Gut immunity Original research Dietary wheat amylase trypsin inhibitors exacerbate CNS inflammation in experimental multiple sclerosis Victor F Zevallos,1,2 Nir Yogev,3,4 Judith Hauptmann,3,5 Alexei Nikolaev,3,5 Geethanjali Pickert,1,5 Valeska Heib,1,5 Nicola Fittler,1,5 Sebastian Steven,6 Felix Luessi,7 Manjusha Neerukonda,1,5 Claudia Janoschka,8 Ann-M arie Tobinski ,3 Luisa Klotz,8 Ari Waisman ,3,5 Detlef Schuppan 1,9 ► Additional supplemental ABSTRACT material is published online Objective Wheat has become a main staple globally. WHAT IS ALREADY KnOWn On THIS TOPIC only. To view, please visit the ⇒ Multiple sclerosis (MS) is an autoimmune journal online (http://d x.d oi.o rg/ We studied the effect of defined pro- inflammatory 10.1 136/g utjnl-2 023- 329562). dietary proteins, wheat amylase trypsin inhibitors (ATI), disease of the central nervous system (CNS) activating intestinal myeloid cells via toll- like receptor 4, with a prevalence that has increased over the For numbered affiliations see end of article. in experimental autoimmune encephalitis (EAE), a model past decades due to genetic and environmental of multiple sclerosis (MS). factors. Correspondence to Design EAE was induced in C57BL/6J mice on ⇒ A Western diet with high caloric intake and Professor Detlef Schuppan, standardised dietary regimes with defined content of high salt worsened experimental autoimmune Institute of Translational gluten/ATI. Mice received a gluten and ATI- free diet with encephalitis (EAE), a mouse model that Immunology, Johannes defined carbohydrate and protein (casein/zein) content, resembles human MS. Gutenberg Universitat Mainz, ⇒ Dietary wheat, the main staple in most Mainz 55131, Rheinland- Pfalz, supplemented with: (a) 25% of gluten and 0.75% ATI; Germany; (b) 25% gluten and 0.19% ATI or (c) 1.5% purified countries, contains amylase trypsin inhibitors D etlef. Schuppan@u nimedizin- ATI. The effect of dietary ATI on clinical EAE severity, on (ATI), non-g luten proteins that activate mainz.d e intestinal, mesenteric lymph node, splenic and central intestinal myeloid cells via toll- like receptor 4. nervous system (CNS) subsets of myeloid cells and VFZ and NY are joint first WHAT THIS STuDY ADDS authors. lymphocytes was analysed. Activation of peripheral blood AW and DS are joint senior mononuclear cells from patients with MS and healthy ⇒ Doses of nutritional ATI, present in normal authors. controls was compared. wheat- based diet, aggravate EAE and CNS Results Dietary ATI dose-d ependently caused inflammation in mice. Received 21 January 2023 ⇒ This is not due to gluten, since gluten, de- Accepted 3 August 2023 significantly higher EAE clinical scores compared with Published Online First mice on other dietary regimes, including on gluten enriched of (contaminating) ATI fails to show 18 August 2023 alone. This was mediated by increased numbers and this EAE aggravating effect. activation of pro- inflammatory intestinal, lymph node, ⇒ Myeloid cell subsets activated by ingested splenic and CNS myeloid cells and of CNS-i nfiltrating ATI are found in intestinal and extraintestinal encephalitogenic T-l ymphocytes. Expectedly, ATI activated organs including the CNS. peripheral blood monocytes from both patients with MS ⇒ These myeloid cells activated by dietary ATI and healthy controls. can be linked to enhanced CNS and effector Conclusions Dietary wheat ATI activate murine and lymphocytes in the CNS. human myeloid cells. The amount of ATI present in an ⇒ ATI enhance the immune activation of average human wheat-b ased diet caused mild intestinal peripheral blood mononuclear cells from inflammation, which was propagated to extraintestinal patients with MS and healthy controls. sites, leading to exacerbation of CNS inflammation HOW THIS STuDY MIGHT AFFECT RESEARCH, and worsening of clinical symptoms in EAE. These PRACTICE OR POLICY results support the importance of the gut- brain axis in inflammatory CNS disease. ⇒ This study suggests that the consumption of wheat and wheat ATI may worsen human MS, ► https://d x.d oi.o rg/1 0.1 136/ and stress the relevance of gut- brain signalling gutjnl-2 023- 330848 in inflammatory CNS disease. InTRODuCTIOn ⇒ Clinical studies should be performed to confirm Multiple sclerosis (MS) is an inflammatory disease the beneficial effect of a wheat (ATI)- free diet © Author(s) (or their of the central nervous system (CNS) with a promi- as adjunctive therapy in patients with MS.employer(s)) 2024. Re- use permitted under CC BY- NC. No nent T cell-d riven autoimmune component, which commercial re- use. See rights is directed towards autoantigens of the myelin and permissions. Published sheath.1 2 First disease manifestations usually occur factors have emerged as important modifiers of MS by BMJ. between the age of 20 and 40 years with visual and manifestation and severity.1 3 To cite: Zevallos VF, Yogev N, sensory disturbances, muscle spasms and limb weak- Recently, nutritional factors have been shown Hauptmann J, et al. Gut ness, progressing to severe disability. While genetics to modulate disease severity in rodent models 2024;73:92–104. clearly determine MS prevalence, environmental of experimental autoimmune encephalomyelitis 92 Zevallos VF, et al. Gut 2024;73:92–104. doi:10.1136/gutjnl-2023-329562 GGGGGGGGGGGGGGGGuuuuuuuuuuuuuuuutttttttttttttttt:::::::::::::::: ffffffffffffffffiiiiiiiiiiiiiiiirrrrrrrrrrrrrrrrsssssssssssssssstttttttttttttttt ppppppppppppppppuuuuuuuuuuuuuuuubbbbbbbbbbbbbbbblllllllllllllllliiiiiiiiiiiiiiiisssssssssssssssshhhhhhhhhhhhhhhheeeeeeeeeeeeeeeedddddddddddddddd aaaaaaaaaaaaaaaassssssssssssssss 11111111111111110000000000000000................1111111111111111111111111111111133333333333333336666666666666666////////////////gggggggggggggggguuuuuuuuuuuuuuuuttttttttttttttttjjjjjjjjjjjjjjjjnnnnnnnnnnnnnnnnllllllllllllllll----------------2222222222222222000000000000000022222222222222223333333333333333----------------333333333333333322222222222222229999999999999999555555555555555566666666666666662222222222222222 oooooooooooooooonnnnnnnnnnnnnnnn 11111111111111118888888888888888 AAAAAAAAAAAAAAAAuuuuuuuuuuuuuuuugggggggggggggggguuuuuuuuuuuuuuuusssssssssssssssstttttttttttttttt 2222222222222222000000000000000022222222222222223333333333333333................ 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PPPPPPPPPPPPPPPPrrrrrrrrrrrrrrrrooooooooooooooootttttttttttttttteeeeeeeeeeeeeeeecccccccccccccccctttttttttttttttteeeeeeeeeeeeeeeedddddddddddddddd bbbbbbbbbbbbbbbbyyyyyyyyyyyyyyyy ccccccccccccccccooooooooooooooooppppppppppppppppyyyyyyyyyyyyyyyyrrrrrrrrrrrrrrrriiiiiiiiiiiiiiiigggggggggggggggghhhhhhhhhhhhhhhhtttttttttttttttt................ Gut immunity (EAE), which share common features with human MS, where Furthermore, we compared the inflammatory effects of wheat components of the Western diet such as a high intake of dietary ATI on peripheral blood mononuclear cells (PBMCs) from salt,4–6 saturated fat7 or refined sugars8 promoted the infiltration patients with MS and healthy controls, confirming transfer- of the CNS with pathogenic T helper (Th)- 17 cells and macro- ability to patients. phages,5 enhancing EAE severity. Diet also affects the intestinal microbiota, with secondary effects on intestinal and peripheral MATERIALS AnD METHODS inflammation.9 However, the mechanisms by which these food Diet preparation components or the intestinal microbiota would influence periph- We prepared a gluten and ATI-f ree (GAF) diet as customised eral immunity remain largely unknown. pellets (Ssniff, Soest, Germany) with a defined carbohydrate Clinical nutritional intervention studies in MS often lack content and either milk casein or maize zein as main protein appropriate sample size, inclusion criteria, patient stratification source (~22% of dry weight) as described before.22–24 Based and control groups.10 There is only one observational study11 on the GAF diet, 3 diet preparations were manufactured, suggesting that a gluten- free diet supplemented with vitamins namely: the gluten and ATI (GA) diet (5.5% of the food dry and minerals might have a positive effect on pain, numbness and weight and 25% of the zein is supplemented with gluten, which mobility in patients with MS. However, this report did not fulfil contains 0.75% associated ATI, equivalent to 0.165% of food the criteria of a standardised clinical trial, lacking controls and dry weight); the gluten (G) diet de- enriched of ATI (5.5% of valid outcome measures. the food dry weight and 25% of the zein is supplemented with Wheat is a major food staple in most societies, mostly used to gluten, which contains 0.19% ATI that remains associated with manufacture baked products, pasta or meat replacements. Wheat the gluten after de-e nrichment, equivalent to 0.042% of food flour contains 9%–13% protein that can be separated into gluten dry weight); the ATI (A) diet (0.15% of the food dry weight) . and non- gluten fractions (85% and 15%, respectively). Gluten Wheat flour contains ~0.4% ATI, and humans consume ~200 g is also a ubiquitous food ingredient, often used to improve the of wheat flour per day, equivalent to 0.8 g ATI daily=11.4 mg/ texture of refined foods. kg/day (body weight 70 kg). Mice consume ~3 g chow per day, Wheat has been systematically grown, bred and introduced into and with the 0.15% pure ATI diet an amount of 3.75 mg per our diet approximately 6000 years ago in central Europe.12 None- 25 g body weight=150 mg/kg/day. Since the division factor for theless, wheat proteins have inflammatory potential, with ~1% of pharmacological dosing (body surface based) for mouse versus most populations being affected by coeliac disease, where gluten man is 12, the human equivalent of the mouse pure ATI dose peptides trigger an intestinal Th1 T cell response in genetically is 150 mg/12=12.5 mg/kg/day. For dietary compositions, see predisposed individuals, leading to small intestinal villous atrophy online supplemental table 1. and a wide range of associated comorbidities.13 Moreover, apart from respiratory and nutritional wheat allergies to gluten and non- Isolation of ATI and ATI bioactivity assays of enzymatically gluten proteins,14 many patients with irritable bowel syndrome suffer from IgE- negative wheat allergy.15 A third inflammatory digested food pellets reaction to wheat is the activation of innate immunity by amylase ATIs were extracted and purified from commercial hexaploidy trypsin inhibitors (ATI), a family of up to 17 structurally related wheat flour (Diamant). Prominent ATI species were dimeric non- gluten proteins that are resistant to intestinal enzymatic diges- 0.19>tetrameric CM3>dimeric 0.28, and tetrameric CM2, 32 33 tion.16 17 ATI activate myeloid cells (monocytes, macrophages and CM16 and CM17. Briefly, wheat or gluten was extracted dendritic cells) via the toll- like receptor 4 (TLR4) in vitro and after using ammonium bicarbonate buffer, dialysed, sterile filtrated oral ingestion in vivo. Unlike the major TLR4 ligand, lipopolysac- and lyophilised. ATI were isolated by fractional salt precipita- charide (LPS), which is derived from (Gram-n egative) bacteria, ATI tion using ammonium sulfate. Further purification of ATI by remain active in the GI tract, where LPS is usually inactivated in fast protein liquid chromatography (FPLC) was used only for 22 the stomach by acid hydrolysis and in the gut by intestinal epithe- in vitro experiments. Food pellets were enzymatically digested 22 lial alkaline phosphatase.18–21 Therefore, ATI survive GI passage in as described and bioactivity tested by adding samples reconsti- their bioactive form to stimulate lamina propria myeloid cells.19 22 23 tuted in phosphate- buffered saline (PBS) to TLR4/MD-2 /CD14/ Importantly, the ATI- induced mild inflammatory signal in the IL-8 Prom/LUCPorter HeLa cells (Novus Biologicals, Wies- gut can be transmitted to the periphery via enhanced myeloid cell baden, Germany) at a concentration of 50–250 µg protein/mL of activation and T cell priming in mesenteric lymph nodes (MLNs) ATI extracts and incubated for 6 hours. Luciferase activity was and extraintestinal organs.19 22 This is accompanied by elevated then analysed 5 min after addition of a luciferase reporter assay circulating inflammatory mediators, such as interleukin (IL)-6 or reagent using a luminometer (Tecan, Männedorf, Switzerland). CC-c hemokine ligand 2 (CCL-) 2, and by exacerbation of experi- mental IBD, intestinal/pulmonary allergies, non- alcoholic steato- Mice hepatitis and liver fibrosis.22–27 Notably, small controlled clinical Female C57BL/6J mice, aged 6–8 weeks, were purchased from studies showed that a wheat- free versus a wheat- containing Harlan (An Venray, The Netherlands) and maintained on stan- diet improved molecular and clinical features of patients with dard chow (Ssniff, Soest, Germany) before being placed on a familial Mediterranean fever, UC and primary sclerosing chol- defined GAF diet for 4 weeks. Two days before EAE induction, angitis.28 29 IgE-n egative wheat allergy and ATI sensitivity can mice continued a GAF diet or changed to a GA, G or A diet (see explain most of the symptoms of non- coeliac wheat (‘gluten’) ‘Diet preparation’ section). sensitivity.30 31 Importantly, food grade gluten can contain up to 5% of ATI,17 22 31 which complicates interpretation of prior chal- Induction of EAE and assessment of EAE severity lenge studies using different gluten preparations. EAE was induced and assessed as previously described.34 Mice Based on prior research on wheat ATI, we tested the puta- were immunised subcutaneously at the tail base with 50 µg of tive disease promoting effect of dietary ATI versus gluten alone myelin oligodendrocyte glycoprotein peptide (MOGP35-5 5), emul- in murine EAE, focusing on clinical disease activity, cellular/ sified in 100 µL complete Freund’s adjuvant, supplemented with molecular components of intestinal and CNS inflammation. 8 mg/mL of heat- inactivated Mycobacterium tuberculosis H37RA Zevallos VF, et al. Gut 2024;73:92–104. doi:10.1136/gutjnl-2023-329562 93 Gut: first published as 10.1136/gutjnl-2023-329562 on 18 August 2023. Downloaded from http://gut.bmj.com/ on March 13, 2024 by guest. Protected by copyright. Gut immunity (Difco Laboratories, Schwerte, Germany). Mice also received variance. Differences among selected experimental groups with 200 ng pertussis toxin (Sigma- Aldrich, Schnelldorf, Germany) p values <0.05 were considered statistically significant. intraperitoneally, on the day of immunisation and 2 days later. Clinical signs of disease were monitored as described,34 based RESuLTS on progressive deterioration of motor skills (from mild to total paralysis), using the following staging criteria: 0, healthy; 1, limp Dietary ATI aggravate CnS autoimmunity in mice tail; 2, partial hindlimb weakness/ataxia; 3, paralysis of at least Mice were kept on a defined GAF diet for 4 weeks, before one hindlimb; 4, complete hindlimb paralysis; 5, partial forelimb studying the immunogenic effect of dietary ATI on CNS inflam- paralysis and 6, moribund/dead. CNS and peripheral organs mation. After this period, mice were either placed on a GA diet were collected for further evaluation. (25% of total protein as crude gluten containing 0.75% ATI) or continued on a GAF diet. Both groups of mice were immunised Cell preparation, flow cytometry staining and acquisition with MOGP35-5 5 and pertussis toxin for the induction of EAE CNS- infiltrating cells were isolated from mice at the peak and and clinical scores were evaluated until day 22, when intes- at onset of disease as described by us,35 stained for surface and tinal and CNS tissues were collected for evaluation (figure 1A). intracellular markers (online supplemental methods) and anal- The pro- inflammatory activity of ATI in the food pellets was ysed using a FACSCanto device, FACSDiva (BD Biosciences, tested in a cell- based TLR4 reporter assay after quantitative 22 38 Heidelberg, Germany) and FlowJo software (Tree Star, Ashland, extraction, confirming that GA pellets had a fourfold higher Oregon, USA). TLR4-s timulating activity than GAF pellets (figure 1B). The mean EAE clinical score as well as the cumulative score Histology and immunohistochemistry (area under the curve) over 22 days were significantly higher in Six μm formalin-f ixed small intestinal sections were stained the group of mice consuming the GA versus GAF diet, reaching with an antibody to F4/80+ (BM4007, diluted 1:100, Acris, significance 14 days post- EAE induction (figure 1C and D), while Herford, Germany) to identify resident macrophages. Immuno- body weight and small intestinal length were comparable in both fluorescence staining on intestinal cryosections was performed groups (online supplemental figure 1A–B). With GA feeding, with the TSA Cy3 System (NEL704A001KT, PerkinElmer, transcripts for key innate inflammatory mediators (KC, kerati- Waltham, Massachusetts, USA) and a fluorescence microscope nocyte chemoattractant or Murine IL- 8; monocyte chenmmoat- (IX70; Olympus, Tokyo, Japan) using primary antibodies against tracrctant protein 1, MCP- 1) were significantly upregulated CD11c (550283, BD, New Jersey, USA, dilution 1:200) and in three representative sections of the intestine (duodenum>- F4/80 (BM8 eBioscience, dilution 1:1000) as described by us36 terminal ileum and mid-c olon) at day 22, with highest expres- (online supplemental methods). sion seen in the duodenum (figure 1E), in line with increased numbers of duodenal and ileal macrophages (figure 2A and B), Quantitative real-time PCR with a trend for upregulation of IL-6 and IL- 15 (online supple- RNA was extracted from duodenum, ileum and colon (Roboklon, mental figure 1C). E3598-0 2, Berlin, Germany) and reverse- transcribed (Bio-R ad, Notably, FACS analysis of inflammatory cell infiltration in Hercules, USA). Quantitative PCR was performed using exon- the CNS revealed a significant increase of CD4 + T cell counts exon boundary- spanning primer sequences for keratinocyte (figure 2C). CNS- infiltrating cells were stimulated with MOGP35- chemoattractant (KC, murine IL-8 ), monocyte chemoattrac- 55 and thereafter gated on MOG-s pecific CD44 +CD40L+ tant protein 1 (MCP- 1), IL- 6 and IL- 15 (online supplemental followed by quantification of T cells as previously described by 39 + table 2), and the SYBR Green methodology on a Step One Plus us. These MOG-s pecific encephalitogenic CD4 T cells, and sequence amplification system (Applied Biosystems, Foster City, particularly the numbers of interferon (INF)γ + IL17a+ CD4+ T USA). The relative messenger RNA expression of the tested gene cells were significantly elevated in the GA compared with the relative to glyceraldehyde- 3-p hosphate dehydrogenase expres- GAF group (figure 2C). No difference between both groups sion was calculated using the 2−ΔCp method, as described.22 was observed for CD8 + T cells and their subsets (online supple- mental figure 1D). Human peripheral blood mononuclear cells PBMCs were isolated from six healthy donors (HD) and six Dietary gluten de-enriched of ATI fails to promote EAE patients with relapsing-r emitting MS (online supplemental To separate the effect of ATI from a potential effect of dietary table 3) using Lymphoprep gradient (STEMCELL Technologies, gluten in worsening EAE, we prepared a diet with 25% gluten (G) Cologne, Germany). Monocytes were isolated by negative selec- de- enriched of ATI (~0.19% ATI of total protein) and compared tion immunomagnetic cell separation (monocyte isolation kit II, it against a GA (~0.75% ATI) and control GAF (0% ATI) diet, Miltenyi Biotec, Bergisch Gladbach, Germany) as described.37 using the same conditions as in the first experiment (figure 3A). ATI de-e nrichment was also confirmed in G pellets, demon- Cell culture and stimulation of human monocytes strating a significantly reduced TLR4- stimulating bioactivity, Human PBMCs were cultured and stimulated with ATI/LPS, comparable to GAF pellets (figure 3B). Importantly, the clinical cell supernatant cytokine/chemokines measured (online supple- symptom score observed over 22 days in all three study groups mental methods) using ELISAs (Ready-S et- Go, eBioscience, correlated with the amount of bioactive ATI present in each diet, Darmstadt, Germany) and transcripts from monocytes (online that is, the GAF group generated a lower disease score, compa- supplemental table 2) were quantified using real-t ime PCR rable to the G group, and both groups (GAF and G) showed a (Bio- Rad). significantly lower cumulative clinical score than the GA group, indicating significant worsening of EAE by the ATI component Statistical analyses (figure 3C and D). As before, there was no significant change Data were statistically analysed using GraphPad Prism V.9.40 in body weight between the three experimental groups (online (GraphPad Software, San Diego, California, USA). Multiple supplemental figure 1E). Again, key inflammatory genes (IL- 6, comparisons were performed by one- way or two- way analysis of MCP- 1 and KC) were upregulated in the duodenum of mice in 94 Zevallos VF, et al. Gut 2024;73:92–104. doi:10.1136/gutjnl-2023-329562 Gut: first published as 10.1136/gutjnl-2023-329562 on 18 August 2023. Downloaded from http://gut.bmj.com/ on March 13, 2024 by guest. Protected by copyright. Gut immunity Figure 1 Dietary amylase trypsin inhibitors (ATI)- containing gluten worsens experimental autoimmune encephalitis (EAE). (A) Schematic view of experimental layout: C57BL/6J mice aged 6 weeks raised on standard chow (SC) were placed on the gluten and ATI-f ree (GAF) diet for 4 weeks prior to myelin oligodendrocyte glycoprotein (MOG)- immunised. Forty- eight hours prior of EAE induction, mice were randomly divided into two groups, which either continue receiving GAF dietary regime or changed to a gluten and ATI (GA) diet containing 25% gluten and 0.75% ATI (n=6 per group). (B) Lipopolysaccharide (LPS)- free toll-l ike receptor 4 (TLR4)- stimulating bioactivity of GAF and GA pellets, quantitatively extracted for ATI and expressed as relative luciferase activity of indicator cells. (C) EAE mean clinical scores, 22 days post-E AE induction. (D) EAE mean cumulative clinical score. (E) KC (IL- 8) and MCP- 1 (CCL- 2) gene expression in duodenum (D), terminal ileum (TI) and mid-c olon (MC), of mice subjected to GAF or GA dietary regiments, as detected 22 days post- MOG immunisation. Results are expressed as mean±SEM (n=6/group, except for figure E having n=4/group). Results are representative of three independent experiments (B and D) or one representative experiment (E). Statistical significance was determined by one- way or two- way analysis of variance. *P<0.05, **p<0.01. GAPDH, glyceraldehyde- 3- phosphate dehydrogenase; mRNA, messenger RNA. Zevallos VF, et al. Gut 2024;73:92–104. doi:10.1136/gutjnl-2023-329562 95 Gut: first published as 10.1136/gutjnl-2023-329562 on 18 August 2023. Downloaded from http://gut.bmj.com/ on March 13, 2024 by guest. Protected by copyright. Gut immunity Figure 2 Dietary amylase trypsin inhibitors (ATI)- containing gluten promotes intestinal and central nervous system (CNS) inflammation. Analyses were performed at day 22 postexperimental autoimmune encephalitis (post-E AE) induction in mice on a gluten and ATI-f ree (GAF) and gluten and ATI (GA) diet. (A) Representative intestinal sections stained for F4/80+ macrophages. (B) F4/80+ cell densities in the villi. (C) Inflammatory cells infiltrating the CNS, including CD4+, CD154+, interferon (INF)γ+, interleukin (IL)- 17a+ and double positive INFγ+IL-1 7a+ cells as quantified by flow cytometry (FACS) analysis (means±SEM; n=4/group; representative of two independent experiments). Analysis by one-w ay or two- way analysis of variance. *P<0.05, **p<0.01, ***p<0.001. 96 Zevallos VF, et al. Gut 2024;73:92–104. doi:10.1136/gutjnl-2023-329562 Gut: first published as 10.1136/gutjnl-2023-329562 on 18 August 2023. Downloaded from http://gut.bmj.com/ on March 13, 2024 by guest. Protected by copyright. Gut immunity Figure 3 Dietary gluten depleted of amylase trypsin inhibitors (ATI) fails to worsen experimental autoimmune encephalitis (EAE). (A) C57BL/6 mice (n=6 per group) were raised on standard chow (SC), myelin oligodendrocyte glycoprotein (MOG)-i mmunised, placed on a control gluten and ATI- free (GAF) diet for 4 weeks and divided in three groups: (1) continued GAF; (2) changed to gluten and ATI (GA) (25% gluten and 0.75% ATI) or (3) changed to gluten (G) (25% gluten and 0.19% ATI). (B) Lipopolysaccharide (LPS)-f ree toll- like receptor 4 (TLR4)- stimulating bioactivity of quantitative extracts of pellets from the three dietary regimes, expressed as relative luciferase activity of indicator cells. (C) Mean clinical scores (22 days postinduction). (D) Mean cumulative clinical scores. (E) Interleukin (IL)-6 , KC and MCP-1 gene expression in duodenal samples analysed after 22 days post- EAE induction (means±SEM; n=6/group, except for (E) with n=4/group). Results are representative of two to three independent experiments for (B), (C) and (D). Analysis by one- way or two- way analysis of variance. *P<0.05, **p<0.01, ***p<0.001. GAPDH, glyceraldehyde- 3- phosphate dehydrogenase; mRNA, messenger RNA. Zevallos VF, et al. Gut 2024;73:92–104. doi:10.1136/gutjnl-2023-329562 97 Gut: first published as 10.1136/gutjnl-2023-329562 on 18 August 2023. Downloaded from http://gut.bmj.com/ on March 13, 2024 by guest. Protected by copyright. Gut immunity the GA group compared with the G and GAF groups (figure 3E), ATI-c onsuming mice compared with those consuming ATI-f ree which was paralleled by increased duodenal numbers of F4/80+, dietary regimes (GAF and G). At the same time, the reverse was CD11c+ and F4/80+CD11c+ macrophages and dendritic cells observed in the mock-t reated control groups exposed to the (figure 4A and B). In the CNS, total infiltrating CD4+ T cells, same dietary regimes (online supplemental figure 2F). This is in and more importantly the number of encephalitogenic, MOG- line with the context-d ependent immune adjuvant role of nutri- specific INFγ+IL- 17A+ CD4+ T cells was significantly increased tional ATI in already established inflammatory diseases.22 24–26 in the GA group and only marginally increased in the G and GAF groups, in alignment with the ATI content and intestinal innate inflammation (figure 4C). Mice on the GAF group had ATI promote the release of innate cytokines and chemokines higher ratios of CNS CD4+ Foxp3+ regulatory T (Treg) cells in peripheral blood mononuclear cells from patients with MS (online supplemental figure 1F), indicating that a gluten and To translate our findings in the rodent model to patients with MS, ATI containing diet shifts the composition of CNS-i nfiltrating we isolated PBMC from fifteen patients with MS (ten females) encephalitogenic CD4+ effector Th1/17 vs suppressive Treg and fifteen healthy controls (HC) (ten females). The PBMCs cells. were cultured and stimulated with ATI and LPS (as a major alternative TLR4 ligand) for 16 hours (figure 6A). The release of Dietary ATI increase CD45+CD11b+ pro-inflammatory myeloid CCL- 2 (MCP- 1), IL- 6 and tumour necrosis factor (TNF)α was cells infiltrating the CnS and decrease Foxp3+CD25+ Treg cells highly increased by exposure to ATI and comparable to levels in mesenteric lymph nodes achieved on LPS stimulation, both in cells from both patients To better capture myeloid cell populations in the CNS and to with MS and HC (figure 6B–D). Furthermore, gene expression confirm the effects of ATI in worsening EAE symptoms, we of the same cytokines and chemokines (IL-6 , CCL-2 , TNFα) and set mice on three dietary regimes: GAF (0% ATI), GA (gluten of IL- 8 was equally upregulated in both patients with MS and containing 0.75% ATI) and A (1.5% purified ATI) using the HC after stimulation with ATI or LPS. While there was a trend same conditions as in the initial experiments but ending the for higher values in the patients with MS, this did not reach experiment at day 15 post-E AE induction, that is, at the peak significance (figure 6E–F). The absence of a major difference of disease (figure 5A). As observed before (figures 1C and 3C), in ATI-r esponsiveness between patients with MS and healthy ATI induced an earlier onset (day 10) of clinical symptoms that controls is in line with the general innate immune-s timulatory continued to increase until day 15, resulting in a significantly activity of ATI. elevated cumulative disease score compared with mice kept on GAF diet (figure 5B, online supplemental figure 2A). We isolated and analysed the different cell populations infiltrating the CNS DISCuSSIOn for whole spinal cord/full brain without isolating specific parts of Dietary wheat ATI that activate TLR4 on myeloid cells (dendritic these organs, following EAE induction, using the gating strategy cells, monocytes, macrophages) residing in the intestinal lamina 19 22 23 26 27 outlined in online supplemental figures 3 and 4, with primary propria promotes CNS inflammation and clinical selection for non- myeloid (CD11b−) and myeloid (CD11b+) symptoms of murine EAE, an established animal model of human CD45+ cells (figure 5C,D and F). ATI feeding increased dose- MS. Our experimental diet containing gluten and ATI (25% dependently (A>GA) T cells infiltrating the CNS (figure 5C), and 0.75% of protein, respectively), corresponds to the average and MHC- II+ cDC2 (CD11c+CD11b+) and macrophages gluten/ATI content in a standard Western diet (15–20 g of gluten (CD11c+/−CD11b+) (figure 5F). Moreover, CNS-i nfiltrating and 0.7–1.5 g of ATI/person/day). 22 We compared this diet with myeloid cells showed a dose- dependent increase of MHC- IIint a control gluten and ATI-f ree diet, and with dietary regimes inflammatory monocytes and enhanced activation of CNS- containing mostly gluten (25% gluten and only 0.19% ATI), or resident myeloid cells (ie, microglia) (figure 5D). In line with ATI (0% gluten and 1.5% ATI). Mice consuming high dietary the aggravated CNS inflammation, we also observed enhanced ATI (1.5% and 0.75%) developed significantly more severe EAE neutrophil infiltration (MHC-I I−Ly6G+), although the numbers and CNS inflammation than those consuming a low ATI (0.19%) of CNS- resident microglia remained unaltered (figure 5E). Such or an ATI- free diet. Notably, gluten alone, did not promote CNS increase in largely pro- inflammatory myeloid cells infiltrating inflammation. Moreover, ATI exerted a dose- dependent effect the CNS was demonstrable both in percentage (data not shown) on innate immune activation, which consecutively had an impact and as total cell counts. on CNS inflammation. The intestinal inflammatory milieu in mice on GA-c ontaining Body weight was not significantly affected, indicating a diets (figures 1E and 2A–B and online supplemental figure 1C) comparable food consumption independent of dietary regime could also promote gut-a ssociated Treg cell accumulation. To or CNS inflammation. Small intestines of mice on the high further explore whether dietary ATI may modulate the number ATI versus low or ATI-f ree diet controls, showed the highest of Treg cells, we performed a short- term comparative study using expression of innate inflammatory genes (KC, MCP- 1, IL- 6 and the three dietary regimes (GAF, GA and G) for 9 days in mice IL- 15) and the highest number of macrophages and dendritic with MOG-p eptide immunisation versus PBS-s ensitised controls cells (F4/80+, CD11c+ and F4/80+CD11c+), particularly in the (online supplemental figure 2B). Expression of key innate duodenum. This increased intestinal inflammatory signal signifi- inflammatory genes (IL-6 and MCP- 1) in the duodenum showed cantly correlated with increased numbers of MOG-r esponsive, only a trend of higher levels in MOG-i mmunised mice kept on a IFNγ-producing and IL- 17A- producing Th1 and Th17 cells and, GA diet compared with the GAF or G diet (online supplemental consequently, a higher cumulative EAE clinical score. These figure 2C). Although total numbers of CD4+Foxp3+ Treg cells inflammatory/encephalitogenic Th cells are key drivers of EAE in the spleen and Peyer’s patches were elevated on EAE induc- pathogenesis, as they are one of the first pathogenic cells trans- tion (day 9), the different dietary regimens had no additive effect migrating across the blood- brain barrier and infiltrating the on peripheral Treg cells (online supplemental figure 2D–E). brain parenchyma.40 Nevertheless, within the intestine-d raining MLNs, the numbers The small intestine has been identified as a key site for of Treg cells were significantly elevated in MOG-s ensitised and the activation of effector Th17 cells with relevance for MS 98 Zevallos VF, et al. Gut 2024;73:92–104. doi:10.1136/gutjnl-2023-329562 Gut: first published as 10.1136/gutjnl-2023-329562 on 18 August 2023. Downloaded from http://gut.bmj.com/ on March 13, 2024 by guest. Protected by copyright. Gut immunity Figure 4 The amylase trypsin inhibitors (ATI) component in dietary gluten promotes intestinal and central nervous system (CNS) inflammation. (A) Representative duodenal sections from the experimental groups in figure 3 stained for F4/80 and CD11c. (B) Numbers of duodenal F4/80+ and CD11c+ cells. (C) CNS- infiltrating inflammatory CD4+ T cells, INFγ+, IL1a+ and double positive INFγ+IL17a+ cells, as determined by FACS analysis 22 days postexperimental autoimmune encephalitis (post- EAE) induction. Results are expressed as means±SEM (n=4/group in figure A and B, n=6/group in figure C). Results are representative of two independent experiments. Statistical significance was determined with one-w ay or two- way analysis of variance. *P<0.05; **p<0.01; ***p<0.001. G, gluten; GA, gluten and ATI; GAF, gluten and ATI-f ree. Zevallos VF, et al. Gut 2024;73:92–104. doi:10.1136/gutjnl-2023-329562 99 Gut: first published as 10.1136/gutjnl-2023-329562 on 18 August 2023. Downloaded from http://gut.bmj.com/ on March 13, 2024 by guest. Protected by copyright. Gut immunity Figure 5 Amylase trypsin inhibitors (ATI) dose- dependently increase pro- inflammatory myeloid CD11b− and CD11b+ cells in the central nervous system (CNS) of experimental autoimmune encephalitis (EAE) mice. (A) C57BL/6 mice (n=6 per group) were raised on standard chow (SC), myelin oligodendrocyte glycoprotein (MOG)-i mmunised, placed on a gluten and ATI- free (GAF) diet for 4 weeks and divided in three groups: (1) continued GAF, (2) change to gluten and ATI (GA) (25% gluten and 0.75% ATI) or (3) change to ATI (1.5% ATI). Mice were sacrificed at day 15 postexperimental autoimmune encephalitis (post-E AE) induction. (B) Mean cumulative clinical score. (C) Total numbers of CNS-i nfiltrating CD45+CD11b− (left) and T cells (right). (D) CD45+CD11b+ cells (left) and MHCIIloCD11c-CD38+ inflammatory monocytes/activated microglia (right). (E) Lineage− CD45+CD11b+Ly6G+ neutrophils (left) and CNS-r esident F4/80hiCXCR1hi microglia (right). (F) CNS-i nfiltrating MHC- II+CD11c+CD38+ cDC/macrophages (left) and MHC-I I+CD11c−CD38+ macrophages/microglia (right). For gating strategy, see online supplemental figure 2. *P<0.05, **p<0.01. 100 Zevallos VF, et al. Gut 2024;73:92–104. doi:10.1136/gutjnl-2023-329562 Gut: first published as 10.1136/gutjnl-2023-329562 on 18 August 2023. Downloaded from http://gut.bmj.com/ on March 13, 2024 by guest. Protected by copyright. Gut immunity Figure 6 Amylase trypsin inhibitors (ATI) induce expression of inflammatory mediators in monocytes from patients with multiple sclerosis (MS) and healthy controls (HC). (A) Blood monocytes were isolated from peripheral blood mononuclear cells (PBMCs) from HC and patients with MS (each n=15), challenged with lipopolysaccharide (LPS) or ATI, or left unstimulated (Con) for 16 hours. (B–D) CCL- 2, interleukin (IL)-6 and tumour necrosis factor (TNF)α released into the medium. (E) IL- 6 and (F) TNFα, CCL- 2 and IL- 8 gene expression in monocytes of all groups. Results are expressed as means±SEM. Significance was determined with one- way or two- way analysis of variance. *P<0.05, **p<0.01, ***p<0.001, ****p<0.0001. mRNA, messenger RNA. Zevallos VF, et al. Gut 2024;73:92–104. doi:10.1136/gutjnl-2023-329562 101 Gut: first published as 10.1136/gutjnl-2023-329562 on 18 August 2023. Downloaded from http://gut.bmj.com/ on March 13, 2024 by guest. Protected by copyright. Gut immunity pathophysiology,41 and in our prior study we provided evidence without gluten (1.5% of total protein), significantly increased that the small intestinal signal of ATI-i nduced innate immune intestinal innate immune activation and worsened all features stimulation was quickly transmitted to the MLNs, with a of EAE. This clearly confirms that (1) dietary gluten itself, highly enhanced expression of myeloid/dendritic cell activation even at high concentrations, can be excluded as trigger of this markers.22 While an interaction of ATI-p rimed myeloid cells with immune activation, and (2) the immune adjuvant effect of ATI is already MOG-s pecific encephalitogenic T cells at the level of dose-d ependent, unlike the other two immune-m ediated wheat MLN lymph nodes is plausible, additional mechanisms may be sensitivities (coeliac disease and wheat allergy), where even low operative; a further migration of these myeloid cells to periph- amounts of wheat protein can cause disease. eral lymph nodes or even the CNS; a general adjuvant role of Early studies evaluating the effects of a gluten-f ree diet on elevated cytokine/chemokine levels originating from the intestine patients with MS,48 49 showed a positive effect on MS- related or the generation/activation of CNS homing Th17 cells within outcomes such as expanded disability scale score, lesion activity the intestine. In the context of CNS autoimmunity, small intes- on MRI, mood, fatigue and quality of life when patients strictly tinal dendritic cells activated by segmented filamentous bacteria avoided gluten-c ontaining foods. However, these studies were have enhanced the pro- inflammatory phenotype of MOG- specific not well-c ontrolled or observational. In addition, dietary compli- (Th17) T cells, facilitating CNS immune activation.42 We previ- ance, placebo/nocebo effects and the lack of end points other ously showed that mice consuming ATI showed a selective increase than symptoms further limit the plausibility of these studies of segmented filamentous bacteria,23 which were implicated to which were performed before the pro- inflammatory activity of favour a pro- inflammatory intestinal environment to drive intes- ATI had not been discovered. Considering the dose- dependent tinal, joint and CNS autoimmunity.23 43 In patients with MS, an effect of dietary ATI and our finding that gluten does not activate expansion of intestinal Th17 cells was linked to similar microbial the intestinal immune system nor worsens EAE, future dietary changes, but the mechanism remains ill-d efined.41 recommendations for dietary studies with patients with MS may To determine the early effects of dietary ATI on CNS inflam- not require a complete wheat-f ree diet but rather a reduction mation, we compared the 1.5% ATI group against the 0.19% low of around 90% of usual wheat (‘gluten’) intake. Based on these ATI and the control ATI-g luten-f ree group (0% ATI). Fifteen days insights, it may be worthwhile to select wheat products that are post- MOG immunisation, an ATI-d ependent worsening of clinical low in ATI and therefore safe for patients with MS.50 51 symptoms accompanied by increase numbers of pro- inflammatory Limitations of our study are that in vivo data were obtained MHC-I Iint, and MHC-I Ihi myeloid cells as well as Ly6G+ neutro- exclusively in the EAE mouse model of MS, and that human phil infiltrating the CNS was clearly observed, indicating that the functional data were based on peripheral PBMC that have a (mild) ATI-i nduced intestinal inflammation affects the differentia- limited predictive value for MS as a CNS disease. Moreover, a tion and acquisition of (peripheral) T cell pathogenicity. clinical study of patients with well-d efined MS on a wheat (ATI)- Furthermore, dietary ATI reduced the number of CD4+C- based versus a wheat (ATI)-f ree diet remains to be done. D25+Foxp3+ Treg in the MLN during the early stage of EAE To conclude, we provided evidence that nutritional wheat (9 days postsensitisation), suggesting an early suppression of ATI dose- dependently exacerbate CNS autoimmunity in murine immune regulation by nutritional ATI. Treg cells are essential EAE. The immune activation is driven by activated myeloid cells for the inhibition of Th1, Th2 and Th17 cell-m ediated inflam- from the gut and from (mesenteric) lymph nodes. Moreover, matory responses, modulated by the complex plasticity of helper ATI can suppress Treg cell numbers, and results are transferable T cells.44 45 Here, IL-1 0-m ediated Stat3 activation in Treg regu- to myeloid cells from patients with MS and controls. Further lates Th17 cells,46 whereas local IL-6 drives differentiation of studies to specify the exact trajectory of signal transduction from the common RORγt precursors to Th17 instead of Treg cells.47 the gut to the periphery including the CNS are on the way. Our In line with the established roles of these cytokines in Treg cell results justify a clinical trial of an ATI- free versus the standard differentiation and the suppression of Treg cells in ATI-f ed EAE ATI-c ontaining diet in patients with MS, including both immu- mice, we previously found that IL-1 0 was downregulated and nological readouts and patient-r elated outcome measures. IL-6 prominently upregulated after incubation of myeloid cells with ATI in vitro and after exposure of mice to ATI in vivo.22 In Author affiliations this line, serum IL-6 , as well as serum MCP-1 (CCL2) and IL-8 1Institute of Translational Immunology, Johannes Gutenberg Universitat Mainz, were increased up to fourfold 2–4 hours after a gavage of ATI.22 Mainz, Germany 2 To translate some of our findings in mice to humans, monocytes Department of Applied and Health Sciences, Northumbria University, Newcastle upon Tyne, UK from patients with MS and healthy controls were challenged with 3Institute for Molecular Medicine, Johannes Gutenberg Universitat Mainz, Mainz, ATI, which triggered the expression and release of a range of pro- Germany inflammatory chemokines and cytokines (CCL- 2, IL-6 , TNFα and 4Department of Dermatology, University of Cologne, Koln, Germany 5 IL-8 ) in both groups. This indicates that, as expected, (dietary) ATI can Research Center for Immunotherapy, Johannes Gutenberg Universitat Mainz, Mainz, directly enhance pro-i nflammatory monocyte- macrophage responses Germany6 19 Department of Cardiology, Johannes Gutenberg Universitat Mainz, Mainz, Germanyvia activation of TLR4 in general, while the extent of this response 7Department of Neurology, Johannes Gutenberg Universitat Mainz, Mainz, Germany was not different in patients with MS versus (healthy) controls. Circu- 8Neurology Department, University Hospital Munster, Munster, Germany lating immune cells cannot be expected to reflect the state of activity 9Division of Gastroenterology, Beth Israel Deaconess Medical Center, Harvard of an autoimmune disease, especially in a low activity and stable CNS Medical School, Boston, Massachusetts, USA disease, as in our patients. However, these results suggest that dietary Twitter Ari Waisman @ariwaisman ATI may promote the severity of murine EAE, and of human MS, Contributors VZ: study design; acquisition of data; analysis and interpretation downstream of modest monocyte- macrophage activation in the gut. of data; drafting and editing the manuscript; statistical analysis. NY: study design; Notably, a diet containing 25% of protein as gluten and a low acquisition of data; analysis and interpretation of data; drafting and editing the amount of ATI (0.19%) only weakly promoted intestinal and manuscript; statistical analysis. JH, GP, AN, VH, NF, MN, CJ, AMT: acquisition of data; CNS inflammation and neurological symptoms (comparable to analysis and interpretation of data; LK: analysis and interpretation of data. SS and FL: interpretation of data, manuscript editing. DS and AW: study design; interpretation of the gluten-f ree and ATI-f ree), whereas a diet with crude gluten data; study supervision; drafting and editing of the manuscript. DS is the guarantor and a higher amount of ATI (0.75% ATI), or purified ATI of this study. All authors revised and edited the manuscript. 102 Zevallos VF, et al. Gut 2024;73:92–104. doi:10.1136/gutjnl-2023-329562 Gut: first published as 10.1136/gutjnl-2023-329562 on 18 August 2023. Downloaded from http://gut.bmj.com/ on March 13, 2024 by guest. Protected by copyright. Gut immunity Funding This work was supported by the German Research Foundation (DFG), 16 Dupont FM, Vensel WH, Tanaka CK, et al. Deciphering the complexities of the wheat Collaborative Research Center on Multiple Sclerosis (SFB TR128/3, projects A03, A07 flour Proteome using quantitative two- dimensional electrophoresis, three proteases and A08) and SFB TR355/1 (project number 490846870) to DS and AW. DS received and Tandem mass Spectrometry. Proteome Sci 2011;9:10. project- related support by DFG grant Schu 646/20- 1/2 (Allergy), Pic/Schu SPP 17 Schuppan D, Pickert G, Ashfaq- Khan M, et al. Non-c eliac wheat sensitivity: 1656 (Intestinal microbiota, to GP and DS), Schu 646/17- 1 (ATI) and by the Leibniz differential diagnosis, triggers and implications. Best Pract Res Clin Gastroenterol Foundation (Wheatscan, SAW-2 016- DFA-2 ). 2015;29:469–76. 18 Sano M, Uchida T, Igarashi M, et al. Increase in the Lipopolysaccharide activity and Competing interests None declared. accumulation of gram- negative bacteria in the stomach with low acidity. Clin Transl Patient and public involvement Patients and/or the public were not involved in Gastroenterol 2020;11:e00190. the design, or conduct, or reporting, or dissemination plans of this research. 1 9 Junker Y, Zeissig S, Kim S- J, et al. Wheat Amylase Trypsin inhibitors drive intestinal Patient consent for publication Consent obtained directly from patient(s). inflammation via activation of toll- like receptor 4. J Exp Med 2012;209:2395–408. 2 0 Lallès JP. Intestinal alkaline phosphatase: novel functions and protective effects. Nutr Ethics approval This study was approved by ethics committee of the State of Rev 2014;72:82–94. North Rhine Westfalia and the University of Münster, Germany (2016- 053- f-S ). All 21 Bates JM, Akerlund J, Mittge E, et al. Intestinal alkaline phosphatase Detoxifies murine studies were approved by the Animal Care and Use Committee of the state Lipopolysaccharide and prevents inflammation in Zebrafish in response to the gut of Rhineland Palatinate, Germany. Participants gave informed consent to participate Microbiota. Cell Host Microbe 2007;2:371–82. in the study before taking part. 2 2 Zevallos VF, Raker V, Tenzer S, et al. Nutritional wheat Amylase-T rypsin inhibitors Provenance and peer review Not commissioned; externally peer reviewed. promote intestinal inflammation via activation of myeloid cells. Gastroenterology 2017;152:1100–13. Data availability statement No data are available. 2 3 Pickert G, Wirtz S, Matzner J, et al. Wheat consumption aggravates colitis Supplemental material This content has been supplied by the author(s). It in mice via Amylase Trypsin inhibitor- mediated Dysbiosis. Gastroenterology has not been vetted by BMJ Publishing Group Limited (BMJ) and may not have 2020;159:257–72. been peer- reviewed. Any opinions or recommendations discussed are solely those 24 Zevallos VF, Raker VK, Maxeiner J, et al. Dietary wheat Amylase Trypsin inhibitors of the author(s) and are not endorsed by BMJ. BMJ disclaims all liability and exacerbate murine allergic airway inflammation. Eur J Nutr 2019;58:1507–14. responsibility arising from any reliance placed on the content. Where the content 25 Bellinghausen I, Weigmann B, Zevallos V, et al. Wheat Amylase-T rypsin inhibitors includes any translated material, BMJ does not warrant the accuracy and reliability exacerbate intestinal and airway allergic immune responses in Humanized mice. J of the translations (including but not limited to local regulations, clinical guidelines, Allergy Clin Immunol 2019;143:201–12. terminology, drug names and drug dosages), and is not responsible for any error 26 Ashfaq-K han M, Aslam M, Qureshi MA, et al. Dietary wheat Amylase Trypsin inhibitors and/or omissions arising from translation and adaptation or otherwise. promote features of murine non- alcoholic fatty liver disease. Sci Rep 2019;9:17463. 2 7 Caminero A, McCarville JL, Zevallos VF, et al. Lactobacilli degrade wheat Amylase Open access This is an open access article distributed in accordance with the Trypsin inhibitors to reduce intestinal dysfunction induced by Immunogenic wheat Creative Commons Attribution Non Commercial (CC BY- NC 4.0) license, which proteins. Gastroenterology 2019;156:2266–80. permits others to distribute, remix, adapt, build upon this work non- commercially, 2 8 Carroccio A, Mansueto P, Soresi M, et al. Wheat consumption leads to immune and license their derivative works on different terms, provided the original work is activation and symptom worsening in patients with familial Mediterranean fever: A properly cited, appropriate credit is given, any changes made indicated, and the use pilot randomized trial. Nutrients 2020;12:1127. is non-c ommercial. See: http://creativecommons.org/licenses/by-nc/4.0/. 29 Liwinski T, Hübener S, Henze L, et al. 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Characterization of 150 wheat Cultivars by LC-M S- the efficacy of a gluten-f ree diet versus a regular diet in a series of relapsing- remitting based label-f ree quantitative Proteomics Unravels possibilities to design wheat better multiple sclerosis patients. Int J Neurol Neurother 2014;1:1–6. for baking quality and human health. Plants (Basel) 2021;10:424. 104 Zevallos VF, et al. Gut 2024;73:92–104. doi:10.1136/gutjnl-2023-329562 Gut: first published as 10.1136/gutjnl-2023-329562 on 18 August 2023. Downloaded from http://gut.bmj.com/ on March 13, 2024 by guest. Protected by copyright. BMJ Publishing Group Limited (BMJ) disclaims all liability and responsibility arising from any reliance Supplemental material placed on this supplemental material which has been supplied by the author(s) Gut DIETARY WHEAT AMYLASE TRYPSIN INHIBITORS EXACERBATE CNS INFLAMMATION IN EXPERIMENTAL MULTIPLE SCLEROSIS SUPPLEMENTARY TABLES Supplementary Table 1. Dietary composition Ingredients GAF (g/kg) GA/G (g/kg) A (g/kg) Zein 221.00 217.66 217.66 ATI * —– —– 5.00 Gluten # —– 55.25 —– Corn starch 459.00 457.34 457.34 Maltodextrin 50.00 50.00 50.00 Sucrose 50.00 50.00 50.00 Cellulose 52.00 52.00 52.00 Amino acid mix # 43.00 43.00 43.00 Mineral mix 60.00 60.00 60.00 Vitamin mix 10.00 10.00 10.00 Choline chloride 2.00 2.00 2.00 Soybean oil 53.00 53.00 53.00 Dietary content: GAF, gluten and ATI-free; GA, gluten containing 0.75% ATI; G, gluten depleted of ATI (containing 0.19% ATI); A, ATI. *ATI, Amylase trypsin inhibitors, purified from wheat; 5.0 g lyophilisate added (containing 1.5 g of pure, bioactive ATI); final concentration: 0.15% of chow weight. # Gluten without or with ATI depletion. 1 Zevallos VF, et al. Gut 2023;0:1–13. doi: 10.1136/gutjnl-2023-329562 BMJ Publishing Group Limited (BMJ) disclaims all liability and responsibility arising from any reliance Supplemental material placed on this supplemental material which has been supplied by the author(s) Gut Supplementary Table 2. Primer sequences. Genes Primers (m, murine; h, human) (F, Forward and R, Reverse) mGAPDH F: 5´-AGGTCGGTGTGAACGGATTTG-3´ R: 5´GGGGTCGTTGATGGCAACA-3´ mKC (IL-8) F: 5′TGTCAGTGCCTGCAGACCAT-3′ R: 5′CCTGAGGGCAACACCTTCA3′ mMCP-1 (CCL-2) F: 5′-CTTCCTCCACCACCATGCA -3′ R: 5′-CCAGCCGGCAACTGTGA-3′ mINF- γ F: 5`-CAGCAACAGCAAGGCGAAA-3` R: 5`-TCAAACTTGGCAATACTCATGAATGCATCCT-3 mIL-15 F: 5’CAG TTG CAG AGT TGG ACG AA3’ R: 5’GTG CTT TGA AGA GCC AGA GG 3’ h18S Cat.No. QT00199367 X03205 (1869 bp) hIL8 Cat.No.QT00000322 NM_000584 (1718 bp) hCCL-2 Cat.No.QT00212730 NM_002982 (760 bp) hTNF-α Cat.No.QT01079561 NM_000594 (1686 bp) hIL-6 Cat.No.QT00083720 NM_000600 (1201 bp) SYBR Green primer sequences (Eurofins, Hamburg, Germany); F (Forward), R (Reverse) and QuantiTect primer sequences (Qiagen, Hilden, Germany). 2 Zevallos VF, et al. Gut 2023;0:1–13. doi: 10.1136/gutjnl-2023-329562 BMJ Publishing Group Limited (BMJ) disclaims all liability and responsibility arising from any reliance Supplemental material placed on this supplemental material which has been supplied by the author(s) Gut Supplementary Table 3. Healthy donors (HD) MS patients (MS) Gender (female, f / male, m) 10f and 5m 10f and 5m Age (years, mean ± SD) 39.1 ± 12.8 (25 – 62) 36.6 ± 10.9 (22 – 52) Treatment N/A Natalizumab Disease course N/A RRMS Disease activity N/A stable Demographics of patients participating in the study. Gender (female, male), age (years ± standard deviation), treatment and disease course/activity. 3 Zevallos VF, et al. Gut 2023;0:1–13. doi: 10.1136/gutjnl-2023-329562 BMJ Publishing Group Limited (BMJ) disclaims all liability and responsibility arising from any reliance Supplemental material placed on this supplemental material which has been supplied by the author(s) Gut DIETARY WHEAT AMYLASE TRYPSIN INHIBITORS EXACERBATE CNS INFLAMMATION IN EXPERIMENTAL MULTIPLE SCLEROSIS SUPPLEMENTARY MATERIALS AND METHODS Cell preparation, Flow Cytometry Staining and Acquisition Briefly, mice were perfused through the left cardiac ventricle with ice-cold saline solution. Next, brain and spinal cord were dissected and digested with collagenase D (2.5 mg/ml) and DNase I (1 mg/ml) (Sigma-Aldrich) at 37°C for 45 min. After passing the tissue through a 70 µm cell strainer, cells were separated by centrifugation on a discontinuous Percoll gradient (30:37:70%), and mononuclear cells were isolated from the interphase. Peripheral lymphoid organs were incubated with 2 mg/ml Collagenase D for 30 min at 37°C. The digested intestine, spleen, and mesenteric lymph nodes (MLN) were mashed through a 70 mm cell strainer. Fluorescence-activated cell sorting (FACS) was performed as detailed before 24, 34. Briefly, Isolated cells were stained with live/dead fixable dyes (Invitrogen, Darmstadt, Germany) and antibodies to surface markers: CD4 (GK1.5 or RM4-5), CD11b (M1/70), CD11c (N418), CD25 (PC61), CD45.1 (A20), CD45.2 (104), CD90.2 (53-2.1), Ly6G (1A8), MHC II (M5/114.15.2), NK1.1 (PK136), CD19 (6D5), CD24 (M1/69), XCR1 (ZET); all BD Biosciences, San Jose, USA; eBioscience, Darmstadt, Germany or BioLegend, San Diego, USA). For intracellular cytokine staining, cells were re-stimulated with T-cell medium containing 20 µg/ml MOGP35- 55 at 37 °C for 6 hrs and additional 1 µl/ml Brefeldin A for the final 4 hrs. Subsequent to live/dead and surface staining, cells were fixed and permeabilized (Cytofix/perm IC staining kit; BD Biosciences), and stained for cytokines IL-17A (TC11-18H10.1; BioLegend), IFN-γ (XMG1.2, eBioscience) and CD154 expression. For intranuclear staining of Foxp3 (FJK-16s, eBioscience), cells were stained for live/dead discrimination and surface markers, fixed and permeabilized, followed by Foxp3 intranuclear staining according to the manufacturer’s instructions (Transcription Factor Staining Buffer Set, eBioscience). Samples were analyzed using a FACSCanto device (BD Biosciences) with FACSDiva software (BD Biosciences). Post-acquisition analysis was performed using FlowJo software (Tree Star, Ashland, Oregon, US). For gating strategies refer to Suppl. Figs.3-4. Zevallos VF, et al. Gut 2023;0:1–13. doi: 10.1136/gutjnl-2023-329562 BMJ Publishing Group Limited (BMJ) disclaims all liability and responsibility arising from any reliance Supplemental material placed on this supplemental material which has been supplied by the author(s) Gut Histology and immunohistochemistry Cryosections were fixed in 4% PFA for 20 min followed by sequential incubation with methanol, avidin/biotin (Vector Laboratories) and protein blocking reagent (T144.1 Roti- ImmunoBlock, Roth, Karlsruhe, Germany) to eliminate unspecific background staining. Slides were then incubated overnight with primary antibody specific for the respective antigen. Subsequently, the slides were incubated for 30 min at room temperature with biotinylated secondary antibodies (Jackson Immunoresearch and BD Pharmingen). All samples were finally treated with streptavidin-horseradish peroxidase and stained with Tyramide (Cy3) according to the manufacturer’s instructions. Nuclei were counterstained with mounting medium for fluorescence with 4,6-diamidino-2-phenylindole (H-1200, Vector Labs, Burlingame, USA). Positive immune cells (F4/80 and CD11c) were quantified using Image J software and counting of positive cells in 5 representative intestinal villi per mouse. Cell culture and stimulation of human monocytes Human PBMC-derived were cultured in X-VIVO 15 medium (Lonza, Basel, Switzerland) at 37°C alone or stimulated with either ATI (100µg/mL) or LPS (50ng/mL) for 16 hours. Then, cell supernatants were collected and cytokine/chemokine release (IL-6, TNFα, CCL2) was assessed by commercial ELISAs (Ready-Set-Go, eBioscience, Darmstadt, Germany). Furthermore, monocyte RNA was isolated using RNeasy (Qiagen), reverse transcribed (Fermentas, Waltham, Massachusetts, USA) and transcripts for IL-6, IL-8, TNFα, and CCL2 quantified in triplicates using gene-specific primers (QuantiTect, Qiagen, Hilden, Germany) (Suppl. Table 2) and the CFX384 Touch™ Real-time PCR Detection System (Bio-Rad, Hercules, USA). Data was quantified using the DDCT method and normalized to 18S RNA expression. Zevallos VF, et al. Gut 2023;0:1–13. doi: 10.1136/gutjnl-2023-329562 BMJ Publishing Group Limited (BMJ) disclaims all liability and responsibility arising from any reliance Supplemental material placed on this supplemental material which has been supplied by the author(s) Gut Zevallos VF, et al. Gut 2023;0:1–13. doi: 10.1136/gutjnl-2023-329562 BMJ Publishing Group Limited (BMJ) disclaims all liability and responsibility arising from any reliance Supplemental material placed on this supplemental material which has been supplied by the author(s) Gut Zevallos VF, et al. Gut 2023;0:1–13. doi: 10.1136/gutjnl-2023-329562 BMJ Publishing Group Limited (BMJ) disclaims all liability and responsibility arising from any reliance Supplemental material placed on this supplemental material which has been supplied by the author(s) Gut Zevallos VF, et al. Gut 2023;0:1–13. doi: 10.1136/gutjnl-2023-329562 BMJ Publishing Group Limited (BMJ) disclaims all liability and responsibility arising from any reliance Supplemental material placed on this supplemental material which has been supplied by the author(s) Gut Zevallos VF, et al. Gut 2023;0:1–13. doi: 10.1136/gutjnl-2023-329562