METTL5 promotes cell proliferation, invasion, and migration by up-regulating Toll-like receptor 8 expression in colorectal cancer.

BACKGROUND: N6-methyladenosine (m6A) modification represents the predominant alteration found in eukaryotic messenger RNA and plays a crucial role in the progression of various tumors. However, despite its significance, the comprehensive investigation of METTL5, a key m6A methyltransferase, in colorectal cancer (CRC) remains limited. AIM: To investigate the role of METTL5 in CRC. METHODS: We assessed METTL5 expression levels in clinical samples obtained from CRC patients as well as in CRC cell lines. To elucidate the downstream targets of METTL5, we performed RNA-sequencing analysis coupled with correlation analysis, leading us to identify Toll-like receptor 8 (TLR8) as a potential downstream target. In vitro functional assessments of METTL5 and TLR8 were conducted using CCK-8 assays, scratch assays, as well as assays measuring cell migration and invasion. RESULTS: Our findings reveal a pronounced upregulation of METTL5 expression in both CRC cells and tissues, which correlated significantly with an unfavorable prognosis. In vitro experiments unequivocally demonstrated the oncogenic role of METTL5, as evidenced by its promotion of CRC cell proliferation, invasion, and migration. Notably, we identified TLR8 as a downstream target of METTL5, and subsequent down-regulation of TLR8 led to a significant inhibition of CRC cell proliferation, invasion, and tumor growth. CONCLUSION: The heightened expression of METTL5 in CRC is strongly associated with clinicopathological features and a poor prognosis, thereby underscoring its potential utility as a critical marker for facilitating early diagnosis and prognostication in CRC.

Porcine TLR8 signaling and its anti-infection function are disturbed by immune checkpoint receptor TIM-3 via inhibition of P13K-AKT pathway.

Toll-like receptor 8 (TLR8), an important innate immune receptor recognizing single stranded RNA and the antiviral imidazoquinoline compounds, can activate intracellular signaling pathway and produce an inflammatory response to kill and eliminate pathogens. However, the molecular regulation mechanisms of TLR8 signaling and its anti-infection activity are not fully elucidated. Our previous transcriptome analysis of porcine TLR8 (pTLR8) signaling suggested the immune checkpoint receptor TIM-3 as the potential regulator for pTLR8. Here we investigated TIM-3 in the regulation of pTLR8 signaling and its anti-infection activity. Our results showed that porcine TIM-3 is upregulated by pTLR8 signaling and TIM-3 inhibits pTLR8 signaling activity in a negative feedback way. Accordingly, TIM-3 disturbs pTLR8 mediated anti-bacterial and anti-viral activity. Mechanistically, TIM-3 suppresses PI3K-AKT pathway by inhibiting the TLR8-PI3K p85 interaction and subsequent AKT phosphorylation which is essential for TLR8 signaling and anti-infection activity. Therefore, our study reveals new insights into innate immune TLR8 signaling and its anti-infection function.

TLR8-activating miR-146a-3p is an intermediate signal contributing to fetal membrane inflammation in response to bacterial LPS.

Preterm birth is the largest contributor to neonatal morbidity and is often associated with chorioamnionitis, defined as inflammation/infection of the fetal membranes (FMs). Chorioamnionitis is characterised by neutrophil infiltration of the FMs and is associated with elevated levels of the neutrophil chemoattractant, interleukin (IL)-8 and the proinflammatory cytokine, IL-1ß. While FMs can respond to infections through innate immune sensors, such as toll-like receptors (TLRs), the downstream mechanisms by which chorioamnionitis arises are not fully understood. A novel group of non-classical microRNAs (miR-21a, miR-29a, miR-146a-3p, Let-7b) function as endogenous danger signals by activating the ssRNA viral sensors TLR7 and TLR8. In this study, the pro-inflammatory roles of TLR7/TLR8-activating miRs were examined as mediators of FM inflammation in response to bacterial lipopolysaccharide (LPS) using an in vitro human FM explant system, an in vivo mouse model of pregnancy, and human clinical samples. Following LPS exposure, miR-146a-3p was significantly increased in both human FM explants and wild-type mouse FMs. Expression of miR-146a-3p was also significantly elevated in FMs from women with preterm birth and chorioamnionitis. FM IL-8 and inflammasome-mediated IL-1ß production in response to LPS was dependent on miR-146a-3p and TLR8 downstream of TLR4 activation. In wild-type mice, LPS exposure increased FM IL-8 and IL-1ß production and induced preterm birth. In TLR7-/-/TLR8-/- mice, LPS exposure was able to initiate but not sustain preterm birth, and FM inflammation was reduced. Together, we demonstrate a novel signalling mechanism at the maternal-fetal interface in which TLR8-activating miR-146a-3p acts as an intermediate danger signal to drive FM inflammasome-dependent and -independent mechanisms of inflammation and, thus, may play a role in chorioamnionitis and subsequent preterm birth.

Chlorogenic acid alleviates crayfish allergy by altering the structure of crayfish tropomyosin and upregulating TLR8.

Studies have shown that high hydrostatic pressure (HHP) processing and chlorogenic acid (CA) treatment can effectively reduce food allergenicity. We hypothesize that these novel processing techniques can help tackle crayfish allergy and examined the impact and mechanism of HHP (300 MPa, 15 min) and CA (CA:tropomyosin = 1:4000, 15 min) on the allergenicity of crayfish tropomyosin. Our results revealed that CA, rather than HHP, effectively reduced tropomyosin's allergenicity, as evident in the alleviation of allergic symptoms in a food allergy mouse model. Spectroscopy and molecular docking analyses demonstrated that CA could reduce the allergenicity of tropomyosin by covalent or non-covalent binding, altering its secondary structure (2.1 % decrease in α-helix; 1.9 % increase in ß-fold) and masking tropomyosin's linear epitopes. Moreover, CA-treated tropomyosin potentially induced milder allergic reactions by up-regulating TLR8. While our results supported the efficacy of CA in alleviating crayfish allergy, further exploration is needed to determine clinical effectiveness.

A new therapeutic target for systemic lupus erythematosus: the current landscape for drug development of a toll-like receptor 7/8 antagonist through academia-industry-government collaboration.

Systemic Lupus Erythematosus (SLE) is an autoimmune disease characterized by inflammation in multiple organs. A few treatments for SLE currently exist, including antimalarials, glucocorticoids, immunosuppressants, and two recently approved antibody agents; however, an unmet medical need remains for SLE. In addition, developing new drugs targeting SLE is a challenge since no specific biomarkers exist for the prediction of disease progression or drug response. A new drug candidate, E6742, is a specific antagonist of the toll-like receptors 7/8. To address the challenges for drug development in SLE, the process of developing E6742 utilizes a unique system of the Japan Agency for Medical Research and Development (AMED), the Cyclic Innovation for Clinical Empowerment (CiCLE) program. In the CiCLE program, a Phase 1 study in healthy adults was completed (NCT04683185) and a Phase 1/2 study in patients with SLE is on-going (NCT05278663). One of the potential benefits of this program is to conduct academia-led clinical research to identify specific biomarkers for E6742 in parallel with clinical studies (UMIN000042037). The aim of this review is to present current progress within the strategic collaboration of the AMED CiCLE program that optimize clinical development for patients with SLE.

Antibody preparation and age-dependent distribution of TLR8 in Bactrian camel spleens.

BACKGROUND: Toll-like receptor 8 (TLR8) can recognize specific pathogen-associated molecular patterns and exert multiple immunological functions through activation of signaling cascades. However, the precise distribution and age-related alterations of TLR8 in the spleens of Bactrian camels have not yet been investigated. This study aimed to prepare a rabbit anti-Bactrian camel TLR8 polyclonal antibody and elucidate the distribution of TLR8 in the spleens of Bactrian camels at different age groups. The methodology involved the construction of the pET-28a-TLR8 recombinant plasmid, followed by the expression of TLR8 recombinant protein via prokaryotic expression. Subsequently, rabbits were immunized with the purified protein to prepare the TLR8 polyclonal antibody. Finally, twelve Alashan Bactrian camels were categorized into four groups: young (1-2 years), pubertal (3-5 years), middle-aged (6-16 years) and old (17-20 years). These camels received intravenous sodium pentobarbital (20 mg/kg) anesthesia and were exsanguinated to collect spleen samples. Immunohistochemical techniques were employed to observe and analyze the distribution patterns and age-related changes of TLR8 in the spleen. RESULTS: The results showed that the TLR8 recombinant protein was expressed in the form of inclusion body with a molecular weight of 52 kDa, and the optimal induction condition involved 0.3 mmol/L IPTG induction for 8 h. The prepared antibody yielded a titer of 1:32 000, and the antibody demonstrated specific binding to TLR8 recombinant protein. TLR8 positive cells exhibited a consistent distribution pattern in the spleen across different age groups of Bactrian camels, primarily scattered within the periarterial lymphatic sheath of the white pulp, marginal zone, and red pulp. The predominant cell type expressing TLR8 was macrophages, with expression also observed in neutrophils and dendritic cells. Statistical analysis revealed that there were significant differences in the distribution density of TLR8 positive cells among different spleen regions at the same age, with the red pulp, marginal zone, and white pulp showing a descending order (P<0.05). Age-related changes indicated that the distribution density in the marginal zone and red pulp exhibited a similar trend of initially increasing and subsequently decreasing from young to old camels. As camels age, there was a significant decrease in the distribution density across all spleen regions (P<0.05). CONCLUSIONS: The results confirmed that this study successfully prepared a rabbit anti-Bactrian camel TLR8 polyclonal antibody with good specificity. TLR8 positive cells were predominantly located in the red pulp and marginal zone of the spleen, signifying their pivotal role in the innate immune response of the spleen. Aging was found to significantly reduce the density of TLR8 positive cells, while leaving their scattered distribution characteristics unaffected. These findings provide valuable support for further investigations into the immunomorphology and immunosenescence of the spleen in Bactrian camels.

TLR8 escapes X chromosome inactivation in human monocytes and CD4+ T cells.

BACKGROUND: Human endosomal Toll-like receptors TLR7 and TLR8 recognize self and non-self RNA ligands, and are important mediators of innate immunity and autoimmune pathogenesis. TLR7 and TLR8 are, respectively, encoded by adjacent X-linked genes. We previously established that TLR7 evades X chromosome inactivation (XCI) in female immune cells. Whether TLR8 also evades XCI, however, has not yet been explored. METHOD: In the current study, we used RNA fluorescence in situ hybridization (RNA FISH) to directly visualize, on a single-cell basis, primary transcripts of TLR7 and TLR8 relative to X chromosome territories in CD14+ monocytes and CD4+ T lymphocytes from women, Klinefelter syndrome (KS) men, and euploid men. To assign X chromosome territories in cells lacking robust expression of a XIST compartment, we designed probes specific for X-linked genes that do not escape XCI and therefore robustly label the active X chromosome. We also assessed whether XCI escape of TLR8 was associated with sexual dimorphism in TLR8 protein expression by western blot and flow cytometry. RESULTS: Using RNA FISH, we show that TLR8, like TLR7, evades XCI in immune cells, and that cells harboring simultaneously TLR7 and TLR8 transcript foci are more frequent in women and KS men than in euploid men, resulting in a sevenfold difference in frequency. This transcriptional bias was again observable when comparing the single X of XY males with the active X of cells from females or KS males. Interestingly, TLR8 protein expression was significantly higher in female mononuclear blood cells, including all monocyte subsets, than in male cells. CONCLUSIONS: TLR8, mirroring TLR7, escapes XCI in human monocytes and CD4+ T cells. Co-dependent transcription from the active X chromosome and escape from XCI could both contribute to higher TLR8 protein abundance in female cells, which may have implications for the response to viruses and bacteria, and the risk of developing inflammatory and autoimmune diseases.

Human endosomal Toll-like receptors TLR7 and TLR8, encoded by two adjacent X-linked genes, recognize self and non-self RNA ligands, and are important mediators of innate immunity and autoimmune pathogenesis. We previously reported that TLR7 evades X chromosome inactivation (XCI) in female immune cells, correlating with enhanced functional properties in B cells harboring biallelic expression of this gene. Here, we conducted a comprehensive single-cell resolution analysis of the transcriptional regulation of both TLR7 and TLR8, in CD14⁺ monocytes and CD4⁺ T lymphocytes. We unequivocally demonstrated that TLR8, like TLR7, escapes XCI in immune cells from female and Klinefelter syndrome males. When we analyzed TLR7 and TLR8 transcripts together, cells from women and KS men exhibited higher frequencies of cells co-transcribing the two genes. Surprisingly, these differences were attributable not only to the ability of TLR7 and TLR8 to be expressed on the Xi, but also to the joint transcriptional behavior of the TLR7­TLR8 gene pair on the active X chromosome specifically. This contrasted with a striking pattern of mutually exclusive transcription on the single X of euploid men. Corroborating our RNA FISH results, we found higher TLR8 protein expression in female than in male leukocytes, including all monocyte subpopulations. In summary, our data suggest that sex-biased co-regulation of the Toll-like receptor locus and XCI escape of TLR8 contribute to the sexual dimorphism in TLR8 expression, which may have important consequences for the functional make-up of monocyte and T cell populations.

Regulation of megakaryo/thrombopoiesis by endosomal toll-like receptor 7 and 8 activation of CD34+ cells in a viral infection model.

Background: CD34+ cells, megakaryocytes (MKs), and platelets express toll-like receptors (TLRs) that enable these cells to amplify the host innate immune response. However, the role of TLR7/TLR8 activation in megakaryopoiesis has not yet been investigated. Objectives: We evaluated the effect of coxsackievirus B3 (CVB3) and synthetic TLR7/TLR8 agonists on the development of human MKs and production of platelets. Methods: CD34+ cells from human umbilical cord were inoculated with CVB3 or stimulated with synthetic TLR7/TLR8 agonists and then cultured in the presence of thrombopoietin. Results: CD34+ cells, MK progenitor cells, and mature MKs expressed TLR7 and TLR8, and exposure to CVB3 resulted in productive infection, as determined by the presence of viral infectious particles in culture supernatants. Cell expansion, differentiation into MKs, MK maturation, and platelet biogenesis were significantly reduced in CD34+-infected cultures. The reduction in MK growth was not due to an alteration in cellular proliferation but was accompanied by an increase in cellular apoptosis and pyroptosis. Impairment of MK generation and maturation of viable cells were also associated with decreased expression of transcription factors involved in these processes. These effects were completely abrogated by TLR7 but not TLR8 antagonists and mimicked by TLR7 but not TLR8 agonists. CVB3 infection of CD34+ cells increased the immunophenotype of MKs characterized as CD148+/CD48+ or CD41+/CD53+ cells. Conclusion: These data suggest a novel role of TLR7 in megakaryo/thrombopoiesis that may contribute to a better understanding of the molecular basis underlying thrombocytopenia and the immunologic role of MKs in viral infection processes.

Glucose metabolism and function of CD4+ Tregs are regulated by the TLR8/mTOR signal in an environment of SKOV3 cell growth.

PURPOSE: To investigate the role of mammalian target of rapamycin (mTOR) signal in Toll-like receptor (TLR) 8-mediated regulation of glucose metabolism and its effect on reversing immunosuppression in CD4+ regulatory T-cells (Tregs) in ovarian cancer (OC). METHODS: Fluorescence-activated cell sorting was used to detect the expression levels of mTOR+ and 4E-BP1+ cells in CD4+ Tregs. The prognosis and immune infiltration analysis of mTOR mRNA in OC were performed using the TIMER and Kaplan-Meier plotter database. Furthermore, real-time polymerase chain reaction (RT-PCR) and western blot (WB) were used to detect expression levels of glucose metabolism-related genes and proteins in CD4+ Tregs. Glucose uptake and glycolysis levels were detected by colorimetry, while the effects of CD4+ Tregs on the proliferation of CD4+ T-effector cells (Teffs) were evaluated by carboxyfluorescein diacetate succinimidyl ester (CFSE). RESULTS: mTOR expression in CD4+ Tregs was significantly higher in patients with OC compared with controls and in CD4+ Tregs than in CD4+ Teffs in OC. Additionally, the expression level of mTOR mRNA was related to prognosis and immune infiltration levels in patients with OC. Blocking the mTOR signal resulted in downregulation of glucose metabolism in CD4+ Tregs. Simultaneous inhibition of the mTOR signal while activation of the TLR8 signal had a coordinated inhibitory effect on glucose metabolism and the immunosuppressive function of CD4+ Tregs. Furthermore, the mTOR signal played an essential role in TLR8-mediated reversal of immunosuppressive function in CD4+ Tregs. CONCLUSION: These findings imply that activation of the TLR8 signal inhibits glucose metabolism in CD4+ Tregs by downregulating mTOR signaling, thereby reversing the immunosuppressive function of these cells in an OC cell growth environment.

Identification of new potent agonists for toll-like receptor 8 by virtual screening methods, molecular dynamics simulation, and MM-GBSA.

Toll-like receptor 8 (TLR8), as an endosomal transmembrane receptor, plays a crucial role in the innate immune response to neoplasia and viruses. Previous studies have shown that TLR8 agonists e.g. Motolimod can be used to treat patients with last-stage cancer. In this study, in order to find new suitable ligands for TLR8, 16 PBD codes related to TLR8 complexes were collected to design the pharmacophore models using the Pharmit server. Then the PubChem, and ZINC databases were screened by them. Subsequently, the ADME-Tox features of the compounds were detected using FAF-Drugs4 and the selected compounds were docked to TLR8 (PDB: 3w3j). Molecular dynamics simulation was used to compare compounds with the best docking scores, with Motolimod in complex with TLR8. Finally, two compounds were identified, PubChem: 124126919 (A) and PubChem: 18559540 (B), each with advantages over Motolimod. As the RMSD results showed that compound A has very good flexibility, in terms of energy calculated using the MM-GBSA method, complex B and TLR8 showed the lowest energy level compared to the rest of the complexes. These observations suggest that these two compounds could be used as TLR8 agonists with the desired pharmacological features in future experimental studies.Communicated by Ramaswamy H. Sarma.

First-in-Human Study of the Safety, Tolerability, Pharmacokinetics, and Pharmacodynamics of E6742, a Dual Antagonist of Toll-like Receptors 7 and 8, in Healthy Volunteers.

The first-in-human phase I study for E6742, a dual toll-like receptor (TLR) 7 and TLR8 antagonist, has been conducted to assess the safety, tolerability, and pharmacokinetics of E6742 in healthy volunteers. In a single ascending dose (SAD) study, 42 subjects received 10-800 mg of E6742 in the fasted state, as well as a 100-mg cohort in the fed state for evaluating the effect of food. In a multiple ascending dose (MAD) study, 18 subjects received 100-400 mg of E6742 twice daily for 7 days. E6742 was rapidly absorbed with a median tmax ranging from 1.50 to 2.50 hours across dose groups under the fasted condition, and eliminated with a median t½ ranging from 2.37 to 14.4 hours. After multiple oral doses, a steady state was reached by day 7. In the SAD study, dose proportionality was observed for Cmax , AUC(0-t) , and AUC(0-inf) values of E6742 up to 800 mg, but these values were slightly less than dose proportional at 10 mg. In the MAD study, the Cmax and AUC(0-12h)ss of E6742 appeared to be almost dose proportionally increased between 100 and 200 mg, while these parameters showed more than a dose proportional increase at 400 mg. In addition to safety and good tolerability, this study demonstrated cytokine concentrations in cultured peripheral blood in response to E6742 were suppressed in a dose-dependent manner. Further clinical studies targeting systemic lupus erythematosus patients are currently underway.

COVID-19 Demonstrates That Inflammation Is a Hyperviscous State.

Many of the complications of severe coronavirus disease-2019 (COVID-19) are caused by blood hyperviscosity driven by marked hyperfibrinogenemia. This results in a distinctive hyperviscosity syndrome which affects areas of high and low shear. A change in blood viscosity causes a threefold inverse change in blood flow, which increases the risk of thrombosis in both arteries and veins despite prophylactic anticoagulation. Increased blood viscosity decreases perfusion of all tissues, including the lungs, heart, and brain. Decreased perfusion of the lungs causes global ventilation-perfusion mismatch which results in silent hypoxemia and decreased efficacy of positive pressure ventilation in treating pulmonary failure in COVID-19. Increased blood viscosity causes a mismatch in oxygen supply and demand in the heart, resulting in myocarditis and ventricular diastolic dysfunction. Decreased perfusion of the brain causes demyelination because of a sublethal cell injury to oligodendrocytes. Hyperviscosity can cause stasis in capillaries, which can cause endothelial necrosis. This can lead to the rarefaction of capillary beds, which is noted in "long-COVID." The genome of the virus which causes COVID-19, severe acute respiratory syndrome coronavirus 2, contains an extraordinarily high number of the oligonucleotide virulence factor 5'-purine-uridine-uridine-purine-uridine-3', which binds to toll-like receptor 8, hyperactivating innate immunity. This can lead to a marked elevation in fibrinogen levels and an increased prevalence of neutrophil extracellular traps in pulmonary failure, as seen in COVID-19 patients.

AMP5A modulates Toll-like receptors 7 and 8 single-stranded RNA immune responses in PMA-differentiated THP-1 and PBMC.

Background: Dysregulation of antiviral immunity has been implicated in the progression of acute respiratory syndrome coronavirus 2 infection into severe cases of coronavirus disease of 2019 (COVID-19). Imbalances in the inflammatory response drive the overabundant production of pro-inflammatory cytokines and chemokines. The low molecular weight fraction of 5% human serum albumin commercial preparation (AMP5A) is a novel biologic drug currently under clinical investigation for the treatment of osteoarthritis and the hyperinflammatory response associated with COVID-19. This study aims to elucidate AMP5A effects following the activation of immune cells with agonists of Toll-like receptor (TLR) 7 and/or 8, which detect ssRNA viral sequences. Methods: CXCL10 ELISAs were used to evaluate the dynamics of myeloid cells activated with CL075 and CL307, agonists of TLR7/8 and TLR7, respectively. In addition, enrichment analysis of gene sets generated by ELISA arrays was utilized to gain insight into the biologic processes underlying the identified differentially expressed cytokine profiles. Finally, relative potency (REP) was employed to confirm the involvement of mechanisms of action paramount to AMP5A activity. Results: AMP5A inhibits the release of CXCL10 from both CL075- and CL307-activated PMA-differentiated THP-1 and peripheral blood mononuclear cells. Furthermore, AMP5A suppresses a distinct set of pro-inflammatory cytokines (including IL-1ß, IL-6, IL-12, and CXCL10) associated with COVID-19 and pro-inflammatory NF-κB activation. REP experiments using antagonists specific for the immunomodulatory transcription factors, peroxisome proliferator-activated receptor γ, and aryl hydrocarbon receptor, also indicate that these pathways are involved in the ability of AMP5A to inhibit CXCL10 release. Conclusion: Due to the biphasic course of COVID-19, therapeutic approaches that augment antiviral immunity may be more beneficial early in infection, whereas later interventions should focus on inflammation suppression. In this study, we show that AMP5A inhibits TLR 7/8 signaling in myeloid cells, resulting in a decrease in inflammatory mediators associated with hyperinflammation and autoimmunity. Furthermore, data demonstrating that AMP5A activates immunomodulatory transcription factors found to be protective in lung disease is provided. These findings suggest that the modes and mechanisms of action of AMP5A are well suited to treat conditions involving dysregulated TLR 7/8 activation.

Photoactivation of Innate Immunity Receptor TLR8 in Live Mammalian Cells by Genetic Encoding of Photocaged Tyrosine.

The effectiveness of innate immune responses relies on an intricate balance between activation and regulation. TLR8, a member of the Toll-like receptor (TLR) family, plays a fundamental role in host defense by sensing viral single-stranded RNAs (ssRNAs). However, the molecular recognition and regulatory mechanism of TLR8 is not fully understood, especially in a whole-cell environment. Here, we engineer the first light-controllable TLR8 model by genetically encoding a photocaged tyrosine, NBY, into specific sites of TLR8. In the caged forms, the activity of TLR8 is masked but can be restored upon decaging by exposure to UV light. To explain the mechanism clearly, we divide the sites with light responsiveness into three groups. They can separately block the ligands that bind to the pockets of TLR8, change the interaction modes between two TLR8 protomers, and interfere with the interactions between TLR8 cytosolic domains with its downstream adaptor. Specifically, we use this chemical caging strategy to probe and evaluate the function of several tyrosine sites located at the interface of TLR8 homodimers with a previously unknown regulatory mode, which may provide a new strategy for TLR8 modulator development. Effects on downstream signaling pathways are monitored at the transcriptional and translational levels in various cell lines. By photoactivating specific cells within a larger population, this powerful tool can provide novel mechanistic insights, with potential in biotechnological and pharmaceutical applications.

Expression of Toll-like receptor 8 in diffuse large B-cell lymphoma and its clinical significance / 白血病·淋巴瘤

Objective:To investigate the expression of Toll-like receptor 8 (TLR8) in diffuse large B-cell lymphoma (DLBCL) and its correlation with clinicopathological characteristics and prognosis of patients.Methods:The data in the Oncomine database was used to analyze the difference of TLR8 mRNA expression between DLBCL tumor tissues and normal lymphocytes, and the result was verified in two independent subsets GSE 25638 and GSE 32018 of the NCBI-GEO database. The OSDLBCL online survival analysis tool was used to analyze the correlation of TLR8 mRNA relative expression level with overall survival (OS) and progression-free survival (PFS) of DLBCL patients. Gene ontology bioprocess (GO_BP) enrichment analysis was performed by using GSEA software. The correlation of TLR8 mRNA expression with tumor immune cell infiltration degree and immune checkpoint-related molecule expression was analyzed by TIMER online tool website. A total of 53 DLBCL patients who underwent lymph node biopsy in Yancheng No. 1 People's Hospital from June 2020 to June 2021 were selected. Immunohistochemistry was used to detect the expression of TLR8 protein, and its relationship with the clinicopathological characteristics of patients was analyzed.Results:The analysis result of data from Oncomine and GEO databases showed that the relative expression levels of TLR8 mRNA in tumor tissues of patients with DLBCL or activated B cell-like DLCBL were higher than those in normal lymphocytes (all P < 0.001). The results of OSDLBCL online survival analysis indicated that the OS ( P = 0.020) and PFS ( P = 0.004) in DLBCL patients with high TLR8 mRNA expression were worse than those in patients with low TLR8 mRNA expression. The level of TLR8 was related to the abnormal function of immune response, cytokine metabolism and DNA damage monitoring; the result of TIMER online analysis showed that the expression level of TLR8 mRNA was positively related to the degree of neutrophil infiltration ( r = 0.78, P < 0.001) and the expression of immunosuppressive molecules [HAVCR2 ( r = 0.85, P < 0.001), LAG3 ( r = 0.63, P < 0.001), CD274 ( r = 0.77, P < 0.001), TIGIT ( r = 0.32, P = 0.037), and C10ORF54 ( r = 0.34, P = 0.029)]. Among 53 DLBCL patients, 29 patients (54.7%) had low expression of TLR8 protein and 24 patients (45.3%) had high expression of TLR8 protein. There were statistical differences in the expressions of TLR8 protein in DLBCL patients with different serum lactate dehydrogenase and β 2-microglobulin levels (both P < 0.05). Conclusions:TLR8 is highly expressed in DLBCL patients, and TLR8 may be a prognostic marker of DLBCL.

Oral Selective TLR8 Agonist Selgantolimod Induces Multiple Immune Cell Responses in Humans.

TLR8 agonists have the potential for use as immunomodulatory components in therapeutic modalities for viral infections such as chronic HBV (CHB) and HIV. In this study, using peripheral blood samples from a phase 1a clinical trial, we examined the acute effects of a single oral administration of a selective TLR8 agonist on immune cell phenotypes. Administration of the TLR8 agonist selgantolimod (SLGN) in healthy individuals resulted in alteration in frequencies of peripheral blood monocytes, pDCs, mDCs and MAIT cells. Frequencies of mDCs and lymphoid cells significantly reduced after 8 h of SLGN administration, whereas pDC frequencies significantly increased, with changes possibly reflecting migration of different cell types between peripheral and tissue compartments in response to the agonist. Myeloid cell activation was evident by an upregulated expression of co-stimulatory molecules CD40 and CD86 accompanied by the production of IL-6 and IL-18 from these cells. Concomitantly, there was induction of the early activation marker CD69 on innate and adaptive lymphoid cells, including MAIT and NK cell subsets. Further, these activated lymphoid cells had enhanced expression of the effector molecules granzyme B and perforin. Microarray analysis of isolated lymphocytes and monocytes from baseline and post-SLGN treatment revealed changes in expression of genes involved in cellular response to cytokine stimulus, innate immune response, myeloid cell differentiation and antigen receptor-mediated signaling pathway. In a preliminary analysis of samples from CHB patients treated with selgantolimod, activation of innate and adaptive lymphocytes was evident. In conclusion, this first in-human study shows that selgantolimod administration in humans results in activation of multiple immune cell responses with antiviral potential.

Follicular Helper T (TFH) Cell Targeting by TLR8 Signaling For Improving HBsAg-Specific B Cell Response In Chronic Hepatitis B Patients.

Identifying signaling pathways that induce B cell response can aid functional cure strategies for chronic hepatitis B infection (CHB). TLR8 activation with ssRNA was shown to enhance follicular helper T cell (TFH) function leading to improved B cell responses in vitro. We investigated whether this mechanism can rescue an exhausted immune response in CHB infection. Effect of TLR8 agonism on supporting cytokines and TFH and B cells were evaluated using ex vivo and in vitro assays. The ability of an oral TLR8 agonist to promote TFH and B cell response was tested in samples from phase 1b clinical trial. TLR8 agonism induced TFH polarizing cytokine IL-12 in monocytes. Treatment of peripheral blood mononuclear cells (PBMCs) from CHB patients with TLR8 agonists induced cytokine IL-21 by TFH cells with enhanced IL-21+BCL-6+ and ICOS+BCL-6+ co-expression. Mechanistically, incubation of isolated naïve CD4+ T cells with TLR8 triggered monocytes resulted in their differentiation into IL-21+ICOS+BCL-6+ TFH in an IL-12 dependent manner. Furthermore, co-culture of these IL-21 producing TFH with autologous naïve B cells led to enhanced memory (CD19+CD27+) and plasma B cell generation (CD19+CD27++CD38+) and IgG production. Importantly, in TFH from CHB patients treated with an oral TLR8 agonist, HBsAg-specific BCL-6, ICOS, IL-21 and CD40L expression and rescue of defective activation induced marker (AIM) response along with partial restoration of HBsAg-specific B cell ELISPOT response was evident. TLR8 agonism can thus enhance HBV-specific B cell responses in CHB patients by improving monocyte-mediated TFH function and may play a role in achieving HBV functional cure.

Single Nucleotide Polymorphisms in Genes Encoding Toll-Like Receptors 7 and 8 and Their Association with Proviral Load of SRLVs in Goats of Polish Carpathian Breed.

Toll-like receptors (TLRs) 7 and 8 are important in single-stranded viral RNA recognition, so genetic variation of these genes may play a role in SRLVs infection and disease progression. Present study aimed to identify SNPs in genes encoding TLR7 and TLR8 in goats of Carpathian breed and analyze their association with the SRLVs provirus concentration as index of disease progression. A total of 14 SNPs were detected, 6 SNPs in the TLR7 gene locus and 8 SNPs in the TLR8 gene. Nine of the 14 identified polymorphisms, 4 in the TLR7 gene and 5 in TLR8 gene, were significantly associated with the SRLVs proviral concentration. These SNPs were located in 3'UTR, 5'UTR and intron sequences as well as in the coding sequences, but they led to silent changes. Homozygous genotypes of three TLR7 SNPs (synonymous variant 1:50703293, 3'UTR variant 1:50701297 and 5'UTR variant 1:50718645) were observed in goats with lower provirus copy number as well as in seronegative animals. The results obtained in this study suggest that SNPs of TLR7/TLR8 genes may induce differential innate immune response towards SRLVs affecting proviral concentration and thereby disease pathogenesis and progression. These findings support a role for genetic variations of TLR7 and TLR8 in SRLVs infection and warrants further studies on the effect of TLR7/TLR8 polymorphisms on SRLVs infection in different populations.

Identification of imidazoquinoline derivative (IQD) interacting sites of porcine TLR8 and the underlying species specificity.

Toll-like receptor 8 (TLR8), as an important innate immune receptor, can recognize specific ligands, activate intracellular signaling and produce an inflammatory response to kill and eliminate pathogenic microorganisms. Recent studies have resolved the crystal structure of human TLR8 (hTLR8) and two types of ligand binding sites were identified. Among the conserved binding site 1 of hTLR8, the residues interacting with imidazoquinoline derivatives (IQDs) were determined. We previously showed that porcine TLR8 (pTLR8) exhibited species specificity for recognition of the hTLR7 agonist imiquimod (R837). Given the species specificity, the pTLR8 residues interacting with IQDs may be different from hTLR8 counterparts. The present study was aimed to identify the pTLR8 residues interacting with small molecular IQDs. Via molecular docking, the pTLR8 residues interacting with R837 and R848 were predicted. The corresponding mutants were tested for pTLR8 signaling in response to IQDs R837, R848 and CL075, and the results showed that five of nine predicted residues (Y336, K341, K342, F395 and G562) are critical for pTLR8 signaling and these residues are partially different from those reported in hTLR8. Further, we found that the pTLR8 GQKNG motif corresponding to hTLR8 RQSYA exhibited disparity to CL075 stimulation. Our study thus reveals fine TLR8 species specificity which deepens the understanding of TLR8 activation mechanism.