Toll-Like Receptors 7/8: A Paradigm for the Manipulation of Immunologic Reactions for Immunotherapy.

The innate immune system recognizes conserved features of viral and microbial pathogens through pattern recognition receptors (PRRs). Toll-like receptors (TLRs) are one type of PRR used by the innate immune system to mediate the secretion of proinflammatory cytokines and promote innate and adaptive immune responses. TLR family members TLR7 and TLR8 (referred to as TLR7/8 from herein) are endosomal transmembrane receptors that recognize purine-rich single-stranded RNA (ssRNA) and bacterial DNA, eliciting an immunologic reaction to pathogens. TLR7/8 were discovered to mediate the secretion of proinflammatory cytokines by activating immune cells. In addition, accumulating evidence has indicated that TLR7/8 may be closely related to numerous immune-mediated disorders, specifically several types of cancer, autoimmune disease, and viral disease. TLR7/8 agonists and antagonists, which are used as drugs or adjuvants, have been identified in preclinical studies and clinical trials as promising immune stimulators for the immunotherapy of these immune-mediated disorders. These results provided reasoning to further explore immunotherapy for the treatment of immune-mediated disorders. Nevertheless, numerous needs remain unmet, and the therapeutic effects of TLR7/8 agonists and antagonists are poor and exert strong immune-related toxicities. The present review aimed to provide an overview of the TLR family members, particularly TLR7/8, and address the underlying molecular mechanisms and clinical implications of TLR7/8 in immune-mediated disorders. The aim of the work is to discuss the underlying molecular mechanisms and clinical implications of TLR7/8 in immune-mediated disorders.

Toll-like receptor 8 agonists improve NK-cell function primarily targeting CD56brightCD16- subset.

BACKGROUND: Toll-like receptors (TLRs) are pattern-recognition sensors mainly expressed in innate immune cells that directly recognize conserved pathogen structures (pathogen-associated molecular patterns-PAMPs). Natural killer (NK) cells have been described to express different endosomal TLRs triggered by RNA and DNA sequences derived from both viruses and bacteria. This study was addressed to establish which endosomal TLR could directly mediate NK activation and function after proper stimuli. It was also important to establish the most suitable TLR agonist to be used as adjuvant in tumor vaccines or in combined cancer immunotherapies. METHODS: We assessed endosomal TLR expression in total NK cells by using RT-qPCR and western blotting technique. In some experiments, we purified CD56brightCD16- and CD56dimCD16+ cells subsets by using NK Cell Isolation Kit Activation marker, cytokine production, CD107a expression and cytotoxicity assay were evaluated by flow cytometry. Cytokine release was quantified by ELISA. NK cells obtained from ovarian ascites underwent the same analyses. RESULTS: Although the four endosomal TLRs (TLR3, TLR7/8, and TLR9) were uniformly expressed on CD56brightCD16- and CD56dimCD16+ cell subsets, the TLR7/8 (R848), TLR3 (polyinosinic-polycytidylic acid, Poly I:C) and TLR9 (ODN2395) ligands promoted NK-cell function only in the presence of suboptimal doses of cytokines, including interleukin (IL)-2, IL-12, IL-15, and IL-18, produced in vivo by other environmental cells. We showed that R848 rather than TLR3 and TLR9 agonists primarily activated CD56brightCD16- NK cells by increasing their proliferation, cytokine production and cytotoxic activity. Moreover, we demonstrated that R848, which usually triggers TLR7 and TLR8 on dendritic cells, macrophages and neutrophils cells, activated CD56brightCD16- NK-cell subset only via TLR8. Indeed, specific TLR8 but not TLR7 agonists increased cytokine production and cytotoxic activity of CD56brightCD16- NK cells. Importantly, these activities were also observed in peritoneal NK cells from patients with metastatic ovarian carcinoma, prevalently belonging to the CD56brightCD16- subset. CONCLUSION: These data highlight the potential value of TLR8 in NK cells as a new target for immunotherapy in patients with cancer.

High-Intensity Interval Training Improves Physical Function, Prevents Muscle Loss, and Modulates Macrophage-Mediated Inflammation in Skeletal Muscle of Cerebral Ischemic Mice.

Although skeletal muscle is the main effector organ largely accounting for disability after stroke, considerably less attention is paid to the secondary abnormalities of stroke-related skeletal muscle loss. It is necessary to explore the mechanism of muscle atrophy after stroke and further develop effective rehabilitation strategy. Here, we evaluated the effects of high-intensity interval (HIIT) versus moderate-intensity aerobic training (MOD) on physical function, muscle mass, and stroke-related gene expression profile of skeletal muscle. After the model of middle cerebral artery occlusion (MCAO) was successfully made, the blood lactate threshold corresponding speed (S LT) and maximum speed (S max) were measured. Different intensity training protocols (MOD < S LT; S LT < HIIT < S max) were carried out for 3 weeks beginning at 7 days after MCAO in the MOD and HIIT groups, respectively. We found that both HIIT and MOD prevented stroke-related gastrocnemius muscle mass loss in MCAO mice. HIIT was more beneficial than MOD for improvements in muscle strength, motor coordination, walking competency, and cardiorespiratory fitness. Furthermore, HIIT was superior to MOD in terms of reducing lipid accumulation, levels of IL-1ß and IL-6 in paretic gastrocnemius, and improving peripheral blood CD4+/CD8+ T cell ratio, level of IL-10. Additionally, RNA-seq analysis revealed that the differentially expressed genes among HIIT, MOD, and MCAO groups were highly associated with signaling pathways involved in inflammatory response, more specifically the I-kappaB kinase/NF-kappaB signaling. Following the outcome, we further investigated the infiltrating immune cells abundant in paretic muscles. The results showed that HIIT modulated macrophage activation by downregulating CD86+ (M1 type) macrophages and upregulating CD163+ (M2 type) macrophages via inhibiting the TLR4/MyD88/NFκB signaling pathway and exerting an anti-inflammatory effect in paretic skeletal muscle. It is expected that these data will provide novel insights into the mechanisms and potential targets underlying muscle wasting in stroke.

Role of toll-like receptor 7/8 pathways in regulation of interferon response and inflammatory mediators during SARS-CoV2 infection and potential therapeutic options.

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.

Bioinformatic analysis and identification of single-stranded RNA sequences recognized by TLR7/8 in the SARS-CoV-2, SARS-CoV, and MERS-CoV genomes.

During virus infection, host toll-like receptors (TLRs) can recognize different pathogen-associated molecular patterns and trigger the innate immune response. TLR7/8 can identify the single-stranded RNA (ssRNA) of the virus. This study aimed to search ssRNA sequences recognized by TLR7/8 from the SARS-CoV-2, SARS-CoV, and MERS-CoV whole genomes by a bioinformatic technique. The immunoinformatic approach showed that the SARS-CoV-2 genome has more ssRNA fragments that could be recognized by TLR7/8 than the SARS-CoV genome. These findings suggest innate immune hyperactivation by SARS-CoV-2. This activity is possibly able to provoke a robust proinflammatory response via TLR7/8 recognition and cause acute lung injury.

TLR8-Mediated Metabolic Control of Human Treg Function: A Mechanistic Target for Cancer Immunotherapy.

Regulatory T (Treg) cells induce an immunosuppressive microenvironment that is a major obstacle for successful tumor immunotherapy. Dissecting the regulatory mechanisms between energy metabolism and functionality in Treg cells will provide insight toward developing novel immunotherapies against cancer. Here we report that human naturally occurring and tumor-associated Treg cells exhibit distinct metabolic profiles with selectivity for glucose metabolism compared with effector T cells. Treg-mediated accelerated glucose consumption induces cellular senescence and suppression of responder T cells through cross-talk. TLR8 signaling selectively inhibits glucose uptake and glycolysis in human Treg cells, resulting in reversal of Treg suppression. Importantly, TLR8 signaling-mediated reprogramming of glucose metabolism and function in human Treg cells can enhance anti-tumor immunity in vivo in a melanoma adoptive transfer T cell therapy model. Our studies identify mechanistic links between innate signaling and metabolic regulation of human Treg suppression, which may be used as a strategy to advance tumor immunotherapy.

Endosomal TLR3, TLR7, and TLR8 control neuronal morphology through different transcriptional programs.

Neuroinflammation is associated with diverse neurological disorders. Endosomal Toll-like receptors (TLRs) including TLR3, TLR7, and TLR8 cell-autonomously regulate neuronal differentiation. However, the mechanisms by which these three TLRs affect neuronal morphology are unclear. In this study, we compare these TLRs in mouse neurons. By combining in vitro neuronal cultures, in utero electroporation, and transcriptomic profiling, we show that TLR8, TLR7, and TLR3 promote dendritic pruning via MYD88 signaling. However, they induce different transcriptomic profiles related to innate immunity, signaling, and neuronal development. The temporal expression patterns and the effects on neuronal morphology are not identical upon activation of these endosomal TLRs. Pathway analyses and in vitro studies specifically implicate mitogen-activated protein kinase signaling in TLR8-mediated dendritic pruning. We further show that TLR8 is more critical for dendritic arborization at a late development stage in vivo. The activation of TLR8, TLR7, or TLR3 results in dendritic shortening, and TLR7 and TLR3 but not TLR8 also control axonal growth. In-depth transcriptomic analyses show that TLRs use different downstream pathways to control neuronal morphology, which may contribute to neuronal development and pathological responses.

SNP-based susceptibility-resistance association and mRNA expression regulation analyses of tlr7 to grass carp Ctenopharyngodon idella reovirus.

Eleven single nucleotide polymorphisms (SNP) in Ctenopharyngodon idella toll-like receptor 7 (citlr7) gene, containing two in the 5'-flanking region, three within the single intron and six distributed in the coding sequence (CDS), were identified. A case-control study of 73 susceptible individuals and 67 resistant individuals was conducted to test the SNPs-based susceptibility-resistance association and mRNA expression of citlr7 to grass carp reovirus (GCRV), showing that both 820 A/G and 1726 A/G were significantly correlative sites in genotype (P < 0·05). Multifactor dimensionality reduction (MDR) analysis suggested the exertion of antiviral effects of 820 A/G might rely on SNPs interactions of citlr7 and C. idella toll-like receptor 8 (citlr8). Combining the mortality rate and citlr7 mRNA expression, it was suggested that 1726 GG-genotyped individuals might be more resistant than 1726 A/G genotyped individuals, indicating the selection on synonymous mutations in 1726 A/G might be susceptibility-resistance-type specific. In addition, haplotype analysis uncovered no significantly correlative haplotypes in citlr7. These findings may provide an in-depth insight for the further functional research of citlr7. The potential genetic markers identified may contribute to the molecular and transgenic breeding of C. idella.

Impaired expression and function of TLR8 in chronic HBV infection and its association with treatment responses during peg-IFN-α-2a antiviral therapy.

BACKGROUND AND AIM: Toll-like receptor 8 (TLR8) plays an important role in controlling chronic viral infections. However, the role of TLR8 in chronic hepatitis B virus (HBV) infection is poorly understood. In this study, we aimed to investigate the expression and function of TLR8 in peripheral blood mononuclear cells (PBMCs) of chronic hepatitis B (CHB) patients and its alteration during peg-IFN-α-2a therapy. METHODS: We evaluated TLR8 expression and antiviral function in vitro by real-time RT-PCR and flow cytometry analysis using fresh PBMCs obtained from CHB patients compared to healthy controls. We also employed clinical cohorts to investigate TLR8 expression in response to peg-IFN-α-2a therapy. RESULTS: TLR8 was mainly expressed in monocytes, and simulation with its ligand resulted in high levels of IFN-γ and TNF-α production. Compared with healthy controls, PBMCs obtained from CHB patients displayed reduced levels of TLR8 expression and IFN-γ, TNF-α and IL-12 induction. The exposure of HepG2.2.15 cells to conditioned medium from PBMCs stimulated by ssRNA40 strongly reduced the levels of HBV DNA, HBsAg and HBeAg, whereas the addition of IFN-γ or TNF-α neutralizing antibodies could block the antiviral effect. NK cells and T cells were the principal IFN-γ-producing lymphocytes after ssRNA40 stimulation, whereas monocytes were the primary source of TNF-α. Analysis of the temporal dynamics showed that patients who achieved a complete response sustained a significant higher level of TLR8 mRNA than those who did not achieve a complete response beginning at week 12 of peg-IFN-α-2a therapy. CONCLUSIONS: TLR8 expression and function in PBMCs were impaired by chronic HBV infection. Higher TLR8 expression after treatment week 12 could potentially predict complete response to peg-IFN-α-2a therapy.

CCN1/CYR61-mediated meticulous patrolling by Ly6Clow monocytes fuels vascular inflammation.

Inflammation is characterized by the recruitment of leukocytes from the bloodstream. The rapid arrival of neutrophils is followed by a wave of inflammatory lymphocyte antigen 6 complex (Ly6C)-positive monocytes. In contrast Ly6C(low) monocytes survey the endothelium in the steady state, but their role in inflammation is still unclear. Here, using confocal intravital microscopy, we show that upon Toll-like receptor 7/8 (TLR7/8)-mediated inflammation of mesenteric veins, platelet activation drives the rapid mobilization of Ly6C(low) monocytes to the luminal side of the endothelium. After repeatedly interacting with platelets, Ly6C(low) monocytes commit to a meticulous patrolling of the endothelial wall and orchestrate the subsequent arrival and extravasation of neutrophils through the production of proinflammatory cytokines and chemokines. At a molecular level, we show that cysteine-rich protein 61 (CYR61)/CYR61 connective tissue growth factor nephroblastoma overexpressed 1 (CCN1) protein is released by activated platelets and enables the recruitment of Ly6C(low) monocytes upon vascular inflammation. In addition endothelium-bound CCN1 sustains the adequate patrolling of Ly6C(low) monocytes both in the steady state and under inflammatory conditions. Blocking CCN1 or platelets with specific antibodies impaired the early arrival of Ly6C(low) monocytes and abolished the recruitment of neutrophils. These results refine the leukocyte recruitment cascade model by introducing endothelium-bound CCN1 as an inflammation mediator and by demonstrating a role for platelets and patrolling Ly6C(low) monocytes in acute vascular inflammation.

Histone Demethylation and Toll-like Receptor 8-Dependent Cross-Talk in Monocytes Promotes Transdifferentiation of Fibroblasts in Systemic Sclerosis Via Fra-2.

OBJECTIVE: To investigate whether epigenetic changes can modulate monocytes to produce tissue inhibitor of metalloproteinases 1 (TIMP-1) via Fra-2 (an activator protein 1 [AP-1] family member), a novel downstream mediator that promotes fibrogenesis. METHODS: AP-1 transcription factors and TIMP-1 expression were measured in monocytes from systemic sclerosis (SSc) patients and healthy controls. Involvement of Fra-2 in the regulation of TIMP-1 following treatment with Toll-like receptor 8 (TLR-8) agonist was investigated using a luciferase activity assay and chromatin immunoprecipitation (ChIP) analysis. Expression of TIMP-1 and Fra-2 was determined in response to TLR-8 treatment and to different histone modifications, including 3'-deazaneplanocin (DZNep) and apicidin. Fibroblasts from healthy controls were cocultured with DZNep plus TLR-8-treated healthy control monocytes. RESULTS: Up-regulation of Fra-2 was detected in bleomycin-challenged mice and in skin biopsy samples from SSc patients. Enhanced expression of Fra-2 and TIMP-1 was correlated in SSc monocytes (P = 0.021). The expression of Fra-1 was significantly reduced (P = 0.037) in SSc monocytes. Inhibiting AP-1 activity reduced TIMP-1 production in TLR-8-stimulated monocytes from healthy controls and SSc patients. ChIP experiments revealed binding of Fra-2 to the TIMP-1 promoter. Stimulation with DZNep plus TLR-8 enhanced Fra-2 and TIMP-1 expression in healthy control monocytes, whereas TLR-8 plus apicidin repressed Fra-2 and TIMP-1 expression. Finally, healthy control monocytes treated with DZNep plus TLR-8 induced strong production of α-smooth muscle actin in dermal fibroblasts, which was inhibited by TIMP-1-blocking antibody. CONCLUSION: These data demonstrate a novel role of histone demethylation induced by DZNep on Fra-2-mediated TIMP-1 production by monocytes in the presence of TLR-8 agonist. This consequently orchestrates the transdifferentiation of fibroblasts, a key event in the pathogenesis of SSc.

[Expression and significance of toll-like receptors 7 and 8 in brain and lung tissues of death cases caused by EV71 infection].

OBJECTIVE: To study the significance of toll-like receptors (TLR) -7 and -8 in the pathogenesis of infection caused by Enterovirus type 71 (EV71) through measuring the expression of TLR7 and TLR8 in brain and lung tissues from the death cases caused by EV71 infection. METHODS: Nine children who died of EV71 infection (EV71 group) were selected as study subjects, and 7 children who died of accidents or non-infectious diseases were used as the control group. Brain and lung tissues from the death cases in both groups at autopsy were collected, and immunohistochemistry was applied to detect the expression of TLR7 and TLR8 in lung and brain tissues in both groups. Integrated optical density (IOD) was applied for semi-quantitative analysis of the expression of TLR7 and TLR8. RESULTS: Immunohistochemical results showed that the expression of TLR7 and TLR8 in lung and brain tissues was strongly positive in the EV71 group, and the IOD values in the EV71 group were also significantly higher than those in the control group (P<0.05). There was no significant difference in the expression of TLR7 and TLR8 between lung and brain tissues in the EV71 group (P>0.05). CONCLUSIONS: TLR7 and TLR8 are highly expressed in lung and brain tissues from the patients who die of severe EV71 infection, suggesting that TLR7 and TLR8 may be involved in the pathogenesis of brain and lung damages caused by severe EV71 infection.

Viral single stranded RNA induces a trophoblast pro-inflammatory and antiviral response in a TLR8-dependent and -independent manner.

Interest is growing in the role of viral infections and their association with adverse pregnancy outcomes. The trophoblast is permissive to viruses, but little is known about their impact on the placenta. We previously established that viral single stranded RNA (ssRNA), a Toll-like receptor 8 (TLR8) agonist, induces a restricted trophoblast pro-inflammatory cytokine/chemokine response by upregulating the secretion of interleukin (IL)-6 and IL-8. In parallel, the type I interferon, IFNbeta, is produced and acts back on the cell in an autocrine/paracrine manner to trigger caspase-3-dependent apoptosis. In this study, we sought to extend these findings by determining the mechanisms involved, examining whether viral ssRNA could induce a trophoblast antiviral response, and evaluating the influence of viral ssRNA on pregnancy outcome using a mouse model. Viral ssRNA induced human first-trimester trophoblast inflammation, type I interferon production, an antiviral response, and apoptosis in both a TLR8/MyD88-dependent and -independent manner. Furthermore, administration of viral ssRNA to pregnant mice induced placental caspase-3 activation, a pro-inflammatory cytokine/chemokine, type I interferon, and antiviral response as well as immune cell infiltration. Thus, ssRNA viral infections may compromise pregnancy by altering placental trophoblast survival and function through both TLR8 and non-TLR8 signaling pathways, leading to immune changes at the maternal-fetal interface.

Determinants of activity at human Toll-like receptors 7 and 8: quantitative structure-activity relationship (QSAR) of diverse heterocyclic scaffolds.

Toll-like receptor (TLR) 7 and 8 agonists are potential vaccine adjuvants, since they directly activate APCs and enhance Th1-driven immune responses. Previous SAR investigations in several scaffolds of small molecule TLR7/8 activators pointed to the strict dependence of the selectivity for TLR7 vis-à-vis TLR8 on the electronic configurations of the heterocyclic systems, which we sought to examine quantitatively with the goal of developing "heuristics" to define structural requisites governing activity at TLR7 and/or TLR8. We undertook a scaffold-hopping approach, entailing the syntheses and biological evaluations of 13 different chemotypes. Crystal structures of TLR8 in complex with the two most active compounds confirmed important binding interactions playing a key role in ligand occupancy and biological activity. Density functional theory based quantum chemical calculations on these compounds followed by linear discriminant analyses permitted the classification of inactive, TLR8-active, and TLR7/8 dual-active compounds, confirming the critical role of partial charges in determining biological activity.

HIV-1 infection induces interleukin-1ß production via TLR8 protein-dependent and NLRP3 inflammasome mechanisms in human monocytes.

The induction of inflammatory cytokines such as IL-1ß is associated with the progression of human immunodeficiency virus, type 1 (HIV-1) disease or AIDS. Unlike most inflammatory cytokines that are regulated by NF-κB at the transcriptional level, production of mature IL-1ß also depends on inflammasome activation. The mechanism by which HIV-1 induces pro-IL-1ß expression and activates inflammasomes to cleave pro-IL-1ß into its bioactive form is not clearly defined. We report here that HIV-1 infection in human monocytes efficiently induced IL-1ß expression and inflammasome activation. Toll-like receptor 8 (TLR8) was required for inducing pro-IL-1ß expression, whereas the NLRP3 inflammasome was required for IL-1ß maturation and release. Furthermore, the lysosomal protease cathepsin B and HIV-1 induced production of reactive oxygen species were critical for HIV-induced inflammasome activation and IL-1ß production. HIV-1 entry, reverse transcription, and integration were all required for both pro-IL-1ß expression and inflammasome activation. Finally, we show that HIV-1-derived RNA was sufficient to induce both pro-IL-1ß expression and inflammasome activation. We conclude that HIV-1 infection induced the expression of pro-IL-1ß via TLR8-mediated mechanisms and activated caspase-1 through the NLRP3 inflammasome to cleave pro-IL-1ß into bioactive IL-1ß. These findings help to elucidate mechanisms of HIV-1 disease progression and identify novel targets for treating HIV-1 induced inflammation and immune activation.

TLR-mediated STAT3 and ERK activation controls IL-10 secretion by human B cells.

IL-10-producing B cells have a regulatory effect in various mouse models for immune-mediated disorders via secretion of IL-10, a potent immunoregulatory cytokine. However, currently, the signaling pathways that regulate IL-10 production in B cells are not well understood. Here, we show that TLR signaling, but not BCR activation or CD40 ligation, induces potent production of IL-10 in human B cells. We demonstrate that the activation of STAT3 and ERK is required for TLR-induced IL-10 production by B cells, since inhibition of STAT3 or ERK activation abrogates TLR-induced IL-10 production. We also uncover a novel function of the TLR-MyD88-STAT3 pathway in B cells, namely controlling IL-10 production, in addition to the known role for this pathway in antibody production. Furthermore, IFN-α, a member of the type I IFN family, differentially modulates TLR7/8- and TLR9-activated STAT3 and ERK in B cells, which provides an explanation for our findings that IFN-α enhances TLR7/8-induced, but not TLR9-induced IL-10 production. These results yield insights into the mechanisms by which TLR signaling regulates IL-10 production in B cells and how type I IFN modulates TLR-mediated IL-10 production by B cells, therefore providing potential targets to modulate the function of IL-10-producing B cells.

TLR8 on dendritic cells and TLR9 on B cells restrain TLR7-mediated spontaneous autoimmunity in C57BL/6 mice.

Systemic lupus erythematosus (SLE) is a complex autoimmune disease with diverse clinical presentations characterized by the presence of autoantibodies to nuclear components. Toll-like receptor (TLR)7, TLR8, and TLR9 sense microbial or endogenous nucleic acids and are implicated in the development of SLE. In mice TLR7-deficiency ameliorates SLE, but TLR8- or TLR9-deficiency exacerbates the disease because of increased TLR7 response. Thus, both TLR8 and TLR9 control TLR7 function, but whether TLR8 and TLR9 act in parallel or in series in the same or different cell types in controlling TLR7-mediated lupus remains unknown. Here, we reveal that double TLR8/9-deficient (TLR8/9(-/-)) mice on the C57BL/6 background showed increased abnormalities characteristic of SLE, including splenomegaly, autoantibody production, frequencies of marginal zone and B1 B cells, and renal pathology compared with single TLR8(-/-) or TLR9(-/-) mice. On the cellular level, TLR8(-/-) and TLR8/9(-/-) dendritic cells were hyperesponsive to TLR7 ligand R848, but TLR9(-/-) cells responded normally. Moreover, B cells from TLR9(-/-) and TLR8/9(-/-) mice were hyperesponsive to R848, but TLR8(-/-) B cells were not. These results reveal that TLR8 and TLR9 have an additive effect on controlling TLR7 function and TLR7-mediated lupus; however, they act on different cell types. TLR8 controls TLR7 function on dendritic cells, and TLR9 restrains TLR7 response on B cells.

Toll-like receptor 7 rapidly relaxes human airways.

RATIONALE: Toll-like receptors (TLRs) 7 and 8 detect respiratory virus single-stranded RNA and trigger an innate immune response. We recently described rapid TLR7-mediated bronchodilation in guinea pigs. OBJECTIVES: To characterize TLR7 expression and TLR7-induced airway relaxation in humans and in eosinophilic airway inflammation in guinea pigs. To evaluate the relaxant effects of other TLRs. METHODS: Human airway smooth muscle strips were contracted with methacholine in vitro, and responses to TLR7 and TLR8 agonists were assessed. TLR7-mediated nitric oxide production was measured using a fluorescent indicator, and TLR7 expression was characterized using immunofluorescence. TLR7 signaling was also evaluated in ovalbumin-challenged guinea pigs. MEASUREMENTS AND MAIN RESULTS: The TLR7 agonist imiquimod (R837) caused rapid dose-dependent relaxation of methacholine-contracted human airways in vitro. This was blocked by the TLR7 antagonist IRS661 and by inhibiting nitric oxide production but not by inhibiting prostaglandin production. TLR7 activation markedly increased fluorescence of a nitric oxide detector. TLR7 was expressed on airway nerves, but not airway smooth muscle, implicating airway nerves as the source of TLR7-induced nitric oxide production. TLR7-mediated relaxation persisted in inflamed guinea pigs airways in vivo. The TLR8 agonists polyuridylic acid and polyadenylic acid also relaxed human airways, and this was not blocked by the TLR7 antagonist or by blocking nitric oxide or prostaglandin production. No other TLRs relaxed the airways. CONCLUSIONS: TLR7 is expressed on airway nerves and mediates relaxation of human and animal airways through nitric oxide production. TLR7-mediated bronchodilation may be a new therapeutic strategy in asthma.

Impaired toll-like receptor 8 signaling in multiple sclerosis.

BACKGROUND: The etiology and immunopathology of multiple sclerosis (MS) is not well understood. It is recognized that although autoreactive T cells are the main early mediators of disease, other cell types, including cells of the innate immune system contribute to MS pathogenesis. The objective of this study was to determine if Toll-like receptor (TLR) signaling is functionally altered in patients with MS. FINDINGS: Peripheral blood mononuclear cells from healthy donors and patients with relapsing remitting MS were stimulated with specific agonists of TLRs 3, 7, 8 and 9. Using quantitative polymerase chain reaction transcript levels of tumor necrosis factor-α, interferon-α and interleukin (IL)-12ß were quantified from patients with MS and healthy donors. TLR8-induced production of IL12B transcripts and protein was functionally impaired in patients with MS as compared to healthy controls (P <0.05 and P <0.005, respectively). Patients with MS also expressed lower baseline levels of TLR8 as compared to healthy controls (P <0.05). CONCLUSIONS: TLR8 expression and signaling is impaired in peripheral blood mononuclear cells from patients with MS. This finding suggests that loss of TLR8 signaling may be contributing to autoimmune processes in MS.