TLR4 Expression Research Articles

Structural characterization of polysaccharides from Panax ginseng C. A. Meyer root and their triggered potential immunoregulatory and radioprotective activities

With people's increasing awareness of healthy diet, the diverse health-promoting functions of ginseng have been widely recognized. As one of the key functional components, ginseng polysaccharides have attracted increasing research interest. Here, three purified polysaccharide fractions, GPS-1a, GPS-1b, and GPS-2, were obtained from the root extract of Panax ginseng C. A. Meyer. Structurally, GPS-1a and GPS-1b were both linked in a → 6)-α-D-Glcp-(1 → pattern but composed of glucose and galactose in molar ratios of 9.76:0.24 and 9.81:0.19. In contrast, GPS-2 was composed of glucose, galactose, arabinose, rhamnose, and galacturonic acid in a molar ratio of 1.82:1.94:0.79:0.52:4.93. The main backbone consisted of →4)-α-D-GalpA-(1→, →4)-α-D-GalpA-6OMe-(1→, →3, 4)-α-D-GalpA-(1→, →3)-α-L-Rhap-(1 → linages, and its branches are composed of →5)-α-L-Araf-(1→, →4)-β-D-Galp-(1→, →2)-β-D-Glcp-(1→, α-D-GalAp-(1→. Benefitting from this structural variance, GPS-2 exhibited the most significant immunoregulatory and radioprotective efficacies. Specifically, GPS-2 promoted TLR2, NF-κB, and TRAF6 protein expression levels, thereby significantly improving macrophage phagocytosis, splenic lymphocyte proliferation, and stimulation of NO, IL-1β, IL-6, and TNF-α secretion, which activated RAW264.7 and splenic lymphocytes. The following radioprotection activity tests unveiled that GPS-2 increased the organ index, number of peripheral blood cells, cellularity of splenocytes, and bone marrow cell numbers in irradiated mice. This investigation revealed the contribution of polysaccharide structure characteristics to the bioactive expression and elucidated the potential utilization of GPS-2 as a radioprotective agent or immunomodulator.

Investigation of TLR4, TLR6, TLR7, and CD36 expression on T lymphocytes in coronary artery disease

Background: Coronary artery disease (CAD) is among the significant causes of death globally, caused by fatty deposits in blood vessel walls. Increasing evidence indicates that toll-like receptors (TLRs) are pivotal to atherosclerosis progression. The function of CD36 as a glycoprotein in atherosclerosis was also suggested. This study aimed to investigate the levels of TLR4, TLR6, and TLR7, as well as CD36 cell surface markers in CAD. Methods: This study included 64 patients undergoing angiography to determine whether atherosclerosis was present. The patients were categorized into two groups. The three main coronary arteries are all stenosed at over 50% in patients with CAD+ (n=22) as cases and CAD− (n=42) with smooth angiography as controls. Each patient's syntax score (SS) and Gensini (vessel score) were calculated. The TLR-4, TLR-6, TLR-7, and CD36 expression were measured using Flow cytometry. Results: TLR4 and TLR6 showed a significant link with the Gensini score in the subject group. In addition, TLR7 had an association with Syntax scoring in this study. Conclusion: The findings illustrated the association between TLR4, TLR6, and TLR7 cell surface markers with Gensini and Syntax scoring in individuals with coronary artery disease.

Effects of an Extract of Physalis Peruviana Linnaeus on the Expression of Inflammatory Markers in the Caco-2 Intestinal Epithelium-like Cell Line

Objective: Physalis Peruviana Linnaeus (PPL) is an herbaceous species characterized by a wide variety of bioactive compounds to which anti-inflammatory properties have been attributed. This makes this fruit a possible complementary therapy for diseases that involve chronic inflammation, such as inflammatory bowel diseases (IBD). In the present study, the effect of a PPL extract on the expression of inflammatory markers in the Caco-2 cell line was evaluated. Methods: An in vitro gastric digest (50 g PPL pulp) was performed, obtaining an extract that was used to challenge Caco-2 cells for 24 and 72 hours. This extract was characterized by LC-MS/MS. Then, the relative mRNA expression of NF-kB, TLR4, IL-18 and MCP-1 was determined through qRT-PCR and the protein levels of TNF-α, IL-6, IL-8, IL-18 and MCP-1 through Luminex Immunoassay. Results: From the characterization of the extract, compounds with bioactive potential such as isothiocyanates, indoles and coumarins were found. Treatment of Caco-2 cells with PPL extract (80 µg/ml), particularly for 72 hours, produced a reduction of IL-18 and MCP-1 mRNA expression (p < 0.01), in addition to IL-18 (p < 0.01), IL-8 (p < 0.0001) and MCP-1 (p < 0.01) protein levels, however, no effects on NF-kB p65 (p = 0.09) and TLR4 (p = 0.20) mRNA expression were observed. Conclusion: The results obtained in this study open the possibility that the regular consumption of 50 g of PPL could constitute a possible complementary therapy for the treatment of IBD, improving the quality of life of these patients.

Immune mRNA Expression and Fecal Microbiome Composition Change Induced by Djulis (Chenopodium formosanum Koidz.) Supplementation in Aged Mice: A Pilot Study.

Background and Objectives: The aging process has always been associated with a higher susceptibility to chronic inflammatory lung diseases. Several studies have demonstrated the gut microbiome's influence on the lungs through cross-talk or the gut-lungs axis maintaining nutrient-rich microenvironments. Taiwan djulis (Chenopodium formosanum Koidz.) provides antioxidant and anti-inflammatory characteristics that could modulate the gut microbiome. This could induce the gut-lung axis through microbial cross-talk, thus favoring the modulation of lung inflammation. Materials and Methods: Here, we investigate the immune mRNA expression in the spleen, fecal microbiome composition, and hyperplasia of the bronchial epithelium in aged 2-year-old BALB/c mice after 60 days of supplementation of djulis. Results: The pro-inflammatory cytokines IFN-γ, TNF-α, and IL-1β, T; cells CD4 and CD8; and TLRs TLR3, TLR4, TLR5, TLR7, TLR8, and TLR9 were reduced in their mRNA expression levels, while the anti-inflammatory cytokines IL-2, IL-4, and IL-10 were highly expressed in the C. formosanum-treated group. Interestingly, the fecal microbiome composition analysis indicated higher diversity in the C. formosanum-treated group and the presence of butyrate-producing bacteria that are beneficial in the gut microbiome. The histopathology showed reduced hyperplasia of the bronchial epithelium based on the degree of lesions. Conclusions: Our findings suggest that Taiwan djulis can modulate the gut microbiome, leading to microbial cross-talk; reducing the mRNA expression of pro-inflammatory cytokines, T cells, and TLRs; and increasing anti-inflammatory cytokines in the spleen, as cytokines migrate in the lungs, preventing lung inflammation damage in aged mice or the gut-lung axis. Thus, Taiwan djulis could be considered a beneficial dietary component for the older adult population. The major limitation includes a lack of protein validation of cytokines and TLRs and quantification of the T cell population in the spleen as a marker of the gut-lung axis.

Neutralization of TLR2 in combination with either TNF-α or IL-1β antibody reduces the severity of septic arthritis through STAT3/mTOR signalling in lymphocytes

Staphylococcus aureus induced Septic arthritis is considered a medical concern. S.aureus binds TLR2 to induce an array of inflammatory responses. Generation of pro-inflammatory cytokines induces T cell responses and control Th17/Treg cell balance. Regulation of T cell-mediated immunity in response to inflammation is significantly influenced by mTOR. Presence of elevated TNF-α, IL-1β decreases Treg cell activity through STAT3/mTOR, promoting proliferation of T cells towards Th17 cells. Therefore, we postulated, neutralizing TLR2 with either TNF-α or IL-1β in combination could be useful in modifying Th17/Treg cell ratio in order to treat septic arthritis by suppressing expression of mTOR/STAT3. To date, no studies have reported effects of neutralization of TLR2 along with either TNF-α or IL-1β on amelioration of arthritis correlating with mTOR/STAT3 expression. Contribution of T lymphocytes collected from blood, spleen, synovial tissues, their derived cytokines IFN-γ, IL-6, IL-17, TGF-β, IL-10 were noted. Expression of TLR2, TNFR1, TNFR2, NF-κB along with mTOR/STAT3 also recorded. Neutralization of TLR2 along with TNF-α and IL-1β were able to shift Th17 cells into immunosuppressive Treg cells. Furthermore,elevated expression of IL-10, TNFR2 and demoted expression of mTOR/ STAT3 along with NF-κB in lymphocytes confirms its role in resolution of arthritis. It was also effective in reducing oxidative stress via increasing expression of the antioxidant enzymes. As a result, it can be inferred that Treg-derived IL-10, which may mitigate inflammatory effects of septic arthritis by influencing the mTOR/STAT3 interaction in lymphocytes, may be selected as a different therapeutic strategy for reducing the impact of septic arthritis.

Impact of Extreme Prematurity, Chorioamnionitis, and Sepsis on Neonatal Monocyte Characteristics and Functions

Introduction: The innate branch of the immune system is important in early life, in particular for infants born preterm. Methods: We performed a longitudinal analysis of the peripheral monocyte compartment in extremely preterm children from a randomized, placebo-controlled study of probiotic supplementation. PBMCs and fecal samples were collected at several timepoints during the first months of life. Monocyte characteristics were analyzed by flow cytometry, and LPS-stimulated PBMC culture supernatants were analyzed by Luminex or ELISA. Plasma cytokines and gut microbiota composition were analyzed by ELISA and 16S rRNA-sequencing, respectively. Results: The extremely preterm infants had persistent alterations in their monocyte characteristics that were further aggravated in chorioamnionitis cases. They showed a markedly reduced TLR4 expression and hampered LPS-stimulated cytokine responses 14 days after birth. Notably, at later timepoints, TLR4 expression and LPS responses no longer correlated. Sepsis during the first weeks of life strongly associated with increased pro-inflammatory, and reduced IL-10, responses also at postmenstrual week 36. Further, we report a correlation between gut microbiota features and monocyte phenotype and responses, but also that probiotic supplementation associated with distinct monocyte phenotypic characteristics, without significantly influencing their responsiveness. Conclusion: Extremely preterm infants have monocyte characteristics and functional features that deviate from infants born full-term. Some of these differences persist until they reach an age corresponding to full-term, potentially making them more vulnerable to microbial exposures during the first months of life.

PSVII-16 Recombinant bovine lactoferrin expressed in Schizochytrium sp. alleviates inflammation and oxidative damage in calf intestinal epithelial cells

Abstract This study aims to generate recombinant bovine lactoferrin (rbLF) by expressing it in Schizochytrium sp. (SZ) and evaluate its effects on calf intestinal epithelial cells (CIECs) exposed to lipopolysaccharide (LPS) or hydrogen peroxide-induced damage. The bovine lactoferrin expression cassette was transfected into Schizochytrium sp. via 18s rRNA gene recombination. Primary CIECs were utilized in this investigation, and the optimal dose of rbLF was evaluated by cell counting kit-8 assay after being treated with different concentrations of rbLF for 24 h. The optimal dose of rbLF was then used as a medium supplement before or after 24 h of 80 μg/mL LPS challenge or 4 h of 400 μM of hydrogen peroxide (H2O2) challenge. Cell viability, gene expression of tumor necrosis factor (TNF) -α, toll-like receptor (TLR) -4, interleukin (IL) -17A, zonula occludens-1 (ZO-1), and claudin-1, as well as protein levels of TNF-α, IL-17A, ZO-1, and claudin-1, were assessed to elucidate the preventive and therapeutic effects of rbLF. Data analysis was performed using SAS 9.4 by F-test, The statistical significance was declared at P &amp;lt; 0.05. Results indicate that rbLF demonstrated no cytotoxicity within the 50 to 1,000 μg/mL range on CIECs. Treatment with 175 μg/mL rbLF for 24 h increased cell viability, expression, and protein concentrations of ZO-1 and claudin following hydrogen peroxide challenge (P &amp;lt; 0.05). Moreover, rbLF pretreatment for 24 hours, despite decreasing cell viability after the H2O2 challenge, increased the expression and protein concentrations of ZO-1 and claudin-1 compared with non-rbLF-treated groups (P &amp;lt; 0.05). When used to prevent LPS-induced inflammation, rbLF enhanced the gene expression and protein concentrations of TNF-α, ZO-1, and claudin-1 compared with controls (P &amp;lt; 0.05). As a treatment for LPS-induced inflammation, rbLF decreased TLR4, TNF-α, and IL-17A expression levels (P &amp;lt; 0.05). These findings suggest that SZ-expressed rbLF is safe for calve intestinal cells and 175 μg/mL of rbLF may mitigate inflammation-induced intestinal barrier dysfunction.

Platycodin D: A promising anti tilapia lake virus natural compound from Platycodon grandiflorus

Tilapia lake virus (TiLV) poses a significant threat to the worldwide tilapia aquaculture sector, and there currently exists no efficient method of containment for this lethal pathogen. Given the widespread utilization of herbal medicines in the treatment of various aquatic animal diseases, it becomes imperative to evaluate and identify herbs that can be used for developing effective drugs against TiLV. We evaluated the efficacy of 20 herbs to inhibit TiLV proliferation in a TiLV infection model (Oreochromis niloticus) through two rounds of screening, with Platycodon grandiflorus extract showing the strongest inhibitory effect on TiLV replication (IC50 = 72.619 mg/L), survival of TiLV-infected tilapia was also improved in individual experiments. Platycodin D (PD), the principal bioactive compound found in Platycodon grandiflorus, demonstrated a dose-dependent reduction in viral load when used for treatment and enhanced the survival rate of tilapia infected with TiLV (42.3 %). The results of serum enzyme activity showed that PD treatment at a concentration of 5 mg/kg significantly enhance the non-specific parameters in tilapia (GSH, ACP, and AKP) (**p < 0.01). Additionally, immune-related gene analysis indicated that PD up-regulated IFN-I, CC2 and other genes expression while down-regulated the expression of pro-inflammatory factors (TNF-α, IL-1β) and TLR2 in the spleen and kidney of tilapia, implying that PD could induce IFN-I expression resulting in potential antiviral effects and inhibit some pro-inflammatory factors to alleviate oxidative stress and inflammatory responses in tilapia. The aforementioned findings suggest that Platycodon grandiflorus could potentially serve as a promising candidate for the development of drugs targeting TiLV. In particular, the bioactive component PD showed a strong anti-TiLV effect, which needs to be further explored.

Unravelling bisphenol A-induced hepatotoxicity: Insights into oxidative stress, inflammation, and energy dysregulation

Bisphenol A (BPA), a prevalent plastic monomer and endocrine disruptor, negatively impacts metabolic functions. This study examines the chronic effects of eco-relevant BPA concentrations on hepatotoxicity, focusing on redox balance, inflammatory response, cellular energy sensors, and metabolic homeostasis in male Swiss albino mice. Chronic BPA exposure resulted in reactive oxygen species (ROS) accumulation, altered hepatic antioxidant defense, lipid peroxidation, and NOX4 expression, leading to reduced cell viability. Additionally, BPA exposure significantly upregulated hepatic pro-inflammatory cytokine genes (Tnf-α, Il-1β, Il-6), NOS2, and arginase II, correlating with increased TLR4 expression, NF-κB phosphorylation, and a dose-dependent decrease in IκBα levels. BPA-induced NF-κB nuclear localization and inflammasome activation (NLRP3, cleaved caspase-1, IL-1β) established an inflammatory milieu. Perturbations in hepatic AMPKα phosphorylation, SIRT1, and PGC-1α, along with elevated p38 MAPK phosphorylation and ERα expression, indicated BPA-induced energy dysregulation. Furthermore, increased PLA2G4A, COX1, COX2, and PTGES2 expression in BPA-treated liver correlated with hyperlipidemia, hepatic FASN expression, steatosis, and visceral adiposity, likely due to disrupted energy sensors, oxidative stress, and inflammasome activation. Elevated liver enzymes (ALP, AST, ALT) and apoptotic markers indicated liver damage. Notably, N-acetylcysteine (NAC) priming reversed BPA-induced hepatocellular ROS accumulation, NF-κB-inflammasome activation, and intracellular lipid accumulation, while upregulating cellular energy sensors and attenuating ERα expression, suggesting NAC's protective effects against BPA-induced hepatotoxicity. Pharmacological inhibition of the NF-κB/NLRP3 cascade in BAY11-7082 pretreated, or NLRP3 immunodepleted hepatocytes reversed BPA's negative impact on SIRT1/p-AMPKα/PGC-1α and intracellular lipid accumulation, providing mechanistic insights into BPA-induced metabolic disruption.

Effect of nystatin on invasion of &lt;i&gt;Serratia grimesii&lt;/i&gt; and &lt;i&gt;Serratia proteamaculans&lt;/i&gt; bacteria into epithelial cells

BACKGROUND: Bacteria use various endocytic pathways during entering non-phagocytic cells. The involvement of caveolae/lipid rafts in bacterial invasion has been demonstrated for many bacterial pathogens. However, for bacteria of the genus Serratia, the involvement of membrane microdomains in the process of bacterial internalization has been poorly studied. AIM: To evaluate the involvement of caveolae/lipid rafts in the invasion of S. grimesii and S. proteamaculans bacteria into non-phagocytic epithelial Caco-2 and M-HeLa cells using nystatin. MATERIALS AND METHODS: M-HeLa and Caco-2 epithelial cells were incubated with 50 μM nystatin for 1 hour at 37 ºC, after which they were infected with the bacteria S. grimesii strain 30063 and S. proteamaculans strain 94, the multiplicity of infection was 100 bacteria per cell. The number of intracellular bacteria was assessed using gentamicin protection assay. The level of caveolin-1 in cells was visualized using confocal microscopy and Western blotting. The expression of Toll-like receptors genes were measured by real-time RT-PCR. RESULTS: Treatment of epithelial cells with nystatin reduces the internalization of S. grimesii and S. proteamaculans into M-HeLa cells by 30% and does not affect penetration into Caco-2 cells. At the same time, nystatin does not affect the redistribution / the integrity impairment of lipid rafts and does not lead to the cytoskeleton reorganization of eukaryotic cells. The addition of nystatin increases the level of caveolin-1 in M-HeLa cells (caveolin-1 is not expressed in Caco-2), which leads to a change plasma membrane fluidity. Nystatin promotes the secretion of proinflammatory cytokines interleukin-6 and interleukin-8 in both cell lines. Infection of M-HeLa cells pretreated with nystatin with the studied bacteria leads to an increase in the expression of tlr2 and tlr4 genes, but does not exceed the level of their expression in control samples. Therefore, it is impossible to speak unambiguously about the participation of Toll-like receptors in the invasion of Serratia bacteria. CONCLUSIONS: The results obtained suggest that the interaction of bacteria with eukaryotic cells induces the expression of caveolin-1, which leads to a change plasma membrane components mobility. This may be due to the fact that β1-integrin is involved in the invasion of the studied bacteria, which should be stabilized at the plasma membrane upon binding of the ligand due to the formation of a cholesterol- and sphingolipid-rich membrane microenvironment.

Viral reprogramming by nervous necrosis virus alters key metabolites and its pathways in sevenband grouper (Hyporthodus septemfasciatus) gills

Nervous necrosis virus (NNV) which mainly infects sevenband grouper (Hyporthodus Septemfasciatus) is considered a potential threat to the grouper aquaculture industry. The gills being one of the portal of entry and an active site of replication of fish viruses emphasises its role as a key region to study the metabolomic changes caused by viral reprograming and hijacking of metabolic pathways associated with immunity of the host. In the present study, liquid chromatography mass spectrometry (LC-MS) was used to detect changes of endogenous compounds of the grouper after NNV infection. A total of 75 metabolites of ten different pathways were identified. The metabolites were mainly associated with fatty acids, lipids, amino acids and nucleotides. The virus reprogramming lead to the downregulation of majority of the metabolites in their pathways. Arachidonic acid (AA), tryptophan, kynurenine and methandriol were selected as representative metabolites and challenge studies with NNV confirmed the fact that, metabolites controlled the replication of virus in a dose dependent manner. Immune gene expression studies also confirmed the effect of metabolites by upregulated expression of interleukins, cytokines and TLRs which are part of cellular immune response. This study shows the viral reprogramming of NNV in grouper gill cells resulting in alterations in basic metabolic pathways associated with normal functioning of the organism.

Minthostachys verticillata essential oil modulates cytokine synthesis and Staphylococcus aureus internalization in MAC-T cells at least through TLR4/MyD88/NFkB pathway.

The aim of this study was to evaluate the activation pathway(s) triggered by Minthostachys verticillata essential oil (EO) in bovine mammary epithelial cells (MAC-T) challenged with a strain of bovine Staphylococcus aureus. MAC-T cells were stimulated with EO, S. aureus or pre-treated with EO and then challenged with S. aureus. Cytokine's release was measured by ELISA. The mRNA for TLR2, TLR4, NOD2, MyD88 and NFκB was quantified by RT-qPCR. S. aureus adherence and internalization was also evaluated. MAC-T cells stimulated with S. aureus synthesized high levels of IL-1ß and IL-6 were kept up to 48 h, while IL-4 levels were not altered.Cells pre-treated with EO for 2 and 6h and then challenged with S. aureus showed a significant increase of IL-1β and IL-6. However, in these cells, a decrease in IL-1ß and IL-6 levels and an increase of IL-4 values was observed from 24 h. No significant increase in the expression levels of TLR2 or NOD2 was detected in all stimulated cells. However, the expression of TLR4, MyD88 and NFκB was increased in cells stimulated with S. aureus at 2 and 6 h as well as in cells pre-treated with EO between 2 and 6h and then challenged with S. aureus. The NFκB expression levels was similar to control at 24h in all stimulated cells, although pro-inflammatory cytokine levels and TLR4 and MyD88 expression levels remained high in cells stimulated with S. aureus. This results suggested the activation of other pathways independent of MyD88 by the pathogen that involucrated the activation of others transcription factors. Pre-treatment with EO during 2, 6 and 24h did not affect S. aureus adherence but decreased its internalization. In conclusion, pre-treatment with EO increased the IL-1β and IL-6 synthesis during the first hours post-challenged with S. aureus up-regulating TLR4/MyD88/NFκB pathway. Furthermore, EO increased the IL-4 levels from 6 to 24 h down-regulating the NFκB and possibly other transcription factors activated by the pathogen, which decreased its internalization into MAC-T cells.

Subgroup Analysis of TLR2, -3, -4 and -8 in Relation to the Severity of Clinical Manifestations of Cervical HPV Infection.

We present the findings of assessing the expression levels of extracellular TLR2 and TLR4 and intracellular TLR3 and TLR8 correlating with the severity of clinical manifestations of HPV infection. A total of 199 women took part in a single-center prospective comparative research study on TLR2, TLR3, TLR4 and TLR8 expression in HPV-related cervical lesions. TLRs' mRNA expression was analyzed using real-time reverse transcription polymerase chain reaction (RT-PCR). Our results indicate the potential significance of TLR3, TLR4 and TLR8 in responding to HPV infection and its progression to SILs and CC, highlighting the importance of HPV polyinfection in relation to TLR4 and TLR8.

Inclusion of sorghum (Sorghum bicolor (L). Moench) with protease and NSP-ase increases jejunal histomorphology and barrier function gene expression in broiler chickens

The objective of this study was to investigate the effect of sorghum with or without protease and NSP-ase on performance, jejunal histomorphology, caecal short-chain fatty acids (SCFA), and gene expression of barrier function in the broiler chickens. A total of 240 males New Lohmann strain MB 202 broiler chicks (1-day-old) were randomly placed into 12 treatments with 4 replications, 5 birds per replicate for 35 d. The diets were a factorial combination of 3 levels of sorghum (0, 10, and 20%), 2 concentrations of protease (with and without protease), and 2 concentrations of NSP-ase (with and without NSP-ase). Results showed that villus height (VH) was improved (P < 0.05) and villus to crypt ratio (VH:CD) tended (P = 0.05) to increase with inclusion of sorghum. Inclusion of sorghum upregulated (P < 0.05) expression of the gene anti-inflammatory IL-13, and downregulated (P < 0.05) gene of toll receptor (TLR) (TLR-3 and TLR-4), pro-inflammatory (IL-18, IL-6, tumor necrosis factor-alpha (TNF-α) and tight junction zona occluding-1 (ZO-1). The result further indicated a decrease (P < 0.05) in the concentration of SCFA. The birds fed diets with protease had lower (P < 0.05) expression of gene TLR-4 and TLR-3, pro-inflammatory (IL-6 and TNF-α), and anti-inflammatory IL-10. Inclusion of sorghum markedly (P < 0.05) interacted with protease, increasing the total of SCFA, acetate, and VH in the broiler. The bird offered diets with NSP-ase had higher (P < 0.05) expression genes of pro-inflammatory IL-18 and TNF-α. Inclusion of sorghum and NSP-ase significantly (P < 0.05) interacted, upregulating gene of tight junction ZO-1 and anti-inflammatory IL-13 but downregulating gene TLR-3, and pro-inflammatory IL-6, while the SCFA production was decreased. Inclusion of sorghum in the diet with or without enzymes did not affect broiler performance (P > 0.05). In conclusion, a diet with sorghum improved jejunal histomorphology without negatively affecting growth performance. Additionally, supplementation with enzymes enhanced intestinal immune responses by up-regulating anti-inflammatory, and downregulating pro-inflammatory gene expression.

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-1β, 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.

Terminalia bellirica and Andrographis paniculata dietary supplementation in mitigating heat stress-induced behavioral, metabolic and genetic alterations in broiler chickens

BackgroundHeat stress (HS) is one of the most significant environmental stressors on poultry production and welfare worldwide. Identification of innovative and effective solutions is necessary. This study evaluated the effects of phytogenic feed additives (PHY) containing Terminalia bellirica and Andrographis paniculata on behavioral patterns, hematological and biochemical parameters, Oxidative stress biomarkers, and HSP70, I-FABP2, IL10, TLR4, and mTOR genes expression in different organs of broiler chickens under chronic HS conditions. A total of 208 one-day-old Avian-480 broiler chicks were randomly allocated into four treatments (4 replicate/treatment, 52 birds/treatment): Thermoneutral control treatment (TN, fed basal diet); Thermoneutral treatment (TN, fed basal diet + 1 kg/ton feed PHY); Heat stress treatment (HS, fed basal diet); Heat stress treatment (HS, fed basal diet + 1 kg/ton feed PHY).ResultsThe findings of the study indicate that HS led to a decrease in feeding, foraging, walking, and comfort behavior while increasing drinking and resting behavior, also HS increased red, and white blood cells (RBCs and WBCs) counts, and the heterophile/ lymphocyte (H/L) ratio (P < 0.05); while both mean corpuscular volume (MCV), and mean corpuscular hemoglobin (MCH) were decreased (P < 0.05). In addition, HS negatively impacted lipid, protein, and glucose levels, liver and kidney function tests, and oxidative biomarkers by increasing malondialdehyde (MDA) levels and decreasing reduced glutathion (GSH) activity (P < 0.05). Heat stress (HS) caused the upregulation in HSP70, duodenal TLR4 gene expression, and the downregulation of I-FABP2, IL10, mTOR in all investigated tissues, and hepatic TLR4 (P < 0.05) compared with the TN treatment. Phytogenic feed additives (PHY) effectively mitigated heat stress’s negative impacts on broilers via an improvement of broilers’ behavior, hematological, biochemical, and oxidative stress biomarkers with a marked decrease in HSP70 expression levels while all tissues showed increased I-FABP2, IL10, TLR4, and mTOR (except liver) levels (P < 0.05).ConclusionPhytogenic feed additives (PHY) containing Terminalia bellirica and Andrographis paniculata have ameliorated the HS-induced oxidative stress and improved the immunity as well as the gut health and welfare of broiler chickens.

Vibriocidal efficacy of Bifidobacterium bifidum and Lactobacillus acidophilus cell-free supernatants encapsulated in chitosan nanoparticles against multi-drug resistant Vibrio cholerae O1 El Tor

BackgroundCholera is a diarrheal disease recognized for being caused by toxin-producing Vibrio (V.) cholerae. This study aims to assess the vibriocidal and immunomodulatory properties of derived cell-free supernatants (CFSs) of Bifidobacterium (B.) bifidum and Lactobacillus (L.) acidophilus encapsulated in chitosan nanoparticles (CFSb-CsNPs and CFSa-CsNPs) against clinical multi-drug resistance (MDR) isolates of V. cholerae O1 El Tor.MethodsWe synthesized CFSb-CsNPs and CFSa-CsNPs using the ionic gelation technique. The newly nanostructures were characterized for size, surface zeta potential, morphology, encapsulation efficacy (EE), stability in different pH values and temperatures, release profile, and in vitro cytotoxicity. The antimicrobial and antibiofilm effects of the obtained nanocomposites on clinical MDR isolates (N = 5) of V. cholerae E1 Tor O1 were investigated by microbroth dilution assay and crystal violet staining, respectively. We conducted quantitative real-time PCR (qRT-PCR) to analyze the relative gene expressions of Bap, Rbmc, CTXAB, and TCP in response to CFSb-CsNPs and CFSa-CsNPs. Additionally, the immunomodulatory effects of formulated structures on the expression of TLR2 and TLR4 genes in human colorectal adenocarcinoma cells (Caco-2) were studied.ResultsNano-characterization analyses indicated that CFSb-CsNPs and CFSa-CsNPs exhibit spherical shapes under scanning electron microscopy (SEM) imaging, with mean diameters of 98.16 ± 0.763 nm and 83.90 ± 0.854 nm, respectively. Both types of nanoparticles possess positive surface charges. The EE% of CFSb-CsNPs was 77 ± 4.28%, whereas that of CFSa-CsNPs was 62.5 ± 7.33%. Chitosan (Cs) encapsulation leads to increased stability of CFSs in simulated pH conditions of the gastrointestinal tract in which the release rates for CFSb-CsNPs and CFSa-CsNPs were reached at 58.00 ± 1.24% and 55.01 ± 1.73%, respectively at pH = 7.4. The synergistic vibriocidal effects observed from the co-administration of both CFSb-CsNPs and CFSa-CsNPs, as evidenced by a fractional inhibitory concentration (FIC) index of 0.57, resulting in a significantly lower MIC of 2.5 ± 0.05 mg/mL (p < 0.0001) compare to individual administration. The combined antibacterial effect of CFSb-CsNPs and CFSa-CsNPs on Bap (0.14 ± 0.05), Rbmc (0.24 ± 0.01), CTXAB (0.30 ± 0.09), and TCP (0.38 ± 0.01) gene expression was significant (p < 0.001). Furthermore, co-administration of CFSb-CsNPs and CFSa-CsNPs also demonstrated the potency of suppressing TLR 2/4 (0.20 ± 0.01 and 0.12 ± 0.02, respectively) gene expression (p = 0.0019) and reduced Caco-2 cells attached bacteria to 526,000 ± 51,46 colony-forming units/mL (11.19%) (p < 0.0001).ConclusionOur study revealed that encapsulating CFSs within CsNPs enhances their vibriocidal activity by improving stability and enabling a controlled release mechanism at the site of interaction between the host and bacteria. Additionally, the simultaneous use of CFSb-CsNPs and CFSa-CsNPs exhibited superior vibriocidal potency against MDR V. cholerae O1 El Tor strains, indicating these combinations as a potential new approach against MDR bacteria.

Multifunctional natamycin modified chondroitin sulfate eye drops with anti-inflammatory, antifungal and tissue repair functions possess therapeutic effects on fungal keratitis in mice.

Fungal keratitis (FK) is recognized as a stubborn ocular condition, caused by intense fungal invasiveness and heightened immune reaction. The glycosaminoglycan chondroitin sulfate exhibits properties of immunomodulation and tissue regeneration. In prior investigations, oxidized chondroitin sulfate (OCS) ameliorated the prognosis of FK in murine models. To further improve the curative efficacy, we used the antifungal drug natamycin to functionalize OCS and prepared oxidized chondroitin sulfate-natamycin (ON) eye drops. The structure of ON was characterized by FTIR, UV–vis, and XPS, revealing that the amino group of natamycin combined with the aldehyde group in OCS through Schiff base reaction. Antifungal experiments revealed that ON inhibited fungal growth and disrupted the mycelium structure. ON exhibited exceptional biocompatibility and promoted the proliferation of corneal epithelial cells. Pharmacokinetic analysis indicated that ON enhanced drug utilization by extending the mean residence time in tears. In murine FK, ON treatment reduced the clinical score and corneal fungal load, restored corneal stroma conformation, and facilitated epithelial repair. ON effectively inhibited neutrophil infiltration and decreased the expression of TLR-4, LOX-1, IL-1β, and TNF-α. Our research demonstrated that ON eye drops achieved multifunctional treatment for FK, including inhibiting fungal growth, promoting corneal repair, enhancing drug bioavailability, and controlling inflammatory reactions.

TLR10 (CD290) Is a Regulator of Immune Responses in Human Plasmacytoid Dendritic Cells.

TLRs are the most thoroughly studied group of pattern-recognition receptors that play a central role in innate immunity. Among them, TLR10 (CD290) remains the only TLR family member without a known ligand and clearly defined functions. One major impediment to studying TLR10 is its absence in mice. A recent study on TLR10 knock-in mice demonstrated its intrinsic inhibitory role in B cells, indicating that TLR10 is a potential drug target in autoimmune diseases. In this study, we interrogated the expression and function of TLR10 in human plasmacytoid dendritic cells (pDCs). We have seen that primary human pDCs, B cells, and monocytes constitutively express TLR10. Upon preincubation with an anti-TLR10 Ab, production of cytokines in pDCs was downregulated in response to stimulation with DNA and RNA viruses. Upon further investigation into the possible mechanism, we documented phosphorylation of STAT3 upon Ab-mediated engagement of TLR10. This leads to the induction of inhibitory molecule suppressor of cytokine signaling 3 (SOCS3) expression. We have also documented the inhibition of nuclear translocation of transcription factor IFN regulatory factor 7 (IRF7) in pDCs following TLR10 engagement. Our data provide the (to our knowledge) first evidence that TLR10 is constitutively expressed on the surface of human pDCs and works as a regulator of their innate response. Our findings indicate the potential of harnessing the function of pDCs by Ab-mediated targeting of TLR10 that may open a new therapeutic avenue for autoimmune disorders.

Glutathione regulates CIA-activated splenic-lymphocytes via NF-κB/MMP-9 and MAPK/PCNA pathways manipulating immune response

Reduced glutathione (GSH) is an antioxidant involved in redox homeostasis, and recently regarded as an inducer of Reductive stress. Its immune-regulatory effects on lymphocytes have not been extensively studied. This study is based on the finding that much increased GSH level in collagen-induced arthritis (CIA) rat spleen, and aimed to investigate the effects of GSH (0, 1, 10, 100 mM) on normal and immune-stimulated spleen lymphocytes respectively. The elevated GSH level is associated with the increased levels of inflammatory factors; especially the increased DPP1 activity indicated immune-granulocytes activation in CIA rat spleen. Exogenous GSH had different influences on normal and CIA lymphocytes, affecting intracellular levels of GSH, Glutathione-S-transferases (GSTs) and Reactive oxygen species (ROS); as well as the expressions of NF-κB, MMP-9, Bcl-2, GST, P38, PCNA and TLR4. The increased extracellular GSH level disturbed redox homeostasis and induces reductive stress to spleen lymphocytes, which decreased intracellular GSH concentration and influenced the MAPK/PCNA and NF-κB/MMP-9 signaling pathways, as well as cell cycles respectively, leading to cell senescence/ferroptosis /apoptosis. This study also revealed the multiple faces of GSH in regulating spleen lymphocytes, which depends on its levels in tissue or in cells, and the activation status of lymphocytes. These findings indicate the immune-regulatory role of GSH on spleen-lymphocytes, and the high level GSH in CIA rat spleens may contribute to CIA development.