Microbiota regulates neonatal disease tolerance to virus-evoked necrotizing enterocolitis by shaping the STAT1-NLRC5 axis in the intestinal epithelium.

Microbiota and feeding modes influence the susceptibility of premature newborns to necrotizing enterocolitis (NEC) through mechanisms that remain unknown. Here, we show that microbiota colonization facilitated by breastmilk feeding promotes NOD-like receptor family CARD domain containing 5 (Nlrc5) gene expression in mouse intestinal epithelial cells (IECs). Notably, inducible knockout of the Nlrc5 gene in IECs predisposes neonatal mice to NEC-like injury in the small intestine upon viral inflammation in an NK1.1+ cell-dependent manner. By contrast, formula feeding enhances neonatal gut colonization with environment-derived tilivalline-producing Klebsiella spp. Remarkably, tilivalline disrupts microbiota-activated STAT1 signaling that controls Nlrc5 gene expression in IECs through a PPAR-γ-mediated mechanism. Consequently, this dysregulation hinders the resistance of neonatal intestinal epithelium to self-NK1.1+ cell cytotoxicity upon virus infection/colonization, promoting NEC development. Together, we discover the underappreciated role of intestinal microbiota colonization in shaping a disease tolerance program to viral inflammation and elucidate the mechanisms impacting NEC development in neonates.

Dietary fiber promotes antigen presentation on intestinal epithelial cells and development of small intestinal CD4+CD8αα+ intraepithelial T cells.

The impact of dietary fiber on intestinal T cell development is poorly understood. Here we show that a low fiber diet reduces MHC-II antigen presentation by small intestinal epithelial cells (IECs) and consequently impairs development of CD4+CD8αα+ intraepithelial lymphocytes (DP IELs) through changes to the microbiota. Dietary fiber supports colonization by Segmented Filamentous Bacteria (SFB), which induces the secretion of IFNγ by type 1 innate lymphoid cells (ILC1s) that lead to MHC-II upregulation on IECs. IEC MHC-II expression caused either by SFB colonization or exogenous IFNγ administration induced differentiation of DP IELs. Finally, we show that a low fiber diet promotes overgrowth of Bifidobacterium pseudolongum, and that oral administration of B. pseudolongum reduces SFB abundance in the small intestine. Collectively we highlight the importance of dietary fiber in maintaining the balance among microbiota members that allow IEC MHC-II antigen presentation and define a mechanism of microbiota-ILC-IEC interactions participating in the development of intestinal intraepithelial T cells.

Transepithelial transport of nanoparticles in oral drug delivery: From the perspective of surface and holistic property modulation.

Despite the promising prospects of nanoparticles in oral drug delivery, the process of oral administration involves a complex transportation pathway that includes cellular uptake, intracellular trafficking, and exocytosis by intestinal epithelial cells, which are necessary steps for nanoparticles to enter the bloodstream and exert therapeutic effects. Current researchers have identified several crucial factors that regulate the interaction between nanoparticles and intestinal epithelial cells, including surface properties such as ligand modification, surface charge, hydrophilicity/hydrophobicity, intestinal protein corona formation, as well as holistic properties like particle size, shape, and rigidity. Understanding these properties is essential for enhancing transepithelial transport efficiency and designing effective oral drug delivery systems. Therefore, this review provides a comprehensive overview of the surface and holistic properties that influence the transepithelial transport of nanoparticles, elucidating the underlying principles governing their impact on transepithelial transport. The review also outlines the chosen of parameters to be considered for the subsequent design of oral drug delivery systems.

Protective Effects of Tea Tree Oil on Inflammatory Injury of Porcine Intestinal Epithelial Cells Induced by Lipopolysaccharide In Vitro.

Tea tree oil (TTO) improves the intestinal mucosal immunity of weaning piglets, but its underlying mechanism is not clear. We hypothesized that TTO may alleviate inflammatory injury by regulating the function of intestinal epithelial cells. Ileum epithelial cells (IPI-2I) were chosen and an inflammatory injury cell model was generated. The cell viability, cytokine secretion, and gene expression of TLR4 and NF-κB were measured to further evaluate the effects of TTO on the inflammatory injury in immune-stressed cells. The results showed that lipopolysaccharide (LPS; content: ≥30 µg/mL; time: 3 h, 6 h, or 9 h) decreased cell viability (p < 0.01), and 50 µg/mL LPS stimulated for 6 h resulted in an increased secretion of proinflammatory cytokines and a dramatically decreased secretion of anti-inflammatory cytokines (p < 0.05) in IPI-2I cells. Concentrations of 0-0.05% of TTO improved cell viability, while the 0.03% TTO treatment resulted in the highest cell viability and alleviated LPS-induced cell death (p < 0.01). In addition, 0.03% TTO alleviated the LPS-induced increase in the gene expression of IL-1ß, TNFα, and IFNγ, as well as the decrease in the expression of IL-10 in IPI-2I cells (p < 0.05). LPS also upregulated the gene expression of TLR4 and NF-κB (p < 0.05); while TTO supplementation alleviated this effect (p < 0.05), 0.03% and 0.05% TTO supplementation had greater effects (p < 0.05). In conclusion, 50 µg/mL LPS stimulated for 6 h can be used to establish an immune-stressed cell model in IPI-2I cell lines, and 0.03% TTO treatment for 6 h alleviated inflammatory injury in the intestinal epithelial cells of pigs.

Selenium alleviates dextran sulfate sodium-induced colitis and inhibits ferroptosis of intestinal epithelial cells via upregulating glutathione peroxidase 4.

BACKGROUND AND AIM: Selenium, an essential micronutrient for humans, has been shown to be protective against ulcerative colitis (UC), but the exact mechanism remains unclear. The role of selenium, protecting against ferroptosis of intestinal epithelial cells (IECs) in colitis, was investigated in this current study. METHODS: Serum selenium level and ferroptosis-related gene expression in the colonic mucosa were measured in UC patients and healthy controls. The effects of sodium selenite supplementation on experimental colitis were investigated in dextran sulfate sodium (DSS)-treated mice. The influence of sodium selenite on IEC ferroptosis was evaluated through assessing cell death rate, intracellular ferrous iron content, lipid reactive oxygen species level, and mitochondrial membrane damage of DSS-treated Caco-2 cells. Moreover, glutathione peroxidase 4 (GPX4) and acyl-CoA synthetase long-chain family member 4, ferroptosis-related genes, were detected in Caco-2 cells and mouse intestines. RESULTS: Serum selenium was decreased in UC patients in comparison with healthy individuals. Additionally, serum selenium level was negatively correlated with disease activity and was associated with clinical inflammation and nutrition indicators. The expression of GPX4 in the mucosa of UC was positively correlated with serum selenium level. The in vivo experiments showed that selenium treatment ameliorated DSS-induced colitis and inhibited ferroptosis in IECs. The in vitro results suggested that selenium supplementation inhibited DSS-induced ferroptosis in Caco-2 cells. GPX4 was upregulated after selenium supplementation both in vivo and in vitro. CONCLUSIONS: Serum selenium level was associated with IEC ferroptosis in UC patients. Selenium supplementation alleviates DSS-induced colitis and inhibits ferroptosis in IECs by upregulating the expression of GPX4.

BPP43_05035 is a Brachyspira pilosicoli cell surface adhesin that weakens the integrity of the epithelial barrier during infection.

The anaerobic spirochete Brachyspira causes intestinal spirochetosis, characterized by the intimate attachment of bacterial cells to the colonic mucosa, potentially leading to symptoms such as diarrhea, abdominal pain, and weight loss. Despite the clinical significance of Brachyspira infections, the mechanism of the interaction between Brachyspira and the colon epithelium is not known. We characterized the molecular mechanism of the B. pilosicoli-epithelium interaction and its impact on the epithelial barrier during infection. Through a proteomics approach, we identified BPP43_05035 as a candidate B. pilosicoli surface protein that mediates bacterial attachment to cultured human colonic epithelial cells. The crystal structure of BPP43_05035 revealed a globular lipoprotein with a six-bladed beta-propeller domain. Blocking the native BPP43_05035 on B. pilosicoli, either with a specific antibody or via competitive inhibition, abrogated its binding to epithelial cells, which required cell surface-exposed N-glycans. Proximity labeling and interaction assays revealed that BPP43_05035 bound to tight junctions, thereby increasing the permeability of the epithelial monolayer. Extending our investigation to humans, we discovered a downregulation of tight junction and brush border genes in B. pilosicoli-infected patients carrying detectable levels of epithelium-bound BPP43_05035. Collectively, our findings identify BPP43_05035 as a B. pilosicoli adhesin that weakens the colonic epithelial barrier during infection.

Hypoxia-inducible lipid droplet-associated protein (HILPDA) and cystathionine ß-synthase (CBS) co-contribute to protecting intestinal epithelial cells from Staphylococcus aureus via regulating lipid droplets formation.

Despite Staphylococcus aureus (S. aureus) being a highly studied zoontic bacterium, its enteropathogenicity remains elusive. Herein, our findings demonstrated that S. aureus infection led to the accumulation of lipid droplets (LDs) in intestinal epithelial cells, accompanied by marked elevation inflammatory response that ultimately decreases intracellular bacterial load. The aforestated phenomenon may be partly attributed to the up-regulation of hypoxia-inducible lipid droplet-associated protein (HILPDA) and the concomitant down-regulation of cystathionine ß-synthase (CBS) protein. Moreover, S. aureus infection up-regulated the expression of HILPDA, thereby promoting LDs accumulation, and down-regulated that of CBS, consequently inhibiting microsomal triglyceride transfer protein (MTTP) expression. This process may suppress the transport of LDs to the extracellular environment, further contributing to the formation of intracellular LDs. In summary, the results of this study provide significant insights into the intricate mechanisms through which the host organism combats pathogens and maintains the balance of sulfur and lipid metabolism. These findings not only enhance our understanding of the host's defense mechanisms but also offer promising avenues for the development of novel strategies to combat intestinal infectious diseases.

ASB3 expression aggravates inflammatory bowel disease by targeting TRAF6 protein stability and affecting the intestinal microbiota.

E3 ubiquitin ligase (E3) plays a vital role in regulating inflammatory responses by mediating ubiquitination. Previous studies have shown that ankyrin repeat and SOCS box-containing protein 3 (ASB3) is involved in immunomodulatory functions associated with cancer. However, the impact of ASB3 on the dynamic interplay of microbiota and inflammatory responses in inflammatory bowel disease (IBD) is unclear. Here, we systematically identify the E3 ligase ASB3 as a facilitative regulator in the development and progression of IBD. We observed that ASB3 exhibited significant upregulation in the lesions of patients with IBD. ASB3-/- mice are resistant to dextran sodium sulfate-induced colitis. IκBα phosphorylation levels and production of proinflammatory factors IL-1ß, IL-6, and TNF-α were reduced in the colonic tissues of ASB3-/- mice compared to WT mice. This colitis-resistant phenotype was suppressed after coprophagic microbial transfer and reversed after combined antibiotics removed the gut commensal microbiome. Mechanistically, ASB3 specifically catalyzes K48-linked polyubiquitination of TRAF6 in intestinal epithelial cells. In contrast, in ASB3-deficient organoids, the integrity of the TRAF6 protein is shielded, consequently decelerating the onset of intestinal inflammation. ASB3 is associated with dysregulation of the colitis microbiota and promotes proinflammatory factors' production by disrupting TRAF6 stability. Strategies to limit the protein level of ASB3 in intestinal epithelial cells may help in the treatment of colitis. IMPORTANCE: Ubiquitination is a key process that controls protein stability. We determined the ubiquitination of TRAF6 by ASB3 in intestinal epithelial cells during colonic inflammation. Inflammatory bowel disease patients exhibit upregulated ASB3 expression at focal sites, supporting the involvement of degradation of TRAF6, which promotes TLR-Myd88/TRIF-independent NF-κB aberrant activation and intestinal microbiota imbalance. Sustained inflammatory signaling in intestinal epithelial cells and dysregulated protective probiotic immune responses mediated by ASB3 collectively contribute to the exacerbation of inflammatory bowel disease. These findings provide insights into the pathogenesis of inflammatory bowel disease and suggest a novel mechanism by which ASB3 increases the risk of colitis. Our results suggest that future inhibition of ASB3 in intestinal epithelial cells may be a novel clinical strategy.

Arf1 promotes porcine intestinal epithelial cell proliferation via the mTORC1 signaling pathway.

The promotion of gut health, a pervasive problem in modern animal husbandry, positively affects organismal health, productivity, and economics. Porcine intestinal epithelial cells (IPEC-J2) continuously proliferate to maintain intestinal homeostasis, including barrier, immune, and absorptive functions. Gut homeostasis is fundamental to organismal health. ADP-ribosylation factor 1 (Arf1), a small GTPase, plays a crucial role in coordinating mTORC1 in response to nutrients, especially amino acid availability in the gut. mTORC1 is the central hub of proliferation. Thus, it seems likely that Arf1 promotes IPEC-J2 cell proliferation. However, the exact role of Arf1 in the porcine gut remains unclear. Therefore, we evaluated the functional role and possible mechanisms of Arf1 in the porcine intestine through Arf1 overexpression and knockdown in IPEC-J2 cells. Arf1 overexpression and knockdown significantly enhanced and inhibited, respectively, IPEC-J2 cell viability, and PCNA expression varied with Arf1 expression. Moreover, the proportion of Ki67-positive cells was significantly greater in the Arf1-overexpressing group than in the control group. These results suggest that Arf1 improves IPEC-J2 cell proliferation. The underlying mechanism was explored by Western blotting. Arf1 overexpression and knockdown significantly enhanced and suppressed, respectively, the levels of p-S6K1 and p-RPS6, which are key downstream targets of the mTORC1 signaling pathway. Collectively, our findings reveal the role of the Arf1-mTORC1 axis in IPEC-J2 cell proliferation and its potential function in regulating intestinal homeostasis and health.

Dietary limonene promotes gastrointestinal barrier function via upregulating tight/adherens junction proteins through cannabinoid receptor type-1 antagonistic mechanism and alters cellular metabolism in intestinal epithelial cells.

Limonene, a dietary monocyclic monoterpene commonly found in citrus fruits and various aromatic plants, has garnered increasing interest as a gastrointestinal protectant. This study aimed to assess the effects of limonene on intestinal epithelial barrier function and investigate the involvement of cannabinoid receptor type-1 (CB1R) in vitro. Additionally, the study focused on examining the metabolomic changes induced by limonene in the intestinal epithelial cells (Caco-2). Initial analysis of transepithelial electrical resistance (TEER) revealed that both l-limonene and d-limonene, isomers of limonene, led to a dose- and time-dependent increase in TEER in normal cells and those inflamed by pro-inflammatory cytokines mixture (CytoMix). Furthermore, both types of limonene reduced CytoMix-induced paracellular permeability, as demonstrated by a decrease in Lucifer yellow flux. Moreover, d-limonene and l-limonene treatment increased the expression of tight junction molecules (TJs) such as occludin, claudin-1, and ZO-1, at both the transcriptional and translational levels. d-Limonene upregulates E-cadherin, a molecule involved in adherens junctions (AJs). Mechanistic investigations demonstrated that d-limonene and l-limonene treatment significantly inhibited CB1R at the protein, while the mRNA level remained unchanged. Notably, the inhibitory effect of d-limonene on CB1R was remarkably similar to that of pharmacological CB1R antagonists, such as rimonabant and ORG27569. d-limonene also alters Caco-2 cell metabolites. A substantial reduction in ß-glucose and 2-succinamate was detected, suggesting limonene may impact intestinal epithelial cells' glucose uptake and glutamate metabolism. These findings suggest that d-limonene's CB1R antagonistic property could effectively aid in the recovery of intestinal barrier damage, marking it a promising gastrointestinal protectant.

Construction of multilayered small intestine-like tissue by reproducing interstitial flow.

Recent advances have made modeling human small intestines in vitro possible, but it remains a challenge to recapitulate fully their structural and functional characteristics. We suspected interstitial flow within the intestine, powered by circulating blood plasma during embryonic organogenesis, to be a vital factor. We aimed to construct an in vivo-like multilayered small intestinal tissue by incorporating interstitial flow into the system and, in turn, developed the micro-small intestine system by differentiating definitive endoderm and mesoderm cells from human pluripotent stem cells simultaneously on a microfluidic device capable of replicating interstitial flow. This approach enhanced cell maturation and led to the development of a three-dimensional small intestine-like tissue with villi-like epithelium and an aligned mesenchymal layer. Our micro-small intestine system not only overcomes the limitations of conventional intestine models but also offers a unique opportunity to gain insights into the detailed mechanisms underlying intestinal tissue development.

NLRP1B allele 2 does not respond to Val-boro-Pro (VbP) in intestinal epithelial cells.

The intestinal mucosa must balance tolerance to commensal microbes and luminal antigens with rapid detection of enteric pathogens in order to maintain homeostasis. This balance is facilitated through the regulation of epithelial layer integrity by innate immune receptors. Certain NOD-like receptors (NLRs) expressed in intestinal epithelial cells, including NLRC4 and NLRP9B, form inflammasomes that protect against pathogens by activating caspase-1 to cause extrusion of infected cells. NLRP1B is a murine NLR encoded by five alleles of a highly polymorphic gene homologous to human NLRP1. NLRP1B forms inflammasomes in response to a variety of pathogens that cause intestinal infections, but it has almost exclusively been studied in immune cells and has not been characterized in cells of the intestinal epithelium. Here, we show that Nlrp1b allele 2 is expressed in ileal and colonic organoids derived for C57BL/6J mice, while the related gene Nlrp1a was not expressed. Nlrp1b was upregulated by interleukin-13 in organoids and by the protozoan Tritrichomonas muris in vivo, suggesting that NLRP1B may be involved in defense against enteric parasites. Surprisingly, while Val-boro-Pro (VbP) activated C57BL/6J-derived bone marrow-derived macrophages, which expressed both Nlrp1a and Nlrp1b, it did not activate intestinal organoids of the same genotype. We furthermore did not detect Nlrp1b in organoids derived from Balb/cJ mice, which express a different allele than the one expressed in C57BL/6J mice. Together, our results suggest that NLRP1B may have an allele-dependent function in murine IECs whose regulation is distinct from that of macrophages, and that the response to VbP might be exclusively driven by NLRP1A in C57BL/6J mice.

Klebsiella pneumoniae increases invasion in intestinal epithelial cells by disrupting the cytoskeleton.

Klebsiella pneumoniae is an opportunistic pathogen and it can cause human mucosal lesions through the intestine, leading to bacteremia and abscess formation in liver and spleen. Previous studies have shown that K. pneumoniae can enter or cross cells through the intestinal epithelium, but the mechanism is unknown. In this study, we treated the intestinal epithelial cell line Caco-2 with KP1195, a clinically isolated strain with high adhesion and invasion of intestinal epithelial cells. The results showed that the treatment of K. pneumoniae could increase the expression of integrin gene and further disrupt the changes of cytoskeleton. Treating Caco-2 with cytoskeletal inhibitor cytorelaxin D can significantly increase the efficiency of K. pneumoniae invading Caco-2 cells. These data suggest that disruption of the cytoskeleton through integrins may be one of the mechanisms by which K. pneumoniae increases intracellular invasion. This study provides a theoretical basis for further understanding of the mechanism of K. pneumoniae entering intestinal epithelial cells.

Mitochondrial perturbation in the intestine causes microbiota-dependent injury and gene signatures discriminative of inflammatory disease.

Mitochondrial dysfunction is associated with inflammatory bowel diseases (IBDs). To understand how microbial-metabolic circuits contribute to intestinal injury, we disrupt mitochondrial function in the epithelium by deleting the mitochondrial chaperone, heat shock protein 60 (Hsp60Δ/ΔIEC). This metabolic perturbation causes self-resolving tissue injury. Regeneration is disrupted in the absence of the aryl hydrocarbon receptor (Hsp60Δ/ΔIEC;AhR-/-) involved in intestinal homeostasis or inflammatory regulator interleukin (IL)-10 (Hsp60Δ/ΔIEC;Il10-/-), causing IBD-like pathology. Injury is absent in the distal colon of germ-free (GF) Hsp60Δ/ΔIEC mice, highlighting bacterial control of metabolic injury. Colonizing GF Hsp60Δ/ΔIEC mice with the synthetic community OMM12 reveals expansion of metabolically flexible Bacteroides, and B. caecimuris mono-colonization recapitulates the injury. Transcriptional profiling of the metabolically impaired epithelium reveals gene signatures involved in oxidative stress (Ido1, Nos2, Duox2). These signatures are observed in samples from Crohn's disease patients, distinguishing active from inactive inflammation. Thus, mitochondrial perturbation of the epithelium causes microbiota-dependent injury with discriminative inflammatory gene profiles relevant for IBD.

CRIg+ macrophages deficiency enhanced inflammation damage in IBD due to gut extracellular vesicles containing microbial DNA.

Gut microbiota-derived extracellular vesicles (mEVs) are reported to regulate inflammatory response by delivering bacterial products into host cells. The complement receptor of the immunoglobulin superfamily macrophages (CRIg+ Mφ) could clear invading bacteria and their derivatives. Here, we investigate the role of CRIg+ Mφ and the mechanism by which mEVs regulate intestinal inflammation. We found that it is exacerbated in IBD patients and colitis mice by mEVs' leakage from disturbed gut microbiota, enriching microbial DNA in the intestinal mucosa. CRIg+ Mφ significantly decrease in IBD patients, allowing the spread of mEVs into the mucosa. The microbial DNA within mEVs is the key trigger for inflammation and barrier function damage. The cGAS/STING pathway is crucial in mEVs-mediated inflammatory injury. Blocking cGAS/STING signaling effectively alleviates inflammation caused by mEVs leakage and CRIg+ Mφ deficiency. Microbial DNA-containing mEVs, along with CRIg+ Mφ deficiency, stimulate inflammation in IBD, with the cGAS/STING pathway playing a crucial role.

Lipocalin-2-mediated intestinal epithelial cells pyroptosis via NF-κB/NLRP3/GSDMD signaling axis adversely affects inflammation in colitis.

Ulcerative colitis (UC) is a major inflammatory bowel disease (IBD) characterized by intestinal epithelium damage. Recently, Lipocalin-2 (LCN2) has been identified as a potential fecal biomarker for patients with UC. However, further investigation is required to explore its pro-inflammatory role in UC and the underlying mechanism. The biological analysis revealed that Lcn2 serves as a putative signature gene in the colon mucosa of patients with UC and its association with the capsase/pyroptosis signaling pathway in UC. In wild-type mice with DSS-induced colitis, LCN2 overexpression in colon mucosa via in vivo administration of Lcn2 overexpression plasmid resulted in exacerbation of colitis symptoms and epithelium damage, as well as increased expression levels of pyroptosis markers (cleaved caspase1, GSDMD, IL-1ß, HMGB1 and IL-18). Additionally, we observed downregulation in the expression levels of pyroptosis markers following in vivo silencing of LCN2. However, the pro-inflammatory effect of LCN2 overexpression was effectively restrained in GSDMD-KO mice. Moreover, single-cell RNA-sequencing analysis revealed that Lcn2 was predominantly expressed in the intestinal epithelial cells (IECs) within the colon mucosa of patients with UC. We found that LCN2 effectively regulated pyroptosis events by modulating the NF-κB/NLRP3/GSDMD signaling axis in NCM460 cells stimulated by LPS and ATP. These findings demonstrate the pro-inflammatory role of LCN2 in colon epithelium and provide a potential target for inhibiting pyroptosis in UC.

Morphological and immunological characterization of primary cultured chicken caecal epithelial cells.

Cell cultures are models in biological and medical research to understand physiological and pathological processes. Cell lines are not always available depending on cell type and required species. In addition, the immortalization process often affects cell biology. Primary cells generally maintain a greater degree of similarity in short-term culture to the cells in tissue. Goal of this study was to verify the suitability of chicken primary epithelial caecal cells (PECCs) for in vitro investigations of host‒pathogen interactions. Epithelial nature of PECCs was confirmed by detection of tight and adherens junctions and cobblestone-like cell morphology. Sialic acids distribution was similar to that in caecal cyrosections. To understand the capacity of PECCs to respond to microbial challenges, the Toll-like receptors (TLRs) repertoire was determined. Exposure of PECCs to polyinosinic-polycytidylic acid (poly(I:C)) or lipopolysaccharide (LPS) led to upregulation of type I and III interferon (IFN) as well as interleukin (IL-) 1ß, IL-6 and IL-8 mRNA expression. Overall, the PECCs showed properties of polarized epithelial cells. The presence of TLRs, their differential expression, as well as pattern recognition receptor dependent immune responses enable PECCs to act as suitable in vitro model for host‒pathogen interaction studies, which are difficult to conduct under in vivo conditions.

A Protein Complex of Liver Origin Activates a Pro-inflammatory Program That Drives Hepatic and Intestinal Injury in Alcohol-Associated Liver Disease.

BACKGROUND & AIMS: There is limited information on how the liver-to-gut axis contributes to alcohol-associated liver disease (AALD). We previously identified that high-mobility group box-1 (HMGB1) undergoes oxidation in hepatocytes and demonstrated elevated serum levels of oxidized HMGB1 ([O] HMGB1) in alcoholic patients. Since interleukin-1 beta (IL-1B) increases in AALD, we hypothesized hepatocyte-derived [O] HMGB1 could interact with IL-1B to activate a pro-inflammatory program that, besides being detrimental to the liver, drives intestinal barrier dysfunction. RESULTS: Alcohol-fed RageΔMye mice exhibited decreased nuclear factor kappa B signaling, a pro-inflammatory signature, and reduced total intestinal permeability, resulting in protection from AALD. In addition, [O] HMGB1 bound and signaled through the receptor for advanced-glycation end-products (RAGE) in myeloid cells, driving hepatic inflammation, intestinal permeability, and increased portal blood lipopolysaccharide in AALD. We identified that [O] HMGB1 formed a complex with IL-1B, which was found in the livers of patients with acute alcoholic hepatitis and mice with AALD. This complex originated from the liver, because it was absent in the intestine when hepatocytes did not produce [O] HMGB1. Mechanistically, the complex bound RAGE in Kupffer cells and macrophages induced a pro-inflammatory program. Moreover, it bound RAGE in intestinal macrophages and epithelial cells, leading to intestinal inflammation, altered intestinal epithelial cell tight junction protein expression, increased intestinal permeability, and elevated portal blood lipopolysaccharide, enhancing AALD pathogenesis. CONCLUSIONS: We identified a protein complex of liver origin that amplifies the pro-inflammatory feedback loop in AALD; therefore, targeting this complex could have significant therapeutic potential.

Cellular evidence and spatial distribution of endosomal biosynthesis and autophagy in intestinal immune barrier cells of crucian carp (Carassiuscarassius).

Crucian carp (Carassius carassius) is an important aquatic economic animal, and the immune barrier function of its intestine has been a focus of research into oral vaccines and drugs. However, the histological structures of the intestinal barrier and its adjacent areas have not been clearly established, and little subcellular evidence is available to elucidate the spatial distribution of intracellular biological processes. In this study, the spatial distribution of autophagy and endosome formation in the intestinal epithelial cells (IECs) of crucian carp were analyzed. These two biological activities are closely related to intestinal homeostasis, immunity, and cell communication. Periodic acid-Schiff (PAS) and Masson's trichrome staining were employed to elucidate the distinctive histological framework of the Crucian carp's myoid cell network, which resides within the subepithelial layer and is characterized by gap junctions. Transmission electron microscopy (TEM), immunohistochemistry (IHC), and immunofluorescence (IF) were used to detect the structural and functional aspects of the IEC in different intestinal segments. TEM and immunohistochemical analyses captured the biogenesis and maturation of early and late endosomes as well as multivesicular bodies (MVBs), as well as the initiation and progression of autophagy, including macroautophagy and mitophagy. The endosome and MVBs-specific marker CD63 and autophagy-related protein LC3 were highly expressed in IECs and were correlated with autophagy and endosome biosynthesis in the apical and basal regions of individual cells, and differed between different intestinal segments. In summary, this study elucidated the ubiquity and morphological characteristics of autophagy and endosome formation across different intestinal segments of crucian carp. A unique myoid cell network beneath the intestinal epithelium in crucian carp was also identified, expanding the histological understanding of this animal's intestinal tract.