Expression Of Toll-like Receptor Research Articles

Neutrophil extracellular DNA traps activate the TLR9 signaling pathway of pancreatic ductal epithelial cells in patients with type 2 autoimmune pancreatitis

The presence of neutrophil infiltration around the pancreatic ducts has been found to be associated with type 2 autoimmune pancreatitis (AIP). However, the functional role and clinical significance of neutrophil migration in the progression of pancreatitis is not fully understood. Here, we found that neutrophil extracellular traps (NETs) are abundant around the pancreatic duct in patients with type 2 AIP. We also observed an increased expression of toll-like receptor 9 (TLR9) in pancreatic ductal epithelial cells (HPDEC) in type 2 AIP patients compared to other pancreatic diseases. TLR9 acts as the DNA component of NETs (NET-DNA) receptor in HPDEC, which senses extracellular DNA and subsequently activates the NF-κB pathway to promote neutrophil recruitment and induce NET formation. In addition, our results indicated that the hydroxychloroquine (HCQ), acting as a TLR9 antagonist, could effectively inhibit the activation of inflammatory pathways, reduce neutrophil migration and block the positive feedback loop. The intervention positions HCQ acts as a potential target drug for the clinical treatment of type 2 AIP.

Training Status Influences Regulation of Muscle and PBMC TLR4 Expression and Systemic Cytokine Responses to Vigorous Endurance Exercise.

A bout of vigorous endurance exercise transiently activates Toll-like receptor 4 (TLR4) and reduces TLR4 protein expressed on peripheral blood mononuclear cells (PBMCs). Endurance training, on the other hand, reduces TLR4-mediated signaling and minimizes the physiological stress imposed by exercise. Less is known about what occurs in skeletal muscle regarding TLR4 regulation and signaling. Therefore, this study aimed to investigate the regulation of TLR4 expressed in different tissue types (PBMCs and skeletal muscle samples) between endurance-trained and untrained men following vigorous endurance exercise and determine the effect of training status on cytokine responses associated with TLR4 activation. Endurance-trained (n = 7) and untrained (n = 5) men cycled for 1-hr at their respiratory compensation point, with blood and skeletal muscle samples collected pre- and 3-hours post-exercise. In response to vigorous exercise, untrained men experienced a decrease in inhibitor of κBα (IκBα) protein (suggesting IκB degradation and the activation of TLR4-associated transcription factor NF-κB) and TLR4 protein levels, along with a simultaneous increase in TLR4 mRNA expression in both skeletal muscle and PBMCs. Moreover, this exercise session led to elevated levels of circulating interleukin-6, tumor necrosis factor-α, and interleukin-1β. Collectively, these results suggest a heightened TLR4-mediated signaling pathway in untrained men. However, no changes in these targets were observed in endurance-trained men, possibly indicating a potential mechanism by which regular endurance training blunts systemic inflammation. These findings highlight the potential of endurance training to mitigate TLR4-mediated signaling, such as systemic inflammation, and shed light on the effects of exercise on TLR4 expression in PBMCs and skeletal muscle.

Differential TLR2 and TLR4 mediated inflammatory and apoptotic responses in asymptomatic and symptomatic Leptospira interrogans infections in canine uterine tissue

Leptospirosis is major zoonotic disease with global implications, affecting both domestic animals and humans. It is caused by Leptospira interrogans (L. interrogans), which can damage multiple organs, including the kidneys, liver, testes, and uterus. Despite this, L. interrogans can also persist asymptomatically in tissues, akin to nonpathogenic strains. The mechanisms driving asymptomatic infections remain poorly understood. This study investigated the role of L. interrogans in asymptomatic infection within the uterine tissue of canines, focusing on the differential expression of Toll-like receptors (TLRs)2 and 4 and their roles in inflammatory and apoptotic pathways. We hypothesized that TLR2 and TLR4 coexpression is crucial for eliciting inflammation and apoptosis, whereas TLR4 alone might be insufficient. Our findings revealed that in symptomatic infections, both TLR2 and TLR4 are coexpressed, leading to markedly elevated levels of the proinflammatory cytokines IL-10, IL-1β, TNF-α, and IL-6. This enhanced inflammatory response is further evidenced by increased CD4 expression, indicating robust T helper cell activation. In contrast, asymptomatic infections are characterized by exclusive TLR4 expression, with inflammatory markers remaining at baseline levels. Additionally, we observed that L. interrogans induces apoptosis in symptomatic animals through TLR2 and TLR4 mediated activation of Caspase 8 and Caspase 3. These findings illustrate that L. interrogans drives both inflammation and apoptosis via the combination of TLR2 and TLR4 actions. When only TLR4 is activated, the immune response is insufficient, resulting in an asymptomatic disease course. This study provides novel insights into the differential roles of TLR receptors in leptospirosis, offering potential directions for targeted therapeutic strategies.

Immunological mechanisms involved in the protection against development of pulmonary tuberculosis in naturally infected goats

Tuberculosis (TB) is a notifiable zoonotic disease caused by bacteria of the Mycobacterium tuberculosis complex (MTBC) that affects a multitude of domestic and wild species. The main lesions caused by these mycobacteria are tuberculous granulomas, which determine the organism's immune response to the disease. Although TB pathogenesis in cattle has been extensively studied, information regarding its progression in other species of interest for the maintenance and transmission of TB such as goats remains limited. This study aimed to characterise the immune response developed in the lungs of goats naturally infected with mycobacteria of MTBC by assessing key cell populations and immunomodulatory molecules involved in defending against TB. Hence, twelve 6–12-month-old Guadarrama kid goats, initially TB-free, were selected and exposed to M. bovis through close contact with other infected goats. Only animals that tested positive by any of the TB diagnostic methods at the end of the experiment were included in the final analysis (n = 9). Gross and microscopic lesions compatible with TB (TBL) in different organs, as well as local response to TB in lungs were evaluated. Our results revealed that after five months of exposure, 44.4 % (4/9) of the M. bovis-infected animals exhibited TBL in the lungs (TBLL+), characterized by a predominance of non-cavitary necrotic granulomas. TBLL+ animals showed significantly higher presence of neutrophils, macrophages (MΦs) and lymphocytes along with greater expression of interferon (IFN)-γ. Conversely, the remaining animals did not present macroscopic or microscopic TBL in the lungs (TBLL-) (5/9). However, these goats displayed elevated expression of toll-like receptors (TLR)2 and TLR4 alongside heightened expression of pro-inflammatory cytokines, inducible nitric oxide synthase (iNOS) and interleukin (IL)-10. These results suggest the potential development of an effective immune response that may suppress or delay of TBL in infected animals. Further research is needed to elucidate how these molecules, which are involved in the defence against MTBC, confer protection and modulate their expression during infection for TB control.

Potential Effect of Etoricoxib in Reducing Inflammation in Methotrexate-Induced Pulmonary Injury in Rats: Role of Oxidative Stress and the TLR4/p38-MAPK/NF-κB Signaling Pathway.

Numerous chemotherapeutic medications can have hazardous effects on the lungs, which can result in severe lung diseases. Methotrexate (MTX) is prescribed for cancer and inflammation-related disorders; nevertheless, it is exceptionally highly toxic and has multiple kinds of adverse reactions, including pulmonary injury. Our work was designed to demonstrate the ability of etoricoxib (ETO) to mitigate MTX-induced lung injury in experimental animals. Adult male Wistar rats were separated into four groups. The first group consisted of healthy controls that received carboxymethyl cellulose (1 ml/day, p.o.), the second group received a single dose of MTX (20 mg/kg/day, i.p.), the third group received ETO (10 mg/kg/day, p.o.) for three weeks, and the fourth group first received a single MTX (20 mg/kg, i.p.) and then was treated with ETO for three weeks. Concomitant treatment with ETO and MTX improved the histological structure of the lung tissue. It significantly altered the levels of oxidant/antioxidant markers, such as malondialdehyde (MDA), heme oxygenase-1 (HO-1), reduced glutathione (GSH), and nuclear factor erythroid 2-related factor 2 (Nrf-2), in favor of antioxidants. Moreover, ETO can normalize the proinflammatory cascade, which includes tumor necrosis factor-alpha (TNF-α) and interleukin-1 beta (IL-1β). At the molecular level, ETO downregulated the protein expression of toll-like receptor 4 (TLR4), nuclear factor kappa-B (NF-κB), and p38 mitogen-activated protein kinase(p38 MAPK) in inflamed rat lungs. In conclusion, our findings indicate that oral administration of ETO ameliorates MTX-induced lung injury by inhibiting oxidative stress and suppressing the TLR4/NF-κB and TLR4/p38-MAPK inflammatory signaling pathways.

Electroacupuncture of "Jiaji" (EX-B2) inhibits inflammatory response by regulating HMGB1/TLR4/NF-κB signaling pathway in rats with spinal cord injury

To observe the effect of electroacupuncture (EA) of "Jiaji" (EX-B2) on expression of high mobility group box 1 (HMGB1)/Toll-like receptor 4 (TLR4)/nuclear factor-κB (NF-κB) related proteins and inflammation-related factors in spinal cord injury (SCI) rats, so as to explore the dynamic process of inhibiting inflammatory response in rats with SCI, and to provide a new idea and theoretical basis of molecular biology for the treatment of SCI. Fifty-four SD rats were randomly divided into sham operation, model, and EA groups (n=18 in each group), which were further divided into 3 d, 7 d and 14 d subgroups, with 6 rats at each time point. The SCI model was established according to the Allen's method. Rats in EA group received EA (1 mA, 100 Hz) intervention at EX-B2 of T9 and T11 24 hours after modeling for 30 min, once a day for 3 d, 7 d, or 14 d, respectively. The motor function of rats' hind limbs was evaluated using BBB scale, the morphological structure of rats' spinal cord tissue was observed by H.E. staining. The contents of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) in spinal cord tissue were detected by ELISA. Western blot and immunofluorescence staining were used to detect the relative protein expression and fluorescence positive expression of HMGB1, TLR4 and NF-κB p65 in spinal cord tissue, respectively. Compared with the sham operation group, the BBB scores after modeling and on day 3, 7 and 14 were all decreased (P<0.05), while the contents of iNOS and COX-2 in spinal cord tissue, the protein expression and fluorescence positive expression of HMGB1, TLR4, and NF-κB p65 were increased (P<0.05) in the model group. In comparison with the model group, the BBB scores on day 3, 7 and 14 were obviously increased (P<0.05), while the contents of iNOS and COX-2, the protein expression and fluorescence positive expression of HMGB1, TLR4, and NF-κB p65 were significantly down-regulated in the EA group (P<0.05). EA of "Jiaji" can promote the recovery of SCI neurological function, which may be related to its function in regulating the HMGB1/TLR4/NF-κB signaling pathway, down-regulating the expression of iNOS and COX-2, and inhibiting the neuroinflammatory response after SCI.

Effects of electroacupuncture on expression of miR-218 and HMGB1/TLR4 signaling pathway in rats with focal cerebral ischemia

To observe the effect of electroacupuncture (EA) on high mobility group box 1 (HMGB1) / toll-like receptor 4 (TLR4) signaling pathway and inflammatory injury in rats with focal cerebral ischemia (FCI), so as to explore its mechanisms underlying amelioration of ischemic stroke. Male SD rats were randomly divided into sham operation (n=18), model (n=18) and EA (n=18) groups. The FCI model was established according to Longa's method. In the EA group, 24 h after modeling, EA stimulation was applied to "Baihui"(GV20), "Fengfu"(GV16), and "Neiguan" (PC6) and "Xinshu" (BL15) on the left side for 20 min, once a day for 1 week. The neurological deficit severity was evaluated by the modified neurological severity score (mNSS), and the morphological changes of neurons were observed after H.E. staining. The expression of HMGB1 and TLR4 proteins were detected by immunofluorescence staining. The contents of tumor necrosis factor(TNF)-α and interleukin(IL)-6 in the ischemic area tissue of the brain were detected using ELISA. The mRNA expressions of HMGB1 and TLR4 as well as the expression of miR-218 in the ischemic cortex were detected by PCR. Compared with the sham operation group, the mNSS at 2 h, 48 h and 7 day (d), IL-6 and TNF-α contents, HMGB1 and TLR4 immunoactivity and mRNA expression at 48 h and 7 d were significantly increased (P<0.01), while miR-218 expression at 48 h and 7 d was obviously down-regulated (P<0.01) in the model group. In comparison with the model group, the mNSS at 7 d, IL-6 and TNF-α contents, HMGB1 and TLR4 immunoactivity and mRNA expression at 7 d not at 48 h were significantly decreased (P<0.01, P<0.05), while the expression of miR-218 at 7 d was apparently up-regulated (P<0.05) in the EA group. H.E. staining showed that there were signs of neuronal necrosis including blurred outline of the cells, disordered internal structure, dense cytoplasm, shrunk nuclei, etc. in the ischemic area of the brain at 48 h and 7 d in the model group, which was evidently milder on day 7 in the EA group. EA can reduce the inflammatory injury of brain tissue and improve the neurological impairment in rats with FCI, which may be associated with its effects in inhibiting HMGB1/TLR4 signaling and down-regulating the level of pro-inflammatory cytokines.

Dietary fiber consumption by sows during pregnancy has effects on gut microbial composition and immunity of offspring

Context Piglets encounter numerous challenges post-birth, and positive maternal influences can significantly aid their survival. Aims This study aimed to investigate the potential impact of dietary fiber (DF) consumption during pregnancy on the establishment of colonic flora and immunity in offspring. Methods Sixty-eight multiparous sows were randomly assigned to either a control diet lacking fiber sources or a diet supplemented with a fiber mixture. The study evaluated the developmental status, intestinal microecology, and immune indices, including the expression of Toll-like receptors and nuclear factor kappa-B, tumor necrosis factor α, interleukins 6 and 10, and interferon γ, as well as the concentrations of complement components 3 and 4, and immunoglobulins G and M in the offspring. Key results The findings revealed a significant reduction in Toll-like receptor 4 and nuclear factor kappa-B messenger RNA levels in the colon and tumor necrosis factor α levels in the serum of 21-day-weaned piglets from the fiber group, indicating a decrease in inflammation. Moreover, there was a notable increase in the abundance of Roseburia and Lactobacillus in the colons of weaned piglets from the fiber-supplemented group, whereas Odoribacter showed a substantial decrease. This indicates that sows transfer beneficial microorganisms to their piglets, and fiber supplementation further enhances these positive microbial changes. Conclusion This study highlights the positive impact on the microbiota profile and immunity of piglets of fiber supplementation in sow diets during pregnancy, using a 3% purified fiber mixture. These findings hold implications for the enhanced development of weaned piglets, providing valuable theoretical support.

Differential TLR-ERK1/2 Activity Promotes Viral ssRNA and dsRNA Mimic-Induced Dysregulated Immunity in Macrophages.

RNA virus-induced excessive inflammation and impaired antiviral interferon (IFN-I) responses are associated with severe disease. This innate immune response, also referred to as "dysregulated immunity" is caused by viral single-stranded RNA (ssRNA)- and double-stranded-RNA (dsRNA)-mediated exuberant inflammation and viral protein-induced IFN antagonism. However, key host factors and the underlying mechanism driving viral RNA-mediated dysregulated immunity are poorly defined. Here, using viral ssRNA and dsRNA mimics, which activate toll-like receptor 7 (TLR7) and TLR3, respectively, we evaluated the role of viral RNAs in causing dysregulated immunity. We observed that murine bone marrow-derived macrophages (BMDMs), when stimulated with TLR3 and TLR7 agonists, induced differential inflammatory and antiviral cytokine response. TLR7 activation triggered a robust inflammatory cytokine/chemokine induction compared to TLR3 activation, whereas TLR3 stimulation induced significantly increased IFN/IFN stimulated gene (ISG) response relative to TLR7 activation. To define the mechanistic basis for dysregulated immunity, we examined cell-surface and endosomal TLR levels and downstream mitogen-activated protein kinase (MAPK) and nuclear factor kappa B (NF-kB) activation. We identified significantly higher cell-surface and endosomal TLR7 levels compared to TLR3, which were associated with early and robust MAPK (p-ERK1/2, p-P38, and p-JNK) and NF-kB activation in TLR7-stimulated macrophages. Furthermore, blocking EKR1/2 and NF-kB activity reduced TLR3/7-induced inflammatory cytokine/chemokine levels, whereas only ERK1/2 inhibition enhanced viral RNA mimic-induced IFN/ISG responses. Collectively, our results illustrate that high cell-surface and endosomal TLR7 expression and robust ERK1/2 activation drive viral ssRNA mimic-induced excessive inflammatory and reduced IFN/ISG response and blocking ERK1/2 activity would likely mitigate viral-RNA/TLR-induced dysregulated immunity.

Effects of amount of lactose in milk replacer on gastrointestinal function of dairy calves

The objective of this study was to evaluate the effects of feeding milk replacer (MR) at different lactose amount while keeping osmolality constant on gastrointestinal function, blood parameters, and inflammation-related mRNA expression in the livers of dairy calves. Fifteen Holstein bull calves were assigned to one of three dietary treatments differing in MR lactose content (L: 38 %, M: 41 %, and H: 46 %). Feeding of the test diets was started at 1 day of age and gradually increased to a maximum feeding rate at 20 days of age (L: 1.16 kg/d, M: 1.21 kg/d, and H: 1.26 kg/d DM). Under these conditions, the lactose dosages for the treatments were 441 g/d, 496 g/d, and 580 g/d, respectively. The MR were prepared to ensure isocaloric and iso-osmotic (451 mOsm/kg) conditions. Fecal scores were recorded daily, and at 14 and 28 days of age, blood and breath samples were collected before and after MR feeding. In addition, feces and urine were collected for 2 consecutive days. Then, the calves were slaughtered to evaluate intestinal permeability and liver mRNA expression. The permeability in the duodenum and ileum was lower in H and M than in L; the permeability in the jejunum was also lower in H than in L. The hepatic mRNA expressions of toll-like receptor-2, IL-1β, and tumor-necrosis factor-α were lower in H and M than L. Nitrogen retention was higher in H than in L, and linear and quadratic increasing trends were observed in tissue ratio of gastrointestinal tract with the increase in lactose amount. Postprandial increase in plasma glucose concentration was smaller and postprandial increase in TG concentration was higher in H than in L. Fecal properties, digestibility, and breath hydrogen concentrations were not affected by treatment. These results indicate that feeding high-lactose MR may increase gastrointestinal weight and decrease permeability in the small intestine of calves.

Different infant formulas can activate toll-like receptor 9 in vitro and inhibit interleukin 6 in human primary intestinal epithelial cells

PurposeNecrotizing enterocolitis (NEC) is the most severe gastrointestinal disease in preterm infants caused by an exaggerated intestinal epithelial immune activation. Several studies show that modulation of toll-like receptor 9 (TLR9) activity may have positive effects on preventing intestinal inflammatory mechanisms ultimately resulting in NEC development. In this study, the effect of various infant formulas (IF) and the probiotic strain Limosilactobacillus fermentum CECT5716 on TLR9 activation were analyzed in vitro.MethodsFirst, TLR4 and TLR9 expression was analyzed on human primary intestinal epithelial cells (P-IECs) by qPCR and Western blot analysis. Then genetically designed HEK-Dual™ hTLR9 (NF/IL8) reporter cells (HEK-Dual) were treated with different IFs, L. fermentum CECT5716, and different functional components to measure TLR9 activation via luminescence. Finally, the IFs were investigated in P-IECs to analyze TLR downstream signaling by Western blot analysis.ResultsIFs containing intact protein and L. fermentum CECT5716 activated TLR9 in HEK-Dual cells, but the functional components lactoferrin, L-5-methyltetrahydrofolate, and hydrolyzed whey proteins failed to activate TLR9. In P-IECs, the IFs induced increased phosphorylation of MAPK8/9 of the TLR signaling pathway and significantly reduced IL6 levels. Consistently, IL6 levels were increased in P-IECs when TLR9-signaling was inhibited. Interestingly, L. fermentum CECT5716 enhanced TLR9-signaling and increased NF-kappa-B inhibitor alpha-phosphorylation.ConclusionWe found out that the used control formula, prebiotic formula, prebiotic formula with hydrolyzed-protein, and L. fermentum CECT5716 reduce IL6 levels in human P-IECs through TLR9 activation. L. fermentum CECT5716 and the here tested IFs could be a promising approach for modulation of gut health in infants.

Intestinal fatty acid binding protein is associated with coronary artery disease in long-term type 1 diabetes—the Dialong study

BackgroundIndividuals with type 1 diabetes are at increased risk of accelerated atherosclerosis, causing coronary artery disease (CAD). The underlying mechanisms remain unclear, but new theories proposed are damage of gut mucosa causing leakage and translocation of gut microbiota products into the circulation, leading to inflammatory responses and atherosclerosis. We therefore aimed to study the associations between gut related inflammatory biomarkers and coronary atherosclerosis in individuals with long-term type 1 diabetes.MethodsIn this cross-sectional, controlled study of 102 participants with type 1 diabetes and 63 control subjects, we measured circulating levels of intestinal fatty acid binding protein (I-FABP), soluble cluster of differentiation 14 (sCD14), lipopolysaccharide binding protein (LBP) and interleukin 18 (IL-18) by enzyme-linked immunosorbent assay (ELISA), and further gene expression of CD14 and toll-like receptor 4 (TLR4) by real time PCR in circulating leukocytes and peripheral blood mononuclear cells (PBMCs). The participants had either established coronary heart disease (CHD) or underwent computed tomography coronary angiography (CTCA) to assess for coronary atherosclerosis, including total, calcified and soft/mixed plaque volumes.ResultsIn the diabetes group, the levels of I-FABP were significantly higher in participants with established CHD or significant stenosis on CTCA compared to the participants with normal arteries or non-significant stenosis, with median 1.67 ng/ml (interquartile range [IQR] 1.02–2.32) vs. median 1.09 ng/ml (IQR 0.82–1.58), p = 0.003. I-FABP was associated with significant coronary artery stenosis by CTCA (> 50%) or previously established CHD in the adjusted analysis (odds ratio [OR] = 2.32, 95% confidence interval [CI]: 1.09–4.95; p = 0.029). The levels of I-FABP correlated also to total coronary plaque volume (r = 0.22, p < 0.05). This association remained significant after adjusting for age, sex, persistent albuminuria, eGFR, statin treatment, diabetes duration and mean time-weighted variables; HbA1c, LDL-cholesterol and systolic blood pressure (OR = 1.97, 95% CI: 1.28–3.01; p = 0.002).ConclusionsIn this cohort of individuals with long-term type 1 diabetes I-FABP associated significantly with coronary artery stenosis, suggesting a potential role of gut mucosa damage in the process of atherosclerosis in type 1 diabetes.

Can the Analysis of Toll-like Receptors (TLR) on NK and NKT-like Cells Improve Gastric Cancer Diagnostics and Treatment?

Background/Objectives: The aim of this study was to determine the assessment of the percentage of NK and NKT-like cells expressing Toll-like receptors (TLR-2, TLR-3, TLR-4, and TLR-9) in patients with gastric cancer (GC) compared with healthy volunteers (HV) and to investigate differences according to cancer subtype. We also assessed TLR gene expression by RT-qPCR to assess whether TLRs could be diagnostic and prognostic biomarkers. Methods: The study included 86 patients with histologically confirmed gastric cancer and 30 healthy volunteers. Peripheral blood samples were collected from the participants, and TLR expression on NK and NKT-like cells was assessed by flow cytometry and RT-qPCR. The expression of TLR2, TLR3, TLR4, and TLR9 genes was assessed using genetic material derived from NK and NKT-like cells sourced from PBMC. The obtained results were statistically analyzed using Mann-Whitney U and Kruskal-Wallis tests, and the predictive ability of variables was assessed using ROC curve analysis. Results: A significantly higher expression of TLR receptors (TLR-2, TLR-3, TLR-4, and TLR-9) was found in patients with gastric cancer compared to healthy volunteers (p < 0.05). TLR expression also differed depending on the cancer subtype, and higher expression was observed in more advanced GC subtypes. RT-qPCR analysis showed significantly increased expression of TLR genes in the group of GC patients. ROC curves indicate a high ability of TLRs to differentiate between GC patients and healthy individuals. Conclusions: The expression of TLRs on NK and NKT-like cells is clearly increased in patients with gastric cancer, especially in more advanced subtypes of the tumor. The results suggest that TLRs could potentially be used as diagnostic and prognostic biomarkers and represent potential targets for immune therapies in GC. However, further studies are needed to determine the functional role of TLRs in disease progression and the possibility of their use in personalized treatment.

Effects of adding niacinamide to diets with normal and low protein levels on the immunity, antioxidant, and intestinal microbiota in growing-finishing pigs

This study aimed to investigate the effects of nicotinamide (NAM) applied to diets with different crude protein levels on immune function, antioxidant capacity, and intestinal flora in growing-finishing pigs. Forty barrows (37.0±1.0 kg) were randomly allocated to one of four dietary treatments (n=10 per group). The diets in the two phases consisted of a basal diet with 30 mg/kg NAM, a basal diet with 360 mg/kg NAM, a low-protein diet with 30 mg/kg NAM, and a low-protein diet with 360 mg/kg NAM. The results showed that dietary addition of 360 mg/kg NAM decreased IL-12, malondialdehyde, IgG and IgM contents in the plasma and increased total superoxide dismutase activity and total antioxidant capacity in the colonic mucosa (P < .05). Supplementing the diet with 360 mg/kg NAM increased mRNA expression of the nucleotide-binding oligomerization domain containing 2 and nuclear factor erythroid 2-related factor 2 and protein expression of nuclear factor kappa-B and toll-like receptor 4 in the colonic mucosa (P < .05). The concentrations of acetic acid and butyric acid in the colonic contents and the abundance of Actinobacteriota in the colon at the phylum level were significantly decreased by feeding low-protein diets (P < .05). Additionally, the addition of 360 mg/kg NAM to diets increased (P < .05) the Sobs, Ace, and Chao indices of colonic microorganisms in pigs. Overall, the rational use of NAM can improve inflammatory status, enhance antioxidant capacity and intestinal barrier function, and increase colonic microbial diversity in growing-finishing pigs.

Aβ1-42 promotes microglial activation and apoptosis in the progression of AD by binding to TLR4

Alzheimer's disease (AD) is one of the most common age-related neurodegenerative diseases and the most devastating form of senile dementia. It has a complex mechanism and no effective treatment. Exploring the pathogenesis of AD and providing ideas for treatment can effectively improve the prognosis of AD. Microglia were incubated with β-amyloid protein 1-42 (Aβ1-42) to construct an AD cell model. After microglia were activated, cell morphology changed, the expression level of inflammatory factors increased, cell apoptosis was promoted, and the expression of microtubule-associated protein (Tau protein) and related proteins increased. By up-regulating and down-regulating Toll-like receptor 4 (TLR4), the cells were divided into TLR4 knockdown negative control group(Lv-NC group), TLR4 knockdown group(Lv-TLR4 group), TLR4 overexpression negative control group(Sh-NC group), and TLR4 overexpression group(Sh-TLR4 group). The expression of inflammatory factors was detected again. It was found that compared with the Lv-NC group, the expression of various inflammatory factors in the Lv-TLR4 group decreased, cell apoptosis was inhibited, and the expression of Tau protein and related proteins decreased. Compared with the Sh-NC group, the expression of inflammatory factors in the Sh-TLR4 group increased, cell apoptosis was promoted, and the expression of Tau protein and related proteins increased. These results indicate that Aβ1-42 may promote microglial activation and apoptosis by binding to TLR4. Reducing the expression of TLR4 can reduce the occurrence of inflammatory response in AD cells and slow down cell apoptosis. Therefore, TLR4 is expected to become a new target for the prevention and treatment of AD.

Dietary bile acids supplementation protects against Salmonella Typhimurium infection via improving intestinal mucosal barrier and gut microbiota composition in broilers

BackgroundSalmonella Typhimurium (S. Typhimurium) is a common pathogenic microorganism and poses a threat to the efficiency of poultry farms. As signaling molecules regulating the interaction between the host and gut microbiota, bile acids (BAs) play a protective role in maintaining gut homeostasis. However, the antibacterial effect of BAs on Salmonella infection in broilers has remained unexplored. Therefore, the aim of this study was to investigate the potential role of feeding BAs in protecting against S. Typhimurium infection in broilers.MethodsA total of 144 1-day-old Arbor Acres male broilers were randomly assigned to 4 groups, including non-challenged birds fed a basal diet (CON), S. Typhimurium-challenged birds (ST), S. Typhimurium-challenged birds treated with 0.15 g/kg antibiotic after infection (ST-ANT), and S. Typhimurium-challenged birds fed a basal diet supplemented with 350 mg/kg of BAs (ST-BA).ResultsBAs supplementation ameliorated weight loss induced by S. Typhimurium infection and reduced the colonization of Salmonella in the liver and small intestine in broilers (P < 0.05). Compared to the ST group, broilers in ST-BA group had a higher ileal mucosal thickness and villus height, and BAs also ameliorated the increase of diamine oxidase (DAO) level in serum (P < 0.05). It was observed that the mucus layer thickness and the number of villous and cryptic goblet cells (GCs) were increased in the ST-BA group, consistent with the upregulation of MUC2 gene expression in the ileal mucosa (P < 0.05). Moreover, the mRNA expressions of Toll-like receptor 5 (TLR5), Toll-like receptor 4 (TLR4), and interleukin 1 beta (IL1b) were downregulated in the ileum by BAs treatment (P < 0.05). 16S rDNA sequencing analysis revealed that, compared to ST group, BAs ameliorated the decreases in Bacteroidota, Bacteroidaceae and Bacteroides abundances, which were negatively correlated with serum DAO activity, and the increases in Campylobacterota, Campylobacteraceae and Campylobacter abundances, which were negatively correlated with body weight but positively correlated with serum D-lactic acid (D-LA) levels (P < 0.05).ConclusionsDietary BAs supplementation strengthens the intestinal mucosal barrier and reverses dysbiosis of gut microbiota, which eventually relieves the damage to the intestinal barrier and weight loss induced by S. Typhimurium infection in broilers.

Buttermilk and Whey as Functional Foods to Ameliorate Clindamycin-Induced Changes in Mouse Intestine: Modulation of Intestinal Motility and Toll-like Receptors Expression.

Antibiotic treatment is one of the main causes of intestinal dysbiosis, leading, in turn, to other intestinal alterations given the multiple relationships of the microbiota with gut health. Whey and buttermilk are two by-products from the dairy industry with numerous bioactive components. This study aimed to assess the potential of two formulas, containing a mixture of lactoferrin, milk fat globule membrane (MFGM), and whey or buttermilk, to reverse the negative effects of clindamycin on gut motility, Toll-like receptors (TLRs) expression, and oxidative stress in the intestine. For this purpose, a murine model of intestinal dysbiosis was established by clindamycin treatment. Male C57BL/6 mice were treated with saline (Control), clindamycin (Clin), a formula containing whey (F1), or buttermilk (F2) supplemented with lactoferrin and MFGM, Clin+F1, or Clin+F2. Clin delayed the whole gut transit, reduced the response to acetylcholine, decreased TLR2 expression, and increased TLR4 expression in the intestine. F1 and F2 formulas reversed the effects of Clin, restoring TLR2 receptor levels and normalizing intestinal dysmotility. These results indicate that whey- and buttermilk-based formulas supplemented with lactoferrin and MFGM could be used as functional foods to prevent or treat motility disorders and restore some components of the immune system after antibiotic treatment.

Comparison of TLR4 Genotype and TLR4 Pathway-Related Cytokines in Different Strains of Mice in Response to Pertussis Toxin Challenge.

The genetic background of Toll-like receptor 4 (TLR4) proved to be important in the induction of immune protection against Bordetella pertussis infection in humans. Currently, the evaluation of the acellular pertussis (aP) vaccine depends largely on using different mouse strains, while the TLR4 genotype of different mouse strains in response to pertussis toxin (PT) is not carefully determined. The current study was designed to determine the differences in TLR4 genotype and TLR4 pathway-related cytokines in response to PT stimulation among mouse strains of ICR, NIH, and BALB/c. We first determined the single-nucleotide polymorphisms (SNPs) in the TLR4 gene by using first-generation sequencing. Then, the cellular response, including the TLR4 mRNA expression and TLR4 signaling-related cytokines, of immune cells from different mouse strains after PT stimulation was determined. Three missense mutation sites (rs13489092, rs13489093, rs13489097) of the TLR4 gene were found. ICR mice were homozygous without mutation, NIH mice were partially heterozygous, and BALB/c mice were homozygous with a missense mutation. The expression of TLR4 was repressed while the downstream cytokines were upregulated after PT stimulation differently among mouse strains. The IFN-β cytokine of the TRIF pathway was significantly increased in ICR mice (p < 0.05). The IL-6 cytokine of the MyD88-dependent pathway was significantly increased in BALB/c mice (p < 0.05). The identified SNPs of the TLR4 gene in different mouse strains might account for the differences in cytokines levels determined after PT stimulation. Our studies might provide useful referees to reduce the mouse-derived difference in the determination of vaccine titer and increase the comparability of the vaccine from different origins, as different mouse strains were used for vaccine development in different countries.

Neutrophil-activating protein in Bacillus spores inhibits casein allergy via TLR2 signaling.

Milk allergy commonly occurs in children, mainly caused by bovine-derived casein (CAS) protein. Neutrophil-activating protein (NAP) of Helicobacter pylori plays an immunomodulatory role with potential to suppress Th2-type immune responses. Bacillus subtilis (B. subtilis) spores are commonly used as oral vectors for drug delivery. To investigate whether recombinantly expressed NAP on B. subtilis spores could be an effective treatment for CAS allergy in mouse. After CAS sensitization, mice were orally administered B. subtilis spores expressing recombinant NAP for 6 weeks. Allergic symptoms and parameters were evaluated after CAS challenge oral gavage, including allergic inflammation, splenic cytokines, and serum-specific antibodies. Protein levels of Toll-like receptor 2 (TLR2) and c-JUN in the jejunum tissue were measured by western blot. Bone marrow-derived macrophages (BMDMs) were stimulated with inactivated NAP spores to measure the influence on cytokine profiles in vitro. NAP recombinant spore treatment significantly reduced allergic symptoms and intestinal inflammation. Interleukin-12 and interferon-gamma levels increased, whereas serum CAS-specific IgG1 and IgE levels decreased. TLR2 and c-JUN expression levels were elevated in the jejunal tissue. Inactivated NAP spores polarized BMDMs to the M1 phenotype and enhanced cytokine expression, which were inhibited by a TLR2 neutralizing antibody. NAP offers a new strategy in the treatment of CAS allergy by inhibiting the Th2 response, while eliciting macrophages to promote Th1 immune responses.

Therapeutic Potential of Ocimum basilicum L. Extract in Alleviating Autistic-Like Behaviors Induced by Maternal Separation Stress in Mice: Role of Neuroinflammation and Oxidative Stress.

A confluence of genetic, environmental, and epigenetic factors shapes autism spectrum disorder (ASD). Early-life stressors like MS play a contributing role in this multifaceted neurodevelopmental disorder. This research was to explore the efficacy of Ocimum basilicum L. (O.B.) extract in mitigating behaviors reminiscent of autism prompted by maternal separation (MS) stress in male mice, focusing on its impact on neuroinflammation and oxidative stress. MS mice were treated with O.B. extract at varying dosages (20, 40, and 60 mg/kg) from postnatal days (PND) 51-53 to PND 58-60. Behavioral experiments, including the Morris water maze, three-chamber test, shuttle box, and resident-intruder test, were conducted post-treatment. The method of maternal separation involved separating the pups from their mothers for 3 h daily, from PND 2 to PND 14. Molecular analysis of hippocampal tissue was performed to assess gene expression of Toll-like receptor 4 (TLR4), tumor necrosis factor-α (TNF-α), and interleukin-1β (IL-1β). Hippocampal and serum malondialdehyde (MDA) levels and total antioxidant capacity (TAC) were measured. O.B. extract administration resulted in the amelioration of autistic-like behaviors in MS mice, as evidenced by improved spatial and passive avoidance memories and social interactions, as well as reduced aggression in behavioral tests. O.B. extract attenuated oxidative stress and neuroinflammation, as indicated by decreased MDA and increased TAC levels, as well as downregulation of TLR4, TNF-α, and IL-1β expression in the hippocampus. O.B. extract may offer a novel therapeutic avenue for ASD, potentially mediated through its anti-inflammatory and antioxidant properties.