Salmonella Flagellin-Dependent Proinflammatory Responses Are Localized to the Conserved Amino and Carboxyl Regions of the Protein

Cell cultures

DLD-1 and Caco-2BBe (American Type Culture Collection, Manassas, VA) human adenocarcinoma cell lines, were used between passages 5 and 15. These cell lines were maintained in DMEM supplemented with 10% FBS, 110 mg/L sodium pyruvate, and antibiotics (Life Technologies, Grand Island, NY). DLD-1 cells were grown to confluence on 96-well plates for NO analysis and Caco-2BBe cells were grown to confluency on 6-well plates or 10-cm plates for IκBα, IL-8, and NF-κB analysis. Before treatment, the growth medium was removed and replaced with DMEM without FBS.

The human myelomonocytic cell line, THP-1 (American Type Culture Collection) was cultured in RPMI 1640 medium containing 2 mM of l-glutamine (Life Technologies) supplemented with 10 mM of HEPES, 1 mM of sodium pyruvate, 4.5 g/L glucose, 1.5 g/L sodium bicarbonate, 10% FBS, and 0.05 mM of 2-ME at 37°C in 5% CO₂. For experimental purposes, cells were resuspended in supplemented RPMI 1640 without FBS then were seeded at 5 × 10⁶ cells/well in 24-well tissue-culture plates.

Generation of SD flagellin protein and protein fragment constructs

Oligonucleotide primers were designed (Table I⇓) based upon the SD fliC flagellin gene sequence (GenBank accession number M84973) and used for PCR amplimer generation. The amplimers were generated from SD genomic DNA templates under the following conditions: 30 cycles of 94°C for 30 s, 55°C for 30 s, and 72°C for 60 s. A final 72°C 7-min extension completed amplification. PCR mixes included 15 mM of MgCl₂, 100 mM of dNTPs, and 10 U of Taq polymerase (Life Technologies). Using primer-introduced restriction enzyme sites, the amplimers were cloned into the pPROEX vector (Life Technologies). The cloning strategy allowed for unidirectional amplimer insertion downstream of and in-frame with a 6HIS residue tag and an isopropyl β-d-thiogalactoside-inducible promoter present in pPROEX. Resulting plasmids were transformed into competent E. coli (Protein Express, Cincinnati, OH), selected, and then confirmed by sequence analysis (Cleveland Genomics, Cleveland, OH).

A panel of fusion proteins was generated, purified, and immunoblotted for confirmation of identity. Some of the smaller flagellin protein constructs were predicted to improperly fold so selected constructs were provided a centrally positioned “hinge” element from the E. coli MukB protein (20). This hinge region has been used by others (20) to bring amino and carboxyl protein regions into hairpin stem structures. A separate 6HIS-tagged E. coli hinge (ECH) fusion protein was purified and tested for bioactivity. The recombinant flagellin proteins are depicted in Fig. 1⇓.

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FIGURE 1.

Recombinant SD flagellin protein constructs generated by PCR. Note the nomenclature of the recombinant flagellin proteins is as follows: N is used to designate the amino terminus of the protein and C designates the C terminus. The D1, D2, and D3 designations are used to describe the flagellin domains 1, 2, and 3. Finally, ECH represents the E. coli hinge element.

Affinity purification and quantitation of flagellin constructs

E. coli, containing selected flagellin constructs, were grown overnight at 37°C in Luria-Bertani broth with 100 μg/ml ampicillin. Stationary phase bacteria were transferred to fresh broth, grown to log phase, and then supplemented with 1 μl/ml of 0.1 M of isopropyl β-D-thiogalactoside to induce fusion protein synthesis. After 4–6 h of induction, the bacteria were lysed in a buffer containing 50 mM of NaH₂PO₄, 300 mM of NaCl, 20 mM of imidazole (pH 7.5). The fusion proteins then were purified from the lysate by affinity chromatography on a Ni-NTA column (Qiagen, Valencia, CA). Bound 6HIS-tagged protein was eluted with a buffer containing 50 mM of NaH₂PO₄ (pH 8.0), 300 mM of NaCl, and 250 mM of imidazole. Each preparation was analyzed by SDS-PAGE. Quantification of the purified fusion proteins was completed by fluorescence using a NanoOrange Protein Quantitation kit (Molecular Probes, Eugene, OR).

Purification of flagellin directly from SD

Flagellin was purified from SD as previously described (7, 21). Briefly, SD (American Type Culture Collection) isolates were incubated in brain-heart infusion broth at 35°C in an orbital shaker incubator at 80 rpm for ∼16 h. Bacteria were collected by centrifugation at 5,000 × g for 30 min then mixed with saline solution to form a moderately thick suspension. The suspension was adjusted to pH 2.0 with 1 M of HCl and maintained at that pH under constant stirring at room temperature for 30 min. The bacteria, devoid of flagella, then were separated by centrifugation at 5,000 × g for 30 min. The supernatant that contained detached flagellin in monomeric form, was further centrifuged at 100,000 × g for 1 h at 4°C. The pH of the supernatant was adjusted to 7.2 with 1 M of NaOH. Ammonium sulfate was added slowly with vigorous stirring to achieve two-thirds saturation (2.67 M) and then the mixture was centrifuged at 15,000 × g for 15 min at 4°C. The precipitate was dissolved in ∼5 ml of distilled water and dialyzed for 18 h at 4°C against distilled water using 50,000 m.w. cutoff dialysis tubing. The dialyzed flagellin then was lyophilized, resuspended in sterile water, and stored at −70°C until used. This flagellin will be referred to as purified native SD flagellin throughout the text.

LPS detection

The chromogenic Limulus amebocyte test (BioWhittaker, Walkersville, MD) was used to determine the level of LPS present in the purified flagellin fusion proteins. Any LPS present in the fusion protein preparations was eliminated by passage over an endotoxin removing gel column (Pierce, Rockford, IL). To further control for potential LPS effects, some studies included IEC pretreatment with 10 μg/ml polymyxin B (PB), an LPS scavenger, before the addition of the flagellin fusion proteins.

NO₂⁻/NO₃⁻ detection

DLD-1 cells were pretreated with IFN-γ (100 U/ml) 2 h before the addition of selected concentrations of flagellin proteins. Cell supernatants were collected 18 h after the addition of the flagellin proteins. The combined concentration of nitrate and nitrite, the degradation products of NO, was determined in the supernatants by the Griess reaction following nitrate reduction as described previously (22). In the text total nitrate/nitrite is referred to as NO.

IκBα Western blot analysis

Caco-2BBe cells, grown in 6-well plates, were stimulated with purified SD flagellin, the full-length 6HIS SD flagellin (6HIS flag) fusion protein, or one of the other flagellin region proteins for selected times. Caco-2BBe cells then were lysed in 4°C buffer containing 50 mM of Tris (pH 8.0), 110 mM of NaCl, 5 mM of EDTA, 1% Triton X-100, and 0.1 mM of PMSF. The amount of protein in each sample was determined by the Bradford assay (Bio-Rad, Hercules, CA). Individual cell lysates were boiled in 10 μl of loading buffer (4% SDS, 20% glycerol, 125 mM Tris-HCl (pH 6.8), and 10% 2-ME), and 50 μg of each protein sample was loaded per lane on an 8–16% Tris-glycine gradient gel (NOVEX, San Diego, CA). Electrophoresed proteins were transferred to a nitrocellulose membrane (NOVEX) with the NOVEX X-cell MiniGel system. Membranes were blocked with 10% nonfat dried milk resuspended in TBS for 30 min before incubation with rabbit polyclonal anti-IκBα antiserum (Santa Cruz Biotechnology, Santa Cruz, CA) at a dilution of 1/200 for 3 h. Blots were washed twice for 7 min in TBS supplemented with 0.1% Tween 20 + TBS, followed by the addition of peroxidase-conjugated anti-rabbit IgG (Sigma-Aldrich, St. Louis, MO) at a dilution of 1/10,000 for 30 min. Blots were washed three times for 5 min each with Tween 20 + TBS and then incubated for 1 min in ECL reagents (ECL kit; Amersham, Little Chalfont, Buckinghamshire, U.K.). Processed blots were placed on x-ray film (Kodak, Rochester, NY) for empirically optimized exposures.

Nuclear protein extraction

Nuclear protein extracts were prepared from Caco-2BBe cells incubated with purified SD flagellin, the 6HIS flag fusion protein, or one of the other flagellin region proteins for 1 h. Cells were washed twice with ice-cold PBS and harvested by scraping into 1 ml of ice-cold PBS. After pelleting at 6,000 rpm for 5 min, cells were washed twice with cold PBS, resuspended in 1 pellet vol of lysis buffer (1.5 mM of MgCl₂, 0.2% v/v Nonidet P-40, 10 mM of HEPES (pH 7.9), 10 mM of KCl, 0.1 mM of EDTA, 1 mM of DTT, and 0.1 mM of PMSF), and incubated for 5 min on ice. Nuclei were collected from the cell lysates by centrifuging at 6,000 rpm for 5 min and then were resuspended in 1 pellet vol of extraction buffer containing 1.5 mM of MgCl₂, 25% v/v glycerol, 20 mM of HEPES (pH 7.9), 420 mM of NaCl, 0.1 mM EDTA, 1 mM of DTT, and 0.5% PMSF. After incubation on ice for 15 min, nuclear proteins were collected by centrifuging at 14,000 rpm for 15 min. To ensure the elimination of nuclear debris, supernatants were also collected.

EMSA

An NF-κB oligonucleotide probe (5′-AGT TGA GGG GAC TTT CCC AGG-3′; Santa Cruz Biotechnology) was labeled with [γ-³²P]ATP by using T4 polynucleotide kinase (Life Technologies) and then purified on a Bio-Spin chromatography column (Bio-Rad). Nuclear protein extracts were preincubated with EMSA buffer (1 mM of EDTA, 1 mM of DTT, 12 mM of HEPES (pH 7.9), 4 mM of Tris-HCl (pH 7.9), 5 mM of MgCl₂, 25 mM of KCl, 12% glycerol, 50 ng/ml poly(dI-dC), and 0.2 mM of PMSF) on ice for 10 min, followed by a 20 min incubation with the radiolabeled probe. The specificity of the binding reaction was determined by incubating duplicate nuclear protein samples with a 100-fold excess of unlabeled probe. Samples were resolved on a nondenaturing 5% polyacrylamide gel and run in 0.5 × Tris/boric acid/EDTA buffer (1 mM of EDTA, 45 mM of boric acid, and 45 mM of Tris-HCl) at a constant current (30 mA) for 1 h. Gels were transferred to 3M paper (Whatman, Clifton, NJ), dried under a vacuum for 1 h at 80°C, and exposed to film overnight at −70°C with the addition of an intensifying screen.

IL-8 and TNF-α ELISA

Following the stimulation of Caco-2BBe or THP-1 cells with individual SD flagellin fusion protein constructs at a concentration of 1 μg/ml, cell supernatants were collected at 22 h poststimulation of Caco-2BBe cells for IL-8 detection and 4 h poststimulation in the THP-1 cells for TNF-α detection. The amount of IL-8 and TNF-α in the supernatants was quantified by an ELISA (Pierce and Endogen, Woburn, MA).

Statistical Analyses

Where applicable, results are expressed as the mean ± SEM of three to four experiments. The Student’s t test was used to compare mean values. Differences were considered significant when p values were <0.05.

The full-length rSD flagellin protein induces proinflammatory mediators

To begin the molecular analysis of SD flagellin, 6HIS flag was tested for biological activity. Confluent Caco-2BBe cells were stimulated with 1 μg/ml of 6HIS flag; in previous studies (7), this dose of native purified SD flagellin consistently provided easily detectable activity and, therefore, was used throughout these experiments with all recombinant flagellin fusion proteins. Western blotting of stimulated lysates showed that Caco-2BBe cells underwent the loss of IκBα at both 20 and 30 min poststimulation with replenishment at 60 min comparable to the native purified SD flagellin protein (Fig. 2⇓A). To confirm nuclear translocation of NF-κB, an expected consequence of IκBα degradation, EMSA analyses were completed and showed that 6HIS flag induced NF-κB activation in Caco-2BBe cells at 1 h poststimulation (Fig. 2⇓B). When IFN-γ-primed DLD-1 cells were incubated with 1 μg/ml of 6HIS flag for 18 h, similar levels of NO production were observed when compared with the purified native SD flagellin (1 μg/ml) or the combination of IL-1β (0.5 ng/ml) and IFN-γ (100 U/ml; Fig. 2⇓C). These levels were significantly greater than untreated control DLD-1 cells (p < 0.001; Fig. 2⇓C). Further, Caco-2BBe cells secreted elevated levels of IL-8 following stimulation for 22 h with 1 μg/ml of 6HIS flag that was equal to purified native SD flagellin at the same dose (Fig. 2⇓D).

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FIGURE 2.

6HIS flag induced IκBα degradation, NF-κB activation, NO, and IL-8 production in two IEC. A, IκBα degradation and replenishment was observed in a time-dependent manner in Caco-2BBe cells stimulated with 1 μg/ml purified SD flagellin or SD 6HIS flagellin for 10, 20, 30, or 60 min. Unstimulated cells (C) showed no IκBα degradation at 30 min. Cells stimulated with purified native SD flagellin or 6HIS flag showed a loss of IκBα at 30 min with replenishment at 60 min. B, Representative EMSA demonstrating NF-κB activation at 60 min in Caco-2BBe cells. Nuclear extracts from unstimulated cells (C) showed no NF-κB activation. Caco-2BBe cells stimulated with a combination of IL-1β (0.5 ng/ml) and IFN-γ (100 U/ml) or 1 μg/ml 6HIS flag for 60 min induced prominent NF-κB activation. C, NO production in DLD-1 cells was induced by 6HIS flag. DLD-1 cells were pretreated for 2 h with IFN-γ (100 U/ml) followed by the addition of 1 μg/ml 6HIS flag. Cell supernatants were collected 18 h later and the combination of nitrite and nitrate were measured by the Griess reaction following nitrate reduction. There are six samples per experimental group. Data are expressed as mean ± SEM. Unstimulated cells (C) produced no detectable NO. Cells treated with 1 μg/ml 6HIS flag or LPS induced high levels of NO production comparable to IL-1β and IFN-γ or purified SD flagellin. IFN-γ-primed DLD-1 cells pretreated with an LPS-binding agent, PB (10 μg/ml), blocked the induction of NO by 1 μg/ml LPS but did not affect NO production in cells treated with 6HIS flag (Student’s t test; p < 0.01). D, Caco-2BBe cells secreted IL-8 in response to 6HIS flag fusion protein stimulation. Supernatants collected 22 h following stimulation of Caco-2BBe cells with a 1 μg/ml 6HIS flag induced high levels of IL-8 comparable to the combination of IL-1β and IFN-γ and 1 μg/ml of purified SD flag. IL-8 secretion was not detected in unstimulated cells (C).

The potential for LPS contribution to these findings also was addressed. IFN-γ-primed DLD-1 cells that were pretreated with 10 μg/ml LPS scavenger, PB, followed by the addition of 1 μg/ml 6HIS flag or Salmonella LPS, had indistinguishable NO levels when compared with cells treated with 6HIS flag (Fig. 2⇑C). However, PB pretreatment abolished the induction of NO (Fig. 2⇑C) in DLD-1 cells stimulated with 1 μg/ml LPS.

Together the data indicate that the 6HIS flag protein retained biological activity comparable to native flagellin establishing the basic construct for further dissection of the flagellin gene.

A flagellin with a D3-region deletion retained biological activity; however, truncated flagellin protein constructs did not induce proinflammatory responses

Using the active 6HIS flag construct as a starting point, an internal deletion was made by eliminating ∼100 aa in the D3 region. This particular deletion was chosen due to convenient restriction enzyme sites in the fliC gene. The resulting protein was designated N/C (Fig. 1⇑). Complete IκBα degradation was observed in Caco-2BBe cells stimulated with 1 μg/ml 6HIS flag at 20 and 30 min with replenishment at 60 min as analyzed by immunoblotting (Fig. 3⇓A). Treatment of cells with the N/C flag also resulted in the loss of IκBα at 30 min (Fig. 3⇓A), although not as completely as 6HIS flag, and similarly treated IFN-γ-primed DLD-1 cells showed NO production comparable to the 6HIS flag and the combination of IL-1β and IFN-γ (Fig. 3⇓B). In addition, substantial IL-8 secretion also was observed in Caco-2BBe cells treated for 22 h with 1 μg/ml N/C flag comparable to the full-length 6HIS flag (Fig. 3⇓C). These data show that a SD flagellin protein containing a deletion of a portion of the D3 region retains bioactivity evidenced by the loss of IκBα and the production of NO.

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FIGURE 3.

A flagellin fusion protein lacking 100 aa in the D3 region, designated N/C flag, retains proinflammatory activity in IEC. A, Cells stimulated with 1 μg/ml 6HIS flag showed complete loss of IκBα at 20 and 30 min with replenishment at 60 min as indicated by immunoblotting. N/C flag (1 μg/ml) induced a loss of IκBα at 30 min in Caco-2BBe cells with reappearance at 60 min. No IκBα degradation was detected in unstimulated cells (C). B, NO production in DLD-1 cells was induced by N/C flag. IL-1β and IFN-γ induced the highest amount of NO detected in IFN-γ-primed DLD-1 cells. Comparable production of NO was observed in cells stimulated with 1 μg/ml 6HIS flag, purified SD flagellin, or N/C flag. Cells that were unstimulated (C) had minimal measurable NO. C, N/C flag induced IL-8 secretion in Caco-2BBe cells. Unstimulated cells (C) had minimal levels of IL-8 in cell supernatants. Cells stimulated with 1 μg/ml N/C flag showed high levels of IL-8 secretion nearly equal to the combination of IFN-γ and IL-lβ, purified SD flag, or 6HIS flag.

Fusion proteins representing the amino (24–222 aa), middle (175–380 aa), and carboxyl (317–506 aa) thirds of SD flagellin protein were constructed and tested (Fig. 1⇑). None of these three proteins induced IκBα degradation (data not shown), NO production (detection of nitrite/nitrate expressed as micromoles; amino third, 10 ± 0.67 μM; middle third, 15 ± 0.54 μM; and carboxyl third, 7 ± 0.56 μM vs 6HIS flag, 58 ± 0.85 μM), or IL-8 production (amino third, 10 ± 0.07 pg/ml; middle third, 10 ± 0.04 pg/ml; and carboxyl third, 3 ± 0.03 pg/ml vs 6HIS flag, 994 ± 0.85 pg/ml) in either of the IEC lines tested. Two possible conclusions for these results were considered. First, improper folding of the truncated proteins may have prevented the linear bioactive domains from forming. Alternatively, a nonlinear bioactive domain was not formed because the two portions were not contained in one of the three proteins. These possibilities were tested with a final set of constructs.

Constructs containing the D3, ND1 and CD1, or ND2 and CD2 regions did not induce proinflammatory responses

The fusion proteins containing the ND1 and CD1 or the ND2 and CD2 regions, without the D3 region, did not induce IκBα degradation or NO induction (data not shown). This could have resulted from an inability to fold correctly or the lack of region formation due to missing aa sequences. The data suggest that flagellin bioactivity resides in a discontinuous domain of the protein formed by juxtaposition of the amino and carboxyl protein regions. To better establish whether a discontinuous domain generated by protein folding was responsible for bioactivity, fusion proteins were generated from constructs containing the D3 region or an ECH (20) cloned between the ND1 and CD1 (designated ND1/D3/CD1 or ND1/ECH/CD1; Fig. 1⇑) or ND2 and CD2 regions (designated ND2/D3/CD2 or ND2/ECH/CD2; Fig. 1⇑). The ECH element has been used successfully in other protein engineering designs to bring the amino and carboxyl elements into a stem structure (20). The D3 region and ECH alone did not induce IκBα degradation (Fig. 4⇓A), NO production (Fig. 4⇓B), or IL-8 secretion (Fig. 4⇓C) in either of the IEC lines stimulated with 1 μg/ml of the fusion proteins. No IκBα degradation (data not shown), NO production (Fig. 4⇓B), or IL-8 secretion (Fig. 4⇓C) was induced in IEC by stimulation with ND1/ECH/CD1, ND2/ECH/CD2, ND1/D3/CD1, or ND2/D3/CD2.

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FIGURE 4.

Recombinant flagellin proteins representing the D3, ND1 and CD1, or ND2 and CD2 regions did not induce proinflammatory mediators in IEC. A, The SD flagellin D3 region, and ECH, did not induce IκBα degradation. Caco-2BBe cells incubated with 1 μg/ml D3 fusion protein or ECH alone showed no IκBα degradation compared with 6HIS flag at 20 and 30 min as analyzed by immunoblotting. Unstimulated cells (C) showed no loss of IκBα. B, IFN-γ-primed DLD-1 cells did not produce NO following exposure to 1 μg/ml fusion proteins representing the D3 region, ECH alone, ND1/D3/CD1, ND1/ECH/CD1, ND2/D3/CD2, or ND2/ECH/CD2 compared with significantly high levels of NO in cells stimulated with IL-1β and IFN-γ, purified SD flag or 6HIS SD flag (Student’s t test; p < 0.01). Control cells (C) incubated with growth medium alone showed no induction of NO. C, Caco-2BBe cells secreted minimal levels of IL-8 into the medium following a 22 h incubation with 1 μg/ml flagellin fusion proteins representing the D3 region, ECH alone, ND1/D3/CD1, ND1/ECH/CD1, ND2/D3/CD2, or ND2/ECH/CD2 vs significant levels of IL-8 detected in cells stimulated with IL-1β and IFN-γ, purified SD flag, or 6HIS flag (Student’s t test; p < 0.01).

These data suggested that the bioactive region may be contained within or formed by an ND1 and CD2 or ND2 and CD1 interaction.

A construct containing the D1 and D2 regions with the ECH retained flagellin inflammatory activities

Based on the results obtained from N/C flag, ND1/ECH/CD1, and ND2/ECH/CD2 proteins, it was hypothesized that the bioactive domain was contained in a region generated by a combination of the amino and carboxyl D1 and D2 regions. A chimeric SD flagellin fusion protein was produced that contained a combination of the ND1 and ND2 and CD1 and CD2 regions separated by the ECH. This fusion protein lacks the entire D3 region and was designated ND1–2/ECH/CD2–1 (Fig. 1⇑). Similar to Caco-2BBe cells stimulated with 1 μg/ml 6HIS flag, ND1–2/ECH/CD2–1 stimulation induced IκBα degradation at 20 and 30 min (Fig. 5⇓A) and NF-κB activation at 1 h poststimulation (Fig. 5⇓B). IFN-γ-primed DLD-1 cells stimulated for 18 h with ND1–2/ECH/CD2–1 also showed significantly higher levels of NO production vs untreated control cells (p < 0.001; Fig. 5⇓C). This trend continued when substantial levels of IL-8 were detected 22 h poststimulation with 1 μg/ml ND1–2/ECH/CD2–1 comparable to purified native SD flagellin, equal to 6HIS flag, and significantly higher than unstimulated cells (p < 0.001; Fig. 5⇓D).

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FIGURE 5.

SD flagellin fusion protein representing the combination of the ND1 and ND2 and CD1 and CD2 regions, separated by the ECH, designated ND1–2/ECH/CD 2–1, induced IκBα degradation and NF-κB activation, NO induction, and IL-8 secretion. A, A loss of IκBα was observed at 20 and 30 min in Caco-2BBe cells following incubation with 1 μg/ml ND1–2/ECH/CD2–1 comparable to 6HIS flag as determined by immunoblotting. Unstimulated cells (C) showed no IκBα loss. B, EMSA demonstrating NF-κB activation in Caco-2BBe cells. Nuclear extractions from unstimulated cells (C) showed no NF-κB activation. NF-κB activa-tion was observed in Caco-2BBe cells following 60 min of stimulation with a combination of IL-1β (0.5 ng/ml) and IFN-γ (100 U/ml), 1 μg/ml 6HIS flag, or ND1–2/ECH/CD2–1. However, no NF-κβ activation was seen in cells incubated with 1 μg/ml ND2/D3/CD2 fusion protein. C, Substantial NO production was generated by the combination of IL-1β and IFN-γ DLD-1 cells. ND1–2/ECH/CD2–1, at a dose of 1 μg/ml, induced high levels of NO production nearly equal to cells stimulated with the same dose of purified SD flagellin or 6HIS flag in IFN-γ-primed DLD-1 cells. NO production could not be detected in unstimulated cells (C). D, High IL-8 secretion was detected in Caco-2BBe cells incubated with 1 μg/ml ND1–2/ECH/CD2–1 (954.5 pg/ml) comparable and nearly equal to the positive control of IL-1β and IFN-γ (1000 pg/ml) purified SD flag (1000 pg/ml), or 6HIS flag (994 pg/ml). IL-8 secretion in unstimulated cells (C) was undetectable.

6HIS flag and ND1–2/ECH/CD2–1 stimulated TNF-α secretion in a human monocyte cell line

The bioactivity of the purified native SD flagellin, 6HIS flag, ND1–2/ECH/CD2–1, and each of the other recombinant flagellin proteins also was established by stimulation of 5 × 10⁶ THP-1 monocytes. Following 4 h of stimulation, significantly higher levels of TNF-α were detected in THP-1 cells stimulated with purified native SD flagellin, 6HIS flag, ND1–2/ECH/CD2–1, and N/C flag vs unstimulated control cells (p < 0.001; Fig. 6⇓). N/C flag, however, stimulated significantly less TNF-α production in cells compared with SD flagellin, 6HIS flag, and ND1–2/ECH/CD2–1 (p < 0.005; Fig. 6⇓). None of the other constructs induced TNF-α production in these cells (Fig. 6⇓), a finding consistent with the IEC results.

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FIGURE 6.

TNF-α induction in human monocytes was stimulated by the SD flagellin fusion protein, ND1–2/ECH/CD2–1, that represents the combination of the conservative ends separated by an ECH element. THP-1 cells were stimulated with 1 μg/ml of the various recombinant flagellin proteins. Supernatants collected 4 h poststimulation were analyzed by ELISA to determine TNF-α levels. Cells (5 × 10⁶) exposed to 1 μg/ml of ND1–2/ECH/CD2–1 produced high levels of TNF-α (900 pg/ml,) comparable to purified SD flagellin (991.5 pg/ml), or 6HIS flag (987 pg/ml). Moderate levels of TNF-α were produced in monocytes stimulated with 1 μg/ml N/C flag (587.3) vs the positive control of purified SD flag or 6HIS flag. Unstimulated cells (C) or cells stimulated with the remaining flagellin fusion proteins stimulated production of minimal levels of TNF-α in THP-1 cells.