Search Results (152)

Systemic lupus erythematosus (SLE) is characterized by autoimmune dysregulation, elevated autoantibody production, and persistent inflammation, predisposing patients to atherosclerosis (AS). Atherogenesis is dependent on lipid homeostasis and inflammatory processes, with the formation of lipid-laden, macrophage-derived foam cells (MDFC) essential for atherosclerotic lesion progression. Elevated cholesterol levels within lipid rafts trigger heightened pro-inflammatory responses in macrophages via Toll-like receptor 9 (TLR9). Artesunate (ART), an artemisinin derivative sourced from Artemisia annua, exhibits therapeutic potential in modulating inflammation and autoimmune conditions. Nonetheless, its impact and mechanisms in SLE-associated AS (SLE-AS) remain largely unexplored. Our investigation demonstrated that ART could effectively ameliorate lupus-like symptoms and atherosclerotic plaque development in SLE-AS mice. Moreover, ART enhanced cholesterol efflux from MDFC by upregulating ABCA1, ABCG1, and SR-B1 both in vivo and in vitro. Moreover, ART reduced cholesterol accumulation in bone marrow-derived macrophages (BMDMs), thereby diminishing TLR9 recruitment to lipid rafts. ART also suppressed TLR9 expression and its downstream effectors in the kidney and aorta of SLE-AS mice, attenuating the TLR9-mediated inflammatory cascade in CPG2395 (ODN2395)-stimulated macrophages. Through bioinformatics analysis and experimental validation, PPARγ was identified as a pivotal downstream mediator of ART in macrophages. Depleting PPARγ levels reduced the expression of ABCA1, ABCG1, and SR-B1 in macrophages, consequently impeding cholesterol efflux. In conclusion, these findings suggest that ART ameliorates SLE-AS by restoring cholesterol homeostasis through the PPARγ-ABCA1/ABCG1/SR-B1 pathway and suppressing lipid raft-driven TLR9/MyD88 inflammation. Full article

B cells contribute to innate and adaptive immunity. In the former, Toll-like receptor (TLR) activation promotes the expansion of inflammatory B cells. In the latter, B cell receptor (BCR) activation results in the production of antibodies or autoantibodies. Antigen processing and presentation are closely associated with major histocompatibility class II (MHC-II) and its companion protein, class II invariant peptide (CLIP). The impact of autophagy on the regulation of these unique mechanisms of B cell activation and subset expansion has not been fully explored. The results from the current study show that activating autophagy with rapamycin (RAPA) or inhibiting autophagy with hydroxycholoroquine (HCQ) differentially influences the TLR9 and BCR activation of B cells. These differences include the selective expansion of B1 and B2 B cell subsets, the regulation of the cell-surface expression of MHC-II and CLIP, and the ability of distinct B cell subsets to present peptide antigens. These novel findings demonstrate that the unique B cell activation mechanisms induced by TLR9 and BCR activation are differentially influenced by RAPA and HCQ, owing to the selective modulation of B cell subset expansion, and antigen processing and presentation by MHC-II proteins. Full article

Background/Objectives: Inflammatory bowel disease (IBD) is a chronic inflammatory condition encompassing ulcerative colitis (UC) and Crohn’s disease (CD). The role of environmental factors in the pathogenesis of IBD remains elusive. Nevertheless, evidence suggests a pivotal role of viruses, specifically Epstein–Barr virus (EBV), in the progression of IBD through mechanisms such as molecular mimicry and bystander activation. Our previous findings demonstrate EBV DNA’s significant role in exacerbating colitis symptoms and elevating the levels of the pro-autoimmune cytokine interleukin-17A (IL-17A) in an IBD mouse model via toll-like receptor 9 (TLR9). Therefore, we aimed to examine the role of EBV particles in the pathogenesis of IBD, and the potential role of TLR9 inhibition in ameliorating disease outcomes. Methods: Three days post colitis induction, EBV particles were intra-rectally injected into female C57BL/6J mice, followed by the intra-peritoneal administration of TLR9 inhibitor. Thereupon, mice were monitored daily and the disease activity index (DAI), colon lengths, and damage scores, as well as the number of cells, double-positive for IL-17A+ and IFN-γ+, and triple-positive for IL-17A+, IFN-γ+, and FOXP3+, were evaluated. Results: Our findings revealed a significant role of TLR9 inhibition in mitigating colitis features in an EBV-injected IBD mouse model compared to the control group. Conclusions: These results indicate an essential role of TLR9 in initiating immune responses against recurrent EBV reactivation events, which ultimately contributes to inflammation aggravation in IBD patients. Consequently, TLR9 could serve as a potential therapeutic target to alleviate the severe symptoms of IBD in EBV-infected individuals. Full article

Previously, we reported that Lactobacillus paragasseri SBT2055 (LG2055) activates plasmacytoid dendritic cells (pDCs) and induces interferon alpha (IFN-α) in vitro. Our clinical trial suggested that LG2055 intake may enhance pDC activity, supporting immune maintenance and reducing subjective common cold symptoms. However, the precise mechanisms remain unclear. In this study, we investigated how LG2055 engages with pDCs to stimulate IFN-α production. We evaluated LG2055-induced pDC activation using flow cytometry, ELISA, and phagocytosis assays. Human peripheral blood mononuclear cells (PBMCs) were stimulated with LG2055 and its components to evaluate immune responses. An in vitro M cell model was used to examine LG2055 translocation. We found that DNA extracted from LG2055 activated pDCs and enhanced IFN-α production via Toll-like receptor 9 (TLR9). Phagocytosis assays demonstrated that LG2055 DNA was internalized by PBMC-derived pDCs, enabling TLR9-mediated signaling. Additionally, LG2055 translocated across M cells in vitro, suggesting potential transport into Peyer’s patches, where it may interact with pDCs. These findings demonstrate that intestinal LG2055 can translocate across M cells, interact with pDCs, and exert immune-stimulatory effects to enhance host antiviral immunity. This study provides mechanistic insight into how dietary components support immune health and could inform the development of novel functional foods or therapeutic strategies. Full article

After weaning, piglets no longer consume breast milk, and their immune system is not yet fully developed. At this time, if weaned piglets are infected with E. coli, their subsequent growth will be seriously affected. In the present study, 48 healthy 28-day-old weaned piglets (6.65 ± 1.19 kg, Duroc × Landrace × Large White) were randomly divided into an LPS group and control group. Piglets in the LPS group were intraperitoneally injected with an LPS solution (LPS was dissolved in sterile saline to form a solution of 100 μg/mL and injected at a dose of 1 mL per kilogram of body weight) for 13 consecutive days. Piglets in the control group were injected with the same volume of sterile saline. On days 1, 5, 9, and 13 of the experiment, six piglets from each group were randomly selected for dissection, the blood and heart samples were collected, and then cardiac function-related indicators were detected. A portion of the heart tissue was fixed in 4% paraformaldehyde and further used to make paraffin sections; then, hematoxylin–eosin (H&E) staining was performed. Masson staining was used to detect the changes in collagen fibers in the hearts. The other parts of the heart tissues were frozen in liquid nitrogen and stored in a refrigerator at −80 °C for the detection of tissue antioxidant indices. The mRNA expression levels of the toll-like receptor 4 (TLR4) signaling pathway, transforming growth factor-β (TGF-β) signaling pathway, and inflammatory cytokines in heart tissues were detected by real-time PCR. The results showed that catalase (CAT) and superoxide dismutase (SOD) contents in the heart tissue homogenates increased significantly on days 1 and 5 in LPS-induced piglets (p < 0.01, p < 0.05), while total antioxidant capacity (T-AOC) and glutathione peroxidase (GSH-Px) contents decreased significantly on day 5 (p < 0.05). On day 5, the contents of serum cardiac function indicators lactate dehydrogenase (LDH), creatine kinase isoenzymes (CK-MB), and cardiac troponin I (cTn-I) were significantly increased in LPS-induced piglets (p < 0.01). On the 1st and 5th days, the heart tissue showed obvious pathological damage, which was manifested as the disordered arrangement of myocardial fibers, depression of myocardial cells, infiltration of inflammatory factors, congestion of capillaries, and significant increase in cardiac collagen fibers. On the 1st day, the mRNA expression levels of tumor necrosis factor-alpha (TNF-α) and interleukin 6 (IL-6) were significantly increased in LPS-induced piglets with heart injury (p < 0.01). On the 5th day, the mRNA expression levels of the TLR4 signaling pathway [TLR4, myeloid differentiation primary response gene 88 (MyD88), nuclear factor kappa-B (NF-κB)], TNF-α, and interleukin 10 (IL-10) were also significantly increased in LPS-induced piglets with heart injury (p < 0.01, p < 0.05). The mRNA expression levels of the TGF-β signaling pathway (TGF-β, Smad2, and Smad4) in cardiac fibrosis-related genes were significantly increased on days 5 and 9 (p < 0.01, p < 0.05). The mRNA expression levels of Smad3 and Smad7 in cardiac fibrosis-related genes were also significantly increased on day 9 (p < 0.01). These results indicate that oxidative stress occurs in the heart tissue of LPS-induced piglets on the 1st and 5th days, leading to cardiac tissue damage. However, on the 9th and 13th days, the degree of heart damage in the piglets was less than that on the 1st and 5th days, which may be due to the tolerance of piglets’ tissues and organs because of multiple same-dose LPS stimulations. Full article

Background: The cell-free DNA (cfDNA) is an extracellular fragmented DNA found in body fluids in physiological and pathophysiological contexts. In cancer, cfDNA has been pointed out as a marker for disease diagnosis, staging, and prognosis; however, little is known about its biological role. Methods: The role of cfDNA released by ES-2 ovarian cancer cells was investigated, along with the impact of glucose bioavailability and culture duration in the cfDNA-induced phenotype. The effect of cfDNA on ES-2 cell proliferation was evaluated by proliferation curves, and cell migration was assessed through wound healing. We explored the impact of different cfDNA variants on ES-2 cells’ metabolic profile using nuclear magnetic resonance (NMR) spectroscopy and cisplatin resistance through flow cytometry. Moreover, we assessed the protein levels of DNA-sensitive Toll-like receptor 9 (TLR9) by immunofluorescence and its colocalization with lysosome-associated membrane protein 1 (LAMP1). Results: This study demonstrated that despite inducing similar effects, different variants of cfDNA promote different effects on cells derived from the ES-2 cell line. We observed instant reactions of adopting the metabolic profile that brings back the cell functioning of more favorable culture conditions supporting proliferation and resembling the cell of origin of the cfDNA variant, as observed in unselected ES-2 cells. However, as a long-term selective factor, certain cfDNA variants induced quiescence that favors the chemoresistance of a subset of cancer cells. Conclusions: Therefore, different tumoral microenvironments may generate cfDNA variants that will impact cancer cells differently, orchestrating the disease fate. Full article

Background: The clinical relevance of circulating cell-free DNA (cfDNA) in oncology has gained significant attention, with its potential as a biomarker for cancer diagnosis and monitoring. However, its precise role in cancer biology and progression remains unclear. cfDNA in cancer patients’ blood has been shown to activate signaling pathways, such as those mediated by toll-like receptors (TLRs), suggesting its involvement in cancer cell adaptation to the tumor microenvironment. Methods: This impact of cfDNA released from MDA-MB-231, a triple-negative breast cancer (TNBC) cell line was assessed, focusing on glucose availability and culture duration. The impact of cfDNA on the proliferation of MDA-MB-231 cells was investigated using proliferation curves, while cellular migration was evaluated through wound healing assays. The metabolic alterations induced by distinct cfDNA variants in MDA-MB-231 cells were investigated through nuclear magnetic resonance (NMR) spectroscopy, and their effect on cisplatin resistance was evaluated using flow cytometry. Furthermore, the expression levels of DNA-sensitive Toll-like receptor 9 (TLR9) were quantified via immunofluorescence, alongside its colocalization with lysosome-associated membrane protein 1 (LAMP1). Results: This study indicates that cfDNA facilitates metabolic adaptation, particularly under metabolic stress, by modulating glucose and glutamine consumption, key pathways in tumor cell metabolism. Exposure to cfDNA induced distinct metabolic shifts, favoring energy production through oxidative phosphorylation. The anti-cancer activity of cfDNA isolated from conditioned media of cells cultured under stressful conditions is influenced by the culture duration, emphasizing the importance of adaptation and se-lection in releasing cfDNA that can drive pro-tumoral processes. Additionally, cfDNA exposure influenced cell proliferation, quiescence, and migration, processes linked to metastasis and treatment resistance. These findings underscore cfDNA as a key mediator of metabolic reprogramming and adaptive responses in cancer cells, contributing to tumor progression and therapy resistance. Furthermore, the activation of TLR9 signaling suggests a mechanistic basis for cfDNA-induced phenotypic changes. Conclusions: Overall, cfDNA serves as a crucial signaling molecule in the tumor microenvironment, orchestrating adaptive processes that enhance cancer cell survival and progression. Full article

Sepsis is a leading cause of death in hospitalized patients. The underlying pathophysiologic mechanisms of sepsis have not been fully elucidated thus far. The receptor of programmed cell death 1 (PD-1) and its ligand (PD-L1), in combination with the Toll-like receptors (TLRs), seem to contribute considerably in systematic responses during sepsis. Investigating the relationship between them and identifying potential target pathways is important in the future management of sepsis, especially in relation to acute lung injury. This study investigated the interactions between TLR-5, -6, and -9 and PD-1/PD-L1 expression in a septic mouse model. Sixty C57BL/6J mice were included and categorized in six study groups. Three sepsis (S) groups (24 h, 48 h, and 72 h) and three sham (Sh) groups (24 h, 48 h, and 72 h) were created. Cecal ligation and puncture (CLP) was utilized to simulate sepsis in the S groups. Hematological analysis and lung tissue histopathological analysis were performed after 24 h, 48 h, and 72 h. Significant decreases in S groups compared to Sh groups in WBC and lymphocyte counts at 24, 48, and 72 h were observed. Significant increases in S groups compared to Sh groups in RBC and monocyte counts, IL-6 and IL-10 levels, alveolar flooding, and alveolar collapse were demonstrated by histopathological analysis. This study suggested a strong correlation between TLR expression and PD-1/PD-L1 up-regulation in lung tissue during sepsis. These molecules, also, seem to contribute to the histopathological changes in lung tissue during sepsis, leading to acute lung injury. Full article

Proprotein convertase subtilisin/kexin type 9 (PCSK9) has emerged as a novel target for reducing low-density lipoprotein cholesterol. PCSK9 activates the atherosclerosis process through pro-inflammation signaling. Furthermore, the serum level of PCSK9 is positively correlated with mortality in patients with heart failure (HF). Cardiac fibrosis plays a crucial role in the pathophysiology of HF. In this study, we intended to examine whether PCSK9 can increase fibroblast activities and explore what its underlying mechanisms are. Migration, proliferation analyses, and Western blotting were used on human cardiac fibroblasts with and without PCSK9. Alirocumab (a PCSK9 inhibitor, 10 mg/kg/week intra-peritoneally for 28 consecutive days) was treated in isoproterenol (100 mg/kg, subcutaneous injection)-induced HF rats. PCSK9 (50, 100 ng/mL) increased proliferation, myofibroblast differentiation capability, and collagen type I production. Compared with control cells, PCSK9 (100 ng/mL)-treated cardiac fibroblasts showed higher nucleotide-binding domain (NOD)-like receptor protein 3 (NLRP3), interleukin (IL)-1, myofibroblast differentiation, and collagen production capabilities, which were attenuated by MCC950 (an NLRP3 inhibitor, 100 μmol/L). PCSK9 upregulated Myd88 and NF-κB signaling, which were reduced by TAK242 (a toll-like receptor (TLR) 4 inhibitor, 10 μmol/L). Moreover, alirocumab significantly improved left ventricular systolic function and attenuated fibrosis in HF rats. In conclusion, PCSK9 upregulates NLRP3 signaling and the profibrotic activities of cardiac fibroblasts through the activation of TLR4/Myd88/NF-κB signaling. Full article

Synthetic cytosine-phosphate-guanine oligodeoxynucleotides (CpG ODNs) are promising candidates for vaccine adjuvants, because they activate immune responses through the Toll-like receptor 9 (TLR9) pathway. However, unmodified CpG ODNs are quickly degraded by serum nucleases, and their negative charge hinders cellular uptake, limiting their clinical application. Our group previously reported that guanine-quadruplex (G4)-forming CpG ODNs exhibit enhanced stability and cellular uptake. G4 structures can form in parallel, anti-parallel, or hybrid topologies, depending on strand orientation, but the effects of these topologies on CpG ODNs have not yet been explored. In this study, we designed three distinct G4 topologies as scaffolds for CpG ODNs. Among the three topology, the parallel G4 CpG ODN demonstrated the highest serum stability and cellular uptake, resulting in the strongest immune response from macrophage cells. Additionally, we investigated the binding affinities of the different G4 topologies to macrophage scavenger receptor-1 and TLR9, both of which are key to immune activation. These findings provide valuable insights into the development of CpG ODN-based vaccine adjuvants. Full article

Neurodegenerative diseases are characterized by progressive loss of neurons and persistent inflammation. Neurons are terminally differentiated cells, and lost neurons cannot be replaced since neurogenesis is restricted to only two neurogenic niches in the adult brain, whose neurogenic potential decreases with age. In this regard, the astrocytes reprogramming into neurons may represent a promising strategy for restoring the lost neurons and rebuilding neural circuits. To date, many anti-inflammatory agents have been shown to reduce neuroinflammation; however, their potential to restore neuronal loss was poorly investigated. This study investigates the anti-inflammatory effects of lactoferrin on DI-TNC1 astrocyte cell line and its ability to induce astrocyte reprogramming in a context of sustained inflammation. For this purpose, astrocytes were pre-treated with lactoferrin (4 μg/mL) for 24 h, then with lipopolysaccharide (LPS) (400 ng/mL), and examined 2, 9 and 16 days from treatment. The results demonstrate that lactoferrin attenuates astrocyte reactivity by reducing Toll-like receptor 4 (TLR4), Glial fibrillary acidic protein (GFAP) and IL-6 expression, as well as by upregulating Interleukin-10 (IL-10) cytokine and NRF2 expression. Moreover, lactoferrin promotes the reprogramming of reactive astrocytes into proliferative neuroblasts by inducing the overexpression of the Sex determining region Y/SRY-box 2 (SOX2) reprogramming transcription factor. Overall, this study highlights the potential effects of lactoferrin to attenuate neuroinflammation and improve neurogenesis, suggesting a future strategy for the treatment of neurodegenerative disorders. Full article

The neonatal period is a critical phase for the development of the intestinal immune system, marked by rapid adaptation to the external environment and unique nutritional demands. Breast milk plays a pivotal role in this transition, yet the mechanisms by which it influences neonatal mucosal immunity remain unclear. This review examines the potential mechanisms by which cell-free DNA (cfDNA) in breast milk may impact neonatal immune development, particularly through Toll-like receptor 9 (TLR9) signalling and gut microbiota interactions. We propose that cfDNA in breast milk interacts with TLR9 on the apical surface of neonatal intestinal epithelial cells, potentially serving as an initial anti-inflammatory stimulus before the establishment of commensal bacteria. This hypothesis is supported by the high concentration and stability of cfDNA in breast milk, as well as the known activation of TLR9 by mitochondrial DNA in breast milk. The review emphasises the need for further empirical research to validate these interactions and their implications for neonatal health, suggesting that understanding these dynamics could lead to improved strategies for neonatal care and disease prevention. Full article

A CpG oligodeoxynucleotide (CpG-ODN), iSN40, was originally identified as promoting the mineralization and differentiation of osteoblasts, independent of Toll-like receptor 9 (TLR9). Since CpG ODNs are often recognized by TLR9 and inhibit osteoclastogenesis, this study investigated the TLR9 dependence and anti-osteoclastogenic effect of iSN40 to validate its potential as an osteoporosis drug. The murine monocyte/macrophage cell line RAW264.7 was treated with the receptor activator of nuclear factor-κB ligand (RANKL) to induce osteoclast differentiation, then the effect of iSN40 on was quantified by tartrate-resistant acid phosphatase (TRAP) staining and real-time RT-PCR. iSN40 completely inhibited RANKL-induced differentiation into TRAP⁺ multinucleated osteoclasts by suppressing osteoclastogenic genes and inducing anti-/non-osteoclastogenic genes. Treatment with a TLR9 inhibitor, E6446, or a mutation in the CpG motif of iSN40 abolished the intracellular uptake and anti-osteoclastogenic effect of iSN40. These results demonstrate that iSN40 is subcellularly internalized and is recognized by TLR9 via its CpG motif, modulates RANKL-dependent osteoclastogenic gene expression, and ultimately inhibits osteoclastogenesis. Finally, iSN40 was confirmed to inhibit the osteoclastogenesis of RAW264.7 cells cocultured with the murine osteoblast cell line MC3T3-E1, presenting a model of bone remodeling. This study demonstrates that iSN40, which exerts both pro-osteogenic and anti-osteoclastogenic effects, may be a promising nucleic acid drug for osteoporosis. Full article

The endogenous neurosteroid (3α,5α)-3-hydroxypregnan-20-one (3α,5α-THP) modulates inflammatory and neuroinflammatory signaling through toll-like receptors (TLRs) in human and mouse macrophages, human blood cells and alcohol-preferring (P) rat brains. Although it is recognized that 3α,5α-THP inhibits TLR4 activation by blocking interactions with MD2 and MyD88, the comprehensive molecular mechanisms remain to be elucidated. This study explores additional TLR4 activation sites, including TIRAP binding to MyD88, which is pivotal for MyD88 myddosome formation, as well as LPS interactions with the TLR4:MD2 complex. Both male and female P rats (n = 8/group) received intraperitoneal administration of 3α,5α-THP (15 mg/kg; 30 min) or a vehicle control, and their hippocampi were analyzed using immunoprecipitation and immunoblotting techniques. 3α,5α-THP significantly reduces the levels of inflammatory mediators IL-1β and HMGB1, confirming its anti-inflammatory actions. We found that MyD88 binds to TLR4, IRAK4, IRAK1, and TIRAP. Notably, 3α,5α-THP significantly reduces MyD88-TIRAP binding (Males: −31 ± 9%, t-test, p < 0.005; Females: −53 ± 15%, t-test, p < 0.005), without altering MyD88 interactions with IRAK4 or IRAK1, or the baseline expression of these proteins. Additionally, molecular docking and molecular dynamic analysis revealed 3α,5α-THP binding sites on the TLR4:MD2 complex, targeting a hydrophobic pocket of MD2 usually occupied by Lipid A of LPS. Surface plasmon resonance (SPR) assays validated that 3α,5α-THP disrupts MD2 binding of Lipid A (Kd = 4.36 ± 5.7 μM) with an inhibition constant (Ki) of 4.5 ± 1.65 nM. These findings indicate that 3α,5α-THP inhibition of inflammatory mediator production involves blocking critical protein-lipid and protein-protein interactions at key sites of TLR4 activation, shedding light on its mechanisms of action and underscoring its therapeutic potential against TLR4-driven inflammation. Full article

(1) Introduction: Toll-like receptors (TLRs) are key in immune response by recognizing pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs). In gastric cancer (GC), TLR2, TLR3, TLR4, and TLR9 are crucial for modulating immune response and tumor progression. (2) Objective: This study aimed to assess the percentage of dendritic cells and monocytes expressing TLR2, TLR3, TLR4, and TLR9, along with the concentration of their soluble forms in the serum of GC patients compared to healthy volunteers. Factors such as disease stage, tumor type, age, and gender were also analyzed. (3) Materials and Methods: Blood samples from newly diagnosed GC patients and healthy controls were immunophenotyped using flow cytometry to assess TLR expression on dendritic cell subpopulations and monocytes. Serum-soluble TLRs were measured by ELISA. Statistical analysis considered clinical variables such as tumor type, stage, age, and gender. (4) Results: TLR expression was significantly higher in GC patients, except for TLR3 on classical monocytes. Soluble forms of all TLRs were elevated in GC patients, with significant differences based on disease stage but not tumor type, except for serum TLR2, TLR4, and TLR9. (5) Conclusions: Elevated TLR expression and soluble TLR levels in GC patients suggest a role in tumor pathogenesis and progression, offering potential biomarkers and therapeutic targets. Full article