Anti-inflammatory effects of steroidal alkaloids of Solanum lyratum Thunb.

Solanum lyratum Thunb., a traditional Chinese herbal medicine, has a promising background. However, the anti-inflammatory effects of its component steroid alkaloid have not been explored. In this study, animal and cell experiments were performed to investigate the anti-inflammatory effects and mechanism of action of Solanum lyratum Thunb steroid alkaloid (SLTSA), in order to provide evidence for its potential utilization. SLTSA effectively inhibited ear swelling and acute abdominal inflammation of mice. We observed concentration-dependent inhibition of pro-inflammatory cytokines by SLTSA, as confirmed by the ELISA and RT-qPCR results. Flow cytometry, immunofluorescence and RT-qPCR analyses revealed that SLTSA suppressed TLR4 expression. Western blot results indicated that SLTSA inhibited the activation of the TLR4/MyD88/NF-κB signaling pathway. Our study demonstrated that SLTSA possesses anti-inflammatory properties.

Solanum nigrum L. berries extract ameliorated the alcoholic liver injury by regulating gut microbiota, lipid metabolism, inflammation, and oxidative stress.

Solanum nigrum L. (SN) berry is an edible berry containing abundant polyphenols and bioactive compounds, which possess antioxidant and antiinflammatory properties. However, the effects of SN on alcohol-induced biochemical changes in the enterohepatic axis remain unclear. In the current study, a chronic ethanol-fed mice ALD model was used to test the protective mechanisms of SN berries. Microbiota composition was determined via 16S rRNA sequencing, we found that SN berries extract (SNE) improved intestinal imbalance by reducing the Firmicutes to Bacteroides ratio, restoring the abundance of Akkermansia microbiota, and reducing the abundance of Allobaculum and Shigella. SNE restored the intestinal short-chain fatty acids content. In addition, liver transcriptome data analysis revealed that SNE primarily affected the genes involved in lipid metabolism and inflammatory responses. Furthermore, SNE ameliorated hepatic steatosis in alcohol-fed mice by activating AMP-activated protein kinase (AMPK), acetyl-CoA carboxylase (ACC), peroxisome proliferator-activated receptor α (PPAR-α). SNE reduced the expression of toll-like receptor 4 (TLR4), myeloid differentiation factor-88 (MyD88) nuclear factor kappa-B (NF-κB), which can indicate that SNE mainly adjusted LPS/TLR4/MyD88/NF-κB pathway to reduce liver inflammation. SNE enhanced hepatic antioxidant capacity by regulating NRF2-related protein expression. SNE alleviates alcoholic liver injury by regulating of gut microbiota, lipid metabolism, inflammation, and oxidative stress. This study may provide a reference for the development and utilization of SN resources.

Mogroside Alleviates Diabetes Mellitus and Modulates Intestinal Microflora in Type 2 Diabetic Mice.

Mogroside, the main component of Siraitia grosvenorii (Swingle) C. Jeffrey (Cucurbitaceae) is a natural product with hypoglycemic and intestinal microbiota regulating properties. However, whether the alteration of intestinal microbiota is associated with the antidiabetic effect of mogroside remains poorly understood. This study investigated the mechanism underlying the hypoglycemic effect of mogroside in regulating intestinal flora and attenuating metabolic endotoxemia. Kunming mice with type 2 diabetes mellitus (T2DM) induced by high-fat diet and intraperitoneal injection of streptozotocin were randomly divided into model, pioglitazone (2.57 mg/kg) and mogroside (200, 100, and 50 mg/kg) groups. After 28 d of administration, molecular changes related to glucose metabolism and metabolic endotoxemia in mice were evaluated. The levels of insulin receptor substrate-1 (IRS-1), cluster of differentiation 14 (CD14) and toll-like receptor 4 (TLR4) mRNAs were measured, and the composition of intestinal microflora was determined by 16s ribosomal DNA (rDNA) sequencing. The results showed that mogroside treatment significantly improved hepatic glucose metabolism in T2DM mice. More importantly, mogroside treatment considerably reduced plasma endotoxin (inhibition rate 65.93%, high-dose group) and inflammatory factor levels, with a concomitant decrease in CD14 and TLR4 mRNA levels. Moreover, mogroside treatment reduced the relative abundance of Firmicutes and Proteobacteria (the inhibition rate of Proteobacteria was 85.17% in the low-dose group) and increased the relative abundance of Bacteroidetes (growth rate up to 40.57%, high-dose group) in the intestines of diabetic mice. This study reveals that mogroside can relieve T2DM, regulating intestinal flora and improving intestinal mucosal barrier, indicating that mogroside can be a potential therapeutic agent or intestinal microbiota regulator in the treatment of T2DM.

Statin prevents cancer development in chronic inflammation by blocking interleukin 33 expression.

Chronic inflammation is a major cause of cancer worldwide. Interleukin 33 (IL-33) is a critical initiator of cancer-prone chronic inflammation; however, its induction mechanism by environmental causes of chronic inflammation is unknown. Herein, we demonstrate that Toll-like receptor (TLR)3/4-TBK1-IRF3 pathway activation links environmental insults to IL-33 induction in the skin and pancreas inflammation. An FDA-approved drug library screen identifies pitavastatin to effectively suppress IL-33 expression by blocking TBK1 membrane recruitment/activation through the mevalonate pathway inhibition. Accordingly, pitavastatin prevents chronic pancreatitis and its cancer sequela in an IL-33-dependent manner. The IRF3-IL-33 axis is highly active in chronic pancreatitis and its associated pancreatic cancer in humans. Interestingly, pitavastatin use correlates with a significantly reduced risk of chronic pancreatitis and pancreatic cancer in patients. Our findings demonstrate that blocking the TBK1-IRF3-IL-33 signaling axis suppresses cancer-prone chronic inflammation. Statins present a safe and effective prophylactic strategy to prevent chronic inflammation and its cancer sequela.

Microbiome dysbiosis in patients with chronic endometritis and Clostridium tyrobutyricum ameliorates chronic endometritis in mice.

Chronic endometritis is associated with the imbalance of female reproductive tract microbiota and pathogenic microbial infection. This study aimed to identify the specific changes in the endometrial microbiome in patients with endometritis and to explore how Clostridium tyrobutyricum (C.t) influences the progression of endometritis in mice for further elucidating endometritis pathogenesis. For this purpose, endometrial tissues from 100 participants were collected and divided into positive, weakly positive, and negative groups based on CD138 levels, while endometrial microbiome differences were detected and analyzed using 16S rRNA gene sequencing. Staphylococcus aureus (S. aureus)-induced endometritis mouse model was established, followed by treatment with C.t, and inflammatory response, epithelial barrier, and TLR4/NF-κB pathway were evaluated. Results showed that α- and ß-diversity was significantly lower in the positive group compared with the weakly positive or negative groups, where the negative group had more unique operational taxonomic units. The abundance of Proteobacteria was found to be increased, while that of Actinobacteria, Firmicutes, and Bacteroidetes was found to be reduced in the positive group, while the area under the curve value was found to be 0.664. Furthermore, C.t treatment resulted in the alleviation of S. aureus-induced inflammatory response, epithelial barrier damage, and activation of the TLR4/NF-κB pathway in mice. Clinical samples analysis revealed that the diversity and abundance of microbiota were altered in patients with endometritis having positive CD138 levels, while mechanistic investigations revealed C.t alleviated S. aureus-induced endometritis by inactivating TLR4/NF-κB pathway. The findings of this study are envisaged to provide a diagnostic and therapeutic potential of microbiota in endometritis.

Diverse roles of SARS-CoV-2 Spike and Nucleocapsid proteins in EndMT stimulation through the TGF-ß-MRTF axis inhibited by aspirin.

BACKGROUND: The SARS-CoV-2 virus causes severe COVID-19 in one-fifth of patients. In addition to high mortality, infection may induce respiratory failure and cardiovascular complications associated with inflammation. Acute or prolonged inflammation results in organ fibrosis, the cause of which might be endothelial disorders arising during the endothelial-mesenchymal transition (EndMT). METHODS: HUVECs and HMEC-1 cells were stimulated with SARS-CoV-2 S (Spike) and N (Nucleocapsid) proteins, and EndMT induction was evaluated by studying specific protein markers via Western blotting. Wound healing and tube formation assays were employed to assess the potential of SARS-CoV-2 to stimulate changes in cell behaviour. MRTF nuclear translocation, ROS generation, TLR4 inhibitors, TGF-ß-neutralizing antibodies, and inhibitors of the TGF-ß-dependent pathway were used to investigate the role of the TGF-ß-MRTF signalling axis in SARS-CoV-2-dependent EndMT stimulation. RESULTS: Both viral proteins stimulate myofibroblast trans-differentiation. However, the N protein is more effective at EndMT induction. The TGF-ß-MRTF pathway plays a critical role in this process. The N protein preferentially favours action through TGF-ß2, whose secretion is induced through TLR4-ROS action. TGF-ß2 stimulates MRTF-A and MRTF-B nuclear translocation and strongly regulates EndMT. In contrast, the Spike protein stimulates TGF-ß1 secretion as a result of ACE2 downregulation. TGF-ß1 induces only MRTF-B, which, in turn, weakly regulates EndMT. Furthermore, aspirin, a common nonsteroidal anti-inflammatory drug, might prevent and reverse SARS-CoV-2-dependent EndMT induction through TGF-ß-MRTF pathway deregulation. CONCLUSION: The reported study revealed that SARS-CoV-2 infection induces EndMT. Moreover, it was demonstrated for the first time at the molecular level that the intensity of the EndMT triggered by SARS-CoV-2 infection may vary and depend on the viral protein involved. The N protein acts through TLR4-ROS-TGF-ß2-MRTF-A/B, whereas the S protein acts through ACE2-TGF-ß1-MRTF-B. Furthermore, we identified aspirin as a potential anti-fibrotic drug for treating patients with SARS-CoV-2 infection.

Improving stroke prognosis by TLR4 KO to enhance N2 neutrophil infiltration and reduce M1 macrophage polarization.

Cerebral ischemic stroke remains a leading cause of mortality and morbidity worldwide. Toll-like receptor 4 (TLR4) has been implicated in neuroinflammatory responses poststroke, particularly in the infiltration of immune cells and polarization of macrophages. This study aimed to elucidate the impact of TLR4 deficiency on neutrophil infiltration and subsequent macrophage polarization after middle cerebral artery occlusion (MCAO), exploring its role in stroke prognosis. The objective was to investigate how TLR4 deficiency influences neutrophil behavior poststroke, its role in macrophage polarization, and its impact on stroke prognosis using murine models. Wild-type and TLR4-deficient adult male mice underwent MCAO induction, followed by various analyses, including flow cytometry to assess immune cell populations, bone marrow transplantation experiments to evaluate TLR4-deficient neutrophil behaviors, and enzyme-linked immunosorbent assay and Western blot analysis for cytokine and protein expression profiling. Neurobehavioral tests and infarct volume analysis were performed to assess the functional and anatomical prognosis poststroke. TLR4-deficient mice exhibited reduced infarct volumes, increased neutrophil infiltration, and reduced M1-type macrophage polarization post-MCAO compared to wild-type mice. Moreover, the depletion of neutrophils reversed the neuroprotective effects observed in TLR4-deficient mice, suggesting the involvement of neutrophils in mediating TLR4's protective role. Additionally, N1-type neutrophils were found to promote M1 macrophage polarization via neutrophil gelatinase-associated lipocalin (NGAL) secretion, a process blocked by TLR4 deficiency. The study underscores the protective role of TLR4 deficiency in ischemic stroke, delineating its association with increased N2-type neutrophil infiltration, diminished M1 macrophage polarization, and reduced neuroinflammatory responses. Understanding the interplay between TLR4, neutrophils, and macrophages sheds light on potential therapeutic targets for stroke management, highlighting TLR4 as a promising avenue for intervention in stroke-associated neuroinflammation and tissue damage.

[Experimental study on the inhibition of the NLRP3 signaling pathway with Shengsan Jiedu Huayu decoction to alleviate inflammatory injury in rats with acute-on-chronic liver failure].

Objective: To observe the therapeutic effect of Shengsan Jiedu Huayu decoction in alleviating inflammatory liver injury in rats with acute-on-chronic liver failure (ACLF) and its effect on the activation intensity for the NLRP3 signaling pathway. Methods: 63 SD rats were randomly divided into a blank group, a model group, and low-, medium-, and high-dose groups of Shengsan Jiedu Huayu decoction (7.29 g/kg/d, 14.58 g/kg/d, and 29.16 g/kg/d). The ACLF rat model was replicated using carbon tetrachloride combined with d-galactosamine and lipopolysaccharide. Different dose gradients of the Shengsan Jiedu Huayu decoction were used for a five-day intervention treatment, and then rat serum and tissue samples were collected. A biochemical analyzer was used to detect the serum levels of ALT, AST, and TBIL in rats. ELISA was used to detect serum IL-18 and IL-1ß content. HE staining was used to observe histomorphological changes in liver tissue. Immunohistochemistry was used to detect GSDMD expression in liver tissue. Western blot and PCR were used to detect NLRP3, Caspase1, ASC, TLR4, IL-1ß, IL-18 protein, and mRNA expression levels.The groups were compared using analysis of variance and the rank-sum test. Results: Compared with the blank group, the model group's rat liver tissue was severely injured. Serum levels of ALT, AST, and TBIL, inflammatory factors IL-1ß and IL-18, and the GSDMD protein expression level, NLRP3 expression level, TLR4, caspase 1, ASC, IL-1ß, IL-18 protein, and mRNA (P<0.01) were all significantly increased in the model than the blank group (P<0.01). Additionally, compared with the model group, the low-, medium-, and high-dose groups of Shengsan Jiedu Huayu decoction had improved liver tissue injury in ACLF rats, while the serum levels of ALT, AST, TBIL, IL-1ß, IL-18, liver tissue GSDMD protein, NLRP3, TLR4, caspase 1, and ASC expressions were all lower in the different dose gradients of the Shengsan Jiedu Huayu decoction than the model group, with the most evident reduction in the high-dose group (P<0.01). Conclusion: Shengsan Jiedu Huayu decoction can weaken the activation intensity of the NLRP3 signaling pathway, alleviate liver tissue pathological injury, reduce inflammatory factor release, and alleviate inflammatory liver injury in ACLF rats.

Capsaicin combined with stem cells improved mitochondrial dysfunction in PIG3V cells, an immortalized human vitiligo melanocyte cell line, by inhibiting the HSP70/TLR4/mTOR/FAK signaling axis.

BACKGROUND: Vitiligo is a common autoimmune skin disease. Capsaicin has been found to exert a positive effect on vitiligo treatment, and mesenchymal stem cells (MSCs) are also confirmed to be an ideal cell type. This study aimed to explore the influence of capsaicin combined with stem cells on the treatment of vitiligo and to confirm the molecular mechanism of capsaicin combined with stem cells in treating vitiligo. METHODS AND RESULTS: PIG3V cell proliferation and apoptosis were detected using CCK-8 and TUNEL assays, MitoSOX Red fluorescence staining was used to measure the mitochondrial ROS level, and JC-1 staining was used to detect the mitochondrial membrane potential. The expression of related genes and proteins was detected using RT‒qPCR and Western blotting. Coimmunoprecipitation was used to analyze the protein interactions between HSP70 and TLR4 or between TLR4 and mTOR. The results showed higher expression of HSP70 in PIG3V cells than in PIG1 cells. The overexpression of HSP70 reduced the proliferation of PIG3V cells, promoted apoptosis, and aggravated mitochondrial dysfunction and autophagy abnormalities. The expression of HSP70 could be inhibited by capsaicin combined with MSCs, which increased the levels of Tyr, Tyrp1 and DCT, promoted the proliferation of PIG3V cells, inhibited apoptosis, activated autophagy, and improved mitochondrial dysfunction. In addition, capsaicin combined with MSCs regulated the expression of TLR4 through HSP70 and subsequently affected the mTOR/FAK signaling pathway CONCLUSIONS: Capsaicin combined with MSCs inhibits TLR4 through HSP70, and the mTOR/FAK signaling pathway is inhibited to alleviate mitochondrial dysfunction and autophagy abnormalities in PIG3V cells.

Prevotella copri promotes vascular calcification via lipopolysaccharide through activation of NF-κB signaling pathway.

Emerging evidence indicates that alteration of gut microbiota plays an important role in chronic kidney disease (CKD)-related vascular calcification (VC). We aimed to investigate the specific gut microbiota and the underlying mechanism involved in CKD-VC. We identified an increased abundance of Prevotella copri (P. copri) in the feces of CKD rats (induced by using 5/6 nephrectomy followed by a high calcium and phosphate diet) with aortic calcification via amplicon sequencing of 16S rRNA genes. In patients with CKD, we further confirmed a positive correlation between abundance of P. copri and aortic calcification scores. Moreover, oral administration of live P. copri aggravated CKD-related VC and osteogenic differentiation of vascular smooth muscle cells in vivo, accompanied by intestinal destruction, enhanced expression of Toll-like receptor-4 (TLR4), and elevated lipopolysaccharide (LPS) levels. In vitro and ex vivo experiments consistently demonstrated that P. copri-derived LPS (Pc-LPS) accelerated high phosphate-induced VC and VSMC osteogenic differentiation. Mechanistically, Pc-LPS bound to TLR4, then activated the nuclear factor κB (NF-κB) and nucleotide-binding domain, leucine-rich-containing family, pyrin domain-containing-3 (NLRP3) inflammasome signals during VC. Inhibition of NF-κB reduced NLRP3 inflammasome and attenuated Pc-LPS-induced VSMC calcification. Our study clarifies a novel role of P. copri in CKD-related VC, by the mechanisms involving increased inflammation-regulating metabolites including Pc-LPS, and activation of the NF-κB/NLRP3 signaling pathway. These findings highlight P. copri and its-derived LPS as potential therapeutic targets for VC in CKD.

Synergistic effect of mesenchymal stem cell-derived extracellular vesicle and miR-137 alleviates autism-like behaviors by modulating the NF-κB pathway.

Autism spectrum disorder (ASD) is a multifaceted neurodevelopmental disorder predominant in childhood. Despite existing treatments, the benefits are still limited. This study explored the effectiveness of mesenchymal stem cell-derived extracellular vesicles (MSC-EVs) loaded with miR-137 in enhancing autism-like behaviors and mitigating neuroinflammation. Utilizing BTBR mice as an autism model, the study demonstrated that intranasal administration of MSC-miR137-EVs ameliorates autism-like behaviors and inhibits pro-inflammatory factors via the TLR4/NF-κB pathway. In vitro evaluation of LPS-activated BV2 cells revealed that MSC-miR137-EVs target the TLR4/NF-κB pathway through miR-137 inhibits proinflammatory M1 microglia. Moreover, bioinformatics analysis identified that MSC-EVs are rich in miR-146a-5p, which targets the TRAF6/NF-κB signaling pathway. In summary, the findings suggest that the integration of MSC-EVs with miR-137 may be a promising therapeutic strategy for ASD, which is worthy of clinical adoption.

Designing of a multi-epitopes based vaccine against Haemophilius parainfluenzae and its validation through integrated computational approaches.

Haemophilus parainfluenzae is a Gram-negative opportunist pathogen within the mucus of the nose and mouth without significant symptoms and has an ability to cause various infections ranging from ear, eye, and sinus to pneumonia. A concerning development is the increasing resistance of H. parainfluenzae to beta-lactam antibiotics, with the potential to cause dental infections or abscesses. The principal objective of this investigation is to utilize bioinformatics and immuno-informatic methodologies in the development of a candidate multi-epitope Vaccine. The investigation focuses on identifying potential epitopes for both B cells (B lymphocytes) and T cells (helper T lymphocytes and cytotoxic T lymphocytes) based on high non-toxic and non-allergenic characteristics. The selection process involves identifying human leukocyte antigen alleles demonstrating strong associations with recognized antigenic and overlapping epitopes. Notably, the chosen alleles aim to provide coverage for 90% of the global population. Multi-epitope constructs were designed by using suitable linker sequences. To enhance the immunological potential, an adjuvant sequence was incorporated using the EAAAK linker. The final vaccine construct, comprising 344 amino acids, was achieved after the addition of adjuvants and linkers. This multi-epitope Vaccine demonstrates notable antigenicity and possesses favorable physiochemical characteristics. The three-dimensional conformation underwent modeling and refinement, validated through in-silico methods. Additionally, a protein-protein molecular docking analysis was conducted to predict effective binding poses between the multi-epitope Vaccine and the Toll-like receptor 4 protein. The Molecular Dynamics (MD) investigation of the docked TLR4-vaccine complex demonstrated consistent stability over the simulation period, primarily attributed to electrostatic energy. The docked complex displayed minimal deformation and enhanced rigidity in the motion of residues during the dynamic simulation. Furthermore, codon translational optimization and computational cloning was performed to ensure the reliability and proper expression of the multi-Epitope Vaccine. It is crucial to emphasize that despite these computational validations, experimental research in the laboratory is imperative to demonstrate the immunogenicity and protective efficacy of the developed vaccine. This would involve practical assessments to ascertain the real-world effectiveness of the multi-epitope Vaccine.

Ghrelin Alleviates Inflammation, Insulin Resistance, and Reproductive Abnormalities in Mice with Polycystic Ovary Syndrome via the TLR4-NF-κB Signaling Pathway.

BACKGROUND: Polycystic ovary syndrome (PCOS) commonly impacts fertile females with potentially severe effects on fertility and metabolism. Blood ghrelin levels are lower in PCOS patients, and exogenous supplements have been proposed for their potential to trigger anti-inflammatory effects at the cellular level. This study aimed to investigate whether pretreatment with ghrelin reduced inflammation, insulin resistance, and reproductive abnormalities in PCOS and the underlying mechanism of this disorder. METHODS: Ghrelin supplementation was first tested in an inflammation model using human ovarian granulosa cells (KGN cells) that were built by treated with Lipolyaccharide. KGN cells were pretreated with ghrelin and exposed to lipopolysaccharide (LPS). Inflammatory gene expression and cytokine production were analyzed by Enzyme-linked immunosorbent assay (ELISA). Based on these results, the PCOS mice model was built with Dehydroepiandrosterone (DHEA) and a high-fat diet. The mRNA and protein expressions of inflammatory factors including Toll-like receptor 4 (TLR4), nuclear factor kappa-B-p65 (NF-κB-p65), Phospho-NF-κB-p65 (p-NF-κB-p65) and myeloid differentiation factor 88 (MYD88) related to the TLR4/NF-κB signaling pathway were evaluated in KGN cells and mouse ovarian tissues using Real-Time Quantitative Polymerase Chain Reaction (RT-qPCR) and western blot, respectively. Lipid metabolism was quantified via an automated biochemical analyzer. RESULTS: The mRNA and protein expressions of interleukin-6 (IL-6), interleukin-1 beta (IL-1ß), and tumor necrosis factor alpha (TNF-α) in ghrelin pretreated KGN cells were lower than the LPS group (p < 0.05). Protein expression was reduced for TLR4, NF-κB-p65, and MYD88 within KGN cells of ghrelin groups compared to the LPS group (p < 0.05). Ghrelin treatment restored the estrous cycle and slowed weight gain and abdominal fat weight of PCOS mice (p < 0.05). Ghrelin treatment decreased the serum concentrations of testosterone, luteinizing hormone, insulin, IL-6, IL-1ß, and TNF-α compared to the PCOS group (p < 0.05). Estradiol concentrations of mice treated with ghrelin were higher than the PCOS group (p < 0.05). The concentrations of low and high-density lipoprotein, triglyceride, and cholesterol in mice treated with ghrelin were lower than in the PCOS mice (p < 0.05). Inflammatory gene expression for IL-6, IL-1ß, TNF-α, TLR4, NF-κB-p65, and MYD88 decreased in the ovarian tissues of ghrelin-treated mice compared to the PCOS group (p < 0.05), along with reduced protein expression of TLR4, p-NF-κB-p65, and MYD88 (p < 0.05). CONCLUSIONS: In the present study, ghrelin treatment effectively reduced inflammation in vitro, and attenuated insulin resistance and reproductive abnormalities in PCOS mice through the TLR4/NF-κB signaling pathway, highlighting potential therapeutic avenues for future PCOS treatments and research directions.

A TLR4 ligand-based adjuvant for promoting the immunogenicity of typhoid subunit vaccines.

None of the typhoid Vi Polysaccharide (ViPS) subunit vaccines incorporate adjuvants, and the immunogenicity of ViPS vaccines (e.g. Typbar TCV® and Typhim Vi®) is in part due to associated TLR4 ligands such as endotoxin present in these vaccines. Since endotoxin content in vaccines is variable and kept very low due to inherent toxicity, it was hypothesized that incorporating a defined amount of a non-toxic TLR4-ligand such as monophosphoryl lipid A in ViPS vaccines would improve their immunogenicity. To test this hypothesis, a monophosphoryl lipid A-based adjuvant formulation named Turbo was developed. Admixing Turbo with Typbar TCV® (ViPS-conjugated to tetanus toxoid) increased the levels of anti-ViPS IgM, IgG1, IgG2b, IgG2a/c, and IgG3 in inbred and outbred mice. In infant mice, a single immunization with Turbo adjuvanted Typbar TCV® resulted in a significantly increased and durable IgG response and improved the control of bacterial burden compared to mice immunized without Turbo. Similarly, when adjuvanted with Turbo, the antibody response and control of bacteremia were also improved in mice immunized with Typhim Vi®, an unconjugated vaccine. The immunogenicity of unconjugated ViPS is inefficient in young mice and is lost in adult mice when immunostimulatory ligands in ViPS are removed. Nevertheless, when adjuvanted with Turbo, poorly immunogenic ViPS induced a robust IgG response in young and adult mice, and this was observed even under antigen-limiting conditions. These data suggest that incorporation of Turbo as an adjuvant will make typhoid vaccines more immunogenic regardless of their intrinsic immunogenicity or conjugation status and maximize the efficacy across all ages.

Vascular endothelial-derived SPARCL1 exacerbates viral pneumonia through pro-inflammatory macrophage activation.

Inflammation induced by lung infection is a double-edged sword, moderating both anti-viral and immune pathogenesis effects; the mechanism of the latter is not fully understood. Previous studies suggest the vasculature is involved in tissue injury. Here, we report that expression of Sparcl1, a secreted matricellular protein, is upregulated in pulmonary capillary endothelial cells (EC) during influenza-induced lung injury. Endothelial overexpression of SPARCL1 promotes detrimental lung inflammation, with SPARCL1 inducing 'M1-like' macrophages and related pro-inflammatory cytokines, while SPARCL1 deletion alleviates these effects. Mechanistically, SPARCL1 functions through TLR4 on macrophages in vitro, while TLR4 inhibition in vivo ameliorates excessive inflammation caused by endothelial Sparcl1 overexpression. Finally, SPARCL1 expression is increased in lung ECs from COVID-19 patients when compared with healthy donors, while fatal COVID-19 correlates with higher circulating SPARCL1 protein levels in the plasma. Our results thus implicate SPARCL1 as a potential prognosis biomarker for deadly COVID-19 pneumonia and as a therapeutic target for taming hyperinflammation in pneumonia.

Evaluation of Toll-like Receptor 4 (TLR4) Involvement in Human Atrial Fibrillation: A Computational Study.

In the present study, we have explored the involvement of Toll-like Receptor 4 (TLR4) in atrial fibrillation (AF), by using a meta-analysis of publicly available human transcriptomic data. The meta-analysis revealed 565 upregulated and 267 downregulated differentially expressed genes associated with AF. Pathway enrichment analysis highlighted a significant overrepresentation in immune-related pathways for the upregulated genes. A significant overlap between AF differentially expressed genes and TLR4-modulated genes was also identified, suggesting the potential role of TLR4 in AF-related transcriptional changes. Additionally, the analysis of other Toll-like receptors (TLRs) revealed a significant association with TLR2 and TLR3 in AF-related gene expression patterns. The examination of MYD88 and TICAM1, genes associated with TLR4 signalling pathways, indicated a significant yet nonspecific enrichment of AF differentially expressed genes. In summary, this study offers novel insights into the molecular aspects of AF, suggesting a pathophysiological role of TLR4 and other TLRs. By targeting these specific receptors, new treatments might be designed to better manage AF, offering hope for improved outcomes in affected patients.

Modulation of Serotonin-Related Genes by Extracellular Vesicles of the Probiotic Escherichia coli Nissle 1917 in the Interleukin-1ß-Induced Inflammation Model of Intestinal Epithelial Cells.

Inflammatory bowel disease (IBD) is a chronic inflammatory condition involving dysregulated immune responses and imbalances in the gut microbiota in genetically susceptible individuals. Current therapies for IBD often have significant side-effects and limited success, prompting the search for novel therapeutic strategies. Microbiome-based approaches aim to restore the gut microbiota balance towards anti-inflammatory and mucosa-healing profiles. Extracellular vesicles (EVs) from beneficial gut microbes are emerging as potential postbiotics. Serotonin plays a crucial role in intestinal homeostasis, and its dysregulation is associated with IBD severity. Our study investigated the impact of EVs from the probiotic Nissle 1917 (EcN) and commensal E. coli on intestinal serotonin metabolism under inflammatory conditions using an IL-1ß-induced inflammation model in Caco-2 cells. We found strain-specific effects. Specifically, EcN EVs reduced free serotonin levels by upregulating SERT expression through the downregulation of miR-24, miR-200a, TLR4, and NOD1. Additionally, EcN EVs mitigated IL-1ß-induced changes in tight junction proteins and oxidative stress markers. These findings underscore the potential of postbiotic interventions as a therapeutic approach for IBD and related pathologies, with EcN EVs exhibiting promise in modulating serotonin metabolism and preserving intestinal barrier integrity. This study is the first to demonstrate the regulation of miR-24 and miR-200a by probiotic-derived EVs.

In Silico Analyses Indicate a Lower Potency for Dimerization of TLR4/MD-2 as the Reason for the Lower Pathogenicity of Omicron Compared to Wild-Type Virus and Earlier SARS-CoV-2 Variants.

The SARS-CoV-2 Omicron variants have replaced all earlier variants, due to increased infectivity and effective evasion from infection- and vaccination-induced neutralizing antibodies. Compared to earlier variants of concern (VoCs), the Omicron variants show high TMPRSS2-independent replication in the upper airway organs, but lower replication in the lungs and lower mortality rates. The shift in cellular tropism and towards lower pathogenicity of Omicron was hypothesized to correlate with a lower toll-like receptor (TLR) activation, although the underlying molecular mechanisms remained undefined. In silico analyses presented here indicate that the Omicron spike protein has a lower potency to induce dimerization of TLR4/MD-2 compared to wild type virus despite a comparable binding activity to TLR4. A model illustrating the molecular consequences of the different potencies of the Omicron spike protein vs. wild-type spike protein for TLR4 activation is presented. Further analyses indicate a clear tendency for decreasing TLR4 dimerization potential during SARS-CoV-2 evolution via Alpha to Gamma to Delta to Omicron variants.

Du-moxibustion ameliorates depression-like behavior and neuroinflammation in chronic unpredictable mild stress-induced mice.

BACKGROUND: Neuroinflammation is involved in the advancement of depression. Du-moxibustion can treat depression. Here, we explored whether Du-moxibustion could alleviate neuroglia-associated neuro-inflammatory process in chronic unpredictable mild stress (CUMS) mice. METHODS: C57BL/6J mice were distributed into five groups. Except for the CON group, other four groups underwent CUMS for four consecutive weeks, and Du-moxibustion was given simultaneously after modeling. Behavioral tests were then carried out. Additionally, Western blot was conducted to measure the relative expression levels of high-mobility group box 1 (HMGB1), toll-like receptor 4 (TLR4), myeloid differentiation factor 88 (MyD88), and nuclear factor-kappa B (NF-κB). Immunofluorescence was employed to evaluate the positive cells of ionized calcium binding adapter molecule 1 (Iba-1) and glial fibrillary acidic protein (GFAP). Furthermore, interleukin-1 beta (IL-1ß) and tumor necrosis factor-alpha (TNF-α) were analyzed using an ELISA assay. RESULTS: We found that CUMS induced depression-like behaviors, such as reduced sucrose preference ratio, decreased locomotor and exploratory activity, decreased the time in open arms and prolonged immobility. Furthermore, versus the CON group, the expression of HMGB1, TLR4, MyD88, NF-κB, positive cells of Iba-1, IL-1ß and TNF-α were increased but positive cells of GFAP were decreased in CUMS group. However, the detrimental effects were ameliorated by treatment with CUMS+FLU and CUMS+DM. LIMITATIONS: A shortage of this study is that only CUMS model of depression were used, while other depression model were not included. CONCLUSIONS: Du-moxibustion alleviates depression-like behaviors in CUMS mice mainly by reducing neuroinflammation, which offers novel insights into the potential treatment of depression.

Exploring the mechanism of Nav1.3 in the ION-CCI rat model based on the TLR4/TRAF6/NF-κB pathway.

BACKGROUND: Trigeminal neuralgia (TN) is a common and difficult-to-treat neuropathic pain disorder in clinical practice. Previous studies have shown that Toll-like receptor 4 (TLR4) modulates the activation of the NF-κB pathway to affect neuropathic pain in rats. Voltage-gated sodium channels (VGSCs) are known to play an important role in neuropathic pain electrical activity. OBJECTIVE: To investigate whether TLR4 can regulate Nav1.3 through the TRAF6/NF-κB p65 pathway after infraorbital nerve chronic constriction injury (ION-CCI). STUDY DESIGN: ION-CCI modeling was performed on SD (Sprague Dawley) rats. To verify the success of the modeling, we need to detect the mechanical pain threshold and ATF3. Then, detecting the expression of TLR4, TRAF6, NF-κB p65, p-p65, and Nav1.3 in rat TG. Subsequently, investigate the role of TLR4/TRAF6/NF-κB pathway in ION-CCI model by intrathecal injections of LPS-rs (TLR4 antagonist), C25-140 (TRAF6 inhibitor), and PDTC (NF-κB p65 inhibitor). RESULTS: ION-CCI surgery decreased the mechanical pain threshold of rats and increased the expression of ATF3, TLR4, TRAF6, NF-κB p-p65 and Nav1.3, but there was no difference in NF-κB p65 expression. After inject antagonist or inhibitor of the TLR4/TRAF6/NF-κB pathway, the expression of Nav1.3 was decreased and mechanical pain threshold was increased. CONCLUSION: In the rat model of ION-CCI, TLR4 in the rat trigeminal ganglion regulates Nav1.3 through the TRAF6/NF-κB p65 pathway, and TLR4 antagonist alleviates neuropathic pain in ION-CCI rats.