Dietary wheat amylase trypsin inhibitors exacerbate CNS inflammation in experimental multiple sclerosis.

OBJECTIVE: Wheat has become a main staple globally. We studied the effect of defined pro-inflammatory dietary proteins, wheat amylase trypsin inhibitors (ATI), activating intestinal myeloid cells via toll-like receptor 4, in experimental autoimmune encephalitis (EAE), a model of multiple sclerosis (MS). DESIGN: EAE was induced in C57BL/6J mice on standardised dietary regimes with defined content of gluten/ATI. Mice received a gluten and ATI-free diet with defined carbohydrate and protein (casein/zein) content, supplemented with: (a) 25% of gluten and 0.75% ATI; (b) 25% gluten and 0.19% ATI or (c) 1.5% purified ATI. The effect of dietary ATI on clinical EAE severity, on intestinal, mesenteric lymph node, splenic and central nervous system (CNS) subsets of myeloid cells and lymphocytes was analysed. Activation of peripheral blood mononuclear cells from patients with MS and healthy controls was compared. RESULTS: Dietary ATI dose-dependently caused significantly higher EAE clinical scores compared with mice on other dietary regimes, including on gluten alone. This was mediated by increased numbers and activation of pro-inflammatory intestinal, lymph node, splenic and CNS myeloid cells and of CNS-infiltrating encephalitogenic T-lymphocytes. Expectedly, ATI activated peripheral blood monocytes from both patients with MS and healthy controls. CONCLUSIONS: Dietary wheat ATI activate murine and human myeloid cells. The amount of ATI present in an average human wheat-based diet caused mild intestinal inflammation, which was propagated to extraintestinal sites, leading to exacerbation of CNS inflammation and worsening of clinical symptoms in EAE. These results support the importance of the gut-brain axis in inflammatory CNS disease.

Multiomics Based Association Mapping in Wheat Reveals Genetic Architecture of Quality and Allergenic Related Proteins.

Wheat is an important staple crop since its proteins contribute to human and animal nutrition and are important for its end-use quality. However, wheat proteins can also cause adverse human reactions for a large number of people. We performed a genome wide association study (GWAS) on 114 proteins quantified by LC-MS-based proteomics and expressed in an environmentally stable manner in 148 wheat cultivars with a heritability > 0.6. For 54 proteins, we detected quantitative trait loci (QTL) that exceeded the Bonferroni-corrected significance threshold and explained 17.3−84.5% of the genotypic variance. Proteins in the same family often clustered at a very close chromosomal position or the potential homeolog. Major QTLs were found for four well-known glutenin and gliadin subunits, and the QTL segregation pattern in the protein encoding the high molecular weight glutenin subunit Dx5 could be confirmed by SDS gel-electrophoresis. For nine potential allergenic proteins, large QTLs could be identified, and their measured allele frequencies open the possibility to select for low protein abundance by markers as long as their relevance for human health has been conclusively demonstrated. A potential allergen was introduced in the beginning of 1980s that may be linked to the cluster of resistance genes introgressed on chromosome 2AS from Triticum ventricosum. The reported sequence information for the 54 major QTLs can be used to design efficient markers for future wheat breeding.

Hybrid QconCAT-Based Targeted Absolute and Data-Independent Acquisition-Based Label-Free Quantification Enables In-Depth Proteomic Characterization of Wheat Amylase/Trypsin Inhibitor Extracts.

Wheat amylase/trypsin inhibitors (ATIs) have gained significant relevance as inducers of intestinal and extra-intestinal inflammation. In this study, we present a novel hybrid data-independent acquisition (DIA) liquid chromatography-mass spectrometry (LC-MS) approach, combining QconCAT technology with short microflow LC gradients and DIA and apply the method toward the quantitative proteome analysis of ATI extracts. The presented method is fast, robust, and reproducible and provides precise QconCAT-based absolute quantification of major ATI proteins while simultaneously quantifying the proteome by label-free quantification (LFQ). We analyzed extracts of 60 varieties of common wheat grown in replication and evaluated the reproducibility and precision of the workflow for the quantification of ATIs. Applying the method to analyze different wheat species (i.e., common wheat, spelt, durum wheat, emmer, and einkorn) and comparing the results to published data, we validated inter-laboratory and cross-methodology reproducibility of ATI quantification, which is essential in the context of large-scale breeding projects. Additionally, we applied our workflow to assess environmental effects on ATI expression, analyzing ATI content and proteome of same varieties grown at different locations. Finally, we explored the potential of combining QconCAT-based absolute quantification with DIA-based LFQ proteome analysis for the generation of new hypotheses or assay development.

Genetic architecture underlying the expression of eight α-amylase trypsin inhibitors.

KEY MESSAGE: Wheat cultivars largely differ in the content and composition of ATI proteins, but heritability was quite low for six out of eight ATIs. The genetic architecture of ATI proteins is built up of few major and numerous small effect QTL. Amylase trypsin inhibitors (ATIs) are important allergens in baker's asthma and suspected triggers of non-celiac wheat sensitivity (NCWS) inducing intestinal and extra-intestinal inflammation. As studies on the expression and genetic architecture of ATI proteins in wheat are lacking, we evaluated 149 European old and modern bread wheat cultivars grown at three different field locations for their content of eight ATI proteins. Large differences in the content and composition of ATIs in the different cultivars were identified ranging from 3.76 pmol for ATI CM2 to 80.4 pmol for ATI 0.19, with up to 2.5-fold variation in CM-type and up to sixfold variation in mono/dimeric ATIs. Generally, heritability estimates were low except for ATI 0.28 and ATI CM2. ATI protein content showed a low correlation with quality traits commonly analyzed in wheat breeding. Similarly, no trends were found regarding ATI content in wheat cultivars originating from numerous countries and decades of breeding history. Genome-wide association mapping revealed a complex genetic architecture built of many small, few medium and two major quantitative trait loci (QTL). The major QTL were located on chromosomes 3B for ATI 0.19-like and 6B for ATI 0.28, explaining 70.6 and 68.7% of the genotypic variance, respectively. Within close physical proximity to the medium and major QTL, we identified eight potential candidate genes on the wheat reference genome encoding structurally related lipid transfer proteins. Consequently, selection and breeding of wheat cultivars with low ATI protein amounts appear difficult requiring other strategies to reduce ATI content in wheat products.

Functional evaluation of a monotreme-specific antimicrobial protein, EchAMP, against experimentally induced mastitis in transgenic mice.

EchAMP, the tenth most abundant transcript expressed in the mammary gland of echidna, has in vitro broad-spectrum antibacterial effects. However, the effects of EchAMP on mastitis, a condition where inflammation is triggered following mammary gland infection, has not been investigated. To investigate the impact of EchAMP against mastitis, EchAMP transgenic mice were generated. In antibacterial assays, the whey fractions of milk from transgenic mice significantly reduced growth of Staphylococcus aureus, Bacillus subtilis, Escherichia coli and Pseudomonas aeruginosa compared with whey fractions from wildtype mice. Furthermore, a mastitis model created by infecting mammary gland with these four bacterial strains displayed a significant reduction in bacterial load in transgenic mice injected with S. aureus and B. subtilis. On further confirmation, histomorphologic analysis showed absence of necrosis and cell infiltration in the mammary glands of transgenic mice. To understand the role of EchAMP against inflammation, we employed an LPS-injected mastitis mouse model. LPS is known to induce phopshorylation of NF-κB and MAPK pathways, which in turn activate downstream proinflammatory signaling mediators, to promote inflammation. In LPS-treated EchAMP transgenic mice, phosphorylation levels of NF-κB, p38 and ERK1/2 were significantly downregulated. Furthermore, in mammary gland of transgenic mice, there was a significant downregulation of mRNA levels of proinflammatory cytokines, namely TNF-α, IL-6 and IL-1ß. Taken together, these data suggest that EchAMP has an antiinflammatory response and is effective against S. aureus and B. subtilis. We suggest that EchAMP may be a potential prophylactic protein against mastitis in dairy animals by expressing this gene in their mammary gland.

Short-term reduction of dietary gluten improves metabolic-dysfunction associated steatotic liver disease: A randomised, controlled proof-of-concept study.

BACKGROUND: The current management of metabolic dysfunction-associated steatotic liver disease (MASLD) relies on lifestyle intervention. Prior studies have shown that nutritional wheat amylase trypsin inhibitors (ATI) activate toll-like receptor 4 on intestinal myeloid cells to enhance intestinal and extra-intestinal inflammation, including the promotion of murine MASLD, insulin resistance and liver fibrosis. AIMS: We aimed to assess the impact of ATI (gluten)-free diet in liver as well as metabolic parameters of biopsy-proven MASLD patients. METHODS: We performed a 6-week, proof-of-concept 1:1 randomised controlled trial of an ATI-free diet. The controls followed a balanced diet recommended by the German Nutrition Society. We assessed changes in controlled attenuation parameter (CAP), body mass index (BMI) and homeostatic model assessment of insulin resistance (HOMA-IR). Patient-reported outcomes were assessed by the CLDQ-NASH questionnaire. Forty-five patients were consecutively enrolled (21 in the intervention arm and 24 in the control arm). RESULTS: Three patients from each arm discontinued the study. In the ATI-free diet group, a significant decrease in BMI (p = 0.018), CAP (p = 0.018) and HOMA-IR (p = 0.042) was observed at 6 weeks. The mean difference in CAP between the two arms at week 6 was 30.5 dB/m (p = 0.039), with a delta significantly higher in the ATI-free diet group (p = 0.043). Only an ATI-free diet could achieve a significant improvement in CLDQ-NASH domains (p value for total scoring: 0.013). CONCLUSIONS: A short-term ATI-free diet leads to significant improvements in liver and metabolic parameters, as well as patient-reported outcomes with good tolerability. A larger follow-up study is justified to corroborate these findings. CLINICAL TRIAL NUMBER: NCT04066400.

Breeding from 1891 to 2010 did not increase the content of amylase/trypsin-inhibitors in wheat (Triticum aestivum).

The prevalence of hypersensitivities towards wheat has increased in the last decades. Apart from celiac disease these include allergic and other inflammatory reactions summarized under the term non-celiac wheat sensitivity. One suspected trigger is the family of amylase/trypsin-inhibitors (ATIs), non-gluten proteins that are prominent wheat allergens and that activate the toll-like receptor 4 on intestinal immune cells to promote intestinal and extra-intestinal inflammation. We therefore quantified 13 ATIs in 60 German hexaploid winter wheat cultivars originating from 1891 to 2010 and harvested in three years by targeted liquid chromatography-tandem mass spectrometry combined with stable isotope dilution assay using specific marker peptides as internal standards. The total ATI content and that of the two major ATIs 0.19 and CM3 did not change from old cultivars (first registered from 1891 to 1950) to modern cultivars (1951-2010). There were also no significant changes in ATI distribution.

Characterization of 150 Wheat Cultivars by LC-MS-Based Label-Free Quantitative Proteomics Unravels Possibilities to Design Wheat Better for Baking Quality and Human Health.

Wheat (Triticum aestivum ssp. aestivum) contributes to 20% of the human protein supply, delivers essential amino acids and is of fundamental importance for bread and pasta quality. Wheat proteins are also involved in adverse human reactions like celiac disease (CD), wheat allergy (WA) and non-celiac wheat sensitivity (NCWS). Using liquid chromatography-mass spectrometry (LC-MS)-based label-free quantitative (LFQ) proteomics of aqueous flour extracts, we determined 756 proteins across 150 wheat cultivars grown in three environments. However, only 303 proteins were stably expressed across all environments in at least one cultivar and only 89 proteins thereof across all 150 cultivars. This underlines the large influence of environmental conditions on the expression of many proteins. Wheat cultivars varied largely in their protein profile, shown by high coefficients of variation across different cultivars. Heritability (h2) ranged from 0-1, with 114 proteins having h² > 0.6, including important proteins for baking quality and human health. The expression of these 114 proteins should be amenable to targeted manipulation across the wheat supply chain by varietal choice and breeding for designing healthier wheat with better quality. Further technical development is urgently required to assign functions to identifiable proteins labeled yet uncharacterized in databases and speeding up detection methods to routinely use proteomics in wheat supply chains.

Attenuation of non-enzymatic thermal glycation of bovine serum albumin (BSA) using ß-carotene.

Although heat treatment of proteins is believed to promote and accelerate glycation, the accompanying structural changes in proteins because of heat denaturation and/or glycation are not completely understood. In addition, there is an increasing interest for inhibitors of thermal glycation in food processing. ß-Carotene is a naturally occurring carotenoid found in many vegetables, fruits and herbs, with known antioxidant activity. However, it has not been identified if ß-carotene possesses anti-glycation activity. We have tested the anti-glycation capacity of ß-carotene in the bovine serum albumin (BSA)/glucose system that was heated to 55 and 65 °C for 3 days and studied the level of glycation, conformational alterations in BSA, and changes in hydrophobicity, due to thermal treatment and/or glycation. ß-Carotene exhibited significant inhibitory effects on the formation of advanced glycation end products (AGEs) and also prevented the secondary structural changes in BSA due to thermal glycation. Our results represent the anti-glycative properties of ß-carotene in food systems where such thermal conditions prevail.