Deoxyguanosine is a TLR7 agonist

Toll-like receptor 7 (TLR7) is an innate immune sensor for single-strand RNA (ssRNA). Recent structural analysis revealed that TLR7 has an additional binding site for nucleosides such as guanosine, and is activated when both guanosine and ssRNA bind. The nucleoside binding site also accommodates imidazoquinoline derivatives such as R848, which activate TLR7 in the absence of ssRNA. Here, we report that deoxyguanosine (dG) triggered cytokine production in murine bone marrow derived macrophages and plasmacytoid dendritic cells, as well as in human peripheral blood mononuclear cells, including type I interferons and pro-inflammatory factors such as TNF and IL-6. This signalling activity of dG was dependent on TLR7 and its adaptor MyD88 and did not require amplification via the type I interferon receptor. dG-triggered cytokine production required endosomal maturation but did not depend on the concurrent provision of RNA. We conclude that dG induces an inflammatory response through TLR7 and propose that dG is an RNA-independent TLR7 agonist.     

TLR3激动剂对B细胞功能的影响

目的观察TLR3激动剂Poly I:C对B细胞功能的影响,包括细胞增殖、共刺激分子表达、细胞因子和抗体的分泌情况。方法利用免疫磁珠法分选小鼠脾脏CD19+B细胞,RT-PCR法证实其表达TLR3。体外用Poly I:C刺激B细胞一定时间后,CFSE分裂法检测B细胞增殖,流式细胞术(flow cytometry,FCM)检测B细胞表面共刺激分子表达,CBA(cytometric bead ar-ray)法或ELISA法检测培养上清中细胞因子含量,CBA法检测B细胞分泌Ig的亚型。结果 Poly I:C可以促进B细胞增殖,上调B细胞表面CD40、CD80、CD86和MHC II类分子的表达,促进IL-6和TNF-α的高分泌,诱导IgG1κ抗体的产生。结论 Poly I:C可以通过促进增殖、细胞因子分泌,诱导抗体产生和上调共刺激分子表达等多方面调节B细胞功能。     

Analysis of NOD1, NOD2, TLR1, TLR2, TLR4, TLR5, TLR6 and TLR9 genes in anal furunculosis of German shepherd dogs

Anal furunculosis (AF) primarily affects German shepherd dogs (GSD) and is characterised by inflammation and ulceration of the perianal tissues with development of cutaneous sinuses or rectocutaneous fistulae. Investigation of pattern recognition receptor (PRR) function has suggested that defective responses might occur in AF-affected GSD. The aim of the current study was to investigate whether canine PRR genes are involved in determining susceptibility to AF in this breed. Chromosomal location and coding sequences for NOD1, NOD2, TLR1, TLR2, TLR4, TLR5, TLR6 and TLR9 were determined and microsatellite markers identified for each gene. Microsatellite genotyping of 100 control GSD and 47 AF-affected GSD showed restricted allelic variation for AHT H91 (associated with TLR5) and REN216 NO5 (associated with both TLR1 and TLR6) compared with non-GSD dogs. Genotyping of single nucleotide polymorphisms identified in canine TLR1, TLR5, TLR6 and NOD2 genes failed to show any significant associations between PRR polymorphisms and AF. The highly restricted PRR genotypes seen in GSD are likely to have resulted from selective breeding and might influence innate immune responses in this breed.     

Treatment with the TLR7 agonist R848 induces regulatory T-cell-mediated suppression of established asthma symptoms

The evolution of allergic asthma is tightly controlled by effector and regulatory cells, as well as cytokines such as IL-10 and/or TGF-β, and it is widely acknowledged that environmental exposure to allergens and infectious agents can influence these processes. In this context, the recognition of pathogen-associated motifs, which trigger TLR activation pathways, plays a critical role with important consequences for disease progression and outcome. We addressed the question whether the TLR7 ligand resiquimod (R848), which has been shown to be protective in several experimental allergic asthma protocols, can also suppress typical asthma symptoms once the disease is established. To this end, we used an OVA-induced experimental model of murine allergic asthma in which R848 was injected after a series of challenges with aerosolized OVA. We found that the treatment attenuated allergic symptoms through a mechanism that required Tregs, as assessed by the expansion of this population in the lungs of mice having received R848, and the loss of R848-mediated suppression of allergic responses after in vivo Treg depletion. IL-10 provided only a minor contribution to this suppressive effect that was largely mediated through a TGF-β-dependent pathway, a finding that opens new therapeutic opportunities for the pharmacological targeting of Tregs.     

Identification of a novel TLR5 agonist derived from the P97 protein of Mycoplasma hyopneumoniae

By modulating specific immune responses against antigens, adjuvants are used in many vaccine preparations to enhance protective immunity. The C-terminal domain of the protein P97 (P97c) of Mycoplasma hyopneumoniae, which is the etiologic agent of porcine enzootic pneumonia, has been shown to increase the specific humoral response against an antigen when this antigen is merged with P97c and delivered by adenovectors. However, the immunostimulating mechanism of this protein remains unknown. In the present study, recombinantly expressed P97c triggered a concentration-dependent TLR5 activation and stimulates the production of interleukin-8 from HEK-Blue mTLR5 cells. Circular dichroism spectroscopy and prediction of 3-dimensional conformation exposed a relevant secondary and tertiary structural homology between P97c and flagellin, the known potent TLR5 agonist. P97c adjuvanticity was evaluated by fusing the conserved epitope of the ectodomain matrix 2 protein (M2e) of the influenza A virus to the protein. Mice immunized with P97c-3M2e revealed a high antibody titer against the M2e epitope associated with a mixed Th1/Th2 immune response. Overall, this study identifies a novel agonist of the pattern recognition receptor TLR5 and reveals that P97c is a potential adjuvant through the activation of the innate immune system.     

TLR7激动剂增强肾癌患者PBMCs抗肿瘤能力

目的:探讨Toll样受体7(TLR7)激动剂刺激肾癌患者外周血单个核细(PBMCs)胞后其抗肿瘤活性的变化。方法:将经TLR7激动剂刺激的肾癌患者外周血单个核细胞与来源于该患者术后标本的原代肾癌细胞共培养。ELISA检测培养基中细胞因子γ-干扰素(IFN-γ)、肿瘤坏死因子α(TNF-α)和白细胞介素2(IL-2)水平,流式细胞术检测肾癌细胞周期,[~(51)Cr]释放实验检测PBMCs抗肿瘤活性变化,Western blot检测肾癌细胞Skp2及其下游通路的蛋白水平。结果:TLR7激动剂刺激外周血单个核细胞后,共培养的培养基中IFN-γ、TNF-α和IL-2表达增加,肾癌细胞的增殖指数明显受抑制、PBMCs对肾癌细胞的杀伤率显著提高(P0.05)。肾癌细胞Skp2蛋白水平在刺激后有明显的下降,下降的规律和细胞增殖指数的变化一致;下游p27蛋白水平明显增加,与Skp2蛋白水平变化的规律相反,而p21和p53无明显变化。结论:TLR7激动剂能够有效增强肾癌患者外周血单个核细胞抗肿瘤活性,使肾癌细胞生长受抑制,其机制可能与抑制Skp2/p27通路使细胞周期停滞有关。     

TLR7 激动剂替代IFN鄄酌诱导CIK 细胞杀伤肿瘤的研究

目的:探讨TLR7 激动剂(Toll like receptor 7 agonist,Tlr7a)替代IFN体外培养细胞因子诱导杀伤性细胞(Cytokine-induced killer cell,CIK)抗肿瘤的免疫效应。方法:分离健康人外周血单个核细胞,在体外诱导CIK。分两组:CIK 组和Tlr7a-CIK 组。对各组分别进行免疫杀伤性研究,检测细胞免疫表型并分析对K562 肿瘤细胞的杀伤活性。结果:CD56+ 细胞在Tlr7a-CIK 组显著增加(P<0.05),Tlr7a-CIK 组杀伤活性较CIK 组显著增强(P<0.05)。结论:Tlr7a 可以替代IFN促进CIK 细胞杀伤肿瘤。     

Detection of a TLR2 agonist by hematopoietic stem and progenitor cells impacts the function of the macrophages they produce

Several groups have shown that detection of microbial components by TLRs on hematopoietic stem and progenitor cells (HSPCs) instructs myeloid cell generation, raising interest in the possibility of targeting TLRs on HSPCs to boost myelopoiesis. However, although “TLR‐derived” cells exhibit myeloid cell characteristics (phagocytosis, cytokine production, antigen presentation), it is not clear whether they are functionally equivalent to macrophages derived in the absence of TLR activation. Our in vitro and in vivo studies show that macrophages derived from mouse and human HSPC subsets (including stem cells) exposed to a TLR2 agonist prior to or during macrophage differentiation produce lower levels of inflammatory cytokines (TNF‐α, IL‐6, and IL‐1β) and reactive oxygen species. This is in contrast to prior exposure of differentiated macrophages to the TLR2 agonist (“tolerance”), which suppresses inflammatory cytokine production, but elevates reactive oxygen species. Soluble factors produced following exposure of HSPCs to a TLR2 agonist can also act in a paracrine manner to influence the function of macrophages derived from unexposed HSPCs. Our data demonstrate that macrophage function can be influenced by TLR signaling in the HSPCs from which they are derived, and that this may impact the clinical utility of targeting TLRs on HSPCs to boost myelopoiesis.     

Peptide-stimulated DO11.10 T cells divide well but accumulate poorly in the absence of TLR agonist treatment

Immunological adjuvants increase the clonal burst size of antigen-specific T cell populations by mechanisms that remain incompletely understood. Using the DO11.10 adoptive transfer system to study peptide-stimulated T cell responses, we found that TLR agonist treatment increased the extent of cellular division undergone by responding T cells, but not by enough to explain the net increases in T cell yield that were achieved. Two novel analyses involving CFSE dye dilution analysis were used to characterize the shortfall, both of which were consistent with the idea that DO11.10 T cells are frequently lost during proliferation unless TLR agonists are present. T cell loss during clonal expansion was correlated with decreased levels of Bcl-2, but TLR agonists did not appear to afford protection by restoring levels of Bcl-2 or of cell surface IL-7Ralpha chain expression. TLR-mediated protection also failed to correlate with increased expression of Bcl-x or decreased expression of pro-apoptotic Bim. Our findings suggest that DO11.10 T cells stimulated by antigenic peptide in vivo divide well, but fail to accumulate efficiently unless TLR agonists are present.     

Pharmacology of the pyrazolo-type compounds: agonist, antagonist and inverse agonist actions

Five compounds that bind to the benzodiazepine (BZ) receptor, but show different pharmacological characteristics from the classical BZs, are profiled. CGS 8216 is a BZ antagonist/inverse agonist that reverses the effects of diazepam and also acts as a proconvulsant. CGS 9895 is also a potent BZ antagonist. In addition, this compound shows an anxiolytic profile. CGS 9896, CGS 17867A and CGS 20625 are BZ agonists (i.e., anxiolytics and anticonvulsants) which produce varying magnitudes of antagonist effect. All of these compounds are unique from the classical BZs in that each has a reduced propensity to produce the sedative and/or muscle relaxant effects characteristically associated with BZs.     

Porcine TLR8 and TLR7 are both activated by a selective TLR7 ligand, imiquimod

Toll-like receptors (TLRs) are a family of highly conserved germline-encoded pattern-recognition receptors (PRR), which are utilized by the innate immune system to recognize microbial components, known as pathogen-associated molecular patterns (PAMP). We cloned and characterized porcine TLR7 and TLR8 genes from pig lymph node tissue. Sequence analysis showed that the aa sequence identities of porcine TLR7 with human, mouse and bovine TLR7 are 85, 78 and 90%, respectively, whereas porcine TLR8 aa sequence identities with human, mouse and bovine TLR8 are 73, 69 and 79%, respectively. Both porcine TLR7 and TLR8 proteins were expressed in cell lines and were N-glycosylated. The stimulatory activity of TLR7 and TLR8 ligands to porcine and human TLR7 and TLR8 in transiently transfected Cos-7 and 293T cells were analyzed using a NF-kappaB reporter assay. Two imidazoquinoline molecules, imiquimod and gardiquimod, markedly activated both porcine TLR7 and TLR8 whereas only human TLR7, but not TLR8, was activated by the ligands. Therefore, receptor specificity for porcine TLR8 is clearly species specific. We further showed that porcine TLR7 and TLR8 are located intracellularly and are mainly within the endoplasmic reticulum. Moreover, activation of transfected cells and porcine PBMC by TLR7 ligands was inhibited by bafilomycin A(1) indicating the requirement of endosomal/lysosomal acidification for activation of the receptors.     

Brucella abortus induces Irgm3 and Irga6 expression via type-I IFN by a MyD88-dependent pathway,without the requirement of TLR2, TLR4, TLR5 and TLR9

The innate immune system senses bacterial pathogens by pattern recognition receptors, such as the well-characterised Toll-like Receptors (TLR). The activation of TLR signalling cascades depends on several adaptor proteins, among which MyD88 plays a key role in triggering innate immune responses. Here, we show in murine macrophages that Brucella abortus triggers expression of the interferon-inducible resistance proteins (IRGs, p47 GTPases) via type-I IFN secretion at late time points, when Brucella has reached its replication niche. This induction requires the adaptor molecule MyD88 but does not involve the TLRs normally implicated in sensing Gram-negative bacteria, namely TLR2, TLR4, TLR5 and TLR9. Brucella mutants lacking the functional VirB type-IV secretion system were not capable of inducing Irgm3 and Irga6 expression, suggesting that the type-IV secretion system is part of the triggering of the activation process. Our data suggest that Brucella is recognized intracellularly by an unknown receptor, different from the conventional ones used for Gram-negative sensing, but one that nevertheless signals through MyD88.     

Expression and subcellular distribution of toll-like receptors TLR4, TLR5 and TLR9 on the gastric epithelium in Helicobacter pylori infection

Toll-like receptors (TLRs) expressed by mucosal epithelium play an essential role in the defense against microbes by recognizing conserved bacterial molecules. For the first time TLR4, TLR5 and TLR9 have been microanatomically localized in patients with noninflamed gastric mucosa and Helicobacter pylori gastritis by immunohistochemistry. Because polarized expression of TLRs in apical and basolateral epithelial compartments is thought to modulate mucosal immunity, subcellular TLR distribution by gastric epithelium was investigated using confocal microscopy. TLR4, TLR5 and TLR9 were expressed by gastric epithelium in antrum and corpus of all patients with H. pylori gastritis (n = 14) and with noninflamed gastric mucosa (n = 5). TLR4 was expressed at the apical and the basolateral pole of the gastric epithelium as well in noninflamed gastric mucosa as in H. pylori gastritis. TLR5 and TLR9 expression in the noninflamed gastric mucosa was identical to that of TLR4 with localization at the apical and the basolateral epithelial pole. However, in H. pylori gastritis TLR5 and TLR9 expression on the gastric epithelium changed to an exclusive basolateral localization without detectable expression at the apical pole. In the human stomach, the gastric epithelium expressed TLR4, TLR5 and TLR9, which gives it the possibility to interact with H. pylori. Furthermore, gastric epithelial TLR4 expression is highly polarized in an apical and a basolateral compartment, whereas TLR5 and TLR9 polarization seems to be a process dynamically influenced by H. pylori infection. This polarized and dynamically regulated gastric epithelial expression of TLRs supports a sentinel role for these receptors in the mucosal immunity to H. pylori.     

Squalene emulsion potentiates the adjuvant activity of the TLR4 agonist,GLA, via inflammatory caspases,IL‐18, and IFN‐γ

The synthetic TLR4 agonist glucopyranosyl lipid adjuvant (GLA) is a potent Th1‐response‐inducing adjuvant when formulated in a squalene oil‐in‐water emulsion (SE). While the innate signals triggered by TLR4 engagement are well studied, the contribution of SE remains unclear. To better understand the effect of SE on the adjuvant properties of GLA‐SE, we compared the innate and adaptive immune responses elicited by immunization with different formulations: GLA without oil, SE alone or the combination, GLA‐SE, in mice. Within the innate response to adjuvants, only GLA‐SE displayed features of inflammasome activation, evidenced by early IL‐18 secretion and IFN‐γ production in memory CD8⁺ T cells and neutrophils. Such early IFN‐γ production was ablated in caspase‐1/11^?/? mice and in IL‐18R1^?/? mice. Furthermore, caspase‐1/11 and IL‐18 were also required for full Th1 CD4⁺ T‐cell induction via GLA‐SE. Thus, we demonstrate that IL‐18 and caspase‐1/11 are components of the response to immunization with the TLR4 agonist/squalene oil‐in‐water based adjuvant, GLA‐SE, providing implications for other adjuvants that combine oils with TLR agonists.     

Poly (γ-glutamic acid) based combination of water-insoluble paclitaxel and TLR7 agonist for chemo-immunotherapy

Advanced anti-cancer regimens are being introduced for more effective cancer treatment with improved life expectancy. In this research, immuno-stimulating agent toll-like receptor-7 (TLR-7) agonist-imiquimod and low dose chemotherapeutic agent-paclitaxel were synergized to demonstrate tumor therapy along with anti-tumor memory effect. Both therapeutic agents being water insoluble were dispersed in water with the help of water soluble polymer: poly (γ-glutamic acid) (γ-PGA) using a co-solvent systems leading to formation of micro-dispersions of drugs. Paclitaxel and imiquimod formed crystalline microstructures in the size range of 2–3 μm and were stably dispersed in γ-PGA matrix for more than 6 months. Paclitaxel and combination of paclitaxel and imiquimod had significant tumor killing effect in-vitro on various tumor cell lines, while antigen presenting cells (dendritic cells-DCs) treated with the same concentration of imiquimod along with the combination led to enhanced proliferation (250%). In DCs, enhanced secretion of pro-inflammatory and Th1 cytokines was observed in cells co-treated with paclitaxel and imiquimod dispersed in γ-PGA. When administered by intra-tumoral injection in mouse melanoma tumor model, the treatment with combination exemplified drastic inhibition of tumor growth leading to 70% survival as compared to individual components with 0% survival at day 41. The anti-tumor response generated was also found to have systemic memory response since the vaccinated mice significantly deferred secondary tumor development at distant site 6 weeks after treatment. The relative number and activation status of DCs in-vivo was found to be dramatically increased in case of mice treated with combination. The dramatic inhibition of tumor treated with combination is expected to be mediated by both chemotherapeutic killing of tumor cells followed by uptake of released antigen by the DCs and due to enhanced proliferation and activation of the DCs.     

Age-related changes and distribution of T cell markers (CD3 and CD4) and toll-like receptors(TLR2, TLR3,TLR4 and TLR7) in the duck lymphoid organs

T lymphocytes and Toll-like receptors have been confirmed to have correlation with the ability to resistance to pathogenic challenges and play an important role in duck immune system. However, the information of ontogeny of T lymphocytes and Toll-like receptors is scarcely in duck. Therefore, to address these questions, we report the development and distribution of CD3 and CD4 by immunocytochemistry and the age-related mRNA level of duck T cell markers (CD3 and CD4) and Toll-like receptors (TLR2, TLR3, TLR4 and TLR7) by real time quantitative PCR in duck lymphoid organs (thymus, bursa of Fabricius and spleen). Results indicated that CD3 and CD4 positive cells can be observed in all test organs and partly change in an age-related way. CD4 positive T cell of duck spleen mainly distributed in periarterial lymphatic sheaths and red pulp, not in white pulp. Both of CD3 and CD4 were experienced significant increased wave twice in duck lymphoid organs and T cell dependent cellular immunity of duck may well established until 5 weeks old. The mRNA expression levels of duck TLRs were age and organ dependent, and duck TLR3 and TLR7 were significantly lower abundance in the spleen but higher in thymus and bursa of Fabricius, respectively. This study provide the essential knowledge of the ontogeny of T cells and Toll-like receptors in duck, which may shed lights on the T-cell mediate immunity and innate immunity in duck.     

A new synthetic TLR4 agonist, GLA, allows dendritic cells targeted with antigen to elicit Th1 T-cell immunity in vivo

Protein‐based vaccines offer safety and cost advantages but require adjuvants to induce immunity. Here we examined the adjuvant capacity of glucopyranosyl lipid A (GLA), a new synthetic non‐toxic analogue of lipopolysaccharide. In mice, in comparison with non‐formulated LPS and monophosphoryl lipid A, formulated GLA induced higher antibody titers and generated Type 1 T‐cell responses to HIV gag‐p24 protein in spleen and lymph nodes, which was dependent on TLR4 expression. Immunization was greatly improved by targeting HIV gag p24 to DCs with an antibody to DEC‐205, a DC receptor for antigen uptake and processing. Subcutaneous immunization induced antigen‐specific T‐cell responses in the intestinal lamina propria. Immunity did not develop in mice transiently depleted of DCs. To understand how GLA works, we studied DCs directly from vaccinated mice. Within 4 h, GLA caused DCs to upregulate CD86 and CD40 and produce cytokines including IL‐12p70 in vivo. Importantly, DCs removed from mice 4 h after vaccination became immunogenic, capable of inducing T‐cell immunity upon injection into naïve mice. These data indicate that a synthetic and clinically feasible TLR4 agonist rapidly stimulates full maturation of DCs in vivo, allowing for adaptive immunity to develop many weeks to months later.     

Relationship of TLR2, TLR4 and tissue remodeling in chronic rhinosinusitis

In order to explore the role of innate immunity in the remodeling of CRS (chronic rhinosinusitis), we investigated the correlation between TLR2, TLR4 and remodeling involved cytokines and histopathological features. Immunohistochemical staining was applied to detect the expression of TLR2, TLR4 and TGF-β1. Masson staining was used for observing the collagen deposition. The other histopathologic features of remodeling were observed by hemotoxylin and eosin (HE) staining. Nasal epithelial cell culture was used to elucidate the effect of TLR2, TLR4 agonists and inhibitors on the expression of TGF-β1 and MMP-9. The association study showed that the significantly higher expression of TLR2, TLR4, TGF-β1 and collagen appeared in CRSsNP (chronic rhinosinusitis without nasal polyps) patients compared with CRSwNP (chronic rhinosinusitis with nasal polyps) patients. In CRSsNP, patients with a severe epithelial damage (score 3) had a significantly higher expression of TLR2 than patients with mild epithelial damage (score ≤ 2) (P < 0.05). Moreover the expression of TLR2 correlated negatively with squamous hyperplasia in CRSsNP, and positively with gland hyperplasia in CRSwNP. The expression of TLR2 and TLR4 was closely related to neutrophil infiltration in CRSsNP (P < 0.01). TGF-β1 was downregulated by TLR2 agonist in CRSwNP and upregulated by TLR4 agonist in CRSsNP (P < 0.05). MMP-9 was upregulated by TLR4 agonist in CRSwNP (P < 0.05). TLR2 and TLR4 had close relationship with TGF-β1 and the histologic features of remodeling, especially collagen deposition and neutrophil infiltration in CRSsNP. The innate immunity could influence the histologic characteristics and involved cytokines through TLR2 and TLR4 in the remodeling of CRS.