Protocol for measuring transcytosis and recycling of IgG in intestinal epithelial Caco-2 cells and primary human intestinal organoids.

Transcytosis is the primary mechanism by which macro-molecules transverse epithelial cell barriers. Here, we present an assay for measuring transcytosis and recycling of IgG in intestinal epithelial Caco-2 cells and primary human intestinal organoids. We describe steps for establishing human enteroids or Caco-2 cells and plating monolayers. We then provide procedures for a transcytosis and recycling assay and a luciferase assay. The protocol facilitates quantification of membrane trafficking and can be used to probe endosomal compartments unique to polarized epithelia. For complete details on the use and execution of this protocol, please refer to Maeda K et al. (2022).1.

Membrane Lipids in Epithelial Polarity: Sorting out the PIPs.

The development of cell polarity in epithelia, is critical for tissue morphogenesis and vectorial transport between the environment and the underlying tissue. Epithelial polarity is defined by the development of distinct plasma membrane domains: the apical membrane interfacing with the exterior lumen compartment, and the basolateral membrane directly contacting the underlying tissue. The de novo generation of polarity is a tightly regulated process, both spatially and temporally, involving changes in the distribution of plasma membrane lipids, localization of apical and basolateral membrane proteins, and vesicular trafficking. Historically, the process of epithelial polarity has been primarily described in relation to the localization and function of protein 'polarity complexes.' However, a critical and foundational role is emerging for plasma membrane lipids, and in particular phosphoinositide species. Here, we broadly review the evidence for a primary role for membrane lipids in the generation of epithelial polarity and highlight key areas requiring further research. We discuss the complex interchange that exists between lipid species and briefly examine how major membrane lipid constituents are generated and intersect with vesicular trafficking to be preferentially localized to different membrane domains with a focus on some of the key protein-enzyme complexes involved in these processes.

The epithelial-specific ER stress sensor ERN2/IRE1ß enables host-microbiota crosstalk to affect colon goblet cell development.

Epithelial cells lining mucosal surfaces of the gastrointestinal and respiratory tracts uniquely express ERN2/IRE1ß, a paralogue of the most evolutionarily conserved endoplasmic reticulum stress sensor, ERN1/IRE1α. How ERN2 functions at the host-environment interface and why a second paralogue evolved remain incompletely understood. Using conventionally raised and germ-free Ern2-/- mice, we found that ERN2 was required for microbiota-induced goblet cell maturation and mucus barrier assembly in the colon. This occurred only after colonization of the alimentary tract with normal gut microflora, which induced Ern2 expression. ERN2 acted by splicing Xbp1 mRNA to expand ER function and prevent ER stress in goblet cells. Although ERN1 can also splice Xbp1 mRNA, it did not act redundantly to ERN2 in this context. By regulating assembly of the colon mucus layer, ERN2 further shaped the composition of the gut microbiota. Mice lacking Ern2 had a dysbiotic microbial community that failed to induce goblet cell development and increased susceptibility to colitis when transferred into germ-free WT mice. These results show that ERN2 evolved at mucosal surfaces to mediate crosstalk between gut microbes and the colonic epithelium required for normal homeostasis and host defense.

Depletion of the apical endosome in response to viruses and bacterial toxins provides cell-autonomous host defense at mucosal surfaces.

Polarized epithelial cells form an essential barrier against infection at mucosal surfaces. Many pathogens breach this barrier to cause disease, often by co-opting cellular endocytosis mechanisms to enter the cell through the lumenal (apical) cell surface. We recently discovered that the loss of the cell polarity gene PARD6B selectively diminishes apical endosome function. Here, we find that in response to the entry of certain viruses and bacterial toxins into the epithelial cells via the apical membrane, PARD6B and aPKC, two components of the PARD6B-aPKC-Cdc42 apical polarity complex, undergo rapid proteasome-dependent degradation. The perturbation of apical membrane glycosphingolipids by toxin- or virus-binding initiates degradation of PARD6B. The loss of PARD6B causes the depletion of apical endosome function and renders the cell resistant to further infection from the lumenal cell surface, thus enabling a form of cell-autonomous host defense.

Tetraspanin CD82 is necessary for muscle stem cell activation and supports dystrophic muscle function.

BACKGROUND: Tetraspanins are a family of proteins known to assemble protein complexes at the cell membrane. They are thought to play diverse cellular functions in tissues by modifying protein-binding partners, thus bringing complexity and diversity in their regulatory networks. Previously, we identified the tetraspanin KAI/CD82 as a prospective marker for human muscle stem cells. CD82 expression appeared decreased in human Duchenne muscular dystrophy (DMD) muscle, suggesting a functional link to muscular dystrophy, yet whether this decrease is a consequence of dystrophic pathology or a compensatory mechanism in an attempt to rescue muscle from degeneration is currently unknown. METHODS: We studied the consequences of loss of CD82 expression in normal and dystrophic skeletal muscle and examined the dysregulation of downstream functions in mice aged up to 1 year. RESULTS: Expression of CD82 is important to sustain satellite cell activation, as in its absence there is decreased cell proliferation and less efficient repair of injured muscle. Loss of CD82 in dystrophic muscle leads to a worsened phenotype compared to control dystrophic mice, with decreased pulmonary function, myofiber size, and muscle strength. Mechanistically, decreased myofiber size in CD82-/- dystrophic mice is not due to altered PTEN/AKT signaling, although increased phosphorylation of mTOR at Ser2448 was observed. CONCLUSION: Basal CD82 expression is important to dystrophic muscle, as its loss leads to significantly weakened myofibers and impaired muscle function, accompanied by decreased satellite cell activity that is unable to protect and repair myofiber damage.

Extracellular cyclic dinucleotides induce polarized responses in barrier epithelial cells by adenosine signaling.

Cyclic dinucleotides (CDNs) are secondary messengers used by prokaryotic and eukaryotic cells. In mammalian cells, cytosolic CDNs bind STING (stimulator of IFN gene), resulting in the production of type I IFN. Extracellular CDNs can enter the cytosol through several pathways but how CDNs work from outside eukaryotic cells remains poorly understood. Here, we elucidate a mechanism of action on intestinal epithelial cells for extracellular CDNs. We found that CDNs containing adenosine induced a robust CFTR-mediated chloride secretory response together with cAMP-mediated inhibition of Poly I:C-stimulated IFNß expression. Signal transduction was strictly polarized to the serosal side of the epithelium, dependent on the extracellular and sequential hydrolysis of CDNs to adenosine by the ectonucleosidases ENPP1 and CD73, and occurred via activation of A2B adenosine receptors. These studies highlight a pathway by which microbial and host produced extracellular CDNs can regulate the innate immune response of barrier epithelial cells lining mucosal surfaces.

Actions of Trace Amines in the Brain-Gut-Microbiome Axis via Trace Amine-Associated Receptor-1 (TAAR1).

Trace amines and their primary receptor, Trace Amine-Associated Receptor-1 (TAAR1) are widely studied for their involvement in the pathogenesis of neuropsychiatric disorders despite being found in the gastrointestinal tract at physiological levels. With the emergence of the "brain-gut-microbiome axis," we take the opportunity to review what is known about trace amines in the brain, the defined sources of trace amines in the gut, and emerging understandings on the levels of trace amines in various gastrointestinal disorders. Similarly, we discuss localization of TAAR1 expression in the gut, novel findings that TAAR1 may be implicated in inflammatory bowel diseases, and the reported comorbidities of neuropsychiatric disorders and gastrointestinal disorders. With the emergence of TAAR1 specific compounds as next-generation therapeutics for schizophrenia (Roche) and Parkinson's related psychoses (Sunovion), we hypothesize a therapeutic benefit of these compounds in clinical trials in the brain-gut-microbiome axis, as well as a potential for thoughtful manipulation of the brain-gut-microbiome axis to modulate symptoms of neuropsychiatric disease.

Trace amine associated receptor 1 (TAAR1) expression and modulation of inflammatory cytokine production in mouse bone marrow-derived macrophages: a novel mechanism for inflammation in ulcerative colitis.

Context: Tissue resident macrophages and peripherally infiltrating macrophages play a prominent role in maintaining homeostasis in the gastrointestinal tract (GIT), though aberrant activation is implicated in inflammatory conditions, including ulcerative colitis (UC). Recent metabolomic studies indicate that tyramine (TYR) is elevated in the stool of patients with UC. TYR activates the mammalian trace amine associated receptor 1 (TAAR1). Our previous work identified TAAR1 expression in mixed populations of immune cells, whereas a limited number of other studies have identified TAAR1-dependent effects in cytokine secretion and gene expression in T-cells and B-cells.Objective: To investigate whether TAAR1 may serve as a novel target for an anti-inflammatory therapeutic in UC, we explored TAAR1 expression in mouse bone marrow-derived macrophages (BMDMs), and its upregulation and activation in response to LPS and TYR.Results: Here, we demonstrate for the first time that TAAR1 is expressed in BMDM and undergoes agonist-induced upregulation. Additionally, TYR elicits significant increases in inflammatory cytokine gene expression in non-polarized and LPS-polarized BMDM, and the TAAR1 antagonist EPPTB inhibits the TYR-mediated upregulation of TAAR1 and inflammatory cytokine gene expression in BMDM. Conclusions: Our data suggest that TAAR1 is a mediator of macrophage inflammation and a potential therapeutic target to attenuate UC symptomology.

Transgenic zebrafish model of DUX4 misexpression reveals a developmental role in FSHD pathogenesis.

Facioscapulohumeral dystrophy type 1 (FSHD-1) is the most common autosomal dominant form of muscular dystrophy with a prevalence of ∼1 in 8000 individuals. It is considered a late-onset form of muscular dystrophy and leads to asymmetric muscle weakness in the facial, scapular, trunk and lower extremities. The prevalent hypothesis on disease pathogenesis is explained by misexpression of a germ line, primate-specific transcription factor DUX4-fl (double homeobox 4, full-length isoform) linked to the chromosome 4q35. In vitro and in vivo studies have demonstrated that very low levels of DUX4-fl expression are sufficient to induce an apoptotic and/or lethal phenotype, and therefore modeling of the disease has proved challenging. In this study, we expand upon our previously established injection model of DUX4 misexpression in zebrafish and describe a DUX4-inducible transgenic zebrafish model that better recapitulates the expression pattern and late onset phenotype characteristic of FSHD patients. We show that an induced burst of DUX4 expression during early development results in the onset of FSHD-like phenotypes in adulthood, even when DUX4 is no longer detectable. We also utilize our injection model to study long-term consequences of DUX4 expression in those that fail to show a developmental phenotype. Herein, we introduce a hypothesis that DUX4 expression during developmental stages is sufficient to induce FSHD-like phenotypes in later adulthood. Our findings point to a developmental role of DUX4 misexpression in the pathogenesis of FSHD and should be factored into the design of future therapies.