scholarly journals P102 Host gene background and concentration influence the butyrate effect on modulating IECs proliferation

2020 ◽  
Vol 14 (Supplement_1) ◽  
pp. S186-S187
Author(s):  
H He ◽  
J Hu ◽  
W Wang ◽  
M Zhang ◽  
M Zheng ◽  
...  
Keyword(s):  
Immune Cell ◽  
Imaging System ◽  
Cell Types ◽  
Pathological Feature ◽  
Intestinal Epithelial ◽  
Barrier Dysfunction ◽  
Cross Sectional ◽  
Mucosal Regeneration ◽  
Intestinal Organoids ◽  
Stability And Accuracy

Abstract Background Intestinal epithelial barrier dysfunction is a major pathological feature of inflammatory bowel disease (IBD). Stability and accuracy of intestinal epithelial cells (IECs) self-renew are the fundament of intestinal mucosal regeneration and repair. Fermentable dietary fibre and short-chain fatty acid (SCFA) play a role in cell proliferation, histone acetylation and immune responses. However, the results about SCFA modulating IECs proliferation are not coincided. There are several reason that SCFA can have an impact on IECs through immune cell in animal experiment and that also include animal gene background, SCFA concentration, cell types used in cell experiment l. So we adopted intestinal organoids to investigate the effect of butyrate on IECs self-renew to avoid the immune cell influence and signal type cell experiments limitation. Methods Six to 8 weeks wide-type mice and IL10−/− mice were used. Mouse intestinal crypt isolation and organoid culture were according to previously published methods (H. Clevers et al., Gastroenterology 2011). Re-suspended isolated ISCs were seeded onto 10 ml MatrigelTM (Corning 356237) in CellCarrier-96 Ultra Microplates per well (Perkinelmer 6055302). Sodium butyrate (SB) was administered at a final concentration (from 0 to 0.2mM) at Day1. Organoids’ cross-sectional area was measured and calculated, as well as all images were captured using an Operetta High-Content Imaging System and Harmony software 4.8. Results SB promoted intestinal organoids proliferation in a narrow effective concentration from 0.02 to 0.09 mM (Figure 1). The effective SB concentration applied to WT mice-derived organoids other than IL10−/− mice-derived organoids. SB can effectively and rapidly promote organoid proliferation (Figures 2 and 4). SB can continuously promote the proliferation of organoids-derived organoids (Figures 3 and 4). Conclusion There is a narrow effective SB concentrationpromotes WT mice-derived organoids other than IL10−/− mice-derived organoids. This result may be used to illustrate the phenomenon that IBD-associated microbiota and metabolites, butyrate, has different effects on IBD patient and IBD model mice.

scholarly journals Bacteriophage EK99P-1 Alleviates Enterotoxigenic Escherichia Coli K99-Induced Barrier Dysfunction and Inflammation

2021 ◽  
Author(s):  
Narae Kim ◽  
Min jeong Gu ◽  
Yoon-Chul Kye ◽  
Young-Jun Ju ◽  
Rira Hong ◽  
...  
Keyword(s):  
Immune Cell ◽  
Inflammatory Responses ◽  
Intestinal Barrier ◽  
Enterotoxigenic Escherichia Coli ◽  
Intestinal Epithelial ◽  
Barrier Dysfunction ◽  
Barrier Disruption ◽  
Host Immune Responses ◽  
Basolateral Side ◽  
Potential Alternative

Abstract Bacteriophages have long been used as a potential alternative to antibiotics for livestock due to their ability to specifically kill ETEC, which are a major cause of diarrhea in piglets. However, the control of ETEC infection by phages within intestinal epithelial cells, and their relationship with host immune responses, remain poorly understood. In this study, we evaluated the effect of phage EK99P-1 against ETEC K99-infected IPEC-J2. Phage EK99P-1 prevented ETEC K99-induced barrier disruption by attenuating the increased permeability mediated by the loss of tight junction proteins such as ZO-1, occludin, and claudin-3. ETEC K99-induced inflammatory responses, such as IL-8 secretion, were decreased by treatment with phage EK99P-1. We used a IPEC-J2/PBMC Transwell co-culture system to investigate whether the modulation of barrier disruption and chemokine secretion by phage EK99P-1 in ETEC K99-infected IPEC-J2 would influence basolateral immune cells. The results showed that phage EK99P-1 reduced the mRNA expression of ETEC K99-induced pro-inflammatory cytokines, IL-1β and IL-8, from PBMC collected on the basolateral side. Together, these results suggest that phage EK99P-1 prevented ETEC K99-induced barrier dysfunction in IPEC-J2 and alleviated inflammation caused by ETEC K99 infection. Reinforcement of the intestinal barrier by phage EK99P-1 also modulates the immune cell inflammatory response.

scholarly journals The Ghost of the IEL: A Halloween Photoshop Exercise

2008 ◽  
Vol 16 (6) ◽  
pp. 75-75
Author(s):  
Emily Bradford ◽  
Gary Shull ◽  
Marian Miller
Keyword(s):  
Cell Activation ◽  
Immune Cell ◽  
Structural Characteristics ◽  
Cell Types ◽  
Cytoskeletal Proteins ◽  
Calcium Handling ◽  
Intestinal Epithelial ◽  
Immune Cell Activation ◽  
Human Intestinal Epithelial Cells ◽  
Apical Membranes

Image of an intraepithelial lymphocyte (IEL) from a CLIC5 mutant mouse small intestine. The CLIC (Chloride Intracellular Channel) family of proteins is expressed in a wide variety of cell types, and several isoforms are known to cycle between soluble and membranebound forms. As well as being widely expressed, the CLICs are involved in diverse functions, including tubulogenesis, immune cell activation, apoptosis and calcium handling. CLIC5 has been shown to associate with cytoskeletal proteins in placental microvilli and inner ear cells, and is required for proper maintenence of hair cell steriocilia. It has also been localized to the cytosol of human intestinal epithelial cells, though its function there remains unclear. The study in which this particular “IEL” was found, involved a search to see what function CLIC5 played in the modulation of tubulovesicles and microvillar apical membranes in the process of acid secretion. In particular, the relative amounts and structural characteristics of these two membrane types was quantified in parietal cells.

scholarly journals Intestinal epithelial CD98 synthesis specifically modulates expression of colonic microRNAs during colitis

2012 ◽  
Vol 302 (11) ◽  
pp. G1282-G1291 ◽  
Author(s):  
Moiz A. Charania ◽  
Saravanan Ayyadurai ◽  
Sarah A. Ingersoll ◽  
Bo Xiao ◽  
Emilie Viennois ◽  
...  
Keyword(s):  
Epithelial Cell ◽  
Epithelial Cells ◽  
Mirna Expression ◽  
Target Genes ◽  
Immune Cell ◽  
Intestinal Inflammation ◽  
Intestinal Epithelial Cells ◽  
Cell Types ◽  
Intestinal Epithelial ◽  
Small Noncoding Rnas

The transmembrane glycoprotein CD98 is known to be involved in intestinal inflammation. In the present study, we found that CD98 overexpression in intestinal epithelial cells does not normally affect the expression of colonic (epithelial and immune cell) microRNAs (miRNAs), small noncoding RNAs that posttranscriptionally regulate a wide variety of biological processes. However, upon dextran sulfate sodium (DSS) treatment, the expression of several colonic miRNAs, but not miRNAs from other tissues such as liver and spleen, were differentially regulated in mice overexpressing CD98 in epithelial cells compared with wild-type (WT) animals. For example, the level of colonic miRNA 132 was not affected by DSS treatment in WT animals but was upregulated in mice overexpressing CD98 in intestinal epithelial cells. Other colonic miRNAs, including colonic miRNA 23a and 23b, were downregulated in WT animals after DSS treatment but not in colonic epithelial cell CD98-overexpressing mice. Interestingly, the expression of potential miRNA target genes affected intestinal epithelial cells that overexpress CD98 and cell types that did not overexpress CD98 but were in close proximity to CD98-overexpressing intestinal epithelial cells. Taken together, these observations show that the combination of an inflammatory context and intestinal epithelial cell expression of CD98 affects the regulation of miRNA expression in colonic epithelial and immune cells. This is new evidence that protein expression modulates miRNA expression and suggests the existence of regulatory crosstalk between proteins and miRNAs in diseases such as colitis.

scholarly journals T helper cells modulate intestinal stem cell renewal and differentiation

2017 ◽  
Author(s):  
Moshe Biton ◽  
Adam L. Haber ◽  
Semir Beyaz ◽  
Noga Rogel ◽  
Christopher Smillie ◽  
...  
Keyword(s):  
Stem Cells ◽  
T Cells ◽  
Epithelial Cells ◽  
Immune Cell ◽  
Intestinal Epithelial Cells ◽  
Cell Types ◽  
Intestinal Epithelial ◽  
T Helper ◽  
Self Renewal

AbstractIn the small intestine, a cellular niche of diverse accessory cell types supports the rapid generation of mature epithelial cell types through self-renewal, proliferation, and differentiation of intestinal stem cells (ISCs). However, not much is known about interactions between immune cells and ISCs, and it is unclear if and how immune cell dynamics affect eventual ISC fate or the balance between self-renewal and differentiation. Here, we used single-cell RNA-seq (scRNA-Seq) of intestinal epithelial cells (IECs) to identify new mechanisms for ISC–immune cell interactions. Surprisingly, MHC class II (MHCII) is enriched in two distinct subsets of Lgr5+ crypt base columnar ISCs, which are also distinguished by higher proliferation rates. Using co-culture of T cells with intestinal organoids, cytokine stimulations, and in vivo mouse models, we confirm that CD4+ T helper (Th) cells communicate with ISCs and affect their differentiation, in a manner specific to the Th subtypes and their signature cytokines and dependent on MHCII expression by ISCs. Specific inducible knockout of MHCII in intestinal epithelial cells in mice in vivo results in expansion of the ISC pool. Mice lacking T cells have expanded ISC pools, whereas specific depletion of Treg cells in vivo results in substantial reduction of ISC numbers. Our findings show that interactions between Th cells and ISCs mediated via MHCII expressed in intestinal epithelial stem cells help orchestrate tissue-wide responses to external signals.

DEVELOPMENT OF A PERSONALIZED MODEL OF INTESTINAL FIBROSIS USING HUMAN INTESTINAL ORGANOIDS DERIVED FROM INDUCED PLURIPOTENT STEM CELLS

2021 ◽  
Vol 27 (Supplement_1) ◽  
pp. S35-S35
Author(s):  
Hannah Estrada ◽  
Shachi Patel ◽  
Shervin Rabizadeh ◽  
Stephan Targan ◽  
Robert Barrett
Keyword(s):  
Stem Cells ◽  
Epithelial Cells ◽  
Pluripotent Stem Cells ◽  
Cell Types ◽  
Mesenchymal Cells ◽  
Intestinal Epithelial ◽  
Intestinal Fibrosis ◽  
Intestinal Organoids ◽  
Induced Pluripotent

Abstract Background Intestinal fibrosis is a serious complication of inflammatory bowel disease (IBD) with > 20% of Crohn’s disease patients developing this complication within 10 years of diagnosis. Despite improvements in anti-inflammatory medication, its incidence remains stubbornly high and thus far surgical intervention remains the only treatment option. Numerous cell types including intestinal epithelial and mesenchymal cells are implicated in this process, yet studies are hampered by the lack of personalized in vitro models. One potential avenue that would permit a personalized approach is to utilize human intestinal organoids (HIOs) derived from induced pluripotent stem cells (iPSCs). iPSCs can be generated from any individual, faithfully recapitulate the genetics of the host and can be directed to form HIOs that contain both epithelial and mesenchymal cells. Our goal was to determine the feasibility of utilizing iPSC-HIO technology to model intestinal fibrotic responses in vitro. Methods iPSCs from two control individuals and two very early onset-IBD (VEOIBD) patients with stricturing complications were obtained and directed to form HIOs. Given HIOs are heterogeneous in terms of size, shape and ratio of mesenchymal to epithelial cells, they were firstly dissociated to a single cell suspension and EpCAM was used to positively select for epithelial cells using magnetic activated cellular sorting. These EpCAM+ cells were then seeded onto transwells and EpCAM- cells were seeded as monolayers in 10% serum containing media. Both cell types were treated with the profibrotic cytokine TGFβ, and changes in the expression of selected genes were analyzed. Results iPSCs from all 4 individuals could be directed to form HIOs containing both epithelial (E-cadherin+) and mesenchymal (vimentin+) cells (see Fig. 1). In the TGFβ-treated mesenchymal cell population, expression of N-cadherin and Col1a1 was significantly increased in all four lines after 8 and 48hrs respectively, with the highest increase occurring in cells derived from VEOIBD patient 2 (see Table 1). In the TGFβ-treated epithelial cell population, Col1a1 and fibronectin expression was increased in all lines after 96hrs with the highest fold change occurring in cells derived from VEOIBD patient 1 (fibronectin) and 2 (Col1a1). Conclusion We demonstrate the feasibility of utilizing iPSC-HIO technology to model intestinal fibrotic responses in vitro. We show that iPSCs generated from all selected individuals could be directed to form HIOs and that responses to the profibrotic cytokine TGFβ can be examined in both intestinal epithelial and mesenchymal cells. This now permits the generation of near unlimited quantities of patient specific cells that could be used to reveal cell and environmental specific mechanisms underpinning intestinal fibrosis which may ultimately lead to personalized treatments. Fluorescent images of human intestinal organoids generated from A) Control 1, B) Control 2, C) VEOIBD patient 1 and D) VEOIBD patient 2 that were immunostained with vimentin (green), E-cadherin (red) and counterstainied with DAPI (blue). All images X20

scholarly journals Bacteriophage EK99P-1 Alleviates Enterotoxigenic Escherichia Coli K99-Induced Barrier Dysfunction and Inflammation

2021 ◽  
Author(s):  
Narae Kim ◽  
Min jeong Gu ◽  
Yoon-Chul Kye ◽  
Young-Jun Ju ◽  
Rira Hong ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Immune Cell ◽  
Inflammatory Responses ◽  
Enterotoxigenic Escherichia Coli ◽  
Epithelial Cell Line ◽  
Intestinal Epithelial ◽  
Barrier Dysfunction ◽  
Peripheral Blood Mononuclear ◽  
Barrier Disruption ◽  
Basolateral Side

Abstract Background Bacteriophages have long been used as a potential alternative to antibiotics for livestock due to their ability to specifically kill enterotoxigenic Escherichia coli (ETEC), which are a major cause of diarrhea in piglets. However, the control of ETEC infection by phages within intestinal epithelial cells, and their relationship with host immune responses, remain poorly understood. Results In this study, we evaluated the effect of phage EK99P-1 against ETEC K99-infected porcine intestinal epithelial cell line (IPEC-J2). Phage EK99P-1 prevented ETEC K99-induced barrier disruption by attenuating the increased permeability mediated by the loss of tight junction proteins such as zonula occludens-1 (ZO-1), occludin, and claudin-3. ETEC K99-induced inflammatory responses, such as IL-8 secretion, were decreased by treatment with phage EK99P-1. We used a IPEC-J2/peripheral blood mononuclear cell (PBMC) Transwell co-culture system to investigate whether the modulation of barrier disruption and chemokine secretion by phage EK99P-1 in ETEC K99-infected IPEC-J2 would influence basolateral immune cells. The results showed that phage EK99P-1 reduced the mRNA expression of ETEC K99-induced pro-inflammatory cytokines, interleukin (IL)-1β and IL-8, from pPBMC collected on the basolateral side. Conclusion Together, these results suggest that phage EK99P-1 prevented ETEC K99-induced barrier dysfunction in IPEC-J2 and alleviated inflammation caused by ETEC K99 infection. Reinforcement of the intestinal barrier by phage EK99P-1 also modulates the immune cell inflammatory response.

scholarly journals A simple strategy for sample annotation error detection in cytometry datasets

2021 ◽  
Author(s):  
Megan Smithmyer ◽  
Alice E Wiedeman ◽  
David A.G. Skibinski ◽  
Adam K Savage ◽  
Carolina Acosta-Vega ◽  
...  
Keyword(s):  
Error Detection ◽  
Immune Cell ◽  
Hla Class I ◽  
Cell Types ◽  
Class I ◽  
Cross Sectional ◽  
Simple Strategy ◽  
Three Samples ◽  
Allele Expression

Mislabeling samples or data with the wrong participant information can impact study integrity and lead investigators to draw inaccurate conclusions. Quality control to prevent these types of errors is commonly embedded into the analysis of genomic datasets, but a similar identification strategy is not standard for cytometric data. Here, we present a method for detecting sample identification errors in cytometric data using expression of HLA class I alleles. We measured HLA-A*02 and HLA-B*07 expression in 3 longitudinal samples from 41 participants using a 33-marker CyTOF panel designed to identify major immune cell types. 3/123 samples (2.4%) showed HLA allele expression that did not match their longitudinal pairs. Furthermore, these same three samples cytometric signature did not match qPCR HLA class I allele data, suggesting that they were accurately identified as mismatches. We conclude that this technique is useful for detecting sample labeling errors in cytometric analyses of longitudinal data. This technique could also be used in conjunction with another method, like GWAS or PCR, to detect errors in cross-sectional data. We suggest widespread adoption of this or similar techniques will improve the quality of clinical studies that utilize cytometry.

scholarly journals Interleukin-17 and Th17 Lymphocytes Directly Impair Motoneuron Survival of Wildtype and FUS-ALS Mutant Human iPSCs

2021 ◽  
Vol 22 (15) ◽  
pp. 8042
Author(s):  
Mengmeng Jin ◽  
Katja Akgün ◽  
Tjalf Ziemssen ◽  
Markus Kipp ◽  
Rene Günther ◽  
...  
Keyword(s):  
Motor Neurons ◽  
Th17 Cells ◽  
Immune Cell ◽  
Cell Types ◽  
Positive Control ◽  
Interleukin 17 ◽  
Fused In Sarcoma ◽  
Th17 Lymphocytes ◽  
Human Ipscs ◽  
Als Patients

Amyotrophic lateral sclerosis (ALS) is a progressive disease leading to the degeneration of motor neurons (MNs). Neuroinflammation is involved in the pathogenesis of ALS; however, interactions of specific immune cell types and MNs are not well studied. We recently found a shift toward T helper (Th)1/Th17 cell-mediated, pro-inflammatory immune responses in the peripheral immune system of ALS patients, which positively correlated with disease severity and progression. Whether Th17 cells or their central mediator, Interleukin-17 (IL-17), directly affects human motor neuron survival is currently unknown. Here, we evaluated the contribution of Th17 cells and IL-17 on MN degeneration using the co-culture of iPSC-derived MNs of fused in sarcoma (FUS)-ALS patients and isogenic controls with Th17 lymphocytes derived from ALS patients, healthy controls, and multiple sclerosis (MS) patients (positive control). Only Th17 cells from MS patients induced severe MN degeneration in FUS-ALS as well as in wildtype MNs. Their main effector, IL-17A, yielded in a dose-dependent decline of the viability and neurite length of MNs. Surprisingly, IL-17F did not influence MNs. Importantly, neutralizing IL-17A and anti-IL-17 receptor A treatment reverted all effects of IL-17A. Our results offer compelling evidence that Th17 cells and IL-17A do directly contribute to MN degeneration.

747 Evaluation of immune microenvironment of primary lung cancer and synchronous liver metastasis with multispectral imaging system

2020 ◽  
Vol 8 (Suppl 3) ◽  
pp. A795-A795
Author(s):  
Hyeonbin Cho ◽  
Jae-Hwan Kim ◽  
Ji-Hyun Kim
Keyword(s):  
T Cells ◽  
Liver Metastasis ◽  
Multispectral Imaging ◽  
Immune Cell ◽  
Imaging System ◽  
Cytotoxic T Cells ◽  
Primary Lung Cancer ◽  
Helper T Cells ◽  
Synchronous Liver Metastasis ◽  
Immune Microenvironment

BackgroundCancer immunotherapy (CIT) has substantially improved the survival of cancer patients. However, according to recent studies, liver metastasis was reported to predict worse outcomes for CIT. The main objective of the study is to evaluate the differences in the immune microenvironment (IME) between the primary lung cancer (PL) and synchronous liver metastasis (LM) using a multispectral imaging system.MethodsSix immune markers (CD4, CD8, CTLA-4, granzyme B (GZB), Foxp3 and PD-L1) were analyzed using a multiplex IHC system and inForm program (Akoya) on paired lung-liver samples of 10 patients. Cells were categorized into tumor nest and stroma, and cell counts per unit area were measured for comparison.ResultsThe number of tumor-infiltrating cytotoxic T cells (TIL) in PL (262.5 cells/mm2) was higher than that of LM (113.3 cells/mm2). Additionally, the ratio between the number of TIL and non-TIL was greater in PL (0.31) compared to that of LM (0.26). A similar trend appeared for Helper T cells and regulatory T cells (Treg), as PL consisted of higher numbers of T cells (791.8 Helper T cells/mm2, 195.7 Treg/mm2) than LM (626.3 Helper T cells/mm2, 121.3 Treg/mm2). However, cytotoxic T cells exhibiting GZB+ and CTLA-4- were fewer in PL (140.2 cells/mm2) than in LM (203.3 cells/mm2), and the ratio is 0.69. The mean number of GZB+ TIL in PL (32.5 cells/mm2) was lower than in LM (35.3 cells/mm2), and their proportions among total TIL counts were 0.12 and 0.31, respectively. In PL, GZB+: GZB- ratio is 0.16 while the ratio is 1.91 for LM. A fewer number of TILs exhibiting GZB suggests that PL has lower efficiency in immune response than LM. Another crucial checkpoint receptor that inhibits immune response, CTLA-4, was more prevalent in PL, with CTLA-4+: CTLA-4- ratio in Treg being 0.36 in PL, compared to 0.11 in LM. The tumor proportion score (TPS) of PD-L1 was higher in PL than LM (40.0 vs. 6.6).ConclusionsIn our study, we showed the differences in the numbers of TIL or regulatory T cells and expressions of immune checkpoint receptors (PD-L1, CTLA-4), which significantly influence outcomes for CIT. The study is ongoing to confirm different IME between the PL and LM groups in a larger tumor cohort.ReferencesPeng, Jianhong, et al., Immune Cell Infiltration in the Microenvironment of Liver Oligometastasis from Colorectal Cancer: Intratumoural CD8/CD3 Ratio Is a Valuable Prognostic Index for Patients Undergoing Liver Metastasectomy. Cancers 2019 Dec; 11(12): 1922. https://doi.org/10.3390/cancers11121922Tumeh, Paul C., et al., Liver Metastasis and treatment outcome with Anti-PD-1 monoclonal antibody in patients with melanoma and NSCLC. Cancer Immunol Res 2017 May; 5(5): 417–424. doi: 10.1158/2326-6066.CIR-16-0325Parra, E.R., Immune Cell Profiling in Cancer Using Multiplex Immunofluorescence and Digital Analysis Approaches; Streckfus, C.F., Ed.; IntechOpen: London, UK, 2018; pp. 1–13. doi: 10.5772/intechopen.80380Ribas, A., Hu-Lieskovan, S., What does PD-L1 positive or negative mean?. The Journal of Experimental Medicine 2016;213(13):2835–2840. https://doi.org/10.1084/jem.20161462

scholarly journals The microbiome’s relationship with congenital heart disease: more than a gut feeling

2021 ◽  
Vol 5 (1) ◽  
Author(s):  
Dan Feng ◽  
Jason T. Christensen ◽  
Anji T. Yetman ◽  
Merry L. Lindsey ◽  
Amar B. Singh ◽  
...  
Keyword(s):  
Congenital Heart Disease ◽  
Heart Disease ◽  
Patient Population ◽  
Congenital Heart ◽  
Basic Research ◽  
Intestinal Epithelial ◽  
Barrier Dysfunction ◽  
Intestinal Dysbiosis ◽  
Epithelial Barrier Dysfunction

AbstractPatients with congenital heart disease (CHD) are at risk for developing intestinal dysbiosis and intestinal epithelial barrier dysfunction due to abnormal gut perfusion or hypoxemia in the context of low cardiac output or cyanosis. Intestinal dysbiosis may contribute to systemic inflammation thereby worsening clinical outcomes in this patient population. Despite significant advances in the management and survival of patients with CHD, morbidity remains significant and questions have arisen as to the role of the microbiome in the inflammatory process. Intestinal dysbiosis and barrier dysfunction experienced in this patient population are increasingly implicated in critical illness. This review highlights possible CHD-microbiome interactions, illustrates underlying signaling mechanisms, and discusses future directions and therapeutic translation of the basic research.

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