Toll-like Receptor 9 Contains Two DNA Binding Sites that Function Cooperatively to Promote Receptor Dimerization and Activation.

Toll-like receptor 9 (TLR9) recognizes DNA containing CpG motifs derived from bacteria and viruses and activates the innate immune response to eliminate them. TLR9 is known to bind to CpG DNA, and here, we identified another DNA binding site in TLR9 that binds DNA containing cytosine at the second position from the 5' end (5'-xCx DNA). 5'-xCx DNAs bound to TLR9 in the presence of CpG DNA and cooperatively promoted dimerization and activation of TLR9. Binding at both sites was important for efficient activation of TLR9. The 5'-xCx DNA bound the site corresponding to the nucleoside binding site in TLR7 and TLR8 as revealed by the structural analysis. This study revealed that TLR9 recognizes two types of DNA through its two binding sites for efficient activation. This information may contribute to the development of drugs that control the activity of TLR9.

Innate IRE1α-XBP1 activation by viral single-stranded RNA and its influence on lung cytokine production during SARS-CoV-2 pneumonia.

The utilization of host-cell machinery during SARS-CoV-2 infection can overwhelm the protein-folding capacity of the endoplasmic reticulum and activate the unfolded protein response (UPR). The IRE1α-XBP1 arm of the UPR could also be activated by viral RNA via Toll-like receptors. Based on these premises, a study to gain insight into the pathogenesis of COVID-19 disease was conducted using nasopharyngeal exudates and bronchioloalveolar aspirates. The presence of the mRNA of spliced XBP1 and a high expression of cytokine mRNAs were observed during active infection. TLR8 mRNA showed an overwhelming expression in comparison with TLR7 mRNA in bronchioloalveolar aspirates of COVID-19 patients, thus suggesting the presence of monocytes and monocyte-derived dendritic cells (MDDCs). In vitro experiments in MDDCs activated with ssRNA40, a synthetic mimic of SARS-CoV-2 RNA, showed induction of XBP1 splicing and the expression of proinflammatory cytokines. These responses were blunted by the IRE1α inhibitor MKC8866, the TLR8 antagonist CU-CPT9a, and knockdown of TLR8 receptor. In contrast, the IRE1α-XBP1 activator IXA4 enhanced these responses. Based on these findings, the TLR8/IRE1α system seems to play a significant role in the induction of the proinflammatory cytokines associated with severe COVID-19 disease and might be a druggable target to control cytokine storm.

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.

miRNAs: possible regulators of toll like receptors and inflammatory tumor microenvironment in colorectal cancer.

BACKGROUND: Colorectal cancer (CRC) is ranked as the third most commonly diagnosed cancer and the third cause of cancer related deaths. CRC is greatly attributed to genetic and epigenetic mutations and immune dysregulation. Tumor aberrant expression of Toll-like Receptors (TLRs) can contribute to tumorigenesis. Recent studies suggested that microRNAs act as direct ligands of TLRs altering their expression and signaling pathways. AIM: To prove our concept that specific miRNA mimics may act as antagonists of their specific toll like receptors inhibiting their expression that could limit the release of pro-inflammatory and pro-tumorigenic cytokines leading to apoptosis of tumor cells. METHODS: From public microarray databases, we retrieved TLRs and miRNAs related to CRC followed by in silico docking of the selected miRNA ligands into the TLRs. Clinical validation after co-immunoprecipitation of TLRs and their interacting miRNA ligands was done. Expression of TLRs 1, 7,8 was determined by ELISA while miRNAs was measured by RT-qPCR. In addition, microRNA mimics of the down regulated miRNAs were transfected into human CRC cell lines. RESULTS: Our data demonstrate that TLRs 1, 7, 8 are up regulated in CRC compared to controls. Further, three miRNAs (-122, -29b and -15b) are relatively downregulated, while 4 miRNAs (-202, miRNA-98, -21 and -let7i) are upregulated in CRC patients compared to those with benign tumor and healthy controls. Transfection of down regulated miRNA mimics into CRC cell lines resulted in a significant reduction of the number and viability of cells as well as down regulating the expression of TLRs 1, 7 and 8 with ultimate reduction of downstream effector IL6 protein, suggesting that these miRNAs are negative regulators of carcinogenesis. CONCLUSION: MicroRNAs could act as antagonistic ligands of TLRs limiting the inflammatory tumor microenvironment.

MiR-574-5p activates human TLR8 to promote autoimmune signaling and lupus.

Endosomal single-stranded RNA-sensing Toll-like receptor-7/8 (TLR7/8) plays a pivotal role in inflammation and immune responses and autoimmune diseases. However, the mechanisms underlying the initiation of the TLR7/8-mediated autoimmune signaling remain to be fully elucidated. Here, we demonstrate that miR-574-5p is aberrantly upregulated in tissues of lupus prone mice and in the plasma of lupus patients, with its expression levels correlating with the disease activity. miR-574-5p binds to and activates human hTLR8 or its murine ortholog mTlr7 to elicit a series of MyD88-dependent immune and inflammatory responses. These responses include the overproduction of cytokines and interferons, the activation of STAT1 signaling and B lymphocytes, and the production of autoantigens. In a transgenic mouse model, the induction of miR-574-5p overexpression is associated with increased secretion of antinuclear and anti-dsDNA antibodies, increased IgG and C3 deposit in the kidney, elevated expression of inflammatory genes in the spleen. In lupus-prone mice, lentivirus-mediated silencing of miR-574-5p significantly ameliorates major symptoms associated with lupus and lupus nephritis. Collectively, these results suggest that the miR-574-5p-hTLR8/mTlr7 signaling is an important axis of immune and inflammatory responses, contributing significantly to the development of lupus and lupus nephritis.

tRF-His-GTG-1 enhances NETs formation and interferon-α production in lupus by extracellular vesicle.

BACKGROUND: Hyperactive neutrophil extracellular traps (NETs) formation plays a crucial role in active severe systemic lupus erythematosus (SLE). However, what triggers the imbalance in dysregulated NETs formation in SLE is elusive. Transfer RNA-derived small RNAs (tsRNAs) are novel non-coding RNAs, which participate in various cellular processes. We explore the role of tsRNAs on NETs formation in SLE. METHODS: We analyzed the levels of NETs DNA and platelet-derived extracellular vesicles (pEVs) from 50 SLE patients and 20 healthy control subjects. The effects of pEVs on NETs formation were evaluated by using immunofluorescence assay and myeloperoxidase-DNA PicoGreen assay. The regulatory mechanism of pEVs on NETs formation and inflammatory cytokines production were investigated using an in vitro cell-based assay. RESULTS: Increased circulating NETs DNA and pEVs were shown in SLE patients and were associated with disease activity (P < 0.005). We demonstrated that SLE patient-derived immune complexes (ICs) induced platelet activation, followed by pEVs release. ICs-triggered NETs formation was significantly enhanced in the presence of pEVs through Toll-like receptor (TLR) 8 activation. Increased levels of tRF-His-GTG-1 in pEVs and neutrophils of SLE patients were associated with disease activity. tRF-His-GTG-1 interacted with TLR8 to prime p47phox phosphorylation in neutrophils, resulting in reactive oxygen species production and NETs formation. Additionally, tRF-His-GTG-1 modulated NF-κB and IRF7 activation in neutrophils upon TLR8 engagement, resulting IL-1ß, IL-8, and interferon-α upregulation, respectively. CONCLUSIONS: The level of tRF-His-GTG-1 was positively correlated with NETs formation in SLE patients; tRF-His-GTG-1 inhibitor could efficiently suppress ICs-triggered NETs formation/hyperactivation, which may become a potential therapeutic target.

Neutrophils and platelets are key members in the immunopathogenesis of SLE. EVs play a key role in intercellular communication. Abnormal NETs formation promotes vascular complications and organ damage in SLE patients. tsRNA is a novel regulatory small non-coding RNA and participates in diverse pathological processes. Herein, we showed that SLE patient-derived ICs activates platelets directly, followed by intracellular tRF-His-GTG-1 upregulation, which is loaded into pEVs. The pEV-carried tRF-His-GTG-1 could interact with TLR8 in neutrophils, followed by activation of the downstream signaling pathway, including p47phox-NOX2-ROS, which causes NETs enhancement, while IRF7 promotes the expression of IFN-α. The tRF-His-GTG-1 inhibitor could suppress efficiently SLE ICs-induced NETs formation and pEVs primed NETs enhancement. This study offers new molecular machinery to explain the association between the platelets-derived tsRNAs, pEVs, and hyperactive NETs formation in lupus. tRF-His-GTG-1 may serve as a potential therapeutic target and help to advance our understanding of tsRNAs in SLE pathogenesis.

Human and Murine Toll-like Receptor-Driven Disease in Systemic Lupus Erythematosus.

The pathogenesis of systemic lupus erythematosus (SLE) is linked to the differential roles of toll-like receptors (TLRs), particularly TLR7, TLR8, and TLR9. TLR7 overexpression or gene duplication, as seen with the Y-linked autoimmune accelerator (Yaa) locus or TLR7 agonist imiquimod, correlates with increased SLE severity, and specific TLR7 polymorphisms and gain-of-function variants are associated with enhanced SLE susceptibility and severity. In addition, the X-chromosome location of TLR7 and its escape from X-chromosome inactivation provide a genetic basis for female predominance in SLE. The absence of TLR8 and TLR9 have been shown to exacerbate the detrimental effects of TLR7, leading to upregulated TLR7 activity and increased disease severity in mouse models of SLE. The regulatory functions of TLR8 and TLR9 have been proposed to involve competition for the endosomal trafficking chaperone UNC93B1. However, recent evidence implies more direct, regulatory functions of TLR9 on TLR7 activity. The association between age-associated B cells (ABCs) and autoantibody production positions these cells as potential targets for treatment in SLE, but the lack of specific markers necessitates further research for precise therapeutic intervention. Therapeutically, targeting TLRs is a promising strategy for SLE treatment, with drugs like hydroxychloroquine already in clinical use.

Subgroup Analysis of TLR2, -3, -4 and -8 in Relation to the Severity of Clinical Manifestations of Cervical HPV Infection.

We present the findings of assessing the expression levels of extracellular TLR2 and TLR4 and intracellular TLR3 and TLR8 correlating with the severity of clinical manifestations of HPV infection. A total of 199 women took part in a single-center prospective comparative research study on TLR2, TLR3, TLR4 and TLR8 expression in HPV-related cervical lesions. TLRs' mRNA expression was analyzed using real-time reverse transcription polymerase chain reaction (RT-PCR). Our results indicate the potential significance of TLR3, TLR4 and TLR8 in responding to HPV infection and its progression to SILs and CC, highlighting the importance of HPV polyinfection in relation to TLR4 and TLR8.

Immunodeficiency and bone marrow failure with mosaic and germline TLR8 gain of function.

Inborn errors of immunity (IEI) are a genetically heterogeneous group of disorders with a broad clinical spectrum. Identification of molecular and functional bases of these disorders is important for diagnosis, treatment, and an understanding of the human immune response. We identified 6 unrelated males with neutropenia, infections, lymphoproliferation, humoral immune defects, and in some cases bone marrow failure associated with 3 different variants in the X-linked gene TLR8, encoding the endosomal Toll-like receptor 8 (TLR8). Interestingly, 5 patients had somatic variants in TLR8 with <30% mosaicism, suggesting a dominant mechanism responsible for the clinical phenotype. Mosaicism was also detected in skin-derived fibroblasts in 3 patients, demonstrating that mutations were not limited to the hematopoietic compartment. All patients had refractory chronic neutropenia, and 3 patients underwent allogeneic hematopoietic cell transplantation. All variants conferred gain of function to TLR8 protein, and immune phenotyping demonstrated a proinflammatory phenotype with activated T cells and elevated serum cytokines associated with impaired B-cell maturation. Differentiation of myeloid cells from patient-derived induced pluripotent stem cells demonstrated increased responsiveness to TLR8. Together, these findings demonstrate that gain-of-function variants in TLR8 lead to a novel childhood-onset IEI with lymphoproliferation, neutropenia, infectious susceptibility, B- and T-cell defects, and in some cases, bone marrow failure. Somatic mosaicism is a prominent molecular mechanism of this new disease.

Effect of Royal Jelly on Gene Expression of Toll-like Receptors 1-9 in Patients with Hepatitis B.

BACKGROUND: By damaging the liver, hepatitis B can result in acute and chronic diseases, such as cirrhosis or hepatocellular carcinoma. Viable treatments for such diseases using natural products and determinative biomarkers have been proposed but require evaluation to improve their effects. Therefore, this study aims to examine how effectively a specific natural product (namely, royal jelly) protects the body from the copy number of the virus, as well as TLR1 to TLR9 gene expressions. METHODS: The effectiveness of royal jelly was tested by giving it (orally) to 30 hepatitis B patients for one month. HBV copy number and mRNA levels of TLRs were explored using Real Time PCR technique, and liver enzymes were evaluated too. RESULTS: Orally treatment with royal jelly led to a significant decrease in HBV-DNA copy number, down-regulation of TLR2 and TLR8, and up-regulation of TLR3. However, mRNA levels of the TLRs were not altered in the female, while TLR1, TLR2, and TLR5 were significantly decreased in the male participants. CONCLUSIONS: It seems that royal jelly has anti-viral and anti-inflammatory roles in the in vivo conditions in a dependent manner in TLR3, TLR2, and TLR8. Therefore, it can be suggested as a safe complementary agent for patients with hepatitis B.

The differential expression of toll like receptors and RIG-1 in the placenta of neonates with in utero infections.

Novel biomarkers of in utero infections are needed to help guide early therapy. The toll like receptors (TLRs) and retinoic acid-inducible gene 1 (RIG-1) are proteins involved in the initial reaction of the innate immune system to infectious diseases. This study tested the hypothesis that a panel of TLRs and RIG-1 in the placenta could serve as an early biomarker of in utero infections. The TLRs and RIG-1 expression as determined by immunohistochemistry was scored in 10 control placentas (normal delivery or neonatal damage from known non-infectious cause), 8 placentas from documented in utero bacterial infection, and 7 placentas from documented in utero viral infections blinded to the clinical information. The non-infected placentas showed the following profile: no expression (TLR1, TLR3, TLR4, TLR7, TLR8), moderate expression (TLR2), and strong expression (RIG-1). The bacterial and viral infection cases shared the following profile: no to mild expression (TLR 2, TLR7, and RIG1), moderate expression (TLR4), and strong expression (TLR1, TLR3, and TLR8). The histologic findings in the chorionic villi were equivalent in the infected cases and controls, underscoring the need for molecular testing by the surgical pathologist when in utero infection is suspected. The results suggest that a panel of TLRs/RIG-1 analyses can allow the pathologist and/or clinician to diagnose in utero infections soon after birth. Also, treatments to antagonize the effects of TLR1, 3, and 8 may help abrogate in utero neonatal damage.

Rare variants in Toll-like receptor 7 results in functional impairment and downregulation of cytokine-mediated signaling in COVID-19 patients.

Toll-like receptors (TLR) are crucial components in the initiation of innate immune responses to a variety of pathogens, triggering the production of pro-inflammatory cytokines and type I and II interferons, which are responsible for innate antiviral responses. Among the different TLRs, TLR7 recognizes several single-stranded RNA viruses including SARS-CoV-2. We and others identified rare loss-of-function variants in X-chromosomal TLR7 in young men with severe COVID-19 and with no prior history of major chronic diseases, that were associated with impaired TLR7 signaling as well as type I and II IFN responses. Here, we performed RNA sequencing to investigate transcriptome variations following imiquimod stimulation of peripheral blood mononuclear cells isolated from patients carrying previously identified hypomorphic, hypofunctional, and loss-of-function TLR7 variants. Our investigation revealed a profound impairment of the TLR7 pathway in patients carrying loss-of-function variants. Of note, a failure in IFNγ upregulation following stimulation was also observed in cells harboring the hypofunctional and hypomorphic variants. We also identified new TLR7 variants in severely affected male patients for which a functional characterization of the TLR7 pathway was performed demonstrating a decrease in mRNA levels in the IFNα, IFNγ, RSAD2, ACOD1, IFIT2, and CXCL10 genes.

Global interpretation of novel alternative splicing events in human congenital pulmonary airway malformations: A pilot study.

Little is known about differentially expressed genes (DEGs) and alternative splicing (AS) landscapes in congenital lung malformations (CLMs). We applied reference-based assembly of sequencing reads from RNA sequencing (RNA-seq) libraries to identify DEGs and AS landscapes in the lesions and normal lung tissue from the most common types of CLMs, including congenital pulmonary airway malformation-Ⅰ (CPAM-Ⅰ), CPAM-Ⅱ, intralobar sequestration (ILS), and ILS with CPAM (ILS-CPAM). We analyzed the expression profiles and related biological functions of AS events (ASEs). We further constructed a co-expression regulatory network between RNA binding protein (RBP) genes and corresponding ASEs to explore the related pathways in the regulated network. Ten DEGs were identified in the four types of CLMs, including eight upregulated genes and two downregulated genes. Additionally, 16 differential ASEs were detected, including the genes MACF1, RFX2, and FBXL4. Gene ontology (GO) enrichment was mainly observed in embryonic visual malformation and apoptotic process, and the KEGG pathway mainly enriched in the PI3K/AKT signaling pathway. We also detected 13 differentially expressed RBPs among 1979 DEGs in CPAM-I, in which ASEs in the MACF1 gene and RBP genes TLR8 and PTRH1 were closely associated. Moreover, we confirmed that the expression levels of PTRH1, NSUN7, and DZIP1L abundantly increased and the expression levels of TLR8, MEF2A, and NIPBL decreased in the CPAM-I lung tissue compared with the controls. It is suggested that ASEs in different types of CLMs is prominently different from normal controls, and ASEs differences occurring in CPAM-I malformation tissue are dramatically different from other types, which demonstrates the complex pathogenesis of CLMs and provides foundations for future studies to elucidate the mechanisms of developing CLMs.

TLR7 and TLR8 evolution in lagomorphs: different patterns in the different lineages.

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.

TLR-8, TNF-α, and ESR-1α Gene Polymorphism Susceptibility in Onset of Arthritis.

Arthritis is a genetic disorder characterized by bones and joint degradation assisted by severe pain and inflammation. It is evident by the studies that 0 candidate genes variations play vital role in its development and progression. Therefore, we investigated the genetic variation of TLR-8, TNF, and ESR-1α genes in the Pakistani population. A case-control study comprising 300 RA, 316 OA, and 412 control subjects was conducted. PCR-RFLP and direct sequencing methods were used for determining genetic variations. Analysis was performed by using PLINK and MEGA 6.0 software. Allelic and genetic frequencies of polymorphisms identified on rs3764879 (TLR-8), rs3764880 (TLR-8), rs5744080 (TLR-8), rs1800629 (TNF), rs2228480 (ESR-1α), and rs1451501590 (ESR-1α) were significantly varied among RA, OA, and controls. Novel functional mutations SCV000844945 and SCV000844946 on TLR-8 as well as a non-functional SCV000804801 and functional variation SCV000804802 on ESR-1α were also identified and reported for the first time in the studied population. Multiple site analyses indicated that polymorphisms on TLR-8 and ESR-1α genes were significant risk factors in disease onset to the next generation. In conclusion, TLR-08 and ESR-1α were significant in the onset of arthritis whereas the TNF was not found as a significant risk factor in the onset of RA and OA.

Activation of Toll-like receptor 7/8 encoded by the X chromosome alters sperm motility and provides a novel simple technology for sexing sperm.

In most mammals, the male to female sex ratio of offspring is about 50% because half of the sperm contain either the Y chromosome or X chromosome. In mice, the Y chromosome encodes fewer than 700 genes, whereas the X chromosome encodes over 3,000 genes. Although overall gene expression is lower in sperm than in somatic cells, transcription is activated selectively in round spermatids. By regulating the expression of specific genes, we hypothesized that the X chromosome might exert functional differences in sperm that are usually masked during fertilization. In this study, we found that Toll-like receptors 7/8 (TLR7/8) coding the X chromosome were expressed by approximately 50% of the round spermatids in testis and in approximately 50% of the epididymal sperm. Especially, TLR7 was localized to the tail, and TLR8 was localized to the midpiece. Ligand activation of TLR7/8 selectively suppressed the mobility of the X chromosome-bearing sperm (X-sperm) but not the Y-sperm without altering sperm viability or acrosome formation. The difference in sperm motility allowed for the separation of Y-sperm from X-sperm. Following in vitro fertilization using the ligand-selected high-mobility sperm, 90% of the embryos were XY male. Likewise, 83% of the pups obtained following embryo transfer were XY males. Conversely, the TLR7/8-activated, slow mobility sperm produced embryos and pups that were 81% XX females. Therefore, the functional differences between Y-sperm and X-sperm motility were revealed and related to different gene expression patterns, specifically TLR7/8 on X-sperm.

TLR8/TLR7 dysregulation due to a novel TLR8 mutation causes severe autoimmune hemolytic anemia and autoinflammation in identical twins.

Our study presents a novel germline c.1715G>T (p.G572V) mutation in the gene encoding Toll-like receptor 8 (TLR8) causing an autoimmune and autoinflammatory disorder in a family with monozygotic male twins, who suffer from severe autoimmune hemolytic anemia worsening with infections, and autoinflammation presenting as fevers, enteritis, arthritis, and CNS vasculitis. The pathogenicity of the mutation was confirmed by in vitro assays on transfected cell lines and primary cells. The p.G572V mutation causes impaired stability of the TLR8 protein, cross-reactivity to TLR7 ligands and reduced ability of TLR8 to attenuate TLR7 signaling. This imbalance toward TLR7-dependent signaling leads to increased pro-inflammatory responses, such as nuclear factor-κB (NF-κB) activation and production of pro-inflammatory cytokines IL-1ß, IL-6, and TNFα. This unique TLR8 mutation with partial TLR8 protein loss and hyperinflammatory phenotype mediated by TLR7 ligands represents a novel inborn error of immunity with childhood-onset and a good response to TLR7 inhibition.

mRNA expression of toll-like receptors 3, 7, 8, and 9 in the nasopharyngeal epithelial cells of coronavirus disease 2019 patients.

BACKGROUND: The etiopathogenesis of coronavirus disease 2019 (COVID-19) stem partially from the abnormal activation of the innate and adaptive immune systems. Here in the current investigation, the mRNA expression levels of toll-like receptors (TLRs) were evaluated in the nasopharyngeal epithelial cells from COVID-19 patients. METHODS: Epithelial cells were obtained using nasopharyngeal swab samples from 90 COVID-19 patients and 50 controls. COVID-19 cases were classified into those without symptoms, with symptoms but not hospitalized, and with symptoms and hospitalized. To determine the mRNA expression levels of TLRs, first RNA was extracted and cDNA was synthesized, and finally Real-time PCR was exerted. RESULTS: It was seen that the transcript levels of TLR3, TLR7, TLR8, and TLR9 were overexpressed in the COVID-19 patients with clinical symptoms needing hospitalization as well as in those with clinical symptoms without needing for hospitalization compared to controls. Upregulation of TLRs was associated with clinical presentations of the patients. CONCLUSIONS: Modulation of TLR3, TLR7, TLR8, TLR9 in the epithelial cells of COVID-19 cases may estimate the disease severity and requirement for hospitalization.

LncRNA TLR8-AS1 suppresses miR-34a maturation in hepatocellular carcinoma to suppress cell proliferation and migration.

TLR8-AS1 has been characterized as an oncogenic lncRNA in ovarian cancer, while its role in hepatocellular carcinoma (HCC) is unknown. This study aimed to explore the role of TLR8-AS1 in HCC. TLR8-AS1 expression in HCC and paired non-tumor tissues from 62 HCC patients was determined by RT-qPCR. The prognostic value of TLR8-AS1 for HCC was analyzed by performing a 5-year follow-up. Correlations between TLR8-AS1 and mature miR-34a and miR-34a precursor were analyzed by Pearson's correlation coefficient. The roles of TLR8-AS1 and miR-34a in regulating the proliferation and migration were explored by CCK-8 assay and Transwell migration assay. We found that TLR8-AS1 was upregulated in HCC and predicted poor survival. Across HCC tissues, TLR8-AS1 was inversely correlated with mature miR-34a, but not miR-34a precursor. In HCC cells, TLR8-AS1 overexpression downregulated mature miR-34a, but not miR-34a precursor. Cell proliferation and Transwell migration assay showed that TLR8-AS1 overexpression reduced the enhancing effects of miR-34a on cell proliferation and migration. TLR8-AS1 may suppress miR-34a maturation in HCC to suppress cell proliferation and migration.

Variety of endosomal TLRs and Interferons (IFN-α, IFN-ß, IFN-γ) expression profiles in patients with SLE, SSc and MCTD.

We investigated Toll-like receptor (TLR)-3/-7/-8/-9 and interferon (IFN)-α/ß/γ mRNA expression in whole blood and serum IFN-α/ß/γ levels in patients with mixed connective tissue disease (MCTD), systemic lupus erythematosus (SLE) and systemic sclerosis (SSc) and in healthy subjects to assess the association between the TLR-IFN expression and severity of and susceptibility to diseases, and identify potential biomarkers. Expression of the IFN-γ, TLR-3 and TLR-8 was detected only in SLE patients. TLR-7, IFN-α and IFN-ß expression was highest in SLE, while TLR-9 expression was highest in SSc patients. In SLE and MCTD patients a strong correlation was observed between TLR-7 and IFN-α expression and IFN-ß and IFN-α expression. In MCTD patients, negative correlation between IFN-α and TLR-9 and TLR-7 and TLR-9 was revealed. TLR-9 expression in anti-U1-70k-negative, anti-C negative and anti-SmB-negative MCTD patients was higher than in MCTD-positive patients. We observed negative correlations between serum IFN-α levels and TLR-7 expression and C3 and C4 levels in SLE patients. In SLE patients we observed that with increased IFN-γ, TLR-3 and TLR-8 expression increased the value of C3 and C4. Our results confirmed that the endosomal TLR-IFN pathway seems to be more important in SLE than in MCTD or SSc, and that IFN-α and IFN-ß may be possible biomarkers for SLE.