In vitro model of inflamed intestinal barrier

Abstract

The present invention relates to an intestinal epithelium model faithfully reproducing the pathophysiological mechanisms observed in vivo in the context of an inflammatory state of the intestinal barrier. The model according to the present invention comprises two compartments separated by a semi-permeable membrane. The first compartment, corresponding to the apical pole of the intestinal epithelium, comprises a coculture of Caco-2 cells differentiated into enterocytes and HT29-MTX cells differentiated into goblet cells. The second compartment, corresponding to the basolateral pole of the intestinal epithelium, comprises a culture of THP-1 monocytic cells differentiated into macrophages.The model according to the present invention is characterized in that the cells contained in the first compartment produce interleukin (IL-6) at a concentration greater than 100 pg / mL, and interleukin 8 (IL-8) at a concentration greater than 150 pg / mL, and the cells contained in the second compartment produce tumor necrosis factor α (TNF-α) at ​​a concentration greater than 40 pg / mL and interleukin 1β (IL-1β) at a concentration greater than 90 pg / mL. The present invention also relates to the method for obtaining this model as well as to a method for selecting candidate compounds for the treatment of inflammatory bowel diseases by verifying their ability to stop inflammation upon increase in permeability or to verify the restoration of permeability and the mucus layer.

Description

Title of the invention: In vitro model of inflamed intestinal barrier Technical field

[0001] The present invention relates to the field of cellular models and particularly to an in vitro model of an inflamed intestinal barrier. The invention also relates to the method for obtaining this model as well as to its uses. Prior art

[0002] The intestinal epithelium forms a protective and permeable barrier that plays a major role in host defense against the luminal microenvironment, while maintaining selective permeability and absorption of nutrients. The epithelium is covered by a monolayer composed of different intestinal epithelial cells, such as enterocytes, goblet cells, M cells or Paneth cells, and cooperates with immune cells in the lamina propria. Good cooperation between all these cells maintains intestinal homeostasis and mucosal integrity, which are challenged daily by external factors in the luminal environment. Defects in intestinal barrier function are associated with diseases of the gastrointestinal tract, such as inflammatory bowel disease (IBD).

[0003] The exact etiology of IBD is unknown, although multiple causes are involved. Studies have reported that in IBD, the role of the intestinal epithelium as a physical and immune barrier may be disrupted (Ramos and Papadakis, Mayo Clinic proceedings (2019), 94(1), 155-165). In particular, an inappropriate inflammatory response is observed, which can be explained by an increase in the permeability of the intestinal barrier, an alteration of the immune barrier, and abnormal cooperation between the intestinal microflora and the intestinal barrier (Rogler, The Lancet. Gastroenterology & Hepatology (2017), 2(7), 521-530).

[0004] Several in vitro, ex vivo and in vivo models representing the pathophysiological manifestations of the disease have been developed for the study and development of drug treatments. Although very interesting for studying the physiology of an organism as a whole, in vivo models have many limitations due in particular to inter-species differences which make extrapolations to humans difficult, or to ethical questions. In vitro models using human cells have the advantage of reducing animal experimentation and offering the possibility of testing treatments in a simple and reproducible manner.

[0005] Originally used in cancer research, monoculture of Caco-2 cells, an immortalized human colorectal adenocarcinoma cell line, are now recognized as a valid model of small intestinal epithelium. In particular, it has been shown that Caco-2 cells are capable of producing inflammatory markers in response to other cytokines such as interleukin IL-1, tumor necrosis factor TNF-α and / or interferon IFN-γ (Rodriguez-Ramiro et al., The British Journal of Nutrition (2013), 110(2), 206-215; Hollebeeck et al., Food & Function (2012), 3(8), 875-885) and / or in some cases, in response to external stimuli such as contact with lipopolysaccharides (LPS) present on the membranes of microorganisms (Weglarz et al., Digestive Diseuses and Sciences (2007), 52(1), 93-102).

[0006] Unfortunately, using a monoculture of Caco-2 as the sole intestinal cell type does not allow for faithful reproduction of the complex composition of the intestinal barrier. In particular, such a monoculture does not allow for obtaining a permeability similar to the intestinal barrier as well as a mucus layer on the luminal side, which severely limits absorption and permeability studies. In this context, several models based on cocultures have been developed to better represent the complex composition of the intestinal barrier (Béduneau et al., European Journal of Pharmaceutics and Biopharmaceutics (2014), 87(2), 290-298). Among these, we can notably cite the Caco-2 / HT29-MTX coculture model, which provides a more physiologically relevant system, since it includes mucus-producing HT29-MTX cells that mimic the function of goblet cells (Lesuffleur et al., Cancer Research (1990), 50(19), 6334-6343).

[0007] In vitro coculture models frequently use insertion systems composed of rigid porous membranes to perform permeability studies (Pan et al., International Journal of Food Sciences and Nutrition (2015), 66(6), 680-685).

[0008] Subsequently, a tri-culture model based on the combination of the Caco-2 / HT29-MTX coculture model with human THP-1 monocytic cells differentiated into an adherent macrophage-like phenotype was developed. A state corresponding to an inflammatory state was obtained by stimulating the THP-1 cells with a cocktail of inflammatory activators including LPS and the pro-inflammatory cytokines IL-1 [> and TNF-a (Kaulmann et al., Molecular Nutrition & Food Research (2016), 60(5), 992-1005). However, this type of model does not take into account the modification of mucus composition and cell monolayer permeability, as described in in vivo models (Michielan and DTncà, Mediators of Inflammation (2015), 2015 628157) or found in patients with IBD (Sôderholm et al., Gastroenterology (1999), 117(1), 65-72). The inflamed intestinal barrier is characterized by various features including a modification of the mucus composition, a modification of intercellular junctions causing an increase in membrane permeability associated with exposure to bacterial antigens which maintains the inflammatory state, a modification of the composition of the intestinal microbiota, and a recruitment of inflammatory cells secreting pro-inflammatory chemokines which fuel the immune response. A "good" model must therefore be able to find all these characteristics.

[0009] Unfortunately, there are currently no in vitro models that can faithfully and comprehensively model the state of the inflamed intestinal barrier. In this context, the development of anti-inflammatory drugs for the treatment of IBD and inflammation of intestinal tissues requires evaluation in animals to verify the efficacy of these substances.

[0010] There is therefore a real need for a reliable intestinal epithelium model that can faithfully reproduce a state of inflammation of the intestinal barrier. Such a model must in particular make it possible to observe a modification of the composition of the mucus as well as an alteration of the permeability of the cell monolayer. This would make it possible to limit the use of animal experimentation in the study of inflammatory bowel diseases, particularly in the search for potential therapeutic candidates that can be applied to these diseases. Summary

[0011] The invention is defined by the claims.

[0012] The present inventors have succeeded in developing an intestinal epithelium model faithfully reproducing the physiopathological mechanisms observed in vivo in the context of an inflammatory state of the intestinal barrier.

[0013] This new model makes it possible to take into account the modification of the composition of the mucus, the effect of the permeability of the intestinal mucosa on the capacity of molecules to pass through the inflamed epithelium and their capacity to interact with the epithelial cells, but also to mimic the presence of immune cells infiltrated into the intestinal epithelium during inflammation. The model according to the present invention can be used to identify and select molecules or systems carrying them having a therapeutic effect in the context of inflammatory bowel diseases, and thus makes it possible to limit the use of animal models.

[0014] The inventors have demonstrated that the present model is stable over time and very easily reproducible. It includes the pathophysiological mechanisms observed in IBD, including the modification of the composition of the mucus, the increase in membrane permeability linked to the modification of intercellular junctions and the secretion of pro-inflammatory cytokines.

[0015] The inflamed intestinal epithelium model according to the present invention is based on a tri-culture of Caco-2 / HT29-MX / THP-1 cells that were treated with different compounds. The inventors demonstrated that treatment of a tri-culture of Caco-2 / HT29-MX / THP-1 cells with compounds such as DSS and LPS faithfully recreated the conditions observed in an inflamed intestinal barrier. Although used in certain models to induce inflammation in vivo, the mechanism by which DSS acts on intestinal permeability remains unclear. Studies using DSS to induce inflammation in certain rodent strains include several days of exposure to DSS. However, in the context of the present invention, the inventors demonstrated that a short application of DSS in vitro made it possible to obtain a faithful model of an inflamed intestinal epithelium.

[0016] Treatment of the tri-culture of Caco-2 / HT29-MX / THP-1 cells with compounds such as DSS and LPS makes it possible to obtain a model with well-defined characteristics, in particular from the point of view of the expression of pro-inflammatory cytokines, the expression of mucins, and the measured transepithelial electrical resistance.

[0017] Thus, according to a first aspect, the present invention relates to an intestinal epithelium model comprising two compartments separated by a semi-permeable membrane, said model comprising:

[0018] - in the first compartment, corresponding to the apical pole of the epithelium in testinal, a coculture of Caco-2 cells differentiated into enterocytes and HT29-MTX cells differentiated into goblet cells; and

[0019] - in the second compartment, corresponding to the basolateral pole of the epithelium intestinal, a culture of THP-1 monocytic cells differentiated into macrophages,

[0020] said model being characterized in that

[0021] the cells contained in the first compartment produce interleukin (IL-6) at a concentration greater than 100 pg / mL, and interleukin 8 (IL-8) at a concentration greater than 150 pg / mL; and

[0022] The cells contained in the second compartment produce tumor necrosis factor-α (TNF-α) at ​​a concentration greater than 40 pg / mL and interleukin I [> (IL-113) at a concentration greater than 90 pg / mL.

[0023] The model according to the present invention also has a transepithelial electrical resistance lower than 85% compared to the transepithelial electrical resistance measured in a control non-inflamed intestinal epithelium model, and an altered expression of mucins. The expression of the mucin MUC2 is notably reduced by at least 30% compared to a control non-inflamed intestinal epithelium model, and the expression of the mucin MUC5AC is increased by at least 50% compared to a control non-inflamed intestinal epithelium model.

[0024] According to a second aspect, the present invention also relates to a method for obtain a model of intestinal epithelium including the following steps:

[0025] - the culture, in a support comprising two compartments separated by a semi-permeable membrane, Caco-2 cells differentiated into enterocytes and HT29-MTX cells differentiated into goblet cells in the first compartment, and THP-1 monocytic cells differentiated into macrophages in the second compartment; and

[0026] - bringing into contact the differentiated Caco-2 and HT29-MTX cells contained in the first compartment with at least one compound selected from the group consisting of dextran sodium sulfate (DSS), trinitrobenzenesulfonic acid (TNBS) and dinitrobenzenesulfonic acid (DNBS);

[0027] Then

[0028] - bringing into contact the differentiated THP-1 monocytic cells contained in the second compartment with a compound selected from the group consisting of lipopolysaccharides (LPS), TNF-a, interferon-y (IFN-y) and 1TL-1 [3;

[0029] or

[0030] - the replacement of said at least one compound chosen from DSS, TNBS and DNBS by LPS, TNF-a, ITFN-y and / or IL-1 [3.

[0031] The inventors have demonstrated that the model according to the present invention, by faithfully reproducing the state of the inflamed epithelium, makes it possible to reliably select compounds having therapeutic activity in the context of inflammatory bowel diseases. The inventors have in fact demonstrated that the model according to the present invention makes it possible, for example, to observe the effects of betamethasone, a synthetic corticosteroid used mainly for its anti-inflammatory effect, in the treatment of IBD.

[0032] Also, according to a third aspect, the present invention relates to a method for the selection of candidate compounds for the treatment of inflammatory bowel diseases, said method comprising the steps of:

[0033] A) contacting a candidate compound with the intestinal epithelium model according to the present invention;

[0034] B) selection of the compound if it makes it possible to obtain, in said model, at least one of the following effects a) to e): a) A decrease of at least 10% in the level of IL-6 and / or IL-8 in the first compartment; b) A decrease of at least 10% in the level of TNF-a and / or IL-1 [3 in the second compartment; c) An increase in MUC2 expression of at least 5%; d) A decrease in MUC5AC expression of at least 5%; e) An increase in transepithelial electrical resistance of at least 10%. Brief description of the drawings Fig.l

[0035] [Fig.l]: Effect of DSS stimulation (5% w / v) at the apical level and LPS exposure (100 ng / mL) at the basolateral level on the synthesis of pro-inflammatory cytokines TNF-α / IL-1[3 synthesized by macrophages and IL-6 / IL-8 by intestinal cells. This figure shows the synthesis of pro-inflammatory cytokines TNF-α / IL-1[3 by macrophages and IL-6 / IL-8 by intestinal cells, after contacting Caco-2 / HT29-MTX cells with DSS and THP-1 cells with LPS. Fig. 2

[0036] [Fig.2]: Effect of DSS stimulation (5% w / v) at the apical level and LPS exposure (100 ng / mL) at the basolateral level on the expression of MUC2, MUC5AC, ZO-1 and Claudin-1 by Caco-2 / HT29-MTX cells. This figure shows the expression levels of mucins MUC2 and MUC5AC and tight junctions ZO-1 and Claudin-1, after contacting Caco-2 / HT29-MTX cells with DSS and THP-1 cells with LPS. Fig. 3

[0037] [Fig.3]: Effect of betamethasone (100 pM) at the apical level on the synthesis of pro-inflammatory cytokines TNF-a / IL-l[3 synthesized by macrophages and IL-6 / IL-8 by intestinal cells, after induction of inflammation. This figure shows the synthesis of pro-inflammatory cytokines TNF-a / IL-l[3 by macrophages and IL-6 / IL-8 by intestinal cells, after induction of inflammation and treatment at the apical level with betamethasone. Fig. 4

[0038] [Fig.4]: Effect of betamethasone (100 pM) at the apical level on the expression of MUC2, MUC5AC, ZO-1 and Claudin-1 by Caco-2 / HT29-MTX cells, after induction of inflammation. This figure shows the expression levels of the mucins MUC2 and MUC5AC and the tight junctions ZO-1 and Claudin-1, after induction of inflammation and treatment at the apical level with betamethasone. Detailed description of the invention

[0039] A first aspect of the present invention relates to an intestinal epithelium model comprising two compartments separated by a semipermeable membrane, said model comprising:

[0040] - in the first compartment, corresponding to the apical pole of the intestinal epithelium, a co-culture of differentiated Caco-2 cells into enterocytes and differentiated HT29-MTX cells into goblet cells; and

[0041] - in the second compartment, corresponding to the basolateral pole of the intestinal epithelium, a culture of THP-1 monocytic cells differentiated into macrophages,

[0042] said model being characterized in that

[0043] the cells contained in the first compartment produce interleukin (IL-6) at a concentration greater than 100 pg / mL, and interleukin 8 (IL-8) at a concentration greater than 150 pg / mL; and

[0044] The cells contained in the second compartment produce tumor necrosis factor-α (TNF-α) at ​​a concentration greater than 40 pg / mL and interleukin I [> (IL-113) at a concentration greater than 90 pg / mL.

[0045] The present invention aims to provide a model of intestinal epithelium. The model according to the invention is advantageously a model of vertebrate intestinal epithelium, particularly human.

[0046] The model according to the invention therefore comprises two compartments. Each compartment represents a specific pole of the intestinal epithelium. As is known to those skilled in the art, the wall of the vertebrate gastrointestinal tract comprises several characteristic layers and is therefore oriented. The innermost layer is the intestinal epithelium (and therefore the part reproduced within the framework of the model according to the present invention) which borders the interior of the tract, the lumen. The underlying tissue layer, the submucosa, is made of connective tissue and is backed by layers of smooth muscle. Another layer, the serosa, composed of epithelium and connective tissue, covers the external surface of the tract. The so-called "apical" pole is the pole which is in contact with the lumen, as opposed to the "basolateral" pole which corresponds to the pole on the side of the external surface of the digestive tract.

[0047] The model according to the present invention aims to reproduce the intestinal epithelium. It comprises two compartments: one corresponding to the apical pole of the intestinal epithelium, i.e. the part in contact with the intestinal lumen, and the other corresponding to the basolateral pole, i.e. the part in contact with the submucosa.

[0048] Each compartment is its own culture medium. In the context of the present invention, the two compartments are separated by a "semi-permeable membrane

[0049] Those skilled in the art know numerous supports that can be used in the context of the present invention. According to a particularly advantageous embodiment, the compartments used are cell culture inserts. A technology particularly suitable for this purpose is Transwell® technology. Such inserts are commonly used as supports in the context of intestinal epithelium models (see for example Rahman et al., International Journal of Molecular Sciences, 22(24), 13472).

[0050] In the context of the present invention, the compartment corresponding to the apical pole of the intestinal epithelium (upper compartment of the Transwell® when this technology is used) comprises a coculture of Caco-2 and HT29-MTX cells. "coculture" here means a joint culture, in the same medium, of these two cell lines.

[0051] “Caco-2 cells” correspond to an immortalized cell line of human colorectal adenocarcinoma cells. Caco-2 cells are capable of spontaneously differentiating into enterocytes, and after differentiation express the main enzymatic systems found in the enterocytes of the intestinal epithelium. This is the cell line most used as an intestinal model for absorption, transport and bioavailability studies. Such cells can easily be obtained by those skilled in the art. They are, for example, available under the reference HTB-37™ from the American Type Culture Collection (ATCC).

[0052] In the context of the present invention, the Caco-2 cells used in the intestinal epithelium model are differentiated into enterocytes. A person skilled in the art knows perfectly well which conditions to use to obtain complete differentiation of a population of Caco-2 cells into enterocytes. A method for doing this is for example disclosed in the experimental part of the present application.

[0053] "HT29-MTX cells" correspond to a stable subpopulation of mucus-secreting cells obtained after treating HT29 human colorectal cancer cells with methotrexate (Lesuffleur et al., Cancer research 50.19 (1990): 6334-6343). HT29-MTX cells spontaneously differentiate into goblet cells. They have the capacity to produce MUC2-type mucus, which is predominantly produced in the small intestine and colon. These cells are widely used in coculture systems with the Caco-2 cell line to mimic the human intestinal barrier in absorption and permeability studies. Those skilled in the art can very easily obtain these cells from suppliers specializing in the field.

[0054] In the context of the present invention, the HT29-MTX cells used in the intestinal epithelium model are differentiated into goblet cells. A person skilled in the art knows perfectly well which conditions to use to obtain complete differentiation of a population of HT29-MTX cells into goblet cells. A method for doing this is for example disclosed in the experimental part of the present application.

[0055] According to a preferred embodiment, the coculture of Caco-2 and HT29-MTX cells is in the form of a monolayer of polarized cells, with a heterogeneous distribution of the two cell types at the membrane level.

[0056] In the context of the present invention, Caco-2 cells were typically seeded at a concentration half that of HT29-MTX cells. For example, Caco-2 cells were seeded at a concentration of 5,000 cells per compartment and HT29-MTX cells were seeded at a concentration of 10,000 cells per compartment.

[0057] Typically, Caco-2 cells are seeded at a concentration of 10,000 to 20,000 cells per cm2, preferably at a concentration of 14,000 and 16,000 cells per cm2, even more preferably at a concentration of about 15,000 cells per cm2.

[0058] Typically, HT29-MTX cells are seeded at a concentration of 20,000 to 40,000 cells per cm2, preferably at a concentration of 28,000 and 32,000 cells per cm2, even more preferably at a concentration of about 30,000 cells per cm2.

[0059] In the context of the present invention, the compartment corresponding to the basolateral pole of the intestinal epithelium (lower compartment of the Transwell® when this technology is used) comprises a culture of THP-1 monocytic cells differentiated into macrophages.

[0060] “THP-1 monocytic cells” correspond to a monocyte line of human origin from acute monocytic leukemia. This line can differentiate into macrophages under treatment with phorbol-12-myristate-13-acetate (PMA). This is a line widely used in inflammatory models. Those skilled in the art can easily obtain such cells. They are, for example, available under the reference TIB-202™ from the ATCC. Those skilled in the art will know what conditions to apply to obtain a differentiated macrophage population from THP-1 monocytic cells. Those skilled in the art will be able, for example, to treat the THP-1 monocytic cells with PMA at a concentration of 200 ng / mL for 48 to 72 hours to obtain a fully differentiated macrophage population.

[0061] Typically, THP-1 monocytic cells are seeded at a concentration of 10,000 to 20,000 cells per cm2, preferably at a concentration of 14,000 and 16,000 cells per cm2, even more preferably at a concentration of about 15,000 cells per cm2.

[0062] A person skilled in the art knows what conditions and what medium to use for the culture of these different cell lines. The culture medium can be any physiologically acceptable medium allowing the growth of the lines used herein. Physiologically acceptable media are media comprising electrolytes such as sodium, potassium, magnesium and / or calcium salts, including anions such as chloride, carbonate, hydroxide or caprylate. A suitable culture medium according to the present invention can comprise D-glucose, non-essential amino acids, vitamins, inorganic salts and albumin. A suitable culture medium that can be used to cultivate the coculture of Caco-2 and HT29-MTX cells is, for example, DMEM (Dulbecco's Modified Eagle Medium) GlutaMAX™ marketed by TermoFisher Scientific. In parallel, a medium of Suitable culture media that can be used to grow differentiated THP-1 monocytic cells is, for example, RPMI (Roswell Park Memorial Institute) / RPMI-1640 medium marketed by TermoFisher Scientific. These media can be supplemented with albumin, for example, through the addition of fetal bovine serum and / or antibiotics such as penicillin and / or streptomycin.

[0063] The model according to the present invention is characterized in that the cells contained in the first compartment produce interleukin (IL-6) at a concentration greater than 100 pg / mL, preferably at a concentration greater than 300 pg / mL, even more preferably at a concentration greater than 600 pg / mL; and interleukin 8 (IL-8) at a concentration greater than 150 pg / mL, preferably at a concentration greater than 500 pg / mL, even more preferably at a concentration greater than 1000 pg / mL.

[0064] The model according to the present invention is characterized in that the cells contained in the second compartment produce tumor necrosis factor a (TNF-a) at a concentration greater than 40 pg / mL, preferably at a concentration greater than 400 pg / mL, even more preferably at a concentration greater than 700 pg / mL; and interleukin I [> (IL-1|3) at a concentration greater than 90 pg / mL, preferably at a concentration greater than 200 pg / mL, even more preferably at a concentration greater than 250 pg / mL

[0065] IL-6, IL-8, TNF-a and 1TL-1 [3 are well-known inflammatory cytokines of the person skilled in the art. Their human sequences are accessible in the UniProt database under the references P05231 for interleukin 6, P10145 for interleukin 8, P01375 for TNF-a and P01584 for interleukin 1 [3.

[0066] A person skilled in the art also knows perfectly well how to measure these proteins and evaluate the concentration of these cytokines produced in a cell culture medium. The experimental part of the present application provides an example of a method allowing such a measurement within the framework of the present invention. Typically, the concentration of the different cytokines can be evaluated in the supernatant of the cell culture (in the first or second compartment as the case may be) by immunoenzymatic assay, for example by ELISA.

[0067] The model according to the present invention can also be characterized according to the transepithelial electrical resistance measured therein. “Transepithelial electrical resistance” or “TEER” is a measurement for assessing the degree of impermeability of a tissue. It consists of measuring the electrical resistance across a cell layer in order to assess its integrity and permeability. TEER measurement is a well-known technique commonly used by those skilled in the art, particularly in the context of in vitro models (Srinivasan, Balaji, et al. Journal of la-boratory automation 20.2 (2015): 107-126). A method for measuring TEER is presented in the experimental part of the present invention.

[0068] In the context of the model according to the present invention, the transepithelial electrical resistance is less than 85%, preferably less than 50%, even more preferably less than 35% compared to the transepithelial electrical resistance measured in a control non-inflamed intestinal epithelium model.

[0069] The model according to the present invention can also be characterized according to the expression of mucins by the cell cultures constituting it, and in particular by the HT29-MTX cells contained in the first compartment. Mucins are macromolecules entering into the composition of numerous mucuses. They cover the epithelia in contact with the external environment and allow their protection against numerous attacks of endogenous or exogenous origin. At least 21 genes coding for mucins have been identified to date in humans.

[0070] In the context of the model according to the present invention, the expression of the mucin MUC2 (whose amino acid sequence is accessible via the reference Q02817 in the Uniprot database) is reduced by at least 30%, preferably by at least 40%, even more preferably by at least 50% compared to a control non-inflamed intestinal epithelium model.

[0071] Similarly, in the context of the model according to the present invention, the expression of the mucin MUC5AC (whose amino acid sequence is accessible via the reference P98088 in the Uniprot database) is increased by at least 50%, preferably by at least 75%, even more preferably by at least 100% compared to a control non-inflamed intestinal epithelium model.

[0072] By "control non-inflamed intestinal epithelium model" is meant here any intestinal barrier model reproducing a healthy intestinal barrier. In the context of the present invention, said control model corresponds to the tri-culture described above, but in which the concentrations of inflammatory cytokines are lower than the thresholds indicated in the context of the model according to the invention. Also, in the control non-inflamed intestinal epithelium model, the cells contained in the first compartment can produce IL-6, but at a concentration lower than 100 pg / mL, and / or IL-8, but at a concentration lower than 150 pg / mL. The cells contained in the second compartment can produce TNF-a, but at a concentration lower than 40 pg / mL and / or ITL-1 [>, but at a concentration lower than 90 pg / mL.Such a model is typically obtained by the same method as described below, but in which the cell cultures have not been contacted with DSS, TNBS, DNBS, LPS, TNF-a, ITFN-y and / or IL-1 [3. .

[0073] According to another aspect, the present invention also relates to the method for obtaining the inflamed epithelium model as described above. Such The process typically includes the following steps:

[0074] - the culture, in a support comprising two compartments separated by a semi-permeable membrane, Caco-2 cells differentiated into enterocytes and HT29-MTX cells differentiated into goblet cells in the first compartment, and THP-1 monocytic cells differentiated into macrophages in the second compartment; and

[0075] - bringing into contact the differentiated Caco-2 and HT29-MTX cells contained in the first compartment with at least one compound selected from the group consisting of dextran sodium sulfate (DSS), trinitrobenzenesulfonic acid (TNBS) and dinitrobenzenesulfonic acid (DNBS);

[0076] Then

[0077] - bringing into contact the differentiated THP-1 monocytic cells contained in the second compartment with a compound selected from the group consisting of lipopolysaccharides (LPS), TNF-a, interferon-y (IFN-y) and 1TL-1 [3;

[0078] or

[0079] - the replacement of said at least one compound chosen from DSS, TNBS and DNBS by LPS, TNF-a, ITFN-y and / or IL-1 [3.

[0080] The definitions and conditions mentioned above for the model itself also apply to the method for obtaining it.

[0081] Also, the culture media used in step a) are the same as those mentioned above. Similarly, the support used is advantageously made up of cell culture inserts such as, for example, the Transwell® system. The first compartment is the upper compartment and corresponds to the apical pole of the intestinal epithelium. The second compartment is the lower compartment and corresponds to the basolateral pole of the intestinal epithelium. The semi-permeable membrane is also as defined above.

[0082] Sodium "DSS" or "dextran sulfate" is a sulfated polysaccharide whose in vivo administration is used to induce models of ulcerative colitis in mice. It can be easily obtained from specialized suppliers such as Sigma-Aldrich.

[0083] Both "TNBS" or "trinitrobenzenesulfonic acid" and "DNBS" or "dinitrobenzenesulfonic acid" are acids used to induce models of ulcerative colitis and intestinal inflammation in mice. They can be readily obtained from specialized suppliers such as Sigma-Aldrich.

[0084] "LPS" or "lipopolysaccharides", also called lipoglycans or endotoxins, are the major component of the outer membrane of Gram-negative bacteria. LPS is a molecule that comprises three parts: a lipid A, a core oli- saccharide and an O antigen. LPS are very commonly used to induce inflammation in cultured cells or organs. They can be easily obtained from specialized suppliers such as Sigma-Aldrich.

[0085] "Interferon-y" or "IFN-y" is a well-known cytokine mediating innate immunity. The protein sequence of interferon-y is accessible under the reference P01579 in the UniProt database.

[0086] There are therefore two different ways to obtain the model according to the present invention.

[0087] According to a first embodiment, the method comprises contacting the differentiated Caco-2 and HT29-MTX cells contained in the first compartment with at least one compound selected from the group consisting of DSS, TNBS and DNBS; and contacting the differentiated THP-1 monocytic cells contained in the second compartment with a compound selected from the group consisting of LPS, TNF-a, IFN-y and IL-1[3.

[0088] According to this embodiment, the method therefore comprises the following steps: i. the culture, in a support comprising two compartments separated by a semi-permeable membrane, of Caco-2 cells differentiated into enterocytes and HT29-MTX cells differentiated into goblet cells in the first compartment, and of THP-1 monocytic cells differentiated into macrophages in the second compartment; ii. bringing the differentiated Caco-2 and HT29-MTX cells contained in the first compartment into contact with at least one compound selected from the group consisting of DSS, TNBS and DNBS; iii. contacting the differentiated THP-1 monocytic cells contained in the second compartment with a compound selected from the group consisting of LPS, TNF-α, IFN-γ and IL-1 [3.

[0089] This embodiment makes it possible to recreate the acute phase and therefore makes it possible, for example, to test the effect of compounds on an already inflamed state.

[0090] According to one embodiment, the coculture of Caco-2 and HT29-MTX cells contained in the first compartment can advantageously be brought into contact with DSS, TNBS or DNBS before the culture of THP-1 monocytic cells differentiated into macrophages contained in the second compartment has been brought into contact with LPS, TNF-a, IFN-y and / or IL-1 [3. In other words, step ii. above is carried out before step iii. According to this embodiment, the coculture of Caco-2 and HT29-MTX cells has therefore been pretreated with DSS.

[0091] Alternatively, the coculture of Caco-2 and HT29-MTX cells contained in the first compartment can be brought into contact with DSS, TNBS or DNBS at the same time as the culture of differentiated THP-1 monocytic cells in ma crophages contained in the second compartment are brought into contact with LPS, TNF-a, IFN-y and / or FIL-1 [3. In this context, step ii. above is carried out at the same time as step iii.

[0092] According to another embodiment, the method according to the present invention comprises bringing the differentiated Caco-2 and HT29-MTX cells contained in the first compartment into contact with at least one compound chosen from the group consisting of DSS, TNBS and DNBS and then replacing this compound with LPS, TNF-a, IFN-y and / or 1TL-1[3.

[0093] According to this embodiment, the method therefore comprises the following steps: i') the culture, in a support comprising two compartments separated by a semi-permeable membrane, of Caco-2 cells differentiated into enterocytes and HT29-MTX cells differentiated into goblet cells in the first compartment, and of THP-1 monocytic cells differentiated into macrophages in the second compartment; ii) bringing the differentiated Caco-2 and HT29-MTX cells contained in the first compartment into contact with at least one compound chosen from the group consisting of DSS, TNBS and DNBS; iii) replacing said at least one compound with LPS, TNF-a, IFN-y and / or 1TL-1 [3.

[0094] This embodiment makes it possible to recreate the steps of establishing inflammation and therefore makes it possible, for example, to test the preventive effects of compounds in the installation of an inflamed state.

[0095] By replacement is meant here that the culture medium of the cells in which they had been placed in contact with DSS, TNBS or DNBS is changed, with a new culture medium into which, this time, LPS, TNF-a, IFN-y and / or 1TL-1 [3] is introduced. Preferably, DSS, TNBS or DNBS is therefore not eliminated by rinsing, but simply by replacing the medium.

[0096] In the context of the present invention, whatever the method used, the DSS, TNBS and / or DNBS is brought into contact with the coculture of differentiated Caco-2 and HT29-MTX cells at concentrations ranging from 1 to 10% by weight per unit volume (w / V), preferably at a concentration of 5% w / V. The coculture was typically incubated with the DSS, TNBS and / or DNBS, for a period of 6 to 72 hours, preferably for a period of approximately 12 hours.

[0097] In the context of the present invention, LPS, TNF-a, IFN-y and / or IL-1 [3 is brought into contact with the culture of differentiated THP-1 monocytic cells or into contact with the coculture of differentiated Caco-2 and HT29-MTX cells at concentrations ranging from 0.1 to 1,000 ng / mL, preferably at a concentration of 100 ng / mL. The culture of differentiated THP-1 monocytic cells, or, depending on the embodiment, the Coculture of differentiated Caco-2 and HT29-MTX cells was typically incubated with LPS, TNF-α, IFN-γ and / or IL-1 [3 for a period of 12 to 72 hours, preferably for a period of about 24 hours. Once this incubation is complete, the epithelial model is ready for use.

[0098] A person skilled in the art knows how to obtain the various cell cultures used in the context of the method according to the present invention. Typically, these cell cultures are obtained as follows:

[0099] - on D1 (day 1), seeding of Caco-2 cells at a concentration ranging from 5000 to 100,000 cells / cm2, preferably 15,000 cells / cm2, in the first compartment (apical compartment);

[0100] - between D1 and D7, preferably on D5, seeding of HT29-MTX cells at a concentration ranging from 5000 to 100,000 cells / cm2, preferably 30,000 cells / cm2, in the first compartment;

[0101] - between D18 and D30, preferably on D18, seeding of THP1 cells monocytic cells at a concentration ranging from 1000 to 1,000,000 cells / cm2, preferably 800,000 cells / cm2, in the second compartment (basolateral compartment) in the presence of phorbol-12-myristate-13-acetate (PMA) at a concentration ranging from 100 to 300, preferably 200 ng / mL, to obtain a population completely differentiated into macrophages;

[0102] - incubation of the different cultures until complete differentiation is obtained. Caco-2 in enterocytes (21 days of culture are generally necessary);

[0103] - bringing the two compartments into contact through the semi-permeable membrane to carry out the method according to the invention.

[0104] Finally, the present invention also relates to the use of the model according to the invention for screening candidate compounds in the context of the treatment of chronic inflammatory bowel diseases (IBD) or acute inflammatory colitis. IBD includes Crohn's disease (CD) and ulcerative colitis (UC) which are characterized by inflammation of the wall of a part of the digestive tract, due to deregulation of the immune system. The inflamed intestinal epithelium model according to the present invention faithfully represents the state of the intestinal epithelium observed in the context of IBD. Also, it makes it possible to observe the effects obtained by applying a test compound or "candidate" compound to an IBD intestinal epithelium.It is thus possible to see whether the said compound has beneficial, anti-inflammatory effects in the context of IBD and intestinal inflammation in general, and therefore to identify compounds with a potential therapeutic action in the context of intestinal inflammation.

[0105] Also, according to another aspect, the present invention relates to a method for the selection of candidate compounds for the treatment of inflammatory diseases of the intestine, said method comprising the steps of:

[0106] A) contacting a candidate compound with the inflamed intestinal epithelium model according to the present invention; and

[0107] B) selection of the compound if it makes it possible to obtain, in said model, at least one of the following effects a) to e): a) A decrease of at least 10%, preferably at least 20%, even more preferably at least 50% of the level of IL-6 and / or IL-8 in the first compartment; b) A decrease of at least 10%, preferably at least 20%, even more preferably at least 50% of the level of TNF-a and / or IL-1 [3 in the second compartment; c) An increase in MUC2 expression of at least 5%, preferably at least 20%, even more preferably at least 40%; d) A decrease in the expression of MUC5AC of at least 5%, preferably at least 20%, even more preferably at least 40%; e) An increase in transepithelial electrical resistance of at least 10%, preferably at least 20%.

[0108] The test compound or "candidate" compound may be of any type. It may be of natural origin or have been produced by chemical synthesis. It may be a library of structurally defined chemical compounds, uncharacterized compounds or substances, or a mixture of compounds.

[0109] The level of expression and synthesis of pro-inflammatory cytokines, as well as the study of the permeability of the intestinal barrier by measuring transepithelial resistance and the expression of mucins, make it possible to evaluate the level of inflammation of the epithelium.

[0110] The present invention is presented in more detail in the examples below. Examples [YES] Materials and methods

[0112] Cells and reagents

[0113] Enterocyte-like Caco-2 cells and human monocyte-like THP-1 cells were obtained from the American Type Culture Collection (ATCC). Goblet-differentiated HT29-MTX cells were kindly provided by a member of INSERM UMR S 938 (Paris, France).

[0114] All cell culture reagents and betamethasone were purchased from Fisher Scientific (Illkirch, France). Dextran sulfate sodium (DSS) and lipopolysaccharides (LPS) were obtained from Sigma-Aldrich (Saint Quentin-Fallavier, France). The SuperMApo was kindly provided by a member of Med'Inn'Pharma in Besançon, France.

[0115] Cell culture methods

[0116] Caco-2 HT29-MTX co-culture

[0117] The culture medium for Caco-2 and HT29-MTX cells consisted of Dulbecco's Modified Eagle Medium Gluta-Max™ containing 15% inactivated fetal bovine serum (FBS), 1% non-essential amino acids, and 1% antibiotics (100 μg / ml streptomycin and 100 IU / ml penicillin). The cells were maintained in an incubator (Binder, Tuttlingen, Germany) at 37°C and 5% CO2 in a water-saturated atmosphere.

[0118] After thawing, Caco-2 and HT29-MTX cells were seeded separately into 25 cm2 flasks. After 3 passages using 0.25% Trypsin-EDTA, Caco-2 and HT29-MTX cells were seeded at a density of 5000 cells and 10000 cells per insert, respectively, into 0.33 cm2 Transwell® -24-well permeable support plates with a polycarbonate membrane (0.4 μm pores, Corning Costar, Cambridge). After 21 days of incubation, the coculture is fully differentiated.

[0119] THP-1 cell culture

[0120] The culture medium of the THP-1 cell line consisted of Roswell Park Memorial Institute medium containing 10% inactivated FCS and 1% antibiotics (100 μg / mL streptomycin and 100 IU / mL penicillin). The cells were maintained in an incubator (Binder, Tuttlingen, Germany) at 37 °C and 5% CO2 in a water-saturated atmosphere.

[0121] After 6 passages in 75 cm2 flasks, the cells were then seeded into 24-well culture plates at a density of 1.5 × 106 cells per well. Monocytes were differentiated into macrophage-like cells after 3 days of incubation with 100 ng / mL of phorbol 12-myristate 13-acetate (PMA).

[0122] Caco-2 tri-culture. HT29-MTX. THP-1

[0123] The insert containing the Caco-2 / HT29-MTX co-culture was transferred, 21 days after seeding, into a 24-well plate containing differentiated THP-1 (after 3 days of incubation with PMA). The tri-culture model combining differentiated Caco-2 / HT29-MTX cells (located on the insert membrane) and THP-1 cells (adhering to the surface of the wells) was ready for use.

[0124] Inflamed tri-culture model

[0125] Stimulation of differentiated THP-1 and Caco-2 / HT29-MTX cells

[0126] For the characterization of the tri-culture, THP-1 cells and Caco-2 / HT29-MTX cells were stimulated with 0.1, 1, 10, 100 and 1000 ng / mL of LPS solution (24-48-72h) and 1, 3, 5, 7 and 10% w / v of DSS (24-48-72h) respectively. For the kinetic studies, Caco-2 / HT29-MTX cells were stimulated first with 5% w / v DSS (6-12-24h) and then, after removal of the medium, with 100 ng / mL LPS solution (24h).

[0127] After stimulation, all supernatants were collected and stored at -80 °C for cytokine assay. THP-1 cells and cocultured cells (Caco-2 and HT29-MTX cells) were harvested separately and stored in RLT lysis buffer at -80 °C for total RNA isolation, reverse transcription (RT), and quantitative polymerase chain reaction (qPCR) analysis.

[0128] Anti-inflammatory treatments for inflamed tri-culture

[0129] 100 pM betamethasone (BTM) in the culture medium or pro-factors Resolvents produced by macrophages after efferocytosis (SuperMApo) were added to the apical compartment for 24 h after induction of inflammation, as described above.

[0130] After stimulation and treatment, all supernatants were collected and stored at -80 °C for cytokine assay. THP-1 cells and cocultured cells (Caco-2 and HT29-MTX cells) were harvested separately and stored in RLT lysis buffer at -80 °C for total RNA isolation, RT and qPCR analysis.

[0131] Cell viability test

[0132] The metabolic activity of intestinal epithelial cells (IECs) was tested using the MTS assay. After removal of the supernatant, the cells were incubated with 200 μL of MTS solution. The formazan content was quantified spectrophotometrically at 450 nm using the FLUOstar Omega spectrophotometer (BMG Labtech, Champigny-sur-Marne, France). All reactions were performed in triplicate.

[0133] Permeability study

[0134] The integrity of the coculture monolayer was monitored by measuring transepithelial electrical resistance (TEER) and lucifer yellow (LY) penetration through the IECs.

[0135] TEER was evaluated using the Millicell electrical resistance system (Millipore, USA). Triplicate measurements were performed and the values ​​were normalized by subtracting the resistance value of an empty insert (membrane) and expressed as follows:

[0136] [Math. 1]:

[0137] %TEER= (TEERf-TEERe)

[0138] TEERe is the resistance value of an insert without cells, TEERf is the final resistance value and TEER; is the initial resistance value.

[0139] Paracellular permeability was assessed by measuring the transport of lucifer yellow (LY) from the apical to basolateral compartments. LY concentrations were determined by fluorescence (e.g., 405 nm and em: 535 nm) using the FLUOstar Omega spectrophotometer (BMG Labtech, Champigny-sur-Marne, France). Triplicate measurements were performed and the permeability coefficient Papp (cm / s) was estimated using the following formula:

[0140] [Math. 2]:

[0141] Papp = f AX Co

[0142] dQ / dt is the flux with Q, the quantity of molecules transported during the time t of the incubation, A is the surface area of ​​the polycarbonate membrane (0.33 cm2), and Co is the initial concentration in the donor compartment.

[0143] Cytokine assay by enzyme-linked immunosorbent assay

[0144] The concentrations of different pro-inflammatory cytokines (interleukin (IL)-1 [3, IL-6, IL-8 and tumor necrosis factor (TNF)-a) were assessed in the apical and basolateral supernatants. The Human IL-1 [> ELISA Kit (Life Technologies-ThermoFisher), the Human IL-6 ELISA Kit (Diaclone), the Human IL-8 ELISA Kit (Life Technologies-ThermoFisher) and the Human TNF-a ELISA Kit (Diaclone) were used according to the manufacturer's instructions. All assays were performed in triplicate.

[0145] Quantification of protein expression by real-time PCR

[0146] Total RNA was isolated from cultured cells using the RNeasy Mini Kit with on-column DNase I treatment (Qiagen) according to the manufacturer's instructions, and quantified using the Nanodrop 2000 (Thermo Fisher Scientific, Waltham, USA).

[0147] cDNA was synthesized by reverse transcription of equal amounts of total RNA (2 pg) from each sample using a High-Capacity RNA-to-cDNA kit (Applied Biosystems) and measured by qRT-PCR using the TaqMan Universal Master Mis II kit (Applied Biosystems), according to the manufacturer's protocol.

[0148] Gene expression levels of each sample were quantified by the comparative 2_AACt method and normalized to the endogenous gene expression of [3-actin. The reference mRNA level was designated as 1 and the relative levels of gene transcripts in the samples were expressed as fold change. All quantifications were performed in triplicate.

[0149] Statistical analysis

[0150] Statistical analysis was performed using Graphpad Prism® 8 software. Data were expressed as mean ± standard deviation (SD). For statistical significance analysis, ANOVA on ranks was applied, followed by Dunn's test for all pairwise comparisons. In all cases, P < 0.05 was considered as significant.

[0151] Results

[0152] Effects of DSS and LPS on IEC and THP-1

[0153] The metabolic activity of CEI and THP-1 exposed to different concentrations of DSS (1 to 10% w / v) or LPS (0.1 to 1000 ng / mL) after 24 hours of incubation was studied. The cell viability of IECs remained stable at 100% and decreased for concentrations above 5% w / v (81.95 and 68.21% for 7 and 10% w / v of DSS, respectively). Below 7% w / v of DSS, the metabolic activity of THP-1 was not influenced. Below 1000 ng / mL, LPS did not modify the viability of IECs and THP-1.

[0154] The integrity of the IECs was monitored by TEER and was not influenced by the tested LPS concentration. On the other hand, TEER was reduced to 68% at a DSS concentration of 1% w / v and decreased to a minimum of 19% at a DSS concentration of 10% w / v. These results were confirmed by evaluating the apparent permeability of lucifer yellow (Papp) under the same conditions to determine the paracellular transport of the IECs. After 24 h of exposure, Papp was increased 4-fold (2.22 x 106 cm / s) with 5% w / v DSS compared to the control (0.51 x 106 cm / s) or LPS at 100 ng / mL (0.57 x 106 cm / s).

[0155] The ability of 5% w / v DSS to induce the secretion of pro-inflammatory cytokines by IECs and THP-1 was studied separately. A slight secretion of IL-6 and IL-8 by IECs and of TNF-a and IL-1 [> by THP-1 cells was induced after 24 hours of direct exposure of these cells to DSS. The secretion of these inflammatory cytokines was not changed when IECs were combined with THP-1 in the tri-culture condition and DSS was applied on the apical side (TNF-a: 41.09 pg / mL; IL-1 [3: 101.02 pg / mL; IL-6: 84.91 pg / mL and IL-8: 284.26 pg / mL). However, if DSS is deposited on the basolateral side, the secretion of IL-6 and IL-8 is slightly but significantly reduced (IL-6: 32.24 pg / mL; IL-8: 197.62 pg / mL).

[0156] After 24 hours of incubation with LPS, the secretions of inflammatory cytokines were completely different. When LPS was in direct contact with THP-1, all cytokines reached the highest concentrations, 5 to 7 times higher than those obtained with apical / basolateral DSS and apical LPS (TNF-a: 699.12 pg / mL; IL-1 [3: 199.29 pg / mL; IL-6: 459.00 pg / mL and IL-8: 861.86 pg / mL). When LPS was added on the apical side, the concentration of cytokines was of the same order as those obtained with DSS on the apical side.

[0157] Permeability and secretion of inflammatory cytokines in a tri-culture model after exposure to a combination of DSS and LPS.

[0158] To achieve an increase in epithelial barrier permeability and a significant release of pro-inflammatory cytokines, co-stimulation with DSS and LPS was studied. DSS was set at 5% w / v to induce an increase in IEC permeability and LPS at 100 ng / mL to induce inflammatory cytokine secretion by THP-1 and without any change in cell viability.

[0159] The TEER value obtained with DSS was not modified by LPS deposition (respectively 37.84% and 31.54% for the conditions DSS (A) + LPS (A) and DSS (A) + LPS (B) versus 33.84% for DSS (A); respectively 97.20% and 99.91% for the conditions DSS (B) + LPS (A) and DSS (B) + LPS (B) versus 96.99% for DSS (B)). While apical DSS is necessary to increase the permeability of IECs, the TEER is independent of the location of LPS deposition in this condition. These results are consistent with those obtained with the apparent permeability of JL. Indeed, this parameter is also independent of LPS localization but largely increased by DSS on the apical side (respectively 2.21 and 2.00 * 10 6 cm / s for induction by DSS (A) + LPS (A) and DSS (A) + LPS (B) vs 0.51 * 106 cm / s compared to the unstimulated control, p < 0.05).

[0160] The concentrations of inflammatory cytokines secreted by THP-1 cells (TNF-a and IL-1 [3]) and by IECs (IL-6 and IL-8) increased under all conditions. When LPS was deposited on the apical surface with or without DSS, the expression and secretion of these cytokines were slightly increased. On the other hand, LPS in the basolateral side induced high secretions and a maximum was reached for apical DSS and basolateral LPS [Fig.l] (for induction by DSS (A) + LPS (B) and DSS (B) + LPS (B): respectively 802.12 and 789.25 pg / mL vs 10.17 pg / mL for TNF-a, p<0.05; respectively 301.82 and 291.99 pg / mL vs 26.31 pg / mL for IL-1 [3, p<0.05; respectively 684.91 and 500.49 pg / mL vs 31.02 pg / mL for IL-6, p<0.05; respectively 1084.26 and 878.09 pg / mL vs 90.23 pg / mL for IL-8, p<0.05).

[0161] The effect of the apical DSS / basolateral LPS combination on IECs was studied. First, apical and basolateral LPS did not induce changes in mucin expression (for LPS (A) and LPS (B) induction: 0.89- and 0.91-fold for MUC2, respectively; 0.94- and 1.12-fold for MUC5AC, respectively) compared to the control. The DSS-A / LPS-B combination induced MUC2 underexpression and MUC5AC overexpression (MUC2: 0.44-fold, p<0.05; MUC5AC: 2.01-fold, p<0.05) [Fig.2]. Tight junction protein expression was also assessed, and ZO-1 and Claudin-1 were downregulated in the DSS-A / LPS-B condition compared to the other conditions (ZO-1: 0.10-fold, p<0.05; Claudin-1: 0.21-fold, p<0.05) [Fig. 2]. Occludin was also downregulated, but not significantly, compared to the control (occludin: 0.96-fold).

[0162] Permeability and secretion of inflammatory cytokines in a tri-culture model after exposure to DSS pretreatment

[0163] To reproduce the kinetics of acute inflammation characterized by an increase- To assess the permeability of the epithelial layer allowing the passage of bacterial endotoxins, DSS and LPS were applied to the apical side of the tri-culture, and different exposure sequences were explored.

[0164] DSS at 5% w / v led to a significant reduction in TEER after 6 h (79.21% TEER) and to a minimum after 12 h of incubation (46.15% at 12 h and 38.64% at 24 h TEER). Removal of DSS and replacement with LPS-containing culture medium for 24 h did not modify these TEER values ​​(for DSS-6, 12 or 24 h (A) + LPS-24 h (A): respectively 72.81%, 42.84% and 33.93% TEER). The modification of TEER values ​​appears to depend on the duration of exposure to DSS and is not influenced by LPS.

[0165] After 6 h of DSS incubation or 24 h of DSS / LPS co-incubation, a very slight increase in inflammatory cytokines secreted by macrophages was observed compared to the reference (without DSS / LPS). The secretion of IL-1 [3 and TNF-a is increased by the duration of exposure to DSS (for 12 h and 24 h: respectively 549.01 and 773.92 pg / mL for TNF-a; respectively 261.69 and 291.21 pg / mL for IL-1|3) to reach the same level of expression as the positive control DSS (A) + LPS (B) - 24 h (802.12 pg / mL for TNF-a; 301.82 pg / mL for IL-1 [3).

[0166] 12 h of pre-incubation with DSS allowed the induction of mediator secretion of inflammation (IL-6 and IL-8) by IECs (669.99 pg / mL for IL-6; 1100.01 pg / mL for IL-8) at the same level as the positive control. For a shorter exposure time, cytokine secretion by IECs is consistent with that of DSS (A) + LPS (A) - 24 h (respectively 289.12 and 111.03 pg / mL for IL-6; respectively 420.02 and 163.03 pg / mL for IL-8).

[0167] Effect of anti-inflammatory treatments on a tri-culture model after exposure to DSS and LPS

[0168] The glucocorticoid BTM was chosen as the reference anti-inflammatory drug for its well-known inhibitory effects on cytokine synthesis. BTM 100 pM significantly reduced cytokine release after 24 h from both activated macrophages (TNF-a: 314.52 pg / mL vs 802.12 pg / mL; IL-1 [3: 160.90 pg / mL vs 301.82 pg / mL, p<0.05 vs without anti-inflammatory treatment) and IECs (IL-6: 200.20 pg / mL vs 684.91 pg / mL; IL-8: 657.91 pg / mL vs 1084.26 pg / mL, p<0.05 vs without anti-inflammatory treatment) [Fig.3]. This anti-inflammatory effect was also evident after exposure to DSS and LPS for 48 hours or after pre-stimulation with DSS for 12 hours and exposure to LPS for 24 hours.

[0169] The TEER values ​​(after DSS / LPS 24 or 36 h of exposure: respectively 73.79% and 69.98% TEER vs 36.72% TEER, p<0.05) and Papp of JL (after DSS / LPS 24 or 36 h of exposure: respectively 1.01*106 and 0.92*10 6 vs 2.23*106 cm / s, p<0.05) have were significantly altered with BTM compared to the untreated reference. The effect of 100 mM BTM on IEC permeability is consistent with mucin expression and tight junction protein expression (Claudin-1; ZO-1) by IECs [Fig. 4]. BTM treatment induces a barrier stabilizing effect after DSS and LPS stimulation for 24 h, 36 h, or 48 h.

[0170] The secretome, called SuperMApo, was composed of high molecular weight molecules called pro-resolving factors produced by macrophages after induction of efferocytosis (Bonnefoy F, Gauthier T, Vallion R, et al. Front. Immunol. 2018;9:2586). These pro-resolving factors exert therapeutic effects in experimental colitis and were chosen here to assess the drug permeability of this model. SuperMApo treatment after DSS / LPS stimulation for 24 h decreased the secretion of IL-6 and IL-8 (IL-6: 222.17 pg / mL vs 684.91 pg / mL; IL-8: 581.16 pg / mL vs 1084.26 pg / mL, p<0.05) and TNF-a and IL-1 [3] (TNF-a: 302.99 pg / mL vs 802.12 pg / mL; IL-1 [3]: 149.57 pg / mL vs 301.82 pg / mL, p<0.05) compared with the reference. As with BTM, these effects were also observed after 48-hour exposure to DSS and LPS or after 12-hour DSS prestimulation and 24-hour LPS exposure.Moreover, this biological treatment restored the normal percentage of TEER (after DSS / LPS 24 or 36 h of exposure: respectively 77.33% and 63.23% TEER) and JL transport (after DSS / LPS 24 or 36 h of exposure: respectively 0.77*106 and 0.96*106 cm / s). These results are in agreement with the modification of the relative expressions of mucins and tight junction proteins.

[0171] Discussion

[0172] The intestinal barrier of IBD patients, a physical barrier formed by the epithelium, mucus, and the underlying host immune system, is impaired. Impaired intestinal barrier function has been implicated in the pathogenesis of IBD, as demonstrated in the DSS colitis model (Ramos GP, Papadakis KA. Mayo Clin. Proc. 2019;94:155-165). The development of an in vitro model mimicking the permeability of inflamed epithelium and cytokine secretion observed in humans and animals will strengthen the relevance of in vitro models of IBD.

[0173] Altered intestinal permeability can be induced directly on IECs by a chemical agent (Wirtz S, Neufert C, Weigmann B, et al. Nat. Protoc. 2007;2:541-546) or indirectly via an inflammatory secretome stimulating TNF receptor 2-mediated signaling on IECs (Su L, Nalle SC, Shen L, et al. Gastroenterology 2013;145:407-415). LPS did not show direct or indirect effects on permeability, probably due to the low level of TNF receptor 2 expression in Caco-2 cells (Vamadevan AS, Fukata M, Arnold ET, et al. Innate Immun. 2010;16:93-103). Unlike LPS, DSS in the range of 1–5% induced an increase in IEC permeability by reducing TEER and increasing Papp without any change in barrier integrity. This action could be a direct physical action on the membrane. Chassaing et al hypothesize that the interaction of DSS with IECs could be explained by the negative charge of DSS, contributed by sulfate groups, which damage negatively charged cell membranes by repulsive forces and increase the permeability of the colonic epithelium (Chassaing B, Aitken JD, Malleshappa M, et al. Curr. Protoc. Immunol. 2014;104:15–25.1–15.25.14). This physical interaction could be adapted to in vitro conditions, but remains uncertain in vivo when DSS is diluted in stool and at concentrations often lower than those in our study.The effect of DSS on permeability could also be attributed to an indirect effect via a complex formed between DSS and medium-chain fatty acids (MCFAs). Laroui et al suggest that this complex, which is metabolized by IEs, triggers intestinal inflammatory signaling cascades. In this in vitro model, medium-chain fatty acids contained in FCS of the cellular medium could generate this complex and participate in the reduction of Caco-2 / HT29-MTX cell monolayer functions (Laroui H, Ingersoll SA, Liu HC, et al. PloS One 2012;7:e32084). These results correlate with the low expression obtained after DSS exposure on IECs of genes encoding tight junction (TJ) proteins (ZO-1, Claudin-1, Occludin), which play an important role in maintaining intestinal permeability and regulate paracellular permeability (Suzuki T. Cell. Mol. Life Sci. CMLS 2013;70:631-659).Deregulation of genes encoding SJ components (Bischoff SC, Barbara G, Buurman W, et al. BMC Gastroenterol. 2014;14:189) could lead to long-term alteration of SJ structure, as observed in mouse models and human IBD (Poritz LS, Garver Kl, Green C, et al. J. Surg. Res. 2007;140:12-19; ​​Capaldo CT, Powell DN, Kalman DJ Mol. Med. Berl. Ger. 2017;95:927-934; Shaoul R, Okada Y, Cutz E, et al. J. Pediatr. Gastroenterol. Nutr. 2004;38:488-493), and could therefore be responsible for the concentration- and time-dependent increase in IEC monolayer permeability in this model of tri-culture. .

[0174] The effect of DSS on IECs generates a modification of the permeability of the intestinal barrier, which could also be regulated by mucins (Bischoff SC, Barbara G, Buurman W, et al. BMC Gastroenterol. 2014;14:189) as has been demonstrated in IBD (Capaldo CT, Powell DN, Kalman DJ Mol. Med. Berl. Ger. 2017;95:927-934). The expression of genes encoding mucins (MUC2 and MUC5AC) was deregulated in the tri-culture, as observed in IBD patients or in the in vivo colitis model, after direct exposure to DSS on IECs. An increase in the expression of MUC5AC (Shaoul R, Okada Y, Cutz E, et al. J. Pediatr. Gastroenterol. Nutr. 2004;38:488-493; Olli KE, Rapp C, O'Connell L, et al. Inflamm. Bowel Dis. 2020;26:1353-1367) and a decrease in MUC2 (Van der Sluis M, De Koning BAE, De Bruijn ACJM, et al. Gastroenterology 2006;131:117-129; Hansson GC, Johansson ME. Gut Microbes 2010;1:51-54) were observed and these changes in expression level were reversible after BTM treatment. In the long term, these changes could induce modifications in the composition of mucus, similar to what is observed in IBD (McGuckin MA, Eri R, Simms LA, et al. Inflamm. Bowel Dis. 2009;15:100-113).

[0175] Unlike DSS, LPS did not alter permeability, but induced the secretion of inflammatory cytokines at higher levels than DSS. The mechanism of action by which LPS induces the release of inflammatory molecules, such as TNF-a, IL-1 [3, IL-6, and IL-8, is well known (Bryant CE, Spring DR, Gangloff M, et al. Nat. Rev. Microbiol. 2010;8:8-14). TLR4, expressed on the cell membrane, is associated with the MD-2 coreceptor via its extracellular fragment. This combination is essential for LPS recognition and binding.The interaction between LPS and its recognition site causes dimerization of the complex receptor, resulting in the recruitment of adaptor proteins to the intracellular domain of TLR4 and the activation of an intracellular cascade that leads to the translocation of the transcription factor nuclear factor (NF)-kB into the nucleus and the production of inflammatory cytokines (Ciesielska A, Matyjek M, Kwiatkowska K. Cell. Mol. Life Sci. CMLS 2021;78:1233-1261).

[0176] Incubation of LPS with IECs alone or in tri-culture induced inflammatory cytokines at relatively low levels in the same range as in vitro models with Caco-2 cells or Caco-2 and HT29-MTX coculture described in the literature. This hyporesponsiveness of IECs to LPS stimulation is probably due to the limited expression of the MD-2 coreceptor (Vamadevan AS, Fukata M, Arnold ET, et al. Innate Immun. 2010;16:93-103.) involved in LPS recognition and essential for NF-kB activation and subsequent secretion of inflammatory cytokines (Shimazu R, Akashi S, Ogata H, et al. J. Exp. Med. 1999;189:1777-1782). To increase these secretions, immune cells can be associated with IECs to enhance cellular crosstalk. Indeed, the effect of LPS in contact with macrophages generated significantly greater secretion of cytokines (IL-6 and IL-8) by IECs.The sensitizing effect on intestinal cell functions was likely mediated by soluble mediators. TNF-α is one of the mediators involved in the initiation of the immune response by IECs and responsible for increasing the levels of pro-inflammatory cytokines (Haller D, Bode C, Hammes WP, et al. Gut 2000;47:79-87).

[0177] Direct exposure to DSS slightly increased the expression and synthesis of the inflammatory cytokines TNF-a and IL-1 [3 by THP-1 as well as IL-6 and IL-8 by CIE or in tri-culture. This slight increase in gene expression of these cytokines could suggest an intracellular action of DSS and not only a disruption of the intestinal barrier. The mechanism of intracellular action of DSS remains unclear, but the production of inflammatory cytokines by CIE and THP-1 could be explained by the activation of the Nlrp3 inflammasome by endocytosed DSS, which induces the cleavage of cytokines into their biologically active forms and their subsequent secretion, thus triggering and maintaining an inflammatory cascade (Bauer C, Duewell P, Lehr HA, et al. Dig. Dis. Basel Switz. 2012;30 Suppl 1:82-90).However, unlike LPS, DSS did not produce sufficient cytokines to induce sufficient inflammation on IECs, especially TNF-α. Its concentration is 10 times lower than that of LPS. Thus, DSS at a nontoxic dose had a weak effect on cytokine synthesis by IECs or macrophages and was insufficient to generate inflammation, whereas LPS did not alter the permeability of the coculture. To recover the acute phase characteristics of an in vivo inflammatory model in patients, it would be necessary to have a DSS / LPS combination. The combination of basolateral LPS and apical DSS incubation was useful to obtain both a modification of the permeability of the epithelial barrier and a release of pro-inflammatory cytokines in the cell supernatants. Similarly, under these conditions, an alteration in the expression of mucins and SJ proteins was observed.This tri-culture model could mimic an acute inflammatory phase of IBD which can be used to test treatments for evaluation of curative action and is called curative model (CU).

[0178] DSS was applied, under different conditions, on the apical side to study the increase in IEC layer permeability upon inflammation induction. LPS was deposited for 24 h to induce maximal inflammatory cytokine production as reported (Shimba A, Ikuta K. 2020;42:669-680). DSS induced membrane permeability and allowed JS opening only after 6 h. Under these conditions, the secretion of inflammatory cytokines by IECs and THP-1 cells was not of the same order as those obtained in the CU model. The amount of LPS released for 24 h was much lower than that which stimulates THP-1 cells in the CU model. This low cytokine synthesis could be explained by a low concentration of LPS in contact with THP-1 cells and therefore probably by insufficient permeability of the monolayer to allow a sufficient diffusion rate of LPS.Longer incubation (6-24 h) with DSS was required to increase suf . significantly the permeability of the co-culture and allow LPS to diffuse from the apical compartment to the basolateral compartment in sufficient quantities to induce the secretion of inflammatory cytokines by THP-1 cells after 24 hours. DSS pretreatment confirmed that maximum permeability was reached between 6 h and 12 h and that the amount of LPS crossed and the exposure time in contact with macrophages were sufficient to induce an inflammatory secretome allowing the secretion of inflammatory mediators (IL-6 and IL-8) by IECs. This tri-culture intestinal model, called a preventive model (PR), could also allow the evaluation of the capacity of active molecules to diffuse through IECs, to restore intestinal permeability and / or to reduce the inflammatory secretome.

[0179] Thus, we developed two in vitro models, the CU model to mimic an acute inflammatory model, and the PR model to break down the different phases of inflammation, with an increase in permeability then a secretion of cytokines.

[0180] As barrier dysfunction is an important factor in the pathogenesis of IBD (Turner JR. Nat. Rev. Immunol. 2009;9:799-809), drug development and research into treatments aimed at restoring the IBD barrier are a major challenge. Glucocorticoids are the mainstay for inducing IBD remission and treating collagenous colitis (Baumgart DC, Sandbom WJ. Lancet Lond. Engl. 2007;369:1641-1657) and restoring increased intestinal permeability in CD patients (Wild GE, Waschke KA, Bitton A, et al. Aliment. Pharmacol. Ther. 2003;18:309-317). The ability of the active glucocorticoid BTM was used to assess the reversibility of the in vitro model. BTM significantly reduced cytokine release from activated macrophages (TNF-a and IL-1 [3]) or IECs (IL-6 and IL-8), similarly after 24 h of incubation (in the CU model) or 36 h (in the PR model).Indeed, glucocorticoids are known to suppress several inflammatory cytokines from IECs and macrophages, including IL-1, IL-6, IL-8, TNF-a (Shimba A, Ikuta K, Semin. Immunopathol. 2020;42:669-680). These effects are mediated by the binding of glucocorticoids to the cytoplasmic glucocorticoid receptor (Auphan N, DiDonato JA, Rosette C, et al. Science 1995;270:286-290; Ramamoorthy S, Cidlowski JA. Rheum. Dis. Clin. North Am. 2016;42:15-31, vii; Jonat C, Rahmsdorf HJ, Park KK, et al. Cell 1990;62:1189-1204).Besides its anti-inflammatory activity, BTM exerts a stabilizing effect on the CIS barrier and restores intestinal barrier permeability in our in vitro model, correlating with glucocorticoid therapy that restores increased intestinal permeability in CD, by inducing the expression of the cytosolic inhibitor IkB to impair intestinal translocation of NFkB to the nucleus and inhibit its activation in vivo and in vitro (Wild GE, Waschke . KA, Bitton A, et al. Food. Pharmacol. Ther. 2003;18:309-317).

[0181] The anti-inflammatory effect of the biological complex, called SuperMApo, was also studied in this tri-culture. This drug contains all the pro-resolving factors released by efferocytic macrophages, involved in the resolution of inflammation and in tissue healing (Bonnefoy F, Gauthier T, Vallion R, et al. Front. Immunol. 2018;9:2586). SuperMApo has been shown to exert a therapeutic effect by stimulating the healing, proliferative and migratory properties of the main cell types orchestrating the tissue repair process, in particular IECs, and by promoting the healing of the intestinal mucosa in a DSS-induced in vivo colitis model (Martin-Rodriguez O, Gauthier T, Bonnefoy F, et al. Front. Immunol. 2021;12:754475).Since the molecular weight of SuperMApo components is very high (including Transforming Growth Factor (GF)-beta ~ 28 kDa; Vascular Endothelial GF ~ 40 kDa and Insulin-Like GF-1 ~ 8 kDa), they certainly cannot cross an intact intestinal barrier. In the absence of sufficient barrier permeability, the effect of SuperMApo on THP-1 cells was not obtained. After apical incubation, the therapeutic effect of SuperMApo was confirmed in both RA and UC models after 24 h (UC model) or 36 h (RA model), suggesting that the permeability of the IEC monolayer by DSS incubation was sufficient to allow this biological complex to cross the barrier and exert its effect. These results are in agreement with the in vivo properties of SuperMApo (Martin-Rodriguez O, et al (2021)).

[0182] This study suggests the relevance of these two models for the preselection of therapeutic agents for the treatment of IBD, by evaluating their anti-inflammatory and pro-healing effects before their use in in vivo models.

[0183] Conclusion

[0184] A novel tri-culture model, characterized by altered mucus layer composition and expression of cell junction and inflammatory crosstalk between immune and epithelial cells, has been developed to increase the relevance of in vitro models in the development of IBD treatments. The anti-inflammatory effect of two well-known anti-inflammatory agents, betamethasone and a cocktail of pro-resolving factors, was confirmed in this tri-culture model, reducing the secretion of inflammatory cytokines and partially restoring the permeability of the intestinal monolayer, suggesting its value in the selection of therapeutic agents for the treatment of IBD.

Claims

Claims

1. An intestinal epithelium model comprising two compartments separated by a semipermeable membrane, said model comprising: - in the first compartment, corresponding to the apical pole of the intestinal epithelium, a coculture of Caco-2 cells differentiated into enterocytes and HT29-MTX cells differentiated into goblet cells; and - in the second compartment, corresponding to the basolateral pole of the intestinal epithelium, a culture of THP-1 monocytic cells differentiated into macrophages, said model being characterized in that the cells contained in the first compartment produce interleukin (IL-6) at a concentration greater than 100 pg / mL, and interleukin 8 (IL-8) at a concentration greater than 150 pg / mL;and the cells contained in the second compartment produce tumor necrosis factor-α (TNF-α) at ​​a concentration greater than 40 pg / mL and interleukin-1 [> (IL-113) at a concentration greater than 90 pg / mL.;

2. The model of claim 1, wherein the transepithelial electrical resistance is less than 85% relative to the transepithelial electrical resistance measured in a control non-inflamed intestinal epithelium model.

3. Model according to claim 1 or 2, characterized in that the expression of the mucin MUC2 is reduced by at least 30% compared to a control non-inflamed intestinal epithelium model, and / or the expression of the mucin MUC5AC is increased by at least 50% compared to a control non-inflamed intestinal epithelium model.

4. A method for obtaining a model of inflamed intestinal epithelium, said method comprising the following steps: i. culturing, in a support comprising two compartments separated by a semi-permeable membrane, Caco-2 cells differentiated into enterocytes and HT29-MTX cells differentiated into goblet cells in the first compartment, and THP-1 monocytic cells differentiated into macrophages in the second compartment; ii. bringing the differentiated Caco-2 and HT29-MTX cells contained in the first compartment into contact with at least one compound selected from the group consisting of dextran sodium sulfate (DSS), trinitrobenzenesulfonic acid (TNBS) and dinitrobenzenesulfonic acid (DNBS); iii. contacting the differentiated THP-1 monocytic cells contained in the second compartment with a compound selected from the group consisting of lipopolysaccharides (LPS), TNF-a, interferon-y (IFN-y) and IL-1 [3.

5. A method for obtaining a model of inflamed intestinal epithelium, said method comprising the following steps: i'. culturing, in a support comprising two compartments separated by a semi-permeable membrane, Caco-2 cells differentiated into enterocytes and HT29-MTX cells differentiated into goblet cells in the first compartment, and THP-1 monocytic cells differentiated into macrophages in the second compartment; ii'. contacting the differentiated Caco-2 and HT29-MTX cells contained in the first compartment with at least one compound selected from the group consisting of DSS, TNBS and DNBS; iii'. replacing said at least one compound with LPS, TNF-a, IFN-y and / or IL-1[3.

6. The method of claim 4 or 5, wherein the Caco-2 cells were seeded at a concentration half that of the HT29-MTX cells.

7. A method according to any one of claims 4 to 6, wherein the Caco-2 cells were seeded at a concentration of 5,000 cells per compartment and the HT29-MTX cells were seeded at a concentration of 10,000 cells per compartment.

8. A method according to any one of claims 4 to 7, wherein the DSS, TNBS and / or DNBS has been brought into contact with the coculture of differentiated Caco-2 and HT29-MTX cells at concentrations ranging from 1 to 10% by weight per unit volume (w / V), preferably at a concentration of 5% w / V.

9. A method according to any one of claims 4 to 8, wherein LPS, TNF-α, ITFN-γ and / or IL-1 [3 has been brought into contact with the culture of THP-1 monocytic cells or into contact with the coculture of Caco-2 and HT29-MTX cells at concentrations ranging from 0.1 to 1000 ng / mL, preferably at a concentration of 100 ng / mL

10. A method for the selection of candidate compounds for the treatment of inflammatory bowel diseases, said method comprising the steps of: A) contacting a candidate compound with the intestinal epithelium model according to any one of claims 1 to 3; B) selection of the compound if it makes it possible to obtain, in said model, at least one of the following effects a) to e) a) A decrease of at least 10% in the level of IL-6 and / or IL-8 in the first compartment; b) A decrease of at least 10% in the level of TNF-a and / or IL-1 [3 in the second compartment; c) An increase in MUC2 expression of at least 5%; d) A decrease in MUC5AC expression of at least 5%; e) An increase in transepithelial electrical resistance of at least 10%.

Images