The role of thymic epithelium in thymus development and age-related thymic involution.

The establishment of an adaptive immune system is critical for protecting our bodies from neoplastic cancers and invading pathogens such as viruses and bacteria. As a primary lymphoid organ, the thymus generates lymphoid T cells that play a major role in the adaptive immune system. T cell generation in the thymus is controlled by interactions between thymocytes and other thymic cells, primarily thymic epithelial cells. Thus, the normal development and function of thymic epithelial cells are important for the generation of immunocompetent and self-tolerant T cells. On the other hand, the degeneration of the thymic epithelium due to thymic aging causes thymic involution, which is associated with the decline of adaptive immune function. Herein we summarize basic and current knowledge of the development and function of thymic epithelial cells and the mechanism of thymic involution. J. Med. Invest. 71 : 29-39, February, 2024.

Paraspeckles / CARM1 mediates the regulation of OEVs on cell differentiation during in vitro embryonic development of yak.

Mammalian embryos produced in vitro have poor embryo quality and low developmental ability compared with in vivo embryos. The main manifestations are the low number of blastocysts, the low ratio of the number of inner cell mass cells to the number of trophoblastic cells, and the high apoptosis rate of blastocysts, resulting in low embryo implantation rate. Therefore, optimizing in vitro culture conditions has become a key technology to im-prove the quality of preimplantation embryos. Oviduct Epithelial cells exosomes (OEVs) can be absorbed and internalized by embryos to improve the blastocyst rate and blastocyst quality of embryos in vitro. As a special nuclear structure, Paraspeckles are involved in the fate determination of mammalian early embryonic mammalian cells. However, the regulation of embryonic cell differentiation by OEVs remains unknown. We aimed to investigate the effects of OEVs on paraspeckle formation and cell fate determination in yak in vitro fertilization (IVF) of em-bryos. To simulate the in vivo oviduct environment after ovulation, we used follicular fluid exosomes (FEVs) to stimulate yak oviduct epithelial cells and collect OEVs. OEVs were added to the yak IVF embryo culture system. Paraspeckle formation, cell differentiation, and blastocyst quality in yak embryos were determined. Our results show that, development of yak embryos is unique compared to other bovine species, and OEVs can be used as a supplement to the in vitro culture system of yak embryos to improve embryonic development and blas-tocyst quality. And also Paraspeckles/CARM1 mediated the regulation of OEVs on cell differentiation during in vitro yak embryo production. These results provide new insights into the study of yak embryonic development and the role of OEVs in embryonic development.

Sentinels of the airways.

Epithelial cells in the larynx and trachea sense harmful cues and trigger protective reflexes.

SimuCell3D: three-dimensional simulation of tissue mechanics with cell polarization.

The three-dimensional (3D) organization of cells determines tissue function and integrity, and changes markedly in development and disease. Cell-based simulations have long been used to define the underlying mechanical principles. However, high computational costs have so far limited simulations to either simplified cell geometries or small tissue patches. Here, we present SimuCell3D, an efficient open-source program to simulate large tissues in three dimensions with subcellular resolution, growth, proliferation, extracellular matrix, fluid cavities, nuclei and non-uniform mechanical properties, as found in polarized epithelia. Spheroids, vesicles, sheets, tubes and other tissue geometries can readily be imported from microscopy images and simulated to infer biomechanical parameters. Doing so, we show that 3D cell shapes in layered and pseudostratified epithelia are largely governed by a competition between surface tension and intercellular adhesion. SimuCell3D enables the large-scale in silico study of 3D tissue organization in development and disease at a great level of detail.

After wounding, a G-protein coupled receptor promotes the restoration of tension in epithelial cells.

The maintenance of epithelial barrier function involves cellular tension, with cells pulling on their neighbors to maintain epithelial integrity. Wounding interrupts cellular tension, which may serve as an early signal to initiate epithelial repair. To characterize how wounds alter cellular tension we used a laser-recoil assay to map cortical tension around wounds in the epithelial monolayer of the Drosophila pupal notum. Within a minute of wounding, there was widespread loss of cortical tension along both radial and tangential directions. This tension loss was similar to levels observed with Rok inactivation. Tension was subsequently restored around the wound, first in distal cells and then in proximal cells, reaching the wound margin ∼10 min after wounding. Restoring tension required the GPCR Mthl10 and the IP3 receptor, indicating the importance of this calcium signaling pathway known to be activated by cellular damage. Tension restoration correlated with an inward-moving contractile wave that has been previously reported; however, the contractile wave itself was not affected by Mthl10 knockdown. These results indicate that cells may transiently increase tension and contract in the absence of Mthl10 signaling, but that pathway is critical for fully resetting baseline epithelial tension after it is disrupted by wounding.

Mesenchyme instructs growth while epithelium directs branching in the mouse mammary gland.

The mammary gland is a unique organ that undergoes dynamic alterations throughout a female's reproductive life, making it an ideal model for developmental, stem cell and cancer biology research. Mammary gland development begins in utero and proceeds via a quiescent bud stage before the initial outgrowth and subsequent branching morphogenesis. How mammary epithelial cells transit from quiescence to an actively proliferating and branching tissue during embryogenesis and, importantly, how the branch pattern is determined remain largely unknown. Here, we provide evidence indicating that epithelial cell proliferation and onset of branching are independent processes, yet partially coordinated by the Eda signaling pathway. Through heterotypic and heterochronic epithelial-mesenchymal recombination experiments between mouse mammary and salivary gland tissues and ex vivo live imaging, we demonstrate that unlike previously concluded, the mode of branching is an intrinsic property of the mammary epithelium whereas the pace of growth and the density of ductal tree are determined by the mesenchyme. Transcriptomic profiling and ex vivo and in vivo functional studies in mice disclose that mesenchymal Wnt/ß-catenin signaling, and in particular IGF-1 downstream of it critically regulate mammary gland growth. These results underscore the general need to carefully deconstruct the different developmental processes producing branched organs.

Effects of L-Leu-L-Leu peptide on growth, proliferation, and apoptosis in broiler intestinal epithelial cells.

Small peptides are nutrients and bioactive molecules that have dual regulatory effects on nutrition and physiology. They are of great significance for maintaining the intestinal health and production performance of broilers. We here cultured the primary small intestinal epithelial cells (IEC) of chicken in a medium containing L-Leu (Leu) and L-Leu-L-Leu (Leu-Leu) for 24 h. The untreated cells were considered as the control group. The growth, proliferation, and apoptosis of IEC were examined. By combining RNA-seq and label-free sequencing technology, candidate genes, proteins, and pathways related to the growth, proliferation, and apoptosis of IEC were screened. Immunofluorescence detection revealed that the purity of the isolated primary IEC was >90%. The Leu-Leu group significantly promoted IEC growth and proliferation and significantly inhibited IEC apoptosis, and the effect was better than those of the Leu and control groups. Using transcriptome sequencing, four candidate genes, CCL20, IL8L1, IL8, and IL6, were screened in the Leu group, and one candidate gene, IL8, was screened in the Leu-Leu group. Two candidate genes, IL6 and RGN, were screened in the Leu-Leu group compared with the Leu group. Nonquantitative proteomic marker sequencing results revealed that through the screening of candidate proteins and pathways, found one growth-related candidate protein PGM3 and three proliferation-related candidate proteins RPS17, RPS11, and RPL23, and two apoptosis-related candidate proteins GPX4 and PDPK1 were found in the Leu-Leu group compared with Leu group. In short, Leu-Leu could promote IEC growth and proliferation and inhibit IEC apoptosis. On combining transcriptome and proteome sequencing technologies, multiple immune- and energy-related regulatory signal pathways were found to be related to IEC growth, proliferation, and apoptosis. Three candidate genes of IL8, IL6, and RGN were identified, and six candidate proteins of PGM3, RPS17, RPS11, RPL23, GPX4, and PDPK1 were involved in IEC growth, proliferation, and apoptosis. The results provide valuable data for preliminarily elucidating small peptide-mediated IEC regulation pathways, improving the small peptide nutrition theoretical system, and establishing small peptide nutrition regulation technology.

Shigella generates distinct IAM subpopulations during epithelial cell invasion to promote efficient intracellular niche formation.

The facultative intracellular pathogen Shigella flexneri invades non-phagocytic epithelial gut cells. Through a syringe-like apparatus called type 3 secretion system, it injects effector proteins into the host cell triggering actin rearrangements leading to its uptake within a tight vacuole, termed the bacterial-containing vacuole (BCV). Simultaneously, Shigella induces the formation of large vesicles around the entry site, which we refer to as infection-associated macropinosomes (IAMs). After entry, Shigella ruptures the BCV and escapes into the host cytosol by disassembling the BCV remnants. Previously, IAM formation has been shown to be required for efficient BCV escape, but the molecular events associated with BCV disassembly have remained unclear. To identify host components required for BCV disassembly, we performed a microscopy-based screen to monitor the recruitment of BAR domain-containing proteins, which are a family of host proteins involved in membrane shaping and sensing (e.g. endocytosis and recycling) during Shigella epithelial cell invasion. We identified endosomal recycling BAR protein Sorting Nexin-8 (SNX8) localized to IAMs in a PI(3)P-dependent manner before BCV disassembly. At least two distinct IAM subpopulations around the BCV were found, either being recycled back to cellular compartments such as the plasma membrane or transitioning to become RAB11A positive "contact-IAMs" involved in promoting BCV rupture. The IAM subpopulation duality was marked by the exclusive recruitment of either SNX8 or RAB11A. Hindering PI(3)P production at the IAMs led to an inhibition of SNX8 recruitment at these compartments and delayed both, the step of BCV rupture time and successful BCV disassembly. Finally, siRNA depletion of SNX8 accelerated BCV rupture and unpeeling of BCV remnants, indicating that SNX8 is involved in controlling the timing of the cytosolic release. Overall, our work sheds light on how Shigella establishes its intracellular niche through the subversion of a specific set of IAMs.

The AMPK-Sirtuin 1-YAP axis is regulated by fluid flow intensity and controls autophagy flux in kidney epithelial cells.

Shear stress generated by urinary fluid flow is an important regulator of renal function. Its dysregulation is observed in various chronic and acute kidney diseases. Previously, we demonstrated that primary cilium-dependent autophagy allows kidney epithelial cells to adapt their metabolism in response to fluid flow. Here, we show that nuclear YAP/TAZ negatively regulates autophagy flux in kidney epithelial cells subjected to fluid flow. This crosstalk is supported by a primary cilium-dependent activation of AMPK and SIRT1, independently of the Hippo pathway. We confirm the relevance of the YAP/TAZ-autophagy molecular dialog in vivo using a zebrafish model of kidney development and a unilateral ureteral obstruction mouse model. In addition, an in vitro assay simulating pathological accelerated flow observed at early stages of chronic kidney disease (CKD) activates YAP, leading to a primary cilium-dependent inhibition of autophagic flux. We confirm this YAP/autophagy relationship in renal biopsies from patients suffering from diabetic kidney disease (DKD), the leading cause of CKD. Our findings demonstrate the importance of YAP/TAZ and autophagy in the translation of fluid flow into cellular and physiological responses. Dysregulation of this pathway is associated with the early onset of CKD.

Mid-old cells are a potential target for anti-aging interventions in the elderly.

The biological process of aging is thought to result in part from accumulation of senescent cells in organs. However, the present study identified a subset of fibroblasts and smooth muscle cells which are the major constituents of organ stroma neither proliferative nor senescent in tissues of the elderly, which we termed "mid-old status" cells. Upregulation of pro-inflammatory genes (IL1B and SAA1) and downregulation of anti-inflammatory genes (SLIT2 and CXCL12) were detected in mid-old cells. In the stroma, SAA1 promotes development of the inflammatory microenvironment via upregulation of MMP9, which decreases the stability of epithelial cells present on the basement membrane, decreasing epithelial cell function. Remarkably, the microenvironmental change and the functional decline of mid-old cells could be reversed by a young cell-originated protein, SLIT2. Our data identify functional reversion of mid-old cells as a potential method to prevent or ameliorate aspects of aging-related tissue dysfunction.

Choroid plexuses carry nodal-like cilia that undergo axoneme regression from early adult stage.

Choroid plexuses (ChPs) produce cerebrospinal fluid and sense non-cell-autonomous stimuli to control the homeostasis of the central nervous system. They are mainly composed of epithelial multiciliated cells, whose development and function are still controversial. We have thus characterized the stepwise order of mammalian ChP epithelia cilia formation using a combination of super-resolution-microscopy approaches and mouse genetics. We show that ChP ciliated cells are built embryonically on a treadmill of spatiotemporally regulated events, starting with atypical centriole amplification and ending with the construction of nodal-like 9+0 cilia, characterized by both primary and motile features. ChP cilia undergo axoneme resorption at early postnatal stages through a microtubule destabilization process controlled by the microtubule-severing enzyme spastin and mitigated by polyglutamylation levels. Notably, this phenotype is preserved in humans, suggesting a conserved ciliary resorption mechanism in mammals.

Force propagation between epithelial cells depends on active coupling and mechano-structural polarization.

Cell-generated forces play a major role in coordinating the large-scale behavior of cell assemblies, in particular during development, wound healing, and cancer. Mechanical signals propagate faster than biochemical signals, but can have similar effects, especially in epithelial tissues with strong cell-cell adhesion. However, a quantitative description of the transmission chain from force generation in a sender cell, force propagation across cell-cell boundaries, and the concomitant response of receiver cells is missing. For a quantitative analysis of this important situation, here we propose a minimal model system of two epithelial cells on an H-pattern ('cell doublet'). After optogenetically activating RhoA, a major regulator of cell contractility, in the sender cell, we measure the mechanical response of the receiver cell by traction force and monolayer stress microscopies. In general, we find that the receiver cells show an active response so that the cell doublet forms a coherent unit. However, force propagation and response of the receiver cell also strongly depend on the mechano-structural polarization in the cell assembly, which is controlled by cell-matrix adhesion to the adhesive micropattern. We find that the response of the receiver cell is stronger when the mechano-structural polarization axis is oriented perpendicular to the direction of force propagation, reminiscent of the Poisson effect in passive materials. We finally show that the same effects are at work in small tissues. Our work demonstrates that cellular organization and active mechanical response of a tissue are key to maintain signal strength and lead to the emergence of elasticity, which means that signals are not dissipated like in a viscous system, but can propagate over large distances.

LIF regulates the expression of miR-27a-3p and HOXA10 in bovine endometrial epithelial cells via STAT3 pathway.

LIF is crucial in regulating embryo implantation, while HOXA10 is a marker gene for uterine receptivity. However, the specific mechanism of LIF regulating HOXA10 during cow embryo implantation has not been fully understood. To address this knowledge gap, the experiment involved treating bovine endometrial epithelial cells (BEECs) with LIF to investigate the relationship between LIF, miRNA, and HOXA10. The experimental findings revealed that applying LIF resulted in a substantial increase in the proliferation of endometrial epithelial cells. Moreover, the expressions of PI3K, AKT, HOXA10, CDK4, cyclinD1, and cyclinE1 were significantly elevated. Conversely, the expression of p21Cipl was significantly reduced. In the group that received a combination of LIF and a STAT3 inhibitor, the expression of PI3K/AKT remained significantly increased, but there was no significant change in the expression of HOXA10. When miRNA-27a-3p was overexpressed, it resulted in a decrease in both the RNA and protein expression of HOXA10. Conversely, inhibiting miRNA-27a-3p increased the RNA and protein expression of HOXA10. In the presence of LIF treatment, the expression of miRNA-27a-3p was reduced, while the expression of HOXA10 was increased. However, when LIF and a STAT3 inhibitor were combined, there was no significant change in the expression of miRNA-27a-3p or HOXA10. Consequently, LIF facilitated cell proliferation by activating the PI3K/AKT pathway. LIF controlled the expression of miRNA-27a-3p and HOXA10 in endometrial epithelial cells through STAT3, with miRNA-27a-3p negatively regulating the expression of HOXA10.

Establishment and characterization of an immortalized bovine intestinal epithelial cell line.

Primary bovine intestinal epithelial cells (PBIECs) are an important model for studying the molecular and pathogenic mechanisms of diseases affecting the bovine intestine. It is difficult to obtain and grow PBIECs stably, and their short lifespan greatly limits their application. Therefore, the purpose of this study was to create a cell line for exploring the mechanisms of pathogen infection in bovine intestinal epithelial cells in vitro. We isolated and cultured PBIECs and established an immortalized BIEC line by transfecting PBIECs with the pCI-neo-hTERT (human telomerase reverse transcriptase) recombinant plasmid. The immortalized cell line (BIECs-21) retained structure and function similar to that of the PBIECs. The marker proteins characteristic of epithelial cells, cytokeratin 18, occludin, zonula occludens protein 1 (ZO-1), E-cadherin and enterokinase, were all positive in the immortalized cell line, and the cell structure, growth rate, karyotype, serum dependence and contact inhibition were normal. The hTERT gene was successfully transferred into BIECs-21 where it remained stable and was highly expressed. The transport of short-chain fatty acids and glucose uptake by the BIECs-21 was consistent with PBIECs, and we showed that they could be infected with the intestinal parasite, Neospora caninum. The immortalized BIECs-21, which have exceeded 80 passages, were structurally and functionally similar to the primary BIECs and thus provide a valuable research tool for investigating the mechanism of pathogen infection of the bovine intestinal epithelium in vitro.

In dairy cattle, the intestine is essential for productivity as it contributes nearly 10% of the total metabolizable energy. The intestinal epithelium is at risk of infection from constant exposure to pathogenic microorganisms, which seriously endangers an animal's health, but no bovine intestinal epithelial cell line has been developed so far for research on intestine -related diseases. Thus, the goal of this study was to create an immortalized cell line from isolated primary bovine intestinal epithelial cells. The expression of an exogenous human telomerase reverse transcriptase (hTERT) gene can circumvent the Hayflick limit by maintaining telomere integrity and we used transfection with a plasmid expressing the hTERT gene to convert primary intestinal epithelial cells into an immortalized cell line, which we then characterized. The results showed that the immortalized cell line (BIECs-21) was structurally and functionally similar to the primary bovine intestinal epithelial cells (BIECs) and thus provided a valuable research tool for investigating the mechanism of pathogen infection of the bovine intestinal epithelium in vitro.

Progenitor hierarchy among parietal epithelial cells depicted at the single-cell level.

The role of parietal epithelial cells (PECs) in kidney function and disease was recently revisited. Building on previous studies of human kidney tissue, in the current issue, Liu et al. further characterize PECs using single-cell RNA sequencing data and confirm the crucial pathophysiological role of PECs in murine kidney biology as a reservoir for different types of progenitors.

Polarity in respiratory development, homeostasis and disease.

The respiratory system is composed of a multitude of cells that organize to form complex branched airways that end in alveoli, which respectively function to guide air flow and mediate gas exchange with the bloodstream. The organization of the respiratory sytem relies on distinct forms of cell polarity, which guide lung morphogenesis and patterning in development and provide homeostatic barrier protection from microbes and toxins. The stability of lung alveoli, the luminal secretion of surfactants and mucus in the airways, and the coordinated motion of multiciliated cells that generate proximal fluid flow, are all critical functions regulated by cell polarity, with defects in polarity contributing to respiratory disease etiology. Here, we summarize the current knowledge of cell polarity in lung development and homeostasis, highlighting key roles for polarity in alveolar and airway epithelial function and outlining relationships with microbial infections and diseases, such as cancer.

Tissue memory relies on stem cell priming in distal undamaged areas.

Epithelial cells that participated in wound repair elicit a more efficient response to future injuries, which is believed to be locally restricted. Here we show that cell adaptation resulting from a localized tissue damage has a wide spatial impact at a scale not previously appreciated. We demonstrate that a specific stem cell population, distant from the original injury, originates long-lasting wound memory progenitors residing in their own niche. Notably, these distal memory cells have not taken part in the first healing but become intrinsically pre-activated through priming. This cell state, maintained at the chromatin and transcriptional level, leads to an enhanced wound repair that is partially recapitulated through epigenetic perturbation. Importantly wound memory has long-term harmful consequences, exacerbating tumourigenesis. Overall, we show that sub-organ-scale adaptation to injury relies on spatially organized memory-dedicated progenitors, characterized by an actionable cell state that establishes an epigenetic field cancerization and predisposes to tumour onset.

Protein Expression Profiles in Exosomes of Bovine Mammary Epithelial Cell Line MAC-T Infected with Staphylococcus aureus.

Mastitis is a common and widespread infectious disease in dairy farms around the world, resulting in reduced milk production and quality. Staphylococcus aureus is one of the main pathogenic bacteria causing subclinical mastitis in dairy cows. S. aureus can activate inflammatory signaling pathways in bovine mammary epithelial cells. Exosomes produced by cells can directly transfer pathogen-related molecules from cell to cell, thus affecting the process of infection. Protein is the material basis of the immune defense function in the body; therefore, a comprehensive comparison of proteins in exosomes derived from S. aureus-infected (SA group) and normal (control group [C group]) bovine mammary epithelial MAC-T cells was performed using shotgun proteomics by a DIA approach. A total of 7,070 proteins were identified and quantified. Compared with the C group, there were 802 differentially expressed proteins (DEPs) identified in the SA group (absolute log2 fold change [|log2FC|] of ≥0.58; false discovery rate [FDR] of <0.05), among which 325 proteins were upregulated and 477 were downregulated. The upregulated proteins, including complement 3 (C3), integrin alpha-6 (ITGA6), apolipoprotein A1 (APOA1), annexin A2 (ANXA2), tripeptidyl peptidase II (TPP2), keratin 8 (KRT8), and recombinant desmoyokin (AHNAK), are involved mostly in host defense against pathogens, inflammation, and cell structure maintenance. KEGG enrichment analysis indicated that DEPs in S. aureus infection were involved in the complement and coagulation cascade, phagosome, extracellular matrix (ECM)-receptor interaction, and focal adhesion pathways. The results of this study provide novel information about proteins in the exosomes of MAC-T cells infected with S. aureus and could contribute to an understanding of the infectious mechanism of bovine mastitis. IMPORTANCE Mastitis is a widespread infectious disease in dairy farms, resulting in reduced milk production and quality. Staphylococcus aureus is one of the main pathogenic bacteria causing subclinical mastitis. Exosomes contain proteins, lipids, and nucleic acids, which are involved in many physiological and pathological functions. The expression of proteins in exosomes derived from bovine mammary epithelial cells infected by S. aureus is still barely understood. These results provide novel information about MAC-T-derived exosomal proteins, reveal insights into their functions, and lay a foundation for further studying the biological function of exosomes during the inflammatory response.

Organ-on-a-Chip Platform with an Integrated Screen-Printed Electrode Array for Real-Time Monitoring Trans-Epithelial Barrier and Bubble Formation.

Cellular tight junctions play a key role in establishing a barrier between different compartments of the body by regulating the selective passage of different solutes across epithelial and endothelial tissues. Over the past decade, significant efforts have been conducted to develop more clinically relevant "organ-on-a-chip" models with integrated trans-epithelial electrical resistance (TEER) monitoring systems to help better understand the fundamental underpinnings of epithelial tissue physiology upon exposure to different substances. However, most of these platforms require the use of high-cost and time-consuming photolithography processes, which limits their scalability and practical implementation in clinical research. To address this need, we have developed a low-cost microfluidic platform with an integrated electrode array that allows continuous real-time monitoring of TEER and the risk of bubble formation in the microfluidic system by using scalable manufacturing technologies such as screen printing and laser processing. The integrated printed electrode array exhibited excellent stability (with less than ∼0.02 Ω change in resistance) even after long-term exposure to a complex culture medium. As a proof of concept, the fully integrated platform was tested with HMT3522 S1 epithelial cells to evaluate the tight barrier junction formation through TEER measurement and validated with standard immunostaining procedures for Zonula occludens-1 protein. This platform could be regarded as a stepping stone for the fabrication of disposable and low-cost organ and tissue-on-a-chip models with integrated sensors to facilitate studying the dynamic response of epithelial tissues to different substances in more physiologically relevant conditions.