Protocol for generating and analyzing organ-on-chip using human and mouse intestinal organoids.

Organ-on-chip technology is a powerful tool for in vitro modeling. Combining it with organoids overcomes lumen inaccessibility while preserving cellular diversity and function of the intestinal epithelium. Here, we present a protocol for generating and analyzing organ-on-chips using human and mouse intestinal organoids. This protocol covers organoid line establishment, single-cell dissociation, chip preparation, and seeding. It outlines procedures for permeability assays, RNA isolation, staining, and imaging. Additionally, we describe independent stimulation and sampling of the apical and basal side.

SLE serum induces altered goblet cell differentiation and leakiness in human intestinal organoids.

Human intestinal epithelial cells are the interface between luminal content and basally residing immune cells. They form a tight monolayer that constantly secretes mucus creating a multilayered protective barrier. Alterations in this barrier can lead to increased permeability which is common in systemic lupus erythematosus (SLE) patients. However, it remains unexplored how the barrier is affected. Here, we present an in vitro model specifically designed to examine the effects of SLE on epithelial cells. We utilize human colon organoids that are stimulated with serum from SLE patients. Combining transcriptomic with functional analyses revealed that SLE serum induced an expression profile marked by a reduction of goblet cell markers and changed mucus composition. In addition, organoids exhibited imbalanced cellular composition along with enhanced permeability, altered mitochondrial function, and an interferon gene signature. Similarly, transcriptomic analysis of SLE colon biopsies revealed a downregulation of secretory markers. Our work uncovers a crucial connection between SLE and intestinal homeostasis that might be promoted in vivo through the blood, offering insights into the causal connection of barrier dysfunction and autoimmune diseases.

The septate junction component bark beetle is required for Drosophila intestinal barrier function and homeostasis.

Age-related loss of intestinal barrier function has been documented across species, but the causes remain unknown. The intestinal barrier is maintained by tight junctions (TJs) in mammals and septate junctions (SJs) in insects. Specialized TJs/SJs, called tricellular junctions (TCJs), are located at the nexus of three adjacent cells, and we have shown that aging results in changes to TCJs in intestines of adult Drosophila melanogaster. We now demonstrate that localization of the TCJ protein bark beetle (Bark) decreases in aged flies. Depletion of bark from enterocytes in young flies led to hallmarks of intestinal aging and shortened lifespan, whereas depletion of bark in progenitor cells reduced Notch activity, biasing differentiation toward the secretory lineage. Our data implicate Bark in EC maturation and maintenance of intestinal barrier integrity. Understanding the assembly and maintenance of TCJs to ensure barrier integrity may lead to strategies to improve tissue integrity when function is compromised.

Neuroglian regulates Drosophila intestinal stem cell proliferation through enhanced signaling via the epidermal growth factor receptor.

The Drosophila intestine is an excellent system for elucidating mechanisms regulating stem cell behavior. Here we show that the septate junction (SJ) protein Neuroglian (Nrg) is expressed in intestinal stem cells (ISCs) and enteroblasts (EBs) within the fly intestine. SJs are not present between ISCs and EBs, suggesting Nrg plays a different role in this tissue. We reveal that Nrg is required for ISC proliferation in young flies, and depletion of Nrg from ISCs and EBs suppresses increased ISC proliferation in aged flies. Conversely, overexpression of Nrg in ISC and EBs promotes ISC proliferation, leading to an increase in cells expressing ISC/EB markers; in addition, we observe an increase in epidermal growth factor receptor (Egfr) activation. Genetic epistasis experiments reveal that Nrg acts upstream of Egfr to regulate ISC proliferation. As Nrg function is highly conserved in mammalian systems, our work characterizing the role of Nrg in the intestine has implications for the treatment of intestinal disorders that arise due to altered ISC behavior.

Ras2, the TC21/R-Ras2 Drosophila homologue, contributes to insulin signalling but is not required for organism viability.

Ras1 (Ras85D) and Ras2 (Ras64B) are the Drosophila orthologs of human H-Ras/N-Ras/K-Ras and R-Ras1-3 genes, respectively. The function of Ras1 has been thoroughly characterised during Drosophila embryonic and imaginal development, and it is associated with coupling activated trans-membrane receptors with tyrosine kinase activity to their downstream effectors. In this capacity, Ras1 binds and is required for the activation of Raf. Ras1 can also interact with PI3K, and it is needed to achieve maximal levels of PI3K signalling in specific cellular settings. In contrast, the function of the unique Drosophila R-Ras member (Ras2/Ras64B), which is more closely related to vertebrate R-Ras2/TC21, has been only studied through the use of constitutively activated forms of the protein. This pioneering work identified a variety of phenotypes that were related to those displayed by Ras1, suggesting that Ras1 and Ras2 might have overlapping activities. Here we find that Ras2 can interact with PI3K and Raf and activate their downstream effectors Akt and Erk. However, and in contrast to mutants in Ras1, which are lethal, null alleles of Ras2 are viable in homozygosis and only show a phenotype of reduced wing size and extended life span that might be related to reduced Insulin receptor signalling.

Intestinal Snakeskin Limits Microbial Dysbiosis during Aging and Promotes Longevity.

Intestinal barrier dysfunction is an evolutionarily conserved hallmark of aging, which has been linked to microbial dysbiosis, altered expression of occluding junction proteins, and impending mortality. However, the interplay between intestinal junction proteins, age-onset dysbiosis, and lifespan determination remains unclear. Here, we show that altered expression of Snakeskin (Ssk), a septate junction-specific protein, can modulate intestinal homeostasis, microbial dynamics, immune activity, and lifespan in Drosophila. Loss of Ssk leads to rapid and reversible intestinal barrier dysfunction, altered gut morphology, dysbiosis, and dramatically reduced lifespan. Remarkably, restoration of Ssk expression in flies showing intestinal barrier dysfunction rescues each of these phenotypes previously linked to aging. Intestinal up-regulation of Ssk protects against microbial translocation following oral infection with pathogenic bacteria. Furthermore, intestinal up-regulation of Ssk improves intestinal barrier function during aging, limits dysbiosis, and extends lifespan. Our findings indicate that intestinal occluding junctions may represent prolongevity targets in mammals.

Keeping it tight: The relationship between bacterial dysbiosis, septate junctions, and the intestinal barrier in Drosophila.

Maladaptive changes in the intestinal flora, typically referred to as bacterial dysbiosis, have been linked to intestinal aging phenotypes, including an increase in intestinal stem cell (ISC) proliferation, activation of inflammatory pathways, and increased intestinal permeability1,2. However, the causal relationships between these phenotypes are only beginning to be unravelled. We recently characterized the age-related changes that occur to septate junctions (SJ) between adjacent, absorptive enterocytes (EC) in the fly intestine. Changes could be observed in the overall level of SJ proteins, as well as the localization of a subset of SJ proteins. Such age-related changes were particularly noticeable at tricellular junctions (TCJ)3. Acute loss of the Drosophila TCJ protein Gliotactin (Gli) in ECs led to rapid activation of stress signalling in stem cells and an increase in ISC proliferation, even under axenic conditions; a gradual disruption of the intestinal barrier was also observed. The uncoupling of changes in bacteria from alterations in ISC behaviour and loss of barrier integrity has allowed us to begin to explore the interrelationship of these intestinal aging phenotypes in more detail and has shed light on the importance of the proteins that contribute to maintenance of the intestinal barrier.

Tricellular junctions regulate intestinal stem cell behaviour to maintain homeostasis.

Ageing results in loss of tissue homeostasis across taxa. In the intestine of Drosophila melanogaster, ageing is correlated with an increase in intestinal stem cell (ISC) proliferation, a block in terminal differentiation of progenitor cells, activation of inflammatory pathways, and increased intestinal permeability. However, causal relationships between these phenotypes remain unclear. Here, we demonstrate that ageing results in altered localization and expression of septate junction proteins in the posterior midgut, which is quite pronounced in differentiated enterocytes (ECs) at tricellular junctions (TCJs). Acute loss of the TCJ protein Gliotactin (Gli) in ECs results in increased ISC proliferation and a block in differentiation in intestines from young flies, demonstrating that compromised TCJ function is sufficient to alter ISC behaviour in a non-autonomous manner. Blocking the Jun N-terminal kinase signalling pathway is sufficient to suppress changes in ISC behaviour, but has no effect on loss of intestinal barrier function, as a consequence of Gli depletion. Our work demonstrates a pivotal link between TCJs, stem cell behaviour, and intestinal homeostasis and provides insights into causes of age-onset and gastrointestinal diseases.

MAP4K3 is a component of the TORC1 signalling complex that modulates cell growth and viability in Drosophila melanogaster.

BACKGROUND: MAP4K3 is a conserved Ser/Thr kinase that has being found in connection with several signalling pathways, including the Imd, EGFR, TORC1 and JNK modules, in different organisms and experimental assays. We have analyzed the consequences of changing the levels of MAP4K3 expression in the development of the Drosophila wing, a convenient model system to characterize gene function during epithelial development. METHODOLOGY AND PRINCIPAL FINDINGS: Using loss-of-function mutants and over-expression conditions we find that MAP4K3 activity affects cell growth and viability in the Drosophila wing. These requirements are related to the modulation of the TORC1 and JNK signalling pathways, and are best detected when the larvae grow in a medium with low protein concentration (TORC1) or are exposed to irradiation (JNK). We also show that MAP4K3 display strong genetic interactions with different components of the InR/Tor signalling pathway, and can interact directly with the GTPases RagA and RagC and with the multi-domain kinase Tor. CONCLUSIONS AND SIGNIFICANCE: We suggest that MAP4K3 has two independent functions during wing development, one related to the activation of the JNK pathway in response to stress and other in the assembling or activation of the TORC1 complex, being critical to modulate cellular responses to changes in nutrient availability.