TLR4 Expression Research Articles

The pro-inflammatory responses of innate immune cells to Leishmania RNA virus 2-infected L. major support the survival and proliferation of the parasites

Infection of Leishmania by Leishmania RNA virus (LRV) has been proposed as a pathogenic factor that induces pro-inflammatory responses through the TLR3/TLR4 signaling pathway. We investigated the effect of L. major infection by LRV2 on innate immune cell responses (human neutrophil (HL-60) and macrophage (THP-1) cell lines). The expression levels of pro- and anti-inflammatory cytokine and chemokine genes as well as genes involved in the amino acid metabolism of arginine were then investigated by RT-qPCR. Moreover, the expression of TLR genes and their downstream signaling pathways were compared in THP-1 cells infected with the two isolates. Apoptosis was also evaluated in infected THP-1 and HL-60 cells using the PI/Annexin V flow cytometry assay. In both cell lines, the expression of pro-inflammatory cytokines increased in response to LRV2+ L. major (Lm+), and the expression of chemokines shifted toward macrophage recruitment. In contrast to LRV2- L. major (Lm-), Lm+ infected THP-1 cells acquired the M2-like phenotype. The presence of LRV2 increased the gene expression of TLRs and their signaling pathways, especially TLR3 and TLR4, which was proportional to the increase in pro-inflammatory cytokines. In addition, Lm+ increased the expression of IL-10 and IFN-β, which contribute to the survival and growth of the parasite in the phagolysosome. Altogether, our results showed that Lm+ could stimulate pro-inflammatory responses that promote parasite replication and stabilization in the host.

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.

Suppressive effects of Toll-Like Receptor 2, Toll-Like Receptor 4, and Toll-Like Receptor 7 on protective responses to Mycobacterium bovis BCG from epithelial cells

Mycobacteria have several mechanisms for evasion of protective responses mounted by the host. In this study, we unravel yet another mechanism that is mediated by Toll-Like Receptors TLR2, TLR4, and TLR7 in epithelial cells. We show that mycobacterial infection of epithelial cells increases the expression of TLR2, TLR4, and TLR7. Stimulation of either TLR along with mycobacterial infection results in an inhibition of oxidative burst resulting in increased survival of mycobacteria inside epithelial cells. TLR stimulation along with mycobacterial infection also inhibits activation of epithelial cells for T cell responses by differentially regulating the activation of ERK-MAPK and p38-MAPK along with inhibition of co-stimulatory molecule CD86 expression. Furthermore, stimulation of either TLR inhibits the induction of apoptosis and autophagy. Knockdown of either TLR by specific siRNAs reverses the inhibition by ROS and apoptosis by mycobacteria and results in reduced intracellular survival of mycobacteria in a MyD88-dependent manner. These results point towards a negative role for TLR2, TLR4, and TLR7 in regulating protective responses to M. bovis BCG infection in epithelial cells.

Sivelestat Sodium Alleviates Ischemia-Reperfusion-Induced Acute Kidney Injury via Suppressing TLR4/Myd88/NF-κB Signaling Pathway in Mice.

We aim to detect the effects of sivelestat on renal ischemia-reperfusion associated with AKI and also explore the underlying mechanism. Mice, aged between 8 and 12weeks, were randomly allocated among four distinct groups, respectively normal saline sham group(C), normal saline surgery group(I), sivelestat (50 mg/kg) sham group(S), sivelestat (50 mg/kg) surgery group(SI) (n=6, each group). In the surgical groups, the renal pedicles of mice were clamped withnon-traumatic micro-aneurysm clamps, resulting in ischemia of the kidneys for 45minutes. This was followed by a period of reperfusion lasting 24hours. Sham group mice underwent the identical surgery produced without clamping renal pedicles. Mice blood was obtained from eyeballs, and Serum creatinine and blood urea nitrogen levels were measured. After a 24-hour period of reperfusion, the mice were euthanized, and their kidneys were gathered for various analyses, including Western Blot (WB) analysis, RT-PCR, immunofluorescence (IF), hematoxylin and eosin (H&E) staining, and Tunel assay. Pretreatments with sivelestat decreased renal Neutrophil elastase (NE), serum creatinine, and blood urea nitrogen levels after renal ischemia-reperfusion. Sivelestat also reduced histological damage and cell apoptosis in kidneys following ischemia-reperfusion injury (IRI). In addition, thesivelestat administrationdiminished the levels of mRNA expression of interleukin 6 (IL-6), Macrophage inflammatory protein-2 (MIP-2), monocyte chemoattractant protein-1 (MCP-1), and tumor necrosis factor (TNF)-α in the kidneys during IRI.The kidney tissues of the SI group had significantly mitigated TLR4, Myd88, and NF-κB p-p65 protein expression levels compared to the I group (all P<0.05). We demonstrated a previously unidentified mechanism that sivelestat effectively attenuates AKI-induced renal dysfunction, possibly through suppressing the TLR4/Myd88/ NF-κB pathway.

TLR-2 and TLR-4 mRNA expression in different grades of histopathological lesions of equine endometrium from follicular phase.

Increased synthesis and deposition of collagen(COL) in the extracellular matrix (ECM) of equine endometrium contributes to endometrosis. Toll-like receptors (TLRs) are transmembrane receptors involved in the innate immune response, recognized for their role in antigen recognition and previously associated with equine endometritis. The TLRs not only recognize pathogen-associated molecular patterns but also regulate inflammations, fibrosis and cancer. The aim of this study was to explore the relationship between TLR expression at different stages of Kenney and Doig's (K-D) grading and COL1 expression during the follicular phase of the oestrous cycle. Forty samples of endometrial tissues were collected post-mortem from mares on the follicular phase of the oestrous cycle (10 samples of each K-D category). Relative mRNA transcription of TLR-2, TLR-4 and COL1A2 genes was assessed using qPCR, and COL1 protein expression by Western blot analysis. The COL1A2 transcription increased in category IIB when compared to categories I, IIA and III endometria (p < .01). The relative protein abundance of COL1 showed no significant differences between endometrial categories (p > .05). As for the TLRs mRNA transcription, TLR-2/-4 transcripts increased in IIA when compared to the other K-D endometria categories (p < .05). Our findings suggest that TLRs may be involved in the initiation of the endometrial inflammatory response. Additional studies are needed to explore TLRs' potential role as diagnostic markers for monitoring inflammation progression and fibrosis development, as well as their involvement in the mechanisms underlying fibrotic pathways.

Evaluation of the Choroid Plexus Epithelium Inflammation TLR4/NF-κB/NKCC1 Signal Pathway Activation in the Development of Hydrocephalus.

Hydrocephalus is characterized by secretion, circulation, and absorption disorder of cerebrospinal fluid (CSF) with high morbidity and complication rate. The relationship between inflammation and abnormal secretion of CSF by choroid plexus epithelium (CPE) had received more attention. In this study, we aim to detect the role of Toll-like receptor 4/nuclear factor-kappa B/Na+/K+/2Cl-cotransporter 1(TLR4/NF-κB/NKCC1) signal pathway in the development of hydrocephalus. Hydrocephalus was induced in adult rats (8 weeks) by intracisternal kaolin injection, then pyrrolidinedithiocarbamate (PDTC) and bumetanide were administrated to the rats mode. Then the rat model was evaluated, and ventricular volume was calculated at different time points. Then CPE, cortex, preventricular tissue, and CSF were obtained. Protein expressions of TLR-4, NKCC/serine-threonine STE20/SPS1-related, proline-alanine-rich kinase (SPAK), pNKCC1, pSPAK, GFAP, AQP1, and AQP4 were measured by RT-PCR, western blot, and immunofluorescence (IF) stains in CPE, respectively. Our data showed that inflammation factors tumor necrosis factor-(TNF-α), interleukin 18(IL-18), and glial fibrillary acidic protein (GFAP) concentrations were significantly higher in the model group than in controls. The TLR4/NF-κB/NKCC1 signal pathway were actived by NF-κB-p65, NKCC1, pNKCC1- pSPAK complex, and Aquaporin1 (AQP1) high expression. PDTC and bumetanide use can help regular TLR4/NF-κB/NKCC1 expression and reduced AQP1 expression by down-regulate NF-B-p65 and inhibiting NKCC1, respectively. As a result, the treatment groups alleviated CPE abnormal secretion and ventricle enlargement. These results confirmed that the inflammatory reaction contributes TLR4/NF-κB/NKCC1 mediated CPE abnormal secretion and consequent hydrocephalus. Regulation of TLR4/NF-κB/NKCC1 and AQP1 can prevent this process. Our study provides a strong rationale for further exploring alleviating CPE abnormal secretion as a therapeutic perspective of hydrocephalus.

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.

Mechanism of Yi-Qi-Bu-Shen Recipe for the Treatment of Diabetic Nephropathy Complicated with Cognitive Dysfunction Based on Network Pharmacology and Experimental Validation.

Diabetic nephropathy (DN) and cognitive dysfunction (CD) are common complications of diabetes. Yi-Qi-Bu-Shen Recipe (YQBS) can effectively reduce blood glucose, improve insulin resistance, and delay the progression of diabetic complications. The underlying mechanisms of its effects need to be further studied. This study elucidates the mechanism of YQBS in DN with CD through network pharmacology and experimental validation. Protein-protein interaction, Gene Ontology (GO), and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were performed. Male Sprague-Dawley (SD) rats were divided into 6 groups: model, YQBS (2, 4, 8 g/kg), positive control (metformin, 200 mg/kg), and control; the DN model was established by high sugar and high fat diet combined with intraperitoneal streptozotocin injection. After the DN model was established, the rats were gavaged for 10weeks. Serum, kidneys, and hippocampus tissues were collected to measure the expression levels of TLR4, NF-κB, TNF-α, and IL-6. The network pharmacology analysis showed that quercetin and kaempferol were the main active components of YQBS. TNF and IL-6 were the key targets, and TLR4/NF-κB pathway was crucial to YQBS in treating DN complicated with CD. Experimental validation showed that the intervention of YQBS can reduce TNF-α and IL-6 in serum, and also significantly decreases the protein expression of TLR4 and NF-κB. YQBS exerts anti-inflammatory effects on DN with CD through TLR4/NF-κB pathway. This study provides a biological basis for the scientific usage of YQBS in inflammation diseases and supplies experimental evidence for future traditional Chinese medicine development.

Neoastilbin ameliorates sepsis-induced liver and kidney injury by blocking the TLR4/NF-κB pathway.

Sepsis frequently causes systemic inflammatory response syndrome and multiple organ failure in patients. Neoastilbin (NAS) is a flavonoid that plays vital functions in inflammation. This work aims to investigate the protective effects of NAS against sepsis-induced liver and kidney injury and elucidate its underlying mechanisms. The mouse model was established using cecal ligation puncture (CLP) induction. NAS was given to mice by gavage for 7 consecutive days before surgery. Liver and kidney function, oxidative stress, and inflammatory factors in serum or tissues were examined by ELISA or related kits. The expression of relevant proteins was assessed by Western blot. Hematoxylin and eosin and/or periodic acid-Schiff staining revealed that NAS ameliorated the pathological damage in liver and kidney tissues of CLP-induced mice. NAS improved liver and kidney functions, as evidenced by elevated levels of blood urea nitrogen, Creatinine, ALT, and AST in the serum of septic mice. TUNEL assay and the expression of Bcl-2 and Bax showed that NAS dramatically reduced apoptosis in liver and renal tissues. NAS treatment lowered the levels of myeloperoxidase and malondialdehyde, while elevated the superoxide dismutase content in liver and kidney tissues of CLP-induced mice. The levels of inflammatory cytokines (IL-6, TNF-α, and IL-1β) in the serum and both tissues of CLP-injured mice were markedly decreased by NAS. Mechanically, NAS downregulated TLR4 expression and inhibited NF-κB activation, and overexpression of TLR4 reversed the protective effects of NAS against liver and kidney injury. Collectively, NAS attenuated CLP-induced apoptosis, oxidative stress, inflammation, and dysfunction in the liver and kidney by restraining the TLR4/NF-κB pathway.

THIOSTREPTON MODULATES TLR4 EXPRESSION AND INDUCES APOPTOSIS IN MDA MB 231 CELLS: AN IN VITRO AND IN SILICO ANALYSIS

Objective: Toll-like receptors (TLRs) are key pattern recognition receptors involved in tumorigenesis, apoptosis, and metastasis. Triple-negative breast cancer (TNBC) is a highly aggressive malignancy with a poor prognosis. Although the role of TLRs in breast cancer remains underexplored, recent studies suggest targeting TLRs in TNBC could be beneficial. In this study Thiostrepton, an antibiotic and novel inhibitor of TLR7-9 in psoriatic inflammation, was investigated for its effects on TLR3, TLR4, and TLR9 expression in TNBC cells (MDA-MB-231). Materials and Methods: The cytotoxicity of thiostrepton was assessed using the MTT assay. RT-PCR was used to measure gene expression levels of TLR3, TLR4, TLR9, Bax, Bcl-2, Nf-κB, and E-cadherin. Cell morphology changes were analyzed with Acridine Orange/Ethidium Bromide (AO/EtBr) staining. Molecular docking and dynamics simulations examined interactions between thiostrepton and the TLR4-MD-2 complex. Results: Thiostrepton led to a concentration- and time-dependent decrease in cell viability. It significantly inhibited TLR4, Bcl-2 gene expression and increased TLR3, Bax, and Nf-κB levels. The changes in Bax and Bcl-2 gene expression, along with alterations in cell morphology, demonstrated that thiostrepton promoted apoptosis in MDA-MB-231 cells. While TLR9 expression reduction was not significant, thiostrepton notably increased TLR3 expression and decreased TLR4 expression. The three independent molecular dynamics simulations demonstrated that thiostrepton binds stably to the TLR4-MD2 domain, exhibiting a high binding affinity as indicated by the binding free energy calculations. Conclusion: Thiostrepton effectively induces apoptosis and reduces cell viability in TNBC cells. In silico analysis suggest thiostrepton could modulate TLR4, highlighting its potential as a candidate for further research and therapeutic development.

Supplementation with Akkermansia muciniphila improved intestinal barrier and immunity in zebrafish (Danio rerio)

Akkermansia muciniphila (Akk), a second-generation probiotic known for its ability to regulate intestinal function in mammals, is not yet fully understood in the context of aquaculture. This study aims to investigate the effects of different forms of Akk on intestinal barrier function and immune response in zebrafish (Danio rerio) under high-fat diet conditions. The experimental groups included a control group, a high-fat diet group, an Akk group, and a group receiving various concentrations of pasteurized Akkermansia muciniphila (P-Akk) along with a high-fat diet. Evaluation methods included histological examination with hematoxylin and eosin staining, ultrastructural analysis using transmission electron microscopy, real-time fluorescence quantitative analysis, and transcriptome sequencing technology. The results showed that both the Akk and P-Akk groups exhibited a significant increase in villi number and length compared to the high-fat group. Furthermore the expression levels of claudin, claudin-2, occludin A, occludin B, and other genes were significantly upregulated, while the expression levels of intestinal proinflammatory factors genes and proteins were significantly downregulated. Compared to the high-fat group, the Akk group showed a more complete and well-preserved nucleus, mitochondria, and tight junction structures. Additionally, the morphology of intestinal epithelial microvilli in the medium and high concentration Akk group was complete and dense. The expressions of tlr2 and nf-κb were upregulated, while the expressions of myd88 and nod2 were downregulated in the medium- and high-concentration Akk groups. Akk may improve immune dysfunction in high-fat fed zebrafish through the TLR2/NF-κB signaling pathway, which requires further study. Transcriptome analysis revealed significant upregulation of the immune-related gene pigr, significant downregulation of stat3, and significant upregulation of the intercellular adhesion molecule f11r. In conclusion, dietary Akk supplementation alleviated intestinal barrier damage and immune dysfunction in high-fat zebrafish. This study provides important insights into the potential use of Akk in fish and lays the foundation for further studies on its role in fish immunity.

Comparative effect of atorvastatin and risperidone on modulation of TLR4/NF-κB/NOX-2 in a rat model of valproic acid-induced autism

BackgroundAutism spectrum disorder (ASD) is a complex neurodevelopmental condition that is significantly increasing, resulting in severe distress. The approved treatment for ASD only partially improves the sympoms, but it does not entirely reverse the symptoms. Developing novel disease-modifying drugs is essential for the continuous improvement of ASD. Because of its pleiotropic effect, atorvastatin has been garnered attention for treating neuronal degeneration. The present study aimed to investigate the therapeutic effects of atorvastatin in autism and compare it with an approved autism drug (risperidone) through the impact of these drugs on TLR4/NF-κB/NOX-2 and the apoptotic pathway in a valproic acid (VPA) induced rat model of autism.MethodsOn gestational day 12.5, pregnant rats received a single IP injection of VPA (500 mg/kg), for VPA induced autism, risperidone and atorvastatin groups, or saline for control normal group. At postnatal day 21, male offsprings were randomly divided into four groups (n = 6): control, VPA induced autism, risperidone, and atorvastatin. Risperidone and atorvastatin were administered from postnatal day 21 to day 51. The study evaluated autism-like behaviors using the three-chamber test, the dark light test, and the open field test at the end of the study. Biochemical analysis of TLR4, NF-κB, NOX-2, and ROS using ELISA, RT-PCR, WB, histological examination with hematoxylin and eosin and immunohistochemical study of CAS-3 were performed.ResultsMale offspring of prenatal VPA-exposed female rats exhibited significant autism-like behaviors and elevated TLR4, NF-κB, NOX-2, ROS, and caspase-3 expression. Histological analysis revealed structural alterations. Both risperidone and atorvastatin effectively mitigated the behavioral, biochemical, and structural changes associated with VPA-induced rat model of autism. Notably, atorvastatin group showed a more significant improvement than risperidone group.ConclusionsThe research results unequivocally demonstrated that atorvastatin can modulate VPA-induced autism by suppressing inflammation, oxidative stress, and apoptosis through TLR4/NF-κB/NOX-2 signaling pathway. Atorvastatin could be a potential treatment for ASD.Graphical

Lentil Waste Extracts for Inflammatory Bowel Disease (IBD) Symptoms Control: Anti-Inflammatory and Spasmolytic Effects

Background/Objectives: In the contest of agro-industrial waste valorization, we focused our attention on lentil seed coats as a source of health-promoting phytochemicals possibly useful in managing inflammatory bowel diseases (IBDs), usually characterized by inflammation and altered intestinal motility. Methods: Both traditional (maceration) and innovative microwave-assisted extractions were performed using green solvents, and the anti-inflammatory and spasmolytic activities of the so-obtained extracts were determined through in vitro and ex vivo assays, respectively. Results: The extract obtained through the microwave-assisted procedure using ethyl acetate as the extraction solvent (BEVa) proved to be the most useful in inflammation and intestinal motility management. In LPS-activated Caco-2 cells, BEVa down-regulated TLR4 expression, reduced iNOS expression and the pro-inflammatory cytokine IL-1 production, and upregulated the anti-inflammatory cytokine IL-10 production, thus positively affecting cell inflammatory responses. Moreover, a significant decrease in the longitudinal and circular tones of the guinea pig ileum, with a reduction of transit speed and pain at the ileum level, together with reduced transit speed, pain, and muscular tone at the colon level, was observed with BEVa. HPLC separation combined with an Orbitrap-based high-resolution mass spectrometry (HRMS) technique indicated that 7% of all the identified metabolites were endowed with proven anti-inflammatory and antispasmodic activities, among which niacinamide, apocynin, and p-coumaric acid were the most abundant. Conclusions: Our results suggest that lentil hull extract consumption could contribute to overall intestinal health maintenance, with BEVa possibly representing a dietary supplementation and a promising approach to treating intestinal barrier dysfunction.

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

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

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

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

Hesperidin attenuates radiation-induced ovarian failure in rats: Emphasis on TLR-4/NF-ĸB signaling pathway

Young women suffering from premature ovarian failure after radiotherapy carry a huge burden in the field of cancer therapy including reproductive loss, emotional stress, and physical troubles that reduce their long-term quality of life. Hesperidin (HSP) exhibited antioxidant, anti-inflammatory, and anti-apoptotic properties. HSP enhanced in vitro follicular maturation and preserved in vivo ovarian stockpile. In this research, the role of HSP in radiation-induced POF in rats was investigated besides ascertaining its underlying mechanisms. Female Sprague-Dawley rats were arbitrarily allocated into four groups: control-group, ϒ-irradiated-group (3.2 Gy once on the 7th day), HSP-group (100 mg/kg, orally for 10 days), and HSP/ϒ-irradiated-group (ϒ-radiation was applied one hour after HSP). At the end of experiment, the whole ovaries were collected for histological and biochemical analyses. Administration of HSP preserved the ovarian histoarchitecture and follicular stock, retained ovarian weight, and conserved serum estradiol and AMH levels following radiation exposure. HSP ameliorated the ovarian oxidative damage mediated by radiation through augmenting the activities of glutathione peroxidase, glutathione reductase, and catalase antioxidant enzymes. HSP exhibited remarkable anti-inflammatory activity by downregulating the expression of ovarian TLR-4, NF-ĸB, and TNF-α. Moreover, HSP suppressed the apoptotic machinery triggered by radiation by reducing p53 and Bax while increasing Bcl-2 mRNA expressions alongside diminishing caspase-3 expression. Additionally, HSP regulated estrous cycle disorder of irradiated rats and improved their reproductive capacity reflected by enhancing pregnancy outcomes. Therefore, HSP represents an appealing candidate as an adjunct remedy for female cancer patients during radiotherapy protocols owing to its antioxidant, anti-inflammatory, anti-apoptotic, and hormone-regulatory effects.

Structural characterization of polysaccharides from Panax ginseng C. A. Meyer root and their triggered potential immunoregulatory and radioprotective activities

With people's increasing awareness of healthy diet, the diverse health-promoting functions of ginseng have been widely recognized. As one of the key functional components, ginseng polysaccharides have attracted increasing research interest. Here, three purified polysaccharide fractions, GPS-1a, GPS-1b, and GPS-2, were obtained from the root extract of Panax ginseng C. A. Meyer. Structurally, GPS-1a and GPS-1b were both linked in a → 6)-α-D-Glcp-(1 → pattern but composed of glucose and galactose in molar ratios of 9.76:0.24 and 9.81:0.19. In contrast, GPS-2 was composed of glucose, galactose, arabinose, rhamnose, and galacturonic acid in a molar ratio of 1.82:1.94:0.79:0.52:4.93. The main backbone consisted of →4)-α-D-GalpA-(1→, →4)-α-D-GalpA-6OMe-(1→, →3, 4)-α-D-GalpA-(1→, →3)-α-L-Rhap-(1 → linages, and its branches are composed of →5)-α-L-Araf-(1→, →4)-β-D-Galp-(1→, →2)-β-D-Glcp-(1→, α-D-GalAp-(1→. Benefitting from this structural variance, GPS-2 exhibited the most significant immunoregulatory and radioprotective efficacies. Specifically, GPS-2 promoted TLR2, NF-κB, and TRAF6 protein expression levels, thereby significantly improving macrophage phagocytosis, splenic lymphocyte proliferation, and stimulation of NO, IL-1β, IL-6, and TNF-α secretion, which activated RAW264.7 and splenic lymphocytes. The following radioprotection activity tests unveiled that GPS-2 increased the organ index, number of peripheral blood cells, cellularity of splenocytes, and bone marrow cell numbers in irradiated mice. This investigation revealed the contribution of polysaccharide structure characteristics to the bioactive expression and elucidated the potential utilization of GPS-2 as a radioprotective agent or immunomodulator.

Investigation of TLR4, TLR6, TLR7, and CD36 expression on T lymphocytes in coronary artery disease

Background: Coronary artery disease (CAD) is among the significant causes of death globally, caused by fatty deposits in blood vessel walls. Increasing evidence indicates that toll-like receptors (TLRs) are pivotal to atherosclerosis progression. The function of CD36 as a glycoprotein in atherosclerosis was also suggested. This study aimed to investigate the levels of TLR4, TLR6, and TLR7, as well as CD36 cell surface markers in CAD. Methods: This study included 64 patients undergoing angiography to determine whether atherosclerosis was present. The patients were categorized into two groups. The three main coronary arteries are all stenosed at over 50% in patients with CAD+ (n=22) as cases and CAD− (n=42) with smooth angiography as controls. Each patient's syntax score (SS) and Gensini (vessel score) were calculated. The TLR-4, TLR-6, TLR-7, and CD36 expression were measured using Flow cytometry. Results: TLR4 and TLR6 showed a significant link with the Gensini score in the subject group. In addition, TLR7 had an association with Syntax scoring in this study. Conclusion: The findings illustrated the association between TLR4, TLR6, and TLR7 cell surface markers with Gensini and Syntax scoring in individuals with coronary artery disease.

Effects of an Extract of Physalis Peruviana Linnaeus on the Expression of Inflammatory Markers in the Caco-2 Intestinal Epithelium-like Cell Line

Objective: Physalis Peruviana Linnaeus (PPL) is an herbaceous species characterized by a wide variety of bioactive compounds to which anti-inflammatory properties have been attributed. This makes this fruit a possible complementary therapy for diseases that involve chronic inflammation, such as inflammatory bowel diseases (IBD). In the present study, the effect of a PPL extract on the expression of inflammatory markers in the Caco-2 cell line was evaluated. Methods: An in vitro gastric digest (50 g PPL pulp) was performed, obtaining an extract that was used to challenge Caco-2 cells for 24 and 72 hours. This extract was characterized by LC-MS/MS. Then, the relative mRNA expression of NF-kB, TLR4, IL-18 and MCP-1 was determined through qRT-PCR and the protein levels of TNF-α, IL-6, IL-8, IL-18 and MCP-1 through Luminex Immunoassay. Results: From the characterization of the extract, compounds with bioactive potential such as isothiocyanates, indoles and coumarins were found. Treatment of Caco-2 cells with PPL extract (80 µg/ml), particularly for 72 hours, produced a reduction of IL-18 and MCP-1 mRNA expression (p < 0.01), in addition to IL-18 (p < 0.01), IL-8 (p < 0.0001) and MCP-1 (p < 0.01) protein levels, however, no effects on NF-kB p65 (p = 0.09) and TLR4 (p = 0.20) mRNA expression were observed. Conclusion: The results obtained in this study open the possibility that the regular consumption of 50 g of PPL could constitute a possible complementary therapy for the treatment of IBD, improving the quality of life of these patients.

Immune mRNA Expression and Fecal Microbiome Composition Change Induced by Djulis (Chenopodium formosanum Koidz.) Supplementation in Aged Mice: A Pilot Study.

Background and Objectives: The aging process has always been associated with a higher susceptibility to chronic inflammatory lung diseases. Several studies have demonstrated the gut microbiome's influence on the lungs through cross-talk or the gut-lungs axis maintaining nutrient-rich microenvironments. Taiwan djulis (Chenopodium formosanum Koidz.) provides antioxidant and anti-inflammatory characteristics that could modulate the gut microbiome. This could induce the gut-lung axis through microbial cross-talk, thus favoring the modulation of lung inflammation. Materials and Methods: Here, we investigate the immune mRNA expression in the spleen, fecal microbiome composition, and hyperplasia of the bronchial epithelium in aged 2-year-old BALB/c mice after 60 days of supplementation of djulis. Results: The pro-inflammatory cytokines IFN-γ, TNF-α, and IL-1β, T; cells CD4 and CD8; and TLRs TLR3, TLR4, TLR5, TLR7, TLR8, and TLR9 were reduced in their mRNA expression levels, while the anti-inflammatory cytokines IL-2, IL-4, and IL-10 were highly expressed in the C. formosanum-treated group. Interestingly, the fecal microbiome composition analysis indicated higher diversity in the C. formosanum-treated group and the presence of butyrate-producing bacteria that are beneficial in the gut microbiome. The histopathology showed reduced hyperplasia of the bronchial epithelium based on the degree of lesions. Conclusions: Our findings suggest that Taiwan djulis can modulate the gut microbiome, leading to microbial cross-talk; reducing the mRNA expression of pro-inflammatory cytokines, T cells, and TLRs; and increasing anti-inflammatory cytokines in the spleen, as cytokines migrate in the lungs, preventing lung inflammation damage in aged mice or the gut-lung axis. Thus, Taiwan djulis could be considered a beneficial dietary component for the older adult population. The major limitation includes a lack of protein validation of cytokines and TLRs and quantification of the T cell population in the spleen as a marker of the gut-lung axis.