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Objectives Innate immunity significantly contributes to SSc pathogenesis. TLR8 is an important innate immune mediator that is implicated in autoimmunity and fibrosis. However, the expression, mechanism of action, and pathogenic role of TLR8 in SSc remain unclear. The aim of this study was to explore the roles and underlying mechanisms of TLR8 in SSc. Methods The expression of TLR8 was analysed, based on a public dataset, and then verified in skin tissues and skin fibroblasts of SSc patients. The role of TLR8 in inflammation and fibrosis was investigated using a TLR8-overexpression vector, activator (VTX-2337), inhibitor (cu-cpt-8m), and TLR8 siRNA in skin fibroblasts. The pathogenic role of TLR8 in skin inflammation and fibrosis was further validated in a bleomycin (BLM)-induced mouse skin inflammation and fibrosis model. Results TLR8 levels were significantly elevated in SSc skin tissues and myofibroblasts, along with significant activation of the TLR8 pathway. In vitro studies showed that overexpression or activation of TLR8 by a recombinant plasmid or VTX-2337 upregulated IL-6, IL-1β, COL I, COL III and α-SMA in skin fibroblasts. Consistently, both TLR8-siRNA and cu-cpt-8m reversed the phenotypes observed in TLR8-activating fibroblasts. Mechanistically, TLR8 induces skin fibrosis and inflammation in a manner dependent on the MAPK, NF-κB and SMAD2/3 pathways. Subcutaneous injection of cu-cpt-8m significantly alleviated BLM-induced skin inflammation and fibrosis in vivo. Conclusion TLR8 might be a promising therapeutic target for improving the treatment strategy for skin inflammation and fibrosis in SSc. [ABSTRACT FROM AUTHOR]

Background: Traumatic cartilage injury is an important cause of osteoarthritis (OA) and limb disability, and toll‐like receptors (TLRs) mediated innate immune response has been confirmed to play a crucial role in cartilage injury. In the previous study, we found that the activation of TLR8 molecules in injured articular cartilage was more obvious than other TLRs by establishing an animal model of knee impact injury in rabbits, and the changes of TLR8 molecules could significantly affect the process of articular cartilage injury and repair. Objective: To verify how mir‐99a‐5p regulates TLR8 receptor mediated innate immune response to treat traumatic cartilage injury. Methods: The impact of a heavy object on the medial condyle of the rabbit's knee joint caused damage to the medial condylar cartilage. Through pathological and imaging analysis, it was demonstrated whether the establishment of an animal model of traumatic cartilage injury was successful. Establishing a cell model by virus transfection of chondrocytes to demonstrate the role of TLR8 in the innate immune response to impact cartilage injury. Through transcriptome sequencing, potential targets of TLR8, mir‐99a‐5p, were predicted, and basic experiments were conducted to demonstrate how they interact with innate immune responses to impact cartilage damage. Results: TLR8 is a receptor protein of the immune system, which is widely expressed in immune cells. In our study, we found that TLR8 expression is localized in lysosomes and endosomes. Mir‐99a‐5p can negatively regulate TLR8 to activate PI3K‐AKT molecular pathway and aggravate cartilage damage. Inhibiting TLR8 expression can effectively reduce the incidence of articular cartilage damage. Conclusion: Based on the results from this study, mir‐99a‐5p may be an effective molecular marker for predicting traumatic cartilage injury and targeting TLR8 is a novel and promising approach for the prevention or early treatment of cartilage damage. [ABSTRACT FROM AUTHOR]

The prognosis of COVID-19 could influence by innate immune sensors such as toll-like receptors (TLRs). The purpose of this data was to investigate TLR3, 7, and 8 expression levels in COVID-19 patients and their relationship to outcome of disease.75 confirm COVID-19 were included sequentially and separated into three groups: mild, severe, and critical. Peripheral blood mononuclear cells were isolated from the whole blood, and RNA was then extracted. The qRT-PCR technique was used to examine the expression of TLR3, TLR7, and TLR8 genes.The patients average ages were 52.69 ± 1.9 and 13 of the 25 individuals in each group were male. TLR3 (p < 0.001), TLR7 (p < 0.001), and TLR8 (p < 0.001) expression levels were considerably greater in COVID-19 patients compared to the control group. The findings also showed that individuals with critical and severe COVID-19 disease had significantly greater TLR7 and TLR8 gene expression levels than patients in mild stage of disease (p < 0.05). The data showed a significant difference (p = 0.01) in the TLR3 transcript levels between critical and mild COVID-19 patients. Furthermore, male severe (p = 0.02) and critical (p = 0.008) patients had significantly higher TLR8 expression levels than female patients in terms of gender. TLR3 (p = 0.2) and TLR7 (p = 0.08) transcripts were more elevated in males than females, but not significantly.

Abstract Background Traumatic cartilage injury is an important cause of osteoarthritis (OA) and limb disability, and toll‐like receptors (TLRs) mediated innate immune response has been confirmed to play a crucial role in cartilage injury. In the previous study, we found that the activation of TLR8 molecules in injured articular cartilage was more obvious than other TLRs by establishing an animal model of knee impact injury in rabbits, and the changes of TLR8 molecules could significantly affect the process of articular cartilage injury and repair. Objective To verify how mir‐99a‐5p regulates TLR8 receptor mediated innate immune response to treat traumatic cartilage injury. Methods The impact of a heavy object on the medial condyle of the rabbit's knee joint caused damage to the medial condylar cartilage. Through pathological and imaging analysis, it was demonstrated whether the establishment of an animal model of traumatic cartilage injury was successful. Establishing a cell model by virus transfection of chondrocytes to demonstrate the role of TLR8 in the innate immune response to impact cartilage injury. Through transcriptome sequencing, potential targets of TLR8, mir‐99a‐5p, were predicted, and basic experiments were conducted to demonstrate how they interact with innate immune responses to impact cartilage damage. Results TLR8 is a receptor protein of the immune system, which is widely expressed in immune cells. In our study, we found that TLR8 expression is localized in lysosomes and endosomes. Mir‐99a‐5p can negatively regulate TLR8 to activate PI3K‐AKT molecular pathway and aggravate cartilage damage. Inhibiting TLR8 expression can effectively reduce the incidence of articular cartilage damage. Conclusion Based on the results from this study, mir‐99a‐5p may be an effective molecular marker for predicting traumatic cartilage injury and targeting TLR8 is a novel and promising approach for the prevention or early treatment of cartilage damage.

Abstract Objective Hypoxia, which can considerably affect the tumor microenvironment, hinders the use of immunotherapy in bladder cancer (BLCA). Therefore, we aimed to identify reliable hypoxia‐related biomarkers to guide clinical immunotherapy in BLCA. Methods Using data downloaded from TCGA‐BLCA cohort, we determined BLCA subtypes which divide 408 samples into different subtypes. Tumor immune infiltration levels of two clusters were quantified using ssGSEA, MCPcounter, EPIC, ESTIMATE, and TIMER algorithms. Next, we constructed a hypoxia score based on the expression of hypoxia‐related genes. The IMvigor210 cohort and SubMap analysis were used to predict immunotherapeutic responses in patients with different hypoxia scores. Hub genes were screened using cytoscape, immunohistochemistry (IHC), and multispectral immunofluorescence were used to detect the spatial distribution of immune markers. Results Patients with BLCA were categorized into cluster1 (n = 227) and Cluster2 (n = 181). Immune infiltration and expression of immune markers were higher in Cluster1. Immune infiltration was also more obvious in the high‐hypoxia score group which related to a better predicted response to immunotherapy. IHC, and multispectral immunofluorescence confirmed the importance of TLR8 in immune infiltration and immune phenotype. Conclusions BLCA subtype can evaluate the infiltration of immune cells in the tumor microenvironment of different patients. Hypoxia score in this study could effectively predict immunotherapeutic responses in patients with BLCA. TLR8 may be a potential target for clinical immunotherapy.

Abstract Background Hyperactive neutrophil extracellular traps (NETs) formation plays a key role in the pathogenesis of severe COVID-19. Extracellular vesicles (EVs) are vehicles which carry cellular components for intercellular communication. The association between COVID-19 patients-derived EVs and NETs formation remains elusive. Methods We explored the roles of EVs in NETs formation from 40 COVID-19 patients with different disease severities as well as 30 healthy subjects. The EVs-carried microRNAs profile was analyzed using next generation sequencing approach which was validated by quantitative reverse transcription PCR. The regulatory mechanism of EVs on NETs formation was investigated by using an in vitro cell-based assay, including immunofluorescence assay, flow cytometry, and immunoblotting. Results COVID-19 patient–derived EVs induced NETs formation by endocytosis uptake. SARS-CoV-2 spike protein-triggered NETs formation was significantly enhanced in the presence of platelet–derived EVs (pEVs) and this effect was Toll-like receptor (TLR) 7/8- and NADPH oxidase-dependent. Increased levels of miR-21/let-7b were revealed in EVs from COVID-19 patients and were associated with disease severity. We demonstrated that the spike protein activated platelets directly, followed by the subsequent intracellular miR-21/let-7b upregulation and then were loaded into pEVs. The pEVs-carried miR-21 interacted with TLR7/8 to prime p47phox phosphorylation in neutrophils, resulting in NADPH oxidase activation to promote ROS production and NETs enhancement. In addition, miR-21 modulates NF-κB activation and IL-1β/TNFα/IL-8 upregulation in neutrophils upon TLR7/8 engagement. The miR-21 inhibitor and TLR8 antagonist could suppress efficiently spike protein-induced NETs formation and pEVs primed NETs enhancement. Conclusions We identified SARS-CoV-2 triggered platelets–derived GU-enriched miRNAs (e.g., miR-21/let-7b) as a TLR7/8 ligand that could activate neutrophils through EVs transmission. The miR-21-TLR8 axis could be used as a potential predisposing factor or therapeutic target for severe COVID-19.

Objective: Hypoxia, which can considerably affect the tumor microenvironment, hinders the use of immunotherapy in bladder cancer (BLCA). Therefore, we aimed to identify reliable hypoxia‐related biomarkers to guide clinical immunotherapy in BLCA. Methods: Using data downloaded from TCGA‐BLCA cohort, we determined BLCA subtypes which divide 408 samples into different subtypes. Tumor immune infiltration levels of two clusters were quantified using ssGSEA, MCPcounter, EPIC, ESTIMATE, and TIMER algorithms. Next, we constructed a hypoxia score based on the expression of hypoxia‐related genes. The IMvigor210 cohort and SubMap analysis were used to predict immunotherapeutic responses in patients with different hypoxia scores. Hub genes were screened using cytoscape, immunohistochemistry (IHC), and multispectral immunofluorescence were used to detect the spatial distribution of immune markers. Results: Patients with BLCA were categorized into cluster1 (n = 227) and Cluster2 (n = 181). Immune infiltration and expression of immune markers were higher in Cluster1. Immune infiltration was also more obvious in the high‐hypoxia score group which related to a better predicted response to immunotherapy. IHC, and multispectral immunofluorescence confirmed the importance of TLR8 in immune infiltration and immune phenotype. Conclusions: BLCA subtype can evaluate the infiltration of immune cells in the tumor microenvironment of different patients. Hypoxia score in this study could effectively predict immunotherapeutic responses in patients with BLCA. TLR8 may be a potential target for clinical immunotherapy. [ABSTRACT FROM AUTHOR]

Regulation of the profile and magnitude of toll-like receptor (TLR) responses is important for effective host defense against infections while minimizing inflammatory toxicity. The chemokine CXCL4 regulates the TLR8 response to amplify inflammatory gene and inflammasome activation while attenuating the interferon (IFN) response in primary monocytes. In this study, we describe an unexpected role for the kinase RIPK3 in suppressing the CXCL4 + TLR8–induced IFN response and providing signal 2 to activate the NLRP3 inflammasome and interleukin (IL)-1 production in primary human monocytes. RIPK3 also amplifies induction of inflammatory genes such as TNF, IL6, and IL1B while suppressing IL12B. Mechanistically, RIPK3 inhibits STAT1 activation and activates PI3K-Akt–dependent and XBP1- and NRF2-mediated stress responses to regulate downstream genes in a dichotomous manner. These findings identify new functions for RIPK3 in modulating TLR responses and provide potential mechanisms by which RIPK3 plays roles in inflammatory diseases and suggest targeting RIPK3 and XBP1- and NRF2-mediated stress responses as therapeutic strategies to suppress inflammation while preserving the IFN response for host defense. RIPK3 modulates CXCL4 and TLR8 costimulation of human monocytes by augmenting inflammasome and inflammatory gene activation, while attenuating the interferon response. [ABSTRACT FROM AUTHOR]

Background: Hyperactive neutrophil extracellular traps (NETs) formation plays a key role in the pathogenesis of severe COVID-19. Extracellular vesicles (EVs) are vehicles which carry cellular components for intercellular communication. The association between COVID-19 patients-derived EVs and NETs formation remains elusive. Methods: We explored the roles of EVs in NETs formation from 40 COVID-19 patients with different disease severities as well as 30 healthy subjects. The EVs-carried microRNAs profile was analyzed using next generation sequencing approach which was validated by quantitative reverse transcription PCR. The regulatory mechanism of EVs on NETs formation was investigated by using an in vitro cell-based assay, including immunofluorescence assay, flow cytometry, and immunoblotting. Results: COVID-19 patient–derived EVs induced NETs formation by endocytosis uptake. SARS-CoV-2 spike protein-triggered NETs formation was significantly enhanced in the presence of platelet–derived EVs (pEVs) and this effect was Toll-like receptor (TLR) 7/8- and NADPH oxidase-dependent. Increased levels of miR-21/let-7b were revealed in EVs from COVID-19 patients and were associated with disease severity. We demonstrated that the spike protein activated platelets directly, followed by the subsequent intracellular miR-21/let-7b upregulation and then were loaded into pEVs. The pEVs-carried miR-21 interacted with TLR7/8 to prime p47phox phosphorylation in neutrophils, resulting in NADPH oxidase activation to promote ROS production and NETs enhancement. In addition, miR-21 modulates NF-κB activation and IL-1β/TNFα/IL-8 upregulation in neutrophils upon TLR7/8 engagement. The miR-21 inhibitor and TLR8 antagonist could suppress efficiently spike protein-induced NETs formation and pEVs primed NETs enhancement. Conclusions: We identified SARS-CoV-2 triggered platelets–derived GU-enriched miRNAs (e.g., miR-21/let-7b) as a TLR7/8 ligand that could activate neutrophils through EVs transmission. The miR-21-TLR8 axis could be used as a potential predisposing factor or therapeutic target for severe COVID-19. Plain English summary: COVID-19 poses a crucial threat to global health. Excessive NETs contribute to microthrombi through platelet-neutrophil interactions, resulting in acute respiratory distress syndrome in severe COVID-19 cases. EVs play a crucial role in intercellular communication during infection. Herein, we showed that the viral spike protein activates platelets directly, followed by intracellular miR21/let7b upregulation, which is loaded into pEVs. The pEV-carried miR-21/let-7b could interact with TLR7/8 in neutrophils, followed by activation of the downstream signaling pathway, including p47phox-NOX2-ROS, which causes NETs enhancement, while NF-κB promotes the expression of proinflammatory cytokines. The miR-21 inhibitor and TLR8 antagonist could efficiently suppress spike protein-induced NETs formation and pEVs primed NETs enhancement. This study offers new molecular machinery to explain the association between platelets–derived miRNAs, NETs formation and SARS-CoV-2 infection. Host-directed therapies are a relatively new and promising approach to COVID-19 treatment. The miR-21-TLR8 axis may be a potential therapeutic target for severe COVID-19. -_k_965S5Tdr4uMAbg8aGR Video Abstract [ABSTRACT FROM AUTHOR]

TLR Agonists have promising activity in preclinical models of viral infection and cancer. However, clinical use is only in topical application. Systemic uses of TLRligands such as Resiquimod, have failed due to adverse effects that limited dose and thus, efficacy. This issue could be related to pharmacokinetic properties that include fast elimination leading to low AUC with simultaneously high cmax at relevant doses. The high cmax is associated with a sharp, poorly tolerated cytokine pulse, suggesting that a compound with a higher AUC/cmax-ratio could provide a more sustained and tolerable immune activation. Our approach was to design TLR7/8-agonist Imidazoquinolines intended to partition to endosomes via acid trapping using a macrolide-carrier. This can potentially extend pharmacokinetics and simultaneously direct the compounds to the target compartment. The compounds have hTLR7/8-agonist activity (EC50 of the most active compound in cellular assays: 75-120 nM hTLR7, 2.8-3.1 µM hTLR8) and maximal hTLR7 activation between 40 and 80% of Resiquimod. The lead candidates induce secretion of IFNa from human Leukocytes in the same range as Resiquimod but induce at least 10-fold less TNFa in this system, consistent with a higher specificity for human TLR7. This pattern was reproduced in vivo in a murine system, where small molecules are thought not to activate TLR8. We found that Imidazoquinolines conjugated to a macrolide or, substances carrying an unlinked terminal secondary amine, had longer exposure compared with Resiquimod. The kinetics of pro-inflammatory cytokine release for these substances in vivo were slower and more extended (for comparable AUCs, approximately half-maximal plasma concentrations). Maximal IFNa plasma levels were reached 4 h post application. Resiquimod-treated groups had by then returned to baseline from a peak at 1 h. We propose that the characteristic cytokine profile is likely a consequence of altered pharmacokinetics and, potentially, enhanced endosomal tropism of the novel substances. In particular, our substances are designed to partition to cellular compartments where the target receptor and a distinct combination of signaling molecules relevant to IFNa-release are located. These properties could address the tolerability issues of TLR7/8 ligands and provide insight into approaches to fine-tune the outcomes of TLR7/8 activation by small molecules. [ABSTRACT FROM AUTHOR]

The clinical treatment of AML is dominated by ''7 + 3'' therapy, but it often shows great toxicity and limited therapeutic efficacy in application. Therefore, it is urgent to develop novel therapeutic strategies to achieve safe and efficient treatment of AML. Small-molecule inhibitors have the characteristics of high specificity, low off-target toxicity and remarkable therapeutic effect, and are receiving more and more attention in tumor therapy. In this study, we screened a library of 1972 FDA-approved small molecular compounds for those that induced the inflammatory death of AML cells, among which the TLR8 agonist Motolimod (MTL) showed stronger anti-AML activity in the animal model but slight affection on normal lymphocytes in control mice. In terms of mechanism, cellular experiments in AML cell lines proved that TLR8 and LKB1/AMPK are the key distinct mechanisms for MTL triggered caspase-3-dependent cell death and the expression of a large number of inflammatory factors. In conclusion, our findings identified the immunoactivator MTL as a single agent exerting significant anti-AML activity in vitro and in vivo, with strong potential for clinical translation.

HIV-1 is a highly host-specific retrovirus that infects humans but not most nonhuman primates. Thus, the lack of a suitable primate model that can be directly infected with HIV-1 hinders HIV-1/AIDS research. In the previous study, we have found that the northern pig-tailed macaques (NPMs) are susceptible to HIV-1 infection but show a nonpathogenic state. In this study, to understand this macaque–HIV-1 interaction, we assembled a de novo genome and longitudinal transcriptome for this species during the course of HIV-1 infection. Using comparative genomic analysis, a positively selected gene, Toll-like receptor 8, was identified with a weak ability to induce an inflammatory response in this macaque. In addition, an interferon-stimulated gene, interferon alpha inducible protein 27, was upregulated in acute HIV-1 infection and acquired an enhanced ability to inhibit HIV-1 replication compared with its human ortholog. These findings coincide with the observation of persistently downregulated immune activation and low viral replication and can partially explain the AIDS-free state in this macaque following HIV-1 infection. This study identified a number of unexplored host genes that may hamper HIV-1 replication and pathogenicity in NPMs and provided new insights into the host defense mechanisms in cross-species infection of HIV-1. This work will facilitate the adoption of NPM as a feasible animal model for HIV-1/AIDS research. [ABSTRACT FROM AUTHOR]

Macrophages promote an early host response to infection by releasing pro-inflammatory cytokines such as interleukin (IL) 1β (IL-1β), tumour necrosis factor (TNF), and IL-6. One of the mechanisms through which cells sense pathogenic microorganisms is through Toll-like receptors (TLRs). IL-1 receptor-associated kinase (IRAK) 1, IRAK2, IRAK3, and IRAK4 are integral to TLR and IL-1 receptor signalling pathways. Recent studies suggest a role for aberrant TLR8 and NLRP3 inflammasome activation during both COVID-19 and HIV-1 infection. Here, we show that pacritinib inhibits the TLR8-dependent pro-inflammatory cytokine response elicited by GU-rich single-stranded RNA derived from SARS-CoV-2 and HIV-1. Using genetic and pharmacologic inhibition, we demonstrate that pacritinib inhibits IRAK1 phosphorylation and ubiquitination which then inhibits the recruitment of the TAK1 complex to IRAK1, thus inhibiting the activation of downstream signalling and the production of pro-inflammatory cytokines.

Summary: Background: Natural killer (NK) cells exhibit a selective deficiency of IFN‐γ production in chronic hepatitis B (CHB). Toll‐like receptor 8 (TLR8) agonists could induce IFN‐γ production in immune cells, although their effects on the deficiency in NK cells remain unclear. Aims: To investigate TLR8 expression in NK cells and the effect of TLR8 agonists in patients with CHB Methods: We enrolled 32 patients with CHB and 19 healthy controls to assess TLR8 expression and IFN‐γ production in NK cells. The sorted NK cells and monocytes were co‐cultured to compare the extent of IFN‐γ and IL‐10 production after TLR8 agonist ssRNA40 stimulation. The synergic effect of NK cells and monocytes was assessed by blocking IL‐12 and IL‐18. We recruited another 22 patients with CHB undergoing nucleotide analogue (NA) therapy to explore the impact of antiviral treatment on the ssRNA40‐mediated response of NK cells. Results: In patients with CHB, TLR8 expression in NK cells was up‐regulated, accompanied by insufficient IFN‐γ production. The enhanced IFN‐γ secretion by ssRNA40 in NK cells depended on monocyte‐derived IL‐12 and IL‐18. NK cells displayed an imbalanced response to ssRNA40 in patients with CHB with a weak increase in IFN‐γ despite a higher IL‐10 production. The response was improved in patients with CHB undergoing NA therapy. Conclusions: In patients with CHB, targeting TLR8 partially rescues the IFN‐γ insufficiency in NK cells. However, NK cells show an inhibitory response to TLR8 agonist stimulation. TLR8 agonist combined with NA may enhance the antiviral effect of NK cells. [ABSTRACT FROM AUTHOR]

Toll‐like receptors (TLRs) have been shown to have anti‐tumor, pro‐tumor, or even dual effects in cancer, and are thus potential prognostic biomarkers and immunotherapeutic targets. The present study aimed to evaluate associations between endosomal TLRs, namely TLR3, TLR7, TLR8, and TLR9, expression and clinicopathological variables and survival in gastric cancer. A total of 564 gastric adenocarcinoma patients were included in this retrospective cohort study. Samples and clinicopathological data were retrieved and organized into tissue microarray blocks. Protein expressions were detected by immunohistochemical staining. The patients were divided into low expression and high expression groups by median values of expression. Cox regression provided hazard ratios (HR) with 95% confidence intervals (CI), adjusted for confounders. Patients with high nuclear TLR3 expression had significantly poorer 5‐year survival than the low nuclear TLR3 expression group in the univariable analysis (crude HR 1.31, 95% CI 1.07–1.60). With radically resected patients, poor prognosis was also seen in the multivariable analysis (adjusted HR 1.38, 95% CI 1.08–1.77). Cytoplasmic TLR3, TLR7, TLR8, and TLR9 were not associated with 5‐year survival. In conclusion, high nuclear TLR3 expression seems to have prognostic impact in gastric cancer, while TLR7, TLR8, and TLR9 do not. [ABSTRACT FROM AUTHOR]

TLR Agonists have promising activity in preclinical models of viral infection and cancer. However, clinical use is only in topical application. Systemic uses of TLR-ligands such as Resiquimod, have failed due to adverse effects that limited dose and thus, efficacy. This issue could be related to pharmacokinetic properties that include fast elimination leading to low AUC with simultaneously high cmax at relevant doses. The high cmax is associated with a sharp, poorly tolerated cytokine pulse, suggesting that a compound with a higher AUC/cmax-ratio could provide a more sustained and tolerable immune activation. Our approach was to design TLR7/8-agonist Imidazoquinolines intended to partition to endosomes via acid trapping using a macrolide-carrier. This can potentially extend pharmacokinetics and simultaneously direct the compounds to the target compartment. The compounds have hTLR7/8-agonist activity (EC50 of the most active compound in cellular assays: 75-120 nM hTLR7, 2.8-3.1 µM hTLR8) and maximal hTLR7 activation between 40 and 80% of Resiquimod. The lead candidates induce secretion of IFNα from human Leukocytes in the same range as Resiquimod but induce at least 10-fold less TNFα in this system, consistent with a higher specificity for human TLR7. This pattern was reproduced in vivo in a murine system, where small molecules are thought not to activate TLR8. We found that Imidazoquinolines conjugated to a macrolide or, substances carrying an unlinked terminal secondary amine, had longer exposure compared with Resiquimod. The kinetics of pro-inflammatory cytokine release for these substances in vivo were slower and more extended (for comparable AUCs, approximately half-maximal plasma concentrations). Maximal IFNα plasma levels were reached 4 h post application. Resiquimod-treated groups had by then returned to baseline from a peak at 1 h. We propose that the characteristic cytokine profile is likely a consequence of altered pharmacokinetics and, potentially, enhanced endosomal tropism of the novel substances. In particular, our substances are designed to partition to cellular compartments where the target receptor and a distinct combination of signaling molecules relevant to IFNα-release are located. These properties could address the tolerability issues of TLR7/8 ligands and provide insight into approaches to fine-tune the outcomes of TLR7/8 activation by small molecules.

These results suggest that LDL-msRNA instigates inflammation associated with atherosclerosis and supports alternative functions of LDL beyond cholesterol transport. Atherosclerosis is the pathophysiological base for most forms of cardiovascular disease (CVD).[2] The disease is driven by a large number of known factors, including inflammation, oxidative stress, and metabolic dysfunction, and many more to be unravelled. Keywords: Macrophage; LDL; TLR8; sRNA; CVD EN Macrophage LDL TLR8 sRNA CVD e146 e148 3 11/01/23 20230901 NES 230901 Commentary on 'LDL delivery of microbial small RNAs drives atherosclerosis through macrophage TLR8' by R.M. Allen I et al. i , I Nature Cell Biology i , https://doi.org/10.1038/s41556-022-01030-7.[1] Atherosclerosis is a complex disease that is characterized by the accumulation of fatty deposits in arteries. Finally, the study investigated the effects of nt-LNA on atherosclerosis in mice, validating a reduction in the development of atherosclerosis in mice fed with Western diet. [Extracted from the article]

COVID-19 disease is characterized by a dysregulation of the innate arm of the immune system. However, the mechanisms whereby innate immune cells, including neutrophils, become activated in patients are not completely understood. Recently, we showed that GU-rich RNA sequences from the SARS-CoV-2 genome (i.e., SCV2-RNA1 and SCV2-RNA2) activate dendritic cells. To clarify whether human neutrophils may also represent targets of SCV2-RNAs, neutrophils were treated with either SCV2-RNAs or, as a control, R848 (a TLR7/8 ligand), and were then analyzed for several functional assays and also subjected to RNA-seq experiments. Results highlight a remarkable response of neutrophils to SCV2-RNAs in terms of TNFα, IL-1ra, CXCL8 production, apoptosis delay, modulation of CD11b and CD62L expression, and release of neutrophil extracellular traps. By RNA-seq experiments, we observed that SCV2-RNA2 promotes a transcriptional reprogramming of neutrophils, characterized by the induction of thousands of proinflammatory genes, similar to that promoted by R848. Furthermore, by using CU-CPT9a, a TLR8-specific inhibitor, we found that SCV2-RNA2 stimulates neutrophils exclusively via TLR8-dependent pathways. In sum, our study proves that single-strand RNAs from the SARS-CoV-2 genome potently activate human neutrophils via TLR8, thus uncovering a potential mechanism whereby neutrophils may contribute to the pathogenesis of severe COVID-19 disease. [ABSTRACT FROM AUTHOR]

Recognition of RNA by receptors of the innate immune system is regulated by various posttranslational modifications. Different single 2′-O-ribose (2′-O-) methylations have been shown to convert TLR7/TLR8 ligands into specific TLR8 ligands, so we investigated whether the position of 2′-O-methylation is crucial for its function. To this end, we designed different 2′-O-methylated RNA oligoribonucleotides (ORN), investigating their immune activity in various cell systems and analyzing degradation under RNase T2 treatment. We found that the 18S rRNA-derived TLR7/8 ligand, RNA63, was differentially digested as a result of 2′-O-methylation, leading to variations in TLR8 and TLR7 inhibition. The suitability of certain 2′-O-methylated RNA63 derivatives as TLR8 agonists was further demonstrated by the fact that other RNA sequences were only weak TLR8 agonists. We were thus able to identify specific 2′-O-methylated RNA derivatives as optimal TLR8 ligands. [ABSTRACT FROM AUTHOR]

Toll-like receptors (TLRs) are one of the most ancient and widely studied innate immune receptors responsible for host defense against invading pathogens. Among the known TLRs, TLR7 and TLR8 sense and recognize single-stranded (ss) RNAs with a dynamic evolutionary history. While TLR8 was lost in birds and duplicated in turtles and crocodiles, TLR7 is duplicated in some birds, but in other tetrapods, there is only one copy. In mammals, with the exception of lagomorphs, TLR7 and TLR8 are highly conserved. Here, we aim to study the evolution of TLR7 and TLR8 in mammals, with a special focus in the order Lagomorpha. By searching public sequence databases, conducting evolutionary analysis, and evaluating gene expression, we were able to confirm that TLR8 is absent in hares but widely expressed in the European rabbit. In contrast, TLR7 is absent in the European rabbit and quite divergent in hares. Our results suggest that, in lagomorphs, more in particular in leporids, TLR7 and TLR8 genes have evolved faster than in any other mammalian group. The long history of interaction with viruses and their location in highly dynamic telomeric regions might explain the pattern observed. [ABSTRACT FROM AUTHOR]

Toll-like receptor 8 (TLR8) is an important component of the human innate immune system that recognizes single stranded RNA (ssRNA). Recent X-ray crystal structures of TLR8 bound to ssRNA revealed a previously unrecognized binding site for a 5'-UpG-3' dinucleotide. Here we use an atomic mutagenesis strategy coupled with a cellular TLR8 activation assay to probe the importance of specific functional groups present on the guanine base in RNA-mediated receptor agonism and antagonism. Results from RNA analogs containing 7-deazaguanosine, 2-aminopurine and inosine confirm the importance of guanine N7, O6 and N2, respectively, in TLR8 activation. Nevertheless, these RNAs each retained TLR8 antagonism activity. RNA containing 7-deaza-8-azainosine (7d8aI) was prepared from a novel phosphoramidite and found to be a weaker TLR8 activator than guanosine-containing RNA. However, 7d8aI-containing RNA also retained TLR8 antagonism activity indicating that removal of multiple TLR8 H-bonding sites on guanine is insufficient for blocking TLR8 antagonism by guanine-containing RNA. We also identified an oligoribonucleotide length dependence on both TLR8 activation and antagonism. These studies extend our understanding of the effects of nucleobase modification on immune stimulation and will inform the design of novel RNA-based therapeutics.

Sjögren’s syndrome (SS) is a chronic systemic autoimmune disease that affects the salivary and lacrimal glands, as well as other organ systems like the lungs, kidneys and nervous system. SS can occur alone or in combination with another autoimmune disease, such as systemic lupus erythematosus (SLE) or rheumatoid arthritis. The etiology of SS is unknown but recent studies have revealed the implication of the activation of innate immune receptors, including Toll-like receptors (TLRs), mainly through the detection of endogenous nucleic acids, in the pathogenesis of systemic autoimmune diseases. Studies on SS mouse models suggest that TLRs and especially TLR7 that detects single-stranded RNA of microbial or endogenous origin can drive the development of SS and findings in SS patients corroborate those in mouse models. In this review, we will give an overview of the function and signaling of nucleic acid-sensing TLRs, the interplay of TLR7 with TLR8 and TLR9 in the context of autoimmunity, summarize the evidence for the critical role of TLR7 in the pathogenesis of SS and present a possible connection between SARS-CoV-2 and SS.

Pathogens trigger multiple pattern recognition receptors (PRRs) that together dictate innate and adaptive immune responses. Understanding the crosstalk between PRRs is important to enhance vaccine efficacy. Abortive HIV‐1 RNA transcripts are produced during acute and chronic HIV‐1 infection and are known ligands for different PRRs, leading to antiviral and proinflammatory responses. Here, we have investigated the crosstalk between responses induced by these 58 nucleotide‐long HIV‐1 RNA transcripts and different TLR ligands. Costimulation of dendritic cells (DCs) with abortive HIV‐1 RNA and TLR7/8 agonist R848, but not other TLR agonists, resulted in enhanced antiviral type I IFN responses as well as adaptive immune responses via the induction of DC‐mediated T helper 1 (TH1) responses and IFNγ+CD8+ T cells. Our data underscore the importance of crosstalk between abortive HIV‐1 RNA and R848‐induced signaling for the induction of effective antiviral immunity. [ABSTRACT FROM AUTHOR]

Leukemogenesis is proposed to result from the continuous interplay between inducive bone marrow (BM) microenvironments and malignant precursor cells. Recent findings point toward an abnormal production of proinflammatory mediators within the BM from acute lymphoblastic leukemia (ALL) patients, although the mechanism underlying this phenomenon is uncertain. Here, we have identified 3 miRNAs, miR‐146a‐5p, miR‐181b‐5p, and miR‐199b‐3p, as potential candidates for TLR8 ligation, which are overexpressed in ALL and show agonist functional binding. When purified from ALL exosomes, they demonstrated their capacity of inducing cytokine production by both, hematopoietic and stromal BM cells. Of note, the exposure of BM cells from ALL patients to the proinflammatory milieu resulting from these miRNAs agonist activity revealed the proliferation of normal progenitors, while poor effects were recorded in the leukemic counterpart. The unconventional roles of the tumor‐secreted miRNAs as TLR8 agonist ligands may provide a novel mechanism contributing a tumor‐microenvironment feedback loop by switching on proinflammatory pathways that further activate normal hematopoietic precursors and support ALL progression. Secreted B‐ALL TLR8‐agonist miRNAs are involved in the promotion of proinflammatory microenvironments that target normal hematopoietic cells. B‐lineage ALL cells secrete exosomes containing miRNAs endowed with the ability of functionally binding TLR8 in hematopoietic and BM mesenchymal stromal cells. Upon TLR8 signaling, the activation of the NF‐kB pathway induces secretion of proinflammatory cytokines that, in turn, promotes cell proliferation in early hematopoietic cell populations, driving a tumor–microenvironment–hematopoietic activation feedback loop that may reduce the normal hematopoietic stem and progenitor cell compartment and facilitate cancer progression. [ABSTRACT FROM AUTHOR]

Toll-like receptor 8 (TLR8) recognizes single-stranded RNA of viral and bacterial origin as well as mediates the secretion of pro-inflammatory cytokines and type I interferons by human monocytes and macrophages. TLR8, as other endosomal TLRs, utilizes the MyD88 adaptor protein for initiation of signaling from endosomes. Here, we addressed the potential role of the Toll-interleukin 1 receptor domain-containing adaptor protein (TIRAP) in the regulation of TLR8 signaling in human primary monocyte-derived macrophages (MDMs). To accomplish this, we performed TIRAP gene silencing, followed by the stimulation of cells with synthetic ligands or live bacteria. Cytokine-gene expression and secretion were analyzed by quantitative PCR or Bioplex assays, respectively, while nuclear translocation of transcription factors was addressed by immunofluorescence and imaging, as well as by cell fractionation and immunoblotting. Immunoprecipitation and Akt inhibitors were also used to dissect the signaling mechanisms. Overall, we show that TIRAP is recruited to the TLR8 Myddosome signaling complex, where TIRAP contributes to Akt-kinase activation and the nuclear translocation of interferon regulatory factor 5 (IRF5). Recruitment of TIRAP to the TLR8 signaling complex promotes the expression and secretion of the IRF5-dependent cytokines IFNβ and IL-12p70 as well as, to a lesser degree, TNF. These findings reveal a new and unconventional role of TIRAP in innate immune defense. [ABSTRACT FROM AUTHOR]

Objective: Septic shock is the severe complication of sepsis, with a high mortality. The inflammatory response regulates the immune status and mediates the progression of septic shock. In this study, we aim to identify the key immune-related genes (IRGs) of septic shock and explore their potential mechanism. Methods: Gene expression profiles of septic shock blood samples and normal whole blood samples were retrieved from the Gene Expression Omnibus (GEO) and Genotype-Tissue Expression Portal (GTEx). The differential expression genes (DEGs) and septic shock-specific immune-related genes (SSSIRGs) were evaluated and identified, along with the immune components by "cell type identification by estimating relative subsets of RNA transcripts (CIBERSORT, version x)" algorithm. Additionally, in order to explore the key regulatory network, the relationship among SSSIRGs, upstream transcription factors (TFs), and downstream signaling pathways were also identified by Gene Set Variation Analysis (GSVA) and co-expression analysis. Moreover, the Connectivity Map (CMap) analysis was applied to find bioactive small molecules against the members of regulation network while Chromatin Immunoprecipitation sequencing (ChIP-seq) and Assay for Targeting Accessible-Chromatin with high-throughput sequencing (ATAC-seq) data were used to validate the regulation mechanism of the network. Results: A total of 14,843 DEGs were found between 63 septic shock blood samples and 337 normal whole blood samples. Then, we identified septic shock-specific 839 IRGs as the intersection of DEGs and IRGs. Moreover, we uncovered the regulatory networks based on co-expression analysis and found 28 co-expression interaction pairs. In the regulation network, protein phosphatase 3, catalytic subunit, alpha isozyme (PPP3CA) may regulate late estrogen response, glycolysis and TNFα signaling via NFκB and HLA; Kirsten rat sarcoma viral oncogene homolog (KRAS) may be related to late estrogen response and HLA; and Toll-like receptor 8 (TLR8) may be associated with TNFα signaling via NFκB. And the regulation mechanisms between TFs and IRGs (TLR8, PPP3CA, and KRAS) were validated by ChIP-seq and ATAC-seq. Conclusion: Our data identify three SSSIRGs (TLR8, PPP3CA, and KRAS) as candidate therapeutic targets for septic shock and provide constructed regulatory networks in septic shock to explore its potential mechanism. [ABSTRACT FROM AUTHOR]

Abstract Toll-like receptors (TLRs) are a family of proteins that recognize pathogen associated molecular patterns (PAMPs). Their primary function is to activate innate immune responses while also involved in facilitating adaptive immune responses. Different TLRs exert distinct functions by activating varied immune cascades. Several TLRs are being pursued as cancer drug targets. We discovered a novel, highly potent and selective small molecule TLR8 agonist DN052. DN052 exhibited strong in vitro cellular activity with EC50 at 6.7 nM and was highly selective for TLR8 over other TLRs including TLR4, 7 and 9. DN052 displayed excellent in vitro ADMET and in vivo PK profiles. DN052 potently inhibited tumor growth as a single agent. Moreover, combination of DN052 with the immune checkpoint inhibitor, selected targeted therapeutics or chemotherapeutic drugs further enhanced efficacy of single agents. Mechanistically, treatment with DN052 resulted in strong induction of pro-inflammatory cytokines in ex vivo human PBMC assay and in vivo monkey study. GLP toxicity studies in rats and monkeys demonstrated favorable safety profile. This led to the advancement of DN052 into phase 1 clinical trials.

Better adjuvants are needed for vaccines against seasonal influenza. TLR7 agonists are potent activators of innate immune responses and thereby may be promising adjuvants. Among the imidazoquinoline compounds, 1-benzyl-2-butyl-1H-imidazo[4,5-c]quinolin-4-amine (BBIQ) was reported to be a highly active TLR7 agonist but has remained relatively unexplored because of its commercial unavailability. Indeed, in silico molecular modeling studies predicted that BBIQ had a higher TLR7 docking score and binding free energy than imiquimod, the gold standard TLR7 agonist. To circumvent the availability issue, we developed an improved and higher yield method to synthesize BBIQ. Testing BBIQ on human and mouse TLR7 reporter cell lines confirmed it to be TLR7 specific with significantly higher potency than imiquimod. To test its adjuvant potential, BBIQ or imiquimod were admixed with recombinant influenza hemagglutinin protein and administered to mice as two intramuscular immunizations 2 weeks apart. Serum anti-influenza IgG responses assessed by ELISA 2 weeks after the second immunization confirmed that the mice that received vaccine admixed with BBIQ had significantly higher anti-influenza IgG1 and IgG2c responses than mice immunized with antigen alone or admixed with imiquimod. This confirmed BBIQ to be a TLR7-specific adjuvant able to enhance humoral immune responses.

Objective: Silymarin is a herbal extract containing flavonolignans, and it has inhibitory effects against the growth of different cancer cell lines by inducing apoptosis. Toll-like receptors are suggested as a novel and attractive target to treat cancer. The current study aimed at examining the mechanism of silymarin-induced apoptosis in Ramos cells and investigating its effects on TLR8 expression. Materials and Methods: The half maximal inhibitory concentration (IC50) of silymarin in Ramos cells was determined via MTT viability test while the type of cell death was tested by annexin V/propidium iodide (PI) double staining method. The activity of caspase-3 and expression of TLR8 were measured in a time-dependent manner (in IC50) by colorimetric assay and real-time polymerase chain reaction (RT-PCR), respectively. Results: The results of MTT showed that IC50 of silymarin in Ramos cells was 100 μg/ml after 48 hr treatment (pConclusion: The results indicated a new mechanism in the anticancer activity ofToll-like receptor (TLR) signaling after silymarin treatment in Ramos cancer cell line. This plant could be used to develop anticancer agents inhibiting TLRs.

Objective: Septic shock is the severe complication of sepsis, with a high mortality. The inflammatory response regulates the immune status and mediates the progression of septic shock. In this study, we aim to identify the key immune-related genes (IRGs) of septic shock and explore their potential mechanism.Methods: Gene expression profiles of septic shock blood samples and normal whole blood samples were retrieved from the Gene Expression Omnibus (GEO) and Genotype-Tissue Expression Portal (GTEx). The differential expression genes (DEGs) and septic shock-specific immune-related genes (SSSIRGs) were evaluated and identified, along with the immune components by “cell type identification by estimating relative subsets of RNA transcripts (CIBERSORT, version x)” algorithm. Additionally, in order to explore the key regulatory network, the relationship among SSSIRGs, upstream transcription factors (TFs), and downstream signaling pathways were also identified by Gene Set Variation Analysis (GSVA) and co-expression analysis. Moreover, the Connectivity Map (CMap) analysis was applied to find bioactive small molecules against the members of regulation network while Chromatin Immunoprecipitation sequencing (ChIP-seq) and Assay for Targeting Accessible-Chromatin with high-throughput sequencing (ATAC-seq) data were used to validate the regulation mechanism of the network.Results: A total of 14,843 DEGs were found between 63 septic shock blood samples and 337 normal whole blood samples. Then, we identified septic shock-specific 839 IRGs as the intersection of DEGs and IRGs. Moreover, we uncovered the regulatory networks based on co-expression analysis and found 28 co-expression interaction pairs. In the regulation network, protein phosphatase 3, catalytic subunit, alpha isozyme (PPP3CA) may regulate late estrogen response, glycolysis and TNFα signaling via NFκB and HLA; Kirsten rat sarcoma viral oncogene homolog (KRAS) may be related to late estrogen response and HLA; and Toll-like receptor 8 (TLR8) may be associated with TNFα signaling via NFκB. And the regulation mechanisms between TFs and IRGs (TLR8, PPP3CA, and KRAS) were validated by ChIP-seq and ATAC-seq.Conclusion: Our data identify three SSSIRGs (TLR8, PPP3CA, and KRAS) as candidate therapeutic targets for septic shock and provide constructed regulatory networks in septic shock to explore its potential mechanism.

COVID-19 disease is characterized by a dysregulation of the innate arm of the immune system. However, the mechanisms whereby innate immune cells, including neutrophils, become activated in patients are not completely understood. Recently, we showed that GU-rich RNA sequences from the SARS-CoV-2 genome (i.e., SCV2-RNA1 and SCV2-RNA2) activate dendritic cells. To clarify whether human neutrophils may also represent targets of SCV2-RNAs, neutrophils were treated with either SCV2-RNAs or, as a control, R848 (a TLR7/8 ligand), and were then analyzed for several functional assays and also subjected to RNA-seq experiments. Results highlight a remarkable response of neutrophils to SCV2-RNAs in terms of TNFα, IL-1ra, CXCL8 production, apoptosis delay, modulation of CD11b and CD62L expression, and release of neutrophil extracellular traps. By RNA-seq experiments, we observed that SCV2-RNA2 promotes a transcriptional reprogramming of neutrophils, characterized by the induction of thousands of proinflammatory genes, similar to that promoted by R848. Furthermore, by using CU-CPT9a, a TLR8-specific inhibitor, we found that SCV2-RNA2 stimulates neutrophils exclusively via TLR8-dependent pathways. In sum, our study proves that single-strand RNAs from the SARS-CoV-2 genome potently activate human neutrophils via TLR8, thus uncovering a potential mechanism whereby neutrophils may contribute to the pathogenesis of severe COVID-19 disease.

The prognosis of COVID-19 could influence by innate immune sensors such as toll-like receptors (TLRs). The purpose of this data was to investigate TLR3, 7, and 8 expression levels in COVID-19 patients and their relationship to outcome of disease. 75 confirm COVID-19 were included sequentially and separated into three groups: mild, severe, and critical. Peripheral blood mononuclear cells were isolated from the whole blood, and RNA was then extracted. The qRT-PCR technique was used to examine the expression of TLR3, TLR7, and TLR8 genes. The patients average ages were 52.69 ± 1.9 and 13 of the 25 individuals in each group were male. TLR3 ( p < 0.001), TLR7 ( p < 0.001), and TLR8 ( p < 0.001) expression levels were considerably greater in COVID-19 patients compared to the control group. The findings also showed that individuals with critical and severe COVID-19 disease had significantly greater TLR7 and TLR8 gene expression levels than patients in mild stage of disease ( p < 0.05). The data showed a significant difference ( p = 0.01) in the TLR3 transcript levels between critical and mild COVID-19 patients. Furthermore, male severe ( p = 0.02) and critical (p = 0.008) patients had significantly higher TLR8 expression levels than female patients in terms of gender. TLR3 ( p = 0.2) and TLR7 ( p = 0.08) transcripts were more elevated in males than females, but not significantly., (© 2024 Published by Elsevier Inc.)

Objectives Evaluation of the anti-Leishmanial activity of imidazoquinoline-based TLR7/8 agonists. Methods TLR7/8-active imidazoquinolines (2 and 3) were synthesized and assessed for activity against Leishmania amazonensis-intracellular amastigotes using mouse peritoneal macrophages. The production of reactive oxygen species (ROS), nitric oxide (NO) and cytokines was determined in infected and non-infected macrophages. Key findings The imidazoquinolines, 2 and 3, were primarily agonists of TLR7 with compound 3 also showing modest TLR8 activity. Docking studies showed them to occupy the same binding pocket on TLR7 and 8 as the known agonists, imiquimod and resiquimod. Compounds 2 and 3 inhibited the growth of L. amazonensis-intracellular amastigotes with the most potent compound (3, IC50 = 5.93 µM) having an IC50 value close to miltefosine (IC50 = 4.05 µM), a known anti-Leishmanial drug. Compound 3 induced macrophages to produce ROS, NO and inflammatory cytokines that likely explain the anti-Leishmanial effects. Conclusions This study shows that activating TLR7 using compounds 2 or 3 induces anti-Leishmanial activity associated with induction of free radicals and inflammatory cytokines able to kill the parasites. While 2 and 3 had a very narrow cytotoxicity window for macrophages, this identifies the possibility to further develop this chemical scaffold to less cytotoxic TLR7/8 agonist for potential use as anti-Leishmanial drug. [ABSTRACT FROM AUTHOR]

Toll‐like receptors (TLRs) are components of innate immunity, but also have a role in carcinogenesis. The prognostic value of TLR5 and TLR8 tumor expression was examined in contrast with known risk markers Ki67 and p53. All HCC patients from Oulu University Hospital with available representative tumor sample were included in this study (n = 182). TLR5, TLR8, Ki67, and p53 expression were investigated by immunohistochemistry. The relation between patient survival and TLR, Ki67, and p53 expression was calculated with Cox regression adjusted for confounding factors. TLR5 cytoplasm intensity was associated with 5‐year overall (strong 0.0% vs weak 23.4%, p < 0.001) and disease‐specific (strong 0.0% vs weak 34.9%, p < 0.001) survival. TLR5 nuclei percentage was associated with poor 5‐year disease‐specific survival (high 16.3% vs low 31.5%, p = 0.022). In adjusted analysis, strong TLR5 cytoplasm intensity was an independent risk factor for poor 5‐year overall (adjusted HR 1.88, 95% CI 1.26–2.81) and disease‐specific (adjusted HR 2.00, 95% CI 1.27–3.15) survival. High Ki67 and p53 expression associated with 5‐year overall‐ and disease‐specific survival. TLR8 was not associated with patient survival. This study suggests that TLR5 expression is independently prognostic in HCC with similar point estimate as previously known p53. [ABSTRACT FROM AUTHOR]

Immune stimulation triggered by siRNAs is one of the major challenges in the development of safe RNAi-based therapeutics. Within an immunostimulatory siRNA sequence, this hurdle is commonly addressed by using ribose modifications (e.g., 2'-OMe or 2'-F), which results in decreased cytokine production. However, as immune stimulation by siRNAs is a sequence-dependent phenomenon, recognition of the nucleobases by the trigger receptor(s) is also likely. Here, we use the recently published crystal structures of Toll-like receptor 8 (TLR8) bound to small-molecule agonists to generate computational models for ribonucleotide binding by this immune receptor. Our modeling suggested that modification of either the Watson-Crick or Hoogsteen face of adenosine would disrupt nucleotide/TLR8 interactions. We employed chemical synthesis to alter either the Watson-Crick or Hoogsteen face of adenosine and evaluated the effect of these modifications in an siRNA guide strand by measuring the immunostimulatory and RNA interference properties. For the siRNA guide strand tested, we found that modifying the Watson-Crick face is generally more effective at blocking TNFα production in human peripheral blood mononuclear cells (PBMCs) than modification at the Hoogsteen edge. We also observed that modifications near the 5'-end were more effective at blocking cytokine production than those placed at the 3'-end. This work advances our understanding of how chemical modifications can be used to optimize siRNA performance.

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV2) is the causative agent of Corona Virus Disease 2019 (COVID-19). Lower production of type I and III interferons and higher levels of inflammatory mediators upon SARS-CoV2 infection contribute to COVID-19 pathogenesis. Optimal interferon production and controlled inflammation are essential to limit COVID-19 pathogenesis. However, the aggravated inflammatory response observed in COVID-19 patients causes severe damage to the host and frequently advances to acute respiratory distress syndrome (ARDS). Toll-like receptor 7 and 8 (TLR7/8) signaling pathways play a central role in regulating induction of interferons (IFNs) and inflammatory mediators in dendritic cells. Controlled inflammation is possible through regulation of TLR mediated response without influencing interferon production to reduce COVID-19 pathogenesis. This review focuses on inflammatory mediators that contribute to pathogenic effects and the role of TLR pathways in the induction of interferon and inflammatory mediators and their contribution to COVID-19 pathogenesis. We conclude that potential TLR7/8 agonists inducing antiviral interferon response and controlling inflammation are important therapeutic options to effectively eliminate SARS-CoV2 induced pathogenesis. Ongoing and future studies may provide additional evidence on their safety and efficacy to treat COVID-19 pathogenesis.

Recognition of RNA by receptors of the innate immune system is regulated by various posttranslational modifications. Different single 2′-O-ribose (2′-O-) methylations have been shown to convert TLR7/TLR8 ligands into specific TLR8 ligands, so we investigated whether the position of 2′-O-methylation is crucial for its function. To this end, we designed different 2′-O-methylated RNA oligoribonucleotides (ORN), investigating their immune activity in various cell systems and analyzing degradation under RNase T2 treatment. We found that the 18S rRNA-derived TLR7/8 ligand, RNA63, was differentially digested as a result of 2′-O-methylation, leading to variations in TLR8 and TLR7 inhibition. The suitability of certain 2′-O-methylated RNA63 derivatives as TLR8 agonists was further demonstrated by the fact that other RNA sequences were only weak TLR8 agonists. We were thus able to identify specific 2′-O-methylated RNA derivatives as optimal TLR8 ligands.

Trigeminal neuropathic pain (TNP) is a significant health problem but the involved mechanism has not been completely elucidated. Toll-like receptors (TLRs) have recently been demonstrated to be expressed in the dorsal root ganglion and involved in chronic pain. Here, we show that TLR8 was persistently increased in the trigeminal ganglion (TG) neurons in model of TNP induced by partial infraorbital nerve ligation (pIONL). In addition, deletion or knockdown of Tlr8 in the TG attenuated pIONL-induced mechanical allodynia, reduced the activation of ERK and p38-MAPK, and decreased the expression of pro-inflammatory cytokines in the TG. Furthermore, intra-TG injection of the TLR8 agonist VTX-2337 induced pain hypersensitivity. VTX-2337 also increased the intracellular Ca2+ concentration, induced the activation of ERK and p38, and increased the expression of pro-inflammatory cytokines in the TG. These data indicate that TLR8 contributes to the maintenance of TNP through increasing MAPK-mediated neuroinflammation. Targeting TLR8 signaling may be effective for the treatment of TNP. [ABSTRACT FROM AUTHOR]

Toll-like receptor 8 (TLR8) recognizes single-stranded RNA of viral and bacterial origin as well as mediates the secretion of pro-inflammatory cytokines and type I interferons by human monocytes and macrophages. TLR8, as other endosomal TLRs, utilizes the MyD88 adaptor protein for initiation of signaling from endosomes. Here, we addressed the potential role of the Toll-interleukin 1 receptor domain-containing adaptor protein (TIRAP) in the regulation of TLR8 signaling in human primary monocyte-derived macrophages (MDMs). To accomplish this, we performed TIRAP gene silencing, followed by the stimulation of cells with synthetic ligands or live bacteria. Cytokine-gene expression and secretion were analyzed by quantitative PCR or Bioplex assays, respectively, while nuclear translocation of transcription factors was addressed by immunofluorescence and imaging, as well as by cell fractionation and immunoblotting. Immunoprecipitation and Akt inhibitors were also used to dissect the signaling mechanisms. Overall, we show that TIRAP is recruited to the TLR8 Myddosome signaling complex, where TIRAP contributes to Akt-kinase activation and the nuclear translocation of interferon regulatory factor 5 (IRF5). Recruitment of TIRAP to the TLR8 signaling complex promotes the expression and secretion of the IRF5-dependent cytokines IFNβ and IL-12p70 as well as, to a lesser degree, TNF. These findings reveal a new and unconventional role of TIRAP in innate immune defense.

While chronic kidney disease is prevalent in adults, obstructive nephropathy (ON) has been reported in both young and old patients. In ON, tubulointerstitial lesions (TILs) have been widely investigated, but glomerular lesions (GLs) have been largely neglected. Here, we show a novel mechanism underlying GL development in ON in young and old mice. TILs develop earlier than GLs owing to infiltration of inflammatory cells in the tubulointerstitium, but GLs develop following the activation of Toll-like receptor 8 (Tlr8) even though the absence of inflammatory cells infiltrating the glomerulus. TLR8 and interleukin 1 beta (IL1β) proteins colocalize with reducing podocyte function markers (PFMs), indicating the activation of TLR8 signaling in injured podocytes. Furthermore, glomerular and serum levels of miR-21, an endogenous ligand for Tlr8, were higher in the ON mouse model than in the sham control. The glomerular expression of Tlr8 positively correlates with miR-21 and the downstream cytokines Il1b and Il6 and negatively correlated with PFMs (Nphs1 and Synpo). We also show the colocalization of TLR8 and IL1β proteins with reducing PFMs in both obstructed and collateral kidney of young and old mice. Furthermore, in vitro study results revealed higher expression of Tlr8 and its downstream cytokines in glomeruli from obstructed kidneys following treatment with miR-21 mimic than in the control. In conclusion, the overexpression of Tlr8 may serve as a plausible mechanism underlying GL development in ON through podocyte injury.

The COVID-19 pandemic is an ongoing global pandemic of coronavirus disease 2019 as an atypical type of viral pneumonia caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Many potential pharmacotherapies are currently being investigated against this disease. This article points to and justifies, the importance of investigating the potential therapeutic value of pharmacological agents acting on Toll-like Receptor (TLR) 7 and/or TLR8 as double-edged swords combating COVID-19. Induction of TLR7 and/or TLR8 may be investigated as a strategy to stimulate immunity and may be added to anti-COVID19 vaccines to cope with their current viral escape challenge. TLR7 stimulation may not only help viral clearance through Th1 antiviral responses but may also provide beneficial broncho- and vaso-dilatory, as well as, anti-inflammatory effects. Pharmacological compounds acting as TLR7 and/or TLR8 agonists may be of value if used by frontline healthcare workers with comorbidities who demonstrate mild symptoms of the disease. On the other hand, TLR7 and/or TLR8 antagonists may be used in combination with immune-modulatory/anti-inflammatory drugs in severe cases of the disease, with potential synergistic effects that could also help in reducing the doses of such therapies, and consequently their adverse effects.

While chronic kidney disease is prevalent in adults, obstructive nephropathy (ON) has been reported in both young and old patients. In ON, tubulointerstitial lesions (TILs) have been widely investigated, but glomerular lesions (GLs) have been largely neglected. Here, we show a novel mechanism underlying GL development in ON in young and old mice. TILs develop earlier than GLs owing to infiltration of inflammatory cells in the tubulointerstitium, but GLs develop following the activation of Toll-like receptor 8 (Tlr8) even though the absence of inflammatory cells infiltrating the glomerulus. TLR8 and interleukin 1 beta (IL1β) proteins colocalize with reducing podocyte function markers (PFMs), indicating the activation of TLR8 signaling in injured podocytes. Furthermore, glomerular and serum levels of miR-21, an endogenous ligand for Tlr8 , were higher in the ON mouse model than in the sham control. The glomerular expression of Tlr8 positively correlates with miR-21 and the downstream cytokines Il1b and Il6 and negatively correlated with PFMs (Nphs1 and Synpo). We also show the colocalization of TLR8 and IL1β proteins with reducing PFMs in both obstructed and collateral kidney of young and old mice. Furthermore, in vitro study results revealed higher expression of Tlr8 and its downstream cytokines in glomeruli from obstructed kidneys following treatment with miR-21 mimic than in the control. In conclusion, the overexpression of Tlr8 may serve as a plausible mechanism underlying GL development in ON through podocyte injury. [ABSTRACT FROM AUTHOR]

Toll-like receptors (TLRs) are the largest pattern recognition receptors responsible for activating the innate and adaptive immune response against viruses through the release of inflammatory cytokines and antiviral mediators. Viruses are recognized by several TLRs, including TLR8, which is known to bind ssRNA structures. However, the similarities between TLR8 and TLR7 have obscured the distinctive characteristics of TLR8 activation and its importance in the immune system. Here we discuss the activation and regulation of TLR8 by viruses and its importance in therapeutical options such as vaccine adjuvants and antiviral stimulators.

Background: Long non-coding RNAs (LncRNAs) are generally reported to participate in the development of Osteoarthritis (OA) by acting as competing endogenous RNAs (ceRNAs). However, the molecular mechanism is largely unknown. This study aimed to investigate the possible mechanisms contributing to osteoarthritis (OA)., Methods: Four gene expression profiles from patients with OA were downloaded from a public database and integrated to screen important RNAs associated with OA. Differentially expressed (DE) lncRNAs, microRNAs (miRNAs), and mRNAs were filtered, and a ceRNA network was constructed. An in vitro OA model was established by treating chondrocytes with IL-1β. The expression levels of MMP-13, COL2A1, aggrecan, and RUNX2 were detected by qRT-PCR and western blot. Cell proliferation ability was detected by CCK-8 assay. Flow cytometry was used for apoptosis assay. A dual luciferase reporter gene was used to confirm the relationship between DLEU1, miR-492, and TLR8 ., Results: An OA-related ceRNA network, including 11 pathways, 3 miRNAs, 7 lncRNAs, and 16 mRNAs, was constructed. DLEU1 and TLR8 were upregulated, and miR-492 was downregulated in IL-1β-induced chondrocytes. Overexpression of DLEU1 suppressed viability and promoted apoptosis and extracellular matrix (ECM) degradation in IL-1β induced chondrocytes. Luciferase reporter assay validated the regulatory relations among DLEU1, miR-492, and TLR8 . Further study revealed that the effects of DLEU1 on chondrocytes could be reversed by miR-492., Conclusion: DLEU1 may be responsible for the viability, apoptosis, and ECM degradation in OA via miR-492/ TLR8 axis., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

• TLR7 and TLR8 play an important role in activating immune response, • TLR7 polymorphisms were not associated with susceptibility to HIV-1 infection, • Met1Val of TLR8 variant modulates HIV-1 infection, • TLR7 rs179009 and TLR8 rs3764880 may modulate HIV-1 RNA viral load in male, • TLR7 and TLR8 polymorphisms not modulate the CD4 cell counts. Toll-like receptors (TLRs) play an important role in activating the innate immune response, inducing inflammation and initiating the adaptive immune response. In this study, we assess the influence of TLR7 and TLR8 gene polymorphisms on HIV-1 susceptibility, AIDS development, and treatment outcomes. The TLR7 and TLR8 single nucleotide polymorphisms (SNPs) were genotyped through real-time PCR in 222 patients living with HIV-1 and 141 healthy controls. Frequencies of the TLR7- IVS2-151 G/A and TLR7 -IVS1 + 1817 G/T genotypes and alleles were not significantly increased in patients with HIV-1 infection compared to healthy controls both in males and females. Whereas, males carrying TLR8 Met allele were twice susceptible to HIV-1 infection compared to subjects with A allele (OR = 2.04, 95 % CI 1.10−3.76; p = 0.021). Interestingly, for TLR8- 129 G/C, both males and females carrying G allele and GG genotype, respectively were significantly associated with HIV-1 infection (p < 0.0001). Moreover, the TLR7 IVS1 + 1817 G/T and the TLR8 rs3764880 were associated with protection to progress the AIDS stage in male and female, respectively (p < 0.05). Males carrying TLR7 IVS2-151-A allele showed a significant increased level of HIV-1 viral load pre-treatment, in comparison with individuals carrying the G allele (p-value = 0.036). Additionally, males carrying TLR8 Met allele showed statistically higher HIV viral load at baseline (p-value = 0.04) and after treatment (p-value = 0.013). Regarding CD4 + T cell counts, no significant association was found with TLR7 and TLR8 SNPs before and after antiretroviral treatment. This data demonstrates that TLR8 polymorphisms could affect HIV-1 infection. Moreover, an association between TLR7 IVS2-151-A and TLR8 Met alleles and plasma HIV viral load level was found. [ABSTRACT FROM AUTHOR]

The IL‐12 family of cytokines plays crucial functions in innate and adaptive immunity. These cytokines include heterodimers sharing distinct α (IL‐12A, IL‐23A, and IL‐27A) with two β (IL‐12B and Epstein‐Barr virus induced gene 3 [EBI3]) chains, respectively, IL‐12 (IL‐12B plus IL‐12A) and IL‐23 (IL‐12B plus IL‐23A) sharing IL‐12B, IL‐27 (EBI3 plus IL‐27A), IL‐35 (EBI3 plus IL‐12A), and IL‐39 (EBI3 plus IL‐23A) sharing EBI3. In this context, we have recently reported that highly pure neutrophils incubated with TLR8 agonists produce functional IL‐23. Previously, we showed that neutrophils incubated with LPS plus IFNγ for 20 h produce IL‐12. Herein, we investigated whether highly pure, TLR8‐activated, neutrophils produce EBI3, and in turn IL‐27, IL‐35, and IL‐39, the IL‐12 members containing it. We report that neutrophils incubated with TLR8 ligands, TNFα and, to a lesser extent, LPS, produce and release remarkable amounts of EBI3, but not IL‐27A, consequently excluding the possibility for an IL‐27 production. We also report a series of unsuccessful experiments performed to investigate whether neutrophil‐derived EBI3 associates with IL‐23A to form IL‐39. Furthermore, we show that neutrophils incubated with IFNγ in combination with either TLR8 or TLR4 ligands express/produce neither IL‐12, nor IL‐35, due to the inability of IFNγ, contrary to previous findings, to activate IL12A transcription. Even IL‐27 was undetectable in supernatants harvested from IFNγ plus R848‐treated neutrophils, although they were found to accumulate IL27A transcripts. Finally, by immunohistochemistry experiments, EBI3‐positive neutrophils were found in discrete pathologies only, including diverticulitis, cholecystitis, Gorham disease, and Bartonella Henselae infection, implying a specific role of neutrophil‐derived EBI3 in vivo. [ABSTRACT FROM AUTHOR]

Introduction: Vaccination is an essential means for prevention of tuberculosis infection, but the effects of various vaccines on the intestinal flora of mice and their response to Mycobacterium tuberculosis (Mtb) infection remain poorly understood. Methodology: In this study, two different vaccinations - ESAT6 and ESAT6 + TLR8 agonists - were administered to mice transgenic for human TLR8 to investigate gut microbiota characteristics following vaccination. Gut microbiota was investigated by next generation sequencing in the MiSeq Sequencing System. Adonis analysis was used to evaluate the effect of variables on gut bacterial community stucture. Chao1, Shannon index, and phylogenetic diversity index were used to explore the gut bacterial diversity. Results: The results showed that different vaccines have significant influence on mice intestinal bacteria (adonis analysis, p < 0.01), with gut bacterial diversity within the ESAT6 + TLR8 agonists group being significantly decreased compared to the ESAT6 treatment group (p < 0.01). Following infection with Mtb via tail vein injection, the bacterial community structure within the control versus vaccinated groups altered significantly (adonis analysis, p < 0.01), and the altered changed genera were markedly different between the groups. Following infection, Bifidobacteria differed between the groups, indicated that they play a vital role in the response to infection. Conclusions: Our results indicated that different vaccines might have distinct influences on intestinal flora, and their role should not be ignored. [ABSTRACT FROM AUTHOR]

Toll-like receptor 8 (TLR8) is a single-stranded RNA sensing receptor and is localized in the cellular compartments, where it encounters foreign or self-nucleic acids and activates innate and adaptive immune responses. However, the mechanism controlling intracellular localization TLR8 is not completely resolved. We previously revealed the intracellular localization of TLR8 ectodomain (ECD), and in this study, we investigated the mechanism of the intracellular localization. Here we found that TLR8 ECDs from different species as well as ECDs from different TLRs are all intracellularly localized, similarly to the full-length porcine TLR8. Furthermore, porcine, bovine, and human TLR8 ECDs are all localized in cell endosomes, reflecting the cellular localization of TLR8. Intriguingly, none of post-translational modifications at single sites, including glycosylation, phosphorylation, ubiquitination, acetylation, and palmitoylation alter porcine TLR8-ECD endosomal localization. Nevertheless, the signal peptide of porcine TLR8-ECD determines its endosomal localization. On the other hand, signaling regulator UNC93B1 also decides the endosomal localization of porcine, bovine, and human TLR8 ECDs. The results from this study shed light on the mechanisms of not only TLR8 intracellular localization but also the TLR immune signaling.