Toll-like Receptor Signaling Pathway Research Articles

Incomplete Knockdown of MyD88 Inhibits LPS-Induced Lung Injury and Lung Fibrosis in a Mouse Model.

Acute lung injury (ALI) is a life-threatening disorder stemmed mainly from an uncontrolled inflammatory response. Lipopolysaccharide (LPS) is commonly used to induce ALI animal models. Toll-like receptor 4 (TLR4) is the main receptor for LPS, and myeloid differentiation factor 88 (MyD88) is a key adaptor protein molecule in the Toll-like receptor (TLR) signaling pathway. Thus, MyD88 knockdown heterozygous mice (MyD88+/-) were used to investigate the effect of incomplete knockout of the MyD88 gene on indirect LPS-induced ALI through intraperitoneal injection of LPS. The LPS-induced ALI significantly upregulated MyD88 expression, and heterozygous mice with incomplete knockout of the MyD88 gene (MyD88+/-) ameliorated LPS-induced histopathological injury and collagen fiber deposition. Heterozygous mice with incomplete knockout of the MyD88 gene (MyD88+/-) inhibited LPS-induced nuclear factor-κB (NF-κB) pathway activation, but TLR-4 expression tended to be upregulated. Incomplete knockdown of the MyD88 gene also downregulated LPS-induced expression of IL1-β, IL-6, TNF-α, TGF-β, SMAD2, and α-SMA. The transcriptome sequencing also revealed significant changes in LPS-regulated genes (such as IL-17 signaling pathway genes) after the incomplete knockdown of MyD88. In conclusion, this paper clarified that LPS activates the downstream NF-κB pathway depending on the MyD88 signaling pathway, which induces the secretion of inflammatory cytokines such as IL-1β/IL-6/TNF-α and ultimately triggers ALI. Incomplete knockdown of the MyD88 reverses LPS-induced lung fibrosis, which confirmed the vital role of MyD88 in LPS-induced ALI.

Immunomodulatory mechanisms of an acidic polysaccharide from the fermented burdock residue by Rhizopus nigricans in RAW264.7 cells and cyclophosphamide-induced immunosuppressive mice

RBAPS is an acidic polysaccharide extracted from the burdock residue fermentation by Rhizopus nigricans. In RBAPS-activated RAW264.7 cells, transcriptome analysis identified a total of 1520 differentially expressed genes (DEGs), including 1223 down-regulated genes and 297 up-regulated genes. DEGs were enriched in the immune-related biological processes, involving in Mitogen-activated protein kinase (MAPK) and Toll-like receptor (TLR) signaling pathway, according to Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis. The results of the confocal laser scanning microscope (CLSM) observation, antibody neutralization and Western blot verified that RBAPS modulated macrophages activation and cytokines secretion mainly via TLR4/MAPK/NF-κB signaling pathway. The immunomodulatory activity in vivo of RBAPS was investigated in cyclophosphamide (CTX)-induced immunosuppressive mice. RBAPS promoted the counts of white blood cells (WBC), red blood cells (RBC) and platelets (PLT) as well as the levels of immunoglobulins and cytokines (IgG, IgM, TNF-α, and IL-2) in immunosuppressive mice. RBAPS protected the spleen and thymus from CTX-induced injury by increasing the organ indexes, attenuating pathological damage, and promoting splenic lymphocytes proliferation. Importantly, RBAPS ameliorated the intestine integrity and function by promoting the expression of Occuldin, Claudin-5, Atg5, and Atg7, activating TLR4/MAPK signaling pathway in CTX-induced mice. This study suggested that RBAPS was a prime candidate of immunologic adjuvant in chemotherapy for the nutraceutical and pharmaceutical application.

Diversity of the bovine genes IRAK1 and IRAK4 in the Toll-like receptor signaling pathway

Diversity of the bovine genes IRAK1 and IRAK4 in the Toll-like receptor signaling pathway

Identification of immunological characteristics and cuproptosis-related molecular clusters in Rheumatoid arthritis

BackgroundRheumatoid arthritis (RA) is a chronic autoimmune disease characterised by progressive articular damage, functional loss, and comorbidities. The relationship between cuproptosis, a form of programmed cell death, and RA remains unknown. Therefore, this study aimed to explore cuproptosis-related molecular clusters in RA. MethodsGene expression profiles of GSE93272 were downloaded from the Gene Expression Omnibus to identify the expression profiles of cuproptosis regulators and the immune infiltration characteristics of RA. The molecular clusters of cuproptosis-related genes and the related immune cell infiltration were explored. Cluster-specific differentially expressed genes were identified using the weighted gene co-expression network analysis. Further, an external dataset (GSE15573) was used, and an enzyme-linked immunosorbent assay was performed to validate the predictive efficiency. ResultsThirteen cuproptosis-related genes and activated immune responses were identified between patients with RA and controls. Immune infiltration revealed significant immunological heterogeneity in the two cuproptosis-related molecular clusters in RA. Functional enrichment indicated that Cluster1 and Cluster2 were predominantly enriched in the toll-like receptor signalling pathway and regulation of autophagy, respectively. Further, the performance of FAM96A and CGRRF1 genes in the external validation dataset was observed to be relatively satisfactory (area under the receiver operating characteristic curve = 0.687 and 0.674, respectively). Based on our serum samples, FAM96A and CGRRF1 both exhibited higher expression levels in patients with RA (p = 0.001; p = 0.000). ConclusionsOur study systematically illustrated the involvement of cuproptosis in the progression of RA, and explored the pathogenic mechanisms and novel therapeutic strategies for RA, targeting FAM96A and CGRRF1.

Fecal microbiota transplantation alleviates experimental colitis through the Toll-like receptor 4 signaling pathway.

Fecal microbiota transplantation (FMT) has shown promising therapeutic effects on mice with experimental colitis and patients with ulcerative colitis (UC). FMT modulates the Toll-like receptor 4 (TLR4) signaling pathway to treat some other diseases. However, it remains unknown whether this modulation is also involved in the treatment of UC. To clarify the necessity of TLR4 signaling pathway in FMT on dextran sodium sulphate (DSS)-induced mice and explain the mechanism of FMT on UC, through association analysis of gut microbiota with colon transcriptome in mice. A mouse colitis model was constructed with wild-type (WT) and TLR4-knockout (KO) mice. Fecal microbiota was transplanted by gavage. Colon inflammation severity was measured by disease activity index (DAI) scoring and hematoxylin and eosin staining. Gut microbiota structure was analyzed through 16S ribosomal RNA sequencing. Gene expression in the mouse colon was obtained by transcriptome sequencing. The KO (DSS + Water) and KO (DSS + FMT) groups displayed indistinguishable body weight loss, colon length, DAI score, and histology score, which showed that FMT could not inhibit the disease in KO mice. In mice treated with FMT, the relative abundance of Akkermansia decreased, and Lactobacillus became dominant. In particular, compared with those in WT mice, the scores of DAI and colon histology were clearly decreased in the KO-DSS group. Microbiota structure showed a significant difference between KO and WT mice. Akkermansia were the dominant genus in healthy KO mice. The ineffectiveness of FMT in KO mice was related to the decreased abundance of Akkermansia. Gene Ontology enrichment analysis showed that differentially expressed genes between each group were mainly involved in cytoplasmic translation and cellular response to DNA damage stimulus. The top nine genes correlating with Akkermansia included Aqp4, Clca4a, Dpm3, Fau, Mcrip1, Meis3, Nupr1 L, Pank3, and Rps13 (|R| > 0.9, P < 0.01). FMT may ameliorate DSS-induced colitis by regulating the TLR4 signaling pathway. TLR4 modulates the composition of gut microbiota and the expression of related genes to ameliorate colitis and maintain the stability of the intestinal environment. Akkermansia bear great therapeutic potential for colitis.

Liposomal co-delivery of toll-like receptors 3 and 7 agonists induce a hot triple-negative breast cancer immune environment

Triple-negative breast cancer (TNBC) is highly aggressive and has no standard treatment. Although being considered as an alternative to conventional treatments for TNBC, immunotherapy has to deal with many challenges that hinder its efficacy, particularly the poor immunogenic condition of the tumor microenvironment (TME). Herein, we designed a liposomal nanoparticle (LN) platform that delivers simultaneously toll-like receptor 7 (imiquimod, IQ) and toll-like receptor 3 (poly(I:C), IC) agonists to take advantage of the different toll-like receptor (TLR) signaling pathways, which enhances the condition of TME from a “cold” to a “hot” immunogenic state. The optimized IQ/IC-loaded LN (IQ/IC-LN) was effectively internalized by cancer cells, macrophages, and dendritic cells, followed by the release of the delivered drugs and subsequent stimulation of the TLR3 and TLR7 signaling pathways. This stimulation encouraged the secretion of type I interferon (IFN-α, IFN-β) and CXCLl0, a T-cell and antigen-presenting cells (APCs) recruitment chemokine, from both cancer cells and macrophages and polarized macrophages to the M1 subtype in in vitro studies. Notably, systemic administration of IQ/IC-LN allowed for the high accumulation of drug content in the tumor, followed by the effective uptake by immune cells in the TME. IQ/IC-LN treatment comprehensively enhanced the immunogenic condition in the TME, which robustly inhibited tumor growth in tumor-bearing mice. Furthermore, synergistic antitumor efficacy was obtained when the IQ/IC-LN-induced immunogenic state in TME was combined with anti-PD1 antibody therapy. Thus, our results suggest the potential of combining 2 TLR agonists to reform the TME from a “cold” to a “hot” state, supporting the therapeutic efficacy of immune checkpoint inhibitors.

Network Pharmacology and Molecular Docking Analysis Exploring the Mechanism of Tripterygium wilfordii in the Treatment of Oral Lichen Planus.

Background: Oral lichen planus (OLP) is an infrequent autoimmune disease of the oral mucosa, which affects up to 2% of the world population. An investigation of Tripterygium wilfordii's mechanism of action for treating OLP was conducted, and a theoretical basis was provided for improving current treatment regimens. Materials and Methods: We used a network pharmacological approach to gain insight into the molecular mechanism of Tripterygium wilfordii in the treatment of OLP. Then, potential protein targets between Tripterygium wilfordii and OLP were analyzed through a drug-target network. This was followed by KEGG enrichment analysis and Gene Ontology (GO) classification. Finally, for molecular docking, AutoDock Vina was used. Results: A protein-protein interaction (PPI) network was constructed by analyzing the common targets of a total of 51 wilfordii-OLP interactions from different databases. The GO and KEGG enrichment analyses showed that the treatment of OLP with Tripterygium wilfordii mainly involves lipopolysaccharide response, bacterial molecular response, positive regulation of cytokine production, and leukocyte proliferation, and the signaling pathways mainly include the AGE-RAGE, NF-κB, Toll-like receptor, IL-17, HIF-1, and TNF signaling pathways. The molecular docking results showed that β-sitosterol, kaempferol, hederagenin, and triptolide have a higher affinity for AKT1, TNF, CASP3, and PTGS2, respectively. Based on the CytoNCA analysis of common targets, 19 key targets, including AKT1, TNF, VEGFA, STAT3, CXCL8, PTGS2, TP53, and CASP3, and their connections were identified. Conclusions: Preliminarily, this study reveals that Tripterygium wilfordii interferes with OLP by interacting with multiple targets through multiple accesses, as validated by molecular docking.

Compound Dihuang Granule Changes Gut Microbiota of MPTP-Induced Parkinson's Disease Mice via Inhibiting TLR4/NF-κB Signaling.

Intestinal microbiota was connected to Parkinson's Disease (PD) pathology. The ancient Chinese medication for PD is Compound Dihuang Granule (CDG), and we found a neuroprotective function in treating the constipation of PD patients. Nevertheless, the mechanism of action still needs to be clarified. We predicted the probable targets of CDG against PD through Traditional Chinese medicine (TCM) network pharmacology and verified the analysis through animal experiments in vivo. The protein-protein interaction (PPI) network analysis screened PD-related genes, including Toll-like receptor 4(TLR4), TANK-binding kinase 1(TBK1), Nuclear Factor- Kappa B (NF-κB), and Tumor necrosis factor (TNF). Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses proved that the NF-κB and toll-like receptor signaling pathways serve a key function in CDG therapy of PD. Molecular docking analysis demonstrated that CDG strongly connected to TLR4/NF-κB. Experiments findings indicated that CDG improved the damage of dopaminergic neurons and gut microbial dysbiosis, ameliorated motor impairments, and suppressed the PD-associated inflammation and oxidative stress in mice induced by 1-methyl-4-phenyl-1,2,3,6-tetrahy dropyridine (MPTP). CDG suppressed the inflammatory proteins in the colon and protected the intestinal barrier. Overall, CDG improved gut microbial in PD by blocking the pathway of TLR4/NF-κB.

Hepatic stellate cells induce an inflammatory phenotype in Kupffer cells via the release of extracellular vesicles.

Liver fibrosis is the response of the liver to chronic liver inflammation. The communication between the resident liver macrophages (Kupffer cells [KCs]) and hepatic stellate cells(HSCs) has been mainly viewed as one-directional: from KCs to HSCs with KCs promoting fibrogenesis. However, recent studies indicated that HSCs may function as a hub of intercellular communications. Therefore, the aim of the present study was to investigate the role of HSCs on the inflammatory phenotype of KCs. Primary rat HSCs and KCs were isolated from male Wistar rats. HSCs-derived conditioned medium (CM) was harvested from different time intervals (Day 0-2: CM-D2 and Day 5-7: CM-D7) during the activation of HSCs. Extracellular vesicles (EVs) were isolated from CM by ultracentrifugation and evaluated by nanoparticle tracking analysis and western blot analysis. M1 and M2 markers of inflammation were measured by quantitative PCR and macrophage function by assessing phagocytic capacity. CM-D2 significantly induced the inflammatory phenotype in KCs, but not CM-D7. Neither CM-D2 nor CM-D7 affected the phagocytosis of KCs. Importantly, the proinflammatory effect of HSCs-derived CM is mediated via EVs released from HSCs since EVs isolated from CMmimicked the effect of CM, whereas EV-depleted CM lost its ability to induce a proinflammatory phenotype in KCs. In addition, when the activation of HSCs was inhibited, HSCs produced less EVs. Furthermore, the proinflammatory effects of CM and EVs are related to activating Toll-like receptor 4 (TLR4) in KCs. In conclusion, HSCs at an early stage of activation induce a proinflammatory phenotype in KCs via the release of EVs. This effect is absent in CM derived from HSCs at a later stage of activation and is dependent on the activation of TLR4 signaling pathway.

Regulation of toll-like receptor (TLR) signaling pathways in atherosclerosis: from mechanisms to targeted therapeutics.

Atherosclerosis, one of the life-threatening cardiovascular diseases (CVDs), has been demonstrated to be a chronic inflammatory disease, and inflammatory and immune processes are involved in the origin and development of the disease. Toll-like receptors (TLRs), a class of pattern recognition receptors that trigger innate immune responses by identifying pathogen-associated molecular patterns (PAMPs) and danger-associated molecular patterns (DAMPs), regulate numerous acute and chronic inflammatory diseases. Recent studies reveal that TLRs have a vital role in the occurrence and development of atherosclerosis, including the initiation of endothelial dysfunction, interaction of various immune cells, and activation of a number of other inflammatory pathways. We herein summarize some other inflammatory signaling pathways, protein molecules, and cellular responses associated with TLRs, such as NLRP3, Nrf2, PCSK9, autophagy, pyroptosisand necroptosis, which are also involved in the development of AS. Targeting TLRs and their regulated inflammatory events could be a promising new strategy for the treatment of atherosclerotic CVDs. Novel drugs that exert therapeutic effects on AS through TLRs and their related pathways are increasingly being developed. In this article, we comprehensively review the current knowledge of TLR signaling pathways in atherosclerosis and actively seek potential therapeutic strategies using TLRs as a breakthrough point in the prevention and therapy of atherosclerosis.

Effects of N-acetyl-L-cysteine polysulfides on periodontitis in a mouse model.

Polysulfides are reported to be involved in various important biological processes. N-acetyl-l-cysteine polysulfide with 2 sulfane sulfur atoms (NAC-S2) regulates diverse toll-like receptor (TLR) signaling pathways. Here, we aimed to determine the role of NAC-S2 in periodontitis and explore the potential mechanism. A periodontitis mouse model was established by ligating the subgingival between the first and second molars in wild-type, TLR4-/- , and Myd88-/- mice. NAC-S2 did not affect the proportion of macrophages (CD11b+ F4/80+ ) or neutrophils (CD11b+ GR-1+ ) in the bone marrow. Mechanically, lipopolysaccharides (LPS), Zymosan A, or poly I: C induced tumor necrosis factor (TNF), interleukin (IL)-6, and IL-1β expression in bone marrow-derived macrophages (BMDMs) could be inhibited by NAC-S2. On the other hand, NAC-S2 suppressed the phosphorylation levels of IκB-α, p65, and IκB kinase (IKK)-β induced by LPS in BMDMs, while LPS induced phosphorylation of ERK1/2, p38, and transforming growth factor β-activated kinase 1 (TAK1) could not be affected by NAC-S2. In wild-type periodontitis mice, NAC-S2 administration decreased the cemento-enamel-junction-alveolar bone crest (CEJ-ABC) distance and the relative mRNA expression of TNF, IL-6, and IL-1β, while such phenomena could not be observed in TLR4 deficiency or Myd88 deficiency mice. All of these results indicate that NAC-S2 ameliorates TLR4/NF-κB pathway mediated inflammation in mouse periodontitis model.

Chrysin and its nanoliposome ameliorated non-alcoholic steatohepatitis via inhibiting TLR4 signalling pathway.

Non-alcoholic steatohepatitis (NASH) is a chronic liver disease histologically characterized by liver steatosis, hepatocellular injury, inflammation and fibrosis, resulting in cirrhosis and hepatocellular carcinoma, but effective measures and obvious pathogenesis for NASH remain elusive. Chrysin (CH) has been reported to have anti-inflammatory effects but shows lower bioavailability. In this study, a chrysin nanoliposome (CH-NL) was first prepared and characterized. Then, we used the methionine-choline-deficient (MCD) diet to induce a mouse model of NASH. Finally, the effects of CH and CH-NL on NASH were evaluated in the liver of NASH mice. The results showed that CH or CH-NL significantly reduced the accumulation of lipids in hepatocytes, alleviated liver injury, decreased the generation of radical oxygen species, and attenuated the accumulation of collagen fibre in the liver of NASH mice. In addition, CH and its nano-liposomes markedly inhibited the production of inflammatory cytokines and inflammatory cell infiltration in the liver of NASH mice. Further studies found that CH-NL and CH-NL downregulated the MCD diet-induced activation of Toll-like receptor 4 (TLR4) signalling pathway in the liver of mice. CH and its nanoliposome alleviated MCD diet-induced NASH in mice, which might be through inhibiting TLR4 signalling pathway.

Constructing and Validating a Network of Potential Olfactory Sheathing Cell Transplants Regulating Spinal Cord Injury Progression.

The pathology of spinal cord injury (SCI), including primary and secondary injuries, primarily involves hemorrhage, ischemia, edema, and inflammatory responses. Cell transplantation has been the most promising treatment for SCI in recent years; however, its specific molecular mechanism remains unclear. In this study, bioinformatics analysis verified by experiment was used to elucidate the hub genes associated with SCI and to discover the underlying molecular mechanisms of cell intervention. GSE46988 data were downloaded from the Gene Expression Omnibus dataset. In our study, differentially expressed genes (DEGs) were reanalyzed using the "R" software (R v4.2.1). Functional enrichment and protein-protein interaction network analyses were performed, and key modules and hub genes were identified. Network construction was performed for the hub genes and their associated miRNAs. Finally, a semi-quantitative analysis of hub genes and pathways was performed using quantitative real-time polymerase chain reaction. In total, 718 DEGs were identified, mainly enriched in immune and inflammation-related functions. We found that Cd4, Tp53, Rac2, and Akt3 differed between vehicle and transplanted groups, suggesting that these genes may play an essential role in the transplantation of olfactory ensheathing cells, while a toll-like receptor signaling pathway was significantly enriched in Gene set enrichment analysis, and then, the differences were statistically significant by experimentally verifying the expression of their associated molecules (Tlr4, Nf-κb, Ikkβ, Cxcl2, and Tnf-α). In addition, we searched for upstream regulatory molecules of these four central genes and constructed a regulatory network. This study is the first to construct a regulatory network for olfactory ensheathing cell transplantation in treating SCI, providing a new idea for SCI cell therapy.

Predicting diagnostic biomarkers associated with immune infiltration in Crohn's disease based on machine learning and bioinformatics

ObjectiveThe objective of this study is to investigate potential biomarkers of Crohn's disease (CD) and the pathological importance of infiltration of associated immune cells in disease development using machine learning.MethodsThree publicly accessible CD gene expression profiles were obtained from the GEO database. Inflammatory tissue samples were selected and differentiated between colonic and ileal tissues. To determine the differentially expressed genes (DEGs) between CD and healthy controls, the larger sample size was merged as a training unit. The function of DEGs was comprehended through disease enrichment (DO) and gene set enrichment analysis (GSEA) on DEGs. Promising biomarkers were identified using the support vector machine-recursive feature elimination and lasso regression models. To further clarify the efficacy of potential biomarkers as diagnostic genes, the area under the ROC curve was observed in the validation group. Additionally, using the CIBERSORT approach, immune cell fractions from CD patients were examined and linked with potential biomarkers.ResultsThirty-four DEGs were identified in colon tissue, of which 26 were up-regulated and 8 were down-regulated. In ileal tissues, 50 up-regulated and 50 down-regulated DEGs were observed. Disease enrichment of colon and ileal DEGs primarily focused on immunity, inflammatory bowel disease, and related pathways. CXCL1, S100A8, REG3A, and DEFA6 in colon tissue and LCN2 and NAT8 in ileum tissue demonstrated excellent diagnostic value and could be employed as CD gene biomarkers using machine learning methods in conjunction with external dataset validation. In comparison to controls, antigen processing and presentation, chemokine signaling pathway, cytokine–cytokine receptor interactions, and natural killer cell-mediated cytotoxicity were activated in colonic tissues. Cytokine–cytokine receptor interactions, NOD-like receptor signaling pathways, and toll-like receptor signaling pathways were activated in ileal tissues. NAT8 was found to be associated with CD8 T cells, while CXCL1, S100A8, REG3A, LCN2, and DEFA6 were associated with neutrophils, indicating that immune cell infiltration in CD is closely connected.ConclusionCXCL1, S100A8, REG3A, and DEFA6 in colonic tissue and LCN2 and NAT8 in ileal tissue can be employed as CD biomarkers. Additionally, immune cell infiltration is crucial for CD development.

Transcriptome profiling reveals the strategy of thermal tolerance enhancement caused by heat-hardening in Mytilus coruscus

The thick-shell mussel Mytilus coruscus serves as a common sessile intertidal species and holds economic significance as an aquatic organism. M. coruscus often endure higher temperatures than their ideal range during consecutive low tides in the spring. This exposure to elevated temperatures provides them with a thermal tolerance boost, enabling them to adapt to high-temperature events caused by extreme low tides and adverse weather conditions. This phenomenon is referred to as heat-hardening. Some related studies showed the phenomenon of heat-hardening in sessile intertidal species but not reported at the mechanism level based on transcriptome so far. In this study, physiological experiments, gene family identification and transcriptome sequencing were performed to confirm the thermotolerance enhancement based on heat-hardening and explore the mechanism in M. coruscus. A total of 2935 DEGs were identified and the results of the KEGG enrichment showed that seven heat-hardening relative pathways were enriched, including Toll-like receptor signal pathway, Arachidonic acid metabolism, and others. Then, 24 HSP70 members and 36 CYP2 members, were identified, and the up-regulated members are correlated with increasing thermotolerance. Finally, we concluded that the heat-hardening M. coruscus have a better thermotolerance because of the capability of maintaining the integrity and the phenomenon of vasodilation of the gill under thermal stress. Further, the physiological experiments yielded the same conclusions. Overall, this study confirms the thermotolerance enhancement caused by heat-hardening and reveals the survival strategy in M. coruscus. In addition, the conclusion provides a new reference for studying the intertidal species' heat resistance mechanisms to combat extreme heat events and the strategies for dealing with extreme weather in aquaculture under the global warming trend.

MYD88L265P augments proximal B-cell receptor signaling in large B-cell lymphomas via an interaction with DOCK8

AbstractDiffuse large B-cell lymphoma (DLBCL) is a clinically and genetically heterogeneous disease with at least 5 recognized molecular subtypes. Cluster 5 (C5)/MCD tumors frequently exhibit concurrent alterations in the toll-like receptor (TLR) and B-cell receptor (BCR) pathway members, MYD88L265P and CD79B, and have a less favorable prognosis. In healthy B cells, the synergy between TLR and BCR signaling pathways integrates innate and adaptive immune responses and augments downstream NF-κB activation. In addition, physiologic TLR9 pathway engagement via MYD88, protein tyrosine kinase 2 (PYK2), and dedicator of cytokinesis 8 (DOCK8) increases proximal BCR signaling in healthy murine B cells. Although C5/MCD DLBCLs are selectively sensitive to Bruton tyrosine kinase (BTK) inhibition in in vitro studies and certain clinical trials, the role of mutated MYD88 in proximal BCR signaling remains undefined. Using engineered DLBCL cell line models, we found that concurrent MYD88L265P and CD79B alterations significantly increased the magnitude and duration of proximal BCR signaling, at the level of spleen tyrosine kinase and BTK, and augmented PYK2-dependent DOCK8 phosphorylation. MYD88L265P DLBCLs have significantly increased colocalization of DOCK8 with both MYD88 and the proximal BCR-associated Src kinase, LYN, in comparison with MYD88WT DLBCLs, implicating DOCK8 in MYD88L265P/proximal BCR cross talk. Additionally, DOCK8 depletion selectively decreased proximal BCR signaling, cellular proliferation, and viability of DLBCLs with endogenous MYD88L265P/CD79BY196F alterations and increased the efficacy of BTK blockade in these lymphomas. Therefore, MYD88L265P/DOCK8-enhanced proximal BCR signaling is a likely mechanism for the increased sensitivity of C5/MCD DLBCLs to BTK blockade.

Analysis of therapeutic mechanism of Liushen Wan against colitis-associated colorectal cancer based on network pharmacology and validation in mice

To explore the therapeutic mechanism of Liushen Wan (LSW) against colitis-associated colorectal cancer (CAC) by network pharmacology. TCMSP, BATMAN-TCM, CNKI, PubMed, Genecards, OMIM, and TTD databases were used to obtain the related targets of LSW and CAC. The common targets of LSW and CAC were obtained using Venny online website. The PPI network was constructed using Cytoscape 3.8.2 to screen the core targets of LSW in the treatment of CAC. GO and KEGG enrichment analysis were conducted using DAVID database. The therapeutic effect of LSW on CAC was evaluated in a C57BL/6J mouse model of AOM/DSS-induced CAC by observing the changes in body weight, disease activity index, colon length, and size and number of the tumor. HE staining and RT-qPCR were used to analyze the effect of LSW on inflammatory mediators. Immunohistochemistry and TUNEL staining were used to evaluate the effect of LSW on the proliferation and apoptosis of AOM/DSS-treated colon tumor cells. Immunohistochemistry and Western blotting were used to detect the effects of LSW on the expression of TLR4 proteins in CAC mice. Network pharmacology analysis identified 69 common targets of LSW and CAC, and 33 hub targets were screened in the PPI network. KEGG pathway enrichment analysis suggested that the effect of LSW on CAC was mediated by the Toll-like receptor signaling pathway. In the mouse model of AOM/DSS-induced CAC, LSW significantly inhibited colitis-associated tumorigenesis, reduced tumor number and tumor load (P < 0.05), obviously improved histopathological changes in the colon, downregulated the mRNA levels of proinflammatory cytokines, and inhibited the proliferation (P < 0.01) and promoted apoptosis of colon tumor cells (P < 0.001). LSW also significantly decreased TLR4 protein expression in the colon tissue (P < 0.05). LSW can inhibit CAC in mice possibly by regulating the expression of TLR4 to reduce intestinal inflammation, inhibit colon tumor cell proliferation and promote their apoptosis.

LncRNA activates immune response against Vibrio anguillarum in the intestine-liver axis of turbot (Scophthalmus maximus L.) by sponging miRNA in a ceRNA regulatory network

Vibriosis, most commonly caused by the bacterium Vibrio anguillarum, is one of the most important diseases affecting the marine aquaculture industry throughout the world. The intestine is the main entry portal of V. anguillarum into host fishes. Although there has been extensive research on the immunity of the gut-associated lymphoid tissue (GALT) in teleost, the mechanisms underlying the immunity of the intestine-liver during bacterial infections are still not well understood. This study conducted an integrative analysis of the transcriptome in turbot (Scopthalmus maximus L.) intestine and liver after V. anguillarum infection. To provide evidence of immunological collaboration in the turbot intestine-liver axis, we constructed the immune gene libraries, and conducted the functional annotation and enrichment analyses of immune genes that were jointly expressed in both turbot intestine and liver. Additionally, we administered the bioinformatic analyses of transcriptomic data to investigate regulatory relationships between non-coding RNAs (ncRNAs) and their target immune genes, based on the ceRNA theory. The recognition and binding abilities to bacteria were activated by the Toll-like receptor (TLR) signaling pathway. Subsequently, NF-κB was activated by the TLR/MyD88 signaling, and its target genes were induced to up-regulate in intestine and associated tissues to the digestive tract to enhance disease resistance through the production of inflammation. Histology of the intestine and liver of infected fish showed pathological changes consistent with inflammation. Bioinformatic analysis suggested that ceRNA regulatory networks play a pivotal role in the regulation of TLR genes, and RT-qPCR analyses and dual-luciferase reporter assays showed confirmed evidence to verify these hypothesized regulatory relationships. Overall, the results in this study demonstrated that a lncRNA (LNC_002468) activated the TLR signaling pathway by upregulating the TLR9 gene through sponging miRNA (novel_709). This study provides a strong indication that ncRNAs play regulatory roles in the antibacterial immune response of teleost fishes. However, further functional verification is required of the key immune genes and their regulatory relationship with miRNAs and lncRNAs.

Toll-like Receptors as Pro-Thrombotic Drivers in Viral Infections: A Narrative Review.

Toll-like receptors (TLRs) have a critical role in the pathogenesis and disease course of viral infections. The induced pro-inflammatory responses result in the disturbance of the endovascular surface layer and impair vascular homeostasis. The injury of the vessel wall further promotes pro-thrombotic and pro-coagulatory processes, eventually leading to micro-vessel plugging and tissue necrosis. Moreover, TLRs have a direct role in the sensing of viruses and platelet activation. TLR-mediated upregulation of von Willebrand factor release and neutrophil, as well as macrophage extra-cellular trap formation, further contribute to (micro-) thrombotic processes during inflammation. The following review focuses on TLR signaling pathways of TLRs expressed in humans provoking pro-thrombotic responses, which determine patient outcome during viral infections, especially in those with cardiovascular diseases.

Copper-Induced Ionoregulatory Disturbance, Histopathology, and Transcriptome Responses in Freshwater Mussel (Anodonta woodiana) Gills

Copper (Cu) contamination has become a severe problem in freshwater environments worldwide. The freshwater mussel Anodonta woodiana is used as a unique bioindicator to monitor Cu contamination in freshwater environments. However, Cu toxicity and response mechanisms in A. woodiana are still largely unknown. A sublethal acute exposure experiment (2.0 mg/L Cu exposure for 72 h) was conducted to investigate the effects of Cu bioaccumulation on ionoregulatory homeostasis, histological features, and transcriptome responses using A. woodiana gills as indicator tissue. The gill bioaccumulation capacity was up to 474. Cu bioaccumulation decreased Na+ and Mg2+ concentrations (p &lt; 0.05) by 82% and 17%, respectively, and induced cilia loss, epithelial desquamation, and filament atrophy of the gills. Transcriptome analysis identified 3160 differentially expressed genes (DEGs), including 1870 upregulated and 1290 downregulated genes. GO enrichment analysis showed that cellular processes, metabolic processes, biological regulation, and responses to stimuli contained the most DEGs in the biological processes. KEGG pathway analysis showed that apoptosis, arginine and proline metabolism, the toll-like receptor signaling pathway, apoptosis-multiple species, histidine metabolism, beta-alanine metabolism, cytokine–cytokine receptor interaction, and the p53 signaling pathway were significantly enriched. These findings provide comprehensive evidence for exploring Cu toxicity and response mechanisms in freshwater mussels.