Nitration of Wheat Amylase Trypsin Inhibitors Increases Their Innate and Adaptive Immunostimulatory Potential in vitro

Abstract

Amylase trypsin inhibitors (ATI) can be found in all gluten containing cereals and are, therefore, ingredient of basic foods like bread or pasta. In the gut ATI can mediate innate immunity via activation of the Toll-like receptor 4 (TLR4) on immune cells residing in the lamina propria, promoting intestinal, as well as extra-intestinal, inflammation. Inflammatory conditions can induce formation of peroxynitrite (ONOO−) and, thereby, endogenous protein nitration in the body. Moreover, air pollutants like ozone (O3) and nitrogen dioxide (NO2) can cause exogenous protein nitration in the environment. Both reaction pathways may lead to the nitration of ATI. To investigate if and how nitration modulates the immunostimulatory properties of ATI, they were chemically modified by three different methods simulating endogenous and exogenous protein nitration and tested in vitro. Here we show that ATI nitration was achieved by all three methods and lead to increased immune reactions. We found that ATI nitrated by tetranitromethane (TNM) or ONOO− lead to a significantly enhanced TLR4 activation. Furthermore, in human primary immune cells, TNM nitrated ATI induced a significantly higher T cell proliferation and release of Th1 and Th2 cytokines compared to unmodified ATI. Our findings implicate a causative chain between nitration, enhanced TLR4 stimulation, and adaptive immune responses, providing major implications for public health, as nitrated ATI may strongly promote inhalative wheat allergies (baker's asthma), non-celiac wheat sensitivity (NCWS), other allergies, and autoimmune diseases. This underlines the importance of future work analyzing the relationship between endo- and exogenous protein nitration, and the rise in incidence of ATI-related and other food hypersensitivities.

Non-celiac wheat sensitivity: rationality and irrationality of a gluten-free diet in individuals affected with non-celiac disease: a review

“…On the other hand, ATIs could be an “interesting” pathogenetic candidate. Although different polymorphisms of Toll-like receptor 4 (TLR4) have not been found in celiac disease [ 119 ], it seems that the TLR4-MD2-CD14 complex may be a potent activator of innate immune responses in monocytes, macrophages, and dendritic cells through Toll-like receptors and may play a role in NCWS [ 60 , 120 , 121 ]. MD2 is a secreted protein that interacts with TLR4, binds the lipid A moiety of lipopolysaccharides, and facilitates the creation of TLR4 dimers that are intimately required for downstream signaling and activation of the NF-κB (nuclear factor kappa light chain enhancer of activated B cells) pathway, while CD14 is a glycosyl-phosphatidylinositol (GPI)-linked protein found on the cell membrane in lipid rafts (microdomains at the plasmatic membrane with definite lipid and protein composition) and functions to transfer lipopolysaccharides from the bacterial cell wall to the MD2/TLR4 complex [ 122 ].…”

Nanoscale distribution of TLR4 on primary human macrophages stimulated with LPS and ATI

“…We focused on ATI and nitrated ATI, which have been identified as stimulating the TLR4/NF-κB pathway in macrophages and dendritic cells. 33,39 Using different quantitative analysis methods, we found that the majority of TLR4 molecules are already located in clusters, whereby pronounced differences existed among the donors already in the unstimulated state. Upon stimulation with LPS or ATI, the overall fraction of clustered TLR4 molecules increased.…”

“…Our prior studies have shown that TLR4 can be stimulated by amylase trypsin inhibitors (ATI) from wheat and thus act as natural activators of innate immunity in monocytes, macrophages and dendritic cells. 7,[33][34][35][36][37][38][39] Moreover, ATI are major allergens in Baker's asthma. 40 ATI are a family of non-gluten proteins found in different cereals containing gluten such as wheat, rye or barley.…”

“…[42][43][44] In a previous study, we have demonstrated that nitrated ATI induced enhanced TLR4 stimulation, pro-inflammatory cytokine secretion and T-cell proliferation. 39 To investigate if the elevated immune reactions are caused by a change in TLR4 surface representation and distribution, we tested native and nitrated ATI as well as the natural TLR4 agonist LPS. We used single molecule localization microscopy (SMLM) to observe their effect on the spatial distribution of TLR4 in primary human macrophages.…”

Reply to “A Gluten Reduction Is the Patients’ Choice for a Dietary ‘Bottom Up’ Approach in IBS—A Comment on “A 5Ad Dietary Protocol for Functional Bowel Disorders” Nutrients 2019, 11, 1938”

“…ATIs represent 2%-4% of total wheat protein and are believed to induce an innate immune response via activation of the Toll-like receptor 4 (TLR4) on immune cells within the intestine. They have also been shown to be involved in an adaptive immune response, have been implicated in coeliac disease, and may also contribute to inflammation in other disorders [48][49][50][51]. Additionally, legumes, which have a higher protein content than cereals, contain significant levels of anti-nutritional factors such as lectins and trypsin inhibitors, and two major storage proteins, vicilins and legumins, which are all resistant to proteolysis in the small intestine [46,52,53].…”