Antigen-Specific Gut Inflammation and Systemic Immune Responses

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Article

Antigen-specific gut inflammation and systemic immune responses induced by pro-longing wheat gluten sensitization in BALB/c murine model Vijaykrishnaraj M, Mohan Kumar BV, Muthukumar SP, Nawneet K Kurrey, and Pichan Prabhasankar J. Proteome Res., Just Accepted Manuscript • DOI: 10.1021/acs.jproteome.7b00199 • Publication Date (Web): 15 Aug 2017 Downloaded from http://pubs.acs.org on August 16, 2017

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Journal of Proteome Research

Antigen-specific gut inflammation and systemic immune responses induced by prolonging wheat gluten sensitization in BALB/c murine model

Vijaykrishnaraj M1,4, Mohan Kumar BV1,4, Muthukumar SP2,3,4, Nawneet K. Kurrey3, and Prabhasankar P1,4* 1

Flour Milling Baking and Confectionery Technology Department 2

3

4

Animal House Facility

Department of Biochemistry

Academy of Scientific and Innovative Research

CSIR-Central Food Technological Research Institute Mysuru – 570020, Karnataka, INDIA

Corresponding author* Dr.P.Prabhasankar Principal Scientist Fax: +91 821 2517233 Tel: +91 821 2517730 E-mail: [email protected] 0 ACS Paragon Plus Environment


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Graphical Abstract

For TOC only

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Abstract

Gluten-related diseases such as wheat allergy, celiac disease, and gluten intolerance are widespread in the globe to genetically pre-disposed individuals. The present study aims to develop wheat-gluten induced BALB/c murine model for addressing wheat-gluten related disorders by sensitizing the wheat gluten through the route of intraperitoneal and oral challenge in prolonged days. During the sensitization, the sera were collected for specific anti-gliadin antibodies response and pro-inflammatory markers quantification. An ex-vivo primary cells and organs were collected for subsequent analysis of inflammatory profile. Prolonging sensitization of gluten can moderate the antigen-specific inflammatory markers such as IL-1β, IL-4, IL-15, IL-6, IFN-ϒ and TNF-α level in mice sera. However, ex-vivo primary cells of splenocytes (SPLs) & intestinal epithelial lymphocytes (IELs) significantly increased the IL-6, IL-15, IL-1β, & IL-4 levels in G + (gliadin and gluten) treated cells. Histopathology staining of jejunum sections indicates that enterocyte degeneration in the apical part of villi and damage of tight junctions in G + (gliadin and gluten) sensitised murine model. Immunohistochemistry of embedded jejunum sections showed significant expression of positive cells of IL-15, tTG and IL-4 in G + sensitised murine model. In contrast, all markers of gluten-related disorders are expressed exclusively such as tTG, ZO-1, IL-15, IL-6, IL-4 and intestinal inflammation was mediated by iNOS, COX-2, TLR-4 and NF-kBp50 signalling mechanism in G+ sensitized mice. Keywords: Gluten, wheat allergy, celiac disease, murine models, cytokines and

Inflammation



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Abbreviations

IgG – Immunoglobulin gamma IgA – Immunoglobulin alpha IgE – Immunoglobulin epsilon WA – Wheat allergy CD – Celiac disease tTG – tissue Transglutaminase HLA – Human leukocyte antigen BSA – Bovine serum albumin PBS – Phosphate buffered saline PBST – Phosphate buffered saline tween 20 GL+ - Gliadin GT+ - Gluten G- - BSA G + - Gliadin and gluten ELISA – Enzyme linked immuno-sorbent assay TMB - 3, 3’, 5, 5’-Tetramethylbenzidine SDS-PAGE – Sodium do-decyl polyacrylamide gel electrophoresis DAB - 3, 3’-Diaminobenzidine PVDF – Polyvinylidene fluoride 3 ACS Paragon Plus Environment

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RT - Room temperature (between 20°C and 25°C) SPLs – Splenocytes IELs – Intraepithelial lymphocytes TLR-4 – Toll-like receptor-4 TER – Trans epithelial electric resistance iNOS – Inducible nitrogen oxide synthase COX-2 – Cyclooxygenase 2 NFKB - Nuclear factor kappa b protein RT-PCR – Reverse transcriptase polymerase chain reaction mRNA – Messenger ribo nucleic acid



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Introduction

Wheat is one of the auspicious crop cereals consumed globally, and it induces acute allergic reactions in susceptible individuals. Ingestion of gluten proteins from wheat and other grains (barley and rye) triggers an inappropriate immune response in genetically predisposed individuals1. The wheat seed contains 80% of storage proteins such as gliadins and glutenins. Gliadins are monomeric proteins with molecular weights around 28,000 – 55,000, and it is soluble in aqueous alcohol. Whereas, glutenin subunit has high molecular weight (HMW) subunits (MW-67,000 – 88,000) and low molecular weight (LMW) subunits (MW- 32,000 – 35, 000). When it is in the condition of reduced disulfide bonds of glutenin subunits, it shows solubility in aqueous alcohol like gliadins2.Gluten is composite of gliadins and glutenins when it is hydrated; it will join to form a network-like structure3. However, these proteins are the repetitive sequence of prolamins and majority of adverse reactions are caused by these sequences in the grains. On the far side, it has potentiality such as viscoelasticity; it plays a fabulous role such as shaping of dough, elasticity and chewy texture in the bakery and pasta products4. Wheat gluten causes skin disorder such as Dermatitis Herpetiformis (DH), it’s an autoimmune disorder. It causes a cutaneous pruritic papulovesicular rash on the elbows, forearms, buttocks, knees, and scalp. The prominent inflammatory cells due to infiltration of neutrophil and eosinophil in 25% cases. Moreover, DH is also associated with gluten-induced enteropathy correlates with villous atrophy and infiltration of intraepithelial lymphocytes5. Celiac disease (CD), is an autoimmune disorder triggered by gliadin in genetically susceptible individuals. Gliadins play a critical role to activate both innate and adaptive immune response, which results in the immune-mediated injury of the intestine such as intestinal permeability, villous atrophy and inflammation infiltration of lamina propria6.


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Ingested gluten gets digested by gastric enzymes, and the gliadin fragments are generated. In spite of Tissue transglutaminase (tTG), an auto antigen against the gliadin, an abnormal immune response is directed, and two autoantibodies such as IgA and IgG are produced. Even though HLA DQ2 and DQ8 are the two genetic factors for CD, those autoantibodies generated are the serological markers to screen this disease4, 7. Pathogenesis of CD results from an interaction between deaminated gliadin peptides (33 amino acid peptide) and HLA DQ2/8 molecules, which induces the activation of CD4+TH1 lymphocytes in the intestinal lamina propria and causes tissue injury. However, the stimulated peptides more fascinate to generate T-cells production in epithelial cells, monocytes and dendritic cells8. At the same time, these cells can produce a number of pro-inflammatory cytokines and inflammatory mediators such as TNF-α, IFN-ϒ, IL-1β, IL-15, COX-2, and NF-kB signalling. Among these, over expression of IL-15 in epithelial cells, plays a crucial role in the activation of NK T cells and subsequent development of enteropathy in CD. However, gliadin may increase intestinal permeability through the subsequent attraction of macrophages in the intestinal sub mucosa9. Celiac disease is a life-long autoimmune disorder, occurring 1 in 100 individuals worldwide. On the epidemiological basis, India is having promising number of gluten sensitive patients in North Indian population. Moreover, 25% of CD patients can be associated with other autoimmune disorders such as type-1-diabetes (in 3% cases), autoimmune thyroiditis (in 10%) and autoimmune hepatitis (in 1% population) 10. To date, the only therapy exists for CD is lifelong gluten-free diet. Alternative therapies are growing to combat this disease such as blocking the inflammatory cascade, modifying the microbiome in gut or intestine, degrading the immunogenic gliadin by enzymes, sourdough technology, and trans amidated wheat11.



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Animal models are playing a vital role in understanding complex immunological reactions and pathophysiological mechanisms of food allergic reactions by researchers in recent days. Currently, several animal models are used for this purpose; including mouse, rat, swine, and dog12. The mouse is one of the extensively used animal models for investigating cellular and molecular mechanisms of various types of IgE-mediated allergy to proteins. Apparently, one of the highest IgE responder mouse strain is BALB/c, and this strain is susceptible for the human phenotype that responds to the allergenic proteins13, 14. The existing animal models are studied mostly in BALB/c mice and gene knock-out or transgenic mice for the gluten sensitization. A few earlier reports show that gluten provokes the specific immune responses in those animal models and the studies were executed in limited period of days. Hence, BALB/c mice is an appropriate model, when compared with a gene knock-out mice for this kind of studies. To understand and develop therapies, the link between innate and humoral immune response to different pathways induced by gluten inflammatory response has to be explored. To overcome this issue, the current study was planned for sensitizing the gluten in BALB/c mice via intraperitoneally and intragastric challenge in prolonged days to mimic the gluten-related inflammatory disorders via mucosal and specific mediated immune response. MATERIALS AND METHODS Materials for animal’s sensitization and diet components

Wheat Flour (Triticum aestivum), (Triticum dicoccum), bakery wheat flour, gluten powder, corn starch and ground-nut oil were procured from the local market (Mysuru, India). BALB/c mice (4-6 weeks old, Female) were approved by the Institutional Animal Ethics Committee (IAEC No: 344/14). Diet ingredients such as casein, dextrinized corn starch, choline bitartrate, L- cysteine were purchased from Hi-Media (Mumbai, India) as per AIN93G diet. Vitamin mix (AIN-93 MX) was purchased from MP Biomedicals (Santa Ana,


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USA), sucrose and salt mixture Bernhart Tommarelli was purchased from SRL, (Mumbai, India). Reagents and antibodies for immuno-reactivity analysis

Gliadin and gluten from wheat, anti-mouse IgG (whole molecule) peroxidase antibody produced in rabbit, protease inhibitor cocktail for use with mammalian cell and tissue extracts and anti-mouse IgA (α-chain specific) peroxidase antibody produced in goat were purchased from Sigma-Aldrich (St. Louis, USA). Goat anti-mouse IgE secondary antibody-HRP was purchased from Thermo Fisher Scientific (Waltham,

USA).

Polyvinylidene fluoride (PVDF) membrane for Western blotting was purchased from Pall Corporation (New York, USA). Antibodies for western blotting such as Anti-Transglutaminase 2, N-Terminal antibody produced in rabbit, Anti-Mouse Tlr4 (N-term) antibody produced in rabbit, AntiCOX2 antibody produced in rabbit and Anti-Actin, N-terminal antibody produced in rabbit were procured from Sigma Aldrich (St. Louis, USA). Anti-IL15 rabbit polyclonal and AntiIL4R antibody were purchased from Abcam (Cambridge, UK). IL-6 (D5W4V) XP® Rabbit mAb, TNF-α (D2D4) XP® Rabbit mAb, IL-1β (3A6) Mouse mAb, NF-κB1 p105/p50 (D4P4D) Rabbit mAb were procured from Cell Signalling Technology (Danvers, USA). Extraction and preparation of wheat gliadins

Extraction and preparation of wheat gliadins were carried out according to Papista et al.15 with slight modification. The wheat flour (10g) was suspended in a known volume of 10% (w/v) NaCl, and it was continuously stirred by using orbital shaker (Genei, Bangalore, India) at 150 rpm for 2 h at room temperature. Later, the suspension was centrifuged at 8000 rpm for 10 min in the cooling centrifuge (Eppendorf, Hamburg, Germany). The pellet was again resuspended with 10% NaCl and repeated for centrifugation. The residue was further extracted twice with 70% (v/v) ethanol and centrifuged as mentioned earlier. The alcohol 8 ACS Paragon Plus Environment


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soluble gliadins were collected in the supernatant, and it was filtered through Whatman filter paper no.1. The lyophilized gliadin powder was used for further analysis. Immunochemical analysis of gliadins and animal diet SDS-PAGE

Prepared gliadins from wheat flour were quantified, and their electrophoretic profile was characterised. Extracted protein profiling was done by SDS-PAGE according to Laemmli16. Fifty µg of extracted proteins were separated on 12% gel in the gel electrophoresis unit (Bio-Rad Mini-protean Tetra system), and protein bands were visualised by Coomassie brilliant blue R-250 staining followed with destaining. The gel was documented by using gel document system (SYNGENE) (Syngene Bio imaging Private Ltd, India). Dot blot

Dot blot analysis was done for gliadins reactivity and levels of gliadins in the animal diet, according to the method Vijaykrishnaraj et al.17. Extracted protein samples (4 µL) were spotted on the nitrocellulose membrane (NCM), then blocking was carried out by adding 1% gelatin for 2 h at room temperature (RT). The blot was washed thrice with PBST (Phosphate buffered saline, pH 7.4 contains 0.05ml Tween 20) and thrice with PBS. Blot was treated with primary antibody, rabbit anti-gliadin (1:1000) and anti-rabbit pep-II (1:1000) in antibody diluting buffer and incubated overnight at 4°C. After washing the blot as mentioned above, secondary antibody goat-anti-rabbit IgG-HRP conjugate (1:5000) was added and it was incubated for 2h at RT. The washed blot was developed by adding (DAB) contains H2O2 and citrate buffer (pH – 5.5). Western blot

Western blot analysis was carried out with 50 µg samples of extracted proteins which were separated on 12% SDS-PAGE and transferred to a PVDF membrane by electro blotting.


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Antibody coating and blotting was carried out as mentioned earlier in the dot blot procedure. The developed blots and reacted proteins were visualised by clarity-Western ECL substrate (Bio-Rad). The developed blot was documented by using gel documentation system (SYNGENE) (Syngene Bio imaging Private Ltd, India). Animal’s sensitization and experimental plan

BALB/c mice (female, 4-6 weeks of age) were maintained in pathogen-free facilities under conditions of 23±2°C RT/Humidity (50±10˚C) with 12 h dark & light cycle in AHF (Animal House Facility, CFTRI). The mice were fed with a purified diet AIN-93G (American Institute of Nutrition) formulated for the growth of rodents according to Reeves et al.18. The absence of gluten was verified by using immunochemical validation, as mentioned above. The mice (n=10/group) were randomly divided into four groups (Control – PBS; GL+ Gliadin; GT+ Gluten; G- BSA) after acclimatisation of 10-14 days with food and water adlibitum. a) Intra peritoneal sensitization

Sensitization of gluten was carried out according to Bodinier et al. & Abe et al.19, 20 with minor modifications. Primary sensitization to the animals were performed via intraperitoneal immunizations (0.2 mg/0.028 kg of bodyweight) of wheat proteins (gluten/gliadins) adsorbed onto alum (aluminum hydroxide 2.52 mg/ml in sterile PBS) were provided from 0th – 56th day (0, 7, 14, 21, 28, 37, and 56) as 0.2 ml per mouse. Control mice received sterile PBS with alum as mentioned earlier. Blood was drawn from each animal by retro-orbital venous plexus fortnightly for serum analysis. b) Oral challenge of wheat gluten

Mice were orally administered (10 mg/0.028 kg of bodyweight) with wheat proteins (gluten/gliadins) in 500 µl of PBS (pH-7.4) through an intragastric feeding needle (20 SWG



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standard bend, B.I.K Industries, Mumbai) every other day for consecutive 60 days. Body weight and animals were health monitored throughout the sensitization. Assessment of specific anti-gliadin IgE, IgG and IgA reactivity

Sensitization efficacy of mice was evaluated by the concentration of anti-gliadin specific IgE, IgG and IgA produced according to Bodinier et al.19. Reactivity from individual serum samples was determined at 15 days interval by in-direct ELISA. Mice sera was used as primary antibody (1:50) ratio for anti-gliadin IgG & IgA measurement. For, anti-gliadin IgE measurement, sera samples were diluted in the ratio of 1:20 in antibody diluting buffer. The respective secondary antibody conjugated with peroxidase-labelled was used. To develop the reactions, TMB (Sigma Aldrich, USA) were used, and the reaction was stopped by the addition of 2N H2SO4. The values were read at 450 nm by ELISA plate reader (Multiskan Go, Thermo Scientific, USA). Detection of inflammatory cytokines in mice sera

Collected mice sera were quantified for inflammatory cytokines (IL-1β, TNF-α, IFNϒ, IL-4, IL-6 and IL-15) by using eBioscience (Affymetrix, San Diego, USA) sandwich ELISA kits according to manufacturer’s instructions. Sera were diluted at 1:25 ratio in antibody diluting buffer in each well for samples. Ex-vivo primary cell culture from sensitized animals a) Isolation of Splenocytes

Cells were isolated according to Papista et al.15 and as per standard protocols. CO2 asphyxiation euthanization was done for animals from each group, followed by spraying 70% alcohol as the disinfectant to the intraperitoneal cavity before dissecting the organs. Spleen and small intestine were dissected aseptically in laminar air flow chamber (BS-II level), and the organs were washed thoroughly using ice-cold PBS-pH-7.4. The spleen was minced properly by surgical scissors in a sterile Petri plate containing a known volume of ice-cold


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PBS-pH-7.4, and the mashed spleen was smashed for the recovery of splenocytes using cell strainer (Pore size: 40µm, Hi-Media, Mumbai). The cell suspension was further used for RBC lysis by RBC lysis buffer (Sigma Aldrich, St. Louis, USA) and isolated cells were counted by Trypan blue dye exclusion method. b) Isolation of Intestinal epithelial lymphocytes

From the same animals, small intestine jejunum section was cut into small pieces, and washed as mentioned earlier. Mincing was performed by sterile 2 ml syringe to remove mucus; the intestine suspension was incubated in isolation medium with 1 mM DTT for 5 min at 37°C with gentle shaking (100 rpm/min). After, centrifugation the pellet was incubated in isolation medium with 0.25 mM EDTA for 10 min at 37°C with gentle shaking. It was repeated 3-4 times, until the supernatant became clear. Supernatants were passed through a loosely packed glass wool column to remove clumps and debris. The enriched population of IELs was centrifuged, and the pellet was suspended in RPMI-1640 complete media. The cell viability was checked at each step using Trypan blue dye exclusion method.

Splenocytes and Intestinal epithelial lymphocytes in-vitro response to wheat gluten

Cells were seeded (1.5 x 106 cells/well) in 1 ml of RPMI-1640 (Sigma Aldrich, St.Louis, USA) with Fetal Bovine Serum (Hi-Media, Mumbai, INDIA) complete medium in a 6 well plate. Peptic-tryptic digestion of wheat gliadin/gluten and BSA were carried out according to Tuckova et al.21 for in-vitro exposure to the cells. A known concentration (150µg) of digested proteins was treated to the cells up to 72 h at 37°C and 5% CO2 atmosphere incubator. Cell proliferation, nitric oxide levels, and cytokines detection were analysed in the cell supernatant15, 21.



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Histopathology of small intestine sections

Small intestine sections (jejunum) were fixed with 10% formaldehyde solution, processed and embedded in paraffin and cut into 4µm cross sections onto a glass slide. The sections were stained with haematoxylin & eosin. The slides were observed under light microscopy (EVOS XL core cell imaging system, Thermo Fisher Scientific, USA) in different magnifications (10x, 40 x and 100 x) for the morphometric evaluation of damage of tight junctions and infiltration of the apical part of villi. Immunohistochemistry of small intestine sections

Immunohistochemistry of paraffin-embedded sections were performed according to Cooper et al.22 with minor modifications. An anti-mouse IL-15, tissue Transglutaminase (tTG), and IL-4 were analysed in fixed tissues by treating with respective antibodies diluted according to manufacturer protocols. Positive cells were determined by using Signal Stain® DAB Substrate Kit Cell Signalling Technology (Danvers, USA) under light microscopy EVOS XL core cell imaging system (Thermo Fisher Scientific, Waltham, USA). RNA isolation and reverse transcriptase-polymerase chain reaction (RT-PCR)

To analyse the mRNA expression of inflammatory cytokines and immune genes, total RNA was extracted from RNA later stored small intestine sections and spleen using TRI reagent (Sigma Aldrich, St. Louis, USA) according to manufacturer instructions. The isolated RNA was quantified by Nano drop (Eppendorf Ltd, Germany) to synthesise single strand cDNA, reverse transcription of 1µg of total RNA was synthesised by using Verso cDNA synthesis kit (Thermo Scientific, USA) according to manufacturer’s protocol. The primer details have been shown in Table 1. The amplified product was detected by the presence of an SYBR green fluorescent signal. Melting curve analysis and the crossing point (Cp) of the long linear portion of the amplification curve was determined. The relative induction of gene


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mRNA expression was calculated using the following equation E∆Cp (control samples – treated samples) and normalised to the expression of β-actin.

Preparation of cell lysates and Western blot

The small intestine and spleen tissues were homogenized in ice-cold RIPA buffer (150mM NaCl, 50 mM Tris-HCl, pH-7.4, 1mM EDTA, 1% Triton-x-100, 1% Sodium deoxycholate, 0.1% SDS and cocktail protease inhibitor) and cell lysates were prepared by tissue homogenizer (T10 Basic Ultra, IKA, India) in 30 sec cycles 2 times. Protein concentration was measured by Brad-ford method23. Samples containing 100µg of total cellular proteins were loaded and separated in 10-15% SDS-PAGE. The resolved proteins were then electrophoresed onto PVDF membrane and subjected to Western blot analysis by using inflammatory markers specific primary antibodies (β-actin, ZO-1, tTG, NFKb-P50/105, TLR-4, iNOS and COX-2) diluted in 2.5% BSA-containing PBS (Phosphate Buffered Saline) pH-7.4 and 0.05% Tween-20. Blot was incubated at 4°C for overnight. Washing was carried out thrice with PBST (PBS pH-7.4 and 0.05% Tween 20) following thrice with PBS pH-7.4. Respective secondary antibodies were diluted in dilution buffer, and the blot was incubated at RT for 2 h. The protein bands were visualised by an enhanced chemiluminescence method using ECL (Clarity-Western ECL, Bio-Rad).

Statistical analysis

Values were expressed as mean ± S.E and comparisons were performed by ANOVA (Stat graphics software) of variance followed by Kruskal-Wallis Test for the null hypothesis. Data were considered statistically significant with the P value less than 0.05. Principal component analysis (PCA) was done to visualise the similarities and dissimilarities of data.



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Results Wheat proteins profile and detection of gluten in prepared animal diet

The gliadin and animal diet extract was analysed by SDS-PAGE for the composition of gliadins and glutenins separation. Gliadins such as α, β, ϒ and ω are major allergenic proteins, which can cause adverse reactions to the immune system. The molecular weight of α, β, ϒ gliadins in the range of 30 to 40 kDa and ω gliadin is in the range of 40 to 50 kDa24. The extracted gliadins SDS-PAGE results show that the separated proteins are in the range 30-40 kDa in the first 4 wells (Fig.1A). Whereas, the animal diet (AIN-93G) have also appeared in the range of 30-40 kDa. For the confirmation of gliadin reactivity, dot-blot and immuno-blot were carried out in the same extract. However, the treated antibodies (Sigma anti-gliadin antibodies & Rabbit PEP-II generated antibodies) did not react to the prepared animal diet. The reacted bands and spot were observed in the wheat gliadin extract of dot-blot and immuno-blot (Fig.1B & Fig.1C). Reactivity of mouse anti-gliadin antibodies response by sensitization of wheat-gluten

Sensitization was done as mentioned in materials and methods section. The animals were divided into four groups (n=10), Control – PBS; GL+ Gliadin; GT+ Gluten; G- BSA. The anti-IgE antibodies response was significantly increased in GL+, GT+ and G- except control groups (Fig.2C; P< 0.05). High IgE antibodies reactivity was observed in 37th, 59th and 73rdday of mice sera. However, the specific anti-gliadin IgG and IgA for gluten-related disorders serological marker also found a significant increase in 93rd day of mice sera in GL+ group (Fig.2A and 2B; P< 0.05). IgE, IgG and IgA markedly increased in GL+ and GT+ groups. These results indicate that the sensitised wheat gluten extract causes immunogenicity to the respective animals by the route of continuous oral challenge. However, the G- group animal sera reactivity was shown significant to IgE, which is due to the albumin proteins cross-reactivity and immunization activity to the respective animals. The Kruskal-Wallis test 15 ACS Paragon Plus Environment

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was done to test the null hypothesis since the P value is less than 0.05, there is a statistically significant difference amongst the medians at the 95% confidence level. Elicitation of inflammatory response and detection of pro-inflammatory cytokines

The pro-inflammatory cytokines such as IL-1β, TNF-α, IFN-ϒ, IL-6, IL-4 and IL-15 were quantified by sand-witch ELISA methods using specific kits. Interleukins level were considerably increased and decreased throughout the sensitization study. GT+ mice group showed significantly increased IL-4 levels, and it was observed as 12000 pg/ml on the 16th day of mice sera. In other groups, the rise was not significant up to 73rd day of sensitised mice sera (Fig.3A). The same pattern of results was noticed for IL-15 levels in GL+, GT+ and Ggroups (Fig. 3B). GL+ group mice sera IL-4 levels were significant at 13000 pg/ml in 93rd day of sensitization (Fig.3A; P< 0.05). IL-15 levels were observed at 15000 pg/ml in GL+ group sera and also observed at 16500 pg/ml in G- group sera. However, IL-15 levels were significantly reduced in GT+ group sera in 93rd day of the sensitization (P< 0.05). IL-15 is an intestinal epithelial marker when intestinal lumen undergoes inflammation and release specific cytokines from dendritic cells. During the 93rd day, the overexpression of IL-15 was observed, it may be due to intraepithelial lymphocytes infiltration by inflammatory dendritic cells. IL-1β levels were significantly increased in consecutive days (37 and 59) of sensitised GL+ and GT+ group mice sera (Fig.3D; P< 0.05). On the other hand, similar levels of IL-1β were observed at G- group mice sera. At the same time, IL-6 levels were significantly observed at 20000 pg/ml in GL+ groups and 28000 pg/ml in GT+ groups vice versa on the121st day (P< 0.05). The same pattern also observed at G- groups at 25000 pg/ml range of IL-6 level (Fig.3E). IFN-ϒ and TNF-α levels were observed the almost similar pattern in consecutive days (16 to 93), at the same time a significant increase in GL+ and GT+ mice sera on the 121st day (P< 0.05). Whereas, G- mice



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sera levels were also showed the same level (Fig.3C & 3F). Moreover, these results suggest that the animals have undergone pro-inflammatory activities to wheat gluten sensitization.

Pro-inflammatory immune response in splenocytes and intestinal intraepithelial lymphocytes cells

After complete sensitization study, at 121st day 3 mice per group were sacrificed to isolate the SPLs and IELs for ex-vivo primary cell culture. Spleen, which is a secondary lymphoid organ of the lymphatic system and has mature naive lymphocytes and serves as an initiative for the adaptive immune response. Moreover, It is also the site to encounter and activate the specific immune cells to respective antigens. The isolated cells were cultured up to 72 h for the in-vitro exposure of peptic-tryptic digested gluten for the secretion of proinflammatory cytokines immune response. The cells were seeded in the same level of population, SPLs cell proliferation assay shows that significant reduction from 0 to 72 h of exposure of GL+ and GT+ sensitised mice groups (Fig.4A). IELs are lymphocytes majorly found in the mucosal lining of the mammalian system such as gastrointestinal tract and reproductive tract. However, similar kind of trend was observed, and it is significantly reduced the proliferation of cells in IELs exposure to GL+ and GT+ sensitised mice groups (Fig.4B). On the other hand, we want to confirm any free radical generation or oxidative changes during exposure. It was confirmed by nitric oxide (NO2) measurement in collected cell supernatant. NO2 levels were observed significant levels in GL+ and GT+ sensitised mice groups (Fig.4C). At the same time, the treated cell supernatant was analysed to detect the proinflammatory cytokines as described earlier. The several quantified cytokines were statistically analysed for multiple comparison analysis via principal component analysis (PCA) showed in Fig.4D & 4E. SPLs cell supernatant content of IL-6, IL-1β, IL-15, and IL-4 17 ACS Paragon Plus Environment

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was discriminately comparable (85%) and relative to GL+ and GT+ sensitised mice groups (Fig.4D). However, the content of IFN-ϒ and TNF-α were only close relative to G- sensitised mice groups (Fig.4D). IELs cell supernatant content of IL-4, IL-15, and IFN- ϒ were discriminately comparable (95%) and relative to GL+ and GT+ sensitised mice groups (Fig.4E). Whereas, the content of IL-6 and TNF-α are closely relative to GT+ and Gsensitised mice groups (Fig.4E). However, in both treated cells all the cytokines content were not closely relative in PBS sensitised mice groups. Gluten induces the intestinal inflammation like intestinal intraepithelial T-cell infiltrates and villous atrophy

The clinical features and morphological changes of small intestine jejunum sections were taken by H&E staining of the fixed tissues shown in Fig.5. Clinically, CD features of villi damage are associated with crypt hyperplasia and IEL infiltrates. Sensitization of gluten to the GL+ and GT+ mice groups induces the villous atrophy and hyperplasia (Fig.5.2 & 5.3), whereas no abnormalities were seen in PBS and G- mice groups (Fig.5.1 & 5.4). However, the H-E staining showing enterocyte degeneration in the apical part of villi and damage of tight junctions in GL+ and GT+ sensitised animals (Fig.5A & 5B). The villi damage score was calculated by observing the villi morphology changes in all groups under the light microscope. Visually estimated the damage in each slide and the values were counted. The microvilli damage score has been shown in Fig.5C. GL+ and GT+ mice groups showed a marked increase of positive IgA cells in earlier. Moreover, immunohistochemistry analysis of small intestine jejunum section showed the significantly increased number of IL-15 cells in GT+ mice groups followed with GL+ and G- mice groups (Fig.6C, 6B & 6D). The above results suggest that sensitization via oral challenge induces the overexpression of IL-15 in intestinal epithelial cells and tight junction. However, the expression of tTG increased in lamina propria and it was detected in GT+ and GL+ groups. (Fig.6Jand 6K). Furthermore, the 18 ACS Paragon Plus Environment


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cytokines IL-15 and IL-4 shown a positive cell expression in GT+ groups (Fig.6G) and, it was evidenced by ELISA and immunohistochemistry. Expression and regulation responses of the bio-markers related to gluten induced inflammation by qRT-PCR assay

To determine whether sensitised mice were tolerated or mimicking the gluten-related disorders and confirm our previous clinical results, we amplified the mRNA with respective gene primers for expression analysis. The cDNA of pro-inflammatory markers (IL-1β, IL-4, IL-6, IL-15, TNF-α, IFN-ϒ, COX-2, iNOS, and NF-kB) were amplified, and their expression profile heat map has been shown in Fig.7A & 8A. A pro-longing gluten sensitization significantly impacted in the expression levels of IL-6, IL-15 and TNF-α in fold changes of 4 in GL+ sensitised mice group of spleen mRNA (Fig.7A; P

Antigen-Specific Gut Inflammation and Systemic Immune Responses

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