Bacterial flagellin is a dominant antigen in Crohn disease (original) (raw)

Isolation of genomic DNA of mouse cecal bacterium. Pelleted bacteria from C3H/HeJBir mouse ceca were inactivated at 80°C for 20 minutes and then were treated with 2 ml lysozyme (20 mg/ml in Tris-EDTA [TE] buffer) for 1 hour at 37°C. This solution was rocked at room temperature for 10 minutes with 40 ∝l proteinase K (10 mg/ml) and 140 ∝l 20% SDS (Sigma-Aldrich, St. Louis, Missouri, USA) and then incubated for 15 minutes at 65°C, then 0.4 ml of 5 M NaCl and 0.32 ml of a 10% cetyltrimethylammonium bromide (CTAB) solution (1 g CTAB [Sigma-Aldrich], 1.4 ml 5M NaCl, and 8.6 ml distilled H2O) was added, followed by incubation at 65°C for 10 minutes. DNA was then extracted twice with phenol, followed by extraction with phenol/chloroform/isoamyl alcohol (24:24:2), and then with chloroform. Finally the DNA was precipitated with 0.6 volumes of isopropanol and resuspended in TE buffer.

Genomic expression library construction. A detailed description of library construction can be found elsewhere (19). Briefly, 20 ∝g of genomic DNA of mouse cecal bacterium was resuspended in 400 ∝l of TE buffer and was sonicated for five seconds at 30% continuous power with a Sonic Dismembrator (Fisher Scientific, Pittsburgh, Pennsylvania, USA) to generate fragments of approximately 0.5–5.0 kb. DNA fragments were blunted with T4 DNA polymerase (Invitrogen, Carlsbad, California, USA) and were ligated to _Eco_RI adaptors (Stratagene, La Jolla, California, USA) with T4 DNA ligase (Stratagene). Adapted inserts were then phosphorylated with T4 polynucleotide kinase (Stratagene) and were selected by size with a Sephacryl 400-HR column (Sigma-Aldrich). Approximately 0.25 ∝g of insert was ligated to 1.0 ∝g Lambda ZAP Express Vector treated with _Eco_RI and calf intestinal alkaline phosphatase (Stratagene), and the ligation mix was packaged with Gigapack III Gold packaging extract (Stratagene) following the manufacturer’s instructions.

Expression screening. Immunoreactive proteins were screened from approximately 6 ∞ 105 plaque-forming units (PFU) of the unamplified cecal bacterium expression lambda library. Briefly, twenty 150-mm petri dishes were plated with E. coli XL1-Blue MRF′ host cells (Stratagene) and approximately 3 ∞ 104 PFU of the unamplified library and were incubated at 42°C until plaques formed. Dry nitrocellulose filters (Schleicher and Schuell, Keene, New Hampshire, USA), pre-wet with 10 mM isopropyl β-thiogalactopyranoside (IPTG), were placed on the plates, which were then incubated overnight at 37°C. Filters were removed and washed three times with PBS containing 0.1% Tween 20 (PBST) (Sigma-Aldrich), blocked with 1.0% BSA (Sigma-Aldrich) in PBST, and washed three times with PBST. Filters were next incubated overnight with E. coli lysate–adsorbed C3H/HeJ Bir mouse serum (1:200 dilution in PBST), washed three times with PBST, and incubated with a goat anti-mouse IgG + IgA + IgM (heavy and light chain) alkaline phosphatase–conjugated secondary antibody (diluted 1:10,000 with PBST; Jackson Laboratories, West Grove, Pennsylvania, USA) for 1 hour. Filters were finally washed three times with PBST and two times with alkaline phosphatase buffer (pH 9.5) and were developed with nitroblue tetrazolium chloride/5-bromo-4-chloro-3-indolylphosphate _p_-toluidine salt (Invitrogen). Reactive plaques were then isolated and a second or third plaque purification was performed. Excision of phagemid followed the Stratagene Lambda ZAP Express protocol, and the resulting plasmid DNA was sequenced with an automated sequencer (ABI, Foster City, California, USA) using M13 forward, reverse, and sequence-specific internal DNA sequencing primers. Nucleic acid and predicted protein sequences were used to search the GenBank nucleotide and translated databases. Protein analysis was performed with the PSORT program (National Institute for Basic Biology, Okazaki, Japan) and with the IDENTIFY program of EMOTIF (Department of Biochemistry, Stanford University). Sequence alignments were produced with the MegAlign program (Clustal) of DNAStar (Madison, Wisconsin, USA). Note that 20 random clones from the lambda library were picked and sequenced prior to serolological expression cloning. None of the 20 were found to be derived from mouse DNA and no flagellins were identified.

Cloning of full-length flagellins representing clones CBir1 and Fla-X. Clone CBir1 contains the conserved NH2 and variable regions of an unknown immunoreactive flagellin. The full-length sequence was obtained by first amplifying the unknown CBir1 carboxy terminus from total genomic cecal bacterium DNA with Expand polymerase (Roche, Indianapolis, Indiana, USA) and the primers CBir1var1 (designed from the variable region of CBir1; CACAATCACAACATCTACCCAG) and CBir1 Carb Z (designed from the carboxy terminus of the related flagellin B of Butyrivibrio fibrisolvens, GenBank accession number AF026812; 5′-TTACTGTAAGAGCTGAAGTACACCCTG-3′). This PCR product was cloned with a Zero Blunt TOPO PCR Cloning Kit (Invitrogen), digested with _Eco_RI, and gel-isolated (carboxy end of CBir1). Clone CBir1 plasmid DNA, which represents the NH2 terminus plus flagellin central variable region and overlaps with the cloned carboxy region, was digested with _Sca_I and then gel-isolated. Both overlapping (181-bp) DNA fragments (approximately 20 ng each) were added to a PCR reaction with the primers CBir1 HIS and CBir1 TERMX (see below), and the amplification product was cloned and expressed as described below.

Fla-X is an immunoreactive full-length flagellin sequence with no known identity in the public databases. Full-length flagellin Fla-X was cloned from total cecal bacterium genomic DNA by PCR amplification with the primers CBir Fla-X HIS (5′-CAATTACATATGCATCACCATCACCATCACGTAGTACAGCACAATC-3′) and CBir1 TERMX (5′-ATAGACTAAGCTTACTGTAAGAGCTGAAGTACACCCTG-3′), and was expressed as described below. The amplification product was cloned with a Zero Blunt TOPO PCR Cloning Kit (Invitrogen), and several clones were sequenced.

Recombinant protein. Recombinant Salmonella muenchen flagellin (≥95% pure by SDS-PAGE) was obtained from Calbiochem (La Jolla, California, USA). Expression of other recombinant flagellin proteins and deletion constructs was accomplished by amplification from the cloned plasmid or genomic DNA (full length Fla-X) with Pfu polymerase (Stratagene) and the following primer pairs: for full-length CBir1, CBir1 HIS (5′-CAATTACATATG_CATCACCATCACCATCAC_GTAGTACAGCACAATTTACAGGC-3′) and CBir1 TERMX (5′-ATAGACTAAGCTTACTGTAAGAGCTGAAGTACACCCTG-3′); for the CBir1 NH2 plus variable regions, CBir1 HIS and CBir1 AV TERM (5′-ATAGACTAAGCTTAAGAAACCTTCTTGATAGCGCCAG-3′); for the CBir1 NH2 terminus, CBir1 HIS and CBir1 A TERM (5′-TAGACTGAATTCTAGTCCATAGCGTCAACGTTCTTTGTGTC-3′); for the CBir1 carboxy terminus, CBir1 C HIS (5′-CAATTACATATG_CATCACCATCACCATCAC_AAGATGAACTTCCATGTAGGTGC-3′) and CBir1 TERMX; for full-length Fla-X, CBir Fla-X HIS (5′-CAATTACATATG_CATCACCATCACCATCAC_GTAGTACAGCACAATC-3′) and CBir1 TERMX (ATAGACTAAGCTTACTGTAAGAGCTGAAGTACACCCTG-3′); for the Fla-X NH2 plus variable regions, Fla-X HIS (5′-CAATTACATATG_CATCACCATCACCATCAC_GTAGTACAGCACAATCTTAGAGC-3′) and Fla-X AV TERM (5′-ATAGACTAAGCTTAGAGGCTGAAATCAATGTCCTCG-3′); for the Fla-X NH2 terminus, Fla-X HIS and Fla-X A TERM (5′-ATAGACTAAGCTTAATGTGCTGAAAGATATCTTGTCAC-3′); and for the Fla-X carboxy terminus, Fla-X C HIS (5′-CAATTACATATG_CATCACCATCACCATCAC_TTCAGCCTCCATGTAGGTGCAGATGC-3′) and CBir1 TERMX. Primers contained restriction sites for cloning (in bold) and a six-histidine tag (in italics) for protein purification (NH2 terminus). The amplification products were digested with the restriction enzymes _Nde_I and _Hin_dIII or _Eco_RI, depending on the primer set used, gel-isolated, and ligated to a pET 17b plasmid vector (Novagen, Madison, Wisconsin, USA) previously cut with _Nde_I and with _Hin_dIII or _Eco_RI and dephosphorylated with alkaline phosphatase (MB grade; Roche). The ligation mix was transformed into XL1 Blue competent cells (Stratagene) and plasmid DNA was prepared for sequencing (Qiagen, Valencia, California, USA). Recombinant protein was expressed by transformation of plasmid DNA into BL21 pLysS competent cells (Novagen) and induction of a single-colony cell culture with 2 mM IPTG (Sigma-Aldrich). Recombinant protein was recovered from cell lysate with nickel-nitrilotriacetic acid agarose beads (Qiagen), following the manufacturer’s instructions, and was dialyzed in 10 mM Tris, pH 4–11 depending on predicted recombinant pI characteristics. Recombinant proteins were “quality-checked” for purity by SDS-PAGE followed by staining with Coomassie blue and by NH2-terminal protein sequencing, and were quantified with a Micro BCA assay (Pierce, Rockford, Illinois, USA). Recombinants were assayed for endotoxin contamination with the Limulus assay (Bio Whittaker, Walkersville, Maryland, USA). Production of the Mycobacterium tuberculosis 38-kDa protein has been described previously (36).

ELISA. Ninety-six–well EIA/RIA microtiter plates (3369; Corning Costar, Cambridge, Massachusetts, USA) were coated overnight at 4°C with 100 ng/well of the recombinant proteins. Solutions were then aspirated from the plates, which were then blocked for 2 hours at room temperature with PBS containing 1% (weight/volume) BSA. This was followed by washing in PBST. Serum diluted in PBS containing 0.1% BSA was added to wells and incubated for 30 minutes at room temperature, followed by washing six times with PBST and then incubation with secondary antibody–HRP conjugate (1:10,000 dilution) for 30 minutes. Plates were then washed six times in PBST and then were incubated with tetramethylbenzidine (TMB) substrate (Kirkegaard and Perry, Gaithersburg, Maryland, USA) for 15 minutes. The reaction was stopped by the addition of 1 N sulfuric acid, and plates were “read” at 450 nm using an ELISA plate reader (Biotek instrument EL311, Hyland Park VA). Background values were determined by reading of reactions that lacked the primary antibody step.

Western blot analysis. Recombinant antigens (50–200 ng/lane) were subjected to SDS-PAGE analysis using 15% polyacrylamide “minigels.” The antigens were transferred to nitrocellulose BA-85 (Schleicher & Schuell, Keene, New Hampshire, USA) and were blocked for 1 hour at room temperature with PBS containing 1% Tween 20. Blots were then washed three times, 10 minutes each wash, in PBST. Next, blots were probed for 1 hour at room temperature with serum diluted 1:500 in PBST followed by washing three times, 10 minutes each wash, in PBST. Blots were then incubated for 30 minutes at room temperature with secondary antibody–HRP diluted 1:10,000 in wash buffer and were again washed three times for 10 minutes each wash in PBST containing 0.5 M sodium chloride. Finally, blots were incubated in chemiluminescent substrate for ECL (Amersham Plc, Little Charlton, UK) for about 1 minute and then were exposed to X-ray film (XAR5) for 10–60 seconds, as required.

CD4+ T cell isolation and culture, and generation of a Cbir1-specific T cell line. CD4+ T cells were isolated from mesenteric lymph nodes (MLNs) of mice with BD IMAG anti-mouse CD4 beads according to the manufacturer’s instructions (BD Biosciences Pharmingen, San Diego, California, USA). Briefly, MLN cells were labeled with anti-CD4 beads and then were placed within the magnetic field of the BD Imagnet. The unlabeled cells in suspension were removed and the cells binding to beads were washed and used in the CD4+ T cell culture. More than 99% of cells were CD4+, as shown by flow cytometry (data not shown). For the generation of a T cell line reactive to CBir1, CD4+ T cells were isolated from MLNs of C3H/HeJBir mice as described above and were cultured with splenic APCs that were pulsed with CBir1 (100 mg/ml) overnight. The cells were restimulated every 10–14 days.

Antigen-specific proliferation of T cells. Spleen and MLN CD4+ T cells, isolated as described above, or a CBir1 flagellin–specific T cell line (4 ∞ 105 cells/well) were incubated in triplicate in the presence of antigen-pulsed, irradiated APCs (4 ∞ 105 cells per well; treated with 1–100 ∝g/ml antigen for 18 hours at 37°C) in 96-well flat-bottomed tissue culture plates (Falcon, Lincoln Park, New Jersey, USA) at 37°C in 5% CO2 humidified air. [3H]thymidine (0.5 ∝Ci) (New England Nuclear, Boston, Massachusetts, USA) was added at day 3 of culture and the cells were harvested at 16 hours after the pulse. The cells were harvested on glass fiber filters on a PHD cell harvester (Cambridge Technology Inc., Watertown, Massachusetts, USA), washed with distilled water, and dried. Proliferation was assessed as the amount of incorporation of [3H]thymidine into cell DNA, as measured by beta scintillation counting (Beckman Instruments, Palo Alto, California, USA) of the harvested samples, and was expressed as cpm ± SD. The preparation of epithelial cell proteins and food antigens has been described previously (14). Ethical approval for animal studies was obtained from the Institutional Animal Care and Use Committee at the University of Alabama (Birmingham, Alabama) and from Corixa Corporation.

Specificity of CD4+ T cell stimulation. APCs were BALB/c spleen cells that were pulsed for 24 hours with nothing, OVA peptide at 2 ∝g/ml, CBir1 at 100 ∝g/ml, or Fla-X at 100 ∝g/ml, alone or in combinations as shown in Table 2. These APCs were washed and irradiated with 3,000 rads prior to culture. CD4+ T cells were isolated from DO11.10 mice and were cultured at a density of 1 ∞ 105 with 4 ∞ 105 prepulsed APCs. [3H]TdR was added at day 3 of culture and the cells were harvested after 16 hours.

Adoptive transfer. CD4+ T cells were cultured with cecal bacterial antigen–pulsed and irradiated C3H/HeJ splenic cells in complete medium at 37°C for 4 days in 5% CO2 air before being transferred intravenously into C3H/HeSnJ scid/scid recipients. Three months later, the recipients were killed and then the cecum and the proximal, medial, and distal portions of the colon were fixed in formalin. Fixed tissues were embedded in paraffin, and sections were stained with hematoxylin and eosin for histological examination. All slides were “read” by an experienced pathologist (A. Lazenby, Department of Pathology, University of Alabama at Birmingham) without knowledge of their origin.

Human subjects. Serum samples from 212 subjects (50 UC patients, 100 CD patients, 22 DCs, and 40 NCs) were obtained from the serum archive of the Cedars-Sinai IBD Research Center. Sera were produced from standard phlebotomy blood specimens and were given an “anonymous” number code, divided into aliquots, and stored at –80°C until use. The UC and CD patient specimens were obtained from a genetic case-control study (37, 38). Each patient’s diagnosis was confirmed by clinical history, endoscopic and radiologic examination, and histopathology findings. The NC group is a collection of environmental controls that contain sera from individuals with no symptoms/signs of disease (i.e., spouses). DC samples include sera from patients with presumed infectious enteritis (stool culture negative for specific pathogens), blastocystis, celiac disease, collagenous colitis, irritable bowel syndrome, radiation proctitis, and acute schistosomiasis. The UC group includes both pANCA-positive and -negative specimens, while the CD group contains samples that are marker-negative, ASCA+; I2+; OmpC+ (I2-positive); OmpC+, I2+, and ASCA+; and pANCA+. Ethical approval for human studies was obtained from the institution review board at Cedars-Sinai Medical Center.

Nucleotide sequence accession numbers. The nucleotide sequence data for the flagellins CBir1 and Fla-X have been assigned GenBank accession numbers AY551005 and AY551006, respectively.