an-optimized-sequential-isolation-crypts-mesenchymal-stromal-cells

An Optimized Sequential Isolation of Crypts and Mesenchymal Stromal Cells from Porcine Intestinal Tissue

The intestinal epithelium closely interacts with the underlying intestinal mesenchymal stromal cells (iMSCs) to regulate development, homeostasis, and ...

University of Maryland

Intraepithelial T lymphocytes (T-IELs), which constitute over 50% of the total T lymphocytes in the animal, patrol the mucosal epithelial lining to defend against pathogen invasion while maintaining gut homeostasis. In addition to expressing T cell markers such as CD4 and CD8, T-IELs display T cell receptors (TCR), including either TCRαβ or TCRγδ. Both humans and mice share similar T-IEL subsets: TCRγδ, TCRαβCD8αα, TCRαβCD4, and TCRαβCD8αβ. Among these subsets, human T-IELs are predominantly TCRαβ (over 80%), whereas those in mice are mostly TCRγδ (~60%). Of note, the majority of the TCRγδ subset expresses CD8αα in both species. Although T-IELs have been extensively studied in humans and mice, their profiles in cattle have not been well examined. Our study is the first to characterize bovine T-IELs using flow cytometry, where we identified several distinct features. The percentage of TCRγδ was comparable to that of TCRαβ T-IELs (both ~50% of CD3), and the majority of bovine TCRγδ T-IELs did not express CD8 (CD8) (above 60%). Furthermore, about 20% of TCRαβ T-IELs were CD4CD8αβ, and the remaining TCRαβ T-IELs were evenly distributed between CD4 and CD8αβ (~40% of TCRαβ T-IELs each) with no TCRαβCD8αα identified. Despite these unique properties, bovine T-IELs, similar to those in humans and mice, expressed a high level of CD69, an activation and tissue-retention marker, and a low level of CD62L, a lymphoid adhesion marker. Moreover, bovine T-IELs produced low levels of inflammatory cytokines such as IFNγ and IL17A, and secreted small amounts of the immune regulatory cytokine TGFβ1. Hence, bovine T-IELs' composition largely differs from that of human and mouse, with the dominance of the CD8 population among TCRγδ T-IELs, the substantial presence of TCRαβCD4CD8αβ cells, and the absence of TCRαβCD8αα T-IELs. These results provide the groundwork for conducting future studies to examine how bovine T-IELs respond to intestinal pathogens and maintain the integrity of the gut epithelial barrier in animals.

Characterization of Bovine Intraepithelial T Lymphocytes in the Gut.

Intestinal epithelial cell activities during homeostasis and regeneration are well described, but their potential interactions with stromal cells remain unresolved. Exploring the functions of these heterogeneous intestinal mesenchymal stromal cells (iMSCs) remains challenging. This difficulty is due to the lack of specific markers for most functionally homogenous subpopulations. In recent years, however, novel clustering techniques such as single-cell RNA sequencing (scRNA-seq), fluorescence-activated cell sorting (FACS), confocal microscope, and computational remodeling of intestinal anatomy have helped identify and characterize some specific iMSC subsets. These methods help researchers learn more about the localization and functions of iMSC populations during intestinal morphogenic and homeostatic conditions. Consequently, it is imperative to understand the cellular pathways that regulate their activation and how they interact with surrounding cellular components, particularly during intestinal epithelial regeneration after mucosal injury. This review provides insights into the spatial distribution and functions of identified iMSC subtypes. It focuses on their involvement in intestinal morphogenesis, homeostasis, and regeneration. We reviewed related signaling mechanisms implicated during epithelial and subepithelial stromal cell crosstalk. Future research should focus on elucidating the molecular intermediates of these regulatory pathways to open a new frontier for potential therapeutic targets that can alleviate intestinal mucosa-related injuries.

Subepithelial Stromal Cells: Their Roles and Interactions with Intestinal Epithelial Cells during Gut Mucosal Homeostasis and Regeneration.

The intestinal epithelial apical junctional complex, which includes tight and adherens junctions, contributes to the intestinal barrier function via their role in regulating paracellular permeability. Myosin light chain II (MLC-2), has been shown to be a critical regulatory protein in altering paracellular permeability during gastrointestinal disorders. Previous studies have demonstrated that phosphorylation of MLC-2 is a biochemical marker for perijunctional actomyosin ring contraction, which increases paracellular permeability by regulating the apical junctional complex. The phosphorylation of MLC-2 is dominantly regulated by myosin light chain kinase- (MLCK-) and Rho-associated coiled-coil containing protein kinase- (ROCK-) mediated pathways. In this review, we aim to summarize the current state of knowledge regarding the role of MLCK- and ROCK-mediated pathways in the regulation of the intestinal barrier during normal homeostasis and digestive diseases. Additionally, we will also suggest potential therapeutic targeting of MLCK- and ROCK-associated pathways in gastrointestinal disorders that compromise the intestinal barrier.

The Regulation of Intestinal Mucosal Barrier by Myosin Light Chain Kinase/Rho Kinases.

Existing gastrointestinal (GI) diagnostic tools are unable to non-invasively monitor mucosal tight junction integrity in vivo beyond the esophagus. In the GI tract, local inflammatory processes induce alterations in tight junction proteins, enhancing paracellular ion permeability. Although transepithelial electrical resistance (TEER) may be used in the laboratory to assess mucosal barrier integrity, there are no existing methodologies for characterizing tight junction dilation in vivo. Addressing this technology gap, intraluminal bioimpedance sensing may be employed as a localized, non-invasive surrogate to TEER electrodes used in cell cultures. Thus far, bioimpedance has only been implemented in esophagogastroduodenoscopy (EGD) due to the need for external electronics connections. In this work, we develop a novel, noise-resilient Bluetooth-enabled ingestible device for the continuous, non-invasive measurement of intestinal mucosal "leakiness." As a proof-of-concept, we validate wireless impedance readout on excised porcine tissues in motion. Through an animal study, we demonstrate how the device exhibits altered impedance response to tight junction dilation induced on mice colonic tissue through calcium-chelator exposure. Device measurements are validated using standard benchtop methods for assessing mucosal permeability.

An ingestible bioimpedance sensing device for wireless monitoring of epithelial barriers.

: Larazotide acetate (LA) is a synthetic octapeptide under development as a therapeutic candidate for celiac disease, acting to reduce intestinal permeability and regulate tight junctions (TJs). Although several studies have shown barrier-protective effects, the cellular mechanisms underlying LA's actions in the intestinal epithelium remain unclear. This study aimed to elucidate the mechanistic roles of LA in maintaining intestinal epithelial integrity during cellular injury.  C2BBe1 and leaky IPEC-J2 cell monolayers were pretreated with 10 mM LA and subjected to anoxia/reoxygenation (A/R) injury. Transepithelial electrical resistance (TEER), TJ protein localization, and phosphorylation of myosin light chain-2 (MLC-2) were analyzed. In addition, RNA sequencing was conducted to identify differentially expressed genes and signaling pathways affected by LA treatment.  LA pretreatment significantly increased TEER and preserved TJ protein organization during A/R injury. Transcriptomic analysis revealed enrichment of genes related to barrier regulation, small GTPase signaling, protein phosphorylation, proliferation, and migration. LA pretreatment markedly reduced MLC-2 phosphorylation, likely through modulation of the ROCK pathway, consistent with RNA-seq findings. Moreover, LA enhanced cellular proliferation, validating transcriptomic predictions.  LA exerts a protective effect on intestinal epithelial integrity by stabilizing tight junctions, reducing MLC-2 phosphorylation, and promoting epithelial proliferation. These findings highlight a novel mechanism for LA and support its therapeutic potential in treating gastrointestinal disorders associated with "leaky gut" and mucosal injury.

Article	Year
Characterization of Bovine Intraepithelial T Lymphocytes in the Gut. Pathogens (Basel, Switzerland)\| PubMed ID: 37764981	2023
Subepithelial Stromal Cells: Their Roles and Interactions with Intestinal Epithelial Cells during Gut Mucosal Homeostasis and Regeneration. Biomedicines\| PubMed ID: 38540281	2024
The Regulation of Intestinal Mucosal Barrier by Myosin Light Chain Kinase/Rho Kinases. International journal of molecular sciences\| PubMed ID: 32443411	2020
An ingestible bioimpedance sensing device for wireless monitoring of epithelial barriers. Microsystems & nanoengineering\| PubMed ID: 39915452	2025
Larazotide Acetate Protects the Intestinal Mucosal Barrier from Anoxia/Reoxygenation Injury via Various Cellular Mechanisms. Biomedicines\| PubMed ID: 41153766	2025

Younggeon Jin

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