Intestinal Epithelial Cells Research Articles

Ginseng-DF ameliorates intestinal mucosal barrier injury and enhances immunity in immunosuppressed mice by regulating MAPK/NF-κB signaling pathways.

Dietary fiber (DF) has a good application prospect in effectively restoring the integrity of the intestinal mucosal barrier. Ginseng-DF has good physicochemical properties and physiological activity and shows positive effects in enhancing immunity. The aim of this study was to investigate the protective effect of Ginseng-DF on intestinal mucosal barrier injury induced by cyclophosphamide (CTX) in immunosuppressed mice and its possible mechanism. The effects of Gginseng-DF on immune function in mice were studied by delayed-type hypersensitivy, lymphocyte proliferation assay and NK cytotoxicity assay, the T lymphocyte differentiation and intestinal barrier integrity were analyzed by flow cytometry and western blot. Ginseng-DF (2.5% and 5%) could attenuate the inhibition of DTH response by CTX, promote the transformation and proliferation of lymphocytes, and stimulate NK effector cell activity. At the same time, Ginseng-DF could restore the proportion of CD4+/CD8+ T lymphocytes induced by CTX to different extents, improved spleen tissue damage, promoted the secretion of immunoglobulin IgG, and enhanced body immunity. More importantly, Ginseng-DF could up-regulate the contents of TNF-α, IFN-γ, IL-6 and IL-1β in serum and intestine of immunosuppressed mice to maintain the balance between Th1/Th2 cytokines, and improve the permeability of intestinal mucosal barrier. Meanwhile, Ginseng-DF could reduce intestinal epithelial cell apoptosis and improve intestinal adaptive immunity in CTX-induced immunosuppressed mice by regulating MAPK/NF-κB signaling pathway. Ginseng-DF can be used as a safe dietary supplement to enhance body immunity and reduce intestinal mucosal injury caused by CTX.

CCR2/CCR5 antagonist cenicriviroc reduces colonic inflammation and fibrosis in experimental colitis.

Cenicriviroc (CVC) is a CCR2/CCR5 antagonist that has been shown to be effective in the treatment of inflammatory and fibrotic diseases. Our study evaluated its efficacy in colitis. Mouse models of DSS-induced acute and chronic colitis were established. The efficacy of CVC in colitis was assessed by disease activity index (DAI) scores, histological assessment of inflammation and fibrosis, and expression assays of key molecules. In in vitro experiments, HT29 cell line was exposed to TNFα to study inflammatory signaling in intestinal epithelial cells. CCD-18Co colonic myofibroblasts and human primary colonic fibroblasts were activated by TGFβ1 to mimic fibroblast activation. In HT29 cells, CVC significantly reduced mRNA expression of CCL5 (P<0.01) but had no effect on CCL2. Furthermore, CVC reduced downstream CX3CL1 (P<0.01) and TNFα (P<0.05) expression, thereby inhibiting inflammatory progression. In acute colitis mice, CVC significantly reduced DAI scores and serum TNFα levels (P<0.05) and attenuated colonic inflammation as shown by HE staining. Meanwhile, CVC had no adverse effects on the liver, heart, and kidney of mice. On the other hand, in cellular models of chronic colitis, CVC decreased the expression of fibrosis markers, including FN, CTGF, α-SMA, and MMP9, and inhibited TGFβ1-induced fibrotic activation (P<0.01). In addition, CVC attenuated colonic fibrosis in chronic colitis mice. Moreover, CVC significantly promoted autophagy, which contributed to its regulation of inflammation. CVC significantly inhibited inflammation through CCL5/CCR5 signaling without damaging vital organs and suppressed fibrotic activation in chronic colitis, suggesting its great potential to relieve colonic inflammation and fibrosis.

HO-1-induced autophagy establishes a HO-1-p62-Nrf2 positive feedback loop to reduce gut permeability in cholestatic liver disease.

The gut-liver axis disruption is a unified pathogenetic principle of cholestatic liver disease (CSLD). Increased gut permeability is the leading cause of gut-liver axis disruption. HO-1 is capable of protecting against gut-liver axis injury. However, it has rarely been reported whether autophagy is involved in HO-1 protecting gut-liver barrier integrity and the underlying mechanism. Mice underwent bile duct ligation (BDL) was established as CSLD model in vivo. Caco-2 cells with LPS treatment was established as in vitro cell model. Immunofluorescence, western blot and transepithelial electrical resistance (TER) assay were used to observe epithelial tight junction (TJ) and autophagy. Liver injury and fibrosis were evaluated as well through H&E staining, masson staining, sirius red staining and ELISA. Our study demonstrated that the epithelial TJ and TER were notably reduced both in BDL mice and in LPS treated intestinal epithelial cells. Increased HO-1 expression could significantly induce intestinal epithelial cell autophagy. Additionally, this increased autophagy level reversed the reduction effects of BDL or LPS on epithelial TJ and TER in vivo and in vitro, therefore decreased transaminase level in serum and relieved liver fibrosis in BDL mice. Besides, increased autophagy level in turn upregulated the expression of HO-1 by p62 degradation of Keap1 and subsequent activation of Nrf2 pathway. Collectively, these results indicate that HO-1 reduces gut permeability by enhancing autophagy level in CSLD, the increased autophagy establishes a HO-1-p62-Nrf2 positive feedback loop to further improve gut-liver axis disruption. Therefore, our study confirms the critical role of autophagy in HO-1 ameliorating gut-liver axis injury during CSLD, highlighting HO-1 as a promising therapeutic target.

Deciphering infected cell types, hub gene networks and cell-cell communication in infectious bronchitis virus via single-cell RNA sequencing.

Infectious bronchitis virus (IBV) is a coronavirus that infects chickens, which exhibits a broad tropism for epithelial cells, infecting the tracheal mucosal epithelium, intestinal mucosal epithelium, and renal tubular epithelial cells. Utilizing single-cell RNA sequencing (scRNA-seq), we systematically examined cells in renal, bursal, and tracheal tissues following IBV infection and identified tissue-specific molecular markers expressed in distinct cell types. We evaluated the expression of viral RNA in diverse cellular populations and subsequently ascertained that distal tubules and collecting ducts within the kidney, bursal mucosal epithelial cells, and follicle-associated epithelial cells exhibit susceptibility to IBV infection through immunofluorescence. Furthermore, our findings revealed an upregulation in the transcription of proinflammatory cytokines IL18 and IL1B in renal macrophages as well as increased expression of apoptosis-related gene STAT in distal tubules and collecting duct cells upon IBV infection leading to renal damage. Cell-to-cell communication unveiled potential interactions between diverse cell types, as well as upregulated signaling pathways and key sender-receiver cell populations after IBV infection. Integrating single-cell data from all tissues, we applied weighted gene co-expression network analysis (WGCNA) to identify gene modules that are specifically expressed in different cell populations. Based on the WGCNA results, we identified seven immune-related gene modules and determined the differential expression pattern of module genes, as well as the hub genes within these modules. Our comprehensive data provides valuable insights into the pathogenesis of IBV as well as avian antiviral immunology.

Modulation of Serotonin-Related Genes by Extracellular Vesicles of the Probiotic Escherichia coli Nissle 1917 in the Interleukin-1β-Induced Inflammation Model of Intestinal Epithelial Cells

Modulation of Serotonin-Related Genes by Extracellular Vesicles of the Probiotic Escherichia coli Nissle 1917 in the Interleukin-1β-Induced Inflammation Model of Intestinal Epithelial Cells

Studying the cellular basis of small bowel enteropathy using high-parameter flow cytometry in mouse models of primary antibody deficiency

BackgroundPrimary immunodeficiencies are heritable defects in immune system function. Antibody deficiency is the most common form of primary immunodeficiency in humans, can be caused by abnormalities in both the development and activation of B cells, and may result from B-cell-intrinsic defects or defective responses by other cells relevant to humoral immunity. Inflammatory gastrointestinal complications are commonly observed in antibody-deficient patients, but the underlying immune mechanisms driving this are largely undefined.MethodsIn this study, several mouse strains reflecting a spectrum of primary antibody deficiency (IgA-/-, Aicda-/-, CD19-/- and JH-/-) were used to generate a functional small-bowel-specific cellular atlas using a novel high-parameter flow cytometry approach that allows for the enumeration of 59 unique cell subsets. Using this cellular atlas, we generated a direct and quantifiable estimate of immune dysregulation. This estimate was then used to identify specific immune factors most predictive of the severity of inflammatory disease of the small bowel (small bowel enteropathy).ResultsResults from our experiments indicate that the severity of primary antibody deficiency positively correlates with the degree of immune dysregulation that can be expected to develop in an individual. In the SI of mice, immune dysregulation is primarily explained by defective homeostatic responses in T cell and invariant natural killer-like T (iNKT) cell subsets. These defects are strongly correlated with abnormalities in the balance between protein (MHCII-mediated) versus lipid (CD1d-mediated) antigen presentation by intestinal epithelial cells (IECs) and intestinal stem cells (ISCs), respectively.ConclusionsMultivariate statistical approaches can be used to obtain quantifiable estimates of immune dysregulation based on high-parameter flow cytometry readouts of immune function. Using one such estimate, we reveal a previously unrecognized tradeoff between iNKT cell activation and type 1 immunity that underlies disease in the small bowel. The balance between protein/lipid antigen presentation by ISCs may play a crucial role in regulating this balance and thereby suppressing inflammatory disease in the small bowel.

Mitochondrial function and gastrointestinal diseases.

Mitochondria are dynamic organelles that function in cellular energy metabolism, intracellular and extracellular signalling, cellular fate and stress responses. Mitochondria of the intestinal epithelium, the cellular interface between self and enteric microbiota, have emerged as crucial in intestinal health. Mitochondrial dysfunction occurs in gastrointestinal diseases, including inflammatory bowel diseases and colorectal cancer. In this Review, we provide an overview of the current understanding of intestinal epithelial cell mitochondrial metabolism, function and signalling to affect tissue homeostasis, including gut microbiota composition. We also discuss mitochondrial-targeted therapeutics for inflammatory bowel diseases and colorectal cancer and the evolving concept of mitochondrial impairment as a consequence versus initiator of the disease.

Impact of PEDV infection on the biological characteristics of porcine intestinal exosomes

Porcine epidemic diarrhea (PED) is a highly contagious intestinal infection primarily affecting pigs. It is caused by the porcine epidemic diarrhea virus (PEDV). PEDV targets the villus tissue cells in the small intestine and mesenteric lymph nodes, resulting in shortened intestinal villi and, in extreme cases, causing necrosis of the intestinal lining. Moreover, PEDV infection can disrupt the balance of the intestinal microflora, leading to an overgrowth of harmful bacteria like Escherichia coli. Exosomes, tiny membrane vesicles ranging from 30 to 150 nm in size, contain a complex mixture of RNA and proteins. MicroRNA (miRNA) regulates various cell signaling, development, and disease progression processes. This study extracted exosomes from both groups and performed high-throughput miRNA sequencing and bioinformatics techniques to investigate differences in miRNA expression within exosomes isolated from PEDV-infected porcine small intestine tissue compared to healthy controls. Notably, two miRNA types displayed upregulation in infected exosomes, while 12 exhibited downregulation. These findings unveil abnormal miRNA regulation patterns in PEDV-infected intestinal exosomes, shedding light on the intricate interplay between PEDV and its host. This will enable further exploration of the relationship between these miRNA changes and signaling pathways, enlightening PEDV pathogenesis and potential therapeutic targets.

Alternariol Monomethyl-Ether Induces Toxicity via Cell Death and Oxidative Stress in Swine Intestinal Epithelial Cells

Alternariol monomethyl-ether (AME), together with altenuene and alternariol, belongs to the Alternaria mycotoxins group, which can contaminate different substrates, including cereals. The aim of the present study was to obtain a deeper understanding concerning the effects of AME on pig intestinal health using epithelial intestinal cell lines as the data concerning the possible effects of Alternaria toxins on swine are scarce and insufficient for assessing the risk represented by Alternaria toxins for animal health. Our results have shown a dose-related effect on IPEC-1 cell viability, with an IC50 value of 10.5 μM. Exposure to the toxin induced an increase in total apoptotic cells, suggesting that AME induces programmed cell death through apoptosis based on caspase-3/7 activation in IPEC-1 cells. DNA and protein oxidative damage triggered by AME were associated with an alteration of the antioxidant response, as shown by a decrease in the enzymatic activity of catalase and superoxide dismutase. These effects on the oxidative response can be related to an inhibition of the Akt/Nrf2/HO-1 signaling pathway; however, further studies are needed in order to validate these in vitro data using in vivo trials in swine.

Isolation of Myenteric and Submucosal Plexus from Mouse Gastrointestinal Tract and Subsequent Co-Culture with Small Intestinal Organoids

Intestinal homeostasis results from the proper interplay among epithelial cells, the enteric nervous system (ENS), interstitial cells of Cajal (ICCs), smooth muscle cells, the immune system, and the microbiota. The disruption of this balance underpins the onset of gastrointestinal-related diseases. The scarcity of models replicating the intricate interplay between the ENS and the intestinal epithelium highlights the imperative for developing novel methods. We have pioneered a sophisticated tridimensional in vitro technique, coculturing small intestinal organoids with myenteric and submucosal neurons. Notably, we have made significant advances in (1) refining the isolation technique for culturing the myenteric plexus, (2) enhancing the isolation of the submucosal plexus—both yielding mixed cultures of enteric neurons and glial cells from both plexuses, and (3) subsequently co-culturing myenteric and submucosal neurons with small intestinal organoids. This co-culture system establishes neural innervations with intestinal organoids, allowing for the investigation of regulatory interactions in the context of gastrointestinal diseases. Furthermore, we have developed a method for microinjecting the luminal space of small intestinal organoids with fluorescently labeled compounds. This technique possesses broad applicability such as the assessment of intestinal permeability, transcytosis, and immunocytochemical and immunofluorescence applications. This microinjection method could be extended to alternative experimental setups, incorporating bacterial species, or applying treatments to study ENS-small intestinal epithelium interactions. Therefore, this technique serves as a valuable tool for evaluating the intricate interplay between neuronal and intestinal epithelial cells (IECs) and shows great potential for drug screening, gene editing, the development of novel therapies, the modeling of infectious diseases, and significant advances in regenerative medicine. The co-culture establishment process spans twelve days, making it a powerful asset for comprehensive research in this critical field.

Corn protein hydrolysate with glutamine-rich peptides protects intestinal barrier in Caco-2 cells: Insights into structural characteristics of identified glutamine peptides

Intestinal barrier is an important basis for intestinal function, which can strictly control the exchange of substances between the host and the external environment. An intact intestianl barrier is also the guardian of human health. In this study, the protective effect of corn protein hydrolysate with glutamine-rich peptides (APH) on intestinal epithelial cell barrier was evaluated in Caco-2 cell inflammatory damage model induced by lipopolysaccharide (LPS). Then, the sequences of glutamine peptides in APH were identified by Nano LC-MS/MS, and the structural characteristics of the identified glutamine peptides were analyzed. The results showed that the hydrolysate effectively alleviated LPS-induced cell damage and decreased the inflammatory response by down-regulating the levels of pro-inflammatory cytokines (TNF-α, IL-8, and IL-1β) and up-regulating the levels of anti-inflammatory cytokine (IL-10). Moreover, the hydrolysate also up-regulated the gene expression of three tight junction proteins (ZO-1, Occludin, Claudin-1) to maintain the integrity of the intestinal epithelial barrier. More importantly, the hydrolysate protected the intestinal barrier function via down-regulating the key genes expression (TLR4, MyD88, NF-κB) in the NF-κB signaling pathway and inhibiting the overexpression of MLCK gene in the MLCK signaling pathway. Finally, 374 corn glutamine peptides were identified from APH, and their structural characteristics showed that corn glutamine peptides had high affinity with membrane proteins. Taken together, these results indicated that APH has excellent potential to protect intestinal barrier function and can be developed into a functional health food.

MiRNA Expression Analysis of IPEC-J2 Cells Damaged by Soybean 7S Globulin Reveals ssc-miR-221-5p as the Factor Alleviating Cell Damage

miRNA Expression Analysis of IPEC-J2 Cells Damaged by Soybean 7S Globulin Reveals ssc-miR-221-5p as the Factor Alleviating Cell Damage

Analysis of intestinal epithelial cell responses to Cryptosporidium highlights the temporal effects of IFN-γ on parasite restriction.

The production of IFN-γ is crucial for control of multiple enteric infections, but its impact on intestinal epithelial cells (IEC) is not well understood. Cryptosporidium parasites exclusively infect epithelial cells and the ability of interferons to activate the transcription factor STAT1 in IEC is required for parasite clearance. Here, the use of single cell RNA sequencing to profile IEC during infection revealed an increased proportion of mid-villus enterocytes during infection and induction of IFN-γ-dependent gene signatures that was comparable between uninfected and infected cells. These analyses were complemented by in vivo studies, which demonstrated that IEC expression of the IFN-γ receptor was required for parasite control. Unexpectedly, treatment of Ifng-/- mice with IFN-γ showed the IEC response to this cytokine correlates with a delayed reduction in parasite burden but did not affect parasite development. These data sets provide insight into the impact of IFN-γ on IEC and suggest a model in which IFN-γ signalling to uninfected enterocytes is important for control of Cryptosporidium.

MiR-329b-5p Affects Sheep Intestinal Epithelial Cells against Escherichia coli F17 Infection

Diarrhea is the most common issue in sheep farms, typically due to pathogenic Escherichia coli (E. coli) infections, such as E. coli F17. microRNA, a primary type of non-coding RNA, has been shown to be involved in diarrhea caused by pathogenic E. coli. To elucidate the profound mechanisms of miRNA in E. coli F17 infections, methods such as E. coli F17 adhesion assay, colony counting assay, relative quantification of bacterial E. coli fimbriae gene expression, indirect immune fluorescence (IF), Cell Counting Kit-8 (CCK-8), 5-ethynyl-2′-deoxyuridine (EdU), Western blotting (WB), and scratch assay were conducted to investigate the effect of miR-329b-5p overexpression/knock-down on E. coli F17 susceptibility of sheep intestinal epithelial cells (IECs). The findings indicated that miR-329b-5p enhances the E. coli F17 resistance of sheep IECs to E.coli F17 by promoting adhesion between E. coli F17 and IEC, as well as IEC proliferation and migration. In summary, miR-329b-5p plays a crucial role in the defense of sheep IECs against E. coli F17 infection, providing valuable insights into its mechanism of action.

MiRNA-192-5p-targeted activated leukocyte cell adhesion molecule improved inflammatory injury of neonatal necrotizing enterocolitis.

Neonatal necrotizing enterocolitis (NEC) is a common gastrointestinal emergency in neonates. MiRNA-192-5p was found associated with ulcerative colitis (UC) progression, also with aberrant expression in intestinal cancer tissue. However, the effects of miRNA-192-5p on NEC have not been reported. Based on the bioinformatics analysis of the GEO dataset, miR-192-5p was identified as the differentially expressed miRNA in NEC, and activated leukocyte cell adhesion molecule (ALCAM) was predicted as its target. After that, in vitro, rat intestinal epithelial cell-6 (IEC-6) were stimulated with LPS to construct a cell model of NEC. IEC-6 cells were transfected with miRNA-192-5p mimics, miRNA-192-5p inhibitors, or miRNA-192-5p inhibitors + sh-ALCAM, and relevant negative control. In vivo, SD rats were treated with artificial feeding, hypoxic reoxygenation, cold stimulation, and LPS gavage to induce NEC, followed by injection of agomiR-NC or agomiRNA-192-5p. Then effects of miRNA-192-5p on NEC model IEC-6 cell viability, apoptosis, ALCAM expression, Interleukin (IL)-1β and IL-6 levels, intestinal injury, intestinal permeability were detected. MiRNA-192-5p expression was downregulated in NEC IEC-6 cells, whose overexpression increased IEC-6 cell viability. MiRNA-192-5p inhibitors increased IL-1β, IL-6 levels and promoted IEC-6 cell apoptosis. MiRNA-192-5p targeting of ALCAM decreased ALCAM expression, IL-1β, and IL-6 levels. AgomiRNA-192-5p decreased ALCAM, IL-1β, and IL-6 levels in intestinal tissue and pathological damage and increased miRNA-192-5p levels. MiR-192-5p protects against intestinal injury by inhibiting ALCAM-mediated inflammation and intestinal epithelial cells, which would provide a new idea for NEC treatment.

Neutrophil-derived PAD4 induces citrullination of CKMT1 exacerbates mucosal inflammation in inflammatory bowel disease.

Peptidyl arginine deiminase 4 (PAD4) plays a pivotal role in infection and inflammatory diseases by facilitating the formation of neutrophil extracellular traps (NETs). However, the substrates of PAD4 and its exact role in inflammatory bowel disease (IBD) remain unclear. In this study, we employed single-cell RNA sequencing (scRNA-seq) and substrate citrullination mapping to decipher the role of PAD4 in intestinal inflammation associated with IBD. Our results demonstrated that PAD4 deficiency alleviated colonic inflammation and restored intestinal barrier function in a dextran sulfate sodium (DSS)-induced colitis mouse model. scRNA-seq analysis revealed significant alterations in intestinal cell populations, with reduced neutrophil numbers and changes in epithelial subsets upon PAD4 deletion. Gene expression analysis highlighted pathways related to inflammation and epithelial cell function. Furthermore, we found that neutrophil-derived extracellular vesicles (EVs) carrying PAD4 were secreted into intestinal epithelial cells (IECs). Within IECs, PAD4 citrullinates mitochondrial creatine kinase 1 (CKMT1) at the R242 site, leading to reduced CKMT1 protein stability via the autophagy pathway. This action compromises mitochondrial homeostasis, impairs intestinal barrier integrity, and induces IECs apoptosis. IEC-specific depletion of CKMT1 exacerbated intestinal inflammation and apoptosis in mice with colitis. Clinical analysis of IBD patients revealed elevated levels of PAD4, increased CKMT1 citrullination, and decreased CKMT1 expression. In summary, our findings highlight the crucial role of PAD4 in IBD, where it modulates IECs plasticity via CKMT1 citrullination, suggesting that PAD4 may be a potential therapeutic target for IBD.

Engineered probiotics with sustained release of interleukin-2 for the treatment of inflammatory bowel disease after oral delivery

Inflammatory bowel disease (IBD) is a kind of auto-immune disease characterized by disrupted intestinal barrier and mucosal epithelium, imbalanced gut microbiome and deregulated immune responses. Therefore, the restoration of immune equilibrium and gut microbiota could potentially serve as a hopeful approach for treating IBD. Herein, the oral probiotic Escherichia coli Nissle 1917 (ECN) was genetically engineered to express secretable interleukin-2 (IL-2), a kind of immunomodulatory agent, for the treatment of IBD. In our design, probiotic itself has the ability to regulate the gut microenvironment and IL-2 at low dose could selectively promote the generation of regulatory T cells to elicit tolerogenic immune responses. To improve the bioavailability of ECN expressing IL-2 (ECN-IL2) in the gastrointestinal tract, enteric coating Eudragit L100-55 was used to coat ECN-IL2, achieving significantly enhanced accumulation of engineered probiotics in the intestine. More importantly, L100-55 coated ECN-IL2 could effectively activated Treg cells to regulate innate immune responses and gut microbiota, thereby relieve inflammation and repair the colon epithelial barrier in dextran sodium sulfate (DSS) induced IBD. Therefore, genetically and chemically modified probiotics with excellent biocompatibility and efficiency in regulating intestinal microflora and intestinal inflammation show great potential for IBD treatment in the future.

Lipidomics of Microplasma-Irradiated Cells at Optimized Discharge Conditions for the Absorption of High-Molecule Drug

Microplasma irradiation is a promising technique for the transdermal delivery of high-molecular-weight drugs. In this technique, microplasma components interact with the skin surface or cell membranes, allowing the drugs to penetrate. For efficient and safe drug delivery, it is crucial to understand these interactions. To this end, this study investigated the effects of microplasma irradiation on cellular lipids, particularly those associated with cell membranes. Rat intestinal epithelial cells were treated with microplasma irradiation at two different voltages (4.0 kV or 4.5 kV). An untargeted lipidomic was conducted using liquid chromatography–mass spectrometry (LC/MS) technique. The results revealed that microplasma irradiation at 4.0 kV induces a significant increase in cell membrane lipids within 10 min post-irradiation. All major cell membrane lipids, including phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, and sphingomyelin, exhibited increases of over 25% within this short timeframe. Notably, this effect is transient, as lipid levels return to their baseline after 12 h. Furthermore, no significant differences in live and apoptotic cell percentages were observed between the control and 12 h post-irradiated cells. In contrast, irradiation at 4.5 kV did not elicit significant changes in cell membrane lipids, correlating with the absence of drug absorption under this condition. Hence, our study unveiled a correlation between the rapid increase in cell membrane lipids and enhanced drug absorption in microplasma-irradiated cells. This lipid augmentation potentially enhances membrane fluidity and permeability, thus facilitating drug absorption. Beyond elucidating the mechanisms and safety of microplasma-based drug delivery, our research provides valuable insights for advancing various microplasma-based biomedical technologies.

Ethanolamine enhances adhesion, promotes microcompartment formation, and modulates gene expression in Levilactobacillus brevis ATCC 14869

ABSTRACT Ethanolamine is an abundant compound in the gastrointestinal tract and a valuable source of carbon and nitrogen for pathogenic bacteria harboring ethanolamine utilization (eut) genes. Eut-positive pathogens can consume free ethanolamine to outcompete commensal microbes, which often lack eut genes, and establish infection. Ethanolamine can also act as a host recognition signal for eut-positive pathogens to upregulate virulence genes during colonization. Therefore, reducing free ethanolamine titers may represent a novel approach to preventing infection by eut-positive pathogens. Interestingly, the commensal microorganism Levilactobacillus brevis ATCC 14869 was found to encode over 18 eut genes within its genome. This led us to hypothesize that L. brevis can compete with eut-positive pathogens by clearing free ethanolamine from the environment. Our results demonstrate that despite being unable to metabolize ethanolamine under most conditions, L. brevis ATCC 14869 responds to the compound by increasing the expression of genes encoding proteins involved in microcompartment formation and adhesion to the intestinal epithelial barrier. The improved intestinal adhesion of L. brevis in the presence of ethanolamine also enhanced the exclusion of eut-positive pathogens from adhering to intestinal epithelial cells. These findings support further studies to test whether L. brevis ATCC 14869 can counter enteric pathogens and prevent or reduce the severity of infections. Overall, the metabolic capabilities of L. brevis ATCC 14869 offer a unique opportunity to add to the armamentarium of antimicrobial therapies as well as our understanding of the mechanisms used by beneficial microbes to sense and adapt to host microenvironments.

Identification of moonlighting adhesins of highly-adhesive Lactobacillus plantarum PO23 isolated from the intestine of Paralichthys olivaceus

At present, feeding probiotics has been an effective method to prevent and control enterogenous diseases of cultured fish. It is very important and urgent to study the molecular mechanism of the adhesion and colonization of probiotics to improve the use efficiency accurately. In our previous research, highly-adhesive Lactobacillus plantarum PO23 (LP-PO23) was isolated from the intestine of olive flounder (Paralichthys olivaceus). To clarify the specific adhesion of LP-PO23 to intestinal epithelial cells (IECs), six adhesins, d-lactate dehydrogenase (LDH), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), elongation factor Tu (EF-Tu), enolase (ENO), 60 kDa chaperonin (Cpn60) and chaperone protein DnaK (DnaK) were identified as moonlighting proteins by far-Western blot and liquid chromatography-mass spectrometry in this research. The recombinant proteins rLDH, rGAPDH, rEF-Tu, rENO, rCpn60 and rDnak containing FLAG tag were expressed and employed as antigens to produce mouse polyclonal antibodies (pAbs). Using pAbs, Western blot, immunofluorescence (IF) and immunoelectron microscopy (IEM) were carried out to confirm the surface distribution of six adhesins on LP-PO23 cells respectively. Finally, the specific binding of rLDH, rGAPDH, rEF-Tu, rENO, rCpn60 and rDnak to IECs was verified by IF. In binding inhibition assay, the mouse anti-GAPDH pAb displayed the strongest inhibition activity among six pAbs. These results confirmed that six moonlighting adhesins participated in the specific adhesion of LP-PO23 to IECs and would facilitate understanding the adhesion mechanism of lactobacilli.