Toll-like Receptor Research Articles

Repurposing celecoxib as adjuvant therapy in patients with Parkinsonian disease: a new therapeutic dawn: randomized controlled pilot study.

The clinical presentations of Parkinson's disease (PD), a chronic neurodegenerative condition, include bradykinesia, hypokinesia, stiffness, resting tremor, and postural instability. Recently, neuroinflammation is involved in pathogenesis of PD. Application of nonsteroidal anti-inflammatory drugs captured attention to treat these neuroinflammation. To investigate the possible effectiveness of celecoxib in patients with PD treated with conventional treatment. Sixty outpatients who fulfilled the inclusion requirements for PD were enrolled in this randomized, prospective, and controlled study. The patients were allocated into two groups at random (n = 30); the control group received standard PD treatment, consisting of levodopa/carbidopa, and the celecoxib group received standard PD treatment plus celecoxib. A neurologist evaluated each patient at the beginning of the treatment and after 6months. Assessment of Unified Parkinson's disease rating scale (UPDRS) for each patient. Before and after treatment, α -synuclein (α-Syn), tumor necrosis factor alpha (TNF-α), Toll like receptors-4 (TLR-4), nuclear factor erythroid 2-related factor 2 (Nrf-2) and brain-derived neurotropic factor (BDNF) were assessed. Paired and unpaired t tests were used to assess statistical significance within and between groups respectively. The celecoxib group exhibited a significant and statistical reduction in the level of measured parameters by unpaired t test as followed: TLR-4 (p = 0.004), TNF-α (p = 0.042), and α-Syn (p = 0.004) apart from a significant increase in BDNF (p = 0.0005) and Nrf-2 (p = 0.004), in comparison with the control group. Also, UPDRS was significantly decreased in celecoxib group (p < 0.05). Celecoxib could be a promising adjuvant therapy in managing patients with PD. NCT05962957.

Nano-selenium ameliorates microplastics-induced injury: Histology, antioxidant capacity, immunity and intestinal microbiota of grass carp (Ctenopharyngodon idella)

Microplastics (MPs) are pollutants widely distributed in the aquatic environments and causing various degrees of aquatic toxicity to aquatic organisms, which has attracted global attention in recent years. Nano-selenium (NSe) has been shown to have the potential to mitigate the harmful impacts of toxic substances. However, there is currently no reported evidence regarding the protective influence of NSe against the adverse effects of MPs. The aim of this study is to determine whether NSe could ameliorate the polystyrene (PS)-MPs-induced injury in grass carp (Ctenopharyngodon idella). The individuals of grass carp were assigned into three groups: (1) the control group fed with basal diet, (2) the PS group fed with basal diet and exposed to PS-MPs, and (3) the NSe group fed with diet supplemented with NSe and exposed to PS-MPs. Our results indicated that NSe administration significantly alleviated the histological damage caused by the PS-MPs in the liver and intestine with lower goblet cell count and larger villus height in the intestine, and significantly lower damage score in the liver. Moreover, NSe mitigated PS-MPs-induced oxidative stress through restoring the activities of antioxidant enzymes (superoxide dismutase (SOD), catalase (CAT), and malondialdehyde (MDA)) except the intestinal CAT activity. Furthermore, NSe supplementation could help fish maintain lower transcriptional level of the immune-related genes (Toll-like receptor 4 (TLR4) and myeloid differentiation factor 88 (MyD88)), inflammation-related genes (major histocompatibility complex class II (MHC-II) and interleukin 8 (IL-8)) and antioxidant enzyme-related genes (nuclear factor (erythroid-derived 2)-like 2 (Nrf-2) and kelch-like ECH-associated protein 1 (Keap-1)) after PS-MPs exposure. Besides, NSe supplementation dramatically helped maintain the intestinal microbial composition, for example, the proportion of Proteobacteria in the grass carp intestine of the NSe group (41 %) was similar to that of the control group (34 %) while 85 % of the PS group. NSe also played a significant protective role in intestinal microbial diversity, effectively resisting the damage on intestinal microbial diversity due to PS-MPs exposure. PS-MPs reduced the beneficial bacteria and increased the pathogenic microorganism like Aeromonas, which was undeniable signs of intestinal dysbiosis. Functional analysis indicated that PS-MPs affected intestinal microbiota functions like inhibition of metabolism, while NSe could significantly alleviate the damage. Our findings suggested that NSe could ameliorate PS-MPs-induced injury, which could contribute to the better understanding of the ecotoxicological effects of MPs on fish and help develop relevant mitigation strategies.

Therapeutic potential of monoterpene molecules acts against 7KCh-mediated oxidative stress and neuroinflammatory amyloidogenic signalling pathways

Alzheimer’s disease (AD) is a degenerative disorder characterised by amyloid-beta aggregates activated by the accumulation of lipid molecules and their derivatives, especially 7-ketocholesterol (7KCh), an oxidised lipid that plays a great part in the progression of AD. The current therapeutics need bio-potential molecules and their biomedical application preventing 7KCh-induced cytotoxicity. In this study, bornyl acetate (BA) and menthol (ME), the natural monoterpenes were investigated for their neuroprotective effects against 7KCh-induced SH-SY5Y cells and their effects were compared to the standard drug galantamine (GA). 7KCh-induced changes like lipid accumulation, amyloid generation, free radical generation, acetylcholinesterase levels, calcium accumulation and mitochondrial membrane integrity were analysed in SH-SY5Y cells with or without BA and ME treatment. Furthermore, various mediators involved in the amyloidogenic, inflammatory and apoptotic pathways were studied. In our results, the cells induced with 7KCh upon co-treatment with BA and ME significantly reduced lipid accumulation and amyloid generation through toll-like receptor (TLR) 4 suppression and enhanced ATP binding cassette (ABCA) 1-mediated clearance. Co-treatment with BA and ME concurrently regulated oxidative stress, acetylcholinesterase activity, mitochondrial membrane potential and intracellular calcification altered by 7KCh-induced SH-SY5Y cells. Moreover, 7KCh-induced cells showed elevated mRNA levels of misfolded protein markers and apoptotic mediators which were significantly downregulated by BA and ME co-treatment. In addition, the protein expression of amyloidogenic, proinflammatory as well as pro-apoptotic markers was decreased by BA and ME co-treatment in 7KCh-induced cells. Overall, BA and ME mediated inhibition of amyloidogenic activation and cell survival against 7KCh-induced inflammation, thereby preventing the onset and progression of AD in comparison to GA.

Signaling Pathways (TNF-α-NF-κB, TLR2-TLR4 as well as ROS-MDA) and Cardiac Damages during Cardiac Surgeries (Coronary stenting, Permanent Pacemaker Implantations, Radiofrequency Ablations).

The mutual activations of multiple signaling pathways are the key factors in the development and progression of myocardial cell injuries. This research aimed to compare the different degrees of myocardial injury after coronary stenting, permanent pacemaker implantations, or cardiac radiofrequency ablation and to investigate the effects of the mutual activation of TNF-α/NF-κB, TLR2/TLR4, and ROS/MDA signaling pathways on myocardial injury in elderly patients after coronary stents or permanent pacemakers or radiofrequency ablation. We determined reactive oxygen species (ROS), malondialdehyde (MDA), toll-like receptor 2 (TLR2), toll-like receptor 4 (TLR4), nuclear factor kappa B (NF-κB), tumor necrosis factor- α (TNF-α) and high-sensitive cardiac troponin T (hs-cTnT) as a marker of myocardial injury in patients. The levels of ROS, MDA, TLR2, TLR4, NF-κB, TNF-α, and hs-cTnT were increased in patients with permanent pacemaker implantations when compared to patients with cardiac radiofrequency ablation (P < 0.01) at 6 months and were further increased in patients with coronary stenting compared to patients with cardiac radiofrequency ablation and permanent pacemaker implantations at 6 months, respectively (P < 0.01). This research confirmed that ROS, MDA, TLR2, TLR4, NF-κB, and TNF-α predicted myocardial injury severity. Oxidative stress (ROS/MDA signaling pathway) may be linked to immune response (TLR2/TLR4 signaling pathway) and pro-inflammatory response (TNF-α/NF-κB signaling pathway) in myocardial injury, and ROS/MDA signaling may play a dominant role.

Fusobacterium Nucleatum Promotes Microsatellite Instability in Colorectal Carcinoma Through Up-regulation of miRNA-155-5p-Targeted Inhibition of MSH6 via the TLR4/NF-κB Signaling Pathway.

Fusobacterium nucleatum (Fn) is significantly associated with poor prognosis in colorectal carcinoma (CRC), however, mechanisms of Fn in DNA mismatch repair (MMR) and microsatellite instability (MSI) in CRC have not been fully elucidated. Clinical samples are collected to analyze the relationship between Fn abundance and microsatellite stability. Tumor cells are treated with Fn to detect the expression of proteins related to toll-like receptor 4 (TLR4), myeloid differentiation factor 88 (Myd88), mutS homolog 6 (MSH6), and nuclear factor-κB (NF-κB) signaling pathways, respectively. Combined with the prediction results from TargetScan, the regulatory role of microRNA upstream of MSH6 is demonstrated. The effect of this regulatory axis on CRC development is demonstrated using a nude mouse tumor model. Compared with microsatellite stability (MSS)-type CRC patients, MSI-type showed higher Fn abundance. Fn treatment of CRC cells activated TLR4/Myd88/NF-κB signaling pathway, transcriptionally activating miRNA-155-5p expression, thereby negatively regulating MSH6. Fn treatment accelerated the malignant progression of CRC in mice, and this process is inhibited by miRNA-155-5p antagomir. Fn in CRC upregulated miRNA-155-5p by activating TLR4/NF-κB signaling to inhibit MSH6, and this regulatory pathway may affect MSS of cancer cells.

Chondroitin Sulfate from Halaelurus burgeri Skin Inhibits Hepatic Endoplasmic Reticulum Stress and Inflammation, and Regulates Gut Microbiota.

Previous study has demonstrated the chemical structure of chondroitin sulfate (CHS) from Halaelurus burgeri skin and its effects on insulin resistance. However, the precise impact of this phenomenon on endoplasmic reticulum (ER) stress and inflammation, which contribute to insulin resistance, remains unclear. This study is to investigate the impact of CHS on ER stress, inflammatory response and signaling, and gut microbiota in high-fat diet (HFD)-fed mice. HFD-fed C57BL/6J mice receive dietary gavage intervention of CHS for 18 weeks. Blood, liver tissue, and feces are harvested for further investigation. Results show that CHS inhibits ER stress, accompanied by lowered blood glucose, nitric oxide (NO), reactive oxygen (ROS), and free fatty acids (FFA) levels, and increases hepatic glycogen accumulation. Moreover, hepatic inflammation is improved by CHS treatment via inactivation of Toll-like receptor 4 (TLR4) signaling and its downstream c-jun N-terminal kinase (JNK) and nuclear factor kappa B (NFκB) pathways. Additionally, CHS regulates gut microbiota, particularly the decline in the Firmicutes to Bacteroidetes ratio. CHS also lowers fecal lipopolysaccharide and elevates several fecal short chain fatty acids. These findings suggest that CHS from H. burgeri skin may be an alternative functional food supplement for anti-ER stress, anti-inflammtion, and regulation of gut microbiota.

Update on Biomarkers of Chronic Inflammatory Processes Underlying Diabetic Neuropathy.

There is an increasing prevalence of diabetes mellitus (DM), particularly type 2 DM (T2DM), and its associated complications. T2DM is linked to insulin resistance, chronic inflammation, and oxidative stress, which can lead to both macrovascular and microvascular complications, including peripheral diabetic neuropathy (PDN). Inflammatory processes play a key role in the development and progression of T2DM and its complications, with specific markers like C-reactive protein (CRP), interleukins (ILs), and tumor necrosis factor (TNF)-α being associated with increased risk. Other key inflammatory markers such as nuclear factor kappa B (NF-κB) are activated under hyperglycemic and oxidative stress conditions and contribute to the aggravation of PDN by regulating inflammatory gene expression and enhancing endothelial dysfunction. Other important roles in the inflammatory processes are played by Toll-like receptors (TLRs), caveolin 1 (CAV1), and monocyte chemoattractant protein 1 (MCP1). There is a relationship between vitamin D deficiency and PDN, highlighting the critical role of vitamin D in regulating inflammation and immune responses. The involvement of macrophages in PDN is also suspected, emphasizing their role in chronic inflammation and nerve damage in diabetic patients. Vitamin D supplementation has been found to reduce neuropathy severity, decrease inflammatory markers, and improve glycemic control. These findings suggest that addressing vitamin D deficiency could offer therapeutic benefits for PDN. These molecular pathways are critical in understanding the pathogenesis of DM complications and may offer potential biomarkers or therapeutic targets including anti-inflammatory treatments, vitamin D supplementation, macrophage phenotype modulation, and lifestyle modifications, aimed at reducing inflammation and preventing PDN. Ongoing and more extensive clinical trials with the aim of investigating anti-inflammatory agents, TNF-α inhibitors, and antioxidants are needed to advance deeper into the understanding and treatment of painful diabetic neuropathy.

The lipooligosaccharide of the gut symbiont Akkermansia muciniphila exhibits a remarkable structure and TLR signaling capacity

The cell-envelope of Gram-negative bacteria contains endotoxic lipopolysaccharides (LPS) that are recognized by the innate immune system via Toll-Like Receptors (TLRs). The intestinal mucosal symbiont Akkermansia muciniphila is known to confer beneficial effects on the host and has a Gram-negative architecture. Here we show that A. muciniphila LPS lacks the O-polysaccharide repeating unit, with the resulting lipooligosaccharide (LOS) having unprecedented structural and signaling properties. The LOS consists of a complex glycan chain bearing two distinct undeca- and hexadecasaccharide units each containing three 2-keto-3-deoxy-D-manno-octulosonic acid (Kdo) residues. The lipid A moiety appears as a mixture of differently phosphorylated and acylated species and carries either linear or branched acyl moieties. Peritoneal injection of the LOS in mice increased higher gene expression of liver TLR2 than TLR4 (100-fold) and induced high IL-10 gene expression. A. muciniphila LOS was found to signal both through TLR4 and TLR2, whereas lipid A only induced TLR2 in a human cell line. We propose that the unique structure of the A. muciniphila LOS allows interaction with TLR2, thus generating an anti-inflammatory response as to compensate for the canonical inflammatory signaling associated with LOS and TLR4, rationalizing its beneficial host interaction.

Electroacupuncture inhibits TLR4/NF-κB signaling in the dorsal root ganglion of rats with spared nerve injury.

Neuropathic pain can be provoked by high mobility group box 1 (HMGB1) activation of toll-like receptor (TLR)4/nuclear factor (NF)-κB signaling in the dorsal root ganglion (DRG). Electroacupuncture (EA) has been reported to effectively alleviate neuropathic pain with few side effects, but its precise mechanism of action remains unknown. The aim of this study was to explore whether 2 Hz EA stimulation suppresses TLR4/NF-κB signaling in the DRG following spared nerve injury (SNI) in a rat model. In this experiment, SNI rats were given 2 Hz EA once every other day for a total of 21 days. Paw withdrawal threshold (PWT) was measured to assess SNI-induced mechanical hypersensitivity, and western blotting and immunofluorescence staining were used to determine the levels of pain-related signaling molecules and pro-inflammatory mediators in the DRG. SNI up-regulated HMGB1, TLR4, myeloid differentiation factor-88 adaptor protein (MyD88) and NF-κB p65 protein expression in the DRG. In addition, immunofluorescence staining demonstrated that SNI induced higher levels of TLR4 and MyD88 in the DRG. We also demonstrated co-localization of TLR4 and MyD88 with both calcitonin gene-related peptide (CGRP) and isolectin GS-IB4 in the DRG of SNI rats, respectively. Meanwhile, 2 Hz EA stimulation effectively reversed the elevations of HMGB1, TLR4, MyD88 and NF-κB p65 induced by SNI in the DRG, which was coupled with amelioration of SNI-induced mechanical hypersensitivity. The results of this study suggested that inhibition of the TLR4/NF-κB signaling pathway in the DRG by 2 Hz EA might be exploited as a therapeutic option for neuropathic pain.

Immunostimulatory Effects of Gamisoyosan on Macrophages via TLR4-Mediated Signaling Pathways.

This study aimed to analyze the immunostimulatory activity of gamisoyosan (GSS) on the activation of macrophages in RAW 264.7 cells and its underlying mechanisms. The effects of GSS on the secretion of nitric oxide (NO), immunomodulatory mediators, cytokines and mRNAs, and related proteins were assessed using the Griess assay, Western blotting, quantitative polymerase chain reaction, enzyme-linked immunosorbent assay, and H2DCFDA, respectively. The level of phagocytosis was determined by the neutral red method while the immune function of GSS was determined using adhesion and wound-healing assays. GSS-treated macrophages significantly increased the production of NO, immunomodulatory enzymes, cytokines, and intracellular reactive oxygen species without causing cytotoxicity. GSS effectively improved macrophage immune function by increasing their phagocytic level, adhesion function, and migration activity. Mechanistic studies via Western blotting revealed that GSS notably induced the activation of the Toll-like receptor (TLR) 4-mediated mitogen-activated protein kinase, nuclear factor-κB, and protein kinase B signaling pathways. Overall, our results indicated that GSS could activate macrophages through the secretion of immune-mediated transporters via TLR4-dependent signaling pathways. Thus, GSS has potential value as an immunity-enhancing agent.

B cell αv integrin regulates germinal center derived lung-resident IgA B cell responses following influenza virus infection.

Emerging studies have highlighted the importance of tissue-resident B cells in the lungs, for protective immunity against respiratory viruses. However, the mechanisms controlling generation and maintenance of such tissue-resident B cells at respiratory sites remain obscure. We have previously shown that αv integrins limit B cell responses to antigens containing Toll-like receptor ligands, and that deletion of B cell αv integrins, in mice, enhances germinal center (GC)-derived long-lived B cell responses after systemic immunization with viral antigens. Here we investigated whether αv also regulates B cell responses at the respiratory tract during viral infection. Our data show that αv integrin restricts tissue-resident B cell responses in the airway, and that deletion of B cell αv promotes generation of lung-resident IgA B cell responses following influenza A virus (IAV) infection. Investigating the mechanism for this, we found that loss of B cell αv, promotes persistence of GC reactions locally in the lungs, which leads to increases in lung-resident IgA+ memory B cells, cross-reactive to antigenic variants. Thus, these studies reveal how IgA B cells are maintained in the lungs and point to a new strategy to improve the durability of lung-resident IgA B cell responses for IAV vaccine efficacy.

Transcriptomic and Proteomic Analyses of the Immune Mechanism in Pathogenetic and Resistant Chinese Soft-Shelled Turtle (Pelodiscus sinensis) Infected with Aeromonas hydrophila

Background: As intensive aquaculture practices have progressed, the prevalence of bacterial diseases in the Chinese soft-shell turtle (Pelodiscus sinensis) has escalated, particularly infections caused by Aeromonas hydrophila, such as ulcerative dermatitis and abscess disease. Despite this, little is known about their immune defenses against this pathogen. Methods: Our study pioneers an integrated analysis of transcriptomics and proteomics to investigate the immune responses of Chinese soft-shelled turtles to A. hydrophila infection. Results: The investigation revealed significant differences in immune-related pathways between groups susceptible and resistant to A. hydrophila infection after 4 days. A total of 4667 and 3417 differentially expressed genes (DEGs), 763 and 568 differentially expressed proteins (DEPs), and 13 and 5 correlated differentially expressed genes and proteins (cor-DEGs-DEPs) were identified in susceptible and resistant Chinese soft-shelled turtles, respectively. In the resistant group, upregulation of immune-related genes, such as CD3ε and CD45, enhanced T-cell activation and the immune response. The proteomic analysis indicated that immune proteins, such as NF-κB1, were significantly upregulated in the resistant group. The correlation analysis between transcriptomics and proteomics demonstrated that the CD40 gene and protein, differentially expressed in the resistant group compared to the control group, were commonly upregulated within the Toll-like receptor signaling pathway. Conclusions: The transcriptomic and proteomic data obtained from this study provide a scientific foundation for understanding the immune mechanisms that enable the Chinese soft-shelled turtle to resist A. hydrophila infection.

Pharmacological targets and validation of remdesivir for the treatment of COVID-19-associated pulmonary fibrosis: A network-based pharmacology and bioinformatics study.

The objective of this study was to employ bioinformatics and network pharmacology methodologies to investigate the targets and molecular mechanisms of remdesivir in the treatment of coronavirus disease 2019 (COVID-19)-associated pulmonary fibrosis (PF). Several open-source databases were utilized to confirm the shared targets of remdesivir, COVID-19, and PF. Following this, a comprehensive analysis incorporating function enrichment, protein-protein interaction (PPI), transcription factor (TF), and molecular docking was conducted to investigate the potential mechanisms underlying the effectiveness of remdesivir in the treatment of COVID-19-associated PF. The initial validation of these findings was performed using publicly available histological and single-cell sequencing databases. The functional enrichment analysis revealed a strong association between remdesivir and viral defense, inflammatory response, and immune response. The key pathways identified in the study were transforming growth factor (TGF-β), PI3K-Akt, mTOR, MAPK, epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor resistance, HIF-1, and Toll-like receptor signaling pathways. Additionally, the PPI analysis demonstrated the network relationships of 13 important targets, while the TF analysis provided valuable insights into the regulatory networks of these targets. Among the identified TFs, RELA was found to be the most significant. To validate our findings, we utilized publicly available histological and single-cell sequencing databases, successfully confirming the involvement of 8 key targets, including AKT1, EGFR, RHOA, MAPK1, PIK3R1, MAPK8, MAPK14, and MTOR. Furthermore, molecular docking studies were conducted to assess the interaction between remdesivir and the identified key targets, thus confirming its effective targeting effects. Remdesivir has the potential to exert antiviral, anti-inflammatory, and immunomodulatory effects in the context of COVID-19-associated PF.

Lipopolysaccharide Induces Trained Innate Immune Tolerance in the Heart Through Interferon Signaling in a Model of Stress-Induced Cardiomyopathy.

Although the ability of the heart to adapt to environmental stress has been studied extensively, the molecular and cellular mechanisms responsible for cardioprotection are not yet fully understood. We administered Toll-like receptor (TLR) agonists or a diluent to wild-type mice and assessed their potential to induce cardiac protection against injury from a high intraperitoneal dose of isoproterenol (ISO) administered 7 days later. Cardioprotective effects were analyzed through serum cardiac troponin I levels, immune cell profiling via flow cytometry, echocardiography, and multiomic single-nuclei RNA and ATAC sequencing. Pretreatment with the TLR4 agonist lipopolysaccharide (LPS), but not TLR1/2 or TLR3 agonists, conferred cardioprotection against ISO, as demonstrated by reduced cardiac troponin I leakage, decreased inflammation, preservation of cardiac structure and function, and improved survival. Remarkably, LPS-induced tolerance was reversed by β-glucan treatment. Multiomic analysis showed that LPS-tolerized hearts had greater chromatin accessibility and upregulated gene expression compared to hearts treated with LPS and β-glucan (reverse-tolerized). The LPS tolerance was associated with upregulation of interferon response pathways across various cell types, including cardiac myocytes and stromal cells. Blocking both type 1 and type 2 interferon signaling eliminated LPS-induced tolerance against ISO, while pretreatment with recombinant type 1 and 2 interferons conferred cardiac protection. Multiomic sequencing further revealed enhanced cytoprotective signaling in interferon-treated hearts. Analysis of cell-cell communication networks indicated increased autocrine signaling by cardiac myocytes, as well as greater paracrine signaling between stromal cells and myeloid cells, in LPS-tolerized versus reverse-tolerized hearts. LPS pretreatment confers cardiac protection against ISO-induced injury through TLR4 mediated type 1 and 2 interferon signaling, consistent with trained innate immune tolerance. The observation that LPS-induced protection in cardiac myocytes involves both cell-autonomous and non-cell-autonomous mechanisms underscores the complexity of innate immune tolerance in the heart, warranting further investigation into this cardioprotective phenotype. What is new?: The Toll-like receptor 4 (TLR4) agonist lipopolysaccharide (LPS) confers cardiac protection against isoproterenol-mediated injury in a manner consistent with trained innate immune tolerance, which is reversed by β-glucan treatment.Activation of type 1 and 2 interferon signaling, which is downstream of Toll-like receptor 4, is necessary and sufficient for LPS-induced cardiac protection.LPS-tolerized hearts show heightened autocrine signaling by cardiac myocytes and, to a greater degree, increased cell-cell communication between cardiac myocytes and stromal and myeloid cells compared to reverse-tolerized hearts.What are the clinical implications?: TLR4 and interferon signaling play key roles in the establishment of cardiac protection and LPS-induced trained innate immune tolerance.The protective effects of LPS are mediated by cell-autonomous and non-cell-autonomous mechanisms, suggesting that a deeper understanding of the molecular and cellular signatures of innate immune tolerance is required for the development of targeted approaches to modulate trained innate immunity, and consequently cytoprotection, in the heart.

The human milk oligosaccharide 3'sialyllactose reduces low-grade inflammation and atherosclerosis development in mice.

Macrophages contribute to the induction and resolution of inflammation and play a central role in chronic low-grade inflammation in cardiovascular diseases caused by atherosclerosis. Human milk oligosaccharides (HMOs) are complex unconjugated glycans unique to human milk that benefit infant health and act as innate immune modulators. Here, we identify the HMO 3'sialyllactose (3'SL) as a natural inhibitor of Toll-Like Receptor (TLR) 4-induced low-grade inflammation in macrophages and endothelium. Transcriptome analysis in macrophages revealed that 3'SL attenuates mRNA levels of a selected set of inflammatory genes and promotes the activity of Liver X Receptor (LXR) and Sterol Regulatory Element-binding Protein-1 (SREBP). These acute anti-inflammatory effects of 3'SL were associated with reduced histone H3K27 acetylation at a subset of lipopolysaccharide (LPS)-inducible enhancers distinguished by preferential enrichment for CCCTC-binding factor (CTCF), Interferon Regulatory Factor 2 (IRF2), B-cell lymphoma 6 (BCL6), and other transcription factor recognition motifs. In a murine atherosclerosis model, both subcutaneous and oral administration of 3'SL significantly reduced atherosclerosis development and the associated inflammation. This study provides evidence that 3'SL attenuates inflammation by a transcriptional mechanism to reduce atherosclerosis development in the context of cardiovascular disease.

An early Transcriptomic Investigation in Adult Patients with Spinal Muscular Atrophy Under Treatment with Nusinersen

Spinal muscular atrophy (SMA) is a rare degenerative disorder with loss of motor neurons caused by mutations in the SMN1 gene. Nusinersen, an antisense oligonucleotide, was approved for SMA treatment to compensate the deficit of the encoded protein SMN by modulating the pre–mRNA splicing of SMN2, the centromeric homologous of SMN1, thus inducing the production of a greater amount of biologically active protein. Here, we reported a 10-month transcriptomics investigation in 10 adult SMA who received nusinersen to search for early genetic markers for clinical monitoring. By comparing their profiles with age-matched healthy controls (HC), we also analyzed the changes in miRNA/mRNAs expression and miRNA-target gene interactions possibly associated with SMA. A multidisciplinary approach of HT-NGS followed by bioinformatics/biostatistics analysis was applied. Within the study interval, those SMA patients who showed some clinical improvements were characterized by having the SMN2/SMN1 ratio slightly increased over the time, while in the stable ones the ratio decreased, suggesting that the estimation of SMN2/SMN1 expression may be an early indicator of nusinersen efficacy. On the other hand, the expression of 38/147 genes/genetic regions DE at T0 between SMA and HC like TRADD and JUND resulted “restored” at T10. We also confirmed the dysregulation of miR-146a(-5p), miR-324-5p and miR-423-5p in SMA subjects. Of interest, miR-146a-5p targeted SMN1, in line with experimental evidence showing the key role of astrocyte-produced miR-146a in SMA motor neuron loss. Molecular pathways such as NOTCH, NF-kappa B, and Toll-like receptor signalings seem to be involved in the SMA pathogenesis.

Galectin-9 activates host immune response and improve immunoprotection of Onychostoma macrolepis against Aeromonas hydrophila infection

Galectin-9 (Gal-9) belongs to a family of the glycan-binding proteins (GBPs) and is known to restrict bacterial activity via interacting with pathogen associated molecular pattern (PAMPs). However, the underlying immune mechanism of endogenous Gal-9 on fish against bacterial infection is still unclear. In this study, effect of Gal-9 from Onychostoma macrolepis (OmGal-9) on expression of immune related-genes were measured by HEK293T. The immune response of O. macrolepis with OmGal-9 overexpression to Aeromonas hydrophila (A. hydrophila) infection (1.65×108 CFU/mL) were evaluated by tissue bacterial load, fish survival rate and transcriptome analysis. The results showed that OmGal-9 displayed a punctate distribution in the nucleus and cytoplasm of HEK293T cells. Compared to cells transfected with the empty vector (EV) group, recombinant plasmid pEGFP-Gal9 treatment (Gal9) significantly down-regulated the expression of immune-related genes TNFα, STAT3, MyD88, LCK, and p52 of HEK293T cells stimulated with LPS at 24 h, while up-regulated IκBα and caspase1 (P < 0.05). The activities of catalase (CAT), superoxide dismutase (SOD), the total antioxidant capacity (T-AOC), alkaline phosphatase (AKP), acid phosphatase (ACP), and lysozyme (LZM) of O.macrolepis were significantly increased on 7 days in Gal9 group compared to EV group (P < 0.05). The bacterial load of liver, spleen, and kidney of O.macrolepis infected with A. hydrophila in Gal9 group at 24 h significantly lower than that in EV group (P < 0.05), and the survival rate has increased from 15% to 35%. A comparative transcriptome analysis between the Gal9 and EV group identified 305 differentially expressed genes (DEGs). The analysis showed that OmGal-9 might play important regulatory in glycolysis / gluconeogenesis, fatty acid degradation, and ascorbate and aldarate metabolism. Moreover, the immune-related DEGs were predominantly enriched in eleven pathways, with the most important three of them being linked to innate immunity: NOD-like, C-type lectin and Toll-like receptor signaling pathway. Taking together, OmGal-9 can enhance the resistance of fish to bacterial diseases by improving immune system function and activating immune-related pathways.

Exploring common pathogenic association between Epstein Barr virus infection and long-COVID by integrating RNA-Seq and molecular dynamics simulations.

The term "Long-COVID" (LC) is characterized by the aftereffects of COVID-19 infection. Various studies have suggested that Epstein-Barr virus (EBV) reactivation is among the significant reported causes of LC. However, there is a lack of in-depth research that could largely explore the pathogenic mechanism and pinpoint the key genes in the EBV and LC context. This study mainly aimed to predict the potential disease-associated common genes between EBV reactivation and LC condition using next-generation sequencing (NGS) data and reported naturally occurring biomolecules as inhibitors. We applied the bulk RNA-Seq from LC and EBV-infected peripheral blood mononuclear cells (PBMCs), identified the differentially expressed genes (DEGs) and the Protein-Protein interaction (PPI) network using the STRING database, identified hub genes using the cytoscape plugins CytoHubba and MCODE, and performed enrichment analysis using ClueGO. The interaction analysis of a hub gene was performed against naturally occurring bioflavonoid molecules using molecular docking and the molecular dynamics (MD) simulation method. Out of 357 common genes, 22 genes (CCL2, CCL20, CDCA2, CEP55, CHI3L1, CKAP2L, DEPDC1, DIAPH3, DLGAP5, E2F8, FGF1, NEK2, PBK, TOP2A, CCL3, CXCL8, DEPDC1, IL6, RETN, MMP2, LCN2, and OLR1) were classified as hub genes, and the remaining ones were classified as neighboring genes. Enrichment analysis showed the role of hub genes in various pathways such as immune-signaling pathways, including JAK-STAT signaling, interleukin signaling, protein kinase signaling, and toll-like receptor pathways associated with the symptoms reported in the LC condition. ZNF and MYBL TF-family were predicted as abundant TFs controlling hub genes' transcriptional machinery. Furthermore, OLR1 (PDB: 7XMP) showed stable interactions with the five shortlisted refined naturally occurring bioflavonoids, i.e., apigenin, amentoflavone, ilexgenin A, myricetin, and orientin compounds. The total binding energy pattern was observed, with amentoflavone being the top docked molecule (with a binding affinity of -8.3 kcal/mol) with the lowest total binding energy of -18.48 kcal/mol. In conclusion, our research has predicted the hub genes, their molecular pathways, and the potential inhibitors between EBV and LC potential pathogenic association. The in vivo or in vitro experimental methods could be utilized to functionally validate our findings, which would be helpful to cure LC or to prevent EBV reactivation.

Intraoperative guidance of pancreatic cancer resection using a toll like receptor 2 targeted fluorescence molecular imaging agent.

To increase the achievement of negative R0 surgical margins and increase the low survival rates of pancreatic cancer, improvements in assessing tumor margins during surgical resections are needed. This can be accomplished by using pancreatic cancer-targeted fluorescence molecular imaging agents to intraoperatively detect tumor margins in real-time. Since toll like receptor 2 (TLR2) is broadly expressed among many cancer types including pancreatic adenocarcinomas, a high-affinity TLR2-targeted fluorescence molecular imaging agent (TLR2L-800) was developed. We investigate the potential for increased survival by employing real-time intraoperative tumor detection in a preclinical orthotopic human pancreatic xenograft tumor model using TLR2L-800. Three cohorts of nude mice bearing orthotopic human pancreatic xenograft tumors were intravenously injected with TLR2L-800. At 24 h postinjection, one cohort underwent in vivo fluorescence-guided surgical removal of tumors using a real-time fluorescence imaging platform, a second cohort underwent visible light surgery, and a third cohort did not undergo surgery. A fourth, non-tumor-bearing cohort was administered TLR2L-800 with no surgery. At 41 d post-surgery, the survival rates were 53% for the fluorescence-guided surgery group and 0% for both the visible light surgery group and the tumor-bearing no surgery group. The overall 200 d survival rate of 35% for the fluorescence-guided surgery group was significant compared to 0% for the visible light surgery group (p-value=0.0018). This study demonstrates the potential of increasing disease-free survival for patients with pancreatic cancer by increasing the attainment of R0 margins using a novel tumor-targeted lipopeptide ligand-based fluorescence molecular imaging agent, TLR2L-800, during real-time fluorescence-guided surgery.

Neuroprotective effects of Gastrodia elata Blume on promoting M2 microglial polarization by inhibiting JNK/TLR4/T3JAM/NF-κB signaling after transient ischemic stroke in rats.

Gastrodia elata Blume, also called Tian Ma (TM), has been used to treat stroke for centuries. However, its effects on inflammation in acute cerebral ischemic injury and underlying mechanisms involved in microglial polarization remain unknown. The present study explored the effects of the TM extract on the modulation of microglial M1/M2 polarization 2days after transient cerebral ischemia. Male Sprague Dawley rats were intracerebroventricularly administered with 1% dimethyl sulfoxide 25min before cerebral ischemia and subsequently intraperitoneally administered 0.25g/kg (DO + TM-0.25g), 0.5g/kg (DO + TM-0.5g), or 1g/kg (DO + TM-1g) of the TM extract after cerebral ischemia onset. DO + TM-0.5g and DO + TM-1 g treatments downregulated the following: phospho-c-Jun N-terminal kinase (p-JNK)/JNK, tumor necrosis factor (TNF) receptor-associated factor 3 (TRAF3), TRAF3-interacting JNK-activating modulator (T3JAM), p-nuclear factor-kappa B p65 (p-NF-κB p65)/NF-κB p65, ionized calcium-binding adapter molecule 1 (Iba1), CD86, TNF-α, interleukin (IL)-1β, and IL-6 expression and toll-like receptor 4 (TLR4)/Iba1, CD86/Iba1, and p-NF-κB p65/Iba1 coexpression. These treatments also upregulated IL-10, nerve growth factor, and vascular endothelial growth factor A expression and YM-1/2/Iba1 and IL-10/neuronal nuclei coexpression in the cortical ischemic rim. The JNK inhibitor SP600125 exerted similar treatment effects as the DO + TM-0.5g and DO + TM-1 g treatments. DO + TM-0.5g and DO + TM-1 g/kg treatments attenuate cerebral infarction by inhibiting JNK-mediated signaling. TM likely exerts the neuroprotective effects of promoting M1 to M2 microglial polarization by inhibiting JNK/TLR4/T3JAM/NF-κB-mediated signaling in the cortical ischemic rim 2days after transient cerebral ischemia.