European Journal of Nutrition

, Volume 58, Issue 4, pp 1507–1514 | Cite as

Dietary wheat amylase trypsin inhibitors exacerbate murine allergic airway inflammation

  • Victor F. ZevallosEmail author
  • Verena K. Raker
  • Joachim Maxeiner
  • Petra Scholtes
  • Kerstin Steinbrink
  • Detlef Schuppan
Original Contribution



Wheat amylase trypsin inhibitors (ATI) are dietary non-gluten proteins that activate the toll-like receptor 4 on myeloid cells, promoting intestinal inflammation.

Aim of the study

We investigated the effects of dietary ATI on experimental allergic airway inflammation.


Mice on a gluten and ATI-free diet (GAFD), sensitized with PBS or ovalbumin (OVA) and challenged with OVA, were compared to mice on a commercial standard chow, a gluten diet naturally containing ~ 0.75% of protein as ATI (G+AD), a gluten diet containing ~ 0.19% of protein as ATI (G−AD) and a GAFD with 1% of protein as ATI (AD). Airway hyperreactivity (AHR), inflammation in bronchoalveolar lavage (BAL) and pulmonary tissue sections were analyzed. Allergic sensitization was assessed ex vivo via proliferation of OVA-stimulated splenocytes.


Mice on a GAFD sensitized with PBS did not develop AHR after local provocation with methacholine. Mice on a GAFD or on a G−AD and sensitized with OVA developed milder AHR compared to mice fed a G+AD or an AD. The increased AHR was paralleled by increased BAL eosinophils, IL-5 and IL-13 production, and an enhanced ex vivo splenocyte activation in the ATI-fed groups.


Dietary ATI enhance allergic airway inflammation in OVA-challenged mice, while an ATI-free or ATI-reduced diet has a protective effect on AHR. Nutritional wheat ATI, activators of intestinal myeloid cells, may be clinically relevant adjuvants to allergic airway inflammation.


Amylase trypsin inhibitors Gluten Wheat sensitivity Innate immunity Allergic airway inflammation 



Allergic airway inflammation


Airway hyper-reactivity


Amylase trypsin inhibitor


Bronchoalveolar lavage




Gluten-free diet


Human lymphocyte antigen




Non-celiac (non-allergy) wheat sensitivity




Periodic acid–Schiff


Toll-like receptor



This study was supported by Grants of the German Research Foundation (DFG Schu646/17-1) and by the Leibniz-Foundation (Project WheatScan).

Author contributions

VZ: study design; acquisition of data; analysis and interpretation of data; drafting and editing of the manuscript; statistical analysis. VR: acquisition of data and support during experiment and data acquisition/analysis (stimulation of splenocytes). JM and PS: acquisition of data and support during in-vivo experiments (lung measurements). KS and other authors: support in revising the manuscript. DS: study design; interpretation of data; study supervision; drafting and editing of the manuscript.

Compliance with ethical standards

Conflict of interest

On behalf of all authors, the corresponding author states that there is no conflict of interest.

Supplementary material

394_2018_1681_MOESM1_ESM.jpg (737 kb)
Supplementary Figure 1. Standard chow diet contains biological active ATI. (A) Experimental protocol, C57BL/6 mice (n=6) were raised on a standard chow (SC) diet, continued on this diet or placed on a defined GAFD for four weeks and then divided in 4 groups: group 1 and 2, continued on SC, and either mock-sensitised three times with PBS or sensitized with OVA; group 3 and 4, were placed on a GAFD, mock-sensitized with PBS or sensitized with OVA. All groups were challenged with OVA. (B) Differential cell counts in BAL. Mac, macrophages; neut, neutrophils; eos, eosinophils; lymph, lymphocytes. (C) Representative H&E-stained lung tissue from all groups. Scale bars, 200 μm. (D) H&E score of all groups (E) IL-13 levels in BAL. All results are representative of two different experiments and are expressed as the mean ± SEM (n=6). Statistical significance was determined with two-way analysis of variance (ANOVA) (JPG 737 KB)
394_2018_1681_MOESM2_ESM.jpg (690 kb)
Supplementary figure 2. Standard chow diet contains biological active ATI. (A) Representative Periodic acid–Schiff (PAS)-stained lung tissue from all groups of mice. Scale bars, 200 μm. (B) PAS score of all groups. (C) Measurement of AHR, assessed by airway resistance to increasing doses of methacholine. (D) TLR4-stimulating bioactivity in protein extracts from SC and GAFD. All results are representative of data of two different experiments and are expressed as the mean ± SEM (n=6). Statistical significance was determined with two-way analysis of variance (ANOVA). ***p< 0.001 (JPG 690 KB)
394_2018_1681_MOESM3_ESM.docx (14 kb)
Supplementary material 3 (DOCX 13 KB)


  1. 1.
    Trompette A, Gollwitzer ES, Yadava K et al (2014) Gut microbiota metabolism of dietary fiber influences allergic airway disease and hematopoiesis. Nat Med 20:159–166. CrossRefGoogle Scholar
  2. 2.
    De Filippo C, Cavalieri D, Di Paola M et al (2010) Impact of diet in shaping gut microbiota revealed by a comparative study in children from Europe and rural Africa. Proc Natl Acad Sci 107:14691–14696. CrossRefGoogle Scholar
  3. 3.
    Loss G, Depner M, Ulfman LH et al (2015) Consumption of unprocessed cow’s milk protects infants from common respiratory infections. J Allergy Clin Immunol 135:56–62. CrossRefGoogle Scholar
  4. 4.
    Junker Y, Zeissig S, Kim S-J et al (2012) Wheat amylase trypsin inhibitors drive intestinal inflammation via activation of toll-like receptor 4. J Exp Med 209:2395–2408. CrossRefGoogle Scholar
  5. 5.
    Catassi C, Bai JC, Bonaz B et al (2013) Non-celiac gluten sensitivity: the new frontier of gluten related disorders. Nutrients 5:3839–3853. CrossRefGoogle Scholar
  6. 6.
    Fasano A, Sapone A, Zevallos V, Schuppan D (2015) Nonceliac gluten sensitivity. Gastroenterology 148:1195–1204. CrossRefGoogle Scholar
  7. 7.
    Wei X, Song H, Yin L et al (2016) Fatty acid synthesis configures the plasma membrane for inflammation in diabetes. Nature 539:294–298. CrossRefGoogle Scholar
  8. 8.
    Schuppan D, Pickert G, Ashfaq-Khan M, Zevallos V (2015) Non-celiac wheat sensitivity: differential diagnosis, triggers and implications. Best Pract Res Clin Gastroenterol 29:469–476. CrossRefGoogle Scholar
  9. 9.
    Catassi C, Alaedini A, Bojarski C et al (2017) The overlapping area of non-celiac gluten sensitivity (NCGS) and wheat-sensitive irritable bowel syndrome (IBS): an update. Nutrients. Google Scholar
  10. 10.
    Fritscher-Ravens A, Schuppan D, Ellrichmann M et al (2014) Confocal endomicroscopy reveals food-associated changes in the intestinal mucosa of patients with irritable bowel syndrome. Gastroenterology 147:1012–1020. CrossRefGoogle Scholar
  11. 11.
    Biesiekierski JR, Peters SL, Newnham ED et al (2013) No effects of gluten in patients with self-reported non-celiac gluten sensitivity after dietary reduction of fermentable, poorly absorbed, short-chain carbohydrates. Gastroenterology 145(2):320–328. e1-3.CrossRefGoogle Scholar
  12. 12.
    Zevallos VF, Raker V, Tenzer S et al (2017) Nutritional wheat amylase-trypsin inhibitors promote intestinal inflammation via activation of myeloid cells. Gastroenterology 152(5):1100–1113.e12. CrossRefGoogle Scholar
  13. 13.
    Skodje GI, Sarna VK, Minelle IH et al (2017) Fructan, rather than gluten, induces symptoms in patients with self-reported non-celiac gluten sensitivity. Gastroenterology 154(3):529–539.e2. CrossRefGoogle Scholar
  14. 14.
    Schuppan D, Zevallos V (2015) Wheat amylase trypsin inhibitors as nutritional activators of innate immunity. Dig Dis 33:260–263. CrossRefGoogle Scholar
  15. 15.
    Kumar RK, Herbert C, Foster PS (2008) The “classical” ovalbumin challenge model of asthma in mice. Curr Drug Targets 9:485–494. CrossRefGoogle Scholar
  16. 16.
    Raker V, Stein J, Montermann E et al (2015) Regulation of IgE production and airway reactivity by CD4 CD8 regulatory T cells. Immunobiology 220:490–499. CrossRefGoogle Scholar
  17. 17.
    Tomioka S, Bates JHT, Irvin CG (2002) Airway and tissue mechanics in a murine model of asthma: alveolar capsule vs. forced oscillations. J Appl Physiol 93(1):263–270. CrossRefGoogle Scholar
  18. 18.
    Tournoy K, Schou P (2000) Airway eosinophilia is not a requirement for allergen-induced airway hyperresponsiveness. Clin Exp Allergy 30:79–85. CrossRefGoogle Scholar
  19. 19.
    Holgate ST (2012) Innate and adaptive immune responses in asthma. Nat Med 18:673–683. CrossRefGoogle Scholar
  20. 20.
    Iwasaki A, Medzhitov R (2015) Control of adaptive immunity by the innate immune system. Nat Immunol 16:343–353. CrossRefGoogle Scholar
  21. 21.
    Bouchaud G, Gourbeyre P, Bihouee T et al (2015) Consecutive food and respiratory allergies amplify systemic and gut but not lung outcomes in mice. J Agric Food Chem 63(28):6475–6483. CrossRefGoogle Scholar
  22. 22.
    Barnard DE, Lewis SM, Teter BB, Thigpen JE (2009) Open- and closed-formula laboratory animal diets and their importance to research. J Am Assoc Lab Anim Sci 48(6):709–713Google Scholar
  23. 23.
    Libbey JE, Doty DJ, Sim JT et al (2016) The effects of diet on the severity of central nervous system disease: one part of lab-to-lab variability. Nutrition 32(7–8):877–883. CrossRefGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  • Victor F. Zevallos
    • 1
    • 2
    Email author
  • Verena K. Raker
    • 2
    • 3
  • Joachim Maxeiner
    • 2
    • 4
  • Petra Scholtes
    • 2
    • 4
  • Kerstin Steinbrink
    • 2
    • 3
  • Detlef Schuppan
    • 1
    • 2
    • 5
  1. 1.Institute of Translational Immunology, University Medical CenterJohannes Gutenberg University MainzMainzGermany
  2. 2.Research Center for Immunotherapy, University Medical CenterJohannes Gutenberg University MainzMainzGermany
  3. 3.Department of Dermatology, University Medical CenterJohannes Gutenberg University MainzMainzGermany
  4. 4.Asthma Core Facility, University Medical CenterJohannes Gutenberg University MainzMainzGermany
  5. 5.Division of Gastroenterology, Beth Israel Deaconess Medical CenterHarvard Medical SchoolBostonUSA

Personalised recommendations