TLR4 Receptor Research Articles

Study on the inhibition of dexmedetomidine (DEX) on malignant development of breast cancer by synchrotron radiation infrared spectroscopy

ObjectivesThe topic of the relationship between analgesia and cancer recurrence inoncology surgery is a subject of debate in the field. Administering analgesic drugs after breast cancer surgery could potentially impact the long-term outcomes for patients. During breast cancer surgery, DEX, a potent α2 adrenergic receptoragonist, is often administered as an analgesic. Despite its common use, the analgesic effect of DEX and its potential impact on the progression of breast cancer remain uncertain. MethodsTo investigate the impact of DEX on breast cancer cells, cells exposed to DEX were collected and analyzed through cell apoptosis detection and synchrotron radiation micro-infrared spectroscopy. ResultsFollowing DEX treatment, the absorption peaks of MDA-MB-231 and MCF-7 cell lines showed significant redshift at 1230–1250 cm−1 and 1380–1420 cm−1 bands (p<0.05). The absorption peak in the 1500–1700 cm−1 band decreased (p<0.05), while the 2910–2990 cm−1 absorption peak increased, along with other apoptotic characteristics (p<0.05). Our cell experiment results demonstrated that DEX was capable of protecting cell immunity via the down-regulation of TLR-4 receptor and effectively hindered the growth of breast cancer cells. ConclusionThe cell and spectrum experiments' results indicate that DEX can encourage tumor cell apoptosis. These findings suggest that DEX is safe to use for breast cancer surgery and pain management.

Galectin-3 Mediates Endotoxin Internalization and Caspase-4/11 Activation in Tubular Epithelials and Macrophages During Sepsis and Sepsis-Associated Acute Kidney Injury.

Besides being recognized by membrane receptor TLR4, lipopolysaccharide (LPS) can also be internalized into the cytosol and activate Caspase-4/11 pyroptotic pathways to further amplify inflammation in sepsis. The objective of this study was to investigate whether Galectin-3 (Gal3) could promote the uptake of LPS by governing RAGE or administering endocytosis, consequently activating Caspase 4/11 and mediating pyroptosis in sepsis-associated acute kidney injury (SA-AKI). By pinpointing Gal3, LPS, and EEA1 (endosome-marker) or LAMP1 (lysosome-marker) respectively, immunofluorescence discovered that Gal3 and LPS were mainly aggregated in early endosomes initially and translocated into lysosomes afterwards. In cells and animal models, Gal3 and the Caspase-4/11 pathways were simultaneously activated, and the overexpression of Gal3 could exacerbate pyroptosis, whereas inhibition of Gal3 or the knockdown of its expression could ameliorate pyroptosis, reduce the pathological changes of SA-AKI and improve the survival of the animals with SA-AKI. Silencing RAGE reduced pyroptosis in primary tubular epithelial cells (PTCs) activated by Gal3 and LPS but not in cells activated by Gal3 and outer membrane vesicles (with LPS inside), whereas pyroptosis in both was reduced by blockade of Gal3, indicating Gal3 promoted pyroptosis through both RAGE-dependent and RAGE-independent pathways. Our investigation further revealed a positive correlation between serum Gal3 and pyroptotic biomarkers IL-1 beta and IL-18 in patients with sepsis, and that serum Gal3 was an independent risk factor for mortality. Through our collective exploration, we unraveled the significant role of Gal3 in the internalization of LPS and the provocation of more intense pyroptosis, thus making it a vital pathogenic factor in SA-AKI and a possible therapeutic target.Gal3 enabled the internalization of endotoxin into endosomes and lysosomes via both RAGE-dependent (A) and RAGE-independent (B) pathways, leading to pyroptosis. The suppression of Gal3 curbed Caspase4/11 noncanonical inflammasomes and diminished sepsis and SA-AKI.

A DNA-polymer hybrid nanocomplex based bi-adjuvant vaccine for tumor immunotherapy

The immunosuppressive tumor microenvironment (TME) seriously impedes the cancer immunotherapy; Toll-like receptors (TLRs) agonists combination is emerging as vaccine adjuvant in reprogramming tumor immune microenvironment. Herein, we develop a supramolecular DNA-polymer hybrid nanocomplex-based bi-adjuvant vaccine for tumor immunotherapy. In the nanocomplex, the co-assembly of TLR9 agonist (CpG) and TLR7 agonist (loxoribine) was achieved via dynamic DNA assembly and boron chemistry. Poly-N-isopropyl acrylamide (PNIPAM) served as the framework of the nanocomplex, and meanwhile, designer DNA sequences were introduced as cross-linkers in the nanoframework to trigger the hybridization chain reaction (HCR)-based assembly of CpG sequences containing hairpins. The polymer framework was decorated with phenylboronic acid to capture and release loxoribine via reversible formation of cyclic boric acid ester. A lipid shell was coated on the outside of the CpG and loxoribine co-loaded nanocomplex to prevent the leakage of loxoribine in plasma. After intravenous injection, the nanocomplex was internalized by the tumor-associated immune cells via lysosomal related pathways. CpG and loxoribine synergistically stimulated TLR9 and TLR7 receptors, thus leading to the conversion of suppressive M2 macrophage to active M1 macrophage, maturation of dendritic cells, and activation of T cells. The dual activation of TLR9 and TLR7 effectively reprogramed tumor immune microenvironments in vivo, wherein robust CD8+ T cell response and efficient tumor regression were achieved. This work provides a unique design concept for the TLR agonists combination-based nanovaccine for tumor immunotherapy.

Prediction of an immunogenic peptide ensemble and multi-subunit vaccine for Visceral leishmaniasis using bioinformatics approaches

Visceral Leishmaniasis (VL) is a neglected tropical disease of public health importance in the Indian subcontinent. Despite consistent elimination initiatives, the disease has not yet been eliminated and there is an increased risk of resurgence from active VL reservoirs including asymptomatic, post kala azar dermatitis leishmaniasis (PKDL) and HIV-VL co-infected individuals. To achieve complete elimination and sustain it in the long term, a prophylactic vaccine, which can elicit long lasting immunity, is desirable. In this study, we employed immunoinformatic tools to design a multi-subunit epitope vaccine for the Indian population by targeting antigenic secretory proteins screened from the Leishmania donovani proteome. Out of 8014 proteins, 277 secretory proteins were screened for their cellular location and proteomic evidence. Through NCBI BlastP, unique fragments of the proteins were cropped, and their antigenicity was evaluated. B-cell, HTL and CTL epitopes as well as IFN-ɣ, IL-17, and IL-10 inducers were predicted, manually mapped to the fragments and common regions were tabulated forming a peptide ensemble. The ensemble was evaluated for Class I MHC immunogenicity and toxicity. Further, immunogenic peptides were randomly selected and used to design vaccine constructs. Eight vaccine constructs were generated by linking random peptides with GS linkers. Synthetic TLR-4 agonist, RS09 was used as an adjuvant and linked with the constructs using EAAK linkers. The predicted population coverage of the constructs was ∼99.8 % in the Indian as well as South Asian populations. The most antigenic, nontoxic, non-allergic construct was chosen for the prediction of secondary and tertiary structures. The 3D structures were refined and analyzed using Ramachandran plot and Z-scores. The construct was docked with TLR-4 receptor. Molecular dynamic simulation was performed to check for the stability of the docked complex. Comparative in silico immune simulation studies showed that the predicted construct elicited humoral and cell-mediated immunity in human host comparable to that elicited by Leish-F3, which is a promising vaccine candidate for human VL.

Determining zebrafish dorsal organizer size by a negative feedback loop between canonical/non-canonical Wnts and Tlr4/NFκB

In vertebrate embryos, the canonical Wnt ligand primes the formation of dorsal organizers that govern dorsal-ventral patterns by secreting BMP antagonists. In contrast, in Drosophila embryos, Toll-like receptor (Tlr)-mediated NFκB activation initiates dorsal-ventral patterning, wherein Wnt-mediated negative feedback regulation of Tlr/NFκB generates a BMP antagonist-secreting signalling centre to control the dorsal-ventral pattern. Although both Wnt and BMP antagonist are conserved among species, the involvement of Tlr/NFκB and feedback regulation in vertebrate organizer formation remains unclear. By imaging and genetic modification, we reveal that a negative feedback loop between canonical and non-canonical Wnts and Tlr4/NFκB determines the size of zebrafish organizer, and that Tlr/NFκB and Wnts switch initial cue and feedback mediator roles between Drosophila and zebrafish. Here, we show that canonical Wnt signalling stimulates the expression of the non-canonical Wnt5b ligand, activating the Tlr4 receptor to stimulate NFκB-mediated transcription of the Wnt antagonist frzb, restricting Wnt-dependent dorsal organizer formation.

Characterization of exclusively non-commensal Neisseria gonorrhoeae pangenome to prioritize globally conserved and thermodynamically stable vaccine candidates using immune-molecular dynamic simulations

Neisseria gonorrhoeae (Ngo) has emerged as a global threat leading to one of the most common sexually transmitted diseases in the world. It has also become one of the leading antimicrobial resistant organisms, resulting in fewer treatment options and an increased morbidity. Therefore, in recent years, there has been an increased focus on the development of new treatments and preventive strategies to combat its infection. In this study, we have combined the most conserved epitopes from the completely assembled strains of Ngo to develop a universal and a thermodynamically stable vaccine candidate. For our vaccine design, the epitopes were selected for their high immunogenicity, non-allergenicity and non-cytotoxicity, making them the ideal candidates for vaccine development. For the screening process, several reverse vaccinology tools were employed to rigorously extract non-homologous and immunogenic epitopes from the selected proteins. Consequently, a total number of 3 B-cell epitopes and 6 T-cell epitopes were selected and joined by multiple immune-modulating adjuvants and linkers to generate a promiscuous immune response. Additionally, the stability and flexible nature of the vaccine construct was confirmed using various molecular dynamic simulation tools. Overall, the vaccine candidate showed promising binding affinity to various HLA alleles and TLR receptors; however, further studies are needed to assess its efficacy in-vivo. In this way, we have designed a multi-subunit vaccine candidate to potentially combat and control the spread of N. gonorrhoeae.

PSVI-10 Effects of Poor Maternal Nutrition During Gestation on Offspring Basal Immune Status

Abstract Poor maternal nutrition during gestation affects offspring pre- and post-natal growth, decreasing muscularity and increasing adiposity. Chronic inflammation may be a potential mechanism for the alterations reported. The objective of this research was to determine the effects of poor maternal nutrition during gestation on offspring inflammatory status. We hypothesized that offspring of restricted- and over-fed ewes would have an altered inflammatory status which would be dependent on offspring sex. To test this hypothesis, multiparous Dorset ewes (n = 45) pregnant with twins were fed 100% (CON), 60% (RES), or 140% (OVER) of National Research Council requirements from d 30 of gestation until parturition. At parturition, all ewes were fed a control diet, such that the nutritional insult only occurred during gestation. At d 120, whole blood was collected from offspring and RNA was isolated to evaluate 86 genes related to the innate and adaptive immune responses. Data were analyzed in SAS 9.4 using PROC MIXED with fixed effects of offspring sex, maternal diet, and the interaction. Maternal diet affected 7 genes. Bovine major histocompatibility complex, a gene in the innate immune response, was downregulated by 27% and 37% in OVER compared with CON and RES, respectively (P = 0.001). Cluster of differentiation 86, a marker of T cell activation, was upregulated by 17% in OVER compared with CON (P = 0.04). Lymphocyte antigen 96, critical in the response to pathogens, expression was 48% greater in RES compared with CON (P = 0.04). A transcription factor in cytokine signaling, Signal transducer and activator of transcription 3 was upregulated in OVER and RES offspring by 19 and 21%, respectively, compared with CON (P = 0.02). Pattern recognition receptors Toll-like receptor-1 (TLR1), TLR4, and TLR8 were upregulated in RES and OVER compared with CON (P ≤ 0.04). The interaction of offspring sex and maternal diet significantly altered gene expression in 2 genes. A regulator of T cell differentiation, GATA Binding Protein 3 was downregulated in OVER females by 21 to 43% compared with all other offspring and was upregulated in OVER males by 26 to 76% compared with all female offspring (P = 0.04). A gene related to cytokine signaling, Janus Kinase 2 was upregulated in RES and OVER males by 29 to 64% compared with all other offspring (P = 0.05). Sex altered gene expression of 54 genes (P ≤ 0.05). Overall, maternal diet altered gene expression of the innate and adaptive immune responses in a sex-specific manner. Alterations in inflammatory status may contribute to altered body composition and may predispose offspring to impaired response to immune challenges. Future work should focus on the impacts of poor maternal nutrition during gestation on the response to an immune challenge.

Transcriptome profiling of Madin-Darby bovine kidney cells uncover differences in the susceptibility of cattle to Toxoplasma gondii and Neospora caninum

Toxoplasma gondii and Neospora caninum are two major apicomplexan protozoan parasites with heteroxenous life cycles and worldwide distributions. The transplacental transmission of N. caninum causes bovine abortion, which resulting in serious economic losses to the dairy industry. Although T. gondii was also reported to cause abortions in pregnant woman and small ruminants, scarce cases about the symptom to the host cattle and the causality remains unknown. In this study, transcriptome analysis of Madin Darby bovine kidney (MDBK) cells infected with T. gondii and N. caninum was performed to uncover the differences in susceptibility of cattle to the two parasites. The results showed that 256 and 2225 differentially expressed genes (DEGs) were detected in cells infected with N. caninum and T. gondii, respectively. Moreover, significant biological differences were revealed by the functional analysis including GO and KEGG enrichment. One serpin peptidase inhibitor (SEPRINA14), which is associated with immunosuppression during pregnancy, was found to significantly decrease in cells infected with N. caninum and increase in cells infected with T. gondii-infected cells. Pattern recognition receptors TLR3 and NOD2 were also significantly upregulated in N. caninum-infected MDBK cells, but not in T. gondii. They could induce an increased inflammatory response leading to severe tissue damage. In addition, the interleukin 12 receptor subunit beta 2 (IL12β2), which plays an essential role in Th1 and Th2 cell differentiation and inflammatory bowel disease, was also markedly upregulated in the N. caninum infected cells, which led to an imbalance in the Th1 and Th2 cells by promoting the Th1 cellular response. Altogether, our findings recognized a new understanding on the differences between T. gondii and N. caninum infection of MDBK cells, where SEPRINA14, TLR3, NOD2, and IL12β2 may be the key genes that affect the difference in susceptibility of cattle to T. gondii and N. caninum, especially in pregnant animals. This study provides more clues as to why N. caninum is more likely to cause abortions in cattle.

Quantifying the impact of immunotherapy on RNA dynamics in cancer

BackgroundCheckpoint inhibitor (CPI) immunotherapies have provided durable clinical responses across a range of solid tumor types for some patients with cancer. Nonetheless, response rates to CPI vary greatly between cancer...

Exploring Proteome of Crimean-Congo Hemorrhagic Fever Virus to Construct Multiepitope Based Subunit Vaccine: Molecular Docking with Immunoinformatic Framework

Purpose: Crimean-Congo hemorrhagic fever virus (CCHFV), a single-stranded RNA (ssRNA) virus that spreads via tick bites. For the treatment of CCHFV, there is still a lack of vaccine or any antiviral medicine. For this purpose, a study was performed to design a multiepitope based subunit vaccine (MESV) for effective prevention against CCHFV infection.&#x0D; Methodology: The study contains immunoinformatic and docking methodologies to obtain a MESV by choosing highly antigenic and overlapping epitopes comprising of 8 epitopes of both MHC class I and II from viral proteins. Epitopes were chosen which were conserved within the epitopes of IFN- gamma, T-cell and B-cell. Then these epitopes were joined to final peptide by GPGPG and AAY linkers. An adjuvant was added at N terminal of vaccine via EAAAK linker for the improvement of vaccine’s immunogenicity.&#x0D; Findings: Our final construct was comprised of 278 amino acids. To validate the vaccine’s immunogenicity and safety, its physiochemical properties, allergenicity as well as antigenicity were checked and it was predicted to be non-allergenic and antigenic. The construct was further analyzed by molecular docking within vaccine and TLR3 receptor to assure its molecular interaction and binding affinity. In the end, In-silico cloning was also carried out for ensuring its expression efficiency.&#x0D; Recommendations: Nonetheless, the designed construct is proposed to be tested in laboratory settings to confirm its immunogenicity and safety.

Azomethine functionalized platform for the selective detection of Zr(IV) ion, biological evaluation and potent TLR-4 inhibitor

In this study, a rapid-sensing material featuring azomethine functionalized Schiff base has been developed to address the pressing need for zirconium detection. The sensor demonstrates remarkable selectivity for zirconium (IV) ions, surpassing its performance with respect to other relevant metal ions. The elucidation of compound structures was accomplished through X-ray crystallography, unveiling a new approach to zirconium (IV) ion detection with a high binding constant of 8.01 × 104 M−1 and an exceptionally low limit of detection (LOD) of 31.5 nM. Moreover, the binding stoichiometry between Probe 4a and Zr(IV) was determined to be 1:1, as confirmed by both Job's plot analysis and spectroscopy analysis. Successful Zr(IV) ion detection in real samples was achieved using synthesized compound 4a. Furthermore, compound 4a exhibits notable antibacterial and antioxidant properties. Molecular docking analysis with the TLR-4 receptor protein supports the antibacterial efficacy of compound 4a, revealing a robust binding energy of -8.23 kcal mol−1.

The Yeast and Hypha Phases of Candida krusei Induce the Apoptosis of Bovine Mammary Epithelial Cells via Distinct Signaling Pathways.

Infection with Candida spp. is a significant cause of bovine mastitis globally. We previously found that C. krusei was the main pathogen causing mycotic mastitis in dairy cows in Yinchuan, Ningxia, China. However, whether the infection of this pathogen could induce apoptosis in BMECs remained unclear. In this report, we explored the apoptosis and underlying mechanism of BMECs induced by C. krusei yeast and hypha phases using a pathogen/host cell co-culture model. Our results revealed that both the yeast and hypha phases of C. krusei could induce BMEC apoptosis; however, the yeast phase induced more cell apoptosis than the hypha phase, as assessed via electronic microscopy and flow cytometry assays. This finding was further corroborated via the measurement of the mitochondrial membrane potential (MMP) and the TUNEL test. Infection by both the yeast and hypha phases of C. krusei greatly induced the expression of proteins associated with cell death pathways and important components of toll-like receptor (TLR) signaling, including TLR2 and TLR4 receptors, as determined via a Western blotting assay. BMECs mainly underwent apoptosis after infection by the C. krusei yeast phase through a mitochondrial pathway. Meanwhile, BMEC apoptosis induced by the C. krusei hypha phase was regulated by a death ligand/receptor pathway. In addition, C. krusei-induced BMEC apoptosis was regulated by both the TLR2/ERK and JNK/ERK signaling pathways. These data suggest that the yeast phase and hypha phase of C. krusei induce BMEC apoptosis through distinct cell signaling pathways. This study represents a unique perspective on the molecular processes underlying BMEC apoptosis in response to C. krusei infection.

Vitamin B1 via Nrf-2/TLR4 signaling pathway ameliorates scopolamine-induced memory dysfunction in adult mice

Alzheimer's disease (AD) is a progressive neurodegenerative disorder characterized by memory impairment and cognitive decline, with oxidative stress and neuroinflammation playing pivotal roles in its pathogenesis. Among potential candidates, vitamin B1 (Vit. B1) has gained attention for its neuroprotective potential due to anti-inflammatory properties and antioxidant properties. This study investigates the therapeutic potential of Vit. B1 in an AD-like mouse model induced by scopolamine (SCOP). Administering Vit. B1 (300 µg/kg) with SCOP (1 mg/kg) is hypothesized to alleviate memory impairment and neuroinflammation. Behavioral tests, including Morris Water Maze (MWM) and Y-maze tests, revealing Vit. B1 ability to restore both long-term and short-term memory, especially in the prob test compared to SCOP-treated mice. Western blot analysis revealed Vit. B1′s role in reversing SCOP-induced changes by suppressing oxidative stress through NrF-2 and HO-1 proteins and inhibiting TLR4 receptor activation that upregulates TNF-α and NF-kβ, contributing to memory dysfunction. Molecular docking studies explored the binding affinity of TLR4 signalling pathway proteins. Results confirmed VitB1′s inhibitory effect on TLR4/md2 activation and its higher binding preference, underscored by experimental confirmation. Overall, these findings highlight Vit. B1′s neuroprotective potential as a therapeutic approach in AD and suggest a potential avenue for novel therapeutic strategies, warranting further preclinical and clinical research.

Protection of lipopolysaccharide-induced otic injury by a single dose administration of a novel dexamethasone formulation

BackgroundThe blood-labyrinth barrier (BLB) separates the inner ear from the circulation and is critical for maintaining ionic homeostasis and limiting the entry of deleterious agents. BLB integrity is disrupted by bacterial lipopolysaccharide (LPS), which elicits a strong inflammatory response in the inner ear leading to irreversible otic damage. Prolonged administration of systemic corticosteroids is the available treatment, but it shows both limited efficacy and major adverse effects. SPT-2101 is a novel in situ-forming gel formulation of dexamethasone allowing slow and sustained drug release after single intratympanic administration.MethodsWe used a rat model of LPS-induced injury to define the functional, cellular and molecular mechanisms associated to BLB dysfunction and the protection by SPT-2101. Hearing was assessed by auditory brainstem response (ABR) recording, BLB permeability by gadolinium dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) and Evans blue extravasation. Gross cochlear histology and cellular alterations were studied by hematoxylin-eosin staining and immunofluorescence. RT-qPCR, PCR array and western blotting were used to assess transcriptional and protein changes.ResultsLPS-challenged rats showed BLB breakdown and altered permeability as shown by the progressive increase in cochlear gadolinium uptake and Evans blue incorporation. LPS administration increased the cochlear expression of the LPS toll-like receptors Tlr2 and co-receptor Cd14, pro-inflammatory cytokines and receptors such as Il1b and ll1r1, and also the oxidative stress and inflammasome mediators NRF2 and NLRP3. LPS also increased IBA1-positive macrophage infiltration in the lateral wall and spiral ganglion. A single intratympanic injection of SPT-2101 protected BLB integrity and prevented otic injury. Comparable effects were obtained by repeated administration of systemic dexamethasone, but not by a single dose. SPT-2101 administration normalized molecular inflammatory mediators and suppressed macrophage infiltration.ConclusionsOur data indicate that single local administration of dexamethasone formulated as SPT-2101 protects BLB functional integrity during endotoxemia, providing a novel therapeutic opportunity to treat diseases related to BLB dysfunction.

Genetic mechanisms of bacteria recognition by cattle innate immunity receptors

The research identified a list of molecules involved in the mechanisms of innate immunity in cattle and the recognition of bacterial pathogens. The current list of molecular receptors has expanded to include TLR receptors and the recently defined NOD-like receptors (NLRs): NOD, NALP, NAIP, and IPAF. TLR molecules are designed to transmit a ligand-binding signal on the cell surface or endosome and activate specific molecules of bacterial origin in the cytosol, such as peptidoglycans, RNA, toxins and flagellins. The obtained data on the molecular structure of TLR and NLR receptors indicate their anti-inflammatory role, mediated by signals through nuclear transcription factor κB and activation of caspase-1 in the inflammasome. It has been shown that the role of immunosensors of extracellular and intracellular perception of bacteria in regulating inflammation is synergistic. Mutations in TLR and NOD receptors are associated with autoimmune inflammatory syndromes. This review examines the body's ways of recognising intracellular pathogens, describes the problem of their mimicry from the animal immune system, and the molecular mechanisms of such interactions. Variants of molecular interactions of innate immune receptors with peptidoglycans, bacterial DNA and toxins, cell wall compartments, and bacterial flagellin receptors are also considered. This study aimed to analyse the current understanding of the genetic and molecular structure of the immune response to bacterial environmental factors and the mechanisms and characteristics of the reaction of the animal body.

Use of single-cell technology to improve our understanding of the role of TLR2 in macrophage-Mycobacterium tuberculosis interaction.

The interaction between Mycobacterium tuberculosis, the agent of tuberculosis (TB), and its host cell, the macrophage, is multifaceted, dynamic, and involves multiple molecular partners. A better understanding of this interaction could help researchers manipulate the immune system in order to design host-targeted immunotherapies and/or develop a novel vaccine protecting better adults against TB. Jani and coworkers studied the role of the macrophage receptor TLR2 in the response to M. tuberculosis using single-cell technologies (C. Jani, S. L. Solomon, J. M. Peters, and S. C. Pringle, et al., mSystems, https://doi.org/10.1128/msystems.00052-23, 2023). This work addresses the multiple challenges associated with such studies and shows how informative single-cell analysis can be for the study of heterogeneous and complex host-pathogen interactions.

A-246 Desialylation Profiles of Monocytes and Macrophages Upon LPS Stimulation

Abstract Background Cell surface expresses a dense layer of glycans terminated with sialic acids (Sias), known as sialylation, which often represent different cellular statuses. Meanwhile, the removal of Sias from the glycans catalyzed by sialidase (desialylation) is also involved in many biological processes. Previous studies have established that lipopolysaccharide (LPS) can induce the expression of endogenous sialidases in monocytes and macrophages, leading to desialylation of the LPS receptor TLR4. This desialylation of TLR4 has been shown to impact the LPS/TLR4 signaling pathway. In this study, we conduct a profiling of desialylation in THP-1 monocytes and THP-1 macrophages in response to LPS stimulation. This will provide insights on the intricate connection between sialylation, desialylation, and LPS-induced signaling. Methods The activity of sialidases was assessed by utilizing 4-methylumbelliferyl-N-acetylneuraminic acid (4-MU-NANA) and ganglioside GM3 as substrates. The total sialic acid in the THP1 was quantified using liquid chromatography tandem mass spectrometry (LC-MS/MS). Flow cytometry was used to analyze the exposure of galactose on THP-1 monocytes and macrophages after LPS stimulation (100 ng/mL LPS for 24 h), using PNA-FITC staining. The expression level of Neu1 and Neu3 sialidases in cell lysates of THP-1 monocytes and macrophages were assessed by western blot technique after LPS stimulation. ELISA was used to measure the levels of sialidases in the cell supernatant of THP-1 monocytes and macrophages upon LPS stimulation. Result We found a significant reduction in total sialic acid levels in both THP-1 monocytes and macrophages upon LPS stimulation. This was further confirmed by the significant increase in total sialidase activities which measured by both 4-MU-Neu5Ac and GM3 substrates in THP-1 monocytes and macrophages upon LPS stimulation. Surprisingly, we also found that sialidase activities significantly increased in the cell supernatant of both THP-1 monocytes and macrophages, indicting sialidases secretion upon LPS stimulation. Western blot analysis further confirmed the high expression and secretion of both Neu1 and Neu3 sialidases from THP-1 monocytes and macrophages after LPS stimulation. Conclusion The present study conducted the first systematic profiling of desialylation and sialidase expression in monocytes and macrophages upon LPS stimulation. This results demonstrate that endogenous sialidase expression and secretion play a role in the LPS/TLR4 signaling pathway and subsequent cellular processes and functions in the context of inflammation and immune response.

SNASP Mutation Aggravates to the TLR4-Mediated Inflammation in SLE by TAK1 Pathway.

Genetic factors play an important role in the pathogenesis of systemic lupus erythematosus (SLE), and abnormal Toll-like receptor (TLR) signaling pathways are closely related to the onset of SLE. In previous studies, we found that the mutant somatic nuclear autoantigenic sperm protein (sNASP) gene in the mouse lupus susceptibility locus Sle2 can promote the development of lupus model mice, but the mechanism is still unclear. Here, we stimulated mouse peritoneal macrophages with different concentrations of lipopolysaccharide. The results showed that sNASP gene mutations can promote the response of the TLR4-TAK1 signaling pathway but have no significant effect on the TLR4-TBK1 signaling pathway. sNASP mutations enhanced TLR4-mediated nuclear factor-κ-gene binding and mitogen-activated protein kinase activation and IL-6, tumor necrosis factor secretion in murine peritoneal macrophages. Collectively, our study revealed the impact of sNASP gene mutation on the sensitivity of TLR4 receptors in mouse peritoneal macrophages and shed light on potential mechanisms underlying inflammation in autoimmune diseases.

Amphipathic Helical Peptide L37pA Protects against Lung Vascular Endothelial Dysfunction Caused by Truncated Oxidized Phospholipids via Antagonism with CD36 Receptor.

The generation of bioactive truncated oxidized phospholipids (Tr-OxPLs) from oxidation of cell-membrane or circulating lipoproteins is a common feature of various pathological states. Scavenger receptor CD36 is involved in lipid transport and acts as a receptor for Tr-OxPLs. Interestingly, Tr-OxPLs and CD36 are involved in endothelial dysfunction-derived acute lung injury, but the precise mechanistic connections remain unexplored. In the present study, we investigated the role of CD36 in mediating pulmonary endothelial cell (EC) dysfunction caused by Tr-OxPLs. Our results demonstrated that the Tr-OxPLs KOdia-PC, Paz-PC, PGPC, PON-PC, POV-PC, and lysophosphocholine caused an acute EC barrier disruption as revealed by measurements of transendothelial electrical resistance and VE-cadherin immunostaining. More importantly, a synthetic amphipathic helical peptide, L37pA, targeting human CD36 strongly attenuated Tr-OxPL-induced EC permeability. L37pA also suppressed Tr-OxPL-induced endothelial inflammatory activation monitored by mRNA expression of inflammatory cytokines/chemokines and adhesion molecules. In addition, L37pA blocked Tr-OxPL-induced NF-κB activation and tyrosine phosphorylation of Src kinase and VE-cadherin. The Src inhibitor SU6656 attenuated KOdia-PC-induced EC permeability and inflammation, but inhibition of the Toll-like receptors (TLRs) TLR1, TLR2, TLR4, and TLR6 had no such protective effects. CD36-knockout mice were more resistant to Tr-OxPL-induced lung injury. Treatment with L37pA was equally effective in ameliorating Tr-OxPL-induced vascular leak and lung inflammation as determined by an Evans blue extravasation assay and total cell and protein content in BAL fluid. Altogether, these results demonstrate an essential role of CD36 in mediating Tr-OxPL-induced EC dysfunction and suggest a strong therapeutic potential of CD36 inhibitory peptides in mitigating lung injury and inflammation.

Identification of conserved immunogenic peptides of SARS-CoV-2 nucleocapsid protein

The emergence of the new SARS-CoV-2 variants has led to major concern regarding the efficacy of approved vaccines. Nucleocapsid is a conserved structural protein essential for replication of the virus. This study focuses on identifying conserved epitopes on the nucleocapsid (N) protein of SARS-CoV-2. Using 510 unique amino acid sequences of SARS-CoV-2 N protein, two peptides (193 and 215 aa) with 90% conservancy were selected for T cell epitope prediction. Three immunogenic peptides containing multiple T cell epitopes were identified which were devoid of autoimmune and allergic immune response. These peptides were also conserved (100%) in recent Omicron variants reported in Jan-August 2023. HLA analysis reveals that these peptides are predicted as binding to large number of HLA alleles and 71–90% population coverage in six continents. Identified peptides displayed good binding score with both HLA class I and HLA class II molecules in the docking study. Also, a vaccine construct docked with TLR-4 receptor displays strong interaction with 20 hydrogen bonds and molecular simulation analysis reveals that docked complex are stable. Additionally, the immunogenicity of these N protein peptides was confirmed using SARS-CoV-2 convalescent serum samples. We conclude that the identified N protein peptides contain highly conserved and antigenic epitopes which could be used as a target for the future vaccine development against SARS-CoV-2. Communicated by Ramaswamy H. Sarma