were assayed for their ability to activate nf-κb in ht cells and analyzed by emsa. as can be seen from the coomassie blue stained gel increased nf-κb dna binding activity corresponded to the increased abundance of an approximately kda protein. anion exchange chromatography on poros hq matrix and elution of bound proteins with an increasing salt gradient as indicated demonstrated that nf-κb dna binding-inducing activity corresponded to chromatographic fractions containing an increased abundance of the kda protein. eluted fractions observed in fig. 2c were concentrated and fractionated on preparative 12% sds-page gels and bands corresponding to b1-b were cut from the gels and the proteins eluted, precipitated and renatured as described in experimental procedures and used to stimulate ht cells. whole cell extracts from these cells were assayed for nf-κb dna binding-inducing activity by emsa and only band corresponding to the kda protein was able to elicit nf-κb dna binding activity while buffer from the beginning or end of the salt gradient failed to activate nf-κb dna binding activity. proteins corresponding to protein bands b1-b and blank areas of the gel were further processed for peptide sequencing as described in experimental procedures. trypsin digestion of the protein corresponding to b and analysis by electrospray ion trap lc/ms identified the amino acid sequence of twenty-one peptides. flagellin was unambiguously identified as the protein consistent with inducing nf-κb dna binding activity. flagellin is required to activate nf-κb in intestinal epithelial cells to determine if flagellin was indeed the factor that was responsible for triggering activation of nf-κb after exposure of intestinal epithelial cells to direct bacterial infection or to filtered culture broths of pathogenic salmonella sp. we prepared infectious bacteria and boiled and filtered culture broths from the non-flagellated e. coli dh5α, pathogenic s. dublin strain an isogenic s. dublin sope- mutant, isogenic s. dublin sopb- mutant, isogenic s. dublin double sope-/sopb- mutant, s. typhimurium strain and isogenic s. typhimurium flic::tn insertion mutant and a s. typhimurium isogenic double mutant flic-/fljb- and were used to challenge ht cells. bacteria and culture broths were used to challenge ht intestinal epithelial cells and wce extracts were prepared after forty-five minutes and analyzed for nf-κb dna binding activity by emsa. salmonella strains could activate nf-κb, while salmonella strains failing to produce flagellin also failed to activate nf-κb. e. coli dh5α is non-flagellated and does not produce flagellin failed to activate nf-κb. we also noticed through numerous experiments that s. dublin direct infections always activated nf-κb to a greater extent than s. typhimurium as observed in fig. 4a while culture broths from both species activated nf-κb almost equally well. we believe this difference is due perhaps to s. dublin releasing more flagellin into the cell culture media than s. typhimurium during infection since purification of flagellin from both s. dublin and s. typhimurium and addition of equivalent amounts of chromatographically purified flagellin gave similar nf-κb activation profiles. of note is the total failure of the double flagellin gene mutants to activate nf-κb as compared to the very minor activation observed in the single phase i flagellin flic::tn insertion mutant which likely is due to the extremely limited expression of the phase ii flagellin, although the strains of salmonella used here genetically are unable or rarely shift phases of flagellin production. these results are consistent with previous reports identifying flagellin as a potent inducer of the proinflammatory response and il- production. since flagellin appears required for activation of the nf-κb pathway upon direct infection of intestinal epithelial cells it appeared possible that flagellin may also be the major determinant of other major mitogenic and stress activated signaling pathways activated upon pathogenic salmonella infection of intestinal epithelial cells. previously others and we have demonstrated that direct salmonella infection of intestinal epithelial cells results in jnk activation and also the activation of nf-κb via ikk. the identification of flagellin as a potent nf-κb activator is significant since sope had previously been shown to be a pathogenic salmonella bacteriophage encoded protein that is injected into the host cell and acts as an exchange factor for the small rho gtpases rac and cdc initiating cytoskeleton rearrangements and eventual activation of the mapk, sapk and nf-κb pathways, while sopb is a salmonella protein that functions as an inositol phosphate phosphatase and participates in cytoskeletal rearrangements and stimulates host cell chloride secretion. flagellin triggers activation of the mitogen activated protein kinase, stress activated protein kinase and ikk signaling pathways intestinal epithelial cells act as sentinels for invasion of luminal surfaces and orchestrate the attraction of effector immune cells to the area by production of chemokine genes like il- and macrophage chemoattractant protein proinflammatory cytokine genes such as tnfα, il- and il-. expression of these genes primarily depends upon the action of transcription factors that are activated in response to the transmission of signals via the mapk, sapk and ikk signaling pathways. since nf-κb is considered a central regulator/activator of the proinflammatory gene program we decided to examine the effect that non-flagellin producing mutant strains of salmonella had on activation of the mapk, sapk and ikk signaling pathways compared to infection of intestinal epithelial cells with wild-type salmonella or by exposure of the intestinal epithelial cells to purified flagellin. infection of ht cells with wild-type s. typhimurium resulted in activation of mapks erk1& the sapks p jnk and ikk as determined by use of activation-indicating phospho-specific antibodies in immunoblot analysis or antibody-specific immuno-kinase assays for jnk and ikk using their respective substrates gst-cjun 1– and gst-iκbα1–. interestingly, mapk stimulation is transient in nature as activation declines beginning at forty-five minutes while p jnk and ikk activity increases with time through one hour. as seen in fig. the flic-/fljb- double mutant salmonella also failed to induce ikk and nf-κb activity. surprisingly, the flic-/fljb- double mutant salmonella failed to induce the sapks p and jnk and only briefly activated mapk. this result is puzzling since other salmonella proteins such as sope and sope can activate the small gtpases rac and cdc and these rho family gtpases have been linked to jnk and p activation yet appear not to function in the flagellin minus strain. the flic-/fljb- double mutant salmonella failed to invade ht cells compared to the wild-type salmonella strain as determined by gentamycin protection/invasion assay. the flagellin flic-/fljb- double mutant displayed a four orders of magnitude difference in its ability to invade ht cells. to demonstrate this point further, we infected ht cells with either wild-type salmonella or the flic-/fljb- double mutant salmonella, both strains were transformed with the plasmid pfm that encodes gfp under the control of the salmonella ssah promoter and only functions once the bacteria has invaded the host cell. the wild-type salmonella clearly was able to infect ht cells while the flagellin mutant bacteria failed to invade ht cells as evidenced by the lack of gfp expression. to determine if flagellin is sufficient or that other bacterially produced proteins are required for invasion, we added either purified flagellin or sterile-filtered culture broths or a combination of both to ht cells that were challenged with the salmonella flic-/fljb- double mutant and assayed for invasion. intestinal epithelial cells failed to be invaded using all tested combinations of purified flagellin and/or culture broths with the flic-/fljb- double mutant strain. to our knowledge there is no known direct connection between expression of flagellin genes and the effectiveness of the type iii secretion system to deliver bacterially produced proteins such as sope, sope and sipa or other sip or sop proteins that play important roles in initiating bacterial internalization. furthermore, to evaluate the effectiveness of flagellin to stimulate p nuclear localization in intestinal epithelial cells we challenged ht cells with purified flagellin and examined p localization using indirect immunofluorescence and found p nuclear localization in nearly every cell. purified flagellin was added to the culture media of ht cells and wce were prepared at various times as indicated after exposure and assayed for nf-κb dna binding activity in emsas. flagellin potently activated nf-κb in a time dependent manner similar to that observed for tnf treatment of ht cells. analysis of the mapk, sapk and ikk signaling pathways at various times after flagellin treatment of ht cells using activation-specific phospho-antibodies to monitor mapk and p kinase activation or antibody-specific immunoprecipitation kinase assays for jnk and ikk activities demonstrated that jnk and ikk activity increased through time to one-hour while p and mapk activity peaked at thirty minutes and began to decline to noticeably lower levels by one-hour. the activation profile of the mapk, sapk and ikk signaling molecules erk1& p jnk and ikk in intestinal epithelial cells in response to purified flagellin exposure remarkably resembled that of intestinal epithelial cells infected with wild-type salmonella. from these observations we conclude that the temporal activation of the signaling pathways examined here, which reflect early events in salmonella infection, are determined almost exclusively by recognition and response of intestinal epithelial cells to flagellin. we wished to further examine the effect of purified flagellin and flagellin present on salmonella on the temporal pattern of proinflammatory cytokine gene expression in intestinal epithelial cells in order to differentiate the effects of flagellin alone vs. flagellated salmonella or non-flagellated salmonella infection. ht cells were left untreated, stimulated with tnfα, or stimulated with flagellin, or infected with wild-type salmonella typhimurium or the salmonella flic/fljb double mutant. after the indicated times after treatment or infection, ht cells were harvested in ice-cold pbs and the cell pellets lysed in trizol and rna was purified and used to prepare first-strand cdna. aliquots of the cdna were used in semi-quantitative rt-pcr reactions using il1α, il-1β, il- tnfα, mcp and β-actin gene specific primers and the products were fractionated on ethidium bromide containing % agarose gels. expression of the known nf-κb target genes il-1β, il- tnfα and mcp was increased in response to tnfα or purified flagellin exposure. wild-type salmonella infection also led to activation of these same genes although the expression of tnfα and mcp was transient in comparison and occurred immediately after infection. the salmonella flic-/fljb- double mutant failed to induce il-1β, il- and tnfα expression, however mcp expression was induced, although at lower levels than that induced by wild-type salmonella, and also, the expression of mcp was not transient in nature and continued throughout the time course. the expression level of β-actin served as an internal standard for comparison. interestingly, il-1α, which is not an nf-κb target gene was stimulated in response to ht cell challenge by all of the treatments. obviously, the salmonella flic-/fljb- double mutant can activate other signaling pathways leading to il-1α expression. we presently do not know what these signaling pathways are. flagellin activates nf-κb dna binding in a myd88-dependent manner flagellin was capable of activating the requisite signaling pathways consistent with proinflammatory gene activation similar to that of a cytokine like tnfα that activates all cells on which a functional cell surface receptor for it is present we decided to examine the potential of the toll-like receptors, to activate the nf-κb pathway in response to flagellin exposure. to quickly test this hypothesis we examined the effect that a dominant-negative myd expressing adenovirus had on flagellin-mediated nf-κb activation in ht cells. myd is an adapter protein utilized by the il- receptor and all of the known tlrs, which share homology to il- through their cytoplasmic signaling domain and is required for immediate activation of the nf-κb pathway. we found that expression of dn-myd in ht cells blocked the activation of nf-κb dna binding activity assayed by emsa analysis in response to il- or flagellin exposure, consistent with the action of a tlr-mediated activation of nf-κb. to examine this possibility further we initially used wild-type, myd88-/- and tlr2-/-/tlr4-/- mefs to verify the role of myd and to examine the potential role of two of the tlrs to respond to flagellin or to direct wild-type salmonella infection and lead to nf-κb activation. wild-type salmonella infection activates nf-κb potently in both the wild-type and tlr deficient mefs but this activation is somewhat defective in the myd deficient mefs. challenge of all three types of cells with concentrated sterile-filtered wild-type s. dublin or the double sope-/sopb- isogenic mutant s. dublin strain se1sb culture broths activated nf-κb in wild-type mefs and tlr2/ double deficient cells but failed to activate nf-κb in myd deficient cells. nf-κb was potently activated in wild-type mefs by exposure to purified flagellin and therefore eliminated the possibility that lps played a role in nf-κb activation in these experiments. the exclusion of lps as a major contributor to nf-κb activation is also provided by the potent activation of the tlr2/ double deficient mefs. tlrs and respond to bacterial lipopeptides, peptidoglycans, certain lpss and gram negative lps respectively. il- stimulation verified the functional requirement of myd in transmission of il- and flagellin-mediated signals. to further define a possible role for the tlrs in flagellin recognition we assayed for the ability of overexpressed tlrs to activate nf-κb in cells that normally respond poorly to flagellin exposure. choosing cells that responded slightly to purified flagellin ensured that the signaling components and adapters that flagellin uses were present and functional and that the limiting factor was likely only to be the receptor that responds to flagellin. we found that hela cells and hek293t cells activated nf-κb dna binding activity in response to il- stimulation but poorly to flagellin exposure and we chose hek293t cells to use further because of their greater transfection efficiency. amino-terminus flag epitope-tagged tlrs 1– were overexpressed in hek 293t cells in transient transfections along with the 2×-nf-κb-dependent promoter driven luciferase reporter gene and the expression of luciferase in response to no treatment, flagellin or tnfα was determined. tlr was the only tlr whose expression resulted in a noticeable response to flagellin challenge of the cells. 293t cells were transfected with empty vector or the individual listed wild-type tlr alleles in triplicate in 6-well dishes. cells were left untreated, tnfα or flagellin. nf-κb reporter activity was adjusted by normalizing expression to control renilla luciferase activity and fold induction was calculated as reporter gene activity in treated cells/reporter gene activity in non-stimulated cells. nd is not determined. to further determine the likelihood of tlr being the tlr through which flagellin activated nf-κb, we constructed dominant-negative signaling mutations by deletion of the carboxyl portion of each tlr to a conserved tryptophan in the tir domain. a similar mutation in the il- receptor abrogates its ability to lead to nf-κb activation. each dn-tlr along with a reverse cloned tlr were cloned into the mammalian expression vector pcdna. all mutant proteins were expressed well. each dn-tlr mammalian expression vector and empty expression vector along with 2× nf-κb luc was transfected as previously described into ht cells which respond very well to flagellin. the transfected cells were left untreated, stimulated with tnfα or with purified flagellin. reporter gene expression was observed not to be affected by dn-tlr expression in response to tnfα stimulation of transfected cells however, only expression of either the dn-tlr or an antisense tlr construct resulted in a nearly fifty percent and twenty-five percent inhibition of flagellin-mediated reporter gene activation respectively, while dn-tlr also was found to mildly inhibit flagellin-mediated reporter expression. these results imply that tlr takes part in cell surface recognition of flagellin and initiates the signaling pathway leading to nf-κb activation. the effect of dn-tlr on nf-κb-dependent reporter gene activation may be non-specific since its expression also inhibited tnfα-mediated reporter activation as compared to the other dn-tlrs. dn-tlr may also compete for an unknown adapter protein that both tlr and tlr might share. in any event, tlr and tlr were shown by the results presented in fig. not to be required for flagellin-mediated activation of nf-κb. flagellin-mediated activation of nf-κb in intestinal epithelial cells leads to increased and decreased expression of a subset of tlrs stimulation of intestinal epithelial cells with s. typhimurium or with purified flagellin led to activation of the proinflammatory gene program. we wished to examine whether or not expression of tlr genes would also be altered in flagellin stimulated cells. ht cells were treated or not with purified flagellin or with tnfα and total rna was isolated from non-treated and treated cells three hours after stimulation and used to make first-strand cdna. real-time rt-pcr using gene-specific primers for each of the tlrs and first-strand cdna prepared from non-stimulated or flagellin stimulated cells was used to generate sybr-green labeled dna products that were detected in an icycler™. interestingly, flagellin only mildly activated the expression of tlr while expression levels of tlrs and were decreased by 2-fold. contrastingly, tnf stimulation led to increased expression of tlrs and, while tlrs and were decreased by approximately 2-, 5- and 3-fold respectively. gapdh expression served as comparative standard. nd none detected by rt2pcr m none detected above level of minus rt control. reverse transcription and real time pcr -rna was prepared from cells left untreated, stimulated with tnfα or flagellin for h. rt2pcr was performed with an icycler to quantify sybr-green labeled products generated from pcr products of 1st strand cdna prepared from tlr through tlr mrna, 18s rrna, and gapdh mrna. rt2pcr was performed with the appropriate primers in triplicate with hotstart taq dna polymerase at 95°c for min to activate taq and amplified for cycles. rt2pcr was performed on the minus rt controls with tlr primers to detect dna contamination. fold change in mrna expression was expressed as 2ΔΔct. Δct is the difference in threshold cycles for the tlr mrnas and 18s rrna. ΔΔct is the difference between Δct non-simulated control and Δct stimulated sample. tlr is expressed in cells that don't respond well to flagellin this study and others have identified tlr as the likely tlr through which flagellin activates nf-κb. previous reports made no determination on the presence or abundance of tlr in the cells that they used to ascertain its function. we wished to determine if tlr protein abundance was absent or greatly decreased in cells that failed to respond or responded poorly to challenge by flagellin. tlr abundance in a number of cell lines was examined by immunoblot analysis using a tlr5-specific antibody and compared with the ability of purified flagellin to induce nf-κb dna binding activity of wces prepared from them. intestinal epithelial cell lines t and ht were used as was the lung adenocarcinoma cell line a the human cervical adenocarcinoma cell line hela, the human embryonic kidney cell line expressing large t antigen hek293t, and the glioblastoma cell line t98g. tlr protein was detected in all cell lines examined by immunoblot with tlr5-specific antibody. t cells exhibited the highest abundance while expression levels of the other cell lines were similar and appeared not to differ by more than two-fold. nf-κb dna binding activity in non-stimulated, tnfα and flagellin stimulated cells was analyzed by emsa assays of wces prepared from each cell type. ht and a cells responded strongly to flagellin and to tnfα stimulation while hela, 293t and t98g cells responded poorly or not at all to flagellin stimulation. the authenticity of the nf-κb dna binding complex was determined using p65-specific antibody to supershift the nf-κb dna:protein complex. it is of interest that some cells that express tlr either do not respond at all or do so very poorly. this may be due to either lack of receptor presence at the plasma membrane and intracellular localization, inactivating or detrimental mutations in the tlr gene in these cell lines or lack of or low abundance of a required co-receptor or adapter protein. il- can activate nf-κb dna binding activity in all of the examined cell lines so it appears that the signaling apparatus downstream of myd to nf-κb is intact. recently muc a secreted and membrane bound mucin protein was shown to serve as a receptor that bound pseudomonas aeruginosa and its flagellin, leading to activation of the mapk pathway although nf-κb activity was not examined. we examined the muc abundance levels in ht, a, hela, 293t and t98g to determine if its expression correlated with the activation profile of nf-κb and mapk in these cells in response to flagellin. should this be the case then muc might serve as a viable co-receptor for tlr in propagating activation signals leading to nf-κb activation. we observed that only ht cellular proteins gave a strong signal by immunoblot analysis using an muc1-specific antibody while muc was barely detectable in the other cell lines. these results suggest that muc does not serve the role of a tlr co-receptor that leads to nf-κb activation and likely plays little to no role activating mapk pathways in a cells where we have observed similar temporal mapk activation in response to flagellin exposure as we do in ht cells. further examination of muc1's role in ht cells in regards to nf-κb and mapk signaling using sirna is warranted. discussion intestinal epithelial cells at mucosal surfaces serve as innate immune sentinels controlling the innate host defense instruction to the immune effector cells inside the body in response to the external environment. previous studies examining pathogenic salmonella invasion of intestinal epithelial cells demonstrated activation of the proinflammatory gene program and invasion of only a minor portion of the cells. we previously demonstrated that nf-κb is as potently induced in pathogenic salmonella.sp infected cells similar to those treated with the proinflammatory cytokines that are potent nf-κb activators such as tnfα and il-1β and that this activity was ikk-mediated. here we examined how bacterial invasion of only a third of the cells could give rise to nf-κb activity profiles consistent with activation of nf-κb in every cell such as the profile tnfα stimulation provides. we found that bacterial infection activates nuclear translocation of p in nearly all of the intestinal epithelial cells consistent with the hypothesis that a cell surface receptor was recognizing either a soluble product that bacteria were producing, or a bacterial product on the bacteria, or both. we examined bacterial culture broths and found a bacterial product that was protein in composition and when used to challenge intestinal epithelial cells it potently activated nf-κb dna binding activity. we further purified this protein by gel permeation and anion exchange chromatography and found the protein to be flagellin by electrospray ion trap mass spectroscopy. while our studies were in progress, flagellin was identified as being a potent proinflammatory mediator leading to il- production and secretion. we demonstrate in this study that flagellin appears to be exclusively responsible for activating nf-κb in intestinal epithelial cells since flagellin mutant strains do not activate nf-κb nor lead to their internalization. furthermore, flagellin challenge of intestinal epithelial cells leads to p nuclear localization in nearly all of the treated cells. transcription factors like activator protein and nf-κb, which are key regulators/activators of the proinflammatory gene program are activated by engagement of the mapk, sapk and ikk signaling pathways. we demonstrate that the mapk, sapk and ikk signaling pathways activation fails to occur in host cells by infection/exposure to salmonella strains devoid of flagellin or products in the culture broths derived from those mutant salmonella strains. we also demonstrate here that combined mutants of both flic and fljb exhibit a severe lack of invasion and failure to activate stress response signaling, which has not been revealed previously. it is likely that the lack of flagellin production interferes with the functioning of the type iii secretion system although flagellin is not known to effect expression of ttss-required gene products. this hypothesis seems credible since supply of flagellin or bacterial culture components from wild-type salmonella cultures in trans to the double flic-/fljb- mutant bacteria fails to compliment their lack of infectivity in gentamycin invsion assays. in fact, we found the abundance of a subset of sip and sop proteins released into the bacterial culture media to be drastically reduced in the flagellin mutant strains used here. these two proteins have not previously been identified as activators of nf-κb nor are they considered as such here. the ttss translocates the salmonella invasion proteins and the sope proteins into the host cell initiating cytoskeletal rearrangements that ultimately lead to bacterial internalization,. in any event, it is clear that purified flagellin activates a similar cadre of proinflammatory genes as does infection of intestinal epithelial cells with wild-type flagellated salmonella. the temporal expression pattern of these genes was found to be remarkably similar indicating that flagellin-mediated temporal activation of the mapk, sapk and ikk signaling pathways can suffice for signaling pathways activated by sips or sope and sope and largely recapitulates the temporal activation of key proinflammatory genes as does infection of intestinal epithelial cells with wild-type flagellated salmonella. the rapid, and potent activation of the mapk, sapk and ikk signaling pathways by flagellin was consistent with and indicative of the activation of a cell surface receptor. in this study and in other studies tlr has been demonstrated to play an integral role in the recognition of flagellin leading to activation of nf-κb and expression of the il- gene. identification of tlr utilized transfection of tlrs – into cell lines which responded poorly to flagellin or not at all and challenging the transfectants with flagellin to identify which tlr responded to this pamp. previous studies that identified tlr as the receptor for flagellin did not examine the abundance of tlr in these cells or account for the lack of tlr5-mediated signaling in response to flagellin. we demonstrate here that cells which respond poorly or not at all contain tlr in at least equivalent abundance as ht cells which are highly responsive to flagellin. we propose at least three possibilities to account for this discrepancy, first, this may be due to either lack of tlr receptor presence at the plasma membrane and intracellular localization; second, inactivating or detrimental mutations in the tlr gene in these cell lines; and lastly, lack of or low abundance of a required co-receptor or adapter protein required for either efficient ligand recognition and/or signaling. these possibilities are currently being investigated. we favor the last possibility since surface biotinylation experiments indicate that tlr is present on the cell surface in both flagellin responding cells and in non-responders mentioned above. invocation of the second hypothesis would require inactivating mutations be present in three different cell lines, a highly improbable outcome. how do the findings presented here correlate with events during a "normal" salmonella infection? we have indicated in this study that defective type iii secretion system functioning leads to loss of host cell infectivity and underscores the importance of this system in the normal course of infection. in the in vivo setting, polarized epithelial cells express tlr on the basolateral surface and flagellin can only reach the receptor either after either breaching the tight junction barrier by physical damage or by loosening of the junctions in response to sips and sops delivered into the intestinal epithelial cells by the ttss or by delivery of flagellin across the intestinal epithelial cell by the bacteria itself. this scenario would imply the main function of the type iii secretion system would be to trigger stress response signaling facilitating invasion and lead to loosening the tight junctions and result in flagellin/ flagellated bacteria to passing through the junctions and infected cells allowing access the basolateral surface and then systemic dispersion. tlr on the basolateral surface of the intestinal epithelial cells, in response to flagellin, could then lead to activation of nf-κb and the proinflammatory gene program and host protection. this model is consistent with activation of the proinflammatory gene program observed in response to flagellated salmonella sp. infection in many reports too numerous to cite here and would allow the innate host defense system a fail-safe way to recognize pathogen exposure. in instances where infection of intestinal epithelial cells by naturally occurring non-flagellated salmonella occurs, a strong proinflammatory response would not initially be presented but the salmonella would instead lead to systemic infection as is the case in chickens with s. galinarum and s. pollorum and result in typhoid-like disease. infection of chicken epithelial cells does not lead to proinflammatory gene expression by these non-flagellated pathogens but does when infected with s. typhimurium or s. dublin. argument for the existence of an additional tlr co-receptor/adapter being in limited abundance or absent might be in evidence from the transfection results presented in table which demonstrated that overexpression of cell surface localized flag-tagged tlr only resulted in slightly over a two-fold increase in nf-κb reporter gene expression in response to flagellin. if only tlr was required for activation of the signaling pathway should not a much more robust response been observed? we have also used dn-tlr transfections and nf-κb-dependent reporter gene assays or overexpresssion of dn-tlr using recombinant adenoviruses and analysis of resulting nf-κb dna binding activity in response to flagellin to examine its effectiveness to completely inhibit tlr5-mediated flagellin activation of nf-κb. we have found it difficult to gain more than a fifty-percent reduction in either reporter gene activation or nf-κb dna binding activity in ht cells. these results suggest that the resting tlr signaling complex may be quite stable as has recently been suggested. should the endogenous tlr signaling complex be extremely stable it would therefore be expected that titration of a required pre-stimulus bound adapter or co-receptor away would be inefficient and this is what we have observed. expression of a dn-mal, a myd88-related tlr adapter had little to no effect on flagellin-mediated nf-κb activity in transient transfection nf-κb reporter gene assays. recently, tlr has been shown to bind flagellin and that this is likely a direct interaction due to failure of the human tlr to respond to a purified flagellin derived from a mouse-specific salmonella strain. these observations still do not preclude the existence of a co-receptor or adapter that is critical for signal transmission. detailed biochemical characterization of the tlr signaling complex will resolve this issue. muc a recently described flagellin interacting membrane protein by virtue of its ability to trigger activation of the mapk pathway in response to flagellin exposure was considered a viable candidate for such a co-receptor but our observations suggest that it can not serve as the putative tlr co-receptor as it is expressed at similar levels in flagellin non-responding cell lines examined here as it is in a cells which respond strongly to flagellin and both cell line types express similar levels of tlr.  in conclusion, our data clearly demonstrates that flagellin can act as the major determinant in activating key stress response signaling pathways and proinflammatory gene program expression in a temporal and qualitative fashion as observed during infection of intestinal epithelial cells by wild-type salmonella sp. that express flagellin, a point that was not well established until this study. in addition, expression of the fli c gene appears to play an important role in the proper functioning of the ttss since mutants that fail to express fli c are defective in expressing a subset of sip proteins and fail to invade host cells. flagellin added in trans cannot restore the ability of the fli c mutant bacteria to invade intestinal epithelial cells. flagellin is "sensed" by tlr and in response propagates signaling pathways culminating in potent proinflammatory gene expression. interestingly we found that tlr is expressed in weakly responding and also in some flagellin non-responding cells, 293t, hela and t98g cells respectively at levels similar to cells such as ht and a cells that respond strongly to flagellin and can be found on the cell surface, raising a strong possibility that productive tlr signaling may require an additional factor/adaptor other than those already known to be key in the il- signaling pathway, which shares extensive similarities to the tlrs signaling pathways. 
in addition, expression of the fli c gene appears to play an important role in the proper functioning of the ttss since mutants that fail to express fli c are defective in expressing a subset of sip proteins and fail to invade host cells. infection of intestinal epithelial cells by pathogenic salmonella leads to activation of signaling cascades that ultimately initiate the proinflammatory gene program. we explored the mechanism by which salmonella activates nf-κb during infection of cultured intestinal epithelial cells and found that flagellin produced by the bacteria and contained on them leads to nf-κb activation in all the cells; invasion of cells by the bacteria is not required to activate nf-κb. purified flagellin activated the mitogen activated protein kinase, stress-activated protein kinase and ikappa b kinase signaling pathways that lead to expression of the proinflammatory gene program in a temporal fashion nearly identical to that of infection of intestinal epithelial cells by salmonella. flagellin added in trans cannot restore the ability of the fli c mutant bacteria to invade intestinal epithelial cells. these collective results provide the evidence that flagellin acts as the main determinant of salmonella mediated nf-κb and proinflammatory signaling and gene activation by this flagellated pathogen. lastly, tlr expression in weak and non-responding cells indicates that additional factors may be required for efficient signal propagation in response to flagellin recognition. conversely, overexpression of dominant-negative tlr alleles only partially blocks nf-κb activation by flagellin. flagellin expression was required for salmonella invasion of host cells and it activated nf-κb via toll-like receptor. surprisingly, a number of cell lines found to be unresponsive to flagellin express tlr and expression of exogenous tlr in these cells induces nf-κb activity in response to flagellin challenge although not robustly.
 infection of intestinal epithelial cells by pathogenic salmonella leads to activation of signaling cascades that ultimately initiate the proinflammatory gene program. the transcription factor nf-κb is a key regulator/activator of this gene program and is potently activated. we explored the mechanism by which salmonella activates nf-κb during infection of cultured intestinal epithelial cells and found that flagellin produced by the bacteria and contained on them leads to nf-κb activation in all the cells; invasion of cells by the bacteria is not required to activate nf-κb.  purified flagellin activated the mitogen activated protein kinase, stress-activated protein kinase and ikappa b kinase signaling pathways that lead to expression of the proinflammatory gene program in a temporal fashion nearly identical to that of infection of intestinal epithelial cells by salmonella. flagellin expression was required for salmonella invasion of host cells and it activated nf-κb via toll-like receptor. surprisingly, a number of cell lines found to be unresponsive to flagellin express tlr and expression of exogenous tlr in these cells induces nf-κb activity in response to flagellin challenge although not robustly. conversely, overexpression of dominant-negative tlr alleles only partially blocks nf-κb activation by flagellin. these observations are consistent with the possibility of either a very stable tlr signaling complex, the existence of a low abundance flagellin co-receptor or required adapter, or both.  these collective results provide the evidence that flagellin acts as the main determinant of salmonella mediated nf-κb and proinflammatory signaling and gene activation by this flagellated pathogen. in addition, expression of the fli c gene appears to play an important role in the proper functioning of the ttss since mutants that fail to express fli c are defective in expressing a subset of sip proteins and fail to invade host cells. flagellin added in trans cannot restore the ability of the fli c mutant bacteria to invade intestinal epithelial cells. lastly, tlr expression in weak and non-responding cells indicates that additional factors may be required for efficient signal propagation in response to flagellin recognition. 