Toll-like Receptor Research Articles

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.

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.

CD14 gene polymorphism in COVID-19 convalescents: analysis of (C-159)T

Cluster of differentiation 14 (CD14) is a pattern recognition receptor for lipopolysaccharide of gram-negative flora and has a close relationship with toll-like receptor 4 (TLR4). The interaction between CD14 and TLR 4 leads to the synthesis of cytokines such as interleukin-1, interleukin-6 and tumor necrosis factor-á. The TLR4 receptor is associated with the penetration of the new coronavirus infection virus into the cell, additionally stimulating the expression of the angiotensin converting enzyme 2 molecule and suppressing apoptosis in infected cells. Work on the study of the (159C)T polymorphism of the CD14 gene in COVID-19 is represented by only a few publications describing observations only in the Western European region. The purpose of our study was to study the association between single nucleotide polymorphism (SNP) of the CD14 gene – (159C)T (rs2569190) and susceptibility to a new coronavirus infection in the population of the Republic of Crimea. The study included 158 patients (87 women (55%) and 71 (45%) men, average age 45.6±6.14 years) who had COVID-19. Verification of previous COVID-19 was based on anamnestic data and data from studies performed at the time of illness (PCR). The control group consisted of 85 respondents who had not suffered a new coronavirus infection. To analyze the (C-159)T polymorphism of the CD14 gene, allele-specific PCR with electrophoretic detection in a 3% agarose gel (NPF "Litekh", Russia) was used. To compare the frequencies of allele combinations, the ÷2 test and odds ratio (95% CI) were used. The distribution of CD14 mutations followed Hardy-Weinberg equilibrium (p 0.05). The results of the study showed that the distribution of CD14 gene genotypes did not differ significantly in all study groups. The dominant genotype was the heterozygous genotype CT of the (C-159)T polymorphism of the CD14 gene. The analysis showed that the presence of CT and TT SNPs was not associated with the risk of developing a new coronavirus infection (p 0.05). Conclusions. The CD14 SNP (C-159)T is not associated with a higher risk of COVID-19 infection. The distribution of occurrence of SNP variants in the group of recovered individuals did not differ significantly from that in the control group (p 0.05).

Toll-like Receptor 9 in Preclinical Models of Idiopathic Pulmonary Fibrosis.

Toll-like Receptor 9 in Preclinical Models of Idiopathic Pulmonary Fibrosis.

Peripheral Blood IFN Responses to Toll-Like Receptor 1/2 Signaling Associate with Longer Survival in Men with Metastatic Prostate Cancer Treated with Sipuleucel-T.

The identification of factors that determine successful cancer immunotherapy, particularly in refractory tumor types like mCRPC, is urgently needed: both to identify patients that may benefit from such therapies and to uncover routes to sensitize patients with cancer to immunotherapy. Our work links functional peripheral immune responses with race and survival after cellular immunotherapy in men with mCRPC.

Elevated levels of damage-associated molecular patterns HMGB1 and S100A8/A9 coupled with toll-like receptor-triggered monocyte activation are associated with inflammation in patients with myelofibrosis.

Inflammation plays a pivotal role in the pathogenesis of primary and post-essential thrombocythemia or post-polycythemia vera myelofibrosis (MF) in close cooperation with the underlying molecular drivers. This inflammatory state is induced by a dynamic spectrum of inflammatory cytokines, although recent evidence points to the participation of additional soluble inflammatory mediators. Damage-associated molecular patterns (DAMPs) represent endogenous signals released upon cell death or damage which trigger a potent innate immune response. We assessed the contribution of two prototypical DAMPs, HMGB1 and S100A8/A9, to MF inflammation. Circulating HMGB1 and S100A8/A9 were elevated in MF patients in parallel to the degree of systemic inflammation and levels increased progressively during advanced disease stages. Patients with elevated DAMPs had higher frequency of adverse clinical features, such as anemia, and inferior survival, suggesting their contribution to disease progression. Monocytes, which are key players in MF inflammation, were identified as a source of S100A8/A9 but not HMGB1 release, while both DAMPs correlated with cell death parameters, such as serum LDH and cell-free DNA, indicating that passive release is an additional mechanism leading to increased DAMPs. HMGB1 and S100A8/A9 promote inflammation through binding to Toll-like receptor (TLR) 4, whereas the former also binds TLR2. Monocytes from MF patients were shown to be hyperactivated at baseline, as reflected by higher CD11b and tissue factor exposure and increased expression levels of proinflammatory cytokines IL-1β and IL-6. Patient monocytes showed preserved TLR4 and TLR2 expression and were able to mount normal or even exacerbated functional responses and cytokine upregulation following stimulation of TLR4 and TLR2. Elevated levels of endogenous TLR ligands HMGB1 and S100A8/A9 coupled to the finding of preserved or hyperreactive TLR-triggered responses indicate that DAMPs may promote monocyte activation and cytokine production in MF, fueling inflammation. Plasma IL-1β and IL-6 were elevated in MF and correlated with DAMPs levels, raising the possibility that DAMPs could contribute to cytokine generation in vivo. In conclusion, this study highlights that, in cooperation with classic proinflammatory cytokines, DAMPs represent additional inflammatory mediators that may participate in the generation of MF inflammatory state, potentially providing novel biomarkers of disease progression and new therapeutic targets.

Glycation Produces Topologically Different α-Synuclein Oligomeric Strains and Modulates Microglia Response via the NLRP3-Inflammasome Pathway.

α-Synuclein, a key player in Parkinson's disease and other synucleinopathies, possesses an inherently disordered structure that allows for versatile structural changes during aggregation. Microglia, the brain immune cells, respond differently to various α-synuclein strains, influencing their activation and release of harmful molecules, leading to neuronal death. Post-translational modifications, such as glycation in α-synuclein, add a layer of complexity to microglial activation. This study aimed to explore the impact of glycation on α-synuclein aggregation and microglial responses, which have not been studied before. Biophysical analyses revealed that glycated α-synuclein oligomers had distinct morphologies with a more negative and hydrophobic surface, preventing fibril formation and interfering with membrane interactions. Notably, there was increased cytosolic Ca2+ dysregulation, redox stress, and mitochondrial instability compared to cells exposed to unmodified α-synuclein oligomers. Additionally, glycated α-synuclein oligomers exhibited impaired binding to Toll-like receptor 2, compromising downstream signaling. Surprisingly, these oligomers promoted TLR4 endocytosis and degradation. In our experiments with oligomers, glycated α-synuclein oligomers preferred NLRP3 inflammasome-mediated neuroinflammation, contributing differently from unmodified α-synuclein oligomers. In summary, this study unveils the mechanism underlying the effect of glycation on α-synuclein oligomers and highlights the conformation-specific microglial responses toward extracellular α-synuclein.

Targeting mast cell activation alleviates anti-MHC I antibody and LPS-induced TRALI in mice by pharmacologically blocking the TLR3 and MAPK pathway

Transfusion-related lung injury (TRALI) poses a significant risk following blood transfusion and remains the primary cause of transfusion-related morbidity and mortality, primarily driven by the activation of immune cells through anti-major histocompatibility complex class I (anti-MHC I) antibody. However, it remains to be defined how immune microenvironmental cue contributes to TRALI. Here, we uncover that activated mast cells within the immune microenvironment promote lung inflammation and injury in antibody-mediated TRALI, both in vitro and in vivo. This was demonstrated by co-culturing lipopolysaccharide (LPS)-pretreated mast cell line with anti-MHC I antibody and establishing a “two-hit” TRALI mouse model through intratracheal injection of LPS followed by tail-vein injection of anti-MHC I antibody. Importantly, mast cell-deficient KitW-sh/W-sh mice exhibited markedly reduced lung inflammation and injury responses in antibody-mediated TRALI compared with wild-type mice. Mechanistically, activation of toll-like receptor 3 (TLR3)/mitogen-activated protein kinase (MAPK) signaling pathway in mast cells contributes to the enhanced production of proinflammatory factors. These excessive proinflammatory factors produced by activated mast cells contribute to lung inflammation and injury in antibody-mediated TRALI. Pharmacologically targeting the TLR3/MAPK pathway to inhibit mast cell activation normalizes the proinflammatory microenvironment and alleviates lung inflammation and injury in the preclinical TRALI mouse model. Overall, we find that activation of mast cells via the TLR3/MAPK pathway contributes to lung inflammation and injury in antibody-mediated TRALI, providing novel insights into its underlying mechanisms. Furthermore, targeting activated mast cells and the associated pathway offers potential therapeutic strategies for antibody-mediated TRALI.

Toll-like Receptors: Therapeutic Potential in Life Threatening Diseases-Cardiac Disorders.

Toll-like receptors (TLRs) belong to the innate immune system. TLRs identify and respond to invading pathogens by recognizing certain molecular patterns associated with the infections. TLRs are crucial for the host's defence against these diseases. TLRs are capable of detecting several endogenous chemicals through the recognition of damage-associated molecular patterns, which are generated in response to various harmful situations. Recent animal studies have shown that TLR signaling has a significant role in the development of serious heart diseases, such as ischemia myocardial damage, myocarditis, and septic cardiomyopathy, where inflammation of the heart muscle is a key factor. This manuscript examines the animal research findings on (1) TLRs, TLR ligands, and the signal transduction system, and (2) the significant involvement of TLR signaling in these crucial cardiac diseases.

Supercritical CO2 extracts of propolis inhibits tumor proliferation and Enhances the immunomodulatory activity via activating the TLR4-MAPK/NF-κB signaling pathway

Propolis is a natural immunomodulator with anticancer activity. This study investigated the immunomodulatory mechanism and anti-tumor activity of supercritical CO2 extracts of propolis (SEP) in tumor-bearing immunosuppression mice. We used cyclophosphamide (CTX) to construct the immunosuppressive mice model and then inoculated them with CT26 cells to build the CT26 tumor-bearing immunosuppression mice model. Upon treatment with SEP, tumor proliferation in mice was markedly suppressed, with tumor volumes decreasing from 1881.43 mm3 to 1049.95 mm3 and weights reducing from 2.07 g to 1.13 g. Concurrently, the immune system recovered well, and the spleen and thymus indexes increased significantly. The total T lymphocyte (CD3+ T cell) contents in the spleen and blood recovered from 11.88 % to 21.19 % and 15.32 % to 22.19 %, respectively. In addition, the CD4+ /CD8+ ratio has returned to a healthy level, 3.12 in the spleen and 5.42 in the blood. The levels of IL-1β, IL-6, and TNF-α were increased by 2.17, 2.76, and 7.15 times in the spleen, 2.76, 1.92, and 3.02 times in the serum. Moreover, the western blot results showed that SEP treatment increased the expression of toll-like receptor 4 (TLR4) and the phosphorylation of p38, extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p65. These results indicated that SEP activated the immune activity of RAW 264.7 macrophages through the TLR4-mitogen-activated protein kinase (MAPK)/nuclear factor kappa B (NF-κB) signaling pathway to exert immunomodulatory function and inhibit tumor proliferation. This study facilitated the further application of SEP as a potential immunomodulatory and anti-tumor functional food.

AHCC inhibited hepatic stellate cells activation by regulation of cytoglobin induction via TLR2-SAPK/JNK pathway and collagen production via TLR4-NF-κβ pathway.

Cirrhosis, which represents the end stage of liver fibrosis, remains a life-threatening condition without effective treatment. Therefore, prevention of the progression of liver fibrosis through lifestyle habits such as diet and exercise is crucial. The functional food AHCC, a standardized extract of cultured Lentinula edodes mycelia produced by Amino Up Co., Ltd. (Sapporo, Japan)] has been reported to be effective in improving the pathophysiology of various liver diseases. In this study, the aim was to analyze the influence of AHCC on hepatic stellate cells, which are responsible for liver fibrosis. Eight-week-old male C57BL6/j mice were induced with liver fibrosis by intraperitoneal injection of carbon tetrachloride. Simultaneously, they were orally administered 3% AHCC to investigate its impact on the progression of liver fibrosis. Using the human hepatic stellate cell (HHSteC) line, we analyzed the influence of AHCC on the expression of molecules related to hepatic stellate cell activation. The administration of AHCC resulted in reduced expression of collagen1a, α smooth muscle actin (αSMA), and heat shock protein 47 in the liver. Furthermore, the expression of cytoglobin, a marker for quiescent hepatic stellate cells, was enhanced. In vitro study, it was confirmed that AHCC inhibited αSMA by inducing cytoglobin via upregulating the stress-activated protein kinase/Jun NH2-terminal kinase (SAPK/JNK) pathway through Toll-like receptor (TLR) 2. In addition, AHCC suppressed collagen1a production by hepatic stellate cells through TLR4-NF-κβ pathway. AHCC was suggested to suppress hepatic fibrosis by inhibition of hepatic stellate cells activation. Daily intake of AHCC from mild fibrotic stages may have the potential to prevent the progression of liver fibrosis.NEW & NOTEWORTHY AHCC, a standardized extract of cultured Lentinula edodes mycelia, suppresses liver fibrosis progression by induction of cytoglobin via the Toll-like receptor 2 (TLR2)-stress-activated protein kinase/Jun NH2-terminal kinase (SAPK/JNK) pathway and the inhibition of collagen production via the TLR4-NFκβ pathway in hepatic stellate cells. Daily oral administration of AHCC from the stage of MASLD may have the potential to prevent disease progression to MASH with fibrosis.