Name: Autor em destaque: Investigando os efeitos de compostos no tecido intestinal usando modelos de linha celular humana 3D
Uploaded: 2023-09-01T13:00:00
Description: Watch this Scientific Journal Video about Investigating the Effects of Compounds on Intestinal Tissue Using 3D Human Cell Line Models at JoVE.com

Agradecimentos à Fundação Umberto Veronesi por uma bolsa de apoio ao trabalho de pesquisadores.

1. Preparação do modelo básico de mucosa intestinal reconstruída em 3D
OBS: Todo o procedimento deve ser realizado em capela de fluxo laminar estéril. Todas as etapas do procedimento que envolvem o uso da incubadora de células significam que as culturas são incubadas a 37 °C em uma atmosfera umidificada contendo 5% de CO2 (salvo indicação em contrário no protocolo).
<ol><li>Preparo prévio das linhagens celulares utilizadas no sistema modelo intestinal<ol><li...

Construção de um Modelo 3D de Mucosa Intestinal com Incorporação de Parafina

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	<li>Chelakkot, C., Ghim, J., Ryu, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+regulating+intestinal+barrier+integrity+and+its+pathological+implications.">Mechanisms regulating intestinal barrier integrity and its pathological implications.</a> Experimental &amp; Molecular Medicine. 50 (8), 1-9 (2018).</li><li>Schoultz, I., Keita, &. #. 1. 9. 7. ;. 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E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Is+it+time+for+reviewer+3+to+request+human+organ+chip+experiments+instead+of+animal+validation+studies.">Is it time for reviewer 3 to request human organ chip experiments instead of animal validation studies.</a> Advanced Science. 7 (22), 2002030 (2020).</li><li>Jung, S. M., Kim, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+models+of+the+small+intestine+for+studying+intestinal+diseases.">In vitro models of the small intestine for studying intestinal diseases.</a> Frontiers in Microbiology. 12, 767038 (2022).</li><li>Nitsche, K. S., M&#252;ller, I., Malcomber, S., Carmichael, P. L., Bouwmeester, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Implementing+organ-on-chip+in+a+next-generation+risk+assessment+of+chemicals:+a+review.">Implementing organ-on-chip in a next-generation risk assessment of chemicals: a review.</a> Archives of Toxicology. 96 (3), 711-741 (2022).</li><li>Kekilli, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Midkine+level+may+be+used+as+a+noninvasive+biomarker+in+Crohn's+disease.">Midkine level may be used as a noninvasive biomarker in Crohn's disease.</a> Turkish Journal of Medical Sciences. 50, 324-329 (2020).</li><li>Truzzi, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spermidine-eugenol+supplement+preserved+inflammation-challenged+intestinal+cells+by+stimulating+autophagy.">Spermidine-eugenol supplement preserved inflammation-challenged intestinal cells by stimulating autophagy.</a> International Journal of Molecular Sciences. 24 (4), 4131 (2023).</li><li>Truzzi, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Are+supplements+safe?+Effects+of+gallic+and+ferulic+acids+on+in+vitro+cell+models.">Are supplements safe? Effects of gallic and ferulic acids on in vitro cell models.</a> Nutrients. 12 (6), 1591 (2020).</li><li>Buckley, A. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualisation+of+multiple+tight+junctional+complexes+in+human+airway+epithelial+cells.">Visualisation of multiple tight junctional complexes in human airway epithelial cells.</a> Biological Procedures. Online. 20, 3 (2018).</li><li>Ghosh, R., Gilda, J. E., Gomes, A. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+necessity+of+and+strategies+for+improving+confidence+in+the+accuracy+of+western+blots.">The necessity of and strategies for improving confidence in the accuracy of western blots.</a> Expert Review of Proteomics. 11 (5), 549-560 (2014).</li></ol>

O sistema modelo básico de mucosa intestinal reconstruída aqui apresentado (Figura 6) combina complexidade fisiológica (culturas de células 3D mais relevantes fisiologicamente contendo uma monocamada de Caco-2 com um suporte de lâmina própria rico em MEC contendo fibroblastos e monócitos) com simplicidade experimental (usando linhagens celulares humanas comerciais para produzir um sistema padronizado e facilmente repetível)13. Como tal, este sistema modelo é ...

A barreira epitelial intestinal, um revestimento interno de uma célula de espessura contendo diferentes tipos de células epiteliais, constitui a primeira barreira ou interface física defensiva entre o meio externo e o meio interno do corpo 1,2. Os enterócitos do tipo colunar constituem o tipo mais abundante de células epiteliais. Estes são responsáveis pela manutenção da integridade da barreira epitelial por meio de interações entre vários componentes da barreira, incluindo as tight junctions (TJs), desempenhando um papel significativo no aperto da barreira 1,3. A estrutura do TJ consiste em proteínas da placa intracelular, como zonula occludens (ZO) e cingulina, cooperando com proteínas transmembrana, incluindo oclusinas, claudinas e moléculas de adesão juncional (JAMs) que formam uma estrutura semelhante a um zíper ligando firmemente as células vizinhas 3,4. As proteínas transmembrana regulam a difusão paracelular passiva de pequenos compostos e excluem moléculas grandes tóxicas.
Compostos alimentares potencialmente tóxicos e contaminantes alimentares estimulam a produção de citocinas inflamatórias que interrompem a permeabilidade epitelial, ativando células imunes e causando inflamação crônica do tecido intestinal 5,6,7. Em contraste, vários fitoquímicos antioxidantes e anti-inflamatórios têm sido relatados para reduzir a expressão de citocinas inflamatórias e aumentar a integridade da barreira intestinal de TJ através da restauração da expressão e montagem da proteína TJ 4,6,8. Assim, a regulação da integridade da barreira epitelial por compostos benéficos e nocivos tem visto um aumento no uso de modelos in vivo e in vitro com o objetivo de mimetizar a barreira intestinal para triagem farmacêutica e estudos de toxicidade. Isso é particularmente relevante dado o crescente interesse na compreensão da fisiopatologia das doenças intestinais intestinais (DII), enterocolite necrosante e câncer, que podem ser simuladas em modelos experimentais8,9,10.
Tem havido uma demanda para o desenvolvimento de modelos in vitro baseados em células, a fim de atingir o objetivo dos "3Rs" em testes com animais. Entre elas, alternativas de substituição ao uso de animais, redução do número de animais utilizados e refinamento na adoção de métodos que aliviem o sofrimento 11,12,13. Além disso, os mecanismos moleculares, celulares e fisiológicos subjacentes entre modelos humanos e murinos (sendo os roedores a espécie mais utilizada) são distintos, levando a controvérsias quanto à eficácia dos modelos murinos como preditores de respostas humanas12,13. Inúmeras vantagens dos modelos in vitro de linhagem celular humana incluem experimentação com restrição de alvo, observação direta e análise contínua13.
Monocamadas do tipo célula única em culturas bidimensionais (2D) têm servido como modelos poderosos. No entanto, estes não conseguem reproduzir com precisão a complexidade fisiológica dos tecidos humanos 8,13,14. Como resultado, sistemas de cultura 3D estão sendo desenvolvidos com melhorias cada vez maiores para recapitular a complexidade fisiológica de tecidos intestinais saudáveis e doentes como caixas de ferramentas de avaliação de risco de última geração13,14. Esses modelos incluem scaffolds 3D Transwell com diversas linhagens celulares, culturas organoides e dispositivos microfluídicos (intestino-sobre-chip) usando linhagens celulares e organoides (derivados de tecidos saudáveis e doentes)8,13,14.
O protocolo 3D de "tecido saudável" equivalente intestinal apresentado no presente estudo baseou-se no equilíbrio entre complexidade fisiológica e simplicidade experimental13. O modelo é representativo de um arcabouço Transwell 3D, composto por três linhagens celulares (enterócitos [linhagem Caco-2 do adenocarcinoma de cólon padrão-ouro] com componente imune de suporte [monócitos U937 e fibroblastos L929]), constituindo um sistema padronizado e facilmente repetível aplicável para a triagem preliminar de moléculas dietéticas de interesse na integridade da barreira epitelial intestinal e resposta imune. O protocolo inclui a inclusão em parafina para avaliação microscópica da luz da integridade da barreira epitelial usando equivalentes intestinais fixos. A vantagem da presente abordagem é que numerosos cortes dos tecidos embutidos podem ser feitos para corar para múltiplos parâmetros a partir de um único experimento.

basic three-dimensional (3d) intestinal model system with an immune component

Tem havido um aumento no uso de modelos intestinais in vivo e in vitro para estudar a fisiopatologia de doenças inflamatórias intestinais, para a triagem farmacológica de substâncias potencialmente benéficas e para estudos de toxicidade de componentes alimentares potencialmente nocivos. De relevância, existe uma demanda atual para o desenvolvimento de modelos in vitro baseados em células para substituir modelos animais. Aqui, um protocolo para um modelo básico de equivalente intestinal tridimensional (3D) de "tecido saudável" usando linhagens celulares é apresentado com o duplo benefício de fornecer simplicidade experimental (sistema padronizado e facilmente repetível) e complexidade fisiológica (enterócitos Caco-2 com um componente imune de suporte de monócitos U937 e fibroblastos L929). O protocolo também inclui a inclusão em parafina para avaliação microscópica de luz de equivalentes intestinais fixos, proporcionando assim a vantagem de analisar múltiplos parâmetros visuais de um único experimento. Cortes corados pela hematoxilina e eosina (H&E) mostrando as células colunares do Caco-2 formando uma monocamada apertada e regular nos tratamentos controle são usados para verificar a eficácia do modelo como sistema experimental. Usando glúten como um componente alimentar pró-inflamatório, os parâmetros analisados a partir de cortes incluem espessura reduzida da monocamada, bem como ruptura e desprendimento da matriz subjacente (H&E), diminuição da expressão de proteínas de junção apertada como mostrado a partir da coloração de ocludina (quantificável estatisticamente) e ativação imunológica de células U937 migratórias, como evidenciado a partir da coloração de cluster de diferenciação 14 (CD14) e diferenciação relacionada a CD11b em macrófagos. Como demonstrado pelo uso de lipopolissacarídeo para simular a inflamação intestinal, parâmetros adicionais que podem ser medidos são o aumento da coloração de muco e a expressão de citocinas (como midkine) que podem ser extraídas do meio antes da fixação. O modelo básico tridimensional (3D) da mucosa intestinal e cortes fixos podem ser recomendados para estudos do estado inflamatório e integridade da barreira, com a possibilidade de analisar múltiplos parâmetros visuais quantificáveis.

The intestinal epithelial barrier, a one-cell-thick internal lining containing different types of epithelial cells, constitutes the first physical defensive barrier or interface between the outside and the internal milieu of the body1,2. Columnar-type enterocytes constitute the most abundant type of epithelial cells. These are&#160;responsible for maintaining epithelial barrier integrity through interactions between several barrier components, including tight junctions (TJs), playing a significant role in barrier tightening1,3. The TJ structure consists of intracellular plaque proteins, such as zonula occludens (ZO) and cingulin, cooperating with transmembrane proteins, including occludins, claudins, and junctional adhesion molecules (JAMs) that form zipper-like structure tightly linking the neighboring cells3,4. The transmembrane proteins regulate the passive paracellular diffusion of small compounds and exclude toxic large molecules.
Potentially toxic food compounds and food contaminants stimulate inflammatory cytokine production that disrupts the epithelial permeability, activating immune cells and causing chronic intestinal tissue inflammation5,6,7. In contrast, various anti-oxidant and anti-inflammatory phytochemicals have been reported to reduce inflammatory cytokine expression and enhance intestinal TJ barrier integrity through the restoration of TJ protein expression and assembly4,6,8. Hence, the regulation of epithelial barrier integrity by both beneficial and harmful compounds has seen an increase in the use of both in vivo and in vitro models aimed at mimicking the intestinal barrier for pharmaceutical screening and toxicity studies. This is particularly relevant given the increasing interest in understanding the pathophysiology of intestinal bowel diseases (IBD), necrotizing enterocolitis, and cancer, which can be simulated in experimental models8,9,10.
There has been a demand for the development of cell-based in vitro models in order to achieve the objective of the &#8220;3Rs&#8221; in animal testing. These include replacement alternatives to the use of animals, reduction in the number of animals used, and refinement in adopting methods that alleviate distress11,12,13. Moreover, the underlying molecular, cellular, and physiological mechanisms between human and murine models (rodents being the most widely used species) are distinctive, leading to controversy regarding the efficacy of the murine models as predictors in human responses12,13. Numerous advantages of in vitro human cell-line models include target-restricted experimentation, direct observation, and continuous analysis13.
Single-cell-type monolayers in two-dimensional (2D) cultures have served as powerful models. However, these cannot precisely reproduce the physiological complexity of human tissues8,13,14. As a result, 3D&#160;culture systems are being developed with ever-increasing improvements to recapitulate the physiological complexity of both healthy and diseased intestinal tissues as next-generation risk assessment toolboxes13,14. These models include 3D Transwell scaffolds with diverse cell lines, organoid cultures, and microfluidic devices (intestine-on-chip) using both cell lines and organoids (derived from both healthy and diseased tissues)8,13,14.&#160;
The 3D &#8220;healthy tissue&#8221; intestinal equivalent protocol presented in the present study was based on striking a balance between physiological complexity and experimental simplicity13. The model is representative of a 3D Transwell scaffold, comprised of three cell lines (enterocytes [the gold-standard colon adenocarcinoma Caco-2 line] with a supporting immune component [U937 monocytes and L929 fibroblasts]), constituting a standardized and easily repeatable system applicable for the preliminary screening of dietary molecules of interest on intestinal epithelial barrier integrity and immune response. The protocol includes paraffin embedding for light microscopic evaluation of epithelial barrier integrity using fixed intestinal equivalents. The advantage of the present approach is that numerous sections of the embedded tissues can be made to stain for multiple parameters from a single experiment.

Autor em destaque: Investigando os efeitos de compostos no tecido intestinal usando modelos de linha celular humana 3D 

Sistema Modelo Intestinal Tridimensional Básico (3D) com um Componente Imunológico

The scope of the work is to use these in vitro three-dimensional human cell line models to investigate the fact of beneficial as well as helpful compounds on intestinal tissue. The models can be used to further research understanding and also as a basic drug model. Using the plant-derived compounds, spermidine and eugenol, both separately and together in supplement form, we measure beneficial increases in autophagy and decreases in inflammation using these models.Then we will also able to show the potentially harmful effect of gluten extracted from certain wheat varieties on intestinal tissues. We are currently using economically priced, standardized, and easily repeatable in vitro system that better reflect the physiology of the human intestine as an alternative to animal models. Since we also use commercial human cell line rather than plant-derived cells, the responses from the study are more applicable to the general population.The protocol is based entirely on the use of different lines. These renders the model most standardized and reproducible. Furthermore, the simultaneous cultivation of different cell lines, enterocytes, fibroblasts, and immune system cells, allows for cellular responses that are closer to what happens in the patient.

O primeiro aspecto importante é determinar a aceitabilidade da mucosa intestinal equivalente 3D básica para fins experimentais. Esta é realizada com a coloração mais utilizada nos laboratórios de histologia e histopatologia, a saber, hematoxilina (cora material nuclear de cor azul-púrpura profunda) e eosina (cora material citoplasmático variando tons de rosa). A coloração H&E é realizada primeiramente em um controle não tratado, que é cultivado nas mesmas condições e prazos dos tratamentos experimentais. A...

Watch this Scientific Journal Video about Investigating the Effects of Compounds on Intestinal Tissue Using 3D Human Cell Line Models at JoVE.com

Aqui descrevemos a construção de um sistema básico de linhagem celular intestinal tridimensional (3D) e um protocolo de inclusão em parafina para avaliação microscópica de luz de equivalentes intestinais fixos. A coloração de proteínas selecionadas permite a análise de múltiplos parâmetros visuais de um único experimento para uso potencial em estudos pré-clínicos de triagem de drogas.

There has been an increase in the use of in vivo and in vitro intestinal models to study the pathophysiology of inflammatory intestinal diseases, for the ...

O escopo do trabalho é usar esses modelos de linhagem celular humana tridimensional in vitro para investigar o fato de compostos benéficos e úteis no tecido intestinal. Os modelos podem ser usados para aprofundar o entendimento da pesquisa e também como um modelo básico de medicamento. Usando os compostos derivados de plantas, espermidina e eugenol, separadamente e em conjunto na forma de suplemento, medimos aumentos benéficos na autofagia e diminuições na inflamação usando esses modelos.Em seguida, também poderemos mostrar o efeito potencialmente prejudicial do glúten extraído de certas variedades de trigo nos tecidos intestinais. Atualmente, estamos usando sistemas in vitro com preços econômicos, padronizados e facilmente repetíveis que refletem melhor a fisiologia do intestino humano como uma alternativa aos modelos animais. Como também usamos linhagem celular humana comercial em vez de células derivadas de plantas, as respostas do estudo são mais aplicáveis à população em geral.O protocolo é inteiramente baseado no uso de diferentes linhas. Isso torna o modelo mais padronizado e reprodutível. Além disso, o cultivo simultâneo de diferentes linhagens celulares, enterócitos, fibroblastos e células do sistema imunológico permite respostas celulares mais próximas do que acontece no paciente.

basic-three-dimensional-3d-intestinal-model-system-with-an-immune

Research

JoVE Journal

Biology

Basic Three-Dimensional (3D) Intestinal Model System with an Immune Component

There has been an increase in the use of in vivo and in vitro intestinal models to study the pathophysiology of inflammatory intestinal diseases, for the pharmacological screening of potentially beneficial substances, and for toxicity studies on potentially harmful food components. Of relevance, there is a current demand for the development of cell-based in vitro models to substitute animal models. Here, a protocol for a basic, &#8220;healthy tissue&#8221; three-dimensional (3D) intestinal equivalent model using cell lines is presented with the dual benefit of providing both experimental simplicity (standardized and easily repeatable system) and physiological complexity (Caco-2 enterocytes with a supporting immune component of U937 monocytes and L929 fibroblasts). The protocol also includes paraffin embedding for light microscopic evaluation of fixed intestinal equivalents, thereby providing the advantage of analyzing multiple visual parameters from a single experiment. Hematoxylin and eosin (H&#38;E) stained sections showing the Caco-2 columnar cells forming a tight and regular monolayer in control treatments are used to verify the efficacy of the model as an experimental system. Using gluten as a pro-inflammatory food component, parameters analyzed from sections include reduced monolayer thickness, as well as disruption and detachment from the underlying matrix (H&#38;E), decreased tight junction protein expression as shown from occludin staining (quantifiable statistically), and immune-activation of migrating U937 cells as evidenced from the cluster of differentiation 14 (CD14) staining and CD11b-related differentiation into macrophages. As shown by using lipopolysaccharide to simulate intestinal inflammation, additional parameters that can be measured are increased mucus staining and cytokine expression (such as midkine) that can be extracted from the medium prior to fixation. The basic three-dimensional (3D) intestinal mucosa model and fixed sections can be recommended for inflammatory status and barrier integrity studies with the possibility of analyzing multiple visual quantifiable parameters.

Here we describe constructing a basic three-dimensional (3D) intestinal cell line model system and a paraffin embedding protocol for light microscopic evaluation of fixed intestinal equivalents. Staining of selected proteins permits the analysis of multiple visual parameters from a single experiment for potential use in preclinical drug screening studies.

Biologia

Preparation of a Basic 3D Reconstructed Intestinal Mucosa Model for Microscopic Evaluation of Fixed Intestinal Equivalents

To begin, select a 24-well plate containing inserts with 0.4 micron filters. Using a pipette, add 500 microliters of HBSS below and above the filter inserts. Then close the multi-well plate and place it in an incubator for two hours.After incubation, carefully aspirate the HBSS from the plate. Prepare a cell-free collagen solution in a sterile 50 milliliter tube in DMEM on ice. Add 250 microliters of collagen above the membrane filter and place the lid on the plate.Allow the solution to polymerize at room temperature. Next, remove the L-929 fibroblast cell culture from the incubator. Add two milliliters of a pre-warmed trypsin EDTA solution to the flask and place it in an incubator for three to five minutes.Transfer the cell suspension to a sterile 15 milliliter tube and centrifuge at 645 G for five minutes. Using a vacuum pump, aspirate the supernatant and resuspend the pellet in one milliliter of DMEM. Add 20 microliters of cell suspension into a tube containing 20 microliters of Trypan Blue solution.And use a cell counting chamber to evaluate the cell density. Next, remove the U-937 monocyte cell culture from the incubator. Centrifuge the cell suspension and resuspend the pellet in one milliliter of RPMI.After ensuring cells are homogeneously suspended, count them using a cell counting chamber. Next, prepare cell suspensions containing 50, 000 L-929 and 15, 000 U-937 cells respectively in 50 microliters of DMEM. Add each 50 microliter cell suspension to 450 microliters of collagen and mix thoroughly.Overlay the pre-coded cell-free lamina propria with 500 microliters of the collagen cell solution. Close the plate and place it in an incubator for two hours to allow the solution to set. Next, remove the Caco-2 cell culture from the incubator.Using a vacuum pump, aspirate the medium and rinse the cells with 10 milliliters of PBS. Add five milliliters of PBS trypsin EDTA solution and place it in an incubator for five to eight minutes. Then centrifuge the cell suspension and resuspend the pellet in one milliliter of DMEM.After counting, prepare a cell suspension containing 150, 000 Caco-2 cells in 50 microliters of DMEM. Place the plate in a laminar flow hood for 10 minutes before transferring it to the incubator. After 30 minutes, add 500 microliters of the DMEM above the reconstructed model and 500 microliters below the filter and return the plate to the incubator.The next day, carefully replace the medium with 500 microliters of fresh DMEM above and below the filter respectively.

Preparação de um Modelo Básico 3D de Mucosa Intestinal Reconstruída para Avaliação Microscópica de Equivalentes Intestinais Fixos

Paraffin Embedding of a Basic 3D Reconstructed Intestinal Mucosa Model for Immune-Histochemical Staining

For paraffin embedding of cells, set the paraffin machine to 58 degrees Celsius. Using pliers, transfer the membrane inserts to clean wells of a sterile 24-well plate. Add 500 microliters of 37%buffered formalin and PBS above the filter, and one milliliter below the filter.Close the lid and leave the plate in a fume hood for two hours. Once the lamina propria is detached from the membrane insert, transfer the intestinal mucosa into a beaker containing 25 milliliters of 35%ethanol, and incubate for 10 minutes. After dehydration in increasing ethanol concentrations, place the samples in 50 milliliters of xylene or a histological clearing agent for 10 to 20 minutes.Once the samples are transparent, place them in a metal tissue cassette holder and submerge them in liquid paraffin inside a heated machine. After 45 minutes, use a microtome to cut four micron sections. Place the cut section on the slides and dry them in an oven at 37 degrees Celsius for 24 hours.An acceptable 3-D intestinal equivalent mucosa model consists of Caco-2 cells formed in a tight and regular monolayer above the extracellular-matrix-rich lamina propria. Unacceptable models include those showing excessive growth, disorganized epithelial layers, malformation or lack of epithelial layer formation. The proinflammatory effect of wheat protein with a high gluten content disrupted the cellular monolayer and reduced the thickness of the epithelial layer compared to the control group.The occludin protein content was significantly higher in the control group than in the group exposed to gluten protein. CD14 and CD11b staining of U937 monocytes showed that wheat protein with a high gluten content activated the monocytes, and induced their migration and differentiation into macrophages. Under the lipopolysaccharide challenge, Caco-2 cells increased mucus production and the release of the inflammatory marker midkine.