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

Junker Y, Zeissig S, Kim SJ, Barisani D, Wieser H, Leffler DA, Zevallos V, Libermann TA, Dillon S, Freitag TL, Kelly CP, Schuppan D - J. Exp. Med. (2012)

Bottom Line: 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.Mice deficient in TLR4 or TLR4 signaling are protected from intestinal and systemic immune responses upon oral challenge with ATIs.Moreover, ATIs may fuel inflammation and immune reactions in other intestinal and nonintestinal immune disorders.

View Article: PubMed Central - HTML - PubMed

Affiliation: Division of Gastroenterology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA 02215, USA.

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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Related in: MedlinePlus

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.
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fig3: 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.

Mentions: Gliadins from the pure wheat strain “Rektor” were separated into their α, γ, and ω fractions via HPLC, and their PT digests were tested on THP-1 monocytic cells. Neither the α- nor the γ-gliadins, which represent >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).


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

Junker Y, Zeissig S, Kim SJ, Barisani D, Wieser H, Leffler DA, Zevallos V, Libermann TA, Dillon S, Freitag TL, Kelly CP, Schuppan D - J. Exp. Med. (2012)

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.
© Copyright Policy - openaccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC3526354&req=5

fig3: 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.
Mentions: Gliadins from the pure wheat strain “Rektor” were separated into their α, γ, and ω fractions via HPLC, and their PT digests were tested on THP-1 monocytic cells. Neither the α- nor the γ-gliadins, which represent >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).

Bottom Line: 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.Mice deficient in TLR4 or TLR4 signaling are protected from intestinal and systemic immune responses upon oral challenge with ATIs.Moreover, ATIs may fuel inflammation and immune reactions in other intestinal and nonintestinal immune disorders.

View Article: PubMed Central - HTML - PubMed

Affiliation: Division of Gastroenterology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA 02215, USA.

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.

Show MeSH
Related in: MedlinePlus