TLR4 Signaling Research Articles

Role of TLR4 activation and signaling in bone remodeling, and afferent sprouting in serum transfer arthritis

BackgroundIn the murine K/BxN serum transfer rheumatoid arthritis (RA) model, tactile allodynia persists after resolution of inflammation in male and partially in female wild type (WT) mice, which is absent in Toll-like receptor (TLR)4 deficient animals. We assessed the role of TLR4 on allodynia, bone remodeling and afferent sprouting in this model of arthritis.MethodsK/BxN sera were injected into male and female mice with conditional or stable TLR4 deletion and controls. Paw swelling was scored and allodynia assessed by von Frey filaments. At day 28, synovial neural fibers were visualized with confocal microscopy and bone density assayed with microCT. Microglial activity and TLR4 dimerization in spinal cords were examined by immunofluorescence and flow cytometry.ResultsIn the synovium, K/BxN injected WT male and female mice showed robust increases in calcitonin gene related-peptide (CGRP+), tyrosine hydroxylase (TH)+ and GAP43+ nerve fibers. Trabecular bone density by microCT was significantly decreased in K/BxN WT female but not in WT male mice. The number of osteoclasts increased in both sexes of WT mice, but not in Tlr4-/- K/BxN mice. We used conditional strains with Cre drivers for monocytes/osteoclasts (lysozyme M), microglia (Tmem119 and Cx3CR1), astrocytes (GFAP) and sensory neurons (advillin) for Tlr4f/f disruption. All strains developed similar arthritis scores after K/BxN serum injection with the exception being the Tlr4Tmem119 mice which showed a reduction. Both sexes of Tlr4Lyz2, Tlr4Tmem119 and Tlr4Cx3cr1 mice displayed a partial reversal of the chronic pain phenotype but not in Tlr4Avil, and Tlr4Gfap mice. WT K/BxN male mice showed increases in spinal Iba1, but not GFAP, compared to Tlr4-/- male mice. To determine whether spinal TLR4 was indeed activated in the K/BxN mice, flow cytometry of lumbar spinal cords of WT K/BxN male mice was performed and revealed that TLR4 in microglia cells (CD11b+ /TMEM119+) demonstrated dimerization (e.g. activation) and a characteristic increase in lipid rafts.ConclusionThese results demonstrated a complex chronic allodynia phenotype associated with TLR4 in microglia and monocytic cell lineages, and a parallel spinal TLR4 activation. However, TLR4 is dispensable for the development of peripheral nerve sprouting in this model.

Sparstolonin B potentiates the antitumor activity of nanovesicle-loaded drugs by suppressing the phagocytosis of macrophages in vivo

BackgroundExtracellular vesicles (EVs) and extruded nanovesicles (ENVs) are promising nanovesicles (NVs) for drug delivery. However, the application of these NVs is strongly hindered by their short half-life in the circulation. Macrophages (Mφs) in the liver and spleen contribute to the rapid depletion of NVs, but the underlying mechanism is unclear.MethodsBy collecting the supernatant of PANC-1 cells and squeezing PANC-1 cells, EVs and ENVs derived from PANC-1 cells were prepared via ultracentrifugation. NVs were subsequently identified via western blot, particle size measurement, and electron microscopy. The distribution of NVs in mouse bodies was observed with a live animal imaging system. Liver Mφs were extracted and isolated after NVs were administered, and transcriptome profiling was applied to determine differentially expressed genes (DEGs). siRNAs targeting interested genes were designed and synthesized. In vitro experiments, Mφs were transfected with siRNA or treated with the corresponding inhibitor, after which NV uptake was recorded. Doxorubicin (DOX) was encapsulated in ENVs using an ultrasound method. PANC-1 cell-derived tumors were established in nude mice in vivo, inhibitor pretreatment or no treatment was administered before intravenous injection of ENVs-DOX, and the therapeutic efficacy of ENVs-DOX was evaluated.ResultsNVs derived from PANC-1 cells were first prepared and identified. After intravenous injection, most NVs were engulfed by Mφs in the liver and spleen. Seven genes of interest were selected via transcriptome sequencing and validated via RT‒PCR. These results confirmed that the TLR2 signaling pathway is responsible for phagocytosis. siTLR2 and its inhibitor sparstolonin B (SpB) significantly inhibited the internalization of NVs by Mφs and downregulated the activity of the TLR2 pathway. The accumulation of ENVs-DOX in the liver was inhibited in vivo by pretreatment with SpB 40 min before intravenous injection, ultimately delaying tumor progression.ConclusionThe TLR2 pathway plays a crucial role in the sequestration of NVs by Mφs. A novel antiphagocytic strategy in which pretreatment of mice with SpB inhibits the clearance of NVs and prolongs their half-life in vivo, thereby improving delivery efficiency, was identified.Graphical

Potential role of Mappain, a prenylated stilbene, in reducing SARS-CoV-2 envelope protein-induced inflammation

As of right now, more than 776 million people have contracted the COVID-19-causing SARS-CoV-2 virus, which has also resulted in over 7 million deaths. A dysregulated host's innate immune response to the virus exacerbates clinical outcomes and leads to cytokine storm. Developing a drug targeting the multiple aspects of the SARS-CoV-2 virus-host cell interactions is essential for effective treatment. The 75 amino acid-long SARS-CoV-2 envelope (2E) protein is a small structural transmembrane protein that plays a crucial role in the viral life cycle and pathogenesis. Mappain is a plant-derived stilbene from Macaranga species that previously demonstrated antiviral potential against enteroviruses in tissue culture. This study aims to assess the inhibitory potential of Mappain against SARS-CoV-2 key proteins and induced hyperinflammation using computational and in-vitro approaches. Autodock Vina was used to evaluate mappain interaction with SARS- CoV-2 antiviral targets including Spike, Envelope, Membrane, 3CLPro, PLpro, Nucleocapsid N and C termini, RdRP, and host receptors—TLR2, TLR3, TLR7, and TLR8. Afterwards, mappain was subjected to in-vitro bioassay testing using inhibition 2E-induced cytokines (IL-6 and TNF-α) in human peripheral blood mononuclear cells, measured by ELISA; Amantadine as control. KEGG pathway enrichment analysis was further performed using network pharmacology to elucidate the mechanism. Mappain demonstrated a high binding score with all the targets, especially with the envelope protein (− 9.6 kcal/mol) and TLR2 (− 9.7 kcal/mol) compared to Amantadine (− 6.1 kcal/mol and − 5.5 kcal/mol, respectively). Mappain also significantly reduced the expression of 2E-induced Interleukin 6 (p ≤ 0.001) and tumour necrosis factor-alpha (p ≤ 0.01) in the PBMCs, when compared to Amantadine HCl. The KEGG Pathway enrichment analysis supports the anti-inflammatory potential of mappain in the virus-induced cytokine storm by specifically targeting TLR signalling and COVID-19 pathways.

Toll-like receptor 3 signaling attenuated colitis-associated cancer development in mice

Inflammatory bowel disease is associated with a high risk of colitis-associated cancer (CAC). We evaluated the role of TLR3 in CAC using a murine model. Wild-type (WT) and TLR3-knockout (TLR3−/−) mice received azoxymethane (AOM) 12.5 mg/kg intraperitoneally on day zero, followed by three cycles of 2% dextran sulfate sodium (DSS) for five days and free water for two weeks. We evaluated clinical indices, such as weight change, colon length, histological severity of colitis, and tumor number. We performed immunofluorescence assays for phospho-IκB kinase and β-catenin in colon tissues. To elucidate the antitumorigenic mechanism of TLR3 signaling, we injected poly(I: C) or phosphate-buffered saline intraperitoneally into an AOM/DSS-induced tumorigenesis model in WT mice. We also evaluate the direct antitumor effect of TLR signaling in AOM-treated WT and TLR3−/− mice without DSS. TLR3 deficiency increased tumor burden and colitis severity in the colon tissue than in the WT mice. β-catenin immunoreactivity was higher in TLR3−/− mice, while phospho-IκB kinase expression was similar. TLR3 activation by poly(I: C) did not reduce tumor burden in WT mice, but long-term AOM administration without DSS significantly increased tumor burden in TLR3−/− mice. TLR3 signaling attenuates CAC development, suggesting it may be a target for preventing CAC in inflammatory bowel disease.

Polysaccharides isolated from Ampelopsis grossedentata and their immunomodulatory activity.

To explore the immunomodulatory activity of polysaccharides from Ampelopsis grossedentata, two polysaccharides named AGP1 and AGP2 were isolated and purified by DEAE-cellulose 52 column and Sephacryl S-300HR chromatography. AGP1 and AGP2 were composed of fucose, arabinose, rhamnose, galactose, glucose, mannose, galacturonic acid, and glucuronic acid, with a ratio of 0.5: 10.2: 0.9: 31.8: 7.4: 3.4: 21.6: 24.2 and 0.4: 6.0: 0.5: 23.3: 3.3: 6.2: 33.5: 26.8, respectively. The average molecular weights of AGP1 and AGP2 were found to be 6.60 × 105 Da and 7.24 × 105 Da, respectively. AGP1 contained →4,6)-Galp-(1 → glycosidic linkages, while AGP2 contained →2)-Galp-(1 → and →2,3,4)-Glcp-(1 → glycosidic linkages. The structures of AGPs were characterized by Fourier transform infrared spectroscopy, nuclear magnetic resonance, and scanning electron microscope. The immunomodulatory activity of AGPs was investigated in RAW264.7 cells, and the results indicated that AGPs significantly activated macrophages, promoted cells differentiation and NO secretion, increased the expression of IL-6 and TNF-α, and induced macrophage M1 polarization. Transcriptomic analysis indicated that AGP1 and AGP2 regulated a total of 1043 and 970 differentially expressed genes respectively, which were identified in different immune related signaling pathways. Moreover, the immunoblot demonstrated that AGPs exerted immune-promoting effects through the TLR4, MAPK and NF-κB signaling pathways in macrophages. Consequently, AGPs have potent immunomodulatory activity and can be considered as immunomodulators in medical and food industries.

CpG can induce the proliferation and differentiation of B10 cells in the peripheral blood of children with immune thrombocytopenic purpura.

Immune thrombocytopenic purpura (ITP) is a disease with a pathogenesis that remains unclear. Accordingly, this study aims to explore the role of B10 cells in ITP pathogenesis and provide a potential novel target for ITP treatment. We selected 42 children diagnosed with ITP and 29 age- and sex-matched healthy children as study objects. Peripheral blood mononuclear cells were isolated and treated with Golgplug combined with CD40L or CpG combined with CD40L, respectively. After 5h and 48h in vitro culture, the proportion of B10 cells in CD19 + cells was compared by flow cytometry and intracellular staining between the two groups. ITP children's prognosis was followed up to analyze the relationship between the proportion of B10 cells and the prognosis of ITP. Without CpG stimulation, the proportion of B10 cells in the peripheral blood of ITP children and healthy counterparts did not differ after 5-h in vitro culture. After 48-h in vitro culture, the proportion of B10 cells in the peripheral blood of ITP children without CpG stimulation was significantly higher than in the control group. Under CpG stimulation, the proportion of B10 cells in the peripheral blood of ITP children cultured in vitro was higher than that in the control group. In children with ITP, the proportion of peripheral blood B cells increased. Additionally, CD19 + cells were more sensitive to CpG-induced TLR9 signal stimulation in children with ITP than those in their healthy counterparts. These findings provide a research direction for ITP pathogenesis.

Based on network pharmacology and molecular docking, the mechanism of action of Chinese herbal compound on mycoplasma synovial sac of chicken was studied

Mycoplasma synoviae (MS) is a wall-less pathogen primarily transmitted through the respiratory tract, causing synovitis and airsacculitis in poultry, which leads to substantial economic losses in the poultry industry. China has a long-standing tradition of Chinese medicine with abundant medicinal resources, renowned for its safety, efficacy, and holistic regulatory effects. This study aims to screen a traditional Chinese medicine (TCM) compound with anti-inflammatory and immunoregulatory properties and preliminarily validate its in vitro efficacy against MS infections. Based on the research foundation of this experiment and findings from previous studies, this study utilized network pharmacology analysis and molecular docking techniques to identify the anti-inflammatory and immunoregulatory effects of traditional Chinese medicinal herbs, including Rhizoma Coptidis, Honeysuckle (Flos Lonicerae Japonicae), Radix Codonopsis, and Radix Glycyrrhizae.The primary active components and potential targets were identified using the Traditional Chinese Medicine Systems Pharmacology Database (TCMSP) and other platforms. Protein-protein interaction (PPI) networks, Gene Ontology (GO) functional annotation, and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were employed to identify critical therapeutic targets. The AutoDock software was used for molecular docking between active components and core targets. Subsequently, in vitro experiments were conducted to validate the effects of key active components on MS targets. The analysis revealed that this TCM compound contains 122 active components, including flavonoids (e.g., quercetin, trimethylisoflavone), chromones (e.g., anti-inflammatory coumarin A), and triterpenes (e.g., glycyrrhizic acid B). These components act on 739 pharmacological targets, and Venn analysis identified 23 intersection targets related to MS. Further analysis indicated that STAT3, IL6, and IL1B were potential core targets. KEGG pathway enrichment analysis showed that MAPK, FoxO, and Toll-like receptor signaling pathways played critical roles in the pathology and treatment of MS, while GO analysis emphasized the significance of the NF-κB signaling pathway and VEGF receptor pathway in immune regulation, inflammation, and tissue repair. Molecular docking results demonstrated that lignans and quercetin in this TCM compound exhibited strong binding affinities to IL1B, IL6, and STAT3, potentially modulating these key targets to exert anti-inflammatory and immunoregulatory effects. In vitro experiments further validated the significant inhibitory effects of quercetin and glycyrrhizic acid on MS infection. The findings suggest that this TCM compound may exert therapeutic effects on MS infection by regulating targets such as CASP3, TLR4, STAT3, IL6, and IL1B, and modulating signaling pathways including MAPK, NF-κB, and FoxO.

Bactericidal antibiotic treatment induces damaging inflammation via TLR9 sensing of bacterial DNA

The immunologic consequences of using bactericidal versus bacteriostatic antibiotic treatments are unclear. We observed a bacteriostatic (growth halting) treatment was more protective than a bactericidal (bacteria killing) treatment in a murine peritonitis model. To understand this unexpected difference, we compared macrophage responses to bactericidal treated bacteria or bacteriostatic treated bacteria. We found that Gram-negative bacteria treated with bactericidal drugs induced more proinflammatory cytokines than those treated with bacteriostatic agents. Bacterial DNA – released only by bactericidal treatments – exacerbated inflammatory signaling through TLR9. Without TLR9 signaling, the in vivo efficacy of bactericidal drug treatment was rescued. This demonstrates that antibiotics can act in important ways distinct from bacterial inhibition: like causing treatment failure by releasing DNA that induces excessive inflammation. These data establish a novel link between how an antibiotic affects bacterial physiology and subsequent immune system engagement, which may be relevant for optimizing treatments to simultaneously clear bacteria and modulate inflammation.

MiR-574-5p in epigenetic regulation and Toll-like receptor signaling.

miR-574-5p is an unusual microRNA (miRNA) that is often upregulated or downregulated following exposure to irradiation or toxic chemicals; bacterial, parasitic or viral infection; and a variety of other disease conditions. Canonically, miR-574-5p epigenetically regulates the expression of many messenger RNAs (mRNAs) through miRNA-mediated posttranscriptional regulation, thereby affecting cellular physiology or pathophysiology and contributing to the pathogenesis or progression of a variety of diseases. However, recent studies have established that in addition to serving as a fine-tuning repressor of gene expression, miR-574-5p also stimulates gene expression as an endogenous ligand for Toll-like receptor-8/7 (TLR8/7). Indeed, the binding of miR-574-5p to TLR8/7 triggers the TLR signaling pathway, leading to the induction of interferons, inflammatory cytokines and autoimmune signaling. These findings suggest that miR-574-5p is not only an important epigenetic regulator of gene expression, but also an important regulator of immune and inflammatory responses. Abnormal miR-574-5p-TLR8/7 signaling has been shown to be tightly associated with inflammation-related cancers and a number of autoimmune disorders. miR-574-5p can serve as a potential biomarker for many diseases. Most importantly, miR-574-5p is a promising therapeutic target for the treatment or prevention of human disorders, especially infectious diseases, cancers and autoimmune diseases.

Promotion of TLR7-MyD88-dependent inflammation and autoimmunity in mice through stem-loop changes in Lnc-Atg16l1

Uncontrolled TLR signaling can cause inflammatory immunopathology and trigger autoimmune diseases. For example, TLR7 promotes pathogenesis of systemic lupus erythematosus. However, whether RNA structural changes affect nucleic acids-sensing TLRs signaling and impact disease progression is unclear. Here by iCLIP-seq we identify a TLR7-binding long non-coding RNA, Lnc-Atg16l1, and find that it promotes TLR7 and other MyD88-dependent TLRs signaling in various types of immune cells. Depletion of Lnc-Atg16l1 attenuates development of TLR7-linked autoimmune phenotypes in the mouse SLE model. Mechanistically, we find that Lnc-Atg16l1 binds to TLR7 at bases near U84 and MyD88 at bases around A129. The analysis of Lnc-Atg16l1 in situ structures show that it strengthens the interaction between TIR domain of TLR7 and MyD88 through specific stem-loop structure changes as a molecular scaffold after TLR7 activation to promote TLR7 downstream signaling. Therefore, we discover a mechanism for host RNA regulation of innate signaling and autoimmune disease through its structural changes. These findings provide insights into the pro-inflammatory function of self RNA in a structure-dependent manner and suggest a potential target for TLR-related autoimmune disorders.

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.

Respiratory bioenergetics is enhanced in human, but not bovine macrophages after exposure to M. bovis PPD: Exploratory insights into overall similar Cellular Metabolic Profiles.

The role of macrophage (MØ) cellular metabolism and reprogramming during TB infection is of great interest due to the influence of Mycobacterium spp. on MØ bioenergetics. Recent studies have shown that M. tuberculosis induces a TLR2-dependent shift towards aerobic glycolysis, comparable to the established LPS induced pro-inflammatory M1 MØ polarisation. Distinct differences in the metabolic profile of murine and human MØ indicates species-specific differences in bioenergetics. So far, studies examining the metabolic potential of bovine MØ are lacking, thus the basic bioenergetics of bovine and human MØ were explored in response to a variety of innate immune stimuli. Cellular energy metabolism kinetics were measured concurrently for both species on a Seahorse XFe96 platform to generate bioenergetic profiles for the response to the bona-fide TLR2 and TLR4 ligands, FSL-1 and LPS respectively. Despite previous reports of species-specific differences in TLR signalling and cytokine production between human and bovine MØ, we observed similar respiratory profiles for both species. Basal respiration remained constant between stimulated MØ and controls, whereas addition of TLR ligands induced increased glycolysis, as measured by the surrogate parameter ECAR. In contrast to MØ stimulation with M. tuberculosis PPD, another TLR2 ligand, M. bovis PPD treatment significantly enhanced basal respiration rates and glycolysis only in human MØ. Respiratory profiling further revealed significant elevation of ATP-linked OCR and maximal respiration suggesting a strong OXPHOS activation upon M. bovis PPD stimulation in human MØ. Our results provide an exploratory set of data elucidating the basic respiratory profile of bovine vs. human MØ that will not only lay the foundation for future studies to investigate host-tropism of the M. tuberculosis complex but may explain inflammatory differences observed for other zoonotic diseases.

Systemic administration of a viral nanoparticle neoadjuvant prevents lung metastasis development through emergency myelopoiesis

ABSTRACT Cancer presents a significant public health concern, particularly in the context of metastatic disease. Surgical removal of primary tumors, while essential, can inadvertently heighten the risk of metastasis. Thus, there is a critical need for innovative neoadjuvant therapies capable of curtailing metastatic progression before or immediately following tumor resection. Addressing this imperative, the papaya mosaic virus nanoparticle (PapMV) has demonstrated potent immunostimulatory capabilities against both viruses and tumors, effectively hindering their proliferation. Our study reveals that PapMV exerts a protective effect against lung metastasis when administered systemically prior to tumor implantation or during the early stages of metastasis in various mouse models of cancer. This anti-tumor effect is initiated by the recruitment of myeloid cells in the lungs. These cells adopt a pro-inflammatory profile, secreting cytokines such as IFN-α, thus fostering a tumor microenvironment inhospitable to tumor progression. Crucially, this protective mechanism hinges on the presence of macrophages before treatment. TLR7 and IFN-I signaling pathways also play pivotal roles in this process. Furthermore, our findings demonstrate that PapMV triggers the activation of the bone marrow emergency response, which accounts for the influx of myeloid cells into the lungs. This study unveils a novel aspect of PapMV’s functionality. By bolstering the immune system, PapMV confers robust protection against metastasis at an early stage of disease progression. This discovery holds promise for therapeutic intervention, particularly as a preemptive measure prior to or just after surgical intervention.

Modulation of the Gut-Lung Axis by Water Kefir and Kefiran and Their Impact on Toll-like Receptor 3-Mediated Respiratory Immunity.

The beneficial effect of milk kefir on respiratory heath has been previously demonstrated; however, water kefir and kefiran in the context of respiratory viral infections have not been investigated. Water kefir and kefiran could be alternatives to milk kefir for their application in persons with lactose intolerance or milk allergy and could be incorporated into vegan diets. Using mice models, this work demonstrated that the oral administration of water kefir or kefiran can modulate the respiratory Toll-like receptor (TLR3)-mediated innate antiviral immunity and improve the resistance to respiratory syncytial virus (RSV) infection. The treatment of mice with water kefir or kefiran for 6 days improved the production of interferons (IFN-β and IFN-γ) and antiviral factors (Mx2, OAS1, RNAseL, and IFITM3) in the respiratory tract after the activation of the TLR3 signaling pathway, differentially modulated the balance of pro- and anti-inflammatory cytokines, reduced RSV replication, and diminished lung tissue damage. Maintaining a proper balance between anti-inflammatory and pro-inflammatory mediators is vital for ensuring an effective and safe antiviral immune response, and the results of this work show that water kefir and kefiran would help to maintain that balance promoting a controlled inflammatory response that defends against infection while minimizing tissue damage.

Exopolysaccharides from Bifidobacterium longum subsp. infantis and Bifidobacterium adolescentis modulate Toll-like receptor signaling

BackgroundExopolysaccharides (EPS) from probiotic bacteria like bifidobacteria, have gained considerable attention for the beneficial effects they exert in the gastrointestinal environment. Here, we investigated whether EPS isolated from Bifidobacterium longum subsp. infantis and Bifidobacterium adolescentis can interact with Toll-like receptors (TLRs) in a structure-dependent way and subsequently we investigated whether they influence cytokine-production in dendritic cells (DCs). ResultsEPS from both B. infantis and B. adolescentis were found to be structurally different and were able to inhibit signaling of TLR2 and TLR4 in an EPS-type dependent fashion. EPS from B. infantis was shown to have stronger inhibitory effects on TLR2/1, whereas EPS from B. adolescentis showed stronger effects for TLR2/6 and TLR4. Incubation of DCs with EPS alone had no effect, however stimulation of DCs with spend-medium of epithelial cells incubated with EPS reduced production of the cytokines MCP-1/CCL2 and TNFα. ConclusionHere we show that EPS from B. infantis and B. adolescentis have structure-dependent immunomodulatory effects, indicating that EPS might be important effector molecules responsible for the health benefits of bifidobacteria.

Glycine Mitigates Lipopolysaccharide Induced Neurotoxicity via Inhibition of TLR-4 Signaling Pathway in Developing Mice Brain

Background: Lipopolysaccharide (LPS) is an endotoxin of the gram-negative bacteria, playing a crucial role in systematic toxicity and cause neurotoxicity in immature brains. Glycine is non-essential amino acid, acting as a neurotransmitter, antioxidant and immune function regulator.Objective: To investigate the potential use of glycine inhibition of TLR-4 receptor to reduce LPS induced neurodegeneration in the developing brain of post-natal day 7 (PND-7) mice. Materials and Methods: A total of 12 PND-7 mice were included in this experimental study from Feb. 2023 to May 2023 at NMMRC, Peshawar. PND-7 mice were randomly distributed into four groups, a control group, a LPS group, LPS+Gly group and Gly group. LPS (250ìg /kg) was administered to LPS group. Glycine (1g/kg) was injected after LPS administration to LPS+Gly group and Gly group mice. After 4hr of the drug treatment, all the PND-7 mice were sacrificed for Western blot analysis. ImageJ software was used for the densitometry of the blots. One way ANOVA and post-hoc tukey tests through Prism graph-5 were applied for statistical analysis. P-value=0.05 was considered statistically significant. Results: Significant differences were observed in the assessed markers in of PND 7 mice brains. Post hoc tukey test revealed significant changes (p<0.01) in the TLR-4 along with BAX, Casp-3, PARP-1and Bcl-2 levels in LPS group. However, significant decrease was observed in TLR-4 levels along with successful amelioration of BAX, Bcl-2, Casp-3 and PARP-1 level in LPS + Gly group as compared to LPS group. Conclusion: Glycine administration significantly improved LPS induced neurodegeneration in brain of the PND-7 mice. Keywords: Apoptosis, Caspase-3, Glycine, Lipopolysaccharide, Neurodegeneration, PARP-1, TLR-4 signaling pathway.

Mesenchymal stem cells: a novel therapeutic approach for feline inflammatory bowel disease

BackgroundInflammatory bowel disease (IBD) poses a significant and growing global health challenge, affecting both humans and domestic cats. Research on feline IBD has not kept pace with its widespread prevalence in human populations. This study aimed to develop a model of feline IBD by incorporating dextran sulfate sodium (DSS) to evaluate the therapeutic potential of MSCs and to elucidate the mechanisms that enhance their action.MethodsWe conducted a comprehensive clinical assessment, including magnetic resonance imaging (MRI), endoscopy, and histopathological examination. Additionally, alterations in intestinal microbiota were characterized by 16 S rDNA sequencing, and the influence of MSCs on IBD-related gene expression was investigated through transcriptome analysis.ResultsAccording to our findings, MSC treatment significantly mitigated DSS-induced clinical manifestations, reduced inflammatory cell infiltration, decreased the production of inflammatory mediators, and promoted mucosal repair. Regarding the intestinal microbiota, MSC intervention effectively corrected the DSS-induced dysbiosis, increasing the presence of beneficial bacteria and suppressing the proliferation of harmful bacteria. Transcriptome analysis revealed the ability of MSCs to modulate various inflammatory and immune-related signaling pathways, including cytokine-cytokine receptor interactions, TLR signaling pathways, and NF-κB pathways.ConclusionThe collective findings indicate that MSCs exert multifaceted therapeutic effects on IBD, including the regulation of intestinal microbiota balance, suppression of inflammatory responses, enhancement of intestinal barrier repair, and modulation of immune responses. These insights provide a solid scientific foundation for employing MSCs as an innovative therapeutic strategy for IBD and pave the way for future clinical explorations.

Effects of replacing fish meal with pork meal and supplementing with crystalline amino acids and fish oil on growth performance, body composition, enzyme activity, intestinal morphology, and immune response of Penaeus monodon

IntroductionThe present study aimed to evaluate the effects of substituting fish meal with pork meal in feed on the growth performance, feed utilization, intestinal morphology, and immune function of Penaeus monodon.MethodsA total of 600 uniformly sized juvenile shrimp with an initial weight of 1.20 ± 0.03 g were randomly assigned to four groups, with each group consisting of six replicates of 25 shrimp each. Fish meal protein was replaced with 0%, 5%, 10%, and 15% pork meal, and crystalline amino acids and fish oil were supplemented accordingly. The groups were designated as P0, P5, P10, and P15, and the study duration was 70 days.ResultsResults indicated no significant difference in specific growth rate (SGR) between the P5 and P10 groups compared to the P0 group. However, the SGR in the P15 group was significantly lower than that in the P5 and P10 groups. Feed conversion rates (FCRs) for the P5 and P10 groups were comparable to the P0 group, whereas the P15 group showed a significantly higher FCR than the other groups. There were no significant differences in whole body composition among all the groups. Apart from alanine, the amino acid composition of muscles did not differ significantly among all the groups. Results of enzyme activity in plasma revealed that the replacement of fish meal with pork meal reduced levels of aspartate aminotransferase (AST) and superoxide dismutase (SOD). Specifically, levels of AST in the P5 and P15 groups were significantly lower compared to the P0 and P10 groups. Histological examination of midgut tissues showed that villus height initially increased and then decreased with higher replacement ratios. The P5 group had the highest villus height, while the P15 group had the lowest villus height. Analysis of gene expression related to liver immune functions indicated that genes in TLR signaling pathway were significantly elevated in the P15 group compared to the other groups.DiscussionIn conclusion, even with the supplementation of crystalline amino acids and fish oil, pork meal should not replace more than 10% of fish meal in the feed of P. monodon to maintain optimal growth and feed efficiency.

The essential roles of memory B cells in the pathogenesis of systemic lupus erythematosus.

Emerging evidence indicates that memory B cells are dysfunctional in systemic lupus erythematosus (SLE). They are hyporesponsive to signalling through the B cell receptor (BCR) but retain responsiveness to Toll-like receptor (TLR) and type I interferon signalling, as well as to T cell-mediated activation via CD40-CD154. Chronic exposure to immune complexes of ribonucleoprotein (RNP)-specific autoantibodies and TLR-engaging or BCR-engaging cargo is likely to contribute to this partially anergic phenotype. TLR7 or TLR8 signalling and the resulting production of type I interferon, as well as the sustained activation by bystander T cells, fuel a positive feedforward loop in memory B cells that can evade negative selection and permit preferential expansion of anti-RNP autoantibodies. Clinical trials of autologous stem cell transplantation or of B cell-targeted monoclonal antibodies and chimeric antigen receptor (CAR) T cells have correlated replenishment of the memory B cell population with relapse of SLE. Moreover, the BCR hyporesponsiveness of memory B cells might explain the failure of non-depleting B cell-targeting approaches in SLE, including BTK inhibitors and anti-CD22 monoclonal antibodies. Thus, targeting of dysfunctional memory B cells might prove effective in SLE, while also avoiding the adverse events of broad-spectrum targeting of B celland plasma cell subsets that are not directly involved in disease pathogenesis.

Investigation on improving immunologic reconstitution insufficiency using DiwuYanggan capsules in AIDS patients.

This study aimed to explore the mechanism of action of DiWuYangGan (DWYG) capsule in improving Immunological non-responder (INR) by analyzing the active ingredients of DWYG. The study employed a randomized, controlled, double-blind, single-simulation method. Patients were randomly divided into control and trial groups and treated with the primal highly effective antiretroviral therapy. To demonstrate the effect of DWYG on INR, patients in the control group were administered simulated DWYG, whereas patients in the trial group were administered DWYG capsules (ChiCTR1900024673). The chemical composition of DWYG was analyzed using ultra-performance liquid chromatography-high-resolution mass spectrometry. Potential targets of DWYG in the treatment of INR were identified and predicted using network pharmacology and molecular docking. The molecular mechanisms underlying the effects of DWYG were validated using a peripheral blood monocyte model. The CD4:CD8 ratio in the trial group was significantly higher than that in the control group (p < 0.01). A total of 210 DWYG compounds were identified and network pharmacology revealed 182 potential therapeutic targets for DWYG and INR. The results of Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analyses showed that the toll-like receptor signaling pathway is one of the key pathways. This study demonstrated that DWYG reduced the expression level of TLR4 and the levels of IL-2, IL-10, and TNF-α, which are important cytokines involved in the immune response. The efficacy of DWYG in the treatment of INR confirmed the potential practical components of DWYG. Moreover, the results of network pharmacology and experimental validation showed that DWYG could restore the immune function of acquired immune deficiency syndrome patients by inhibiting the expression of TLR4 and related signaling pathways and the overactivation of immune function. https://www.chictr.org.cn/index.html, identifier ChiCTR1900024673.