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. 2012 Dec 17;209(13):2395-408.
doi: 10.1084/jem.20102660. Epub 2012 Dec 3.

Wheat amylase trypsin inhibitors drive intestinal inflammation via activation of toll-like receptor 4

Yvonne Junker  1 , Sebastian Zeissig, Seong-Jun Kim, Donatella Barisani, Herbert Wieser, Daniel A Leffler, Victor Zevallos, Towia A Libermann, Simon Dillon, Tobias L Freitag, Ciaran P Kelly, Detlef Schuppan
Affiliations

Wheat amylase trypsin inhibitors drive intestinal inflammation via activation of toll-like receptor 4

Yvonne Junker et al. J Exp Med. .

Abstract

Ingestion of wheat, barley, or rye triggers small intestinal inflammation in patients with celiac disease. Specifically, the storage proteins of these cereals (gluten) elicit an adaptive Th1-mediated immune response in individuals carrying HLA-DQ2 or HLA-DQ8 as major genetic predisposition. This well-defined role of adaptive immunity contrasts with an ill-defined component of innate immunity in celiac disease. We identify the α-amylase/trypsin inhibitors (ATIs) CM3 and 0.19, pest resistance molecules in wheat, as strong activators of innate immune responses in monocytes, macrophages, and dendritic cells. ATIs engage the TLR4-MD2-CD14 complex and lead to up-regulation of maturation markers and elicit release of proinflammatory cytokines in cells from celiac and nonceliac patients and in celiac patients' biopsies. Mice deficient in TLR4 or TLR4 signaling are protected from intestinal and systemic immune responses upon oral challenge with ATIs. These findings define cereal ATIs as novel contributors to celiac disease. Moreover, ATIs may fuel inflammation and immune reactions in other intestinal and nonintestinal immune disorders.

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Figures

Figure 1.
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 < 0.05 versus negative control (if not indicated otherwise); graphs in A–C, E, and F illustrate representative data from one of at least three independent experiments, all performed in triplicates. Error bars depict standard errors of the mean.
Figure 2.
Figure 2.
Gliadin-induced innate immune responses are mediated via TLR4 and via the MyD88-dependent and -independent pathway. (A and B) KC (murine IL-8; A) and TNF (B) secretion in peritoneal macrophages isolated from TLR4-deficient C3H/HeJ mice compared with C3H/HOuJ wild-type mice stimulated with LPS or PT gliadin. The TLR2 agonist Pam3CSK4 served as cell viability control. (C and D) IL-8 secretion upon PT gliadin and LPS stimulation in 293 cells transfected with the TLR4–MD2–CD14 complex (C) and in untransfected cells (D). LPS and PMA served as positive controls, and the TLR2 agonist Pam3CSK4 served as negative control. (E) IL-8 secretion after preincubation with anti-TLR4 and anti-CD14 antibodies in monocyte-derived DC stimulated with PT gliadin and LPS. TLR2 agonist Pam3CSK4 and TLR3 agonist Poly I:C served as positive controls. (F) TNF secretion in peritoneal macrophages isolated from MyD88−/− mice compared with C57BL/6J wild-type mice upon PT gliadin stimulation. LPS served as positive control for the MyD88 knockdown, and TLR3 agonist Poly I:C served as cell viability control. (G) RANTES secretion in peritoneal macrophages isolated from C57BL/6J mice upon LPS, Poly I:C, and PT gliadin stimulation. *, P < 0.05 versus negative (positive) control (if not indicated otherwise); all graphs illustrate representative data from one of at least three independent experiments, and all experiments were performed in triplicates. Error bars depict standard errors of the mean.
Figure 3.
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 < 0.05 versus negative (positive) control (if not indicated otherwise); all graphs illustrate representative data from one of at least three independent experiments, all performed in triplicates. Error bars depict standard errors of the mean.
Figure 4.
Figure 4.
Identification of ATI CM3 and 0.19 as inducers of innate immune responses. (A) Coimmunoprecipitation of a soluble flag-tagged TLR4/MD2 fusion protein with or without biotinylated ATI. Pull-down was performed by streptavidin-agarose. Western blot (WB) analysis using a rabbit anti-flag antibody was performed to detect the TLR4/MD2 fusion protein. The image illustrates representative data from one of three independent experiments. (B) CM3 and 0.19 were expressed in HEK-293 cells and purified as Flag-tagged molecules, yielding expected molecular masses of 15 kD and 17 kD, respectively, after 15% SDS-PAGE and Western blotting with Flag antibody. There was no expression in mock (vector only)-transfected cells. 0.19 was not secreted into media but could be recovered in SDS/DTT buffer (Cell), deoxycholate and Triton X-100 buffer (Sup), or the remaining cell pellet. (C) Purity of CM3 and 0.19 ATI after affinity purification on Flag-agarose (15% SDS-PAGE) after staining with Coomassie blue or blotting with Flag antibody. (B and C) Arrows denote expected molecular masses of CM3 and 0.19 ATIs. (D) IL-8 secretion by TLR4–CD14–MD2-expressing HEK-293 cells after stimulation with recombinant ATI. Cells were left untreated or stimulated with affinity-purified detergent extracts from mock-transfected cells, from recombinant CM3, 0.19, or with ATI isolated from wheat (all 5 µg/ml). Controls were 5 µg/ml Flag peptide, 100 µg/ml PT gliadin, and 10 ng/ml LPS. (E) Effect of expression of CM3 and 0.19 in HEK-TLR4 cells on downstream signaling of the canonical (pNF-κB/p65) and the alternative (pIRF3) pathways. Western blots of whole cell lysates (50 µg protein). (F) Reduction and alkylation (R/A) of enriched ATIs from spring wheat (ABic: ammonium bicarbonate extract; AA: acetic acid extract). Stimulatory capacity was measured by IL-8 secretion in U-937 cells. LPS served as positive control. *, P < 0.05 versus negative (positive) control. All graphs illustrate representative data from one of at least three independent experiments. D and F were performed in triplicates. Error bars depict standard errors of the mean.
Figure 5.
Figure 5.
Sequence alignment of distinct ATI variants from wheat and barley. (A and B) Sequence alignment between CM3 and 0.19 (A) and between CM3 or 0.19 and other ATIs from wheat (CM1, CM2, CM3, CM16, CMX1, CMX2, 0.19, and 0.53) and barley (CMa, CMb, and CMd; B) using the ClustalW program (2.0.12 version). The highly conserved cysteine residues are boxed. Asterisks, colons, and periods denote identity, high homology, and low homology of amino acid residues, respectively. Similarities are much higher among individual ATIs.
Figure 6.
Figure 6.
ATI induces innate immune responses in vitro, in vivo, and in duodenal biopsies. (A and B) Intraperitoneal injection of LPS (1 µg/g mouse weight), water-soluble (ws) gliadin (500 µg/g mouse weight), or water-soluble zein (500 µg/g mouse weight) into C57BL/6J, MyD88−/− (A) and Rag1−/− (B) mice (n = 4 animals per group). Serum was taken 2 h after injection, and serum cytokine levels were measured by ELISA. (C–E) C57BL/6J (C and E), C3H/HeJ (TLR4 deficient; D), and C3H/HOuJ mice (D) were gavaged with LPS (20 µg/g mouse weight), water-soluble gliadin (2 mg/g mouse weight), ATI (0.075 mg/g mouse weight), water-soluble zein (2 mg/g mouse weight), or PBS. 4 h after gavage, the mice were euthanized, and duodenal samples were snap frozen in liquid nitrogen. Duodenal cytokine mRNA levels were measured by quantitative RT-PCR (n = 4 animals per group). (F and G) IL-8 transcript levels after incubation of duodenal biopsies with PT gliadin (PT), α-gliadin 33mer, ATI, or 33mer plus ATI (results expressed as medians and quartiles). For F, a biopsy obtained from the same patient and incubated with medium alone (M) served as control; for G, an external calibrator (cDNA derived from RNA extracted from human duodenum) was used. P-values were derived by Student’s t test: *, P < 0.05 versus negative control. Error bars depict standard errors of the mean.
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