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.

Intestinal Mucosal Immune Barrier: A Powerful Firewall Against Severe Acute Pancreatitis-Associated Acute Lung Injury via the Gut-Lung Axis.

The pathogenesis of severe acute pancreatitis-associated acute lung injury (SAP-ALI), which is the leading cause of mortality among hospitalized patients in the intensive care unit, remains incompletely elucidated. The intestinal mucosal immune barrier is a crucial component of the intestinal epithelial barrier, and its aberrant activation contributes to the induction of sustained pro-inflammatory immune responses, paradoxical intercellular communication, and bacterial translocation. In this review, we firstly provide a comprehensive overview of the composition of the intestinal mucosal immune barrier and its pivotal roles in the pathogenesis of SAP-ALI. Secondly, the mechanisms of its crosstalk with gut microbiota, which is called gut-lung axis, and its effect on SAP-ALI were summarized. Finally, a number of drugs that could enhance the intestinal mucosal immune barrier and exhibit potential anti-SAP-ALI activities were presented, including probiotics, glutamine, enteral nutrition, and traditional Chinese medicine (TCM). The aim is to offer a theoretical framework based on the perspective of the intestinal mucosal immune barrier to protect against SAP-ALI.

Suitable Cottonseed Protein Concentrate Supplementation in Common Carp (Cyprinus carpio) Serves as an Effective Strategy for Fish Meal Sparing Based on Improvement in Intestinal Antioxidant Capacity, Barrier and Microbiota Composition.

The application of cottonseed protein concentrate (CPC) is an effective strategy to moderate the shortage of fish meal (FM) for the aquafeed industry. However, little attention has been paid to the effects of replacing fishmeal with CPC on cyprinid fish. This study used common carp (Cyprinus carpio) as the biological model and assessed the potential of applying CPC as a substitute for fishmeal in the diet of common carp. The proportion of fish meal substituted with CPC in the six diets was 0% (CPC0), 25% (CPC25), 50% (CPC50), 75% (CPC75), and 100% (CPC100). Each diet was fed to three replicate groups of common carp (4.17 ± 0.02 g) for 56 days. Results revealed that the CPC50 group significantly increased the growth indexes via up-regulating the genes of the GH/IGF axis and the TOR pathway. The intestinal digestive ability was also elevated in the CPC50 group via markedly increasing intestinal villus height, protease and lipase activities in the whole intestine, and the amylase activity of the foregut and midgut. The CPC50 group captured significantly higher activities and gene expressions of antioxidant enzymes and lower malonaldehyde contents via evoking the Nrf2/Keap1 signal pathway. The CPC50 group enhance the intestinal mechanical barrier via up-regulating the gene expressions of tight junction proteins and heighten the intestinal biological barrier by increasing the probiotics (Lactococcus) and decreasing the harmful bacteria (Enterococcus). But excessive substitution levels (75% and 100%) would compromise growth performance, intestinal antioxidant capacity, and immune function. The optimum substitution level was estimated to be 46.47%, 47.72%, and 46.43% using broken-line regression analyses based on mass gain rate, protein efficiency ratio, and feed conversion rate. Overall, the fishmeal in common carp feed could be substituted up to 50% by CPC without negative influence on growth, feed utilization, and or intestinal health.

Mucosal-associated invariant T cells in infectious diseases of respiratory system: recent advancements and applications.

Mucosal-associated invariant T (MAIT) cells are an atypical subset of T lymphocytes, which have a highly conserved semi-constant αß chain of T-cell receptor (TCR) and recognize microbe-derived vitamin B metabolites via major histocompatibility complex class I related-1 molecule (MR1). MAIT cells get activated mainly through unique TCR-dependent and TCR-independent pathways, and express multiple functional and phenotypic traits, including innate-like functionality, T helper (Th) 1 cell immunity, Th 17 cell immunity, and tissue homing. Given the functions, MAIT cells are extensively reported to play a key role in mucosal homeostasis and infectious diseases. In the current work, we review the basic characteristics of MAIT cells and their roles in mucosal homeostasis and development of respiratory infectious diseases as well as their potential therapeutic targets.

The Role of High Mobility Group Box B-1 in the Prognosis of Colorectal Cancer Based on the Changes in the Intestinal Mucosal Barrier.

Background: To investigate the expression of high mobility group box B-1 (HMGB-1) in patients with colorectal cancer (CRC) and its association with clinicopathological features and prognosis in colorectal carcinoma by combining bioinformatics and clinical data analysis, and to clarify the role of HMGB-1. To examine whether HMGB-1 expression is related to the damage of the intestinal mucosal barrier, and then explore the potential HMGB-1-dependent mechanisms affecting the progression of CRC. Methods: CRC datasets of GSE12945, GSE17536, and GSE17537 from the public gene chip database were screened and downloaded. Clinical information and CRC tissue samples from patients with stage I-III CRC from the hospital were collected. Serum samples of patients were applied by enzyme-linked immunosorbent assay on HMGB-1, and were divided into high and low HMGB-1 expression, which was examined by 16S rDNA sequencing. Immunohistochemistry was performed to examine the relationship between the expression of HMGB-1 and tight junction protein, occludin, tumor necrosis factor-α, and interferon-γ. Results: Based on the Cutoff value of 10.24 ng/mL, the CRC patients were divided into high and low expression groups. In the HMGB-1H patient group, the TNM staging, overall survival, disease-free survival, recurrence, and metastasis were inferior to the HMGB-1L group. The results of 16S rDNA sequencing demonstrated that the Providencia genus was found to be enriched in the HMGB-1L group. Immunohistochemical results showed that HMGB-1 expression was negatively correlated with the expression of ZO-1 and occludin (R = 0.035, R = 0.003, P < .05), but was positively correlated with the expression of TNF-α and IFN-γ (R = 0.016, R = 0.001, P < .05). Conclusion: The survival of CRC patients with positive HMGB-1 expression was significantly shortened, which may be related to the decrease of Rovitensis content, the decreased expression of ZO-1 and occludin, and the increased levels of TNF-α and IFN-γ, which in turn damage the intestinal mucosal barrier, leading to the development of CRC.

Chronic exposure to polystyrene nanoplastics induces intestinal mechanical and immune barrier dysfunction in mice.

Micro(nano)plastics are prevalent in the environment, and prolonged exposure to them represents a threat to human health. The goal of this study is to assess the health risk of long-term exposure to nanoplastics (NPs) at environmental concentrations on the intestinal mechanical and immune barrier in mice. In this study, mice were provided drinking water containing polystyrene NPs (PS-NPs; 0.1, 1, and 10 mg·L-1) for 32 consecutive weeks. The levels of endocytosis proteins caveolin and clathrin and of tight junctional proteins claudin-1, occludin, and ZO-1, and morphological changes, proportion of lymphocytes B in MLNs and lymphocytes T in IELs and LPLs were determined by immunohistochemistry, hematoxylin-eosin, and flow cytometry assays in the intestinal tissues of mice at 28 weeks. The activities or concentrations of ROS, SOD, MDA, and GSH-Px and inflammatory factors (IL-1ß, IL-6, and TNF-α) in the intestinal tissues of mice were measured by ELISA at 12, 16, 20, 24, and 32 weeks. Compared with the control group, oral ingested PS-NPs entered the intestinal tissues of mice and upregulated expression levels of the clathrin and caveolin. The intestinal tissue structure of mice in the PS-NPs (1 and 10 mg·L-1) exposure groups showed significant abnormalities, such as villus erosion, decreased of crypts numbers and large infiltration of inflammatory cells. Exposure to 0.1 mg·L-1 PS-NPs decreased occludin protein levels, but not claudin-1 and ZO-1 levels. The levels of these three tight junction proteins decreased significantly in the 1 and 10 mg·L-1 PS-NPs exposed groups. Exposure to PS-NPs led to a significant time- and dose-dependent increase in ROS and MDA levels, and concurrently decreased GSH-Px and SOD contents. Exposure to PS-NPs increased the proportion of B cells in MLNs, and decreased the proportion of CD8+ T cells in IELs and LPLs. The levels of pro-inflammatory cytokines IL-6, TNF-α and IL-1ß were markedly elevated after PS-NPs exposure. Long-term PS-NPs exposure impaired intestinal mechanical and immune barrier, and indicate a potentially significant threat to human health.

From trash to treasure: the role of bacterial extracellular vesicles in gut health and disease.

Bacterial extracellular vesicles (BEVs) have emerged as critical factors involved in gut health regulation, transcending their traditional roles as byproducts of bacterial metabolism. These vesicles function as cargo carriers and contribute to various aspects of intestinal homeostasis, including microbial balance, antimicrobial peptide secretion, physical barrier integrity, and immune system activation. Therefore, any imbalance in BEV production can cause several gut-related issues including intestinal infection, inflammatory bowel disease, metabolic dysregulation, and even cancer. BEVs derived from beneficial or commensal bacteria can act as potent immune regulators and have been implicated in maintaining gut health. They also show promise for future clinical applications in vaccine development and tumor immunotherapy. This review examines the multifaceted role of BEVs in gut health and disease, and also delves into future research directions and potential applications.

Effects of prolonged fluoride exposure on innate immunity, intestinal mechanical, and immune barriers in mice.

The aim of this study is to explore the effects of fluoride on the innate immunity, intestinal mechanical barrier, and immune barrier of C57BL/6 mice, as well as to analyze the degree of structural and tissue damage, providing reference data for related research. Mice were randomly divided into four groups and then treated with 0 mg/L (control), 50 mg/L, 100 mg/L, 125 mg/L sodium fluoride solution, respectively, for 120 days. Histological technique, ELISA, MTT colorimetry methods were used to detect and analyze the effects of different concentrations of fluoride on the intestinal morphology, mechanical barrier and the immune functions and innate immunity of mice. The results showed that compared with the control group, the villi were injured in different degrees of the three fluoride groups, the number of goblet cells, the protein expression levels of connexin ZO-1, Claudin-1 and Occludin, the content of Diamine Oxidase (DAO), endotoxin (ET) and D-lactic acid (D-LA), the activity of natural killer cell (NK cells), the number and percentage of neutrophils and erythrocytes, the phagocytic rate of neutrophils, and the rate of C3bR rosette (which is formed by the adhesion of C3b receptors on the red blood cell membrane to complement sensitized yeast) and IC rosette (which is formed by the adhesion of C3b molecules in the immunecomplex adhered to the red blood cell membrane to non sensitized yeast) of red blood cells, the content of interlenkin 1 beta (IL-1ß) and interlenkin 8 (IL-8), the number and percentage of lymphocytes decreased with the increasing of fluoride concentration. In addition, the content of the Immunoglobulin A (sIgA) showed a trend of increase at first and then decrease in salivary gland and jejunum. It is concluded that excessive intake of fluoride for a long time has a certain damage effect on the intestinal tract, leading to an increase in the permeability of the intestinal tract, thereby destroying the mechanical and immune barrier function of the intestinal tract.

The immune-stealth polymeric coating on drug delivery nanocarriers: In vitro engineering and in vivo fate.

Although essential nanosystems such as nanoparticles and nanocarriers are desirable options for transporting various drug molecules into the biological environment, they rapidly remove from the circulatory system due to their interaction with multiple in vivo barriers, especially the immune barrier, which will result in their short-term effects. In order to improve their effectiveness and durability in the circulatory system, the polymer coatings can use to cover the surface of nanoparticles and nanocarriers to conceal them from the immune system. Due to their different properties (like charge, elasticity, and hydrophilicity/hydrophobicity), these coatings can improve drug delivery nanosystem durability and therapeutic applications. The mentioned coatings have different types and are divided into various categories, such as synthetic polymers, polysaccharides, and zwitterionic polymers. Each of these polymers has unique properties based on its category, origin, and chemical structure that make them suitable for producing stealth drug delivery nanocarriers. In this review article, we have tried to explain the importance of these diverse polymer coatings in determining the fate of drug nanocarriers and then introduced the different types of these coatings and, finally, described various methods that directly and indirectly analyze the nanocoatings to determine the stability of nanoparticles in the body.

Pathogenesis of Nasal Polyposis: Current Trends.

Chronic Rhinosinusitis (CRS) is characterized by edema of the sub-epithelial layers, but, only specific types of CRS are developing polyps. Nasal polyposis may develop under different pathogenetic mechanisms rendering the typical macroscopic classification of CRS, with or without nasal polyps, rather deficient. Currently, we approach nasal polyposis, in terms of diagnosis and treatment, according to its endotype, which means that we focus on the specific cells and cytokines that are participating in its pathogenesis. It appears that the molecular procedures that contribute to polyp formation, initiating with a Th-2 response of the adaptive immune system, are local phenomena occurring in the sub-epithelial layers of the mucosa. Several hypotheses are trying to approach the etiology that drives the immune response towards Th-2 type. Extrinsic factors, like fungi, Staphylococcus superantigens, biofilms, and altered microbiome can contribute to a modified and intense local reaction of the immune system. Some hypotheses based on intrinsic factors like the elimination of Treg lymphocytes, low local vitamin-D levels, high levels of leukotrienes, epithelial to mesenchymal transition (EMT) induced by hypoxia, and altered levels of NO, add pieces to the puzzle of the pathogenesis of nasal polyposis. Currently, the most complete theory is that of epithelial immune barrier dysfunction. Intrinsic and extrinsic conditions can damage the epithelial barrier rendering sub-epithelial layers more vulnerable to invasion by pathogens that trigger a Th-2 response of the adaptive immune system. Th2 cytokines, subsequently, induce the accumulation of eosinophils and IgE together with the remodeling of the stroma in the sub-epithelial layers leading, eventually, to the formation of nasal polyps.

The efficacy of bone marrow mesenchymal stem cells on rat intestinal immune-function injured by ischemia/reperfusion.

Background: Transplantation of bone marrow mesenchymal stem cells (BMSCs) has a promising therapeutic efficiency for varieties of disorders caused by ischemia or reperfusion impairment. It has been shown that BMSCs can mitigate intestinal ischemia/reperfusion (I/R) injuries, but the underlying mechanism is still unclear. This study aimed at investigating the efficacy of BMSCs on the immune function of intestinal mucosal microenvironment after I/R injuries. Methods: Twenty adult Sprague-Dawley rats were randomly assigned to a treatment or a control group. All the rats underwent superior mesenteric artery clamping and unclamping. In the treatment group, BMSCs were implanted into the intestine of ten rats by direct submucosal injection whereas the other ten rats in the control group were injected with the same volume of saline. On the fourth and seventh day after BMSCs transplantation, intestinal samples were examined for the CD4 (CD4-positive T-lymphocytes)/CD8 (CD8-positive T-lymphocytes) ratio of the bowel mucosa via flow cytometry, and for the level of Interleukin-2 (IL-2), Interleukin-4 (IL-4) and Interleukin-6 (IL-6) via ELISA. Paneth cell counts and Secretory Immunoglobulin A (SIgA) level were examined via immunohistochemical (IHC) analysis. Real time PCR (RT-PCR) was used to detect the expression levels of tumor necrosis factor-α (TNF-α) and trypsinogen (Serine 2) (PRSS2) genes. White blood cell (WBC) count was measured by manual counting under the microscope. Results: The CD4/CD8 ratio in the treatment group was significantly lower compared with that in the control group. The concentration of IL-2 and IL-6 was lower in the treatment group compared with the control group, while the level of IL-4 is the reverse between the two groups. The number of Paneth cells in intestinal mucosa increased significantly, while the level of SIgA in intestinal mucosa decreased significantly, after BMSCs transplantation. The gene expression levels of TNF-α and PRSS2 in intestinal mucosa of treatment group were significantly lower than those of control group. The WBC count in the treatment group was significantly lower than that in the control group. Conclusion: We identified immune-relevant molecular changes that may explain the mechanism of BMSCs transplantation efficacy in alleviating rat intestinal immune-barrier after I/R.

Aflatoxin B1 damaged structural barrier through Keap1a/Nrf2/ MLCK signaling pathways and immune barrier through NF-κB/ TOR signaling pathways in gill of grass carp (Ctenopharyngodon idella).

Aquafeeds are susceptible to contamination caused by aflatoxin B1 (AFB1). The gill of fish is an important respiratory organ. However, few studies have investigated the effects of dietary AFB1 exposure on gill. This study aimed to discuss the effects of AFB1 on the structural and immune barrier of grass carp gill. Dietary AFB1 increased reactive oxygen species (ROS) levels, protein carbonyl (PC) and malondialdehyde (MDA) contents, which consequently caused oxidative damage. In contrast, dietary AFB1 decreased antioxidant enzymes activities, relative genes expression (except MnSOD) and the contents of glutathione (GSH) (P < 0.05), which are partly regulated by NF-E2-related factor 2 (Nrf2/Keap1a). Moreover, dietary AFB1 caused DNA fragmentation. The relative genes of apoptosis (except Bcl-2, McL-1 and IAP) were significantly upregulated (P < 0.05), and apoptosis was likely upregulated through p38 mitogen-activated protein kinase (p38MAPK). The relative expressions of genes associated with tight junction complexes (TJs) (except ZO-1 and claudin-12) were significantly decreased (P < 0.05), and TJs were likely regulated by myosin light chain kinase (MLCK). Overall, dietary AFB1 disrupted the structural barrier of gill. Furthermore, AFB1 increased gill sensitivity to F. columnare, increased Columnaris disease and decreased the production of antimicrobial substances (P < 0.05) in grass carp gill, and upregulated the expression of genes involved with pro-inflammatory factors (except TNF-α and IL-8) and the pro-inflammatory response partly attributed to the regulation by nuclear factor κB (NF-κB). Meanwhile, the anti-inflammatory factors were downregulated (P < 0.05) in grass carp gill after challenge with F. columnare, which was partly attributed to the target of rapamycin (TOR). These results suggested that AFB1 aggravated the disruption of the immune barrier of grass carp gill after being challenge with F. columnare. Finally, the upper limit of safety of AFB1 for grass carp, based on Columnaris disease, was 31.10 µg/kg diet.

Contact pattern, current immune barrier, and pathogen virulence determines the optimal strategy of further vaccination.

Background: The current outbreak of novel coronavirus disease 2019 has caused a serious disease burden worldwide. Vaccines are an important factor to sustain the epidemic. Although with a relatively high-vaccination worldwide, the decay of vaccine efficacy and the arising of new variants lead us to the challenge of maintaining a sufficient immune barrier to protect the population. Method: A case-contact tracking data in Hunan, China, is used to estimate the contact pattern of cases for scenarios including school, workspace, etc, rather than ordinary susceptible population. Based on the estimated vaccine coverage and efficacy, a multi-group vaccinated-exposed-presymptomatic-symptomatic-asymptomatic-removed model (VEFIAR) with 8 age groups, with each partitioned into 4 vaccination status groups is developed. The optimal dose-wise vaccinating strategy is optimized based on the currently estimated immunity barrier of coverage and efficacy, using the greedy algorithm that minimizes the cumulative cases, population size of hospitalization and fatality respectively in a certain future interval. Parameters of Delta and Omicron variants are used respectively in the optimization. Results: The estimated contact matrices of cases showed a concentration on middle ages, and has compatible magnitudes compared to estimations from contact surveys in other studies. The VEFIAR model is numerically stable. The optimal controled vaccination strategy requires immediate vaccination on the un-vaccinated high-contact population of age 30-39 to reduce the cumulative cases, and is stable with different basic reproduction numbers ( R 0 ). As for minimizing hospitalization and fatality, the optimized strategy requires vaccination on the un-vaccinated of both aged 30-39 of high contact frequency and the vulnerable older. Conclusion: The objective of reducing transmission requires vaccination in age groups of the highest contact frequency, with more priority for un-vaccinated than un-fully or fully vaccinated. The objective of reducing total hospitalization and fatality requires not only to reduce transmission but also to protect the vulnerable older. The priority changes by vaccination progress. For any region, if the local contact pattern is available, then with the vaccination coverage, efficacy, and disease characteristics of relative risks in heterogeneous populations, the optimal dose-wise vaccinating process will be obtained and gives hints for decision-making.

Identification of a tumour immune barrier in the HCC microenvironment that determines the efficacy of immunotherapy.

BACKGROUND & AIMS: The tumour microenvironment (TME) is a crucial mediator of cancer progression and therapeutic outcome. The TME subtype correlates with patient response to immunotherapy in multiple cancers. Most previous studies have focused on the role of different cellular components in the TME associated with immunotherapy efficacy. However, the specific structure of the TME and its role in immunotherapy efficacy remain largely unknown. METHODS: We combined spatial transcriptomics with single-cell RNA-sequencing and multiplexed immunofluorescence to identify the specific spatial structures in the TME that determine the efficacy of immunotherapy in patients with hepatocellular carcinoma (HCC) receiving anti-PD-1 treatment. RESULTS: We identified a tumour immune barrier (TIB) structure, a spatial niche composed of SPP1+ macrophages and cancer-associated fibroblasts (CAFs) located near the tumour boundary, which is associated with the efficacy of immune checkpoint blockade. Furthermore, we dissected ligand‒receptor networks among malignant cells, SPP1+ macrophages, and CAFs; that is, the hypoxic microenvironment promotes SPP1 expression, and SPP1+ macrophages interact with CAFs to stimulate extracellular matrix remodelling and promote TIB structure formation, thereby limiting immune infiltration in the tumour core. Preclinically, the blockade of SPP1 or macrophage-specific deletion of Spp1 in mice led to enhanced efficacy of anti-PD-1 treatment in mouse liver cancer, accompanied by reduced CAF infiltration and increased cytotoxic T-cell infiltration. CONCLUSIONS: We identified that the TIB structure formed by the interaction of SPP1+ macrophages and CAFs is related to immunotherapy efficacy. Therefore, disruption of the TIB structure by blocking SPP1 may be considered a relevant therapeutic approach to enhance the therapeutic effect of immune checkpoint blockade in HCC. IMPACT AND IMPLICATIONS: Only a limited number of patients with hepatocellular carcinoma (HCC) benefit from tumour immunotherapy, which significantly hinders its application. Herein, we used multiomics to identify the spatial structure of the tumour immune barrier (TIB), which is formed by the interaction of SPP1+ macrophages and cancer-associated fibroblasts in the HCC microenvironment. This structure constrains immunotherapy efficacy by limiting immune cell infiltration into malignant regions. Preclinically, we revealed that blocking SPP1 or macrophage-specific deletion of Spp1 in mice could destroy the TIB structure and sensitize HCC cells to immunotherapy. These results provide the first key steps towards finding more effective therapies for HCC and have implications for physicians, scientists, and drug developers in the field of HCC.

Lactoferrin Alleviates Lipopolysaccharide-Induced Infantile Intestinal Immune Barrier Damage by Regulating an ELAVL1-Related Signaling Pathway.

As the most important intestinal mucosal barrier of the main body, the innate immune barrier in intestinal tract plays especially pivotal roles in the overall health conditions of infants and young children; therefore, how to strengthen the innate immune barrier is pivotal. A variety of bioactivities of lactoferrin (LF) has been widely proved, including alleviating enteritis and inhibiting colon cancer; however, the effects of LF on intestinal immune barrier in infants and young children are still unclear, and the specific mechanism on how LF inhibits infantile enteritis by regulating immune signaling pathways is unrevealed. In the present study, we firstly performed pharmacokinetic analyses of LF in mice intestinal tissues, stomach tissues and blood, through different administration methods, to confirm the metabolic method of LF in mammals. Then we constructed in Vitro and in Vivo infantile intestinal immune barrier damage models utilizing lipopolysaccharide (LPS), and evaluated the effects of LF in alleviating LPS-induced intestinal immune barrier damage. Next, the related immune molecular mechanism on how LF exerted protective effects was investigated, through RNA-seq analyses of the mouse primary intestinal epithelial cells, and the specific genes were analyzed and screened out. Finally, the genes and their related immune pathway were validated in mRNA and protein levels; the portions of special immune cells (CD4+ T cells and CD8+ T cells) were also detected to further support our experimental results. Pharmacokinetic analyses demonstrated that the integrity of LF could reach mice stomach and intestine after oral gavage within 12 h, and the proper administration of LF should be the oral route. LF was proven to down-regulate the expression levels of inflammatory cytokines in both the primary intestinal epithelial cells and mice blood, especially LF without iron (Apo-LF), indicating LF alleviated infantile intestinal immune barrier damage induced by LPS. And through RNA-seq analyses of the mouse primary intestinal epithelial cells treated with LPS and LF, embryonic lethal abnormal vision Drosophila 1 (ELAVL1) was selected as one of the key genes, then the ELAVL1/PI3K/NF-κB pathway regulated by LF was verified to participate in the protection of infantile intestinal immune barrier damage in our study. Additionally, the ratio of blood CD4+/CD8+ T cells was significantly higher in the LF-treated mice than in the control mice, indicating that LF distinctly reinforced the overall immunity of infantile mice, further validating the strengthening bioactivity of LF on infantile intestinal immune barrier. In summary, LF was proven to alleviate LPS-induced intestinal immune barrier damage in young mice through regulating ELAVL1-related immune signaling pathways, which would expand current knowledge of the functions of bioactive proteins in foods within different research layers, as well as benefit preclinical and clinical researches in a long run.

Progress of Studies on Plant-Derived Polysaccharides Affecting Intestinal Barrier Function in Poultry.

As natural bioactive components, plant-derived polysaccharides have many biological functions, such as anti-inflammatory, antioxidant, anticoccidial, and immunity regulation, and have been widely used in poultry production. In this review paper, firstly, the sources and structures of plant-derived polysaccharides are reviewed; secondly, the effects of plant-derived polysaccharides on the intestinal microbiome, permeability, morphology and immune function of poultry are summarized; thirdly, the potential molecular regulation mechanism of plant-derived polysaccharides on the intestinal barrier function of poultry was preliminarily analyzed. The review paper will bring a basis for the scientific utilization of plant-derived polysaccharides in the poultry industry.

FoxP3 expression by retinal pigment epithelial cells: transcription factor with potential relevance for the pathology of age-related macular degeneration.

BACKGROUND: Forkhead-Box-Protein P3 (FoxP3) is a transcription factor and marker of regulatory T cells, converting naive T cells into Tregs that can downregulate the effector function of other T cells. We previously detected the expression of FoxP3 in retinal pigment epithelial (RPE) cells, forming the outer blood-retina barrier of the immune privileged eye. METHODS: We investigated the expression, subcellular localization, and phosphorylation of FoxP3 in RPE cells in vivo and in vitro after treatment with various stressors including age, retinal laser burn, autoimmune inflammation, exposure to cigarette smoke, in addition of IL-1ß and mechanical cell monolayer destruction. Eye tissue from humans, mouse models of retinal degeneration and rats, and ARPE-19, a human RPE cell line for in vitro experiments, underwent immunohistochemical, immunofluorescence staining, and PCR or immunoblot analysis to determine the intracellular localization and phosphorylation of FoxP3. Cytokine expression of stressed cultured RPE cells was investigated by multiplex bead analysis. Depletion of the FoxP3 gene was performed with CRISPR/Cas9 editing. RESULTS: RPE in vivo displayed increased nuclear FoxP3-expression with increases in age and inflammation, long-term exposure of mice to cigarette smoke, or after laser burn injury. The human RPE cell line ARPE-19 constitutively expressed nuclear FoxP3 under non-confluent culture conditions, representing a regulatory phenotype under chronic stress. Confluently grown cells expressed cytosolic FoxP3 that was translocated to the nucleus after treatment with IL-1ß to imitate activated macrophages or after mechanical destruction of the monolayer. Moreover, with depletion of FoxP3, but not of a control gene, by CRISPR/Cas9 gene editing decreased stress resistance of RPE cells. CONCLUSION: Our data suggest that FoxP3 is upregulated by age and under cellular stress and might be important for RPE function.

Dietary mannan oligosaccharides strengthens intestinal immune barrier function via multipath cooperation during Aeromonas Hydrophila infection in grass carp (Ctenopharyngodon Idella).

In recent years, mannose oligosaccharide (MOS) as a functional additive is widely used in aquaculture, to enhance fish immunity. An evaluation of the effect of dietary MOS supplementation on the immune barrier function and related signaling molecules mechanism of grass carp (Ctenopharyngodon idella) was undertaken in the present study. Six diets with graded amounts of MOS supplementation (0, 200, 400, 600, 800, and 1000 mg/kg) were fed to 540 grass carp over 60 days. To examine the immune response and potential mechanisms of MOS supplementation on the intestine, a challenge test was conducted using injections of Aeromonas hydrophila for 14 days. Results of the study on the optimal supplementation with MOS were found as follows (1) MOS enhances immunity partly related to increasing antibacterial substances content and antimicrobial peptides expression; (2) MOS attenuates inflammatory response partly related to regulating the dynamic balance of intestinal inflammatory cytokines; (3) MOS regulates immune barrier function may partly be related to modulating TLRs/MyD88/NFκB and TOR/S6K1/4EBP signalling pathways. Finally, the current study concluded that MOS supplementation could improve fish intestinal immune barrier function under Aeromonas hydrophila infected conditions.

Supplementation of Dietary Crude Lentinan Improves the Intestinal Microbiota and Immune Barrier in Rainbow Trout (Oncorhynchus mykiss) Infected by Infectious Hematopoietic Necrosis Virus.

The effects of crude lentinan (CLNT) on the intestinal microbiota and the immune barrier were evaluated in rainbow trout (Oncorhynchus mykiss) infected by infectious hematopoietic necrosis virus (IHNV). The results showed that supplementary CLNT declined the rainbow trout mortality caused by IHNV, which suggested that CLNT has preventive effects on IHNV infection. IHNV destroyed intestinal integrity, as well as caused the intestinal oxidative and damage in rainbow trout. Supplementary CLNT significantly strengthened the intestinal immune barrier by declining intestinal permeability, as well as enhancing intestinal antioxidant and anti-inflammatory abilities in IHNV-infected rainbow trout (P<0.05). In addition, CLNT modified the aberrant changes of intestinal microbiota induced by IHNV, mainly represented by promoting the growths of Carnobacterium and Deefgea and inhibiting Mycobacterium and Nannocystis. Especially, supplementing with CLNT significantly promoted the growth of short-chain fatty acid-producing bacteria (P<0.05) and consequently increased the production of acetic acid, butanoic acid, and hexanoic acid in the intestine of IHNV-infected rainbow trout. Furthermore, it was speculated that CLNT could regulate the self-serving metabolic pathways of intestinal microbiota induced by IHNV, such as fatty acid metabolism and amino acid metabolism. Together, CLNT played the antiviral effects on IHNV infection through strengthening the intestinal immune barrier, as well as regulating intestinal microbiota and SCFA metabolism in rainbow trout. The present data revealed that CLNT exerted a promising prebiotic role in preventing the rainbow trout from IHNV infection.

A Quantitative Digital Analysis of Tissue Immune Components Reveals an Immunosuppressive and Anergic Immune Response with Relevant Prognostic Significance in Glioblastoma.

Objectives: Immunostimulatory therapies using immune checkpoint blockers show clinical activity in a subset of glioblastoma (GBM) patients. Several inhibitory mechanisms play a relevant role in the immune response to GBM. With the objective of analyzing the tumor immune microenvironment and its clinical significance, we quantified several relevant immune biomarkers. Design: We studied 76 primary (non-recurrent) GBMs with sufficient clinical follow-up, including a subgroup of patients treated with a dendritic cell vaccine. The IDH-mutation, EGFR-amplification, and MGMT methylation statuses were determined. Several relevant immune biomarkers, including CD163, CD8, PD1, and PDL1, were quantified in representative selected areas by digital image analysis and semiquantitative evaluation. The percentage of each immune expression was calculated with respect to the total number of tumor cells. Results: All GBMs were wild-type IDH, with a subgroup of classical GBMs according to the EGFR amplification (44%). Morphologically, CD163 immunostained microglia and intratumor clusters of macrophages were observed. A significant direct correlation was found between the expression of CD8 and the mechanisms of lymphocyte immunosuppression, in such a way that higher values of CD8 were directly associated with higher values of CD163 (p < 0.001), PDL1 (0.026), and PD1 (0.007). In a multivariate analysis, high expressions of CD8+ (HR = 2.05, 95%CI (1.02−4.13), p = 0.034) and CD163+ cells (HR 2.50, 95%CI (1.29−4.85), p = 0.007), were associated with shorter survival durations. The expression of immune biomarkers was higher in the non-classical (non-EGFR amplified tumors) GBMs. Other relevant prognostic factors were age, receipt of the dendritic cell vaccine, and MGMT methylation status. Conclusions: In accordance with the inverse correlation between CD8 and survival and the direct correlation between effector cells and CD163 macrophages and immune-checkpoint expression, we postulate that CD8 infiltration could be placed in a state of anergy or lymphocytic inefficient activity. Furthermore, the significant inverse correlation between CD163 tissue concentration and survival explains the relevance of this type of immune cell when creating a strong immunosuppressive environment. This information may potentially be used to support the selection of patients for immunotherapy.