Toll-like Receptor Signaling Research Articles

A novel biomimetic nanovesicle containing caffeic acid-coupled carbon quantum dots for the the treatment of Alzheimer's disease via nasal administration.

Alzheimer's disease (AD) is a common neurodegenerative disease characterized by progressive cognitive and physical impairment. Neuroinflammation is related to AD, and the misfolding and aggregation of amyloid protein in the brain creates an inflammatory microenvironment. Microglia are the predominant contributors to neuroinflammation, and abnormal activation of microglia induces the release of a large amount of inflammatory factors, promotes neuronal apoptosis, and leads to cognitive impairment. In this study, we used microglial membranes containing caffeic acid-coupled carbon quantum dots to prepare a novel biomimetic nanocapsule (CDs-CA-MGs) for the treatment of AD. The application of CDs-CA-MGs via nasal administration can bypass the blood‒brain barrier (BBB) and directly target the site of inflammation. After treatment with CDs-CA-MGs, AD mice showed reduced inflammation in the brain, decreased neuronal apoptosis, and significantly improved learning and memory abilities. In addition, CDs-CA-MGs affect inflammation-related JAK-STAT and Toll-like receptor signaling pathways in AD mice. CDs-CA-MGs significantly downregulated interleukins (IL-1β and IL-6) and tumor necrosis factor (TNF-α). This finding suggested that CDs-CA-MGs may improve cognitive impairment by modulating inflammatory responses. In conclusion, the use of CDs-CA-MGs provides a possible therapeutic strategy for the treatment of AD.

TMT-based quantitative proteomics unveils the protective mechanism of Polygonatum sibiricum polysaccharides on septic acute liver injury

Polygonatum sibiricum polysaccharides (PSP) has been shown to possess multiple pharmacological functions. Our previous study found that PSP could protect against acute liver injury during sepsis via inhibiting inflammatory response. However, the underlying molecular mechanism by which PSP alleviates septic acute liver injury (SALI) remains unknown. Herein, TMT-based quantitative proteomics was utilized to explore the essential pathways and proteins involved in the protective effects of PSP on SALI. The results revealed that 632 and 176 differentially expressed proteins (DEPs) were identified in Model_vs_Control and PSP_vs_Model, respectively. GO annotation showed similar trends, suggesting that these DEPs were primarily involved in the cellular anatomical entity in Cellular Component, the cellular processe and the biological regulation in Biological Process, the binding and the catalytic activity in Molecular Function. Meanwhile, KEGG enrichment analysis implied that four common pathways, including the NF-κB signaling pathway, the IL-17 signaling pathway, the TNF signaling pathway and the Toll-like receptor signaling pathway, were closely associated with the pathogenesis of sepsis among the top 20 remarkably enriched pathways in Model_vs_Control_up and PSP_vs_Model_down. Moreover, the levels of several common DEPs, including TLR2, IKKi, JunB and CXCL9, were validated by WB, which was in line with the results of proteomics. Therefore, the protective effects of PSP on SALI might exert via blocking the above-mentioned inflammation pathways.Significance: PSP, recognized as a key component of Polygonatum sibiricum, exhibits a range of pharmacological functions. Our previous study found that PSP could protect against SALI, yet failing to clarify the mechanism of action. To reveal the underlying molecular mechanism involved in the protective effects of PSP on SALI, a TMT-based quantitative proteomic analysis was performed to detect and analyse the DEPs in liver tissue among the control group, the model group and the PSP group in this study. The results provide theoretical references for exploring the action mechanism of drugs and facilitate the comprehensive utilization of PSP. SignificancePSP have been identified as the most crucial components of Polygonatum sibiricum with various pharmacological functions. Our previous study found that PSP could protect against SALI, but the mechanism of action remains unknown. To reveal the underlying molecular mechanism involved in the protective effects of PSP on SALI, a TMT-based quantitative proteomic analysis was performed to detect and analyse the DEPs in liver tissue among the control group, the model group and the PSP group in this study. The results provide theoretical references for exploring the action mechanism of drugs and facilitate the comprehensive utilization of PSP.

Anti-Inflammatory Potential of Costus speciosus rhizome Bioactive Phytochemicals: A Combined GC-MS and Computational Approach Targeting TLR-4 Signaling.

Plants represent a rich reservoir of bioactive compounds with established therapeutic value in diverse diseases. Notably, the Toll-like receptor-4 (TLR-4) signaling pathway plays a pivotal role in inflammation. Upon engagement with pro-inflammatory ligands like lipopolysaccharide, TLR-4 triggers downstream cascades involving nuclear factor ĸappa B and mitogen- activated protein kinases. This signaling cascade ultimately dictates the onset and progression of inflammatory diseases. Therefore, targeting TLR-4 signaling offers a promising therapeutic approach for managing inflammatory disorders. This study investigated the potential of Costus speciosus rhizome phytocompounds, a traditional medicinal plant, as novel as modulators of TLR-4 signaling, highlighting their mechanisms of action and potential clinical applications. In the present study, 18 phytocompounds isolated from the rhizome of Costus speciosus, were studied against TLR-4/AP-1 signaling, which is implicated in the inflammatory process using a computational approach. The compounds exhibited binding affinities ranging from -4.087 to -8.93 kcal/mol with the TLR-4 protein due to the formation of multiple intermolecular interactions. Benzenepropanoic acid, 3,5-bis(1,1-dimethylethyl)-4-hydroxy-, methyl ester (compound 7) exhibited exceptional binding energy (-8.93 kcal/mol), indicating strong affinity for the TLR-4 protein. Additionally, compound 7 displayed favorable ADMET properties, suggesting promising drug development potential. Molecular dynamics simulations confirmed the stability of the compound 7-TLR4 complex, further supporting its ability to modulate TLR-4 signaling. These findings highlight the therapeutic potential of Costus speciosus phytocompounds, particularly compound 7, as potent anti-inflammatory modulators. Further research is warranted to validate their anti-inflammatory and neuroprotective effects in pre-clinical models, paving the way for their development as novel therapeutic agents for inflammatory diseases.

Machine learning-derived peripheral blood transcriptomic biomarkers for early lung cancer diagnosis: Unveiling tumor-immune interaction mechanisms.

Lung cancer continues to be the leading cause of cancer-related mortality worldwide. Early detection and a comprehensive understanding of tumor-immune interactions are crucial for improving patient outcomes. This study aimed to develop a novel biomarker panel utilizing peripheral blood transcriptomics and machine learning algorithms for early lung cancer diagnosis, while simultaneously providing insights into tumor-immune crosstalk mechanisms. Leveraging a training cohort (GSE135304), we employed multiple machine learning algorithms to formulate a Lung Cancer Diagnostic Score (LCDS) based on peripheral blood transcriptomic features. The LCDS model's performance was evaluated using the area under the receiver operating characteristic (ROC) curve (AUC) in multiple validation cohorts (GSE42834, GSE157086, and an in-house dataset). Peripheral blood samples were obtained from 20 lung cancer patients and 10 healthy control subjects, representing an in-house cohort recruited at the Sixth People's Hospital of Chengdu. We employed advanced bioinformatics techniques to explore tumor-immune interactions through comprehensive immune infiltration and pathway enrichment analyses. Initial screening identified 844 differentially expressed genes, which were subsequently refined to 87 genes using the Boruta feature selection algorithm. The random forest (RF) algorithm demonstrated the highest accuracy in constructing the LCDS model, yielding a mean AUC of 0.938. Lower LCDS values were significantly associated with elevated immune scores and increased CD4+ and CD8+ T-cell infiltration, indicative of enhanced antitumor-immune responses. Higher LCDS scores correlated with activation of hypoxia, peroxisome proliferator-activated receptor (PPAR), and Toll-like receptor (TLR) signaling pathways, as well as reduced DNA damage repair pathway scores. Our study presents a novel, machine learning-derived peripheral blood transcriptomic biomarker panel with potential applications in early lung cancer diagnosis. The LCDS model not only demonstrates high accuracy in distinguishing lung cancer patients from healthy individuals but also offers valuable insights into tumor-immune interactions and underlying cancer biology. This approach may facilitate early lung cancer detection and contribute to a deeper understanding of the molecular and cellular mechanisms underlying tumor-immune crosstalk. Furthermore, our findings on the relationship between LCDS and immune infiltration patterns may have implications for future research on therapeutic strategies targeting the immune system in lung cancer.

Employing single-cell RNA sequencing coupled with an array of bioinformatics approaches to ascertain the shared genetic characteristics between osteoporosis and obesity.

This study examined the relationship between obesity (OB) and osteoporosis (OP), aiming to identify shared genetic markers and molecular mechanisms to facilitate the development of therapies that target both conditions simultaneously. Using weighted gene co-expression network analysis (WGCNA), we analyzed datasets from the Gene Expression Omnibus (GEO) database to identify co-expressed gene modules in OB and OP. These modules underwent Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment and protein-protein interaction analysis to discover Hub genes. Machine learning refined the gene selection, with further validation using additional datasets. Single-cell analysis emphasized specific cell subpopulations, and enzyme-linked immunosorbent assay (ELISA), protein blotting, and cellular staining were used to investigate key genes. WGCNA revealed critical gene modules for OB and OP, identifying the Toll-like receptor (TLR) signalling pathway as a common factor. TLR2 was the most significant gene, with a pronounced expression in macrophages. Elevated TLR2 expression correlated with increased adipose accumulation, inflammation, and osteoclast differentiation, linking it to OP development. Our study underscores the pivotal role of TLR2 in connecting OP and OB. It highlights the influence of TLR2 in macrophages, driving both diseases through a pro-inflammatory mechanism. These insights propose TLR2 as a potential dual therapeutic target for treating OP and OB.

Salmonella Typhimurium infection inhibits macrophage IFNβ signaling in a TLR4-dependent manner.

Type I Interferons (IFNs) generally have a protective role during viral infections, but their function during bacterial infections is dependent on the bacterial species. Legionella pneumophila, Shigella sonnei and Mycobacterium tuberculosis can inhibit type I IFN signaling. Here we examined the role of type I IFN, specifically IFNβ, in the context of Salmonella enterica serovar Typhimurium (STm) macrophage infections and the capacity of STm to inhibit type I IFN signaling. We demonstrate that IFNβ has no effect on the intracellular growth of STm in infected bone marrow derived macrophages (BMDMs) derived from C57BL/6 mice. STm infection inhibits IFNβ signaling but not IFNγ signaling in a murine macrophage cell line. We show that this inhibition is independent of the type III and type VI secretion systems expressed by STm and is also independent of bacterial phagocytosis. The inhibition is Toll-like receptor 4 (TLR4)-dependent as the TLR4 ligand, lipopolysaccharide (LPS), alone is sufficient to inhibit IFNβ-mediated signaling. Cells downregulated their surface levels of IFNα/β receptor 1 (IFNAR1) in response to LPS, which may be mediating our observed inhibition. Lastly, we examined this inhibition in the context of TLR4-deficient BMDMs as well as TLR4 RNA interference and we observed a loss of inhibition with LPS stimulation as well as STm infection. In summary, we show that macrophages exposed to STm have reduced IFNβ signaling via crosstalk with TLR4 signaling, which may be mediated by reduced host cell surface IFNAR1, and that IFNβ signaling does not affect cell-autonomous host defense against STm.

Evaluation and Analysis of Traditional Chinese Medicine Treatment of Bovine Viral Diarrhea/Mucosal Disease Based on Network Pharmacology and In Vitro Studies

Background Bovine viral diarrhea/mucosal disease (BVD-MD) is widely distributed worldwide. The disease causes serious economic losses to animal husbandry every year. Finding targeted antiviral drugs proves to be an effective strategy. Purpose This study was based on network pharmacology and in vitro studies to analyze the potential of traditional Chinese medicine (TCM) in the treatment of BVD-MD. Methods The intersection targets between TCM and the disease were identified. Network topology analysis and protein–protein interaction (PPIs) networks were performed. Intersection targets were analyzed for gene ontology (GO) and Kyoto encyclopedia of genes and genomes (KEGG) enrichment. The molecular docking and molecular dynamics simulation methods were used to reveal the degree of binding of core components to key target genes. Characterization of the anti-Bovine viral diarrhea virus (BVDV) effect of TCM by cytotoxicity and in vitro studies. Results The results revealed the selection of five key Chinese medicines, along with 206 targets associated with BVD-MD. The toll-like receptor, PI3K-AKT, and tumor necrosis factor (TNF) signaling pathways were closely related to the five TCMs. AKT1, EGFR, HSP90AA1, and MAPK1 had good binding with quercetin, the core component of Chinese medicine. Molecular dynamics analysis showed that quercetin exhibited complex stability with AKT1. In vitro studies have demonstrated that the inhibitory effect of quercetin on BVDV is mainly in the initial phase. Quercetin inhibited the expression of AKT1. Conclusion The mechanism of BVDV inhibition by the core Chinese medicines is closely related to MAPK1, AKT1, TNF, and HSP90AA1, with the reduction of AKT1 ultimately affecting viral expression.

Toll-like receptor 7 protects against intestinal inflammation and restricts the development of colonic tissue-resident memory CD8+ T cells.

The maintenance of intestinal homeostasis depends on a complex interaction between the immune system, intestinal epithelial barrier, and microbiota. Alteration in one of these components could lead to the development of inflammatory bowel diseases (IBD). Variants within the autophagy gene ATG16L1 have been implicated in susceptibility and severity of Crohn's disease (CD). Individuals carrying the risk ATG16L1 T300A variant have higher caspase 3-dependent degradation of ATG16L1 resulting in impaired autophagy and increased cellular stress. ATG16L1-deficiency induces enhanced IL-1β secretion in dendritic cells in response to bacterial infection. Infection of ATG16L1-deficient mice with a persistent strain of murine norovirus renders these mice highly susceptible to dextran sulfate sodium colitis. Moreover, persistent norovirus infection leads to intestinal virus specific CD8+ T cells responses. Both Toll-like receptor 7 (TLR7), which recognizes single-stranded RNA viruses, and ATG16L1, which facilitates the delivery of viral nucleic acids to the autolysosome endosome, are required for anti-viral immune responses. However, the role of the enteric virome in IBD is still poorly understood. Here, we investigate the role of TLR7 and ATG16L1 in intestinal homeostasis and inflammation. At steady state, Tlr7-/- mice have a significant increase in large intestinal lamina propria (LP) granzyme B+ tissue-resident memory CD8+ T (TRM) cells compared to WT mice, reminiscent of persistent norovirus infection. Deletion of Atg16l1 in myeloid (Atg16l1ΔLyz2 ) or dendritic cells (Atg16l1ΔCd11c ) leads to a similar increase of LP TRM. Furthermore, Tlr7-/- and Atg16l1ΔCd11c mice were more susceptible to dextran sulfate sodium colitis with an increase in disease activity index, histoscore, and increased secretion of IFN-γ and TNF-α. Treatment of Atg16l1ΔCd11c mice with the TLR7 agonist Imiquimod attenuated colonic inflammation in these mice. Our data demonstrate that ATG16L1-deficiency in myeloid and dendritic cells leads to an increase in LP TRM and consequently to increased susceptibility to colitis by impairing the recognition of enteric viruses by TLR7. In conclusion, the convergence of ATG16L1 and TLR7 signaling pathways plays an important role in the immune response to intestinal viruses. Our data suggest that activation of the TLR7 signaling pathway could be an attractive therapeutic target for CD patients with ATG16L1 risk variants.

Changes and associations between synovial fluid and magnetic resonance imaging markers of osteoarthritis after high tibial osteotomy

BackgroundMechanobiological mechanisms of osteoarthritis (OA) are unclear. Our objectives were to explore: 1) changes in knee joint physiology using a large panel of synovial fluid biomarkers from before to one year after high tibial osteotomy (HTO) surgery, and 2) the association of changes in the synovial fluid biomarkers with the changes in MRI measures of knee effusion-synovitis and articular cartilage composition.MethodsTwenty-six patients with symptomatic knee OA and varus alignment underwent synovial fluid aspirations and 3 T MRI before and one year after medial opening wedge HTO. Cytokine and growth factor levels in synovial fluid were measured with multiplex assays. Ontology and pathway enrichment was assessed using data protein sets with gene set enrichment analysis (GSEA), and analyzed using linear mixed effects models. MRIs were analyzed for effusion-synovitis and T2 cartilage relaxation time using manual segmentations. Changes in biomarker concentrations were correlated to changes in MRI effusion-synovitis volume and articular cartilage T2 relaxation times.ResultsDecreased enrichment in Toll-like receptor and TNF-α signalling was detected one year after HTO. The leading contributors to this reduction included IL-6, TNF-α and IL-1β, whereas the highest contributors to positive enrichment were EGF, PDGF-BB and FGF-2. Effusion-synovitis volume decreased (mean [95%CI]) one year after HTO (-2811.58 [-5094.40, -528.76mm3]). Effusion-synovitis volume was moderately correlated (r [95% CI]) with decreased MMP-1 (0.44 [0.05; 0.71]), IL-7 (0.41 [0.00; 0.69]) and IL-1β (0.59 [0.25; 0.80]) and increased MIP-1β (0.47 [0.10; 0.73]). Medial tibiofemoral articular cartilage T2 relaxation time decreased (mean [95% CI]) one year after HTO (-0.33 [-2.69; 2.05]ms). Decreased T2 relaxation time was moderately correlated to decreased Flt-3L (0.61 [0.28; 0.81]), IL-10 (0.47 [0.09; 0.73]), IP-10 (0.42; 0.03–0.70) and increased MMP-9 (-0.41 [-0.7; -0.03]) and IL-18 (-0.48 [-0.73; -0.10]).ConclusionsDecreased aberrant knee mechanical loading in patients with OA is associated with decreased biological and imaging measures of inflammation (measured in synovial fluid and on MRI) and increased anabolic processes. These exploratory findings suggest that improvement in knee loading can produce long-term (one year) improvement in joint physiology.

Nanopore-based full-length transcriptome sequencing of the skin in Pseudopleuronectes yokohamae identifies novel antimicrobial peptide genes

The marbled flounder (Pseudopleuronectes yokohamae) is highly esteemed for its exceptional nutritional value and delicious taste. However, this species has extremely limited transcriptome data, which can offer priceless information for disease protection. In the study, the skin transcriptomic sequencing of P. yokohamae revealed 7.72 GB of clean data using the Nanopore sequencing platform. The results revealed 30,498 transcripts of functional annotations in the P. yokohamae transcriptome. All transcripts were searched in eight functional databases. A total of 10,337 ORFs were obtained, of which 6081 complete ORFs accounted for 58.83% of all predicted CDS. Moreover, 10,195 SSRs were detected. Meanwhile, the non-pecific immunity pathways were investigated for better understanding of the immunological reaction in P. yokohamae, and seven innate immune pathways were identified. The innate-immune related genes were highly expressed in the NOD-like receptor signaling pathway, followed by the C-type lectin receptor signaling pathway, Toll-like receptor signaling pathway, RIG-I-like receptor signaling pathway and Cytosolic DNA-sensing pathway. In this study, four families of antimicrobial peptides (AMPs) in P. yokohamae were analysed for the first time, including piscidins, hepcidins, liver-expressed antimicrobial peptide and defensins. Seven AMPs, including Pypleurocidin-like WF3, Pypleurocidin-like WFX, Pyhepcidin 1, Pyhepcidin-like 1, PyLEAP-2, Pybeta-defensin and Pybeta-defensin-like 1, were further identified. The seven AMPs showed a highly identity in their cDNA and genomic structures and an inducible expression pattern preferable to skin in response to pathogens. The transcriptomic data and investigation of AMPs from P. yokohamae promote a deeper awareness of fish mucosal immunity and provide information in the prevention of fish diseases.

Deciphering farnesol's anti-arthritic and immunomodulatory potential by targeting multiple pathways: a combination of network pharmacology guided exploration and experimental verification.

Farnesol (FAR), a sesquiterpene alcohol, has documented FAR's anti-inflammatory and antioxidant activities. Current study was undertaken to assess the efficacy and mechanism of FAR in arthritis by employing network pharmacology and experimental models. Two experimental models comprising formaldehyde- and complete Freund's adjuvant (CFA)-induced arthritis evaluated the efficacy of FAR in treating arthritis. Various parameters were assessed. Then, a network pharmacology approach was applied to gain further insight into the potential mechanism and signaling pathways. FAR significantly reduced paw volume and the arthritic score and improved the hematological and biochemical changes. Radiographic and histological examination showed the anti-arthritic efficacy of FAR, which was associated with down-regulation of pro-inflammatory mediators and upregulation of anti-inflammatory mediators. Network pharmacology analysis revealed that FAR may exert its anti-arthritic effects by targeting specific genes associated with arthritis. Pathway analysis revealed the involvement of three key signaling pathways (IL-17 signaling, TNF signaling, and toll-like receptor signaling) in the development and progression of arthritis. The results pointed out the protective attributes of farnesol against formaldehyde and CFA-induced arthritis via modulation of multiple targets. This study provides a valuable reference for the development of a new treatment or complementary therapy for arthritis.

7975 TLR4 Signaling Modulates Reproductive and Metabolic Outcomes in a Mouse Model of Polycystic Ovary Syndrome

Abstract Disclosure: K.D. Wiggins: None. Z. Del Mundo: None. J.A. Ayala Angulo: None. M. Lopez: None. C. Garcia: None. C. Nguyen: None. J. De La Torre: None. A. Saba: None. D. Trinh: None. D. Nicholas: None. Polycystic Ovarian Syndrome (PCOS), characterized by disruptions in both reproductive and metabolic functions, is associated with abnormal immune system activity and chronic inflammation. Although the precise mechanistic drivers of chronic inflammation in PCOS are not fully elucidated, elevated serum levels of lipopolysaccharide (LPS) have been observed. We propose that Toll-like receptor 4 (TLR4) signaling, influenced by dysregulated LPS levels, is a central driver of chronic inflammation affecting both reproductive and metabolic pathways. To investigate this hypothesis, we subcutaneously implanted a nonsteroidal aromatase inhibitor, Letrozole (LET), in pubertal TLR4 knockout mice (TLR4KO) and C57BL/6J (wild type) mice. Our five-week LET treatment revealed that TLR4KO mice successfully entered all phases of the estrous cycle, with normalized Luteinizing Hormone (LH) and increased Follicle Stimulating Hormone (FSH) levels. These findings highlight TLR4's role in shaping cycling patterns and regulating fundamental hormonal pathways vital for reproductive health. Despite the genetic manipulation of the inflammatory pathway leading to a reduction in overall body weight gain, it had no discernible impact on the overall body composition of fat and lean mass. Additionally, our study found that dysregulation of blood glucose levels over time was independent of reduced body weight. TLR4KO mice demonstrated inefficient glucose clearance compared to wild-type mice, hinting at potential increases in insulin resistance, imbalances in adipose tissue inflammation, or possible alterations in the gut microbiota. Our study underscores the significance of disrupting the pro-inflammatory TLR4 pathway in shaping the reproductive and metabolic phenotypes in Letrozole-induced PCOS. The investigation into TLR4 becomes pivotal for comprehending the role of chronic inflammation in PCOS development or progression, offering valuable insights into potential therapeutic targets. Presentation: 6/2/2024

Skin microRNA transcriptomic and functional analysis revealed novel-m0065-3p regulating antiviral immune responses via targeting IRF7 in rainbow trout (Oncorhynchus mykiss) infected with IHNV

miRNAs are small non-coding RNA that instrumental in host immune response to pathogen infection. However, studies on the involvement of miRNAs in rainbow trout (Oncorhynchus mykiss) antiviral response are still lacking. In this study, miRNA profiles of 48 hpi (T48SKs) compared to control (C48SKs), novel-m0065-3p and interferon regulatory factor 7 (IRF7) expression, and novel-m0065-3p-IRF7 functions were examined in rainbow trout skin following infectious hematopoietic necrosis virus (IHNV) challenge through RNA-seq, qRT-PCR, and overexpression and inhibition assays. Transcriptome analysis identified 23 up-regulated and 25 down-regulated differentially expressed miRNAs (DEMs). Enrichment analysis revealed that target genes were enriched in MAPK, RIG-I-like receptor, and Toll-like receptor signaling pathways. The DEMs (miR-205-z, novel-m0065-3p, novel-m0215-5p, novel-m0384-5p, and novel-m0397‐3p) were identified, and targeted key immune-related genes. Expression patterns suggested that novel-m0065-3p and IRF7 were potential regulators in antiviral immune responses of rainbow trout. Functional analysis revealed that the overexpression of novel-m0065-3p reduced significantly IRF7 expression in liver cells, which was attenuated by the introduction of IRF7, whereas the opposite result was obtained by silencing novel-m0065-3p. Overexpressed novel-m0065-3p promoted liver cell proliferation and inhibited apoptosis, and co-transfection of IRF7 attenuated the effect of novel-m0065-3p. Furthermore, IRF7 overexpression inhibited significantly IHNV replication. In vivo, the injection of agomiR-m0065-3p and antagomiR-m0065-3p changed significantly the expression of IRF7 and downstream genes. This study provided valuable information for drug-targeted diseases research and directed breeding efforts.

Transcriptomic Profiling of Primary Microglia: Effects of miR-19a-3p and miR-19b-3p on Microglia Activation.

Neuropathic pain resulting from spinal cord injury (SCI) is a significant secondary health issue affecting around 60% of individuals with SCI. After SCI, activation of microglia, the immune cells within the central nervous system, leads to neuroinflammation by producing pro-inflammatory cytokines and affects neuropathic pain. This interplay between inflammation and pain contributes to the persistent and intense pain experienced by many individuals with SCI. MicroRNAs (miRs) have been critical regulators of neuroinflammation. Previous research in our laboratory has revealed upregulation levels of circulating miR-19a and miR-19b in individuals with SCI with neuropathic pain compared to those without pain. In this study, we treated primary microglial cultures from mice with miR-19a and miR-19b for 24 h and conducted RNA sequencing analysis. Our results showed that miR-19a and miR-19b up- and downregulate different genes according to the volcano plots and the heatmaps. miR-19a and miR-19b regulate inflammation through distinct signaling pathways. The results showed that miR-19a promotes inflammation via toll-like receptor signaling, TNF signaling, and cytokine-cytokine receptor interactions, while miR-19b increases inflammatory responses through the PI3K-Akt signaling pathway, focal adhesion, and extracellular matrix receptor interactions. The protein-protein interaction (PPI) networks used the STRING database to identify transcription factors associated with genes up- or downregulated by miR-19a and miR-19b. Key transcription factors, such as STAT1, STAT2, and KLF4 for miR-19a, and Nr4a1, Nr4a2, and Nr4a3 for miR-19b, were identified and revealed their roles in regulating neuroinflammation. This study demonstrates that miR-19a and miR-19b modulate diverse patterns of gene expression, regulate inflammation, and induce inflammatory responses in microglia.

TLR2–EGR1 signaling axis modulates TGFβ1-induced differentiation of fibroblasts into myofibroblasts in pulmonary fibrosis

Pulmonary fibrosis is a progressive lung condition characterized by the excessive activation of myofibroblasts. Transforming growth factor beta 1 (TGFβ1) plays a crucial role in the differentiation of fibroblasts into myofibroblasts. In addition, toll-like receptor 2 (TLR2), known for its role in immune responses, contributes to pulmonary fibrosis by promoting myofibroblast differentiation. However, the interplay between TGFβ1 and TLR2 signaling pathways in myofibroblast differentiation has remained elusive. In the present study, we investigated the involvement of TLR2 in TGFβ1-induced fibroblast differentiation into myofibroblasts using IMR-90 human pulmonary fibroblasts as a model cell line. We found that TLR2 activation induced myofibroblast differentiation by enhancing the expression of early growth response 1 (EGR1) via the mitogen-activated protein kinase (MAPK) signaling pathway. Elevated EGR1 levels were detected in the lung tissues of a bleomycin (BLM)-induced mouse model of pulmonary fibrosis. Moreover, the administration of tomaralimab, an antagonistic anti-TLR2 antibody, reduced the EGR1 expression and collagen deposition. Altogether, targeting the TLR2–EGR1 pathway could be a promising therapeutic approach for pulmonary fibrosis by blocking TGFβ1-induced myofibroblast differentiation.

GSP1-111 Modulates the Microglial M1/M2 Phenotype by Inhibition of Toll-like Receptor 2: A Potential Therapeutic Strategy for Depression.

Neuroinflammation plays a vital role in neurodegenerative diseases and neuropsychiatric disorders, and microglia and astrocytes chiefly modulate inflammatory responses in the central nervous system (CNS). Toll-like receptors (TLRs), which are expressed in neurons, astrocytes, and microglia in the CNS, are critical for innate immune responses; microglial TLRs can regulate the activity of these cells, inducing protective or harmful effects on the surrounding cells, including neurons. Therefore, regulating TLRs in microglia may be a potential therapeutic strategy for neurological disorders. We examined the protective effects of GSP1-111, a novel synthetic peptide for inhibiting TLR signaling, on neuroinflammation and depression-like behavior. GSP1-111 decreased TLR2 expression and remarkably reduced the mRNA expression of inflammatory M1-phenotype markers, including tumor necrosis factor (TNF)α, interleukin (IL)-1β, and IL-6, while elevating that of the M2 phenotype markers, Arg-1 and IL-10. In vivo, GSP1-111 administration significantly decreased the depression-like behavior induced by lipopolysaccharide (LPS) in a forced swim test and significantly reduced the brain levels of M1-specific inflammatory cytokines (TNFα, IL-1β, and IL-6). GSP1-111 prevented the LPS-induced microglial activation and TLR2 expression in the brain. Accordingly, GSP1-111 prevented inflammatory responses and induced microglial switching of the inflammatory M1 phenotype to the protective M2 phenotype. Thus, GSP1-111 could prevent depression-like behavior by inhibiting TLR2. Taken together, our results suggest that the TLR2 pathway is a promising therapeutic target for depression, and GSP1-111 could be a novel therapeutic candidate for various neurological disorders.

Exploration of Pharmacological Mechanism of Cinnamomum tamala Essential Oil in Treating Inflammation based on Network Pharmacology, Molecular Modelling, and Experimental Validation.

Cinnamomum tamala (Buch.-Ham.) T.Nees & Eberm., also known as Indian Bay leaf, holds a distinctive position in complementary and alternative medicinal systems due to its anti-inflammatory properties. However, the active constituents and key molecular targets by which C. tamala essential oil (CTEO) exerts its anti-inflammatory action remain unclear. The present study used network pharmacology and experimental validation to investigate the mechanism of CTEO in the treatment of inflammation. GC-MS analysis was used to identify the constituents of CTEO. The key constituents and core targets of CTEO against inflammation were obtained by network pharmacology. The binding mechanism between the active compounds and inflammatory genes was ascertained by molecular docking and molecular dynamics simulation analysis. The pharmacological mechanism predicted by network pharmacology was verified in lipopolysaccharide-stimulated murine macrophage (RAW 264.7) cell lines. Forty-nine constituents were identified by GC-MS analysis, with 44 constituents being drug-like candidates. A total of 549 compounds and 213 inflammation-related genes were obtained, revealing 68 overlapping genes between them. Compound target network analysis revealed cinnamaldehyde as the core bioactive compound with the highest degree score. PPI network analysis demonstrated Il-1β, TNF-α, IL8, IL6 and TLR4 as key hub anti-inflammatory targets. KEGG enrichment analysis revealed a Toll-like receptor signalling pathway as the principally regulated pathway associated with inflammation. A molecular docking study showed that cinnamaldehyde strongly interacted with the Il-1β, TNF-α and TLR-4 proteins. Molecular dynamics simulations and MMPBSA analysis revealed that these complexes are stable without much deviation and have better free energy values. In cellular experiments, CTEO showed no cytotoxic effects on RAW 264.7 murine macrophages. The cells treated with LPS exhibited significant reductions in NO, PGE2, IL-6, TNF-α, and IL-1β levels following treatment with CTEO. Additionally, CTEO treatment reduced the ROS levels and increased the antioxidant enzymes such as SOD, GSH, GPx and CAT. Immunofluorescence analysis revealed that CTEO inhibited LPS-stimulated NF-κB nuclear translocation. The mRNA expression of TLR4, MyD88 and TRAF6 in the CTEO group decreased significantly compared to the LPS-treated group. The current findings suggest that CTEO attenuates inflammation by regulating TLR4/MyD88/NF- κB signalling pathway.

Chiral Intranasal Nanovaccines as Antivirals for Respiratory Syncytial Virus.

This study aimed to develop an intranasal nanovaccine by combining chiral nanoparticles with the RSV pre-fusion protein (RSV protein) to create L-nanovaccine (L-Vac). The results showed that L-NPs increased antigen attachment in the nasal cavity by 3.83 times and prolonged its retention time to 72h. In vivo experimental data demonstrated that the intranasal immunization with L-Vac induced a 4.86-fold increase in secretory immunoglobulin A (sIgA) secretion in the upper respiratory tract, a 1.85-fold increase in the lower respiratory tract, and a 20.61-fold increase in RSV-specific immunoglobin G (IgG) titer levels in serum, compared with the commercial Alum Vac, while the neutralizing activity against the RSV authentic virus is 1.66-fold higher. The mechanistic investigation revealed that L-Vac activated the tumor necrosis factor (TNF) signaling pathway in nasal epithelial cells (NECs), in turn increasing the expression levels of interleukin-6 (IL-6) and C-C motif chemokine ligand 20 (CCL20) by 1.67-fold and 3.46-fold, respectively, through the downstream nuclear factor kappa-B (NF-κB) signaling pathway. Meanwhile, CCL20 recruited dendritic cells (DCs) and L-Vac activated the Toll-like receptor signaling pathway in DCs, promoting IL-6 expression and DCs maturation, and boosted sIgA production and T-cell responses. The findings suggested that L- Vac may serve as a candidate for the development of intranasal medicine against various types of respiratory infections.

Single-cell RNA Sequencing Reveals the Diversity of the Immunological Landscape Response to Genital Herpes

Genital herpes (GH) is a common sexually transmitted disease, which is primarily caused by herpes simplex virus type 2 (HSV-2), and continues to be a global health concern. Although our understanding of the alterations in immune cell populations and immunomodulation in GH patients is still limited, it is evident that systemic intrinsic immunity, innate immunity, and adaptive immunity play crucial roles during HSV-2 infection and GH reactivation. To investigate the mechanisms underlying HSV-2 infection and recurrence, single-cell RNA sequencing (scRNA-seq) was performed on immune cells isolated from the peripheral blood of both healthy individuals and patients with recurrent GH. Furthermore, the systemic immune response in patients with recurrent GH showed activation of classical monocytes, CD4+ T cells, natural killer cells (NK cells), and plasmacytoid dendritic cells (pDCs), especially of genes associated with the Toll-like receptor signaling pathway and T cell activation. Circulating immune cells in GH patients show higher expression of genes associated with inflammation and antiviral responses both in the scRNA-Seq data set and in independent quantitative real-time polymerase chain reaction (qRT-PCR) analysis and ELISA experiments. This study demonstrated that localized genital herpes, resulting from HSV reactivation, may influence the functionality of circulating immune cells, suggesting a potential avenue for future research into the role of systemic immunity during HSV infection and recurrence.

Study on the activity and mechanism of herbal formula anti-infection powder (AIP) against influenza-virus-induced pneumonia through genetic susceptibility genes

Anti-infection powder (AIP), a patented Chinese herbal formulation, is used traditionally in the treatment of upper respiratory tract infections. In this study, an ethanol extract of AIP was demonstrated to inhibit influenza A virus (IAV) infection and IAV-induced pneumonia (IVP), both in vitro and in vivo, highlighting its potential mechanism of action. To determine the anti-IAV activity of AIP and to explore the possible mechanisms of inhibiting IAV-induced pneumonia. An ethanol extract was extracted from AIP and its major ingredients were determined by high-performance liquid chromatography (HPLC). An IAV-infected A549 cell model and an IAV-induced mouse pneumonia model were established to evaluate the therapeutic effects of AIP on IVP in vivo and in vitro. The mice were respectively administered AIP at high- and low-dose in different groups. The anti-IAV activity of AIP was evaluated by detecting viral load, lung lesion, lung index, suvival time, inflammatory cytokines and transcriptomic analysis in the lung tissue. The potential pathways and targets that involved in AIP against IVP were predicted by network pharmacology. Mendelian randomization (MR), colocalization analysis, and molecular docking were employed to identify novel therapeutic targets for IVP. Polymerase chain reaction (PCR) and Western blot (WB) techniques were used to confirm the effect of AIP on the expression of risk target genes in the lungs of IVP mice. In A549 cell line, AIP effectively inhibited IAV infection with IC50 values of 65.49 μg/mL. The anti-IAV activity of AIP was mainly determined by chlorogenic acid, forsythiarin, puerarin, paeoniflorin and prim-o-glucosylcimigin. Moreover, AIP inhibited the neuraminidase activity and the M gene expression in vitro. In vivo, oral administration of AIP significantly reduced viral load and improved lung tissue lesions. AIP decreased the concentration of pro-inflammatory factors such as IL-1β, TNF-α, and IFN-γ, and significantly increased the concentration of the anti-inflammatory factor IL-4. According to network pharmacology analysis, toll-like receptor signaling pathway, chemokine signaling pathway, and TNF signaling pathway may be the possible mechanisms by which AIP inhibits IVP and regulates excessive inflammatory response.Two new genes, LRG1 and PSMA4, associated with genetic susceptibility to influenza and pneumonia, predicted as potential IVP drug target genes by MR and colocalization analysis. The antiviral mechanism of AIP may be to inhibit the expression levels of LRG1 and PSMA4 in lungs of mouse IVP. AIP exhibited anti-IAV activities both in vitro and in vivo. AIP had a protective effect against pneumonia caused by influenza virus and can inhibit the progression of inflammation. This effect may be associated with its ability to inhibit the expression levels of genetic susceptibility genes (LRG1 and PSMA4) in lungs of mouse IVP. The findings of this study enhance our understanding of the role and mechanisms of AIP in the treatment of IVP.