R848 Stimulation Research Articles

PTPN22 Variant R620W Is Associated With Reduced Toll-like Receptor 7-Induced Type I Interferon in Systemic Lupus Erythematosus.

Protein tyrosine phosphatase nonreceptor type 22 (PTPN22) is associated with an increased risk of systemic lupus erythematosus (SLE). PTPN22 encodes Lyp, and a disease-associated coding variant bears an R620W substitution (LypW). LypW carriage is associated with impaired production of type I interferon (IFN) by myeloid cells following Toll-like receptor (TLR) engagement. The aim of this study was to investigate the effects of LypW carriage on TLR signaling in patients with SLE. Plasma IFNα concentrations and whole-blood IFN gene scores were compared in SLE patients who were LypW carriers and those who were noncarriers. TLR-7 agonist R848-stimulated IFNα and tumor necrosis factor levels, IFN-dependent gene expression, and STAT-1 activation were determined in peripheral blood mononuclear cells (PBMCs) and/or plasmacytoid dendritic cells (PDCs) obtained from these patients. The effect of LypW expression on the systemic type I IFN response to R848 stimulation in vivo was assessed in transgenic mice. Plasma IFNα levels and whole-blood IFN gene signatures were comparable in SLE patients who were LypW carriers and those who were noncarriers. However, PBMCs from LypW carriers produced less IFNα and showed reduced IFN-dependent gene up-regulation and STAT-1 activation after R848 stimulation. The frequency of PDCs producing IFNα2 and the per-cell IFNα2 levels were significantly reduced in LypW carriers. LypW-transgenic mice displayed reduced TLR-7-induced circulating type I IFN responses. PDCs from SLE patients carrying the disease-associated PTPN22 variant LypW showed a reduced capacity for TLR-7 agonist-induced type I IFN production, even though LypW carriers displayed systemic type I IFN activation comparable with that observed in noncarriers. LypW carriage identifies SLE patients who may harbor defects in TLR- and PDC-dependent host defense or antiinflammatory functions.

Population diversity in transcriptional responses of macrophages to TLR7/8 signaling (IRM12P.654)

Abstract The Toll-like receptors (TLR) of the innate immune system play a key role in the recognition of pathogens and the initiation of a robust innate immune response. Although an innate immune response is essential for resistance to pathogen infection, the magnitude and qualities of innate immune responses are quite variable in the human population. Here we performed transcriptome sequencing of monocyte-derived macrophages generated from 78 healthy individuals, including 10 replicates from repeat donors, to profile gene expression patterns in responses to TLR7/8 signaling. Extensive qualitative and quantitative diversity was apparent in the response of individuals in this panel. Cluster analysis discriminated nine distinct clusters of genes expressed in response to the TLR7/8 agonist (R848) with correlated expression variations within this panel. Interestingly, the induction of cytokine and chemokine transcription varied among donors and majority of these genes have very reproducible variability of their expression in 10 replicates. In addition, it was shown that donors with high expression of cytokine and chemokine genes after R848 stimulation were also high responder to Respiratory syncytial virus infection. Thus, there are the variations in gene expression among individuals with TLR7/8 stimulation, indicating that genetic polymorphisms in “master” regulators may correlate expression patterns of multiple genes during innate responses.

Identification and Immune Functional Characterization of Pigeon TLR7

Toll-like receptor 7 (TLR7) is activated by single-stranded RNA and synthetic imidazoquinoline components, and induces interferon production. In this study, we cloned the TLR7 gene from King pigeon (Columba livia). The TLR7 open reading frame is 3144 bp and encodes a 1047-amino acid protein, consisting of a canonical TLR composition with 15 leucine-rich repeats (LRRs). Amino acid-inserting modifications were found at position 15 of LRR2, LRR11, LRR13, and LRR14 and position 10 of LRR10. The tissue distribution of pigeon TLR7 suggests that immune-associated tissues, especially the spleen and liver, have high TLR7 expression. HEK293T cells transfected with pigeon TLR7 plasmid responded to the agonist R848, indicating a functional TLR7 homolog. Following R848 stimulation of pigeon peripheral blood mononuclear cells, the levels of IFN-γ, IL-6, IL-8, CCL5, and IL-10 mRNA, assessed using quantitative real-time PCR, were significantly up-regulated. After Newcastle disease virus vaccine strain LaSota inoculation and agonist R848 injection, the level of TLR7 mRNA in the spleen of pigeons increased significantly in the R848-injected group, but decreased in the LaSota-inoculated group at three day post-infection (d.p.i.). The mRNA levels of inflammatory cytokines and chemokines were significantly upregulated in both LaSota-inoculated and R848-injected groups. Triggering pigeon TLR7 leads to robust up-regulation of inflammatory cytokines and chemokines, suggesting an important role in the innate immune response.

The Contribution of Toll-like Receptor (TLR) Pathway Hyper-Reactivity to Clonal Selection in Secondary MDS and AML

Background: Stem cell (HSC) hypersensitivity to inflammatory cytokines and exaggerated TLR-dependent production of such cytokines contribute to bone marrow failure and clonal selection in Fanconi anemia (FA). Clonal neoplasms in FA patients and FA-deficient mice exhibit either resistance or paradoxical proliferative responses to tumor necrosis factor-alpha (TNF) and interferon-gamma (IFN). Because FA MDS/AML shares cytogenetic and clinical features in common with secondary MDS/AML developing after prior MDS or exposure to cytotoxic chemotherapy (sMDS/sAML), we tested the idea that an FA-like TLR/cytokine hypersensitive phenotype might underlie clonal selection in sMDS/sAML. A pilot study of 4 hematologically normal individuals with histories of prior cytotoxic chemotherapy (including alkylating agents) revealed 2 whose committed progenitor cells were hypersensitive to TNF and IFN. Three patients with sMDS/sAML exhibited resistance to IFN and paradoxical growth responses to TNF. To determine the true prevalence of these FA-like phenotypes, we conducted a larger study of patients with sMDS/sAML, quantifying a) progenitor cell growth in response to inflammatory cytokines and b) TLR7/8 or TLR4-dependent cytokine production in peripheral blood monocytes.Methods: Patients with newly diagnosed sMDS or sAML were eligible. All potential subjects with active infections were excluded. Research bone marrow aspirates and peripheral blood (PB) were obtained at the same time. TNF was quantified (ELISA) in the supernatants of PB CD14+ cells cultured in the presence of 1 ng/ml lipopolysaccharide (LPS, a TLR-4 agonist) or 3-5 μM R848 (TLR-7/8 agonist) for 24-hours. BFU-E and CFU-GM were quantified in methylcellulose cultures of low density bone marrow mononuclear cells (LDBMCs) (SCF, IL-3, EPO +/- TNF or IFN). Progenitor responses were classified as resistant/paradoxical if either CFU-GM or BFU-E colonies were >120% of control (normal volunteer bone marrow cells) at TNF 1 ng/ml, or >100% at IFN 0.1 ng/ml; all other responses were categorized as sensitive. TNF production was classified as exaggerated if levels exceeded normal mean values by more than two standard deviations.Results: A total of 22 patients with sMDS/sAML had bone marrow and PB analysis, including 13 patients with complex karyotypes and 9 patients with non-complex karyotypes. An additional 18 patients had PB samples drawn without providing bone marrow samples. Progenitor responses: In colony assays, 18/22 patients demonstrated resistant/paradoxical growth responses to TNF/IFN, a phenomenon seen in patients with both complex and non-complex karyotypes. Interestingly, TNF resistance was observed only in samples that were also IFN resistant. Among patients with a complex karyotype, only 3 demonstrated normal progenitor sensitivity. None of these 3 had received prior chemotherapy (2 with radiation only). Macrophage responses: CD14+ cells from 63% of patients with resistant/paradoxical colony assay responses overproduced TNF in response to either R848 or LPS. Mean TNF levels from CD14+ cells of these patients were higher compared to patients with sensitive colony responses [1756 pg/ml (R848) and 1699 pg/ml (LPS), versus 783 pg/ml (R848) and 959 pg/ml (LPS), for resistant/paradoxical versus sensitive patients respectively]. Among all patients with PB samples (including those with/without marrow results), CD14+ cells from 68% of these patients overproduced TNF. Mean TNF levels in response to R848 or LPS stimulation of patient cells were elevated compared to normal donor controls [1730 pg/ml (R848) and 1795 pg/ml (LPS), versus 719 pg/ml (R848) and 555 pg/ml (LPS), from patient and normal donor CD14+ cells, respectively].Conclusions: In patients with sMDS/sAML there is a high prevalence of TNF resistance in committed progenitors and TLR-hyper-reactivity in CD14+ cells. In FA the MDS/AML clonal resistance phenotype is selected because it possesses a selective advantage over the unfit pool of non-clonal FA HSC. We suggest that prior chemotherapy can create a fixed phenotype of HSC unfitness (e.g. hypersensitivity to inflammatory cytokines) and that the desultory fitness landscape in that pool permits the selection of cytokine resistant clones. Mechanistic studies should provide opportunities to identify patients at risk of sMDS/sAML and to develop pharmacological approaches to leukemia prevention. DisclosuresNo relevant conflicts of interest to declare.

O6.04 * GRANULOCYTIC MYELOID-DERIVED CELLS OBTAINED FROM BLOOD MAINLY SUPPRESS T-CELL PROLIFERATION IN PATIENTS WITH GLIOBLASTOMA

OBJECTIVE: To study the importance of myeloid-derived suppressor cells (MDSC) in primary glioblastomas, we investigated the frequency, phenotype, and immune-suppressive function of different myeloid-derived cells (MDC) in the blood and freshly resected tumors of 40 patients. RESULTS: Multicolour flow cytometric analysis showed that the frequency of CD14lowCD15+ granulocytic CD11bhighCD33b+ MDC was higher in peripheral blood and tumor samples of glioblastoma patients compared to healthy controls and positively correlated with the tumor volume before resection. Granulocytic MDC expressed CD16, CD66b, CD114, CD195 and could be further divided into CD14lowCD15high neutrophilic and CD14lowCD15int eosinophilic MDC. Moreover, we could detect an intratumoral accumulation of CD14highCD15+ monocytic CD11bhighCD33b+ MDC expressing CD115, CD124, CD184, CD206 and CX3CR1. All MDC subsets stained positively for reactive oxygen species, but Arginase 1 was specifically expressed in tumor-infiltrating MDC. T cell suppression assays with FACS-sorted MDC revealed that granulocytic and most prominently eosinophilic MDC obtained from the blood, but not the tumor could efficiently suppress non-specific T cell proliferative responses. Contrary to blood-derived MDC, HLA-DR expression was significantly increased on tumor-infiltrating MDC. Upon LPS or R848 stimulation, blood-derived MDC showed an increased production of IL-10, in contrast to tumor-infiltrating MDC that were resistant to Toll-like-receptor (TLR) stimulation. Although the frequency of granulocytic MDC inversely correlated with CD4+ and CD8+ T memory cells in the blood, no association with time to tumor progression in glioblastoma patients could be found. CONCLUSION: Our findings provide a detailed characterization of the nature and suppressive function of MDC in glioblastoma patients and indicate that granulocytic MDC obtained from the blood but not from the tumor mainly suppress T-cell proliferation.

Immunomodulatory effect of R848 on cytokine production associated with Schistosoma mansoni infection

This work aimed to determine the effect of resiquimod (R848), a low molecular weight imidazoquinolinamine compound with properties as immune response modifiers, on cytokine production in patients infected with Schistosoma mansoni with various degrees of hepatic fibrosis. Schistosoma-infected patients were classified into four groups according to the degree of hepatic fibrosis as examined by ultrasonography group 0 (no fibrosis), group I (incipient fibrosis), group II (moderate fibrosis), and group III (severe fibrosis). Levels of interferon-γ (IFN-γ), interleukin 4 (IL-4), IL-10, and IL-13 were measured by ELISA in the supernatants of cultured peripheral blood mononuclear cells (PBMC) in response to schistosoma egg soluble antigen (SEA) in the presence and absence of R848. R848 induced a significantly higher level of IL-10 in supernatants of SEA-stimulated PBMC of patients in all groups of hepatic fibrosis except group III. Moreover, we significantly found higher levels of IFN-γ in group 0 and group I and significantly lower level of IL-4 in group 0 after R848 stimulation. However, R848 had no effect on IL-13. Taken together, these results suggest that R848 might be used as an immunopotentiating agent in view of novel therapy or vaccine for S. mansoni infection.

Effects of age and R848 stimulation on expression of Toll-like receptor 8 mRNA by foal neutrophils

The innate immune system plays a critical role in protecting neonates against infections early in life and Toll-like receptors (TLRs) are key components of innate immune recognition of pathogens. This study examined the effects of age and stimulation with a TLR 7/8 agonist (R848) on TLR8 mRNA expression by foal neutrophils during the first month of life. We also examined the effects of R848 stimulation on mRNA expression of interleukin (IL)-6 and IL-8 at 1 and 14 days of life. We observed that TLR8 mRNA was constitutively expressed (P<0.05) at all ages examined, and its expression did not change upon stimulation with R848. Stimulation with R848 resulted in significantly increased (P<0.05) expression of IL-6 and IL-8 mRNA at both 1 and 14 days of life. Our results demonstrate that foal neutrophils express constitutive levels of TLR8 mRNA. The increased expression of IL-6 and IL-8, both downstream products of the TLR 7/8 signaling pathway, following stimulation with R848 suggests that additional experiments to confirm the signaling pathway by which R848 stimulates foal neutrophils, and to investigate its ability to stimulate functional responses of foal neutrophils, are merited.

Diclofenac inhibits lactate formation and efficiently counteracts local immune suppression in a murine glioma model

Lactate formation in highly proliferative tumors such as malignant gliomas is associated with poor survival and contributes to the suppression of local immunity. Here, we report that diclofenac used at nontoxic concentrations significantly decreased lactate production in murine glioma cells and inhibited the expression of lactate dehydrogenase-A in vitro. Lactate reduction was accompanied by a dose-dependent inhibition of cell growth and a cell cycle arrest at the G2/M checkpoint. In the presence of diclofenac, murine bone marrow-derived dendritic cells (DCs) showed enhanced IL-12, but decreased IL-10 secretion on Toll-like receptor stimulation with R848 that correlated with reduced lactate levels in the glioma cell coculture and a blockade of signal transducers and activators of transcription 3 phosphorylation. In vivo, diclofenac treatment diminished intratumoral lactate levels and resulted in a significant delay of glioma growth. Ex vivo analyses revealed that tumor-infiltrating DCs regained their capacity to produce IL-12 on R848 stimulation. Moreover, diclofenac reduced the number of tumor-infiltrating regulatory T cells and impaired the upregulation of the Treg activation marker CD25. Nevertheless, a single intratumoral injection of R848 combined with diclofenac failed to induce an additional survival advantage in glioma-bearing mice. Further analyses illustrated that the presence of diclofenac during T-cell activation compromised INF-γ production and T-cell proliferation, indicating that immunotherapeutic approaches have to be carefully timed when combined with diclofenac. In summary, diclofenac appears as an attractive agent for targeting lactate production and counteracting local immune suppression in malignant gliomas.

Plasmacytoid dendritic cell interferon-α production to R-848 stimulation is decreased in male infants

BackgroundSex differences in response to microbial infections, especially viral ones, may be associated with Toll-like receptor (TLR)-mediated responses by plasmacytoid dendritic cells (pDCs).ResultsIn this study, we identified sex differences in human infant pDC interferon-α production following challenge with the TLR7/8 agonist R-848. Male pDC responses were significantly lower than those of females during early infancy. This difference may be attributed to the androgen surge experienced by males during the early infancy period. Pretreatment of human pDCs with dihydrotestosterone produced a significant reduction in interferon-α production following R-848 challenge.ConclusionsAndrogen-mediated regulation of pDC TLR7-driven innate immune responses may contribute to the observed sex differences in response to infections during early infancy.

R-848 triggers the expression of TLR7/8 and suppresses HIV replication in monocytes

BackgroundToll-like receptors (TLR) 7 and 8 are important in single-stranded viral RNA recognition and may play a role in HIV infection and disease progression. We analyzed TLR7/8 expression and signaling in monocytes from HIV-infected and uninfected subjects to investigate a pathway with new potential for the suppression of HIV replication.MethodsEighty-one HIV-infected and uninfected subjects from Liaoning and Henan provinces in China participated in this study. Monocytes were isolated from subjects' peripheral blood mononuclear cells by magnetic bead selection. TLR7 and TLR8 mRNA was measured using quantitative real-time reverse transcriptase PCR. R-848 (resiquimod) was used as a ligand for TLR7 and TLR8 in order to 1) assess TLR7/8-mediated monocyte responsiveness as indicated by IL-12 p40 and TNF-α secretion and 2) to examine HIV replication in cultured monocytes in the presence of R-848.ResultsWe found that expression of TLR7/8 mRNA in peripheral blood monocytes decreased with disease progression. TLR7 expression was decreased with stimulation with the TLR7/8 agonist, R-848, in vitro, whereas TLR8 expression was unaffected. Following R-848 stimulation, monocytes from HIV-infected subjects produced significantly less TNF-α than those from uninfected subjects, but trended towards greater production of IL-12 than stimulated monocytes from uninfected subjects. R-848 stimulation also suppressed HIV replication in cultured monocytes.ConclusionsOur study provides evidence that the TLR7 and TLR8 triggering can suppress HIV replication in monocytes and lead to postpone HIV disease progression, thereby offering novel targets for immunomodulatory therapy.

Kinase Inhibitors Reduce TNF-Alpha Over-Production in Monocytes From Fanconi Anemia Group A Patients

Abstract 2409Fanconi Anemia (FA) is a chromosomal instability syndrome with hypersensitivity to alkylating agents as principal diagnostic feature. Many laboratories have demonstrated the involvement of FA proteins in DNA repair mechanisms. Recent work has also demonstrated other functions of some FA proteins suggesting that they play alternative roles in other regulatory pathways, particularly those that influence hematopoiesis.Eighty percent of FA patients develop bone marrow failure with a high incidence of evolution in myelodysplasia and/or acute leukemia. Each of these abnormalities has been related to TNF-alpha hypersensitivity in the stem and progenitor cell pools and the toll-like receptor dependent overproduction of TNF-alpha by FA macrophages. The TNF-hypersensitive phenotype involves at least two kinases, PKR and ASK1, each of which is hyperactivated in FA cells and induce apoptotic responses both in ground state and after TNF-alpha and IFN-gamma stimulation. Recent evidence showed that R848 (a TLR8 ligand) and endotoxin (LPS, a TLR4 ligand)-induced TNF-alpha gene expression in Fancc-deficient mononuclear phagocytes cells is inhibited by kinase inhibitors dasatinib and BIRB796 We sought to evaluate the activity of these agents in primary mononuclear phagocytes obtained from children with FA-A. Objectives:To determine whether primary monocytes from the peripheral blood of FANCA-deficient patients: (a) exhibit the TNF-overproduction phenotype in response to LPS and the TLR8 ligand R848, and (b) respond to dasatinib and BIRB796 by suppressing TNF-production. Methods:Six FA patients with mild to severe marrow failure on no treatment were included in this study. Healthy subjects were recruited as normal controls that were run in parallel in each case. CD14+ monocytes freshly isolated from peripheral blood were cultured for 24 hours with LPS and R848 with or without dasatinib or BIRB796. Supernatant media were collected and frozen at −80 degrees. After thawing the samples, TNF-alpha content was quantified by ELISA. Results:Baseline TNF-alpha concentration (without any TLR stimulation) was higher in FA patients than control. After LPS or R848 stimulation FA-A monocytes produced substantially more TNF-alpha than did the control samples. Both dasatinib and BIRB796 suppressed TLR-induced (both LPS and R848) TNF-alpha production. Specifically, with R848 as the agonist, BIRB 769 suppressed TNF-alpha production by 60% and dasatinib by 42%. Both inhibitors were even more potent in suppressing LPS induced TNF-alpha expression as both reduced TNF-alpha by 75%. In the absence of TLR stimulation, the presence of BIRB or dasatinib in culture reduced TNF-alpha by >50% compared to baseline in patient samples. The inhibitory effect of kinase inhibitors was observed also in the normal control. Conclusions:These findings: (a) demonstrate for the first time that TLR-induced TNF-alpha gene expression in primary FANCA deficient mononuclear phagocytes is aberrantly regulated and (b) that in FANCA-deficient macrophages the TNF-alpha overproduction phenotype can be controlled by therapeutically achievable doses of BIRB796 and dasatinib. In addition (c), since both agents function in large part to suppress p38 MAPK activation future, our data point to the biochemical roles played by FANCA in modulating upstream pathways that govern p38 activation. Moreover (d), given that in FA patients, TNF hypersensitive stem cells are over-exposed to TNF-alpha, particularly during inflammatory events and that exposure to TNF was shown not only to suppress hematopoiesis in FA but also to favor the emergence of neoplastic clones, these results point to these two agents as potential candidates for preclinical trials seeking to enhance hematopoiesis and suppress clonal evolution. Disclosures:No relevant conflicts of interest to declare.

Role for IL-10 in inducing functional impairment of monocytes upon TLR4 ligation in patients with chronic HCV infections

The consequences of chronic infection with the HCV on immunity to distinct pathogens are not fully appreciated, despite the potent modulatory effects of HCV on the immune system. We observed that upon TLR4 ligation, monocytes from chronic HCV patients demonstrated three to five times lower TNF and IL-12p40 production as compared with healthy individuals. However, augmented production of TNF, IL-12p40, and IL-12p70 by monocytes was observed upon stimulation with R848. Importantly, we observed that the levels of IL-10 in chronic HCV patients are higher in serum and that more IL-10 is produced by monocytes as compared with healthy individuals. The inhibitory effect of IL-10 on the production of proinflammatory cytokines by monocytes was only observed upon LPS stimulation but not upon R848 stimulation, showing that only the TLR4 pathway in monocytes is sensitive to the suppressive effects of IL-10. Interestingly, monocytes stimulated with the TLR4 agonist, but not TLR8 agonist, produced higher levels of IL-10 when exposed to patient serum as compared with serum from healthy individuals. Our results indicate that by differentially affecting TLR4 and TLR8 pathways, IL-10 may mediate highly selective modulation of the function of monocytes observed in chronic HCV patients. This suggests that there is no overall increased susceptibility to pathogens but a specific suppression of the functionality of TLR4 signaling pathway in monocytes, which is, at least partly, mediated via IL-10.

IL-29 and IFNα differ in their ability to modulate IL-12 production by TLR-activated human macrophages and exhibit differential regulation of the IFNγ receptor expression

AbstractThe interferon-λ (IFNλ) family of cytokines, consisting of interleukin-28A (IFNλ2), IL-28B (IFNλ3), and IL-29 (IFNλ1), have been extensively studied for their antiviral activities. However, little is known about the effect of IFNλ on antigen-presenting cells. In the present study, we show for the first time that IL-29 can increase Toll-like receptor (TLR)–induced IL-12p40 production by human monocyte-derived macrophages. In contrast, IL-29 did not affect monocytes or monocyte-derived dendritic cells (DCs) because of restricted IL-28 receptor α chain expression by macrophages. Furthermore, IL-29–treated macrophages were more responsive to IFNγ, because IL-29 enhanced IFNγ-induced IL-12p40 and tumor necrosis factor (TNF) production by macrophages on R848 stimulation. However, IFNα suppressed IFNγ-induced IL-12p40 and tumor necrosis factor TNF production by human macrophages. The differential effects of IL-29 and IFNα on the responsiveness of macrophages to IFNγ could not be explained by an effect on TLR7 or TLR8 mRNA expression or by altered IL-10 signaling. However, we demonstrated that IL-29 up-regulated, whereas IFNα down-regulated, the surface expression of the IFNγ receptor 1 chain on macrophages, thereby resulting in differential responsiveness of TLR-challenged macrophages to IFNγ. Our findings on the differences between IFNα and IL-29 in modulating TLR-induced cytokine production by macrophages may contribute to understanding the role of IFNs in regulating immunity to pathogens.

C/EBPδ and STAT-1 Are Required for TLR8 Transcriptional Activity

Toll-like receptor 8 (TLR8), which is expressed primarily in myeloid cells, plays a central role in initiating immune responses to viral single-stranded RNA. Despite the great interest in the field of TLR8 research, very little is known in terms of TLR8 biology and its transcriptional regulation. Here, we describe the isolation of the hTLR8 promoter and the characterization of the molecular mechanisms involved in its regulation. Reporter gene analysis and ChIP assays demonstrated that the hTLR8 regulation of the basal transcription is regulated via three C/EBP cis-acting elements that required C/EBPδ and C/EBPβ activity. In addition, we observed that R848 stimulation increases TLR8 transcriptional activity via an enhanced binding of C/EBPδ, and not C/EBPβ, to its responsive sites within the TLR8 promoter. Moreover, we showed that IFN-γ also increased TLR8 transcription activity via the binding of STAT1 transcription factor to IFN-γ activated sequence elements on the TLR8 promoter and enhanced TLR8 functionality. These results shed new light on the mechanisms involved during TLR8-mediated innate immune response.

Cellular and Molecular Mechanisms Underlying the Strong Neonatal IL-12 Response of Lamb Mesenteric Lymph Node Cells to R-848

BackgroundComparative studies on the response of neonates and adults to TLR stimulation have been almost exclusively limited to comparisons of human neonatal cord blood cells with peripheral blood from adults, and analyses of spleen cell responses in mice. We need to extend these studies and gain further information regarding such responses at mucosal sites.Methodology/Principal FindingsWe used sheep as a large animal model to study TLR agonist responses in the lymph nodes draining the intestine, an organ that must adapt to profound changes after birth. In response to the imidazoquinoline compound R-848, neonatal mesenteric lymph node (MLN) and spleen cells produced more IL-12 and, consequently, more IFNγ than their adult counterparts. This difference was age-related for both organs, but the preferential IL-12 response decreased more rapidly in the MLN, with young animals producing similar amounts of this cytokine to adults, from the age of 20 days onwards. Intracellular assays and depletion experiments identified CD14+CD11b+CD40+ cells as the main producer of IL-12. These cells accounted for a greater proportion of neonatal than of adult MLN cells, and also produced, in direct response to R-848, more IL-12 after isolation. This strong IL-12 response in neonates occurred despite the production of larger amounts of the regulatory cytokine IL-10 and the stronger upregulation of SOCS-1 and SOCS-3 mRNA levels than in adult cells, and was correlated with an increase in p38/MAPK phosphorylation.Conclusions/SignificanceThis is the first attempt to decipher the mechanism by which neonatal MLN cells produce more IL-12 than adult cells in response to the TLR8 agonist R-848. CD14+CD11b+CD40+ IL-12-producing cells were more numerous in neonate than in adult MLN cells and displayed higher intracellular responsiveness upon R-848 stimulation. This work provides relevant information for future vaccination or immunostimulation strategies targeting neonates.

Identification and Characterization of Novel Bone Marrow Myeloid DEC205+Gr-1+ Cell Subsets That Differentially Express Chemokine and TLRs

Bone marrow-derived immunomodulatory cytokines impart a critical function in the regulation of innate immune responses and hemopoiesis. However, the source of immunomodulatory cytokines in murine bone marrow and the cellular immune mechanisms that control local cytokine secretion remain poorly defined. Herein, we identified a population of resident murine bone marrow myeloid DEC205(+)CD11c(-)B220(-)Gr1(+)CD8alpha(-)CD11b(+) cells that respond to TLR2, TLR4, TLR7, TLR8, and TLR9 agonists as measured by the secretion of proinflammatory and anti-inflammatory cytokines in vitro. Phenotypic and functional analyses revealed that DEC205(+)CD11b(+)Gr-1(+) bone marrow cells consist of heterogeneous populations of myeloid cells that can be divided into two main cell subsets based on chemokine and TLR gene expression profile. The DEC205(+)CD11b(+)Gr-1(low) cell subset expresses high levels of TLR7 and TLR9 and was the predominant source of IL-6, TNF-alpha, and IL-12 p70 production following stimulation with the TLR7 and TLR9 agonists CpG and R848, respectively. In contrast, the DEC205(+)CD11b(+)Gr-1(high) cell subset did not respond to CpG and R848 stimulation, which correlated with their lack of TLR7 and TLR9 expression. Similarly, a differential chemokine receptor expression profile was observed with higher expression of CCR1 and CXCR2 found in the DEC205(+)CD11(+)Gr-1(high) cell subset. Thus, we identified a previously uncharacterized population of resident bone marrow cells that may be implicated in the regulation of local immune responses in the bone marrow.

TLR Ligand-Dependent Activation of Naive CD4 T Cells by Plasmacytoid Dendritic Cells Is Impaired in Hepatitis C Virus Infection

Chronic hepatitis C virus (HCV) infection is characterized by diminished numbers and function of HCV-reactive T cells and impaired responses to immunization. Because host response to viral infection likely involves TLR signaling, we examined whether chronic HCV infection impairs APC response to TLR ligand and contributes to the origin of dysfunctional T cells. Freshly purified myeloid dendritic cells (MDC) and plasmacytoid DC (PDC) obtained from subjects with chronic HCV infection and healthy controls were exposed to TLR ligands (poly(I:C), R-848, or CpG), in the presence or absence of cytokine (TNF-alpha or IL-3), and examined for indices of maturation and for their ability to activate allogeneic naive CD4 T cells to proliferate and secrete IFN-gamma. TLR ligand was observed to enhance both MDC and PDC activation of naive CD4 T cells. Although there was increased CD83 and CD86 expression on MDC from HCV-infected persons, the ability of MDC to activate naive CD4 T cells in the presence or absence of poly(I:C) or TNF-alpha did not differ between HCV-infected and healthy control subjects. In contrast, PDC from HCV-infected persons had reduced activation marker (HLA-DR) and cytokine (IFN-alpha) expression upon R-848 stimulation, and these were associated with impaired activation of naive CD4 T cells. These data indicate that an impaired PDC responsiveness to TLR ligation may play an important role in the fundamental and unexplained failure to induce new T cell responses to HCV Ags and to other new Ags as a consequence of HCV infection.

Toll-Like Receptor-Stimulated CD14+ Monocytes Induce Soluble Factors That Activate Human Mesenchymal Stem Cells To Inhibit T-Cell Alloreactivity.

Human mesenchymal stem cells (MSCs) produce soluble factors that inhibit T-cell proliferation and alloreactivity. We have previously shown that MSCs require activation by CD14+ monocytes in cell culture. Toll-like receptors (TLRs) critically modulate antigen-presenting cell (APC) activation, maturation and function. Therefore, we aimed to determine whether TLR agonists could enhance CD14-mediated MSC activation and subsequent MSC-mediated T-cell inhibition. TLR agonists and human IL-1β were used to stimulate CD14+ cells isolated from peripheral blood of normal donors. TLR agonists included formalin-fixed Staphylococcus aureus Cowan A strain (SAC, TLR-2), Pam3CysSerLys4 (Pam3Cys, TLR-2), Salmonella enteriditis lipopolysaccharide (LPS, TLR-4), and R848 (Resiquimod, TLR7/8) in complete RPMI media (heat-inactivated FBS, glutamine, and antibiotics). TLR-stimulated CD14+ cells and supernatants from TLR-stimulated CD14+ cells were then used to stimulate third- and fourth-passage human MSCs (CD45−CD105+CD90+CD80−CD73+HLA-I+) expanded from normal volunteer bone marrow aspirate specimens. After a 24-hour culture with stimulant cells or supernatants, stimuli were removed, MSCs were washed twice with sterile PBS, and then were cultured for an additional 24 h in FBS-free RPMI media. Supernatants from TLR-stimulated CD14+ cell cultures and from washed MSC cultures were used to measure cytokine and chemokine production and to inhibit T-cell alloreactivity using an established mixed lymphocyte reaction (MLR) IFN-γ ELISPOT. MLR was also performed in the presence of TLR agonists and media alone. TLR stimulation resulted in high-level soluble factor induction (IL-1β, IL-6, TNF-α, and RANTES) from CD14+ cells. For example, levels of inducible IL-6 measured in CD14+ cell culture supernatants following LPS, SAC and R848 stimulation were 5.2 (70.3 ± 26.2 ng/ml), 4.7 (64.1 ± 16.3 ng/ml), and 4.6 (63.4 ± 13.1 ng/ml)-fold higher than after IL-1β stimulation (13.7 ± 4.0 ng/ml) (Mean ± SEM, 4 independent experiments). Supernatants of TLR-stimulated CD14+ cells induced higher levels of soluble factors from MSCs than stimulation with CD14+ cells themselves, suggesting that soluble factors from CD14+ cells activate MSCs. Not all supernatants of TLR-stimulated CD14+ cells were similar in their capacity to activate MSC-mediated inhibition of T-cell alloreactivity. For example, supernatants of Pam3Cys-, R848- and SAC-stimulated CD14+ cells all induced high levels of TNF-α in washed MSC cultures; but only supernatant from Pam3Cys-stimulated CD14+ cells resulted in MSC-mediated inhibition of T-cell alloreactivity (63.7 ± 12.9 % inhibition, Mean ± SEM, 4 independent ELISPOTs). Ongoing studies are being performed to define these immunomodulatory factors. Together, these results suggest that ex vivo TLR stimulation enhances soluble factor production from CD14+ cells, which, in turn, increases MSC production of immunomodulatory factors that mediate inhibition of T-cell alloreactivity.

Human CD4+ CD25high Regulatory T Cells Inhibit Myeloid but Not Plasmacytoid Dendritic Cells Activation.

CD4+CD25+ regulatory T cells (Treg) are essential negative regulators of immune responses. However, the mechanisms of immune suppression and the spectrum of cells they target remain incompletely defined. In particular, although Treg directly suppress conventional T cells in vitro, they might also affect antigen presenting cells (APC).Here, we studied the maturation of human myeloid (mDC) and plasmacytoid (pDC) dendritic cells activated with Toll-like receptor (TLR) ligands in the presence of CD4+ CD25high regulatory T cells in vitro. T cells and DC subsets were purified from normal human peripheral blood. LPS, CpG ODN 2216 and R-848 were used to trigger the maturation of mDC, pDC or both through TLR4, TLR9 and TLR7/8 respectively. Preactivated CD4+ CD25high Treg had no effect on the maturation of pDC. Conversely, they strongly suppressed TLR-triggered mDC costimulatory molecules up-regulation, pro-inflammatory cytokines secretion and their antigen presentation capacity, as opposed to conventionnal T cells (Tconv). At a ratio of 3 Treg for 1 DC, the percentage of mDC acquiring CD80 was reduced 5 fold (from 75% to 16%) while the Mean Fluorescence Intensity was decreased by approximately 65% for CD80 and 35% for CD86 after LPS stimulation and by 50% and 20% after R-848 stimulation. Furthermore, Treg dramatically decreased the secretion of IL-12p40, TNF-α, and IL-6 by mDC (95%, 93% and 50% average inhibition respectively) after LPS activation and to a lesser but still significant extent (38%, 35%, and 38% average inhibition respectively) after R848 stimulation. Finally, we found that Treg-conditionned mDC had a reduced ability to trigger naïve T cell proliferation in a mixed leukocyte reaction.Suppression of mDC activation by Treg appeared to require cell-cell contact. Moreover, the inhibition of pro-inflammatory cytokines secretion, but not of phenotypic maturation, was almost completely restored using an anti-IL10 receptor monoclonal antibody, but not anti-TGFβ nor anti-CTLA-4 blocking antibodies. Those data suggest that Treg prevent the co-stimulatory molecules up-regulation on mDC through contact dependent mechanisms, while the modulation of cytokines secretion appears to be largely mediated by IL-10.Overall, our results provide the first evidence of a direct inhibition of human mDC but not pDC maturation by CD4+ CD25high Treg. Therefore, by restraining the maturation of mDC, human Treg may enlist those cells for the initiation and the amplification of tolerance in vivo.

The Immune Response Modifiers Imiquimod and R-848 Are Potent Activators of B Lymphocytes

Imiquimod and R-848 are members of a family of immune response modifiers that stimulate cytokine production in monocyte/macrophages and dendritic cell cultures. This study evaluated the effects of the imidazoquinolines, imiquimod and R-848, on B lymphocyte activation. Both agents induced proliferation of murine T-cell-depleted and highly purified splenic B cell preparations as well as purified human B cells. Resting and activated B cells responded to these agents, with activated cells responding more efficiently. B cells from the LPS-hyporesponsive C3H/HeJ mice and guanosine-hyporesponsive SJL mice proliferated in response to imiquimod and R-848, indicating a different mechanism of action than lipopolysaccharide and guanine nucleosides. B cells were also stimulated by imiquimod and R-848 to produce increased immunoglobulin levels. Increased expression of a number of B cell activation markers were seen following imiquimod or R-848 stimulation. Finally, R-848 was shown to act as a vaccine adjuvant enhancing OVA-specific IgG2a levels while suppressing total IgE. These results indicate that R-848 and imiquimod are potent activators of B lymphocytes and are capable of augmenting antigen-specific immunoglobulin production.