Previously, we have noted that pathogenic Salmonella sp. infection leads to potent IKK and NF-κB activation and activation of the proinflammatory gene program 3. 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 10. 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 HT29 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 pFM10.1, that encodes green fluorescent protein (GFP) under the control of the Salmonella ssaH promoter and only functions once the bacteria has invaded the host cell 34. 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 p65 (RelA) in non-treated (mock-infected), Salmonella infected or TNFα (10 ng/ml) stimulated cells and found that p65 (RelA) was localized to the cytoplasm in non-treated cells whereas, in Salmonella infected cells or in TNFα treated cells p65 (RelA) had localized to the nucleus (Fig. 1B). 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 3.

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 16. To investigate this possibility sterile-filtered S. dublin culture broth left either untreated or boiled for twenty minutes was used to challenge HT29 intestinal epithelial cells and NF-κB DNA binding activity was monitored by electromobility shift assays (EMSAs) of whole cell extracts (WCE) prepared forty-five minutes after exposure 335. Potent activation of NF-κB in response to the broth under both conditions was observed indicating the activating factor was heat-stable (AD, TT and JD, personal observations) and is not LPS since HT29 cells are not responsive to LPS 335.

The native sterile-filtered concentrated broth was subsequently treated with DNase, RNase, proteinase K or crudely size fractionated on 100 kDa centricon filters. The variously treated broths were then used to challenge HT29 intestinal epithelial cells and WCEs were prepared after forty-five minutes and NF-κB DNA binding activity was analyzed by EMSA (Fig 2A). Direct infection of HT29 cells by S. typhimurium 1103 or exposure to the culture broths (supt), 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 100 kDa filter (Fig. 2A). To further determine the identity of the NF-κB inducing activity, sterile-filtered concentrated broth culture was fractionated by Superose 12 gel permeation chromatography (Fig. 2B) and by anion exchange chromatography (Fig. 2C). Aliquots of chromatography fractions were assayed for their ability to activate NF-κB in HT29 cells and analyzed by EMSA. As can be seen from the Coomassie blue stained gel (Fig. 2B, top panel) increased NF-κB DNA binding activity (Fig. 2B, lower panel lanes 4–6) corresponded to the increased abundance of an approximately 55 kDa protein. Anion exchange chromatography on POROS HQ matrix and elution of bound proteins with an increasing salt gradient as indicated (Fig. 2C) demonstrated that NF-κB DNA binding-inducing activity corresponded to chromatographic fractions containing an increased abundance of the 55 kDa protein (Fig. 2C top panel, and data not shown). Eluted fractions observed in Fig. 2C were concentrated and fractionated on preparative 12% SDS-PAGE gels and bands corresponding to B1-B6 were cut from the gels and the proteins eluted, precipitated and renatured as described in Experimental Procedures and used to stimulate HT29 cells. Whole cell extracts from these cells were assayed for NF-κB DNA binding-inducing activity by EMSA and only band 2 (B2) corresponding to the 55 kDa protein (Fig. 2C lower panel) 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-B6 and blank areas of the gel were further processed for peptide sequencing as described in Experimental Procedures. Trypsin digestion of the protein corresponding to B2 and analysis by electrospray ion trap LC/MS identified the amino acid sequence of twenty-one peptides. Flagellin (seventy-five percent coverage by the twenty-one peptides) was unambiguously identified as the protein consistent with inducing NF-κB DNA binding activity (Fig 3).

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 2229, an isogenic S. dublin 2229 SopE^- mutant, isogenic S. dublin 2229 SopB^- mutant, isogenic S. dublin 2229 double SopE^-/SopB^- mutant (strain SE1SB2), S. typhimurium strain 1103, and isogenic S. typhimurium fliC^::Tn10 insertion mutant (strain 86) and a S. typhimurium 1103 isogenic double mutant fliC^-/fljB^- and were used to challenge HT29 cells. Bacteria and culture broths were used to challenge HT29 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 (fliC and fliC^-/fljB^- mutants as indicated) also failed to activate NF-κB (Fig. 4A &4B). 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 (Fig. 4B). 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 (TT & JD, unpublished observations).

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::Tn10 insertion mutant (next to last lanes in Fig. 4A &4B) which likely is due to the extremely limited expression of the phase II flagellin (from fljB), 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-8 production 16171819. 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 8 and also the activation of NF-κB via IKK 3. 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 Rac1 and CdC42 initiating cytoskeleton rearrangements and eventual activation of the MAPK, SAPK and NF-κB pathways 715, while SopB is a Salmonella protein that functions as an inositol phosphate phosphatase and participates in cytoskeletal rearrangements and stimulates host cell chloride secretion 36.

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-8 and macrophage chemoattractant protein 1 (MCP1) proinflammatory cytokine genes such as TNFα, IL-1 and IL-6 1456. 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 HT29 cells with wild-type S. typhimurium resulted in activation of MAPKs ERK1&2, the SAPKs p38, JNK and IKK (Fig. 5) as determined by use of activation-indicating phospho-specific antibodies in immunoblot (IB) analysis or antibody-specific immuno-kinase assays (KA) for JNK and IKK using their respective substrates GST-cJun 1–79 and GST-IκBα1–54 373839. Interestingly, MAPK stimulation is transient in nature as activation declines beginning at forty-five minutes while p38, JNK and IKK activity increases with time through one hour. As seen in Fig. 4, the fliC^-/fljB^- double mutant Salmonella also failed to induce IKK and NF-κB activity (Fig. 5 as indicated). Surprisingly, the fliC^-/fljB^- double mutant Salmonella failed to induce the SAPKs p38 and JNK and only briefly (fifteen minutes) activated MAPK. This result is puzzling since other Salmonella proteins such as SopE and SopE2 can activate the small GTPases Rac and CdC42, and these Rho family GTPases have been linked to JNK and p38 activation 781415 yet appear not to function in the flagellin minus strain.

The fliC^-/fljB^- double mutant Salmonella failed to invade HT29 cells compared to the wild-type Salmonella strain as determined by gentamycin protection/invasion assay (see Experimental Procedures). The flagellin fliC^-/fljB^- double mutant displayed a four orders of magnitude difference in its ability to invade HT29 cells (TT & JD, unpublished observations). To demonstrate this point further, we infected HT29 cells with either wild-type Salmonella or the fliC^-/fljB^- double mutant Salmonella (strain 134), both strains were transformed with the plasmid pFM10.1 that encodes GFP under the control of the Salmonella ssaH promoter and only functions once the bacteria has invaded the host cell 1034. The wild-type Salmonella clearly was able to infect HT29 cells (GFP, Fig. 5B) while the flagellin mutant bacteria failed to invade HT29 cells as evidenced by the lack of GFP expression (Fig. 5B). 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 HT29 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 (TT & JD, unpublished observations). 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, SopE2 and SipA or other Sip or Sop proteins 714154041 that play important roles in initiating bacterial internalization. Furthermore, to evaluate the effectiveness of flagellin to stimulate p65 (RelA) nuclear localization in intestinal epithelial cells we challenged HT29 cells with purified flagellin and examined p65 (RelA) localization using indirect immunofluorescence and found p65 (RelA) nuclear localization in nearly every cell (Fig. 5B as indicated).

Purified flagellin (0.5 μg/ml) was added to the culture media of HT29 cells and WCE were prepared at various times as indicated after exposure and assayed for NF-κB DNA binding activity in EMSAs (Fig. 6A). Flagellin potently activated NF-κB in a time dependent manner similar to that observed for TNF (10 ng/ml) treatment of HT29 cells (Fig. 6A). Analysis of the MAPK, SAPK and IKK signaling pathways (Fig. 6B) at various times after flagellin treatment of HT29 cells using activation-specific phospho-antibodies to monitor MAPK and p38 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 p38 and MAPK (ERK1&2) activity peaked at thirty minutes and began to decline to noticeably lower levels by one-hour (Fig 6B as indicated). The activation profile of the MAPK, SAPK and IKK signaling molecules ERK1&2, p38, 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 (Fig. 5A). From these observations we conclude that the temporal activation of the signaling pathways examined here (MAPK, SAPK and IKK), 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. HT29 cells were left untreated, stimulated with TNFα (10 ng/ml), or stimulated with flagellin (0.5 ug/ml), or infected with wild-type Salmonella typhimurium or the Salmonella fliC/fljB double mutant (at MOI of 50). After the indicated times after treatment or infection, HT29 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 (see Experimental Procedures). Aliquots of the cDNA were used in semi-quantitative RT-PCR reactions using IL1α, IL-1β, IL-8, TNFα, MCP1 and β-actin gene specific primers (sequences available upon request) and the products were fractionated on ethidium bromide containing 1.2% agarose gels. Expression of the known NF-κB target genes IL-1β, IL-8, TNFα and MCP1 was increased in response to TNFα or purified flagellin exposure (Fig. 6C). Wild-type Salmonella infection also led to activation of these same genes although the expression of TNFα and MCP1 was transient in comparison and occurred immediately after infection. The Salmonella fliC^-/fljB^- double mutant failed to induce IL-1β, IL-8 and TNFα expression, however MCP1 expression was induced, although at lower levels than that induced by wild-type Salmonella, and also, the expression of MCP1 was not transient in nature and continued throughout the time course (9 h) (Fig. 6C). 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 HT29 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 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 (see p65 [RelA] nuclear localization in Fig. 1 and Fig. 5C) 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 MyD88 (aa 152–296) 42 expressing adenovirus had on flagellin-mediated NF-κB activation in HT29 cells. MyD88 is an adapter protein utilized by the IL-1 receptor and all of the known TLRs, which share homology to IL-1 through their cytoplasmic signaling domain and is required for immediate activation of the NF-κB pathway 4344. We found that expression of DN-MyD88 in HT29 cells blocked the activation of NF-κB DNA binding activity assayed by EMSA analysis in response to IL-1 or flagellin exposure, consistent with the action of a TLR-mediated activation of NF-κB (TT & JD, unpublished observations). To examine this possibility further we initially used wild-type, MyD88^-/- and TLR2^-/-/TLR4^-/- MEFs (a gift of S. Akira, Univ. of Osaka, JA) to verify the role of MyD88 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 (Fig. 7). Wild-type Salmonella infection activates NF-κB potently in both the wild-type and TLR deficient MEFs (lanes 2 & 15) but this activation is somewhat defective in the MyD88 deficient MEFs (lane 10). 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 SE1SB2 culture broths activated NF-κB in wild-type MEFs and TLR2/4 double deficient cells but failed to activate NF-κB in MyD88 deficient cells (compare lanes 11 and 12 with lanes 3, 4, 6, 7, 16 and 17). NF-κB was potently activated in wild-type MEFs by exposure to purified flagellin (0.5 μg/ml) 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 TLR2/4 double deficient MEFs (lanes 16 & 17). TLRs 2 and 4 respond to bacterial lipopeptides, peptidoglycans, certain LPSs and gram negative LPS respectively 454647. IL-1 stimulation verified the functional requirement of MyD88 in transmission of IL-1 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-1 stimulation but poorly to flagellin exposure (TT & JD, unpublished observations) (but see Fig. 9B) and we chose HEK293T cells to use further because of their greater transfection efficiency. Amino-terminus FLAG epitope-tagged TLRs 1–9 (kind gifts of R. Medzhitov, Yale Univ. and R. Ulevitch, TSRI) 4849 were overexpressed in HEK 293T cells in transient transfections along with the 2×-NF-κB-dependent promoter driven luciferase reporter gene 50 and the expression of luciferase in response to no treatment, flagellin (0.5 μg/ml) or TNFα (10 ng/ml) was determined. TLR5 was the only TLR whose expression resulted in a noticeable response to flagellin challenge of the cells (Table 1).

To further determine the likelihood of TLR5 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 (see Materials and Methods). A similar mutation in the IL-1 receptor abrogates its ability to lead to NF-κB activation 5152. Each DN-TLR along with a reverse cloned TLR5 (AS-TLR5) were cloned into the mammalian expression vector pCDNA3.1 (Invitrogen, Carlsbad, CA). All mutant proteins were expressed well (TT & JD unpublished observations). Each DN-TLR mammalian expression vector and empty expression vector along with 2× NF-κB Luc was transfected as previously described 3 into HT29 cells which respond very well to flagellin. The transfected cells were left untreated, stimulated with TNFα (10 ng/ml) or with purified flagellin (0.5 μg/ml). Reporter gene expression was observed not to be affected by DN-TLR expression in response to TNFα stimulation of transfected cells (Fig. 8A) however, only expression of either the DN-TLR5 or an antisense TLR5 construct resulted in a nearly fifty percent and twenty-five percent inhibition of flagellin-mediated reporter gene activation respectively (Fig. 8B), while DN-TLR2 also was found to mildly inhibit flagellin-mediated reporter expression. These results imply that TLR5 takes part in cell surface recognition of flagellin and initiates the signaling pathway leading to NF-κB activation. The effect of DN-TLR2 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-TLR2 may also compete for an unknown adapter protein that both TLR2 and TLR5 might share. In any event, TLR2 and TLR4 were shown by the results presented in Fig. 7 not to be required for flagellin-mediated activation of NF-κB.

Stimulation of intestinal epithelial cells with S. typhimurium or with purified flagellin led to activation of the proinflammatory gene program (Fig. 6C). We wished to examine whether or not expression of TLR genes would also be altered in flagellin stimulated cells. HT29 cells were treated or not with purified flagellin (0.5 μg/ml) or with TNFα (10 μg/ml) 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 (Superarray, Frederick, MD) and first-strand cDNA prepared from non-stimulated or flagellin stimulated cells was used to generate SYBR-green (Perkin-Elmer) labeled DNA products that were detected in an iCycler™ (Bio-Rad). Interestingly, flagellin only mildly activated the expression of TLR2, while expression levels of TLRs 5, 6, 9 and 10 were decreased by 2-fold (Table 2). Contrastingly, TNF stimulation led to increased expression of TLRs 3 and 4 (1.6- and 3.5-fold respectively), while TLRs 2, 9 and 10 were decreased by approximately 2-, 5- and 3-fold respectively. GAPDH expression served as comparative standard.

This study and others 2233 have identified TLR5 as the likely TLR through which flagellin activates NF-κB. Previous reports made no determination on the presence or abundance of TLR5 in the cells that they used to ascertain its function 2233. We wished to determine if TLR5 protein abundance was absent or greatly decreased in cells that failed to respond or responded poorly to challenge by flagellin. TLR5 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 T84 and HT29 were used as was the lung adenocarcinoma cell line A549, the human cervical adenocarcinoma cell line HeLa, the human embryonic kidney cell line expressing large T antigen HEK293T, and the glioblastoma cell line T98G. TLR5 protein was detected in all cell lines examined by immunoblot with TLR5-specific antibody (Fig. 9A). T84 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 (Fig. 9A). 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 (Fig 9B). HT29 and A549 cells responded strongly to flagellin and to TNFα stimulation while HeLa, 293T and T98G cells responded poorly (HeLa, 293T) or not at all (T98G) 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 TLR5 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 TLR5 gene in these cell lines or lack of or low abundance of a required co-receptor or adapter protein (as is the case in some cells for TLR4 and its co-receptor/adapter MD2 305354). IL-1 can activate NF-κB DNA binding activity in all of the examined cell lines so it appears that the signaling apparatus downstream of MyD88 to NF-κB is intact.

Recently Muc1 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 5556 although NF-κB activity was not examined. We examined the muc1 abundance levels in HT29 (strong flagellin responder), A549 (strong flagellin responder), HeLa, 293T (both weak flagellin responders) and T98G (no flagellin response) to determine if its expression correlated with the activation profile of NF-κB and MAPK in these cells in response to flagellin 55. Should this be the case then muc1 might serve as a viable co-receptor for TLR5 in propagating activation signals leading to NF-κB activation. We observed that only HT29 cellular proteins gave a strong signal by immunoblot analysis using an muc1-specific antibody while muc1 was barely detectable in the other cell lines (Fig. 9C). These results suggest that muc1 does not serve the role of a TLR5 co-receptor that leads to NF-κB activation and likely plays little to no role activating MAPK pathways in A549 cells where we have observed similar temporal MAPK activation in response to flagellin exposure as we do in HT29 cells (TT and JD, unpublished results). Further examination of muc1's role in HT29 cells in regards to NF-κB and MAPK signaling using siRNA is warranted.

Human tumor necrosis factor alpha (TNFα) and human IL-1 were purchased from R&D Systems (Minneapolis, MN). Tris [hydroxymethyl]aminomethane (Tris) was purchased from Fisher Scientific (Fairlawn, NJ). Fetal calf serum was purchased from US Biotechnologies Inc. (Parkerford, PA). Para-nitro-phenylphosphate (PNPP) was purchased from Aldrich Chemical (Milwaukee, WI). The Polyacrylamide gel electrophoresis (PAGE) supplies: acrylamide, bis-acrylamide, sodium dodecyl sulfate (SDS), TEMED, and ammonium persulfate were purchased from Bio-Rad Laboratories (Hercules, CA). Dulbecco's modified essential medium (DMEM), DMEM:F12, phosphate buffered saline (PBS), glutamine, penicillin G, streptomycin, amphotericin B, and Grace's Insect medium were purchased from Invitrogen (Carlsbad, CA). Luria Broth (LB) was purchased from Becton Dickson and Co (Sparks, MD). The protease inhibitors: aprotinin, bestatin, leupeptin, pepstatin A, and phenylmethylsulfonyl fluoride (PMSF) were purchased from Cal Biochem (La Jolla, CA). Protease inhibitor cocktail contained 10 μg/ml aprotinin, 2.5 μg/ml leupeptin, 8.3 μg/ml bestatin, and 1.7 μg/ml pepstatin A. Phorbol 12-myristate 13 acetate (PMA), N-[2-hydroxyethyl]piperazine-N'-[2-ethanesulfonic acid] (Hepes), anisomycin, and 2-[N-morpholino]ethanesulfonic acid (MES) were purchased from Sigma Chemical (St. Louis, MO). All other reagents were purchased from Sigma Chemical or Fisher Scientific unless stated otherwise.

HT29 human intestinal (colorectal adenocarcinoma) epithelial cells (ATCC HTB-38), HeLa cervical epithelial adenocarcoma cells (ATCC CCL-2), 293T kidney cells (CRL-11268), A549 lung carcinoma cells (ATCC-185), and T98G glioblastoma cells (ATCC CRL-1690) were cultured in DMEM with 2 mM glutamine, 10% Fetal Calf Serum, 100 Units/ml Penicillin G, and 100 μg/ml Streptomycin at 37°C in a humidified 5% CO₂ atmosphere. T84 colorectal carcinoma cells (ATCC CCL-248) were cultured in DMEM:F12 with 2 mM glutamine, 5% Fetal Calf Serum, 100 Units/ml Penicillin G, and 100 μg/ml Streptomycin at 37°C in a humidified 5% CO₂ atmosphere. H5 insect cells (Invitrogen) were cultured in Grace's medium with 2 mM glutamine, 10% Fetal Calf Serum, 100 Units/ml Penicillin G, 100 μg/ml Streptomycin, and 0.25 μg/ml amphotericin B at 28°C. MyD88^-/- & TLR2^-/-/TLR4^-/- double knockout cells were obtained from Shizuo Akira and Osamu Takeuchi (Univ. of Osaka, Japan) and grown in DMEM with 2 mM glutamine, 10% Fetal Calf Serum, 100 Units/ml Penicillin G, and 100 μg/ml Streptomycin at 37°C in a humidified 5% CO₂ atmosphere.

Salmonella typhimurium strain SJW1103 (FliC, phase 1 flagellin, stabilized) 69 is a wild-type Salmonella typhimurium and can only express the Phase I fliC flagellin, SJW86 (SJW1103 FliC::TN10), and SJW134 (SJW1103 FliC and FljB deletions) were obtained from Robert Macnab (Yale Univ., Conn) and have been described 70. Salmonella serovar dublin strain 2229, strain SE1 (2229 SopE mutant), strain SB2 (2229 SopB mutant), and SE1SB2 (2229 SopE and SopB mutant) were obtained from Edward Galyov (Compton Laboratory, Berkshire, UK) and have been described 1415. Salmonella strains for stimulation were grown in LB at 37°C without agitation for 16 hours, centrifuged at 6,000 × g for 1 minute, gently washed with PBS, and gently suspended in DMEM to maintain cells with attached flagella.

Plasmid pFM10.1 (ampicillin resistance), encodes a green fluorescent protein (GFP) expressed after the Salmonella host is internalized by mammalian cells, obtained from Stanley Falkow (Stanford Univ., Stanford, CA) 1034 and was transformed into strains SJW1103 and SJW134 by electroporation. Strains containing pFM10.1 were designated SJW1103G and SJW134G.

Native flagellin was harvested from S. dublin 2229 or S. typhimurium SJW1103. Starter cultures were grown in Luria broth (LB) for 18 hours at 37°C with aeration, diluted 1:5000 in fresh LB, and grown for 12 hours under the same conditions. All subsequent procedures were performed at 4°C. Cells were removed from the medium by centrifugation at 10,000 × g for 5 min and discarded. The supernatant containing flagellin was filtered through a 0.8 micron filter (Millipore, Bedford, MA) to remove residual cells. Supernatant was concentrated 100 fold using an Amicon 100 kiloDalton (kDa) cutoff membrane (Millipore). Initial studies used concentrated culture supernatant from S. dublin strain 2229 that was treated with DNase, RNase, Protease K, boiled for 20 min or 100 mM DTT at 37°C for 2 hours and used for stimulation of cultured cells.

Concentrated S. typhimurium 1103 bacterial culture supernatant was washed 4 times by 1:10 dilution with 50 mM MES, pH 6.0, 50 mM NaCl and re-concentrated. Material not retained by the 100 kDa membrane was discarded. Washed culture supernatant was fractionated by gel permeation or anion exchange chromatography for analysis. For long-term storage, washed culture supernatant was supplemented with protease cocktail and stored at -20°C.

Fractionation by gel permeation chromatography was performed with a Superose 12HR column (Pharmacia) on a Bio-Logic system (Bio-Rad). One-half mililiter of 100× washed supernatant (equivalent of 50 ml original culture supernatant) was separated on the column at 0.4 ml/minute in 50 mM Hepes, pH 7.4, 200 mM NaCl. Fractions (0.5 ml) were collected, and 50 μl was fractionated by SDS-PAGE and stained with Bio-Safe Coomassie (Bio-Rad). Thirty microliters of each fraction was used for stimulation of HT29 cells (60 mm dishes) for 45 min and NF-κB DNA binding activity in the resulting whole cell extracts extracts were assayed by EMSA. The column was standardized with catalase (232 kDa), aldolase (158 kDa), abumin (67 kDa), ovalbumin (43 kDa), and Chymotripsinogen A (25 kDa), all obtained from Amersham-Pharmacia.

Fractionation by anion exchange chromatography was performed with Poros HQ matrix (2 ml column, PerSeptive Biosystems, Farmingham, MA) on a Bio-Logic system. Five mililiters of 100× washed supernatant (equivalent of 500 ml original culture supernatant) was separated at 1 ml/minute in 50 mM Hepes, pH 7.4, and a NaCl gradient from 50–500 mM. Fractions were collected and 5 μl of each fraction was examined by 10% SDS-PAGE. Proteins were fractionated on duplicate 10% SDS-PAGE precast gels (BioRad). One gel was stained with Bio-Safe Coomassie (Bio-Rad) and the protein bands were isolated for Mass Spectroscopy analysis (CCF Mass spectroscopy core facility) from the other identical non-stained gel, by electro-elution with a whole gel eluter (Bio-Rad) and SDS was removed with SDS-Out (Pierce, Rockland, IL) per the manufacturers directions. Proteins isolated from bands B1 to B6 were acetone precipitated by addition of 20 μg Aprotinin and 5 μg of BSA to each eluted fraction, ice-cold acetone (-20°C) was added to 80%, mixed well and precipitated overnight at -20°C. Proteins were pelleted by centrifugation at 14,000 × g in the cold for 30 min, acetone/liquid was removed and the pellets washed 2× with 1 ml acetone (-20°C). After removal of the acetone, protein pellets were air dried and then resuspended and denatured in 5 μl of 6 M guanidinium hydrochloride (Gu-HCl) at room temperature for 30 min. Resuspended proteins were two-fold serially diluted in DMEM to a final Gu-HCl concentration of 55 mM to renature the proteins. Two hundred fifty microliters of individual renatured proteins/DMEM were added per ml to HT29 cells (60 mm dishes) and whole cell extracts were prepared 45 min after stimulation and were assayed for NF-κB DNA binding activity by EMSA.

The washed and concentrated culture supernatant from S. typhimurium 1103 containing flagellin was boiled for 20 minutes and precipitants removed by centrifugation at 15,000 × g. The supernatant containing flagellin was diluted 1:2 with 50 mM MES, pH 6.0, 50 mM NaCl and mixed with 2 ml Poros SP cation exchange matrix (PerSeptive Biosystems) per 1 liter of original culture. The Poros SP matrix was prepared as a 50% slurry and equilibrated with 50 mM MES, pH 6.0. The flagellin preparation and matrix were mixed on a roller at 12 to 14 RPM for 2 hours. The matrix along with bound contaminants was removed by filtration through a 0.85 micron filter and discarded, flagellin failed to bind to the cation exchange matrix at pH 6.0 and eluted in the flowthrough and was collected.

The pH of the flowthrough was adjusted by five-fold dilution of the sample with 50 mM Hepes, pH 7.8, 50 mM NaCl, and loaded onto a Poros HQ anion exchange column (2 ml column, PerSeptive Biosystems) equilibrated with 50 mM Hepes, pH 7.4, 50 mM NaCl. The column was washed with 2 volumes 50 mM Hepes, pH, 7.4, 50 mM NaCl, and eluted with a 10 column volume linear gradient of 50–500 mM NaCl in 50 mM Hepes, pH 7.4. Flagellin eluted from the column between 200–275 mM NaCl. Fractions containing flagellin were pooled and concentrated. The preparation was determined to be pure by electrophoresis of 5 μg protein by SDS-PAGE and stained with Bio-Safe Coomassie (Bio-Rad). Samples were stored at -80°C in 50 mM Hepes, pH 7.4, approx 225 mM NaCl, 10% glycerol and protease cocktail. A 4 liter preparation of culture supernatant yielded 2 mg purified flagellin.

Gels were fixed and stained (Bio-Safe Blue, BioRad). All of the following procedures were performed by the CCF Mass spectroscopy core facility. Excised gel bands were reduced (100 mM DTT), and alkylated (100 mM iodoacetamide). Proteins in the gel bands were digested with modified trypsin (Promega, 20 μg/mL) with an overnight incubation at 37°C. Tryptic peptides were extracted from the gel with 50% acetonitrile, 0.1% acetic acid, concentrated in a SpeedVac (Thermo Savant) to remove acetonitrile, and reconstituted to 20 uL with 0.1% acetic acid. Extracted peptides were subjected to reversed phase (50 uM ID packed with Phenomenex Jupiter C18, 6 cm capillary column) liquid chromatography (2%–70% solvent B; Solvent A, 50 mM acetic acid, aqueous, Solvent B acetonitrile), coupled to a Finnigan LCQ DECA ion trap mass spectrometer for peptide sequencing, as described 38.

IκBα amino acids 1 to 54 fused to GST or cJUN amino acids 1–79 fused to GST were prepared as previously described 373839 and stored in kinase buffer (20 mM Hepes, pH 7.6, 10 MM MgCl2, 10 mM NaCl, 2 mM beta-glycerophosphate, 10 mM PNPP).

HT29 cells for microscopic examination were grown in 6 well plates on sterile cover slips to a density of 50–75%. Cells were stimulated as described above. After stimulation, cover slips with HT29 cells were washed 2 times with ice cold PBS and fixed with 4% w/v formalin at room temperature for 20 minutes. Cells were washed 4 times with PBS prior to mounting for visualization of Salmonella invasion. Cover slips were mounted with Vectashield mounting medium with DAPI (Vector Laboratories, Burlingame, CA), and cover slips sealed to slides.

Cells for antibody staining were treated with absolute methanol for 20 minutes following formalin fixation, then washed 3 times with PBS supplemented with 0.1% BSA (PBSB) and used directly or stored in the cold after azide was added to 0.02%. For p65(RelA) localization, cells on coverslips were blocked for 1 h at 37°C with PBS supplemented with 1% BSA. The PBSB was removed, washed once with PBSB and coverslips were placed cell-side down onto 150 μl of p65 antibody (Zymed, South San Francisco, CA) diluted 1:1500 in PBSB on a square of parafilm and placed in a humidified chamber at 37°C for 1.5 h. Coverslips were removed and placed cell-side up in 6-well dishes and washed 3 × 5 min with PBSB. Coverslips were then removed and placed cell-side down onto 150 μl of FITC-labeled donkey anti-rabbit secondary antibody (Jackson Immunoresearch Laboratories, West Grove, PA) (1:300 in PBSB) on a square of parafilm and placed in a humidified chamber at 37°C for 1.5 h. Coverslips were removed and placed cell-side up in 6-well dishes and washed 5 × 5 min with PBSB, removed and placed cell-side down onto slides mounted with Vectashield (Vector Laboratories, Burlingame, CA) with DAPI and then sealed. NF-κB localization was determined by indirect immunofluorescence. Samples were observed on a Leica DMR upright microscope (Leica Microsystems Inc., Heidelberg, Germany) at 400× with oil immersion and equipped with FITC and UV filters. Images were collected with a MicroMax RS camera (Princeton Instruments Inc., Princeton, NJ), and Image Pro plus, version 4.5, software (Media Cybermetics Inc., Carlsbad, CA). Color enhancements were performed with Image Pro plus software. Visible light plus color overlays for Fig. 1 and Fig. 5B were performed with MetaMorph Software (Universal Imaging Corp., Downington, PA).

Mouse embryo fibroblasts (MEFs) or HT29 cells were grown in DMEM as above to a density of 90% prior to stimulation. All cells were washed with warm PBS and supplemented with DMEM without serum or antibiotics in preparation for stimulation. Cells were stimulated with; 10 ng/ml TNFα, 1 μg/ml flagellin unless specified otherwise, 20 μg/ml Anisomycin, 12.5 ng/ml PMA, or 10⁸ Salmonella/ml at 37°C for desired times and extracts prepared as below. Cells harvested beyond one hour were washed with warm PBS and supplemented with warm DMEM, 2 mM glutamine, and 200 ug/ml gentamycin after 1 hour and returned to 37°C until extract preparation desired.

Cells were washed with ice-cold PBS and all subsequent steps carried out at 4°C or on ice. Cells were scraped from the dish in ice-cold PBS, and collected by centrifugation at 1000 × g for 1 minute. Cells were lysed by suspension in 50 mM Tris-HCl, pH 7.6, 400 mM NaCl, 25 mM beta-glycerol phosphate, 25 mM NaF, 10 mM PNPP, 10 % glycerol, 0.5 mM sodium orthovanadate, 0.5% nonidet-40 (NP-40), 5 mM benzamidine, 2.5 mM metabisulfite, 1 mM PMSF, 1 mM DTT and protease inhibitor cocktail as described 3.

NF-κB DNA binding assays were carried out as previously described 33538. Anti-p65 antibody (Zymed, South San Francisco), anti-p50 antibody (Santa Cruz Biotechnologies, Santa Cruz, CA), and anti-STAT3 antibody (Santa Cruz) were used for EMSA supershifts.

HT29 cells, 90–95% confluent in 35 mm round dishes, were prepared for stimulation as above and treated with a 1 ml suspension of Salmonella SJW1103 or SJW134 or left untreated in triplicate as above. After one hour, HT29 cells were washed 4× with warm PBS, supplemented with warm DMEM, 2 mM glutamine, and 200 μg/ml gentamycin, and incubated at 37°C for 4 hours. Cells were then harvested as above and lysed by suspension in 1 ml sterile distilled water. Ten-fold serial dilutions were prepared in PBS and 100 μl of each dilution was plated on LB agar plates and grown at 37°C for 20 hours. Colonies were counted and averaged.

Whole cell extracts (250 μg) were supplemented with 150 μl of Buffer A (20 mM Hepes, pH 7.9, 20 mM beta-glycerophosphate, 10 mM NaF, 0.1 mM orthovanadate, 5 mM PNPP, 10 mM 2-mercaptoethanol, 0.5 mM PMSF, and protease inhibitor cocktail), and immuno precipitation kinase assays carried out as described 3 using either IKKα monoclonal antibody (PharMingen – Becton Dickson), anti-JNK1 (Santa Cruz Biotechnologies, Santa Cruz, CA), or anti-hemagglutinin (HA) epitope antibody (Covence Antibodies, Princeton, NJ) as indicated. Protein G immunopellets were collected by centrifugation at 500 × g for 30 sec, washed 3 times with Buffer B (Buffer A plus 250 mM NaCl), and one time with Buffer C (Buffer A plus 50 mM NaCl and 10 mM MgCl₂). Immunopellets were resuspended in 30 μl Kinase buffer with 0.1 mM orthovanadate, 50 μM "cold" ATP, 5 μCi γ-³²P-ATP, 2 mM DTT, and 2 μg of soluble GST-IκBα1–54 or GST-cJUN1-79, and incubated at 30°C for 30 minutes. Reactions were stopped by the addition of 15 μl 4× SDS-PAGE loading buffer, heated at 95°C for 5 minutes, and resolved on 10% SDS-PAGE gels by standard procedures. Gels were rinsed, stained with Bio-Safe Coomassie (Bio-Rad) to visualize protein bands, rinsed, photographed then dried and exposed to Kodak X-OMAT AR film (Eastman Kodak Co., Rochester, NY) to detect substrate phosphorylation.

Protein samples (40 μg) were resolved by SDS-PAGE on a 10% acrylamide gels by standard procedures, and proteins transferred to PVDF membrane (Millipore) and probed with antibodies as described 3. Membranes were washed 3× briefly with TBST, incubated with a 1:1000 dilution (1:800 for anti-TLR5) of the primary antibody in TBST, 1% non-fat milk for 1 hour, washed 3 × 5 min with TBST, and then incubated with a 1:2000 dilution of the appropriate HRP-conjugated secondary antibody in TBST, 0.5% non-fat milk for 1 hour. Primary antibodies used were: anti-IKKα/β (H-470, Santa Cruz), anti-JNK1, anti-ERK2 (K-23, Santa Cruz), anti-phospho-ERK (E-4, Santa Cruz), anti-p38MAPK (Cell Signaling Technologies, Beverly, MA), anti-phosopho-p38MAPK (Cell Signaling), anti-TLR5 (H-127, Santa Cruz), anti-muc1 (H-295, Santa Cruz) and anti-actin (C-11, Santa Cruz). Secondary antibodies used were: anti-mouse IgG HRP conjugate (Amersham-Pharmacia), anti-rabbit IgG HRP conjugate (Amersham-Pharmacia), anti-goat IgG-HRP conjugate (Santa Cruz). HRP activity was detected by ECL (Amersham-Pharmacia) as per manufacturers instructions, on Kodak X-OMAT AR film.

All DN-TLRs were constructed using PCR. The universal 5' primer consisted of a 5'KPN I restriction site followed by sequences encoding the kozak sequence, translational start site, and preprotrypsin leader sequence of pCMV-1 (Sigma) that all the wild-type TLRs were initially cloned into. The 3' anti-sense (AS) primers were human TLRgene-specific primers (sequences available upon request) that created a stop codon immediately after a conserved tryptophan in Box 9 of the TLR TIR homology domain according to Bazan 71, thus creating carboxy terminus deletions. The 5' end of the AS primer contained a number of convenient restriction sites to allow directional cloning. PCR was performed with turbo-Pfu polymerase (Stratagene, La Jolla, CA) using standard procedures on individual wild-type TLR pCMV-1 plasmid DNAs (5 ng each, kind gifts of R. Medzhitov, Yale Univ. and R. Ulevitch, TSRI) 4849 with the 150 ng each of the universal 5' sense primer and individual gene-specific TLR 3' primers. PCR products were cleaned-up with PCR cleanup kit (Qiagen, Germany) digested with appropriate restriction enzymes, gel purified and then ligated into the mammalian expression vector pCDNA3.1 (Invitrogen). Positive clones were sequenced to verify the mutations and tested for expression in transient expression assays and detected on immunoblots by probing with anti-FLAG M2 monoclonal antibody (Sigma). All wild-type and DN-TLR alleles are amino terminus FLAG epitope-tagged.

HT29 cells were transfected with Lipofectamine Plus (Invitrogen) as previously described 3. In transfections monitoring reporter gene expression, transfections were performed at least three times in 6 well dishes in triplicate with the total DNA mass kept constant at 4 μg (2 μg effector plasmid DNA, 100 ng 2× NF-κB Luc reporter gene, 50 ng pRL-TK, a thymidine kinase promoter driven Renilla luciferase normalization reporter and 1.85 μg pCDNA3.1 plasmid DNA as bulk filler DNA) and fire-fly luciferase expression was normalized to Renilla luciferase expression using the dual-luciferase assay (Promega, Madison, WI). Fold inductions were calculated and values between experiments did not vary more than 15%, a representative experiment is presented. Transfection of 293T cells was performed with lipofectamine 2000 (Invitrogen) in 6-well dishes in triplicate as per the manufacture's protocol. TLR expression plasmids were added at 2 μg/well, and NF-κB and normalization control plasmids were as above with HT29 cells and pCDNA3.1 plasmid DNA as bulk filler DNA to a final DNA mass of 4 μg/well. Fold inductions were calculated and values between experiments (N of 3) did not vary more than 10%, a representative experiment is presented.

Cells (N = 3) were stimulated 3 hours at 37°C with TNFα or FliC or left untreated and harvested for total RNA isolation. Total cellular RNA was extracted from cells with Trizol reagent (Invitrogen) 3 and reverse transcribed with ReactionReady first strand cDNA synthesis kit (SuperArray Bioscience Corp., Fredrick, MD). RNA (2.5 ug per 20 ul reaction) was reverse transcribed using random primers and Moloney murine leukemia virus reverse transcriptase per manufacturer specified conditions. Controls without reverse transcriptase (minus RT) was also generated for each RNA sample. RT²PCR was performed with an iCycler (Bio-Rad) to quantify TLR1 through TLR10 mRNA, 18S rRNA, and GAPDH mRNA. RT²PCR (25 ul reaction volume) was performed with the appropriate primers (SuperArray) per manufacturers instructions in triplicate with HotStart Taq DNA polymerase (SuperArray) at 95°C for 15 min to activate Taq and amplified for 40 cycles (95°C, 30 sec, 55°C, 30 sec, 72°C, 30 sec). RT²PCR was performed on the minus RT controls with TLR5 primers to detect DNA contamination. Real-time PCR analysis was performed using SYBR-green (Perkin-Elmer) according to manufacture's instructions with the specific primer pairs indicated above and primer pairs for 18S ribosomal RNA as reference RNA (Classic 18S primer pairs – Ambion Inc). Cycle time (Ct) was measured using the iCycler™ and its associated software (Bio-Rad). Relative transcript quantities were calculated by the ΔΔCt method using 18S ribosomal RNA as a reference amplified from samples using the Classic 18S primer pairs from Ambion, Inc (Austin, TX). Normalized samples were then expressed relative to the average ΔCt value for untreated controls to obtain relative fold-change in expression levels. Fold change in mRNA expression was expressed as 2^ΔΔCt. 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. Values for fold-induction varied less than 5% among replicates.