Saffron (Crocus sativus L.) Flower Water Extract Disrupts the Cecal Microbiome, Brush Border Membrane Functionality, and Morphology In Vivo (Gallus gallus)

Saffron (Crocus sativus L.) is known as the most expensive spice. C. sativus dried red stigmas, called threads, are used for culinary, cosmetic, and medicinal purposes. The rest of the flower is often discarded, but is now being used in teas, as coloring agents, and fodder. Previous studies have attributed antioxidant, anti-inflammatory, hepatoprotective, neuroprotective, anti-depressant, and anticancer properties to C. sativus floral bio-residues. The aim of this study is to assess C. sativus flower water extract (CFWE) for its effects on hemoglobin, brush boarder membrane (BBM) functionality, morphology, intestinal gene expression, and cecal microbiome in vivo (Gallus gallus), a clinically validated model. For this, Gallus gallus eggs were divided into six treatment groups (non-injected, 18 Ω H2O, 1% CFWE, 2% CFWE, 5% CFWE, and 10% CFWE) with n~10 for each group. On day 17 of incubation, 1 mL of the extracts/control were administered in the amnion of the eggs. The amniotic fluid along with the administered extracts are orally consumed by the developing embryo over the course of the next few days. On day 21, the hatchlings were euthanized, the blood, duodenum, and cecum were harvested for assessment. The results showed a significant dose-dependent decrease in hemoglobin concentration, villus surface area, goblet cell number, and diameter. Furthermore, we observed a significant increase in Paneth cell number and Mucin 2 (MUC2) gene expression proportional to the increase in CFWE concentration. Additionally, the cecum microbiome analysis revealed C. sativus flower water extract altered the bacterial populations. There was a significant dose-dependent reduction in Lactobacillus and Clostridium sp., suggesting an antibacterial effect of the extract on the gut in the given model. These results suggest that the dietary consumption of C. sativus flower may have negative effects on BBM functionality, morphology, mineral absorption, microbial populations, and iron status.

Aging-Related Impairments to M Cells in Peyer’s Patches Coincide With Disturbances to Paneth Cells

The decline in mucosal immunity during aging increases susceptibility, morbidity and mortality to infections acquired via the gastrointestinal and respiratory tracts in the elderly. We previously showed that this immunosenescence includes a reduction in the functional maturation of M cells in the follicle-associated epithelia (FAE) covering the Peyer’s patches, diminishing the ability to sample of antigens and pathogens from the gut lumen. Here, co-expression analysis of mRNA-seq data sets revealed a general down-regulation of most FAE- and M cell-related genes in Peyer’s patches from aged mice, including key transcription factors known to be essential for M cell differentiation. Conversely, expression of ACE2, the cellular receptor for SARS-Cov-2 virus, was increased in the aged FAE. This raises the possibility that the susceptibility of aged Peyer’s patches to infection with the SARS-Cov-2 virus is increased. Expression of key Paneth cell-related genes was also reduced in the ileum of aged mice, consistent with the adverse effects of aging on their function. However, the increased expression of these genes in the villous epithelium of aged mice suggested a disturbed distribution of Paneth cells in the aged intestine. Aging effects on Paneth cells negatively impact on the regenerative ability of the gut epithelium and could indirectly impede M cell differentiation. Thus, restoring Paneth cell function may represent a novel means to improve M cell differentiation in the aging intestine and increase mucosal vaccination efficacy in the elderly.

Intracellular pH dynamics regulates intestinal stem cell fate

Emerging evidence is revealing critical roles of intracellular pH (pHi) in development, but it remains unclear whether pHi regulates stem cell fate specification. We find that pHi dynamics is a key regulator of cell fate in the mouse intestinal stem cell lineage. We identify a pHi gradient along the intestinal crypt axis and find that dissipating this gradient inhibits crypt budding due to loss Paneth cell differentiation. Mechanistically, decreasing pHi biases intestinal stem cell fate toward the absorptive and away from the secretory lineage, by regulating the activity of the lineage transcription factor Atoh1. Our findings reveal a previously unrecognized role for pHi dynamics in the specification of cell fate within an adult stem cell lineage.

Multi-omic analysis defines the first microRNA atlas across all small intestinal epithelial lineages and reveals novel markers of almost all major cell types

MicroRNA-mediated regulation is critical for the proper development and function of the small intestinal (SI) epithelium. However, it is not known which microRNAs are expressed in each of the cell types of the SI epithelium. To bridge this important knowledge gap, we performed comprehensive microRNA profiling in all major cell types of the mouse SI epithelium. We used flow cytometry and fluorescence-activated cell sorting with multiple reporter mouse models to isolate intestinal stem cells, enterocytes, goblet cells, Paneth cells, enteroendocrine cells, tuft cells and secretory progenitors. We then subjected these cell populations to small RNA-sequencing. The resulting atlas revealed highly enriched microRNA markers for almost every major cell type (https://sethupathy-lab.shinyapps.io/SI_miRNA/). Several of these lineage-enriched microRNAs (LEMs) were observed to be embedded in annotated host genes. We used chromatin-run-on sequencing to determine which of these LEMs are likely co-transcribed with their host genes. We then performed single-cell RNA-sequencing to define the cell type specificity of the host genes and embedded LEMs. We observed that the two most-enriched microRNAs in secretory progenitors are miR-1224 and miR-672, the latter of which we found is deleted in hominin species. Finally, using several in vivo models, we established that miR-152 is a Paneth cell-specific microRNA.

IFN-γ mediates Paneth cell death via suppression of mTOR

Paneth cells constitutively produce antimicrobial peptides and growth factors that allow for intestinal homeostasis, host protection and intestinal stem cell replication. Paneth cells rely heavily on the glycolytic metabolic program, which is in part controlled by the kinase complex Mechanistic target of rapamycin (mTORC1). Yet, little is known about mTOR importance in Paneth cell integrity under steady state and inflammatory conditions. Our results demonstrate that IFN-γ, a crucial mediator of the intestinal inflammation, acts directly on murine Paneth cells to alter their mitochondrial integrity and membrane potential, resulting in an mTORC1-dependent cell death mechanism distinct from canonical cell death pathways including apoptosis, necroptosis, and pyroptosis. These results were established with the purified cytokine and a physiologically relevant common Th1-inducing human parasite Toxoplasma gondii. Given the crucial role for IFN-γ, which is a cytokine frequently associated with the development of inflammatory bowel disease (IBD) and compromised Paneth cell functions, the identified mechanisms underlying mTORC1-dependent Paneth cell death downstream of IFN-γ may provide promising novel approaches for treating intestinal inflammation.

The impact of di-isononyl phthalate exposure on specialized epithelial cells in the colon

Di-isononyl phthalate (DiNP) is a high-molecular-weight phthalate commonly used as a plasticizer for polyvinyl chloride and other end products, such as medical devices and construction materials. Most of our initial exposure to DiNP occurs by ingestion of DiNP-contaminated foods. However, little is known about the effects of DiNP on the colon. Therefore, the goal of this study was to test the hypothesis that DiNP exposure alters immune responses and impacts specialized epithelial cells in the colon. To test this hypothesis, adult female mice were orally dosed with corn-oil vehicle control or doses of DiNP ranging from 20 µg/kg/d to 200 mg/kg/d for 10-14 days. After the dosing period, mice were euthanized in diestrus, and colon tissues and sera were collected for histological, genomic, and proteomic analysis of various immune factors and specialized epithelial cells. Subacute exposure to DiNP significantly increased protein levels of Ki67 and MUC2, expression of a Paneth cell marker (Lyz1), and estradiol levels in sera compared to control. Gene expression of mucins (Muc1, Muc2, Muc3a, and Muc4), Toll-like receptors (Tlr4 and Tlr5), and specialized epithelial cells (ChgA, Lgr5, Cd24a, and Vil1) were not significantly different between treatment groups and control. Cytokine levels of IL-1RA and CXCL12 were also not significantly different between DiNP treatment groups and control. These data reveal that DiNP exposure increases circulating estradiol levels and gene expression in specialized epithelial cells with immune response capabilities (e.g., goblet and Paneth cells) in the mouse colon, which may initiate immune responses to prevent further damage in the colon.

Regulation of Paneth Cell Function by RNA-Binding Proteins and Noncoding RNAs

Paneth cells are specialized intestinal epithelial cells that are located at the base of small intestinal crypts and play a vital role in preserving the gut epithelium homeostasis. Paneth cells act as a safeguard from bacterial translocation across the epithelium and constitute the niche for intestinal stem cells in the small intestine by providing multiple niche signals. Recently, Paneth cells have become the focal point of investigations defining the mechanisms underlying the epithelium-microbiome interactions and pathogenesis of chronic gut mucosal inflammation and bacterial infection. Function of Paneth cells is tightly regulated by numerous factors at different levels, while Paneth cell defects have been widely documented in various gut mucosal diseases in humans. The post-transcription events, specific change in mRNA stability and translation by RNA-binding proteins (RBPs) and noncoding RNAs (ncRNAs) are implicated in many aspects of gut mucosal physiology by modulating Paneth cell function. Deregulation of RBPs and ncRNAs and subsequent Paneth cell defects are identified as crucial elements of gut mucosal pathologies. Here, we overview the posttranscriptional regulation of Paneth cells by RBPs and ncRNAs, with a particular focus on the increasing evidence of RBP HuR and long ncRNA H19 in this process. We also discuss the involvement of Paneth cell dysfunction in altered susceptibility of the intestinal epithelium to chronic inflammation and bacterial infection following disrupted expression of HuR and H19.

Evaluation of Antioxidant Prophylactic Effects of Parsley (Petroselinum Crispum) on Sepsis Following Cecal Ligation and Puncture

Background and Aims. Sepsis causes release of free oxygen radicals that destroy membrane integrity.We evaluated the antioxidant effects of parsley, Petroselinum crispum(Pc), which has been used therapeutically for centuries in Anatolia, using a rat model of sepsis caused by cecal ligation and puncture (CLP).Methods.Wistar albino rats were separated into four groups of eight: a sham group with incised and sutured abdomen, a Pc extract(PcE) group was given 2 g/kg parsley extract for 14 days by gastric gavage, a CLP group with sepsis caused by CLP and a PcE + CLP group given parsley extract for 14 days, then made septic by CLP. PcE is given for 14 days and then sepsis is performed by the CLP procedure. Results. The groups were compared in terms of hemogram, biochemical and histological characteristics. It has been proven that the administration of PcE before CLP-induced sepsis increases neutrophil, PLT counts and TAS levels which decrease with sepsis and decreases biochemical changes (BUN, AST, ALT, LDH, TOS, OSI) which increase with sepsis, to have a protective effect on sepsis . In the PcE + CLP group, the severity of intestinal infiltration was decreased significantly compared to the CLP group; epithelial damage was similar to the CLP group. In the PcE + CLP group, the crypt and villus length was greater and the decrease in Paneth cell degranulation intensity was found to be more than for the CLP group ; also, the morphology of the cells was similar to the Sham group. Discussion. PcE exhibit potential for prophylaxis for sepsis.

Primary Prostatic Carcinoma With De Novo Diffuse Neuroendocrine Differentiation

The 2016 World Health Organization classification of prostate cancer with neuroendocrine (NE) differentiation includes NE cells in usual prostate cancer, adenocarcinoma with Paneth cell-like NE differentiation, well-differentiated NE tumor (carcinoid), small cell NE carcinoma, and large cell NE carcinoma. In this article, we report a rare case of primary prostatic carcinoma with de novo diffuse NE differentiation presenting with bilateral hydronephrosis in a 79-year-old man. This case did not fit into any of the existing classifications. The clinical, radiological, morphological, and immunohistochemical findings and response to androgen deprivation therapy (ADT) are presented. The proposed pathogenesis of NE differentiation via transdifferentiation from conventional prostatic adenocarcinoma whereby genomic alterations, coupled with ADT can induce lineage plasticity resulting in NE differentiation is described.