Orally administered BZL-sRNA-20 oligonucleotide targeting TLR4 effectively ameliorates acute lung injury in mice.

The global COVID-19 pandemic emerged at the end of December 2019. Acute respiratory distress syndrome (ARDS) and acute lung injury (ALI) are common lethal outcomes of bacterial lipopolysaccharide (LPS), avian influenza virus, and SARS-CoV-2. Toll-like receptor 4 (TLR4) is a key target in the pathological pathway of ARDS and ALI. Previous studies have reported that herbal small RNAs (sRNAs) are a functional medical component. BZL-sRNA-20 (Accession number: B59471456; Family ID: F2201.Q001979.B11) is a potent inhibitor of Toll-like receptor 4 (TLR4) and pro-inflammatory cytokines. Furthermore, BZL-sRNA-20 reduces intracellular levels of cytokines induced by lipoteichoic acid (LTA) and polyinosinic-polycytidylic acid (poly (I:C)). We found that BZL-sRNA-20 rescued the viability of cells infected with avian influenza H5N1, SARS-CoV-2, and several of its variants of concern (VOCs). Acute lung injury induced by LPS and SARS-CoV-2 in mice was significantly ameliorated by the oral medical decoctosome mimic (bencaosome; sphinganine (d22:0)+BZL-sRNA-20). Our findings suggest that BZL-sRNA-20 could be a pan-anti-ARDS ALI drug.

Altered expression of DNA methyltransferases and methylation status of the TLR4 and TNF-α promoters in COVID-19.

Epigenetic modifications play a significant role in the host's immune response to viral infection. Two epigenetic events, DNA methylation and histone acetylation, are crucial for modifying the chromatin architecture and the location of regulatory elements such as promoters and enhancers. In this case-control study, we evaluated the expression of genes involved in epigenetic machinery (DNMT1, DNMT3A, DNMT3B, HDAC2, and HDAC3) and the degree of methylation of promoters of immune response genes (IFITM1/2/3, TLR3/4, TNF-α, NF-κB, and MYD88) as well as global methylation (LINE-1 and global 5-mC) in blood samples from 120 COVID-19 patients (30 mild, 30 moderate, 30 severe, and 30 critical) and 30 healthy subjects without COVID-19. In contrast to previous reports, DNMT3A and DNMT3B expression was found to be significantly downregulated in COVID-19 cases, whereas DNMT1, HDAC2, and HDAC3 expression did not change. DNMT1 and DNMT3A were negatively correlated with COVID-19 severity. Critically ill patients had lower HDAC3 expression levels. TLR4 and TNF-α had increased promoter methylation, whereas IFITM1/2/3, TLR3, NF-κB, MYD88, and LINE-1 did not differ between cases and controls. Methylation of the TNF-α promoter increased as disease severity increased. Significantly less methylation of the TLR3 promoter was observed in patients with a positive outcome (recovery). We also found a correlation between the expression of DNMT3B and the methylation level of the TLR4 promoter. In milder cases, the global 5-mC levels were lower than that in more severe cases. Our findings suggest the exclusion of DNMTs inhibitors previously recommended for COVID-19 treatment and the need for additional research in this area.

MicroRNAs and cytokines as potential predictive biomarkers for COVID-19 disease progression.

Host microRNAs can influence the cytokine storm associated SARS-CoV-2 infection and proposed as biomarkers for COVID-19 disease. In the present study, serum MiRNA-106a and miRNA-20a were quantified by real time-PCR in 50 COVID-19 patients hospitalized at Minia university hospital and 30 healthy volunteers. Profiles of serum inflammatory cytokines (TNF-α, IFN-γ, and IL-10) and TLR4 were analyzed by Eliza in patients and controls. A highly significant decrease (P value = 0.0001) in the expressions of miRNA-106a and miRNA-20a was reported in COVID-19 patients compared to controls. A significant decrease in the levels of miRNA-20a was also reported in patients with lymphopenia, patients having chest CT severity score (CSS) > 19 and in patients having O2 saturation less than 90%. Significantly higher levels of TNF-α, IFN-γ, IL-10 and TLR4 were reported in patients compared to controls. IL-10 and TLR4 levels were significantly higher in patients having lymphopenia. TLR-4 level was higher in patients with CSS > 19 and in patients with hypoxia. Using univariate logistic regression analysis, miRNA-106a, miRNA-20a, TNF-α, IFN-γ, IL-10 and TLR4 were identified as good predictors of disease. Receiver operating curve showed that the downregulation of miRNA-20a in patients having lymphopenia, patients with CSS > 19 and patients with hypoxia could be a potential biomarker with AUC = 0.68 ± 0.08, AUC = 0.73 ± 0.07 and AUC = 0.68 ± 0.07 respectively. Also, ROC curve showed accurate association between the increase of serum IL-10 and TLR-4 and lymphopenia among COVID-19 patients with AUC = 0.66 ± 0.08 and AUC = 0.73 ± 0.07 respectively. ROC curve showed also that serum TLR-4 could be a potential marker for high CSS with AUC = 0.78 ± 0.06. A negative correlation was detected between miRNA-20a with TLR-4 (r = - 0.30, P value = 0.03). We concluded that, miR-20a, is a potential biomarker of COVID-19 severity and blockade of IL-10 and TLR4 may constitute a novel therapy for COVID-19 patients.

A comparative study of spike protein of SARS-CoV-2 and its variant Omicron (B.1.1.529) on some immune characteristics.

The emergence of Omicron variant raises great concerns because of its rapid transmissibility and its numerous mutations in spike protein (S-protein). S-protein can act as a pathogen-associated molecular pattern and complement activator as well as antigen. We compared some immune characteristics of trimer S-proteins for wild type (WT-S) and B.1.1.529 Omicron (Omicron-S) to investigate whether the mutations have affected its pathogenicity and antigenic shift. The results indicated that WT-S and Omicron-S directly activated nuclear factor-κB (NF-κB) and induced the release of pro-inflammatory cytokines in macrophages, but the actions of Omicron-S were weaker. These inflammatory reactions could be abrogated by a Toll-like receptor 4 antagonist TAK-242. Two S-proteins failed to induce the production of antiviral molecular interferon-ß. In contrast to pro-inflammatory effects, the ability of two S-proteins to activate complement was comparable. We also compared the binding ability of two S-proteins to a high-titer anti-WT-receptor-binding domain antibody. The data showed that WT-S strongly bound to this antibody, while Omicron-S was completely off-target. Collectively, the mutations of Omicron have a great impact on the pro-inflammatory ability and epitopes of S-protein, but little effect on its ability to activate complement. Addressing these issues can be helpful for more adequate understanding of the pathogenicity of Omicron and the vaccine breakthrough infection.

Polymorphisms in the Genes Coding for TLRs, NLRs and RLRs Are Associated with Clinical Parameters of Patients with Acute Myeloid Leukemia.

Toll-like receptors (TLRs), NOD-like receptors (NLRs), and RIG-I-like receptors (RLRs) are major elements of the innate immune system that recognize pathogen-associated molecular patterns. Single-nucleotide polymorphisms (SNPs) in the TLR, NLR, and RLR genes may lead to an imbalance in the production of pro- and anti-inflammatory cytokines, changes in susceptibility to infections, the development of diseases, and carcinogenesis. Acute myeloid leukemia (AML) is a bone marrow malignancy characterized by uncontrolled proliferation of transformed myeloid precursors. We retrospectively analyzed 90 AML patients. We investigated the effect of fifteen SNPs located in the genes coding for RLR1 (rs9695310, rs10738889, rs10813831), NOD1 (rs2075820, rs6958571), NOD2 (rs2066845, rs2066847, rs2066844), TLR3 (rs5743305, rs3775296, 3775291), TLR4 (rs4986791, rs4986790), and TLR9 (rs187084, rs5743836). We observed that TLR4 rs4986791, TLR9 rs5743836, and NOD2 rs2066847 were associated with CRP levels, while RLR-1 rs10738889 was associated with LDH level. Furthermore, we found TLR3 rs5743305 AA to be more common in patients with infections. We also found TLR9 rs187084 C to be associated with more favorable risk, and RLR-1 rs9695310 GG with higher age at diagnosis. In conclusion, the current study showed that SNPs in the genes encoding TLRs, NLRs, and RLRs may be potential biomarkers in patients with AML.

Diacerein ameliorates acetaminophen hepatotoxicity in rats via inhibiting HMGB1/TLR4/NF-κB and upregulating PPAR-γ signal.

BACKGROUND: Acetaminophen (APAP) is a worldwide antipyretic as well as an analgesic medication. It has been extensively utilized during the outbreak of coronavirus 2019 (COVID-19). APAP misuse would lead to liver injury. Diacerein (DIA), an anthraquinone derivative, has antioxidant and inflammatory properties. Hence, this study attempted to evaluate the impact of DIA treatment on liver injury induced by APAP and its influence on nuclear factor-κB (NF-κB) /toll-like receptor 4 (TLR4)/high mobility group box-1(HMGB-1) signaling as well as the expression of peroxisome proliferator-activated receptor-gamma (PPAR-γ) expression. METHODS: Male albino rats received 25 as well as 50 mg/kg/day DIA orally for seven days. One hour after the last administration, rats received APAP (1gm/kg, orally). For histopathological analysis, liver tissues and blood were collected, immunohistochemical (IHC) assay, biochemical assay, as well as quantitative real-time polymerase chain reaction (qRT-PCR). RESULTS: DIA markedly reduced liver injury markers and ameliorated histopathological changes. Moreover, DIA dose-dependently alleviated oxidative stress status caused by APAP administration along with inflammatory markers, including the level of interleukin-1 beta (IL-1ß), myeloperoxidase (MPO), tumor necrosis factor-alpha (TNF-α), and interleukin 6 (IL-6). Furthermore, DIA downregulated protein levels as well as mRNA of HMGB-1, TLR4, NF-κB p65 expression, and enhanced PPAR-γ expression. Moreover, DIA ameliorated apoptotic (Bax) and caspase-3 expressions and increased the anti-apoptotic (Bcl2) expression. CONCLUSIONS: This study demonstrated that DIA exerts anti-apoptotic, anti-inflammatory, and antioxidant properties against liver injury induced by APAP that is attributed to inhibition of the HMGB1/TLR4/NF-κB pathway, besides upregulation of the expression of PPAR-γ.

A human pluripotent stem cell-based model of SARS-CoV-2 infection reveals an ACE2-independent inflammatory activation of vascular endothelial cells through TLR4.

To date, the direct causative mechanism of SARS-CoV-2-induced endotheliitis remains unclear. Here, we report that human ECs barely express surface ACE2, and ECs express less intracellular ACE2 than non-ECs of the lungs. We ectopically expressed ACE2 in hESC-ECs to model SARS-CoV-2 infection. ACE2-deficient ECs are resistant to the infection but are more activated than ACE2-expressing ones. The virus directly induces endothelial activation by increasing monocyte adhesion, NO production, and enhanced phosphorylation of p38 mitogen-associated protein kinase (MAPK), NF-κB, and eNOS in ACE2-expressing and -deficient ECs. ACE2-deficient ECs respond to SARS-CoV-2 through TLR4 as treatment with its antagonist inhibits p38 MAPK/NF-κB/ interleukin-1ß (IL-1ß) activation after viral exposure. Genome-wide, single-cell RNA-seq analyses further confirm activation of the TLR4/MAPK14/RELA/IL-1ß axis in circulating ECs of mild and severe COVID-19 patients. Circulating ECs could serve as biomarkers for indicating patients with endotheliitis. Together, our findings support a direct role for SARS-CoV-2 in mediating endothelial inflammation in an ACE2-dependent or -independent manner.

Toll-Like Receptors (TLRs) in Health and Disease: An Overview.

Toll-like receptors were discovered as proteins playing a crucial role in the dorsoventral patterning during embryonic development in the Drosophila melanogaster (D. melanogaster) almost 40 years ago. Subsequently, further research also showed a role of the Toll protein or Toll receptor in the recognition of Gram-positive bacterial and fungal pathogens infecting D. melanogaster. In 1997, the human homolog was reported and the receptor was named the Toll-like receptor 4 (TLR4) that recognizes lipopolysaccharide (LPS) of the Gram-negative bacteria as a pathogen-associated molecular pattern (PAMP). Identification of TLR4 in humans filled the long existing gap in the field of infection and immunity, addressing the mystery surrounding the recognition of foreign pathogens/microbes by the immune system. It is now known that mammals (mice and humans) express 13 different TLRs that are expressed on the outer cell membrane or intracellularly, and which recognize different PAMPs or microbe-associated molecular patterns (MAMPs) and death/damage-associated molecular patterns (DAMPs) to initiate the protective immune response. However, their dysregulation generates profound and prolonged pro-inflammatory immune responses responsible for different inflammatory and immune-mediated diseases. This chapter provides an overview of TLRs in the control of the immune response, their association with different diseases, including TLR single nucleotide polymorphisms (SNPs), interactions with microRNAs (miRs), use in drug development and vaccine design, and expansion in neurosciences to include pain, addiction, metabolism, reproduction, and wound healing.

Host cell entry mediators implicated in the cellular tropism of SARS­CoV­2, the pathophysiology of COVID­19 and the identification of microRNAs that can modulate the expression of these mediators (Review).

The pathophysiology of coronavirus disease 2019 (COVID­19) is mainly dependent on the underlying mechanisms that mediate the entry of severe acute respiratory syndrome coronavirus 2 (SARS­CoV­2) into the host cells of the various human tissues/organs. Recent studies have indicated a higher order of complexity of the mechanisms of infectivity, given that there is a wide­repertoire of possible cell entry mediators that appear to co­localise in a cell­ and tissue­specific manner. The present study provides an overview of the 'canonical' SARS­CoV­2 mediators, namely angiotensin converting enzyme 2, transmembrane protease serine 2 and 4, and neuropilin­1, expanding on the involvement of novel candidates, including glucose­regulated protein 78, basigin, kidney injury molecule­1, metabotropic glutamate receptor subtype 2, ADAM metallopeptidase domain 17 (also termed tumour necrosis factor­α convertase) and Toll­like receptor 4. Furthermore, emerging data indicate that changes in microRNA (miRNA/miR) expression levels in patients with COVID­19 are suggestive of further complexity in the regulation of these viral mediators. An in silico analysis revealed 160 candidate miRNAs with potential strong binding capacity in the aforementioned genes. Future studies should concentrate on elucidating the association between the cellular tropism of the SARS­CoV­2 cell entry mediators and the mechanisms through which they might affect the clinical outcome. Finally, the clinical utility as a biomarker or therapeutic target of miRNAs in the context of COVID­19 warrants further investigation.

Possible Link between SARS-CoV-2 Infection and Parkinson's Disease: The Role of Toll-Like Receptor 4.

Parkinson's disease (PD) is the most common neurodegenerative motor disorder characterized by selective degeneration of dopaminergic neurons in the substantia nigra pars compacta (SNpc) of the midbrain, depletion of dopamine (DA), and impaired nigrostriatal pathway. The pathological hallmark of PD includes the aggregation and accumulation α-synuclein (α-SYN). Although the precise mechanisms underlying the pathogenesis of PD are still unknown, the activation of toll-like receptors (TLRs), mainly TLR4 and subsequent neuroinflammatory immune response, seem to play a significant role. Mounting evidence suggests that viral infection can concur with the precipitation of PD or parkinsonism. The recently identified coronavirus named severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of ongoing pandemic coronavirus disease 2019 (COVID-19), responsible for 160 million cases that led to the death of more than three million individuals worldwide. Studies have reported that many patients with COVID-19 display several neurological manifestations, including acute cerebrovascular diseases, conscious disturbance, and typical motor and non-motor symptoms accompanying PD. In this review, the neurotropic potential of SARS-CoV-2 and its possible involvement in the pathogenesis of PD are discussed. Specifically, the involvement of the TLR4 signaling pathway in mediating the virus entry, as well as the massive immune and inflammatory response in COVID-19 patients is explored. The binding of SARS-CoV-2 spike (S) protein to TLR4 and the possible interaction between SARS-CoV-2 and α-SYN as contributing factors to neuronal death are also considered.

The Effects of Insulin-Like Growth Factor I and BTP-2 on Acute Lung Injury.

Acute lung injury (ALI) afflicts approximately 200,000 patients annually and has a 40% mortality rate. The COVID-19 pandemic has massively increased the rate of ALI incidence. The pathogenesis of ALI involves tissue damage from invading microbes and, in severe cases, the overexpression of inflammatory cytokines such as tumor necrosis factor-α (TNF-α) and interleukin-1ß (IL-1ß). This study aimed to develop a therapy to normalize the excess production of inflammatory cytokines and promote tissue repair in the lipopolysaccharide (LPS)-induced ALI. Based on our previous studies, we tested the insulin-like growth factor I (IGF-I) and BTP-2 therapies. IGF-I was selected, because we and others have shown that elevated inflammatory cytokines suppress the expression of growth hormone receptors in the liver, leading to a decrease in the circulating IGF-I. IGF-I is a growth factor that increases vascular protection, enhances tissue repair, and decreases pro-inflammatory cytokines. It is also required to produce anti-inflammatory 1,25-dihydroxyvitamin D. BTP-2, an inhibitor of cytosolic calcium, was used to suppress the LPS-induced increase in cytosolic calcium, which otherwise leads to an increase in proinflammatory cytokines. We showed that LPS increased the expression of the primary inflammatory mediators such as toll like receptor-4 (TLR-4), IL-1ß, interleukin-17 (IL-17), TNF-α, and interferon-γ (IFN-γ), which were normalized by the IGF-I + BTP-2 dual therapy in the lungs, along with improved vascular gene expression markers. The histologic lung injury score was markedly elevated by LPS and reduced to normal by the combination therapy. In conclusion, the LPS-induced increases in inflammatory cytokines, vascular injuries, and lung injuries were all improved by IGF-I + BTP-2 combination therapy.

Toll-like Receptor-4 (TLR4) Agonist-Based Intranasal Nanovaccine Delivery System for Inducing Systemic and Mucosal Immunity.

Eliciting a robust immune response at mucosal sites is critical in preventing the entry of mucosal pathogens such as influenza and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). This task is challenging to achieve without the inclusion of a strong and safe mucosal adjuvant. Previously, inulin acetate (InAc), a plant-based polymer, is shown to activate toll-like receptor-4 (TLR4) and elicit a robust systemic immune response as a vaccine adjuvant. This study investigates the potential of nanoparticles prepared with InAc (InAc-NPs) as an intranasal vaccine delivery system to generate both mucosal and systemic immune responses. InAc-NPs (∼250 nm in diameter) activated wild-type (WT) macrophages but failed to activate macrophages from TLR4 knockout mice or WT macrophages when pretreated with a TLR4 antagonist (lipopolysaccharide-RS (LPS-RS)), which indicates the selective nature of a InAc-based nanodelivery system as a TLR4 agonist. Intranasal immunization using antigen-loaded InAc-NPs generated ∼65-fold and 19-fold higher serum IgG1 and IgG2a titers against the antigen, respectively, as compared to PLGA-NPs as a delivery system. InAc-NPs have also stimulated the secretion of sIgA at various mucosal sites, including nasal-associated lymphoid tissues (NALTs), lungs, and intestine, and produced a strong memory response indicative of both humoral and cellular immune activation. Overall, by stimulating both systemic and mucosal immunity, InAc-NPs laid a basis for a potential intranasal delivery system for mucosal vaccination.

Immunomodulatory Effects of Vitamin D Supplementation in a Deficient Population.

In addition to its canonical functions, vitamin D has been proposed to be an important mediator of the immune system. Despite ample sunshine, vitamin D deficiency is prevalent (>80%) in the Middle East, resulting in a high rate of supplementation. However, the underlying molecular mechanisms of the specific regimen prescribed and the potential factors affecting an individual's response to vitamin D supplementation are not well characterized. Our objective is to describe the changes in the blood transcriptome and explore the potential mechanisms associated with vitamin D3 supplementation in one hundred vitamin D-deficient women who were given a weekly oral dose (50,000 IU) of vitamin D3 for three months. A high-throughput targeted PCR, composed of 264 genes representing the important blood transcriptomic fingerprints of health and disease states, was performed on pre and post-supplementation blood samples to profile the molecular response to vitamin D3. We identified 54 differentially expressed genes that were strongly modulated by vitamin D3 supplementation. Network analyses showed significant changes in the immune-related pathways such as TLR4/CD14 and IFN receptors, and catabolic processes related to NF-kB, which were subsequently confirmed by gene ontology enrichment analyses. We proposed a model for vitamin D3 response based on the expression changes of molecules involved in the receptor-mediated intra-cellular signaling pathways and the ensuing predicted effects on cytokine production. Overall, vitamin D3 has a strong effect on the immune system, G-coupled protein receptor signaling, and the ubiquitin system. We highlighted the major molecular changes and biological processes induced by vitamin D3, which will help to further investigate the effectiveness of vitamin D3 supplementation among individuals in the Middle East as well as other regions.