Wheat has only recently been added to the human diet (since the Neolithic age, i.e., roughly 12,000 years ago; Charmet, 2011). Today wheat has become the world’s major staple, and wheat products are widely used ingredients in processed foods. Wheat consumption correlates with certain disorders like wheat allergies and especially celiac disease (Shewry et al., 2003). Celiac disease is a common small intestinal enteropathy caused by dietary gluten in wheat, barley, and rye and affects ∼1% of most populations (Fasano et al., 2003; Green and Cellier, 2007; Di Sabatino and Corazza, 2009; Schuppan et al., 2009) Wheat gluten represents a family of largely water-insoluble storage proteins, subdivided into gliadins and glutenins, whereas other proteins are extractable as water-soluble albumins and salt-soluble globulins (Shewry et al., 2003; Wieser, 2007). Because of their unusual structure, with a high proline and glutamine content, the gluten proteins are partly resistant to intestinal enzymes, which leads to several nondegraded immunogenic peptides that can be sensed by the intestinal immune system. These gluten peptides are bound by the human lymphocyte antigen HLA-DQ2 or HLA-DQ8 (the major and necessary genetic predisposition for celiac disease) on intestinal antigen-presenting cells, and this binding is potentiated by the ubiquitous enzyme tissue transglutaminase (TG2), the celiac disease autoantigen (Dieterich et al., 1997). TG2 deamidates certain glutamine residues in (immunodominant) gluten peptides to glutamic acid, thereby increasing the peptides’ binding affinity to HLA-DQ2 and HLA-DQ8 and the subsequent T cell activation (Dieterich et al., 1997; van de Wal et al., 1998). This results in villus atrophy and crypt hyperplasia of the intestinal mucosa, the histological hallmarks of celiac disease, and frequent nutrient malabsorption and may promote certain celiac disease–associated autoimmune disorders (Green and Cellier, 2007; Di Sabatino and Corazza, 2009; Schuppan et al., 2009). Although several HLA-DQ2– and HLA-DQ8–restricted gluten peptides that trigger the adaptive immune response in celiac disease have been identified (Molberg et al., 1998; Anderson et al., 2000; Shan et al., 2002), only 2–5% of individuals expressing these HLAs develop the disease, indicating additional mechanisms of celiac disease pathogenesis, especially innate immune activation. The innate immune system provides an early response to many microbial and chemical stimuli and is critical for successful priming of adaptive immunity. Responsive innate cells are primarily macrophages, monocytes, DCs, and polymorphonuclear leukocytes that by means of their pattern-recognition receptors, such as TLRs, induce the release of proinflammatory cytokines and chemokines, resulting in recruitment and activation of additional inflammatory cells (Medzhitov, 2007). Thus, peptides p31-43 or p31-49 from α-gliadin that lack adaptive stimulatory capacity were incriminated as triggers of innate immunity as they induced IL-15 and Cox-2 expression in patients’ biopsies (Maiuri et al., 2003) and MHC class I polypeptide–related sequence A (MICA) on intestinal epithelial cells (Hüe et al., 2004). However, these studies were difficult to reproduce in cell culture, and no receptor responsible for the observed effects could be identified. In cell culture, gliadin was reported to induce increased expression of co-stimulatory molecules and the production of proinflammatory cytokines in monocytes and DCs (Nikulina et al., 2004; Cinova et al., 2007). In addition, the chemokine receptor CXCR3 was implicated in increased intestinal epithelial permeability upon gliadin challenge in a MyD88-dependent manner (Thomas et al., 2006; Lammers et al., 2008). However, no defined gliadin peptide was reproducibly identified. Collectively, a clear picture of the role of the innate immune system in celiac disease has not emerged. In this study, we show that members of the nongluten α-amylase/trypsin inhibitor (ATI) family contained in wheat and related cereals are strong inducers of innate immune responses in human and murine macrophages, monocytes, and DCs. ATI family members activate the TLR4–MD2–CD14 complex and elicit strong innate immune effects not only in vitro but also in vivo after oral or systemic challenge. Our findings have broad implications not only for celiac disease but also for other intestinal inflammatory disorders of the gastrointestinal tract. RESULTS Gliadin digests induce innate immune responses Human monocytic THP-1 and U937 cells were exposed to pepsin/trypsin (PT)-digested gliadin mimicking intestinal digestion. PT-digested zein, a mixture of storage proteins from corn which is functional homologous to gliadin but lacks immunogenic peptides, served as a negative control. PT gliadin but not zein caused a dose-dependent secretion of IL-8, TNF, and MCP-1 (monocyte chemotactic protein-1) in both cell lines (Fig. 1, A and B; and not depicted) in accordance with other studies (Nikulina et al., 2004; Cinova et al., 2007). To rule out LPS contamination as a trigger of innate responses, gliadin, LPS, and TNF were digested with proteinase K to eliminate peptide-induced responses. Both TNF and PT gliadin lost their stimulatory capacity, whereas LPS was still able to induce IL-8 (Fig. 1 C), indicating that the stimulatory effect of gliadin was caused by protein and not LPS contamination. Interestingly, exposure of primary human monocyte-derived DCs to PT gliadin and LPS led to a similar cytokine profile in response to gliadin. In contrast, the intestinal epithelial cell line HT29 showed only a minor response (not depicted). Figure 1. Gliadin leads to secretion of inflammatory cytokines. (A and B) THP-1 cells were treated with PT gliadin, and IL-8 (A) and MCP-1 (B) secretion was measured. LPS and PT zein served as positive and negative controls, respectively. (C) Proteinase K digests of PT gliadin, TNF, and LPS were added to THP-1 cells, and IL-8 secretion was used as read out of innate activation. (D) PT gliadin stimulation of monocyte-derived DCs of healthy controls (n = 10) and of patients with celiac disease on a gluten-free (gfd; n = 8) or regular diet (n = 3). Graph shows one representative patient per group. (E) DCs of healthy controls were stimulated with PT gliadin and subjected to analysis by flow cytometry. The dark gray and dotted histograms represent PT gliadin and LPS stimulation, respectively, the light gray histograms show PT zein stimulation, and the dashed lines represent the unstimulated control. (F) HT29, U937, and THP-1 cells were stimulated with gliadin peptide p31-43 or a scrambled control peptide, and IL-8 secretion was measured. *, P 90% of total gliadin, harbored stimulatory activity, whereas IL-8 release was strongly induced by PT-digested ω1.2- and ω5-gliadins (Fig. 3 A). The lack of stimulation by α- and γ-gliadins was not caused by toxic effects because addition of LPS or whole PT gliadin fully restored the stimulatory capacity (Fig. 3 A). Furthermore, ω1.2- and ω5-gliadins strongly induced IL-8 secretion in TLR4-transfected but not untransfected HEK-293 cells (Fig. 3, B and C). Figure 3. Gliadin-induced innate immune responses are elicited by wheat ATI, a protein copurifying with ω-gliadins. (A) Stimulation of THP-1 cells with α-, γ-, ω1.2-, and ω5-gliadin fractions (all 100 µg/ml) isolated from the pure wheat strain Rektor. Co-incubation of α- and γ-gliadin with 100 µg/ml of regular PT gliadin from Sigma-Aldrich served as cell viability control. LPS was used as positive control, whereas PT or PT zein served as negative control. (B and C) IL-8 secretion after stimulation with 100 µg/ml ω-gliadins in TLR4-transfected (B) and in untransfected HEK-293 cells (C). 10 ng/ml PMA served as cell viability control. 10 ng/ml LPS, 100 µg/ml PT gliadin, or 100 µg/ml of a PT digest of Rektor gliadin (PT Rektor) served as positive control, and 100 µg/ml PT zein, 1 µg/ml Pam3CSK4, or a PT mixture (PT ctrl) served as negative controls. (D) Stimulatory capacity of synthetic overlapping 20mers of ω5-gliadin in TLR4-transfected HEK-293 cells. For illustration purposes, 9 fractions each were pooled in the stimulation experiments (each fraction at a concentration of 100 µg/ml), while also all 43 fractions were tested individually. LPS served as positive and Pam3CSK4 or PT zein as negative controls. (E and F) Dose response of IL-8 release by monocyte-derived DCs stimulated with water-soluble (ws) gliadin (which is enriched in ATI; E) or with purified ATI (F). LPS and water-soluble zein served as positive and negative controls, respectively. (G) Secretion of IL-12 in monocyte-derived DCs from healthy subjects upon stimulation with ATI and PT gliadin in the presence of 1,000 U/ml Interferon-γ as co-stimulatory protein. LPS and PT zein served as positive and negative controls, respectively. (H) Effect of proteinase K digestion of ATI and LPS on IL-8 secretion in DCs. (I) KC secretion in peritoneal macrophages isolated from MyD88−/− mice compared with C57BL/6J wild-type mice upon ATI or water-soluble gliadin stimulation. LPS and water-soluble zein served as positive and negative controls, respectively. (J) IL-8 secretion of monocyte-derived DCs stimulated with ATI and LPS after preincubation with anti-TLR4 or anti-CD14 antibodies. TLR2 agonist Pam3CSK4 served as positive control. *, P 80% according to HPLC and mass spectrometry analysis. 43 20mer peptides with an overlap of 10 aa covering the 439-residue ω5-gliadin were synthesized at 60–80% purity by Primm Biotechnology. Cell culture and in vitro stimulation experiments. THP-1, U937, HEK-293 (all from American Type Culture Collection), and HEK-293 cells stably transfected with the TLR4–CD14–MD2 complex (InvivoGen) were cultured in complete RPMI or DMEM (Cellgro) supplemented with 100 IU/ml penicillin/100 µg/ml streptomycin and 10% fetal calf serum at 37°C in a 5% CO2 atmosphere. All cell lines were tested for mycoplasma contamination on a regular basis and proved to be mycoplasma free. Peripheral monocytes were isolated from 40 ml blood obtained from 11 patients with celiac disease (8 on a gluten-free and 3 on a gluten-containing diet) during their diagnostic workup (median 37 yr, range 18–59 yr) after prior informed consent, and healthy control monocytes were from buffy coats of leukopheresis concentrates of anonymous blood donors (approval #2006-P-000117/4, Institutional Review Board of the Beth Israel Deaconess Medical Center). Peripheral blood mononuclear cells were isolated as described previously (Vissers et al., 1988), and CD14-positive monocytes were purified by MACS separation according to the manufacturer’s protocol (Miltenyi Biotec). For generation of DCs, monocytes were cultured in RPMI supplemented with 10% fetal calf serum, 200 U/ml rhIL-4, and 300 U/ml rhGM-CSF (both from PeproTech) for 6–8 d. Murine resident peritoneal macrophages were isolated by peritoneal lavage using a 3-ml syringe and 18G needles. 3 × 3 ml of sterile PBS was injected into the peritoneal cavity and reaspirated after gentle massage of the abdomen (Fortier et al., 1982). For further purification, MACS separation for CD11b-positive cells was performed according to the manufacturer’s protocol. All animal experiments were approved by the review committee of the Beth Israel Deaconess Medical Center (protocol 031-2008). To elicit IL-12 secretion, human DCs were stimulated with PT gliadin or ATI in the presence of 1,000 U/ml Interferon-γ as co-stimulatory protein. For stimulation, 106/ml cells were seeded in triplicates on polystyrene wells. Unless stated otherwise, supernatants were harvested 16 h after addition of the stimulants. Exclusion of LPS contamination. To prove that stimulatory effects were caused by protein, PT gliadin, wheat ATI, LPS, or TNF was incubated with or without 20 µg/ml proteinase K (Promega) for 4 h at 56°C. After proteinase K inactivation by boiling for 5 min, the digests were used for cell stimulation. Coimmunoprecipitation. A soluble flag-tagged TLR4/MD2 construct (gift of W. Falk, University of Regensburg, Regensburg, Germany; Brandl et al., 2005) was expressed in HEK-293 cells. The supernatant was harvested, and protein was concentrated using a 100-kD size exclusion cartridge (EMD Millipore). Purified wheat ATI was biotinylated using 10 mM N-Hydroxysulfosuccinimide (NHS) esters of biotin solution according to the manufacturer’s protocol (Thermo Fisher Scientific). 1 µg of flag-tagged TLR4/MD2 was then incubated with or without 10 µg ATI for 2 h at 4°C, followed by incubation with streptavidin-agarose for 1 h. Precipitates were boiled at 100°C, and eluates were run on a 4–20% gradient SDS gel followed by Western blot analysis using a horseradish peroxidase–labeled rabbit anti-flag antibody (Sigma-Aldrich). Reduction and alkylation on enriched ATIs from spring wheat. 1 mg of a neutral extract enriched in ATIs from spring wheat (extracted with 50 mM ammonium bicarbonate or 0.05% acetic acid) was reconstituted in 1 ml of 100 mM Tris, pH 8.5, and incubated with 100 mM dithiothreitol (DTT; Promega) at 37°C for 1 h. Samples were alkylated with iodoacetamide (Sigma-Aldrich) at a final concentration of 250 mM for 30 min at room temperature in the dark. Finally, 200 mM DTT was added to quench iodoacetamide, and protein was purified using spin columns (Amicon-Ultra 3K; EMD Millipore). ATI treated in the same way but without the reducing and alkylating agents was used as control. Samples were freeze dried, reconstituted in PBS, and filtrated. Then they were added to the monocytic cell line U937 (106/ml) in triplicates at a concentration of 50 µg/ml and incubated overnight. IL-8 from cell supernatants was measured by ELISA according to the manufacturer’s protocol. Animals. C3H/HeJ, C3H/HeOuJ, and Rag1−/− mice were obtained from the Jackson Laboratory. MyD88−/− mice were a gift from S. Akira (Osaka University, Osaka, Japan; Kawai et al., 1999). Congenic C57BL/6J mice served as experimental controls and were bred under the same conditions in the same facility. All experiments were performed with mice at age 5–7 wk. In vivo experiments. Mice were injected intraperitoneally with water-soluble gliadin, zein (500 µg/g body weight), or LPS (1 µg/g body weight) in 200 µl PBS or PBS alone (negative control). 2 h after injection, mice were euthanized by ketamine/xylazine administration, and blood was drawn by retroorbital bleeding. Mice were either raised on gluten-free diet (C57BL/6J and MyD88−/− mice) or put on gluten-free diet for at least 2 wk (C3H/HeJ and C3H/HeOuJ mice) and starved the night before the experiment. Gluten-free standardized diet AIN-76A was obtained from Research Diets. All stimulants were diluted in PBS. Mice were administered gliadin, zein (2 mg/g mouse weight), LPS (20 µg/g mouse weight), or ATI (0.075 mg/g mouse weight) in 200 µl PBS via gavage. Mice were euthanized after 4 h, and the duodenum was snap frozen in liquid nitrogen. Cytokine/chemokine assays. The concentration of IL-8, TNF, IL-12, MCP-1, RANTES, and mouse IL-8 (KC) in cell culture supernatants and serum samples was determined using validated ELISAs (IL-8 and hTNF [BD]; MCP-1, hRANTES, mRANTES, KC, and IL-12 [R&D Systems]; and mTNF [eBioscience]) according to the manufacturers’ protocols. RNA isolation and quantitative RT-PCR. Samples from the small intestine (0.5-cm segment, 2 cm distal to the pylorus) were collected at sacrifice and snap frozen for further analysis. Total RNA isolation was performed using TRIzol (Invitrogen). Exon–exon boundary-spanning primer sequences were obtained from PrimerBank, and sequences are listed in Table 1. Real-time PCR was performed using 480 SYBR Green I master mix (Roche) and a LightCycler 480 system (Roche). Mouse GAPDH served as endogenous control. PCR was set up in triplicates, and threshold cycle (Ct) values of the target genes were normalized to the endogenous control. Differential expression was calculated according to the 2-ΔΔCT method. Table 1. Primers used in this study Cytokine/chemokine Forward primer Reverse primer mKC 5′-CTGGGATTCACCTCAAGAACATC-3′ 5′-CAGGGTCAAGGCAAGCCTC-3′ mTNF 5′-CCCTCACACTCAGATCATCTTCT-3′ 5′-GCTACGACGTGGGCTACAG-3′ mIL-1β 5′-GCAACTGTTCCTGAACTCAACT-3′ 5′-ATCTTTTGGGGTCCGTCAACT-3′ mIL-6 5′-TAGTCCTTCCTACCCCAATTTCC-3′ 5′-TTGGTCCTTAGCCACTCCTTC-3′ mMCP-1 5′-TTAAAAACCTGGATCGGAACCAA-3′ 5′-GCATTAGCTTCAGATTTACGGGT-3′ hGAPDH 5′-TGGTAAAGTGGATATTGTTGCC-3′ 5′-GGTGAAGACGCCAGTGGAC-3′ hIL-8 5′-GGAAGGAACCATCCTCACTGT-3′ 5′-CCACTCTCAATCACTCTCAG-3′ Blocking experiments. Monocyte-derived DCs were seeded at a concentration of 1 × 106/ml in 96-well plates. Cells were preincubated with blocking antibodies (10 µg/ml rat anti-TLR4 [InvivoGen] and 20 µg/ml goat anti-CD14 [R&D Systems]) for 3 h at 37°C before stimulation. Flow cytometry. Human monocyte-derived DCs were stimulated with LPS, PT gliadin, and PT zein overnight. For flow cytometry, cells were preincubated with FcR blocking reagent (Miltenyi Biotec) for 15 min at 4°C before staining with monoclonal antibodies (final concentration 10 µg/ml; all from eBioscience) for 30 min at 4°C. Cells were then washed with staining buffer (PBS and 1% BSA), cell viability was assessed by DAPI exclusion (0.1 µg/ml; Roche), and only viable cells were analyzed by flow cytometry using a four-laser LSRII (BD) and FlowJo software (Tree Star). Identification of ATI by mass spectrometry. The 15-kD protein band that copurified with ω-gliadins was subjected to in-gel disulfide reduction and tryptic digestion, followed by nanoflow reverse phase chromatography and tandem mass spectrometry on a 4800Plus MALDI-TOF/TOF instrument (AB SCIEX) as described in detail elsewhere (Kornek et al., 2011; Krishnamurthy et al., 2011). Recombinant expression of ATI CM3 and 0.19 proteins. To exclude bacterial contaminants, recombinant flag-tagged ATI CM3 and 0.19 were generated in eukaryotic mycoplasma-free HEK-293 cells, using cDNAs optimized to fit eukaryotic codon usage (Genscript; CM3 and 0.19 with GenBank accession numbers AY436554.1 and AY729672.1, respectively). Subconfluent HEK-293 cells cultured on 10-cm tissue culture dishes were transfected with 10 µg plasmid DNA encoding CM3 and 0.19 using Lipofectamine 2000 (Invitrogen), followed by incubation for 48 h. Media were obtained by centrifugation at 4,500 rpm for 10 min at 4°C. Cells were subjected to lysis and centrifuged at 14,000 rpm for 20 min at 4°C. Aliquots of the detergent-soluble and insoluble fractions were boiled at 100°C, separated on a SDS-15% polyacrylamide gel, and subjected to Western blot analysis. Protein lysates were probed with rabbit anti-flag antibody, followed by horseradish peroxidase–labeled anti–rabbit IgG (Vector Laboratories). Protein bands were visualized using enhanced chemiluminescence (Thermo Fisher Scientific) and X-Oat 2000A processor (Kodak). Although sufficient CM3 protein was secreted into media, 0.19 ATI had to be isolated after cell lysis from the detergent-soluble fraction. 0.19 and CM3 were bound to Flag-M2 agarose (Sigma-Aldrich) and washed, and bound ATIs were eluted with TBS containing Flag peptide (Sigma-Aldrich). Eluted CM3 and 0.19 were also applied to an endotoxin removal column (Norgen Biotek Corp.). ATIs were used at 5 µg/ml to stimulate HEK-293 cells stably transfected with the TLR4–CD14–MD2 complex, and IL-8 secretion was quantified after 24 h. Downstream signaling pathways induced by CM3 and 0.19 overexpression. HEK-293 cells stably expressing TLR4–CD14–MD2 were induced to express CM3 or 0.19, respectively, followed by cell lysis. 50 µg protein from cleared lysates was run on a 15% SDS-gel and subjected to Western blotting to analyze activation of the canonical and the alternative (p-IRF3) pathways using antibodies to phosphorylated and unphosphorylated NF-κB/p65 and IRF3 (Cell Signaling Technology). Stimulation of duodenal biopsies. Duodenal biopsies were obtained after informed consent during diagnostic endoscopy from healthy controls and celiac disease patients in remission (on gluten-free diet for ≥24 mo). All experiments were approved by the Ethical Committee of the IRCCS–Ospedale Maggiore Policlinico, Milan, Italy (protocol 70/2004/584). Biopsies were incubated for 4 h in complete medium alone, with 1 mg/ml PT gliadin, 200 µg/ml ATI, 50 µg/ml of synthetic α-gliadin 33mer, or 33mer and ATI. RNA was extracted, reverse transcribed, and analyzed by quantitative RT-PCR as described in the section RNA isolation and quantitative RT-PCR using the SYBR Green method and the primers listed in Table 1. Statistical analysis. Differences were tested for statistical significance by one-way ANOVA multivariate analysis followed by Dunnett’s post-test using Prism 4.01 software (GraphPad Software). P < 0.05 was considered significant. In all graphs, error bars depict standard errors of the mean. For injection and gavage experiments, at least three mice per group were used. Graphs illustrate representative data from one of at least three independent experiments.
