Direct Toll-like Receptors Research Articles

The multiple functions of miR-574-5p in the neuroblastoma tumor microenvironment.

Neuroblastoma is the most common extracranial solid tumor in childhood and arises from neural crest cells of the developing sympathetic nervous system. Prostaglandin E2 (PGE2) has been identified as a key pro-inflammatory mediator of the tumor microenvironment (TME) that promotes neuroblastoma progression. We report that the interaction between the microRNA miR-574-5p and CUG-binding protein 1 (CUGBP1) induces the expression of microsomal prostaglandin E2 synthase 1 (mPGES-1) in neuroblastoma cells, which contributes to PGE2 biosynthesis. PGE2 in turn specifically induces the sorting of miR-574-5p into small extracellular vesicles (sEV) in neuroblastoma cell lines. sEV are one of the major players in intercellular communication in the TME. We found that sEV-derived miR-574-5p has a paracrine function in neuroblastoma. It acts as a direct Toll-like receptor 7/8 (TLR7/8) ligand and induces α-smooth muscle actin (α-SMA) expression in fibroblasts, contributing to fibroblast differentiation. This is particularly noteworthy as it has an opposite function to that in the TME of lung carcinoma, another PGE2 dependent tumor type. Here, sEV-derived miR-574-5p has an autokrine function that inhibits PGE2 biosynthesis in lung cancer cells. We report that the tetraspanin composition on the surface of sEV is associated with the function of sEV-derived miR-574-5p. This suggests that the vesicles do not only transport miRs, but also appear to influence their mode of action.

Systematic Optimization of Potent and Orally Bioavailable Purine Scaffold as a Dual Inhibitor of Toll-Like Receptors 7 and 9.

Several toll-like receptors (TLRs) reside inside endosomes of specific immune cells-among them, aberrant activation of TLR7 and TLR9 is implicated in myriad contexts of autoimmune diseases, making them promising therapeutic targets. However, small-molecule TLR7 and TLR9 antagonists are not yet available for clinical use. We illustrate here the importance of C2, C6, and N9 substitutions in the purine scaffold for antagonism to TLR7 and TLR9 through structure-activity relationship studies using cellular reporter assays and functional studies on primary human immune cells. Further in vitro and in vivo pharmacokinetic studies identified an orally bioavailable lead compound 29, with IC50 values of 0.08 and 2.66 μM against TLR9 and TLR7, respectively. Isothermal titration calorimetry excluded direct TLR ligand-antagonist interactions. In vivo antagonism efficacy against mouse TLR9 and therapeutic efficacy in a preclinical murine model of psoriasis highlighted the potential of compound 29 as a therapeutic candidate in relevant autoimmune contexts.

Chronic Exposure of Gingival Fibroblasts to TLR2 or TLR4 Agonist Inhibits Osteoclastogenesis but Does Not Affect Osteogenesis.

Chronic exposure to periodontopathogenic bacteria such as Porphyromonas gingivalis and the products of these bacteria that interact with the cells of the tooth surrounding tissues can ultimately result in periodontitis. This is a disease that is characterized by inflammation-related alveolar bone degradation by the bone-resorbing cells, the osteoclasts. Interactions of bacterial products with Toll-like receptors (TLRs), in particular TLR2 and TLR4, play a significant role in this chronic inflammatory reaction, which possibly affects osteoclastic activity and osteogenic capacity. Little is known about how chronic exposure to specific TLR activators affects these two antagonistic activities. Here, we studied the effect of TLR activation on gingival fibroblasts (GF), cells that are anatomically close to infiltrating bacterial products in the mouth. These were co-cultured with naive osteoclast precursor cells (i.e., monocytes), as part of the peripheral blood mononuclear cells (PBMCs). Activation of GF co-cultures (GF + PBMCs) with TLR2 or TLR4 agonists resulted in a weak reduction of the osteoclastogenic potential of these cultures, predominantly due to TLR2. Interestingly, chronic exposure, especially to TLR2 agonist, resulted in increased release of TNF-α at early time points. This effect, was reversed at later time points, thus suggesting an adaptation to chronic exposure. Monocyte cultures primed with M-CSF + RANKL, led to the formation of bone-resorbing osteoclasts, irrespective of being activated with TLR agonists. Late activation of these co-cultures with TLR2 and with TLR4 agonists led to a slight decrease in bone resorption. Activation of GF with TLR2 and TLR4 agonists did not affect the osteogenic capacity of the GF cells. In conclusion, chronic exposure leads to diverse reactions; inhibitory with naive osteoclast precursors, not effecting already formed (pre-)osteoclasts. We suggest that early encounter of naive monocytes with TLR agonists may result in differentiation toward the macrophage lineage, desirable for clearing bacterial products. Once (pre-)osteoclasts are formed, these cells may be relatively insensitive for direct TLR stimulation. Possibly, TLR activation of periodontal cells indirectly stimulates osteoclasts, by secreting osteoclastogenesis stimulating inflammatory cytokines.

Minocycline Prevents the Development of Mechanical Allodynia in Mouse Models of Vincristine-Induced Peripheral Neuropathy.

Vincristine is an antineoplastic substance that is part of many chemotherapy regimens, used especially for the treatment of a variety of pediatric cancers including leukemias and brain tumors. Unfortunately, many vincristine-treated patients develop peripheral neuropathy, a side effect characterized by sensory, motoric, and autonomic symptoms. The sensory symptoms include pain, in particular hypersensitivity to light touch, as well as loss of sensory discrimination to detect vibration and touch. The symptoms of vincristine-induced neuropathy are only poorly controlled by currently available analgesics and therefore often necessitate dose reductions or even cessation of treatment. The aim of this study was to identify new therapeutic targets for the treatment of vincristine-induced peripheral neuropathy (VIPN) by combining behavioral experiments, histology, and pharmacology after vincristine treatment. Local intraplantar injection of vincristine into the hind paw caused dose- and time-dependent mechanical hypersensitivity that developed into mechanical hyposensitivity at high doses, and lead to a pronounced, dose-dependent infiltration of immune cells at the site of injection. Importantly, administration of minocycline effectively prevented the development of mechanical hypersensitivity and infiltration of immune cells in mouse models of vincristine induce peripheral neuropathy (VIPN) based on intraperitoneal or intraplantar administration of vincristine. Similarly, Toll-like receptor 4 knockout mice showed diminished vincristine-induced mechanical hypersensitivity and immune cell infiltration, while treatment with the anti-inflammatory meloxicam had no effect. These results provide evidence for the involvement of Toll-like receptor 4 in the development of VIPN and suggest that minocycline and/or direct Toll-like receptor 4 antagonists may be an effective preventative treatment for patients receiving vincristine.

Targeting Toll-like receptors with soluble Toll-like receptor 2 prevents peritoneal dialysis solution–induced fibrosis

Peritoneal membrane failure due to fibrosis limits the use of peritoneal dialysis (PD). Peritoneal fibrosis may potentially be induced by sterile inflammation caused by ongoing cellular stress due to prolonged exposure to PD solutions (PDS). Effective therapies to prevent this process remain to be developed. Toll-like receptors (TLRs) mediate sterile inflammation by recognizing damage-associated molecular patterns (DAMPs) released by cellular stress. We evaluated the involvement of TLRs and DAMPs in PDS-induced fibrosis models and the therapeutic potential of TLR-DAMP targeting for preventing fibrosis. A range of PDS elicited pro-inflammatory and fibrotic responses from PD patient peritoneal leukocytes, mesothelial cells and mouse peritoneal leukocytes. TLR2/4 blockade of human peritoneal cells or TLR2/4 knockouts inhibited these effects. PDS did not induce rapid ERK phosphorylation or IκB-α degradation, suggesting that they do not contain components capable of direct TLR activation. However, PDS increased the release of Hsp70 and hyaluronan, both TLR2/4 DAMP ligands, by human and mouse peritoneal cells, and their blockade decreased PDS-driven inflammation. Soluble TLR2, a TLR inhibitor, reduced PDS-induced pro-inflammatory and fibrotic cytokine release exvivo. Daily catheter infusion of PDS in mice caused peritoneal fibrosis, but co-administration of soluble TLR2 prevented fibrosis, suppressed pro-fibrotic gene expression and pro-inflammatory cytokine production, reduced leukocyte/neutrophil recruitment, recovered Treg cell levels and increased the Treg:Th17 ratio. Thus, TLR2/4, Hsp70 and hyaluronan showed major roles in PDS-induced peritoneal inflammation and fibrosis. The study demonstrates the therapeutic potential of a TLR-DAMP targeting strategy to prevent PDS-induced fibrosis.

STAT1 plays a role in TLR signal transduction and inflammatory responses.

Activation of the Toll-like receptor (TLR) family of innate immune sensors stimulates multiple signal transduction pathways. Previous studies have suggested that TLR2, TLR4 and TLR9 induce serine phosphorylation of Signal Transducers and Activators of Transcription-1 (STAT1) at residue 727 (S727), although its role in TLR signaling was unclear. We report here that STAT1 rapidly undergoes phosphorylation following TLR4 challenge with lipopolysaccharide (LPS) in a model of LPS hypersensitivity in vivo. Importantly, genetic ablation of STAT1 protected against LPS-induced lethality suggesting that STAT1 may have a key role in TLR-induced inflammation. We have found that multiple TLRs induce Ser727 phosphorylation of STAT1, which is dependent on MyD88 and TRIF signaling, but independent of interferon (IFN) regulatory factor (IRF)-3, IRF7 and the IFN receptor complex, suggesting that activation is a direct TLR response rather than autocrine activation via IFN. Importantly, we found that STAT1 interacts with tumor necrosis factor (TNF) receptor-associated factor-6 (TRAF6), a key mediator of TLR signaling after TLR challenge and that following activation, STAT1 translocates to the nucleus. Critically, macrophages generated from mice in which the S727 residue was replaced with alanine (STAT1 S727A mice) display significantly reduced TNFα protein production, but not reduced interleukin-6 or RANTES protein in response to multiple TLR challenges, as compared with wild-type macrophages. These results clearly demonstrate cross-talk between the TLR and JAK/STAT signaling pathways with direct recruitment of STAT1 by TRAF6 and that the direct activation of STAT1 by TLR signaling suggests a crucial role for STAT1 in TLR-induced inflammation.

Gliadin is an uncatalogued Toll-like receptor ligand

Coeliac disease is a chronic intestinal inflammatory condition, which is caused by an inappropriate immune response to components of wheat family cereals in a genetically susceptible host. Gliadins are the major pathogenic constituent of wheat; their toxicity and immunogenicity depend on their amino acid sequence. They are known to initiate the innate and the adaptive immune response. Nevertheless, it is not yet known how they are recognised by the intestinal epithelium and immune cells. Toll-like receptors (TLRs) are the best-studied group of pattern recognition receptors, which play a critical role in the initiation of innate immunity through the recognition of pathogen- and damage-associated molecular patterns. Exogenous food-derived proteins are not yet recognised as TLR ligands. Gliadin can activate TLR signalling pathway in vitro. The existing evidence is suggestive of the direct contribution of gliadin and/or other wheat components to activating TLR signalling. However, the data available so far are controversial and are mainly focussed on TLR 2 and TLR4. It is hypothesised that gliadin is a direct ligand for one of the TLRs. If indeed gliadin is proven to be a direct ligand of TLRs, our understanding of the pathogenesis of gastrointestinal diseases, such as colorectal cancer, will also be greatly influenced. In order to fully appreciate the role of gliadin as a direct TLR ligand, it should be proven to interact physically with and bind to one or more of the TLR molecules. Furthermore, it should be documented that TLR pathway activation is the downstream effect of gliadin/TLR binding.

A Critical Role of MyD88 Signaling in Donor T Cells for Graft-Versus-Host Disease (GVHD) and Graft-Versus-Leukemia Effect (GVL) After Experimental Allogeneic Hematopoietic Stem Cell Transplantation (allo-HSCT)

AbstractAbstract 1893The therapeutic potential of allogeneic hematopoietic stem cell transplantation (allo-HSCT) relies on the graft-versus-leukemia effect (GVL) to eradicate residual tumor cells by immunologic mechanisms. However, graft-versus-host disease (GVHD) remains the major toxicity of allo-HSCT. Alloreactive donor T cells are important effector cells in the development of GVHD, and proinflammatory cytokines enhance the generation of donor antihost cytotoxic function. Myeloid differentiation factor (MyD88) is a cytoplasmic adaptor molecule essential for integrating and transducing the signals generated by the Toll-like receptor (TLR) family. TLR engagement on professional antigen-presenting cells induces their maturation, resulting in optimal T-cell activation. However, recent advances indicate that the adjuvant effects of certain TLR agonists may also be attributed to the activation of TLRs and MyD88 directly in T cells. Both CD4 and CD8 T cells express functional TLRs. It remains to be defined whether direct TLR signaling on donor T cells is critical for GVHD or GVL activity.We used C57BL/6 (H-2b) → B6D2F1 (H-2b/d) experimental allo-HSCT model, which differs at major and minor histocompatibility loci, to address the role of donor T cell MyD88 signaling on GVHD and GVL. Lethally irradiated recipient mice were transplanted TCD-BM (5 × 106) together with either wild-type (WT) or MyD88 knock out (KO) mice spleen T cells (1 × 106) on day 0 and then host-type P815 mastocytoma or L1210 leukemia (H-2d) cells were injected either intravenously (3 × 103) or subcutaneously (1 × 106) on day 1 to generate a GVHD/GVL model. First of all, clinical GVHD scores were comparable between recipients of WT T cells and MyD88 KO T cells. At 70 days post-allo-HSCT, 50 % of allogeneic recipients of WT T cells died due to severe GVHD, but necropsy showed no evidence of tumor. In contrast, 83.5% of those of MyD88 KO T cells died with gross evidence of tumors (P<.05). Moreover, subcutaneous tumors in the allogeneic recipients receiving MyD88 KO T cells exhibited markedly increased growth in vivo compared to those receiving WT T cells (tumor volume on day 41, 15205.6 vs. 373.9 mm3, P<.01). GVHD mortality is critically dependent on donor CD4 T cells in this donor/recipient strain combination (B6 → B6D2F1) and CD8 T cells that mediate cytotoxicity are more potent effectors of GVL. The percentages of donor T cells to undergo proliferation or apoptosis in response to alloantigens in vivo between the two T cell types was examined; apoptosis of CD8 T cells in recipients of MyD88 KO T cells was significantly enhanced compared to those of WT T cell recipients (P<.01) whereas apoptosis of CD4 T cells was comparable between two groups. Resultingly, the percentages of CD8 T cells in recipients of MyD88 KO T cells were significantly lower (P<.01). We next examined the effects of MyD88 signaling in donor T cells on cytolytic activity to host antigens. Splenocytes harvested from WT mice showed stronger cytolytic activity against P815 targets compared to those from MyD88 KO mice (P<.01). After allogeneic mixed leukocyte reaction, responder T cells from MyD88 KO mice showed markedly reduced IFN-γ, MCP-1 and IL-17A production with a significant augmentation in IL-10 secretion. We further evaluated the effect of T-cell MyD88 deficiency on GVL mediated by the intensity of total body irradiation (TBI) conditioning (1300 vs. 900 cGy, Exp Hematol 2011; 39: 1018–29). Enhanced GVL in the allogeneic recipients receiving 1300 cGy TBI was not shown in the recipients of MyD88 KO T cells.In summary, these results highlight a critical role for MyD88 signaling in T-cell activation and cytotoxicity, offering the opportunity for improving GVL activity by targeting TLR-MyD88 signaling within donor T cells. Furthermore, these data demonstrated that MyD88 deficiency in T cells can impair cytolytic function or subsequent GVL activity of CD8 T cells without significant change in the severity of CD4-dependent GVHD. This difference is attributed to the fact that MyD88 deficiency in T cells causes an enhanced apoptosis of donor CD8 T cells but not donor CD4 T cells in vivo after HSCT. Disclosures:No relevant conflicts of interest to declare.

Herpes Simplex Virus Induces the Early Activation of NK Cell via MyD88- Dependent Signal~!2009-12-24~!2010-05-14~!2010-06-22~!

In the course of a Herpes Simples Virus type-1 (HSV-1) infection, the bidirectional cross-talk between Natural Killer (NK) cells and Dendritic Cells (DCs) is imperative in mounting an efficient immunological response. Enhanced DC maturation by Toll-Like Receptor (TLR) ligands is considered as a pivotal link between innate and adaptive immune responses, yet the impact of direct TLR signaling on NK:DC interaction during an HSV infection has yet to be examined. We demonstrate that HSV mediates the activation of NK cells and the induction of the cooperative relationship between NK cells and DCs via TLR. During the course of an HSV infection, the initial response required both types of innate cells and demonstrated phenotypic and functional activation much like that of TLR-stimulated cells. Using a MyD88 knock-out system diminished the early interaction between DCs and NK after HSV exposure. Our results indicate that HSV TLR- ligands modulate the early NK: DC interaction and may be a mechanism used by these innate immune cells to overcome virus mediated immune evasion and the initiation of a potent acquired immune response.

The mechanism of LPS-induced HIV type I activation in transgenic mouse macrophages

In the course of the development of acquired immunodeficiency syndrome (AIDS), bacterial infection causes deleterious effects on the progression of the disease; bacterial LPS in the circulation activate immune cells, resulting in the acceleration of HIV replication. However, the precise HIV activation mechanisms in infected hosts remain largely unknown. Previously, we generated transgenic (Tg) mice carrying the HIV type I (HIV-1) genome and showed that LPS induces the activation of HIV-1 in splenocytes through the induction of tumor necrosis factor (TNF) and IL-1, although similarly induced IFN-gamma and IL-6 are not involved. In this study, we analyzed the mechanisms of HIV-1 activation in macrophages using these HIV-1 Tg mice, because macrophages are one of the major reservoirs of HIV-1. In contrast to splenocytes, direct Toll-like receptor (TLR) 4 signaling rather than TLR-induced pro-inflammatory cytokines was responsible for the LPS-induced activation of HIV-1 in macrophages, because the time course of HIV-1 activation was earlier than that observed in splenocytes and TNF neutralization did not inhibit the activation. p38 mitogen-activated protein kinase (MAPK) and nuclear factor kappa B (NF-kappaB) activation, but neither extracellular signal-regulated kinase nor c-Jun N-terminal kinase activation, were required for the activation, because only inhibitors for p38 MAPK and NF-kappaB suppressed activation of HIV-1. Furthermore, we showed that myeloid differentiation primary response gene (MyD) 88, rather than Toll/IL-1R domain-containing adaptor inducing IFN-beta (TRIF), was required as an adaptor molecule for this activation using Myd88(-/-) mice and Dynasore, a specific inhibitor for TRIF, and small interfering RNAs specific for Myd88 and Trif. These observations suggest that suppression of these molecules, which are involved in the TLR4-MyD88 pathway and the downstream p38 MAPK and NF-kappaB pathways, should be beneficial to prevent development of AIDS in HIV-1-infected people.

The contribution of direct TLR signaling to T cell responses

It is well established that Toll-like receptors (TLRs) play a critical role in the generation of innate immune responses and thereby also play an important, indirect role in the initiation of subsequent adaptive T cell responses. However, T cells also express certain TLRs, and we have focused on the physiological importance of direct TLR signaling in T cells. TLRs can function as co-stimulatory receptors that complement TCR-induced signals to enhance effector T cell proliferation, survival and cytokine production. We also found that TLR signaling pathways in T cells are required for the effective clonal expansion of antigen-specific T cells during infection in vivo. Thus, the importance of TLRs in T cell-mediated immunity reflects both T cell-extrinsic and T cell-intrinsic components, which warrants a reconsideration of the dogma that restricts germ-line encoded pattern recognition to cells of the innate immune system.

T Cell Activation by TLRs: A Role for TLRs in the Adaptive Immune Response

Toll-like receptor (TLR) activation is primarily thought to affect antigen-presenting cells (APCs) by inducing an innate immune response that can subsequently activate the adaptive immune system. However, there are increasing data that demonstrate expression and activation of TLRs on T cells, thus providing evidence for a direct role for TLRs in the activation of an adaptive immune response. A study recently demonstrated that Pam3CSK {N-palmitoyl-S-[2,3-bis(palmitoloxy)-(2RS)-propyl]-Cys-Ser-Lys(4)}, a TLR2 agonist lipopeptide, activates T helper 1 (T(H)1) cells and induces interferon-gamma (IFN-gamma) production, even in the absence of TLR1, which differs from its mechanism of activation of APCs. Moreover, whereas Pam3CSK-stimulated IFN-gamma production by T(H)1 cells is ablated in the absence of both myeloid differentiation marker 88 (MyD88), an adaptor protein in the TLR pathway, and interleukin-1 receptor (IL-1R)-associated kinase-4 (IRAK4), the mitogen-activated protein kinases p38 and c-Jun N-terminal kinase (JNK) are still phosphorylated. These data suggest that TLR2 activation of T(H)1 cells occurs through a mechanism different from that described for APCs and provides further evidence of direct TLR activation of the adaptive immune system.

Direct Toll-like receptor 2 mediated co-stimulation of T cells in the mouse system as a basis for chronic inflammatory joint disease

The pathogenesis of chronic inflammatory joint diseases such as adult and juvenile rheumatoid arthritis and Lyme arthritis is still poorly understood. Central to the various hypotheses in this respect is the notable involvement of T and B cells. Here we develop the premise that the nominal antigen-independent, polyclonal activation of preactivated T cells via Toll-like receptor (TLR)-2 has a pivotal role in the initiation and perpetuation of pathogen-induced chronic inflammatory joint disease. We support this with the following evidence. Both naive and effector T cells express TLR-2. A prototypic lipoprotein, Lip-OspA, from the etiological agent of Lyme disease, namely Borrelia burgdorferi, but not its delipidated form or lipopolysaccharide, was able to provide direct antigen-nonspecific co-stimulatory signals to both antigen-sensitized naive T cells and cytotoxic T lymphocyte (CTL) lines via TLR-2. Lip-OspA induced the proliferation and interferon (IFN)-γ secretion of purified, anti-CD3-sensitized, naive T cells from C57BL/6 mice but not from TLR-2-deficient mice. Induction of proliferation and IFN-γ secretion of CTL lines by Lip-OspA was independent of T cell receptor (TCR) engagement but was considerably enhanced after suboptimal TCR activation and was inhibitable by monoclonal antibodies against TLR-2.