Activation Of Toll-like Receptor Research Articles

The immunostimulatory activity of sea spray aerosols: bacteria and endotoxins activate TLR4, TLR2/6, NF-κB and IRF in human cells

Frequent exposure to sea spray aerosols (SSA) containing marine microorganisms and bioactive compounds may influence human health. However, little is known about potential immunostimulation by SSA exposure. This study focuses on the effects of marine bacteria and endotoxins in SSA on several receptors and transcription factors known to play a key role in the human innate immune system. SSA samples were collected in the field (Ostend, Belgium) or generated in the lab using a marine aerosol reference tank (MART). Samples were characterized by their sodium contents, total bacterial counts, and endotoxin concentrations. Human reporter cells were exposed to SSA to investigate the activation of toll-like receptor 4 (TLR4) in HEK-Blue hTLR4 cells and TLR2/6 in HEK-Blue hTLR2/6 cells, as well as the activation of nuclear factor kappa B (NF-κB) and interferon regulatory factors (IRF) in THP1-Dual monocytes. These responses were then correlated to the total bacterial counts and endotoxin concentrations to explore dose-effect relationships. Field SSA contained from 3.0 × 103 to 6.0 × 105 bacteria/m3 air (averaging 2.0 ± 1.9 × 105 bacteria/m3 air) and an endotoxin concentration ranging from 7 to 1217 EU/m3 air (averaging 389 ± 434 EU/m3 air). In contrast, MART SSA exhibited elevated levels of total bacterial count (from 2.0 × 105 to 2.4 × 106, averaging 7.3 ± 5.5 × 105 cells/m3 air) and endotoxin concentration from 536 to 2191 (averaging 1310 ± 513 EU/m3 air). SSA samples differentially activated TLR4, TLR2/6, NF-κB and IRF. These immune responses correlated dose-dependently with the total bacterial counts, endotoxin levels, or both. This study sheds light on the immunostimulatory potential of SSA and its underlying mechanisms, highlighting the need for further research to deepen our understanding of the health implications of SSA exposure.

Effect of chronic cold stress on gut microbial diversity, intestinal inflammation and pyroptosis in mice.

Hypothermia is an essential environmental factor in gastrointestinal diseases, but the main molecular mechanisms of pathogenesis remain unclear. The current study sought to better understand how chronic cold stress affects gut damage and its underlying mechanisms. In this work, to establish chronic cold stress (CS)-induced intestinal injury model, mice were subjected to continuous cold exposure (4°C) for 3h per day for 3weeks. Our results indicated that CS led to gut injury via inducing changes of heat shock proteins 70 (HSP70) and apoptosis-related (caspases-3, Bax and Bcl-2) proteins; enhancing expression of intestinal tight-related (ZO-1 and occludin) proteins; promoting releases of inducible nitric oxide synthase (iNOS), tumor necrosis factor-α (TNF-α), cyclooxygenase-2 (COX-2), high mobility group box 1 (HMGB1), interleukin1β (IL-1β), IL-18 and IL-6 inflammatory mediators in the ileum; and altering gut microbial diversity. Furthermore, persistent cold exposure resulted in the cleavage of pyroptosis-related Gasdermin D (GSDMD) protein by regulating the NLRP3/ASC/caspase-1 and caspase-11 pathway, and activation of toll-like receptor 4 (TLR4)/myeloid differentiation factor 88 (MyD88)-mediated nuclear factor kappa B (NF-κB) and mitogen-activated protein kinase (MAPK) signaling pathways, which are strongly associated with changes in gut microbiota diversity. Taken together, these investigations provide new insights into the increased risk of intestinal disorders at extremely low temperatures and establish a theoretical foundation for the advancement of novel pharmaceutical interventions targeting cold-related ailments.

SARS-CoV-2 envelope protein impairs airway epithelial barrier function and exacerbates airway inflammation via increased intracellular Cl− concentration

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection disrupts the epithelial barrier and triggers airway inflammation. The envelope (E) protein, a core virulence structural component of coronaviruses, may play a role in this process. Pathogens could interfere with transepithelial Cl− transport via impairment of the cystic fibrosis transmembrane conductance regulator (CFTR), which modulates nuclear factor κB (NF-κB) signaling. However, the pathological effects of SARS-CoV-2 E protein on airway epithelial barrier function, Cl− transport and the robust inflammatory response remain to be elucidated. Here, we have demonstrated that E protein down-regulated the expression of tight junctional proteins, leading to the disruption of the airway epithelial barrier. In addition, E protein triggered the activation of Toll-like receptor (TLR) 2/4 and downstream c-Jun N-terminal kinase (JNK) signaling, resulting in an increased intracellular Cl− concentration ([Cl−]i) via up-regulating phosphodiesterase 4D (PDE4D) expression in airway epithelial cells. This elevated [Cl−]i contributed to the heightened airway inflammation through promoting the phosphorylation of serum/glucocorticoid regulated kinase 1 (SGK1). Moreover, blockade of SGK1 or PDE4 alleviated the robust inflammatory response induced by E protein. Overall, these findings provide novel insights into the pathogenic role of SARS-CoV-2 E protein in airway epithelial damage and the ongoing airway inflammation during SARS-CoV-2 infection.

Abstract 6743: Identifying new cellular adjuvants for cancer vaccines and immunotherapies

Abstract Background: Cancer vaccines represent a promising strategy for antitumor immunotherapy. Despite considerable research efforts and progress, the effectiveness of cancer vaccines remains a challenge. This is primarily due to relatively low immune responses against tumors, stemming from issues of poor immunogenicity and the immunosuppressive microenvironment commonly found in many solid tumors. Use of an optimal adjuvant can augment immune responses against tumor antigens and guide them towards a specific adaptive immune enhancement. Dendritic cells (DCs) play a primary role in initiating immunes response to vaccines, often through activation of toll-like receptors (TLRs). Identifying novel immunomodulatory proteins that regulate DCs or TLR activation can identify novel adjuvant candidates with applications for cancer immunotherapy. Methods: We constructed a Membrane Proteome Array (MPA) encompassing ~6,000 structurally intact human membrane proteins in their native conformation (94% of the entire membrane proteome) and used it to screen for cellular proteins that can modulate response to vaccination through DCs and TLRs. To identify proteins that interact with TLRs, the library was expressed in live TLR reporter cells, and these cells were tested for response to known activators. In addition, cells expressing the MPA were presented to DCs to identify membrane proteins that modulate DC activity. TLR-expressing cell activation was measuring using an NF-kB-activated luciferase reporter, while DC assays used upregulation of cell surface markers (CD80, CD83, and MHCII) to measure the activation of immature monocyte-derived dendritic cells. Results: Screening of TLR2, TLR3, and TLR5 reporter cell lines identified both known and novel regulators of TLR activation. Activators common to several TLRs were identified as well as activators unique to individual TLRs. Screening of primary human DCs likewise identified known regulators of activation (for example CD40L) and several novel targets. Putative novel regulators of DC activation are being further investigated to determine their biological significance. Conclusion: Discovering novel immunomodulatory targets and proteins that regulate DCs and TLR activation can identify novel adjuvant candidates with targeted immune outcomes, such as immune stimulating and anti-cancer responses. This study identified novel regulators of immune activation which are being further investigated as adjuvant candidates for cancer vaccines. Citation Format: Joseph Rucker, Nnenna Nwogu, Nicholas M. Molino, Ami Snyder, Jonathan T. Sullivan, Benjamin J. Doranz. Identifying new cellular adjuvants for cancer vaccines and immunotherapies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6743.

TLR3 Agonist Amplifies the Anti-Inflammatory Potency of ADSCs via IL-10-Mediated Macrophage Polarization in Acute Pancreatitis.

The immunoregulatory role of mesenchymal stem cells (MSCs) in inflammation is heterogeneous and can exhibit anti-inflammatory or proinflammatory properties depending on the microenvironment. We herein observed that the activation of Toll-like receptor 3 (TLR3) by polyinosinic : polycytidylic acid (poly(I : C)) stimulation facilitated the transformation of adipose-derived stem cells (ADSCs) into an anti-inflammatory phenotype. The enhanced anti-inflammatory properties were assessed in a taurocholate-induced pancreatitis model. The results demonstrated that poly(I : C) pretreated ADSCs exhibited enhanced anti-inflammatory properties than untreated ADSCs in taurocholate-induced pancreatitis. Mechanistically, poly(I : C)-treated ADSCs showed increased production and secretion of interleukin-10 (IL-10), which demonstrates a potent ability to alleviate inflammatory signaling cascades in acinar cells. Simultaneously, the heightened anti-inflammatory effects of poly(I : C)-treated ADSCs in pancreatitis were associated with the regulation of macrophage classical/alternative transformation, thereby mitigating inflammatory factor-mediated damage to the pancreatic acinar cell. We propose that TLR3 activation by poly(I : C) is an effective strategy to enhance the anti-inflammatory properties of MSCs, which offers a valuable consideration for improving the therapeutic efficacy of MSCs in inflammatory diseases.

Monocyte-Mediated Thrombosis Linked to Circulating Tissue Factor and Immune Paralysis in COVID-19.

SARS-CoV-2 infections cause COVID-19 and are associated with inflammation, coagulopathy, and high incidence of thrombosis. Myeloid cells help coordinate the initial immune response in COVID-19. Although we appreciate that myeloid cells lie at the nexus of inflammation and thrombosis, the mechanisms that unite the two in COVID-19 remain largely unknown. In this study, we used systems biology approaches including proteomics, transcriptomics, and mass cytometry to define the circulating proteome and circulating immune cell phenotypes in subjects with COVID-19. In a cohort of subjects with COVID-19 (n=35), circulating markers of inflammation (CCL23 [C-C motif chemokine ligand 23] and IL [interleukin]-6) and vascular dysfunction (ACE2 [angiotensin-converting enzyme 2] and TF [tissue factor]) were elevated in subjects with severe compared with mild COVID-19. Additionally, although the total white blood cell counts were similar between COVID-19 groups, CD14+ (cluster of differentiation) monocytes from subjects with severe COVID-19 expressed more TF. At baseline, transcriptomics demonstrated increased IL-6, CCL3, ACOD1 (aconitate decarboxylase 1), C5AR1 (complement component 5a receptor), C5AR2, and TF in subjects with severe COVID-19 compared with controls. Using stress transcriptomics, we found that circulating immune cells from subjects with severe COVID-19 had evidence of profound immune paralysis with greatly reduced transcriptional activation and release of inflammatory markers in response to TLR (Toll-like receptor) activation. Finally, sera from subjects with severe (but not mild) COVID-19 activated human monocytes and induced TF expression. Taken together, these observations further elucidate the pathological mechanisms that underlie immune dysfunction and coagulation abnormalities in COVID-19, contributing to our growing understanding of SARS-CoV-2 infections that could also be leveraged to develop novel diagnostic and therapeutic strategies.

Knockdown of Tlr3 in dorsal striatum reduces ethanol consumption and acute functional tolerance in male mice

Systemic activation of toll-like receptor 3 (TLR3) signaling using poly(I:C), a TLR3 agonist, drives ethanol consumption in several rodent models, while global knockout of Tlr3 reduces drinking in C57BL/6J male mice. To determine if brain TLR3 pathways are involved in drinking behavior, we used CRISPR/Cas9 genome editing to generate a Tlr3 floxed (Tlr3F/F) mouse line. After sequence confirmation and functional validation of Tlr3 brain transcripts, we injected Tlr3F/F male mice with an adeno-associated virus expressing Cre recombinase (AAV5-CMV-Cre-GFP) to knockdown Tlr3 in the medial prefrontal cortex, nucleus accumbens, or dorsal striatum (DS). Only Tlr3 knockdown in the DS decreased two-bottle choice, every-other-day (2BC-EOD) ethanol consumption. DS-specific deletion of Tlr3 also increased intoxication and prevented acute functional tolerance to ethanol. In contrast, poly(I:C)-induced activation of TLR3 signaling decreased intoxication in male C57BL/6J mice, consistent with its ability to increase 2BC-EOD ethanol consumption in these mice. We also found that TLR3 was highly colocalized with DS neurons. AAV5-Cre transfection occurred predominantly in neurons, but there was minimal transfection in astrocytes and microglia. Collectively, our previous and current studies show that activating or inhibiting TLR3 signaling produces opposite effects on acute responses to ethanol and on ethanol consumption. While previous studies, however, used global knockout or systemic TLR3 activation (which alter peripheral and brain innate immune responses), the current results provide new evidence that brain TLR3 signaling regulates ethanol drinking. We propose that activation of TLR3 signaling in DS neurons increases ethanol consumption and that a striatal TLR3 pathway is a potential target to reduce excessive drinking.

Inflammation in amebiasis and its modulation by the Autonomic Nervous System

Neuroimmune regulation of inflammation is only beginning to be explored in depth. In the case of amebiasis, the inflammatory process prompted by E. histolytica has been well characterized, but the modulation of this process by neurotransmitters is not as well defined. Regarding the inflammatory process, it is known that neutrophils and macrophages are important cellular effectors in the host defense against E. histolytica. The production of proinflammatory cytokines (IL-1β, IL-8, INF-γ and TNF-α) and anti- inflammatory cytokines (IL-10 and TGF-β) is triggered by the activation of Toll-Like Receptors and two types of transcription factors: 1) nuclear factor kappa-light-chain- enhancer of activated B cells, and 2) activator of transcriptions: Toll-Like Receptors and these transcription factors are in turn modulated by the action of three neurotransmitters of the autonomous nervous system acetylcholine, adrenaline and noradrenaline that can activate cholinergic and adrenergic receptors of immune cells, and thus modify the inflammatory process induced by the parasite. A vagotomy of the liver of hamsters inoculated with E. histolytica induces a stronger response in tissues through a more abundant formation of collagen, together with an increase in the levels of IL-10 and INF- γ and a decrease in macrophages, which impedes the dissemination of trophozoites.

Impact of Toll-Like Receptors (TLRs) and TLR Signaling Proteins in Trigeminal Ganglia Impairing Herpes Simplex Virus 1 (HSV-1) Progression to Encephalitis: Insights from Mouse Models.

Herpes simplex virus 1 (HSV-1) or simplexvirus humanalpha 1 is a neurotropic virus that is responsible for orofacial infections in humans. More than 70% of the world's population may have seropositivity for HSV-1, and this virus is a leading cause of sporadic lethal encephalitis in humans. The role of toll-like receptors (TLRs) in defending against HSV-1 infection has been explored, including the consequences of lacking these receptors or other proteins in the TLR pathway. Cell and mouse models have been used to study the importance of these receptors in combating HSV-1, how they relate to the innate immune response, and how they participate in the orchestration of the adaptive immune response. Myeloid differentiation factor 88 (MyD88) is a protein involved in the downstream activation of TLRs and plays a crucial role in this signaling. Mice with functional MyD88 or TLR2 and TLR9 can survive HSV-1 infection. However, they can develop encephalitis and face a 100% mortality rate in a dose-dependent manner when MyD88 or TLR2 plus TLR9 proteins are non-functional. In TLR2/9 knockout mice, an increase in chemokines and decreases in nitric oxide (NO), interferon (IFN) gamma, and interleukin 1 (IL-1) levels in the trigeminal ganglia (TG) have been correlated with mortality.

Functional 2D Nanoplatforms Alleviate Eosinophilic Chronic Rhinosinusitis by Modulating Eosinophil Extracellular Trap Formation.

The therapeutic outcomes of patients with eosinophilic chronic rhinosinusitis (ECRS) remain unsatisfactory, largely because the underlying mechanisms of eosinophilic inflammation are uncertain. Here, it is shown that the nasal secretions of ECRS patients have high eosinophil extracellular trap (EET) and cell-free DNA (cfDNA) levels. Moreover, the cfDNA induced EET formation by activating toll-like receptor 9 (TLR9) signaling. After demonstrating that DNase I reduced eosinophilic inflammation by modulating EET formation, linear polyglycerol-amine (LPGA)-coated TiS2 nanosheets (TLPGA) as functional 2D nanoplatforms with low cytotoxicity, mild protein adsorption, and increased degradation rate is developed. Due to the more flexible linear architecture, TLPGA exhibited higher cfDNA affinity than the TiS2 nanosheets coated with dendritic polyglycerol-amine (TDPGA). TLPGA reduced cfDNA levels in the nasal secretions of ECRS patients while suppressing cfDNA-induced TLR9 activation and EET formation in vitro. TLPGA displayed exceptional biocompatibility, preferential nasal localization, and potent inflammation modulation in mice with eosinophilic inflammation. These results highlight the pivotal feature of the linear molecular architecture and 2D sheet-like nanostructure in the development of anti-inflammation nanoplatforms, which can be exploited for ECRS treatment.

Piperine attenuates hepatic ischemia/reperfusion injury via suppressing the TLR4 signaling cascade in mice

Piperine, the major active substance in black pepper, has been shown to have anti-inflammatory and antioxidant effects in several ischemic diseases. However, the role of piperine in hepatic ischemia/reperfusion injury (HIRI) and its underlying mechanisms remain unclear. In this study, the mice were administered piperine (30 mg/kg) intragastric administration before surgery. After 24 h of hepatic ischemia-reperfusion, liver histopathological evaluation, serum transaminase measurements, and TUNEL analysis were performed. The infiltration of inflammatory cells and production of inflammatory mediators in the liver tissue were determined by immunofluorescence and immunohistochemical staining. The protein levels of toll-like receptor 4 (TLR4) and related proteins such as nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), interleukin-1 receptor-associated kinase 1 (IRAK1), p65, and p38 were detected by western blotting. The results showed that plasma aminotransferase (ALT), aspartate aminotransferase (AST), hepatocyte apoptosis, oxidative stress, and inflammatory cell infiltration significantly increased in HIRI mice. Piperine pretreatment notably repaired liver function, improved the histopathology and apoptosis of liver cells, alleviated oxidative stress injury, and reduced inflammatory cell infiltration. Further analysis showed that piperine attenuated tumor necrosis factor-a (TNF-α) and interleukin 6 (IL-6) production and reduced TLR4 activation and phosphorylation of IRAK1, p38, and NF-κB in HIRI. Piperine has a protective effect against HIRI through the TLR4/IRAK1/NF-κB signaling pathway and may be a safer option for future clinical treatment and prevention of ischemia-related diseases.

RHBDL4-triggered downregulation of COPII adaptor protein TMED7 suppresses TLR4-mediated inflammatory signaling

The toll-like receptor 4 (TLR4) is a central regulator of innate immunity that primarily recognizes bacterial lipopolysaccharide cell wall constituents to trigger cytokine secretion. We identify the intramembrane protease RHBDL4 as a negative regulator of TLR4 signaling. We show that RHBDL4 triggers degradation of TLR4’s trafficking factor TMED7. This counteracts TLR4 transport to the cell surface. Notably, TLR4 activation mediates transcriptional upregulation of RHBDL4 thereby inducing a negative feedback loop to reduce TLR4 trafficking to the plasma membrane. This secretory cargo tuning mechanism prevents the over-activation of TLR4-dependent signaling in an in vitro Mycobacterium tuberculosis macrophage infection model and consequently alleviates septic shock in a mouse model. A hypomorphic RHBDL4 mutation linked to Kawasaki syndrome, an ill-defined inflammatory disorder in children, further supports the pathophysiological relevance of our findings. In this work, we identify an RHBDL4-mediated axis that acts as a rheostat to prevent over-activation of the TLR4 pathway.

SPINK5 inhibits esophageal squamous cell carcinoma metastasis via immune activity.

Esophageal squamous cell carcinoma (ESCC) is a predominant subtype of esophageal cancer with relatively high mortality worldwide. Serine peptidase inhibitor Kazal-type 5 (SPINK5) is reported to be downregulated in ESCC. However, its explicit role in ESCC remains further investigation. The tumor tissues and adjacent non-cancerous tissues were obtained from 196 patients with ESCC for the determination of SPINK5 mRNA levels. Additionally, the relationship between SPINK5 mRNA levels and clinicopathological features of ESCC patients was explored. The effects of SPINK5 on the invasion and migration of ESCC cells were assessed using Transwell assays. Furthermore, SPINK5 mRNA and LEKTI protein were measured in ESCC cell lines after treatment with poly (I:C), lipopolysaccharide (LPS) or unmethylated CpG DNA. Moreover, the correlation between expression of SPINK5 and nuclear factor-kappa B (NF-κB) signaling pathway-related genes was analyzed in the TCGA-ESCC cohort, and the effects of SPINK5 on NF-κB transcription was analyzed using a luciferase reporter gene assay. Finally, the correlations between SPINK5 and infiltration of immune cells, immune scores, stromal scores and ESTIMATE (i.e., Estimation of STromal and Immune cells in MAlignant Tumor tissues using Expression data) scores were explored. SPINK5 mRNA levels were downregulated in tumor tissues, which was significantly correlated with higher lymph node metastases. Overexpressed SPINK5 inhibited cell invasion and migration in ESCC cell lines. Mechanistically, LPS-induced activation of Toll-like receptor 4 (TLR4) decreased SPINK5 mRNA and LEKTI in KYSE150 and KYSE70 cells. Spearman correlation analysis revealed that SPINK5 mRNA was significantly negatively correlated with a total of seven NF-κB signaling pathway-related genes in TCGA-ESCC patients. Moreover, downregulation of SPINK5 increased and upregulation of SPINK5 decreased the activity of the NF-κB promoter in HEK293T cells. Finally, immune cells infiltration analysis revealed that SPINK5 was significantly correlated with the infiltration of various immune cells, stromal scores, immune scores and ESTIMATE scores. SPINK5 plays critical roles in the TLR4/NF-κB pathway and immune cells infiltration, which might contribute to the ESCC metastasis. The findings of the present study may provide a promising biomarker for the diagnosis and treatment of esophageal squamous cell carcinoma.

Atherosclerosis and Toll-Like Receptor4 (TLR4), Lectin-Like Oxidized Low-Density Lipoprotein-1 (LOX-1), and Proprotein Convertase Subtilisin/Kexin Type9 (PCSK9).

Atherosclerosis is a leading cause of death in the world. A significant body of evidence suggests that inflammation and various players are implicated and have pivotal roles in the formation of atherosclerotic plaques. Toll-like receptor 4 (TLR4) is linked with different stages of atherosclerosis. This receptor is highly expressed in the endothelial cells (ECs) and atherosclerotic plaques. TLR4 activation can lead to the production of inflammatory cytokines and related responses. Lectin-like oxidized low-density lipoprotein-1 (LOX-1), an integral membrane glycoprotein with widespread expression on the ECs, is involved in atherosclerosis and has some common pathways with TLR4 in atherosclerotic lesions. In addition, proprotein convertase subtilisin/kexin type9 (PCSK9), which is a regulatory enzyme with different roles in cholesterol uptake, is implicated in atherosclerosis. At present, TLR4, PCSK9, and LOX-1 are increasingly acknowledged as key players in the pathogenesis of atherosclerotic cardiovascular diseases. Herein, we presented the current evidence on the structure, functions, and roles of TLR4, PCSK9, and LOX-1 in atherosclerosis.

MiR-154-5p Is a Novel Endogenous Ligand for TLR7 Inducing Microglial Activation and Neuronal Injury.

Toll-like receptors (TLRs) are a collection of pattern recognition sensors that form a first line of defence by detecting pathogen- or damage-associated molecular patterns and initiating an inflammatory response. TLR activation in microglia, the major immune cells in the brain, can trigger the release of inflammatory molecules, which may contribute to various CNS diseases including Alzheimer's disease. Recently, some microRNAs were shown to serve as signalling molecules for TLRs. Here, we present miR-154-5p as a novel TLR7 ligand. Exposing microglia to miR-154-5p results in cytokine release and alters expression of the TLR signalling pathway dependent on TLR7. Additionally, miR-154-5p causes neuronal injury in enriched cortical neuron cultures and additive toxicity in the presence of microglia. Finally, intrathecal injection of miR-154-5p into mice leads to neuronal injury and accumulation of microglia in the cerebral cortex dependent on TLR7 expression. In conclusion, this study establishes miR-154-5p as a direct activator of TLR7 that can cause neuroinflammation and neuronal injury, which may contribute to CNS disease.

Biomimetic virus-like particles with magnetic core. From bioactivity to an immunodiagnostic tool

In this study, biomimetic virus-like particles (VLPs) composed of superparamagnetic iron oxide nanoparticles (SPIONs) and receptor binding domain (RBD) of the SARS-CoV-2 virus were obtained. The bioactivity of the VLPs was subsequently tested, utilizing natural targets of the RBD protein: angiotensin-converting enzyme 2 (ACE2), toll-like receptor 4 (TLR4), and neutralizing anti-SARS-CoV-2 antibodies. The conducted analyses include both in vitro and in cellulo methods such as dynamic light scattering (DLS), ζ-potential measurement, bio-layer interferometry (BLI), cytotoxicity study, stimulation of the TLR4 activation pathway, spectral flow cytometry, and imaging flow cytometry. The data confirmed the bioactivity of the obtained VLPs, which were subsequently utilized for a proof-of-concept diagnostic tool for the detection of specific anti-SARS-CoV-2 antibodies. The method was proven sensitive and effective, with significantly decreased protocol time compared to commercially available ELISA tests.

Toll-like receptor 3 signaling drives enteric glial cells against dextran sulfate sodium-induced colitis in mice.

The activation of toll-like receptor 3 (TLR3) has been reported to attenuate astrocytes injury in central nervous system, but its effect on enteric glial cells (EGCs) remains unknown. Here, we confirmed that the residence of EGCs was regulated by TLR3 agonist (polyinosinic-polycytidylic acid, PIC) or TLR3/dsRNA complex inhibitor in dextran sulfate sodium (DSS)-induced mice. In vitro, TLR3 signaling prevented apoptosis in EGCs and drove the secretion of EGCs-derived glial cell line-derived neurotrophic factor, 15-hydroxyeicosatetraenoic acid and S-nitrosoglutathione. PIC preconditioning enhanced the protective effects of EGCs against the dysfunction of intestinal epithelial barrier and the development of colitis in DSS-induced mice. Interestingly, PIC stimulation also promoted the effects of EGCs on converting macrophages to an M2-like phenotype and regulating the levels of inflammatory cytokines, including IL-1β, TNF-α and IL-10, in DSS-induced mice. These findings imply that TLR3 signaling in EGCs may provide a potential target for the prevention and treatment of colitis.

Trophic transfer of micro- and nanoplastics and toxicity induced by long-term exposure of nanoplastics along the rotifer (Brachionus plicatilis)-marine medaka (Oryzias melastigma) food chain

Microplastics (MPs) and nanoplastics (NPs) are emerging pollutants in the ocean, but their transfer and toxicity along the food chains are unclear. In this study, a marine rotifer (Brachionus plicatilis)-marine medaka (Oryzias melastigma) food chain was constructed to evaluate the transfer of polystyrene MPs and NPs (70 nm, 500 nm, and 2 μm, 2000 μg/L) and toxicity of 70 nm PS-NPs (0, 20, 200, and 2000 μg/L) on marine medaka after long-term food chain exposure. The results showed that the amount of 70 nm NPs accumulated in marine medaka was 1.24 μg/mg, which was significantly higher than that of 500 nm NPs (0.87 μg/mg) and 2 μm MP (0.69 μg/mg). Long-term food chain exposure to NPs caused microflora dysbiosis, resulting in activation of toll-like receptor 4 (TLR4) pathway, which induced liver inflammation. Moreover, NPs food chain exposure increased liver and muscle tissue triglyceride and lactate content, but decreased the protein, sugar, and glycogen content. NPs food chain exposure impaired reproductive function and inhibited offspring early development, which might pose a threat to the sustainability of marine medaka population. Overall, the study revealed the transfer of MPs and NPs and the effects of NPs on marine medaka along the food chain.

ACOX1 deficiency-induced lipid metabolic disorder facilitates chronic interstitial fibrosis development in renal allografts

Chronic interstitial fibrosis presents a significant challenge to the long-term survival of transplanted kidneys. Our research has shown that reduced expression of acyl-coenzyme A oxidase 1 (ACOX1), which is the rate-limiting enzyme in the peroxisomal fatty acid β-oxidation pathway, contributes to the development of fibrosis in renal allografts. ACOX1 deficiency leads to lipid accumulation and excessive oxidation of polyunsaturated fatty acids (PUFAs), which mediate epithelial–mesenchymal transition (EMT) and extracellular matrix (ECM) reorganization respectively, thus causing fibrosis in renal allografts. Furthermore, activation of Toll-like receptor 4 (TLR4)-nuclear factor kappa-B (NF-κB) signaling induced ACOX1 downregulation in a DNA methyltransferase 1 (DNMT1)-dependent manner. Overconsumption of PUFA resulted in endoplasmic reticulum (ER) stress, which played a vital role in facilitating ECM reorganization. Supplementation with PUFAs contributed to delayed fibrosis in a rat model of renal transplantation. The study provides a novel therapeutic approach that can delay chronic interstitial fibrosis in renal allografts by targeting the disorder of lipid metabolism.

Toll-like receptor 4 mediated autophagy regulates airway smooth muscle cells behavior

Objectives Airway remodeling, a prominent feature of asthma, involves aberrant proliferation, apoptosis, and migration of airway smooth muscle cells (ASMCs). Toll-like receptors (TLRs) are implicated in the regulation of the autophagy pathway. In this study, we aimed to investigate the influence of Toll-like receptor 4 (TLR4) on autophagy and its underlying mechanism in ASMC proliferation, apoptosis, and migration. Methods Histopathological changes in the lungs of asthmatic mice assessed by Hematoxylin-Eosin (HE) and Masson staining. Cell proliferation, apoptosis and migration were evaluated utilizing CCK8, Edu, Flow cytometry and wound heading assays. The effectiveness of siRNA transfection and the expression of TLR4, autophagy, and proliferation-related proteins after siRNA treatment were examined through RT-PCR and Western blot (WB). Conclusion We observed an increase in TLR4 expression and autophagy in a mouse model of OVA-induced asthma. In vitro experiments showed that siRNA-mediated inhibition of TLR4 suppressed autophagy, proliferation, and migration of ASMCs, whereas TLR4 activation by lipopolysaccharide (LPS) had the opposite effect. Furthermore, the autophagy inhibitor 3-Methyladenine (3MA) inhibited ASMCs proliferation and migration while promoting apoptosis. Significantly, our study demonstrated that autophagy inhibition reversed the promotion effect of LPS on ASMC proliferation and migration. These findings suggest that TLR4 may modulate ASMC behavior through the regulation of autophagy.