Development of a human primary gut-on-a-chip to model inflammatory processes

AbstractInflammatory bowel disease (IBD) is a complex multi-factorial disease for which physiologically relevant in vitro models are lacking. Existing models are often a compromise between biological relevance and scalability. Here, we integrated intestinal epithelial cells (IEC) derived from human intestinal organoids with monocyte-derived macrophages, in a gut-on-a-chip platform to model the human intestine and key aspects of IBD. The microfluidic culture of IEC lead to an increased polarization and differentiation state that closely resembled the expression profile of human colon in vivo. Activation of the model resulted in the polarized secretion of CXCL10, IL-8 and CCL-20 by IEC and could efficiently be prevented by TPCA-1 exposure. Importantly, upregulated gene expression by the inflammatory trigger correlated with dysregulated pathways in IBD patients. Finally, integration of activated macrophages offers a first-step towards a multi-factorial amenable IBD platform that could be scaled up to assess compound efficacy at early stages of drug development or in personalized medicine.

Download Full-text

Deficiency in the anti-apoptotic protein DJ-1 promotes intestinal epithelial cell apoptosis and aggravates inflammatory bowel disease via p53

Journal of Biological Chemistry ◽

10.1074/jbc.ra119.010143 ◽

2020 ◽

Vol 295 (13) ◽

pp. 4237-4251 ◽

Cited By ~ 1

Author(s):

Jie Zhang ◽

Min Xu ◽

Weihua Zhou ◽

Dejian Li ◽

Hong Zhang ◽

...

Keyword(s):

Inflammatory Bowel Disease ◽

Epithelial Cell ◽

Bowel Disease ◽

Intestinal Epithelial Cell ◽

Intestinal Inflammation ◽

Intestinal Epithelial ◽

P53 Expression ◽

Inflammatory Bowel

Parkinson disease autosomal recessive, early onset 7 (PARK7 or DJ-1) is involved in multiple physiological processes and exerts anti-apoptotic effects on multiple cell types. Increased intestinal epithelial cell (IEC) apoptosis and excessive activation of the p53 signaling pathway is a hallmark of inflammatory bowel disease (IBD), which includes ulcerative colitis (UC) and Crohn's disease (CD). However, whether DJ-1 plays a role in colitis is unclear. To determine whether DJ-1 deficiency is involved in the p53 activation that results in IEC apoptosis in colitis, here we performed immunostaining, real-time PCR, and immunoblotting analyses to assess DJ-1 expression in human UC and CD samples. In the inflamed intestines of individuals with IBD, DJ-1 expression was decreased and negatively correlated with p53 expression. DJ-1 deficiency significantly aggravated colitis, evidenced by increased intestinal inflammation and exacerbated IEC apoptosis. Moreover, DJ-1 directly interacted with p53, and reduced DJ-1 levels increased p53 levels both in vivo and in vitro and were associated with decreased p53 degradation via the lysosomal pathway. We also induced experimental colitis with dextran sulfate sodium in mice and found that compared with DJ-1−/− mice, DJ-1−/−p53−/− mice have reduced apoptosis and inflammation and increased epithelial barrier integrity. Furthermore, pharmacological inhibition of p53 relieved inflammation in the DJ-1−/− mice. In conclusion, reduced DJ-1 expression promotes inflammation and IEC apoptosis via p53 in colitis, suggesting that the modulation of DJ-1 expression may be a potential therapeutic strategy for managing colitis.

Download Full-text

Mycotoxins: Biotransformation and Bioavailability Assessment Using Caco-2 Cell Monolayer

Toxins ◽

10.3390/toxins12100628 ◽

2020 ◽

Vol 12 (10) ◽

pp. 628

Author(s):

Van Nguyen Tran ◽

Jitka Viktorová ◽

Tomáš Ruml

Keyword(s):

Cell Monolayer ◽

Intestinal Barrier ◽

Intestinal Transport ◽

Human Colon ◽

In Vitro Models ◽

Biologically Active ◽

In Vitro Techniques ◽

In Vivo Cytotoxicity

The determination of mycotoxins content in food is not sufficient for the prediction of their potential in vivo cytotoxicity because it does not reflect their bioavailability and mutual interactions within complex matrices, which may significantly alter the toxic effects. Moreover, many mycotoxins undergo biotransformation and metabolization during the intestinal absorption process. Biotransformation is predominantly the conversion of mycotoxins meditated by cytochrome P450 and other enzymes. This should transform the toxins to nontoxic metabolites but it may possibly result in unexpectedly high toxicity. Therefore, the verification of biotransformation and bioavailability provides valuable information to correctly interpret occurrence data and biomonitoring results. Among all of the methods available, the in vitro models using monolayer formed by epithelial cells from the human colon (Caco-2 cell) have been extensively used for evaluating the permeability, bioavailability, intestinal transport, and metabolism of toxic and biologically active compounds. Here, the strengths and limitations of both in vivo and in vitro techniques used to determine bioavailability are reviewed, along with current detailed data about biotransformation of mycotoxins. Furthermore, the molecular mechanism of mycotoxin effects is also discussed regarding the disorder of intestinal barrier integrity induced by mycotoxins.

Download Full-text

Mucosa-associated microbiota drives pathogenic functions in IBD-derived intestinal iNKT cells

Life Science Alliance ◽

10.26508/lsa.201800229 ◽

2019 ◽

Vol 2 (1) ◽

pp. e201800229 ◽

Cited By ~ 6

Author(s):

Claudia Burrello ◽

Gabriella Pellegrino ◽

Maria Rita Giuffrè ◽

Giulia Lovati ◽

Ilaria Magagna ◽

...

Keyword(s):

Lamina Propria ◽

Intestinal Inflammation ◽

Inflammatory Responses ◽

Ex Vivo ◽

Lymphoid Tissues ◽

Inkt Cells ◽

Inflammatory Processes ◽

Inflammatory Bowel

Inflammatory bowel disease (IBD) pathogenesis has been linked to the aberrant activation of the Gut-associated lymphoid tissues against components of the intestinal microbiota. Although the contribution of CD4+ T helper cells to inflammatory processes is being increasingly acknowledged, the functional engagement of human invariant natural killer T (iNKT) cells is still poorly defined. Here, we evaluated the functional characteristics of intestinal iNKT cells during IBD pathogenesis and to exploit the role of mucosa-associated microbiota recognition in triggering iNKT cells’ pro-inflammatory responses in vivo. Lamina propria iNKT cells, isolated from surgical specimens of active ulcerative colitis and Crohn’s disease patients and non-IBD donors, were phenotypically and functionally analyzed ex vivo, and stable cell lines and clones were generated for in vitro functional assays. iNKT cells expressing a pro-inflammatory cytokine profile were enriched in the lamina propria of IBD patients, and their exposure to the mucosa-associated microbiota drives pro-inflammatory activation, inducing direct pathogenic activities against the epithelial barrier integrity. These observations suggest that iNKT cell pro-inflammatory functions may contribute to the fuelling of intestinal inflammation in IBD patients.

Download Full-text

FDA-Approved Excipient N,N-Dimethylacetamide Attenuates In-Vitro and In-Vivo Inflammatory Bowel Disease

10.21203/rs.3.rs-864635/v1 ◽

2021 ◽

Author(s):

Jagadish Koya ◽

Tong Shen ◽

Geming Lu ◽

Alex Gauthier ◽

Lin Mantell ◽

...

Keyword(s):

Inflammatory Bowel Disease ◽

Intestinal Epithelial Cells ◽

Raw 264.7 Cells ◽

Raw 264.7 ◽

Intestinal Epithelial ◽

Human Monocytes ◽

Human Intestinal Epithelial Cells ◽

Inflammatory Bowel

Abstract Background: Inflammatory bowel disease (IBD) affects almost 7 million people worldwide and is increasing in incidence. While the precise pathogenesis of IBD remains unknown, the production of inflammatory cytokines and chemokines play a central role. We have previously found that N,N-dimethylacetamide (DMA), a widely used non-toxic drug excipient, suppresses cytokine and chemokine secretion in vitro and prevents inflammation-induced preterm birth in vivo. Methods: Using sandwich enzyme-linked immunosorbent assays (ELISAs), we tested whether DMA attenuates cytokine and chemokine secretion from LPS- or TNFa-stimulated human intestinal epithelial cells and human monocytes and HMGB1 release from RAW 264.7 cells. To test our hypothesis that the mechanism of DMA’s effects in in-vitro and in-vivo models of IBD is inhibition of the NF-kB pathway, we used western blotting to track levels of the nuclear factor kappa B (NF-kB) inhibitory molecule I kappa B alpha (IkBa) in THP-1 human monocytes in the absence or presence of DMA. Finally, we induced colitis in C57Bl/6 mice with dextran sodium sulfate (DSS) and then tested whether daily i.p injections of DMA at 2.1 g/kg/day attenuates clinical and histopathologic signs of colitis.Results: DMA attenuated cytokine and chemokine release from human intestinal epithelial cells and human monocytes and HMGB1 release from RAW 264.7 cells. Importantly, DMA prevented degradation of IkBa in THP-1 cells, thereby suggesting one mechanism for DMA’s effects. Finally, we show here, for the first time, that DMA attenuates clinical and histologic features of DSS-induced colitis. Conclusion: DMA should be further explored in preclinical and clinical trials for its potential as novel drug therapy for IBD.

Download Full-text

Coculture Strategy for Developing Lactobacillus paracasei PS23 Fermented Milk with Anti-Colitis Effect

Foods ◽

10.3390/foods10102337 ◽

2021 ◽

Vol 10 (10) ◽

pp. 2337

Author(s):

Kai-Yi Lee ◽

Ying-Chieh Tsai ◽

Sheng-Yao Wang ◽

Yen-Po Chen ◽

Ming-Ju Chen

Keyword(s):

Short Chain Fatty Acids ◽

Fermented Milk ◽

Fecal Calprotectin ◽

Lactobacillus Paracasei ◽

Intestinal Epithelial ◽

Epithelial Barrier Function ◽

Inflammatory Processes ◽

Bleeding Score

Few studies have documented the effects of fermented milk on intestinal colitis, which are mediated by regulating various microbial and inflammatory processes. Here, we investigated the effects of fermented milk with Lactobacillus paracasei PS23 on intestinal epithelial cells in vitro and dextran sulfate sodium (DSS)-induced colitis in vivo. As L. paracasei PS23 grew poorly in milk, a coculture strategy with yogurt culture was provided to produce fermented milk (FM). The results indicated that the coculture exhibited a symbiotic effect, contributing to the better microbial and physicochemical property of the fermented milk products. We further evaluated the anti-colitis effect of fermented milk with L. paracasei PS23 in vitro. Both PS23-fermented milk (PS23 FM) and its heat-killed counterpart (HK PS23 FM) could protect or reverse the increased epithelial permeability by strengthening the epithelial barrier function in vitro by increasing transepithelial electrical resistance (TEER). In vivo analysis of the regulation of intestinal physiology demonstrated that low-dose L. paracasei PS23-fermented ameliorated DSS-induced colitis, with a significant attenuation of the bleeding score and reduction of fecal calprotectin levels. This anti-colitis effect may be exerted by deactivating the inflammatory cascade and strengthening the tight junction through the modification of specific cecal bacteria and upregulation of short-chain fatty acids. Our findings can clarify the role of L. paracasei PS23 in FM products when cocultured with yogurt culture and can elucidate the mechanisms of the anti-colitis effect of L. paracasei PS23 FM, which may be considered for therapeutic intervention.

Download Full-text

Dihydroartemisinin Protects against Dextran Sulfate Sodium-Induced Colitis in Mice through Inhibiting the PI3K/AKT and NF-κB Signaling Pathways

BioMed Research International ◽

10.1155/2019/1415809 ◽

2019 ◽

Vol 2019 ◽

pp. 1-12 ◽

Cited By ~ 3

Author(s):

Ning Li ◽

Wenjing Sun ◽

Xin Zhou ◽

Hao Gong ◽

Yuqing Chen ◽

...

Keyword(s):

Ulcerative Colitis ◽

Signaling Pathways ◽

Dextran Sulfate ◽

Dextran Sulfate Sodium ◽

Body Weight Loss ◽

Intestinal Epithelial ◽

Histological Damage ◽

Inflammatory Bowel

Ulcerative colitis is a common inflammatory bowel disease, and the activation of thePI3K/AKT and NF-κB signaling pathways plays a pivotal role in its pathogenesis. Dihydroartemisinin (DHA) is a widely used antimalarial drug and has shown anticancer effect partially through inhibiting the activation of PI3K/AKT and NF-κB. This study aimed to investigate the effect of dihydroartemisinin on ulcerative colitis and its mechanism. Adult male C57 mice were subjected to 3.0% dextran sulfate sodium (DSS) for seven days; simultaneously, dihydroartemisinin or control saline was administered by oral gavage once a day. In vitro, the intestinal epithelial cell-6 was treated with LPS for 24 hours with or without dihydroartemisinin combined with PI3K/Akt activator 740 Y-P or NF-κB activator phorbol myristate acetate. Western blotting was used to test the activation of PI3K/AKT and NF-κB. Dihydroartemisinin significantly ameliorated body weight loss, shortened colon length, and increased DAI in DSS-induced colitis. Meanwhile, histological damage was improved and was accompanied by decreased expression and secretion of proinflammatory cytokines. Moreover, DSS-induced elevation of phosphorylation of PI3K, AKT, IKKα, IκBα, and NF-κB (p65) was remarkably blunted by dihydroartemisinin both in vivo and in vitro, indicating an inhibitive property on the PI3K/AKT and NF-κB signaling pathways. Furthermore, administration of 740 Y-P or PMA significantly blocked protective activity of dihydroartemisinin against colitis in vitro. In conclusion, dihydroartemisinin can attenuate DSS-induced colitis, and its anticolitis effect might be mediated via the PI3K/AKT and NF-κB signaling pathways. DHA might serve as a promising drug for patients with ulcerative colitis.

Download Full-text

An Intestine-on-a-Chip Model of Plug-and-Play Modularity to Study Inflammatory Processes

SLAS TECHNOLOGY Translating Life Sciences Innovation ◽

10.1177/2472630320924999 ◽

2020 ◽

Vol 25 (6) ◽

pp. 585-597 ◽

Cited By ~ 2

Author(s):

Linda Gijzen ◽

Diego Marescotti ◽

Elisa Raineri ◽

Arnaud Nicolas ◽

Henriette L. Lanz ◽

...

Keyword(s):

Intestinal Inflammation ◽

In Vivo Models ◽

Inflammatory Conditions ◽

Human Situation ◽

Inflammatory State ◽

Inflammatory Processes ◽

Factor Α ◽

Key Aspects

Development of efficient drugs and therapies for the treatment of inflammatory conditions in the intestine is often hampered by the lack of reliable, robust, and high-throughput in vitro and in vivo models. Current models generally fail to recapitulate key aspects of the intestine, resulting in low translatability to the human situation. Here, an immunocompetent 3D perfused intestine-on-a-chip platform was developed and characterized for studying intestinal inflammation. Forty independent polarized 3D perfused epithelial tubular structures were grown from cells of mixed epithelial origin, including enterocytes (Caco-2) and goblet cells (HT29-MTX-E12). Immune cells THP-1 and MUTZ-3, which can be activated, were added to the system and assessed for cytokine release. Intestinal inflammation was mimicked through exposure to tumor necrosis factor-α (TNFα) and interleukin (IL)-1β. The effects were quantified by measuring transepithelial electrical resistance (TEER) and proinflammatory cytokine secretion on the apical and basal sides. Cytokines induced an inflammatory state in the culture, as demonstrated by the impaired barrier function and increased IL-8 secretion. Exposure to the known anti-inflammatory drug TPCA-1 prevented the inflammatory state. The model provides biological modularity for key aspects of intestinal inflammation, making use of well-established cell lines. This allows robust assays that can be tailored in complexity to serve all preclinical stages in the drug discovery and development process.

Download Full-text

FDA-Approved Excipient N,N-Dimethylacetamide Attenuates Inflammatory Bowel Disease in In-Vitro and In-Vivo Models

10.21203/rs.3.rs-948100/v1 ◽

2021 ◽

Author(s):

Jagadish Koya ◽

Tong Shen ◽

Geming Lu ◽

Alex Gauthier ◽

Lin Mantell ◽

...

Keyword(s):

Inflammatory Bowel Disease ◽

Intestinal Epithelial Cells ◽

Raw 264.7 Cells ◽

Raw 264.7 ◽

Intestinal Epithelial ◽

Human Monocytes ◽

Human Intestinal Epithelial Cells ◽

Inflammatory Bowel

Abstract Inflammatory bowel disease (IBD) affects almost 7 million people worldwide and is increasing in incidence. While the precise pathogenesis of IBD remains unknown, the production of inflammatory cytokines and chemokines play a central role. We have previously found that N,N-dimethylacetamide (DMA), a widely used non-toxic drug excipient, suppresses cytokine and chemokine secretion in vitro and prevents inflammation-induced preterm birth in vivo. Using sandwich enzyme-linked immunosorbent assays (ELISAs), we tested whether DMA attenuates cytokine and chemokine secretion from LPS- or TNFa-stimulated human intestinal epithelial cells and human monocytes and HMGB1 release from RAW 264.7 cells. To test our hypothesis that the mechanism of DMA’s effects in in-vitro and in-vivo models of IBD is inhibition of the NF-kB pathway, we used western blotting to track levels of the nuclear factor kappa B (NF-kB) inhibitory molecule I kappa B alpha (IkBa) in THP-1 human monocytes in the absence or presence of DMA. Finally, we induced colitis in C57Bl/6 mice with dextran sodium sulfate (DSS) and then tested whether daily i.p injections of DMA at 2.1 g/kg/day attenuates clinical and histopathologic signs of colitis. DMA attenuated cytokine and chemokine release from human intestinal epithelial cells and human monocytes and HMGB1 release from RAW 264.7 cells. Importantly, DMA prevented degradation of IkBa in THP-1 cells, thereby suggesting one mechanism for DMA’s effects. Finally, we show here, for the first time, that DMA attenuates clinical and histologic features of DSS-induced colitis. Based on these data, DMA should be further explored in preclinical and clinical trials for its potential as novel drug therapy for IBD.

Download Full-text

In Vitro and in Vivo Comparison of Binding of 99m-Tc-Iabeled Anti-CEA MAb F33-104 with 99m-Tc-labeled Anti-CEA MAb BW431/26

Nuklearmedizin ◽

10.1055/s-0038-1632202 ◽

1999 ◽

Vol 38 (04) ◽

pp. 115-119

Author(s):

N. Oriuchi ◽

S. Sugiyama ◽

M. Kuroki ◽

Y. Matsuoka ◽

S. Tanada ◽

...

Keyword(s):

Nude Mice ◽

Binding Capacity ◽

Colon Adenocarcinoma ◽

Human Colon ◽

Cell Binding ◽

Vitro Binding ◽

Labeling Method ◽

Human Colon Adenocarcinoma

Summary Aim: The purpose of this study was to assess the potential for radioimmunodetection (RAID) of murine anti-carcinoembryonic antigen (CEA) monoclonal antibody (MAb) F33-104 labeled with technetium-99m (99m-Tc) by a reduction-mediated labeling method. Methods: The binding capacity of 99m-Tc-labeled anti-CEA MAb F33-104 with CEA by means of in vitro procedures such as immunoradiometric assay and cell binding assay and the biodistribution of 99m-Tc-labeled anti-CEA MAb F33-104 in normal nude mice and nude mice bearing human colon adenocarcinoma LS180 tumor were investigated and compared with 99m-Tc-labeled anti-CEA MAb BW431/26. Results: The in vitro binding rate of 99m-Tc-labeled anti-CEA MAb F33-104 with CEA in solution and attached to the cell membrane was significantly higher than 99m-Tclabeled anti-CEA MAb BW431/261 (31.4 ± 0.95% vs. 11.9 ± 0.55% at 100 ng/mL of soluble CEA, 83.5 ± 2.84% vs. 54.0 ± 2.54% at 107 of LS 180 cells). In vivo, accumulation of 99m-Tc-labeled anti-CEA MAb F33-104 was higher at 18 h postinjection than 99m-Tc-labeled anti-CEA MAb BW431/26 (20.1 ± 3.50% ID/g vs. 14.4 ± 3.30% ID/g). 99m-Tcactivity in the kidneys of nude mice bearing tumor was higher at 18 h postinjection than at 3 h (12.8 ± 2.10% ID/g vs. 8.01 ± 2.40% ID/g of 99m-Tc-labeled anti-CEA MAb F33-104, 10.7 ± 1.70% ID/g vs. 8.10 ± 1.75% ID/g of 99m-Tc-labeled anti-CEA MAb BW431/26). Conclusion: 99m-Tc-labeled anti-CEA MAb F33-104 is a potential novel agent for RAID of recurrent colorectal cancer.

Download Full-text

Modulation of Matrix Metalloproteinases by Plant-Derived Products

Current Cancer Drug Targets ◽

10.2174/1568009620666201120144838 ◽

2020 ◽

Vol 20 ◽

Author(s):

Nur Najmi Mohamad Anuar ◽

Nurul Iman Natasya Zulkafali ◽

Azizah Ugusman

Keyword(s):

Clinical Trials ◽

Natural Products ◽

Matrix Metalloproteinases ◽

Animal Studies ◽

Current Knowledge ◽

In Vitro Models ◽

In Vivo Studies ◽

Extracellular Matrix Components

: Matrix metalloproteinases (MMPs) are a group of zinc-dependent metallo-endopeptidase that are responsible towards the degradation, repair and remodelling of extracellular matrix components. MMPs play an important role in maintaining a normal physiological function and preventing diseases such as cancer and cardiovascular diseases. Natural products derived from plants have been used as traditional medicine for centuries. Its active compounds, such as catechin, resveratrol and quercetin, are suggested to play an important role as MMPs inhibitors, thereby opening new insights into their applications in many fields, such as pharmaceutical, cosmetic and food industries. This review summarises the current knowledge on plant-derived natural products with MMP-modulating activities. Most of the reviewed plant-derived products exhibit an inhibitory activity on MMPs. Amongst MMPs, MMP-2 and MMP-9 are the most studied. The expression of MMPs is inhibited through respective signalling pathways, such as MAPK, NF-κB and PI3 kinase pathways, which contribute to the reduction in cancer cell behaviours, such as proliferation and migration. Most studies have employed in vitro models, but a limited number of animal studies and clinical trials have been conducted. Even though plant-derived products show promising results in modulating MMPs, more in vivo studies and clinical trials are needed to support their therapeutic applications in the future.

Download Full-text