Particulate matter 10 exposure affects intestinal functionality in both inflamed 2D intestinal epithelial cell and 3D intestinal organoid models.

Background: A growing body of evidence suggests that particulate matter (PM10) enters the gastrointestinal (GI) tract directly, causing the GI epithelial cells to function less efficiently, leading to inflammation and an imbalance in the gut microbiome. PM10 may, however, act as an exacerbation factor in patients with inflamed intestinal epithelium, which is associated with inflammatory bowel disease. Objective: The purpose of this study was to dissect the pathology mechanism of PM10 exposure in inflamed intestines. Methods: In this study, we established chronically inflamed intestinal epithelium models utilizing two-dimensional (2D) human intestinal epithelial cells (hIECs) and 3D human intestinal organoids (hIOs), which mimic in vivo cellular diversity and function, in order to examine the deleterious effects of PM10 in human intestine-like in vitro models. Results: Inflamed 2D hIECs and 3D hIOs exhibited pathological features, such as inflammation, decreased intestinal markers, and defective epithelial barrier function. In addition, we found that PM10 exposure induced a more severe disturbance of peptide uptake in inflamed 2D hIECs and 3D hIOs than in control cells. This was due to the fact that it interferes with calcium signaling, protein digestion, and absorption pathways. The findings demonstrate that PM10-induced epithelial alterations contribute to the exacerbation of inflammatory disorders caused by the intestine. Conclusions: According to our findings, 2D hIEC and 3D hIO models could be powerful in vitro platforms for the evaluation of the causal relationship between PM exposure and abnormal human intestinal functions.

Links of Cytoskeletal Integrity with Disease and Aging.

Aging is a complex feature and involves loss of multiple functions and nonreversible phenotypes. However, several studies suggest it is possible to protect against aging and promote rejuvenation. Aging is associated with many factors, such as telomere shortening, DNA damage, mitochondrial dysfunction, and loss of homeostasis. The integrity of the cytoskeleton is associated with several cellular functions, such as migration, proliferation, degeneration, and mitochondrial bioenergy production, and chronic disorders, including neuronal degeneration and premature aging. Cytoskeletal integrity is closely related with several functional activities of cells, such as aging, proliferation, degeneration, and mitochondrial bioenergy production. Therefore, regulation of cytoskeletal integrity may be useful to elicit antiaging effects and to treat degenerative diseases, such as dementia. The actin cytoskeleton is dynamic because its assembly and disassembly change depending on the cellular status. Aged cells exhibit loss of cytoskeletal stability and decline in functional activities linked to longevity. Several studies reported that improvement of cytoskeletal stability can recover functional activities. In particular, microtubule stabilizers can be used to treat dementia. Furthermore, studies of the quality of aged oocytes and embryos revealed a relationship between cytoskeletal integrity and mitochondrial activity. This review summarizes the links of cytoskeletal properties with aging and degenerative diseases and how cytoskeletal integrity can be modulated to elicit antiaging and therapeutic effects.

Limosilactobacillus reuteri DS0384 promotes intestinal epithelial maturation via the postbiotic effect in human intestinal organoids and infant mice.

Little is known about the modulatory capacity of the microbiota in early intestinal development. We examined various intestinal models that respond to gut microbial metabolites based on human pluripotent stem cell-derived human intestinal organoids (hIOs): physiologically relevant in vitro fetal-like intestine, intestinal stem cell, and intestinal disease models. We found that a newly isolated Limosilactobacillus reuteri strain DS0384 accelerated maturation of the fetal intestine using 3D hIO with immature fetal characteristics. Comparative metabolomic profiling analysis revealed that the secreted metabolite N-carbamyl glutamic acid (NCG) is involved in the beneficial effect of DS0384 cell-free supernatants on the intestinal maturation of hIOs. Experiments in an intestinal stem cell spheroid model and hIO-based intestinal inflamed model revealed that the cell-free supernatant from DS0384 comprising NCG promoted intestinal stem cell proliferation and was important for intestinal protection against cytokine-induced intestinal epithelial injury. The probiotic properties of DS0384 were also evaluated, including acid and bile tolerance and ability to adhere to human intestinal cells. Seven-day oral administration of DS0384 and cell-free supernatant promoted the intestinal development of newborn mice. Moreover, NCG exerted a protective effect on experimental colitis in mice. These results suggest that DS0384 is a useful agent for probiotic applications and therapeutic treatment for disorders of early gut development and for preventing intestinal barrier dysfunction.

Generation of Highly Expandable Intestinal Spheroids Composed of Stem Cells.

Many of early findings regarding intestinal stem cells (ISCs) and their niche in the human intestine have relied on colorectal cancer cell lines and labor-intensive and time-consuming mouse models. However, these models cannot accurately recapitulate the physiologically relevant aspects of human ISCs. In this study, we demonstrate a reliable and robust culture method for 3D expanding intestinal spheroids (InSexp) mainly comprising ISCs and progenitors, which can be derived from 3D human intestinal organoids (HIOs). We did functional chararcterization of InSexp derived from 3D HIOs, differentiated from human pluripotent stem cells, and optimization culture methods. Our results indicate that InSexp can be rapidly expanded and easily passaged, and show enhanced growth rates via WNT pathway activation. InSexp are capable of exponential cell expansion and cryopreservation. Furthermore, in vitro-matured HIO-derived InSexp proliferate faster than immature HIO-derived InSexp with preservation of the parental HIO characteristics. These findings may facilitate the development of scalable culture systems for the long-term maintenance of human ISCs and provide an alternative platform for studying ISC biology.

Microtubule Integrity Is Associated with the Functional Activity of Mitochondria in HEK293.

Mitochondria move along the microtubule network and produce bioenergy in the cell. However, there is no report of a relationship between bioenergetic activity of mitochondria and microtubule stability in mammalian cells. This study aimed to investigate this relationship. We treated HEK293 cells with microtubule stabilizers (Taxol and Epothilone D) or a microtubule disturber (vinorelbine), and performed live-cell imaging to determine whether mitochondrial morphology and bioenergetic activity depend on the microtubule status. Treatment with microtubule stabilizers enhanced the staining intensity of microtubules, significantly increased ATP production and the spare respiratory capacity, dramatically increased mitochondrial fusion, and promoted dynamic movement of mitochondria. By contrast, bioenergetic activity of mitochondria was significantly decreased in cells treated with the microtubule disturber. Our data suggest that microtubule stability promotes mitochondrial functional activity. In conclusion, a microtubule stabilizer can possibly recover mitochondrial functional activity in cells with unstable microtubules.

Truncated Neogenin Promotes Hippocampal Neuronal Death after Acute Seizure.

Protecting hippocampal neurons from death after seizure activity is critical to prevent an alteration of neuronal circuitry and hippocampal function. Here, we present a novel target, a truncated form of neogenin that is associated with seizure-induced hippocampal necroptosis, and novel use of the γ-secretase inhibitor N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester (DAPT) as a pharmacological regulator of neogenin truncation. We show that 3 days after pilocarpine-induced status epilepticus in mice, when hippocampal cell death is detected, the level of truncated neogenin is increased, while that of full-length neogenin is decreased. Moreover, phosphorylation of mixed lineage kinase domain-like pseudokinase, a crucial marker of necroptosis, was also markedly upregulated at 3 days post-status epilepticus. In cultured hippocampal cells, kainic acid treatment significantly reduced the expression of full-length neogenin. Notably, treatment with DAPT prevented neogenin truncation and protected cultured neurons from N-methyl-D-aspartate (NMDA)-induced death. These data suggest that seizure-induced hippocampal necroptosis is associated with the generation of truncated neogenin, and that prevention of this by DAPT treatment can protect against NMDA-induced excitotoxicity.

MIT-001 Restores Human Placenta-Derived Mesenchymal Stem Cells by Enhancing Mitochondrial Quiescence and Cytoskeletal Organization.

Inflammation is a major cause of several chronic diseases and is reported to be recovered by the immuno-modulation of mesenchymal stem cells (MSCs). While most studies have focussed on the anti-inflammatory roles of MSCs in stem cell therapy, the impaired features of MSCs, such as the loss of homeostasis by systemic aging or pathologic conditions, remain incompletely understood. In this study, we investigated whether the altered phenotypes of human placenta-derived MSCs (hPD-MSCs) exposed to inflammatory cytokines, including TNF-α and IFN-γ, could be protected by MIT-001, a small anti-inflammatory and anti-necrotic molecule. MIT-001 promoted the spindle-like shape and cytoskeletal organization extending across the long cell axis, whereas hPD-MSCs exposed to TNF-α/IFN-γ exhibited increased morphological heterogeneity with an abnormal cell shape and cytoskeletal disorganization. Importantly, MIT-001 improved mitochondrial distribution across the cytoplasm. MIT-001 significantly reduced basal respiration, ATP production, and cellular ROS levels and augmented the spare respiratory capacity compared to TNF-α/IFN-γ-exposed hPD-MSCs, indicating enhanced mitochondrial quiescence and homeostasis. In conclusion, while TNF-α/IFN-γ-exposed MSCs lost homeostasis and mitochondrial quiescence by becoming over-activated in response to inflammatory cytokines, MIT-001 was able to rescue mitochondrial features and cellular phenotypes. Therefore, MIT-001 has therapeutic potential for clinical applications to treat mitochondrion-related inflammatory diseases.

The mitochondrial-derived peptide MOTS-c promotes homeostasis in aged human placenta-derived mesenchymal stem cells in vitro.

Mesenchymal stem cells (MSCs) are multipotent cells with critical roles in homeostasis and regeneration. MSCs undergo aging in response to various stresses, and this causes many diseases including degenerative disorders. Thus, regulation of aging factors is crucial for healthy aging. Mitochondrial open reading frame of the 12S rRNA-c (MOTS-c) was recently reported to regulate metabolic homeostasis. Here, we investigated the restorative effects of MOTS-c on aged human placenta-derived MSCs (hPD-MSCs). MOTS-c promoted the morphology of old hPD-MSCs. MOTS-c significantly activated AMP-activated protein kinase, which is the main target pathway of MOTS-c, and inhibited its antagonistic effector mTORC1. MOTS-c considerably enhanced mitochondrial homeostasis by decreasing oxygen consumption and reactive oxygen species production. The mitochondrial state of MOTS-c-treated old hPD-MSCs was more similar to that of young hPD-MSCs than the mitochondrial state of non-treated old hPD-MSCs. MOTS-c also decreased lipid synthesis. In conclusion, we demonstrated that MOTS-c promotes homeostasis in aged hPD-MSCs.