TLR3 Agonist Poly Research Articles

IRAK4 kinase-deficient mice exhibit increased susceptibility to S. pneumoniae that is overcome by poly IC:LC treatment (55.23)

Abstract Interleukin-1 receptor-associated kinase 4 (IRAK4) is a critical component of Toll-like receptor signaling, and patients with IRAK4 mutations are extremely susceptible to recurrent bacterial infections. In these studies, we used mice homozygous for a mutant IRAK4 that lacks kinase activity (IRAK4KDKI) to address the role of IRAK4 in response to TLR agonists and infection with whole bacteria. IRAK4KDKI macrophages have a diminished response to the TLR4 agonist LPS, little to no response to the TLR2 agonist Pam3Cys, but normal response to the TLR3 agonist poly I:C when compared to wild-type (WT) macrophages as measured by cytokine mRNA expression and MAPK activation. Thus, diminished responses of IRAK4KDKI cells are specific to TLR pathways that are MyD88-dependent. Importantly, we have identified two proteins downstream of MAPK signaling, MNK1 and MSK1, whose phosphorylation is deficient in IRAK4KDKI macrophages when compared to WT. These data suggest a role for IRAK4 in propagating a TLR signal beyond initial phosphorylation of p38 and ERK. Additionally, IRAK4KDKI mice were more susceptible to infection with S. pneumoniae, demonstrating the usefulness of this murine model for the exploration of strategies to overcome IRAK4 deficiencies. Co-treatment of infected mice with Poly IC:LC, a TLR3 agonist, significantly improved the survival rate of both WT and IRAK4KDKI mice providing a potential treatment strategy in both normal and immunocompromised patients.

Uterine Epithelial Cells Specifically Induce Interferon-Stimulated Genes in Response to Polyinosinic-Polycytidylic Acid Independently of Estradiol

Interferon β (IFNβ) is an antiviral cytokine secreted in response to pathogenic exposure that creates a restrictive intracellular environment through the action of downstream interferon-stimulated genes (ISG). The objective of this study was to examine the expression of IFNβ and ISG in both human uterine epithelial cells (UEC) and the ECC-1 uterine epithelial cell line and determine if expression changes with TLR stimulation and hormone exposure. Stimulation of primary uterine epithelial cells and ECC-1 cells with the TLR3 agonist poly (I∶C) induced the mRNA expression of IFNβ, MxA, OAS2 and PKR. Other TLR agonists including imiquimod and CpG had no effect on either IFNβ or ISG expression. In contrast to ECC-1 cell responses which were slower, maximal IFNβ upregulation in UEC occurred 3 hours post-stimulation and preceded the ISG response which peaked approximately 12 hours after poly (I∶C) exposure. Unexpectedly, estradiol, either alone or prior to treatment with poly (I∶C), had no effect on IFNβ or ISG expression. Blockade of the IFN receptor abrogated the upregulation of MxA, OAS2 and PKR. Furthermore, neutralizing antibodies against IFNβ partially inhibited the upregulation of all three ISG. Estradiol, directly and in the presence of poly (I∶C) had no effect on IFNβ and ISG expression. These results indicate that uterine epithelial cells are important sentinels of the innate immune system and demonstrate that uterine epithelial cells are capable of mounting a rapid IFN-mediated antiviral response that is independent of estradiol and is therefore potentially sustained throughout the menstrual cycle to aid in the defense of the uterus against potential pathogens.

Intradermally administered TLR4 agonist GLA-SE enhances the capacity of human skin DCs to activate T cells and promotes emigration of Langerhans cells

The natural TLR4 agonist lipopolysaccharide (LPS) has notable adjuvant activity. However, it is not useful as a vaccine adjuvant due to its toxicity. Glucopyranosyl lipid A (GLA) is a synthetic derivative of the lipid A tail of LPS with limited cytotoxicity, but strong potential to induce immune responses in mice, guinea pigs, non-human primates, and humans. In this study we determined how this synthetic TLR4 agonist affects the function of different subsets of human skin dendritic cells (DCs). The effect of GLA in an aqueous formulation (GLA-AF) or in an oil-in-water emulsion (GLA-SE) was compared to that of LPS and TLR3 agonist poly(I:C) using a human skin explant model with intradermal injections for the administration of the agonists. Intradermal injection of GLA-SE or LPS, but not GLA-AF, enhanced the emigration of CD1ahigh/langerin+ Langerhans cells (LCs), but not dermal DCs (DDCs). LCs and CD14− DDCs exhibited an enhanced mature phenotype following intradermal administration of either of the two GLA formulations tested, similar to DCs that emigrated from LPS-injected skin. However, only injection of GLA-SE resulted in a significant increase in the production of the wide range of cytokines that is observed with LPS. Moreover, DCs that emigrated from GLA-SE-injected skin induced stronger CD4+ T-cell activation, as indicated by a more pronounced T-cell proliferation, than DCs from skin injected with GLA-AF or LPS. Altogether, our data show that GLA-SE has a notable potency to stimulate the function of skin DCs, indicating that GLA-SE may be a good candidate as adjuvant for vaccines administered via the intradermal route.

Association of TLR3-hyporesponsiveness and functional TLR3 L412F polymorphism with recurrent herpes labialis

HSV-1 persistently infects almost 90% of our population; however, only 30% of the infected subjects suffer from recurrent herpes lesions, most frequently herpes labialis (HL). We hypothesized that variations in toll-like receptor (TLR) functions might contribute to HL susceptibility. In our study, the TLR-2/1,-3, and -7/8 responses of immune cell subsets derived from asymptomatic HSV-1 carriers were compared with responses of subjects with HL history. Remarkably, natural killer (NK) cells isolated from HL subjects showed significantly lower IFN-γ responses selectively to the TLR3 agonist poly(I:C). Furthermore, the TLR3 L412F genetic polymorphism was found to reduce NK cell TLR3-responsiveness and is associated with susceptibility to recurrent HL. The TLR3 response detected in HL total peripheral blood mononuclear cells (PBMCs), however, was not impaired, indicating restoration of NK cell TLR3-deficiency through co-stimulatory functions. In conclusion, our results suggest that decreased TLR3 response of NK cells is associated with HL susceptibility; and potentially explain why symptomatic outbreak of HL usually occurs after stress or prolonged UV light exposure, when host co-stimulatory functions are disturbed.

Abstract 522: Cooperative effect of the TLR3-agonist Poly(A:U) combined to a Smac-mimetic against malignant nasopharyngeal carcinomas cells

Abstract Nasopharyngeal carcinomas (NPC) are malignant epithelial tumors of the nasopharyngeal cavity. They are consistently associated with the Epstein-Barr virus (EBV). Though NPCs are on average more radiosensitive and chemosensitive than other tumors of the upper aero-digestive tract, many therapeutic challenges remain. In this context the development of therapeutic approaches taking better account of biological characteristics of NPC and well defined biological targets remains a priority. We have previously reported a cooperative effect of the TLR3-agonist poly(I:C) combined to a Smac-mimetic against NPC cells. (Friboulet et al., Neoplasia 2008, 10: 1183-1194 and BMC cancer 2010, 10: 327). However Poly(I:C) is a ligand not only for the TLR3 but also other receptors of double-stranded RNAs, like RIG1 and PKR. In contrast Poly(A:U) is much more specific for TLR3. In addition, Poly(A:U) has less secondary effects in patients and has already been successfully used in a phase III clinical trial against breast cancer (Salaun et al., Cancer Res. 201, 71: 1607-14). Therefore one aim of this study was to explore the therapeutic potential of the treatment of NPC cells by Poly(A:U) combined to an inhibitor of IAPs (“inhibitor of apoptosis proteins”). Another aim was to assess the expression of TLR3 by NPC cells, an aspect of their phenotype which - to our knowledge - has never been previously described. The expression of TLR3 was assessed in a series of EBV-positive NPC tumor lines (C666-1, C15, C17, C18) and a series of NPC clinical specimens by Western blot and immunohistochemistry, respectively. In vitro TLR3 was consistently detected in malignant NPC cells at a high level by comparison with non-NPC control lines. In all NPC biopsies, TLR3 expression was detected in a substantial fraction of malignant NPC cells. The effects of poly(A:U) as single agents were tested on the EBV-positive NPC cells lines C666-1 without significant effects on cell growth and cell survival at least at concentrations compatible with clinical use. In contrast, growth inhibition and apoptosis induction were obtained by combination of Poly(A:U) with RMT 5265, an IAP inhibitor based on Smac-mimicry which targets c-IAP1, c-IAP2 and XIAP. It is noteworthy that these effects were obtained using concentrations of Poly(A:U) of 1 μg/ml or less and concentrations of the IAP inhibitor (RMT 5265) of 100 nM or less. These data confirm that TLR3 expression is a factor of vulnerability for NPC cells as it is the case for other head and neck carcinomas (Rydberg et al., Immunology 2009, 128: e601–e611; Umemura et al., Cancer Res. Online 2011). Until now, Smac mimetics and Poly(A:U) have been the subject of separate therapeutic trials. In light of our observations, combined use of both types of compounds should be considered for treatment of EBV-associated nasopharyngeal carcinomas. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 522. doi:1538-7445.AM2012-522

Contribution of immature muscle precursors to myositis pathogenesis: a source of type I interferon and a possible target of the immune attack

ObjectivesTo investigate the contribution of immature muscle precursors to the adaptive and innate immune responses in inflammatory myopathy.MethodMuscle biopsies from eight patients with polymyositis and dermatomyositis were investigated for the...

Immature muscle precursors are a source of interferon‐β in myositis: Role of Toll‐like receptor 3 activation and contribution to HLA class I up‐regulation

To investigate the production of type I interferon (IFN) by myoblasts and to identify its cell source and the link to Toll-like receptor (TLR) and C-type lectin receptor (CLR) expression and function in myositis biopsy sections. Production of IFNβ was assessed in cultured myoblasts after stimulation with the TLR-3 agonist poly(I-C) or with cytokines involved in Th1 and Th17 differentiation. Expression of HLA class I molecules by myoblasts was analyzed by fluorescence-activated cell sorting after activation of TLR-3 and IFNβ neutralization. In muscle biopsy samples from patients with polymyositis or dermatomyositis, expression of IFNβ, CD56 (a marker of immature muscle precursors), and HLA class I was analyzed using immunohistochemistry. Inflammatory infiltrates were characterized for the expression of myeloid dendritic cells (DCs), their associated CLRs, and the products of activated DCs, interleukin-12 (IL-12), and IL-23. In cultured myoblasts, stimulation of TLR-3 induced the production of IFNβ when combined with IFNγ and up-regulated the expression of HLA class I molecules, which was decreased after IFNβ blockade. In myositis biopsy tissues, immature muscle precursors overexpressing HLA class I were identified as a source of IFNβ. CLRs associated with myeloid DCs were broadly expressed in inflammatory infiltrates, in association with IL-12 and IL-23, and with immature muscle precursors. Immature muscle precursors may represent a local source of IFNβ and the target of an immune response involving activated DCs associated with the expression of CLRs and of IL-12 and IL-23, which are implicated in T cell polarization. In turn, such local production of IFNβ after TLR-3 activation in the presence of the Th1 cytokine IFNγ may explain HLA class I overexpression in myositis.

Effects of TLR agonists on maturation and function of 3-day dendritic cells from AML patients in complete remission

BackgroundActive dendritic cell (DC) immunization protocols are rapidly gaining interest as therapeutic options in patients with acute myeloid leukemia (AML). Here we present for the first time a GMP-compliant 3-day protocol for generation of monocyte-derived DCs using different synthetic Toll-like receptor (TLR) agonists in intensively pretreated patients with AML.MethodsFour different maturation cocktails were compared for their impact on cell recovery, phenotype, cytokine secretion, migration, and lymphocyte activation in 20 AML patients and 25 healthy controls.ResultsMaturation cocktails containing the TLR7/8 agonists R848 or CL075, with and without the addition of the TLR3 agonist poly(I:C), induced DCs that had a positive costimulatory profile, secreted high levels of IL-12(p70), showed chemotaxis to CCR7 ligands, had the ability to activate NK cells, and efficiently stimulated antigen-specific CD8+ T cells.ConclusionsOur results demonstrate that this approach translates into biologically improved DCs, not only in healthy controls but also in AML patients. This data supports the clinical application of TLR-matured DCs in patients with AML for activation of innate and adaptive immune responses.

Abstract LB-19: The Smac mimetic LBW242 induces apoptosis in melanoma in a TLR-3-dependent and TNFR-independent manner

Abstract Toll-like Receptor-3 (TLR-3), a pattern recognition receptor family member, has been shown to activate immune system pathways; however, it has also been shown to induce apoptosis in certain cancer cells. Combination of the synthetic TLR-3 ligand, poly I:C, with the Smac mimetic LBW242, an inhibitor of apoptosis protein (IAP) antagonist, has been shown to potently induce apoptosis in melanoma cells which are insensitive to either single agent. To better elucidate the mechanism responsible for this combination induced cell death, we performed a pooled shRNA screen. We identified receptor interacting protein kinase-1 (RIPK1) and as an important component of this process. Constitutive knockdown of RIPK1 prevents the LBW242 and poly I:C combination induced apoptosis. The IAP proteins cIAP1 and cIAP2 have been shown previously to regulate Caspase-8 activation downstream of Tumor Necrosis Factor Receptor-1 (TNFR1). Interestingly, we found that the combination induced apoptosis was independent of TNF signaling. Utilizing both gain of function and loss of function studies, we found that expression of TLR-3 is necessary for the combination induced apoptosis in melanoma cells. Taken together, our data indicate that the combination of the Smac mimetic LBW242 and TLR-3 agonist poly I:C induces a novel type of TLR-3 dependent apoptosis in melanoma cells that is independent of TNFR1. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-19. doi:10.1158/1538-7445.AM2011-LB-19

Macrophage Activation and Differentiation Signals Regulate Schlafen-4 Gene Expression: Evidence for Schlafen-4 as a Modulator of Myelopoiesis

BackgroundThe ten mouse and six human members of the Schlafen (Slfn) gene family all contain an AAA domain. Little is known of their function, but previous studies suggest roles in immune cell development. In this report, we assessed Slfn regulation and function in macrophages, which are key cellular regulators of innate immunity.Methodology/Principal FindingsMultiple members of the Slfn family were up-regulated in mouse bone marrow-derived macrophages (BMM) by the Toll-like Receptor (TLR)4 agonist lipopolysaccharide (LPS), the TLR3 agonist Poly(I∶C), and in disease-affected joints in the collagen-induced model of rheumatoid arthritis. Of these, the most inducible was Slfn4. TLR agonists that signal exclusively through the MyD88 adaptor protein had more modest effects on Slfn4 mRNA levels, thus implicating MyD88-independent signalling and autocrine interferon (IFN)-β in inducible expression. This was supported by the substantial reduction in basal and LPS-induced Slfn4 mRNA expression in IFNAR-1−/− BMM. LPS causes growth arrest in macrophages, and other Slfn family genes have been implicated in growth control. Slfn4 mRNA levels were repressed during macrophage colony-stimulating factor (CSF-1)-mediated differentiation of bone marrow progenitors into BMM. To determine the role of Slfn4 in vivo, we over-expressed the gene specifically in macrophages in mice using a csf1r promoter-driven binary expression system. Transgenic over-expression of Slfn4 in myeloid cells did not alter macrophage colony formation or proliferation in vitro. Monocyte numbers, as well as inflammatory macrophages recruited to the peritoneal cavity, were reduced in transgenic mice that specifically over-expressed Slfn4, while macrophage numbers and hematopoietic activity were increased in the livers and spleens.Conclusions Slfn4 mRNA levels were up-regulated during macrophage activation but down-regulated during differentiation. Constitutive Slfn4 expression in the myeloid lineage in vivo perturbs myelopoiesis. We hypothesise that the down-regulation of Slfn4 gene expression during macrophage differentiation is a necessary step in development of this lineage.

Profound CD8+ T cell immunity elicited by sequential daily immunization with exogenous antigen plus the TLR3 agonist poly(I:C)

The development of vaccines that elicit robust CD8+ T cell immunity has long been a subject of intense investigation. Although whole exogenous protein has not historically been considered as useful for eliciting CD8+ T cell immunity, we report herein that whole, protein antigen is capable of eliciting profound levels of CD8+ T cell immunity if it is administered via repeated, daily subcutaneous immunization in combination with the TLR3 agonist poly(I:C). Mice immunized for four consecutive days with 100μg of either whole exogenous OVA or whole HPV16 E7 protein combined with 10μg of poly(I:C) mounted remarkable antigen-specific CD8+ T cell responses as measured by tetramer staining and ELISPOT analysis of splenocytes and peripheral blood, with up to 30% of peripheral CD8+ T cells being antigen specific within 7–8 days of vaccination. CD8+ T cell immunity elicited using this vaccination approach was critically dependent upon cross presentation, as either whole protein or long synthetic peptides were highly effective immunogens whereas minimal peptide epitopes were not. Vaccine-induced CD8+ T cells were also able to regress large, established tumors in vivo. Together these data suggest that ‘cluster’ vaccination with exogenous antigen combined with TLR3 agonist may constitute a profoundly important advancement in therapeutic vaccine design.

The TLR7/8 Ligand R848 Is Superior In Generation of Monocyte Derived Dendritic Cells From AML Patients for the Induction of Potent T and NK Cell Immune Responses

AbstractAbstract 2195 Introduction:Therapeutic vaccination with dendritic cells (DC) is currently considered as an investigational therapy in acute myeloid leukemia (AML) for eradication of minimal residual disease (MRD). Dendritic cells derived from autologous peripheral blood monocytes have been tested as cellular adjuvants for therapeutic vaccination of malignancies and proven feasibility and safety, but overall clinical response rates remain very low. The vast majority of DCs used for clinical trials were differentiated with a standard maturation cocktail composed of the cytokines TNF-a, IL-1b, IL-6 and PGE2 leading to DCs unable to secrete biologically active IL-12. This cytokine is fervently desired because of its leading role in promoting T helper 1 cell polarization and therefore fostering the appropriate adaptive immune responses needed to combat minimal residual disease. Cocktails containing synthetic Toll-like receptors (TLR) agonists emerged as an attractive alternative for the induction of DC maturation with T helper type 1 polarizing capacity. Our present investigation was designed to study the feasability of a clinical grade DC 3-day mDC generation protocol from nonleukemic monocytes of intensively pretreated AML patients with novel maturation cocktails containing different TLR-agonists in vitro and assessment of their potency to induce adaptive and innate immune responses. Material & Methods:Monocytes isolated from peripheral blood of AML patients in CR and healthy donors were differentiated into immature DC with GM-CSF and IL-4. After 48 hours DC were additionally cultured with TNF-a, IL1-b, INF-g, PGE2 and corresponding to the defined cocktail with the TLR7/8 agonists R848 (R) or CL075 (C) with or without the TLR3 agonist poly(I:C) (P) for 24 hours. mDCs were analyzed for expression of maturation surface markers, costimulatory profile, IL-12(p70)/IL-10 ratio, migratory capacity, NK cell activation and polarization of T cells. Results:No significant difference in absolute monocyte counts and percentage of DC recovery between healthy controls and AML patients in CR was found using different maturation cocktails (C, CP, R, and RP). Phenotype analysis of surface marker expression revealed no substantial differences between the different DC generation protocols used in healthy donors and AML patients in CR. The costimulatory profile assesed by the expression of two members of the B7 family, CD80 (B7.1) and CD274 (B7-H1 or PD-L1), was in healthy donors superior to AML patients, but these differences were not statistically significant. Variations were noted in the capacity of DCs derived from different donors to produce IL-12(p70) and IL-10, but importantly no significant differences between AML patients in CR and healthy controls could be observed. Interestingly, both healthy donor and AML derived DCs secrete a significantly higher proportion of IL-12(p70) with R848 containing cocktails compared to CL075. Treatment with the CP cocktails even leads to a inverse Il12/IL-10 ratio in AML patients. The high CCR7 expression was paralleled by a strong migratory capacity as well as positive chemotactic reponses to CCL19 chemokine signals. DCs matured with these novel cocktails induced potent alloresponses and strongly activated NK cells measured by upregulation of CD69 expression and IFN-g secretion. No differences beetween R848 and CL075 could be observed. Conclusion:Here we report for the first time a clinically applicable, time- and resource saving 3-day TLR-agonist containing maturation protocol for the generation of IL-12(p70) secreting DCs from AML patients in remission validated with healthy controls which allowed efficient generation, easy harvesting, stable maturation and substantial recoveries of mature DCs. Comparison of different TLR7/8 agonists showed superiority of R848 in IL-12(p70) production to CL075. We believe that these studies point the way to improved DCs that will induce better and long lasting immune responses in the vaccination against acute myelo Disclosures:No relevant conflicts of interest to declare.

Biodegradable nanoparticles containing TLR3 or TLR9 agonists together with antigen enhance MHC-restricted presentation of the antigen

The effects of intraphagosomal toll-like receptor (TLR) activation on the MHC-restricted presentation of exogenous antigen were examined in dendritic cells (DCs). For phagosomal targeting, nanoparticles containing both a TLR agonist and a model antigen, ovalbumin (OVA), were prepared using biodegradable polymer poly(D,L-lactic acid-co-glycolic acid) and were then opsonized with mouse IgG. After incubating mouse DCs with the nanoparticles, the efficacy of OVA peptide presentation was evaluated using OVA-specific CD8 and CD4 T cells. Inclusion of either the TLR3 agonist poly(I:C) or the TLR9 agonist CpG oligodeoxynucleotides (ODN) significantly increased and prolonged both MHC class I- and class II-restricted OVA presentation. Accordingly, the DCs that phagocytosed the nanoparticles containing poly(I:C) or CpG ODN together with OVA efficiently induced the proliferation of OVA-specific CD8 and CD4 T cells. The potency levels of poly(I:C) and CpG ODN in increasing the MHC-restricted presentation of the exogenous antigen appeared to be similar. A combination of the 2 TLR agonists was synergistic in increasing the MHC class I-restricted, but not the class II-restricted, presentation of exogenous antigen. These results show that IgG-opsonized biodegradable nanoparticles containing both intraphagosomal TLR agonists and antigens can be efficient carrier materials in inducing antigen-specific T cell responses.

Systemic challenge with the TLR3 agonist poly I:C induces amplified IFN-α/β and IL-1β responses in the diseased brain and exacerbates chronic neurodegeneration

Systemic challenge with the TLR3 agonist poly I:C induces amplified IFN-α/β and IL-1β responses in the diseased brain and exacerbates chronic neurodegeneration

Inhibition of the Type I Interferon Response in Human Dendritic Cells by Dengue Virus Infection Requires a Catalytically Active NS2B3 Complex

Dengue virus (DENV) is the most prevalent arthropod-borne human virus, able to infect and replicate in human dendritic cells (DCs), inducing their activation and the production of proinflammatory cytokines. However, DENV can successfully evade the immune response in order to produce disease in humans. Several mechanisms of immune evasion have been suggested for DENV, most of them involving interference with type I interferon (IFN) signaling. We recently reported that DENV infection of human DCs does not induce type I IFN production by those infected DCs, impairing their ability to prime naive T cells toward Th1 immunity. In this article, we report that DENV also reduces the ability of DCs to produce type I IFN in response to several inducers, such as infection with other viruses or exposure to Toll-like receptor (TLR) ligands, indicating that DENV antagonizes the type I IFN production pathway in human DCs. DENV-infected human DCs showed a reduced type I IFN response to Newcastle disease virus (NDV), Sendai virus (SeV), and Semliki Forest virus (SFV) infection and to the TLR3 agonist poly(I:C). This inhibitory effect is DENV dose dependent, requires DENV replication, and takes place in DENV-infected DCs as early as 2 h after infection. Expressing individual proteins of DENV in the presence of an IFN-alpha/beta production inducer reveals that a catalytically active viral protease complex is required to reduce type I IFN production significantly. These results provide a new mechanism by which DENV evades the immune system in humans.

The viral TLR3 agonist poly(I:C) stimulates phagocytosis and intracellular killing of Escherichia coli by microglial cells

Stimulation of murine primary microglia with Toll-like receptor (TLR) agonists enhances their ability to phagocytose and kill bacteria. Here we show that the viral TLR3 agonist poly(I:C) stimulates the release of cyto-/chemokines and nitric oxide by microglia. Poly(I:C) increases microglial phagocytosis and intracellular killing of Escherichia coli K1, a pathogenic encapsulated bacterial strain, after 30 and 90 min of co-incubation. Stimulation with a viral epitope may strengthen the resistance of the brain to bacterial infections in vivo. Our data encourage animal experiments with poly(I:C) derivatives to assess whether this approach can increase the resistance of the CNS against bacterial infections.

Plasmodium yoelii: Influence of immune modulators on the development of the liver stage

Plasmodium sporozoites suppress the respiratory burst and antigen presentation of Kupffer cells, which are regarded as the portal of invasion into hepatocytes. It is not known whether immune modulation of Kupffer cells can affect the liver stage. In the present study, we found that sporozoites inoculated into Wistar rats could be detected in the liver, spleen, and lung; however, most sporozoites were arrested in the liver. Sporozoites were captured by Kupffer cells lined with endothelial cells in the liver sinusoid before hepatocyte invasion. Pretreatment with TLR3 agonist poly(I:C) and TLR2 agonist BCG primarily activated Kupffer cells, inhibiting the sporozoite development into the exoerythrocytic form, whereas Kupffer cell antagonists dexamethasone and cyclophosphamide promoted development of the liver stage. Our data suggests that sporozoite development into its exoerythrocytic form may be associated with Kupffer cell functional status. Immune modulation of Kupffer cells could be a promising strategy to prevent malaria parasite infection.

Activation of innate immunity influences salivary gland function and development of Sjögren’s Syndrome (93.33)

Abstract Sjögren’s syndrome (SS) is a chronic autoimmune disorder characterized by progressive lymphocytic infiltration within the exocrine glands. Loss of salivary gland (SG) function is a major feature of the disease. This study was undertaken to investigate the role of innate immunity activation in pathogenesis of SS. Mice were repeatedly injected with TLR3 agonist poly(I:C), and monitored for SG function and sialadenitis. Poly(I:C) treatment rapidly induced SG hypofunction. However, SG function was restored, once poly(I:C) treatment was terminated. This phenomenon was independent of the genetic background of the mice, and did not require T and B cells. Mice deficient in IFNAR1 and thereby lacking type I IFN signaling, did not loose SG function to the same extent as the wild type mice. In the autoimmune-prone NZB/W F1 mice, poly(I:C) treatment induced severe and accelerated sialadenitis. The SG in these mice showed presence of T-B cell aggregates and germinal center formation. Some of the T cells in these infiltrates were CXCR5+ICOS+ Tfh cells. Significant up-regulation in the expression of IL-21 and Il-21R genes was seen in the SG of poly(I:C) treated mice. This study demonstrates that activation of innate immunity in a genetically susceptible individual modulates the development of SS. This study also shows that type I IFN can adversely affect SG function and that IL-21 pathway and Tfh cells might be important in the pathogenesis of SS.

Identification, characterization and genetic mapping of TLR7, TLR8a1 and TLR8a2 genes in rainbow trout (Oncorhynchus mykiss)

Induction of the innate immune pathways is critical for early anti-viral defense but there is limited understanding of how teleost fish recognize viral molecules and activate these pathways. In mammals, Toll-like receptors (TLR) 7 and 8 bind single-stranded RNA of viral origin and are activated by synthetic anti-viral imidazoquinoline compounds. Herein, we identify and describe the rainbow trout (Oncorhynchus mykiss) TLR7 and TLR8 gene orthologs and their mRNA expression. Two TLR7/8 loci were identified from a rainbow trout bacterial artificial chromosome (BAC) library using DNA fingerprinting and genetic linkage analyses. Direct sequencing of two representative BACs revealed intact omTLR7 and omTLR8a1 open reading frames (ORFs) located on chromosome 3 and a second locus on chromosome 22 that contains an omTLR8a2 ORF and a putative TLR7 pseudogene. We used the omTLR8a1/2 nomenclature for the two trout TLR8 genes as phylogenetic analysis revealed that they and all the other teleost TLR8 genes sequenced to date are similar to the zebrafish TLR8a, but are distinct from the zebrafish TLR8b. The duplicated trout loci exhibit conserved synteny with other fish genomes extending beyond the tandem of TLR7/8 genes. The trout TLR7 and 8a1/2 genes are composed of a single large exon similar to all other described TLR7/8 genes. The omTLR7 ORF is predicted to encode a 1049 amino acid (aa) protein with 84% similarity to the Fugu TLR7 and a conserved pattern of predicted leucine-rich repeats (LRR). The omTLR8a1 and omTLR8a2 are predicted to encode 1035- and 1034-aa proteins, respectively, and have 86% similarity to each other. omTLR8a1 is likely the ortholog of the only Atlantic salmon TLR8 gene described to date as they have 95% aa sequence similarity. The tissue expression profiles of omTLR7, omTLR8a1 and omTLR8a2 in healthy trout were highest in spleen tissue followed by anterior and then posterior kidney tissues. Rainbow trout anterior kidney leukocytes produced elevated levels of pro-inflammatory and type I interferon cytokines mRNA in response to stimulation with the human TLR7/8 agonist R848 or the TLR3 agonist poly I:C. Only poly I:C-induced IFN2 transcription was significantly suppressed in the presence of chloroquine, a compound known to block endosomal acidification and inhibit endosomal maturation. The effect of chloroquine on R848-induced cytokine expression was equivocal and so it remains questionable whether rainbow trout recognition of R848 requires endosomal maturation. TLR7 and TLR8a1 expression levels in rainbow trout anterior kidney leukocytes were not affected by poly I:C or R848 treatments, but surprisingly, TLR8a2 expression was moderately down-regulated by R848. The down-regulation of omTLR8a2 may imply that this gene has evolved to a new or altered function in rainbow trout, as often occurs when the two duplicated genes remain active.

Enhanced induction of a histamine-forming enzyme, histidine decarboxylase, in mice primed with NOD1 or NOD2 ligand in response to various Toll-like receptor agonists

We investigated the immunopharmacological aspects of innate immune responses via Toll-like receptors (TLRs), NOD1 and NOD2, in terms of induction of the histamine-forming enzyme, histidine decarboxylase (HDC), activity in mice. Intravenous injection of TLR4-agonistic synthetic lipid A definitely induced HDC activity in the liver, spleen, and lungs, especially the lungs, in mice, where maximum activity was induced about 3 h after the injection of lipid A. The TLR2/6 agonistic synthetic diacyl-type lipopeptide FSL-1 and TLR3-agonistic poly I:C were also effective in inducing HDC, while the NOD2-agonistic synthetic muramyldipeptide (MDP) and NOD1-agonistic synthetic FK156 and FK565 exhibited only weak activities in this respect. Mice primed with intravenous injection of NOD1 or NOD2 agonists produced higher HDC activity following the 4-6 h later intravenous challenge with the above TLR agonists. Among the priming agents, FK565 exhibited the strongest activity, and it was effective via various administration routes - intraperitoneal, subcutaneous, intramuscular, as well as intravenous injection; furthermore, oral (gastric) administration was effective, although it needed a dose 10 times higher than that required for other administration routes. These findings suggest that HDC is induced in association with TLRs and NOD1/2, and that the newly formed histamine by the induced HDC might play important roles in the regulation of inflammatory and immune responses in various organs.