Ligand Poly Research Articles

Precision-cut human liver slice cultures as an immunological platform

The liver is the central metabolic organ in the human body, and also plays an essential role in innate and adaptive immunity. While mouse models offer significant insights into immune-inflammatory liver disease, human immunology differs in important respects. It is not easy to address those differences experimentally. Therefore, to improve the understanding of human liver immunobiology and pathology, we have established precision-cut human liver slices to study innate immunity in human tissue. Human liver slices collected from resected livers could be maintained in ex vivo culture over a two-week period. Although an acute inflammatory response accompanied by signs of tissue repair was observed in liver tissue following slicing, the expression of many immune genes stabilized after day 4 and remained stable until day 15. Remarkably, histological evidence of pre-existing liver diseases was preserved in the slices for up to 7 days. Following 7 days of culture, exposure of liver slices to the toll-like receptor (TLR) ligands, TLR3 ligand Poly-I:C and TLR4 ligand LPS, resulted in a robust activation of acute inflammation and cytokine genes. Moreover, Poly-I:C treatment induced a marked antiviral response including increases of interferons IFNB, IL-28B and a group of interferon-stimulated genes. Therefore, precision-cut liver slices emerge as a valuable tool to study human innate immunity.

TLR3 Ligand Poly(I:C) Exerts Distinct Actions in Synovial Fibroblasts When Delivered by Extracellular Vesicles.

Extracellular vesicles (EV) can modulate the responses of cells to toll-like receptor (TLR) ligation; conversely, TLR ligands such as double-stranded RNA (dsRNA) can enhance the release of EV and influence of the composition and functions of EV cargos. Inflamed synovial joints in rheumatoid arthritis (RA) are rich in EV and extracellular RNA; besides, RNA released from necrotic synovial fluid cells can activate the TLR3 signaling in synovial fibroblasts (SFs) from patients with RA. Since EV occur prominently in synovial joints in RA and may contribute to the pathogenesis, we questioned whether EV can interact with dsRNA, a TLR3 ligand, and modify its actions in arthritis. We have used as model the effects on RA SFs, of EV released from monocyte U937 cells and peripheral blood mononuclear cells upon stimulation with Poly(I:C), a synthetic analog of dsRNA. We show that EV released from unstimulated cells and Poly(I:C)-stimulated U937 cells [Poly(I:C) EV] differ in size but bind similar amounts of Annexin V and express comparable levels of MAC-1, the receptor for dsRNA, on the vesicular membranes. Specifically, Poly(I:C) EV contain or associate with Poly(I:C) and at least partially protect Poly(I:C) from RNAse III degradation. Poly(I:C) EV shuttle Poly(I:C) to SFs and reproduce the proinflammatory and antiviral gene responses of SFs to direct stimulation with Poly(I:C). Poly(I:C) EV, however, halt the death receptor-induced apoptosis in SFs, thereby inverting the proapoptotic nature of Poly(I:C). These prosurvival effects sharply contrast with the high toxicity of cationic liposome-delivered Poly(I:C) and may reflect the route of Poly(I:C) delivery via EV or the fine-tuning of Poly(I:C) actions by molecular cargo in EV. The demonstration that EV may safeguard extracellular dsRNA and allow dsRNA to exert antiapoptotic effects on SFs highlights the potential of EV to amplify the pathogenicity of dsRNA in arthritis beyond inflammation (by concurrently enhancing the expansion of the invasive synovial stroma).

Region-specific innate antiviral responses of the human epididymis.

Viral infections of the epididymis are associated with epididymitis, which damages the epithelium and impairs fertility. We showed previously that innate immune response genes were differentially expressed in the corpus and cauda region of the human epididymis in comparison to the caput. Here we investigate the antiviral defense response mechanisms of human epididymis epithelial (HEE) cells. Toll-like receptor (TLR) 3 and retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs) are enriched in HEE cells from the corpus and cauda region. These HEE cells show an enhanced response to antiviral ligands (poly(I:C) and HSV-60), as shown by increased IFN-β mRNA expression and IFN-β secretion. Nuclear translocation of phosphorylated p65 occurs after poly(I:C) exposure. In addition, paired box 2 (PAX2), which was implicated in regulating antiviral response pathways, is required for basal expression of the DNA sensor, Z-DNA binding protein (ZBP1) and type I interferon, in caput but not in cauda cells.

Human CD141+ Dendritic Cell and CD1c+ Dendritic Cell Undergo Concordant Early Genetic Programming after Activation in Humanized Mice In Vivo.

Human immune cell subsets develop in immunodeficient mice following reconstitution with human CD34+ hematopoietic stem cells. These “humanized” mice are useful models to study human immunology and human-tropic infections, autoimmunity, and cancer. However, some human immune cell subsets are unable to fully develop or acquire full functional capacity due to a lack of cross-reactivity of many growth factors and cytokines between species. Conventional dendritic cells (cDCs) in mice are categorized into cDC1, which mediate T helper (Th)1 and CD8+ T cell responses, and cDC2, which mediate Th2 and Th17 responses. The likely human equivalents are CD141+ DC and CD1c+ DC subsets for mouse cDC1 and cDC2, respectively, but the extent of any interspecies differences is poorly characterized. Here, we exploit the fact that human CD141+ DC and CD1c+ DC develop in humanized mice, to further explore their equivalency in vivo. Global transcriptome analysis of CD141+ DC and CD1c+ DC isolated from humanized mice demonstrated that they closely resemble those in human blood. Activation of DC subsets in vivo, with the TLR3 ligand poly I:C, and the TLR7/8 ligand R848 revealed that a core panel of genes consistent with DC maturation status were upregulated by both subsets. R848 specifically upregulated genes associated with Th17 responses by CD1c+ DC, while poly I:C upregulated IFN-λ genes specifically by CD141+ DC. MYCL expression, known to be essential for CD8+ T cell priming by mouse DC, was specifically induced in CD141+ DC after activation. Concomitantly, CD141+ DC were superior to CD1c+ DC in their ability to prime naïve antigen-specific CD8+ T cells. Thus, CD141+ DC and CD1c+ DC share a similar activation profiles in vivo but also have induce unique signatures that support specialized roles in CD8+ T cell priming and Th17 responses, respectively. In combination, these data demonstrate that humanized mice provide an attractive and tractable model to study human DC in vitro and in vivo.

Delivery of the TLR ligand poly(I:C) to liver cells in vitro and in vivo by calcium phosphate nanoparticles leads to a pronounced immunostimulation.

The selective activation of the immune system is a concurrent problem in the treatment of persistent diseases like viral infections (e.g. hepatitis). For the delivery of the toll-like receptor ligand poly(I:C), an immunostimulatory action has been discovered earlier by hydrodynamic injection. However, this technique is not clinically transferable to human patients. We have developed a modular system where poly(I:C) was incorporated into calcium phosphate nanoparticles. The uptake into relevant liver cells was studied both in vitro and in vivo. After intravenous injection into mice, the in vivo uptake was especially prominent in lung and liver, 1 and 3h after the injection. The corresponding strong immune reaction proves their high potential to turn up the immune system, e.g. against viral infections, without adverse side reactions.

Altered expression of complement factors in patients with nasal polyps in response to the TLR3 ligand PolyI:C

Altered expression of complement factors in patients with nasal polyps in response to the TLR3 ligand PolyI:C

Coordinated Role of Toll-Like Receptor-3 and Retinoic Acid-Inducible Gene-I in the Innate Response of Bovine Endometrial Cells to Virus.

Bovine herpesvirus-4 (BoHV-4) and bovine viral diarrhea virus (BVDV) infect the uterus of cattle, often resulting in reduced fertility, or abortion of the fetus, respectively. Here, exposure of primary bovine endometrial cells to BoHV-4 or BVDV modulated the production of inflammatory mediators. Viral pathogen-associated molecular patterns (PAMPs) are detected via pattern-recognition receptors (PRRs). However, the relative contribution of specific PRRs to innate immunity, during viral infection of the uterus, is unclear. Endometrial epithelial and stromal cells constitutively express the PRR Toll-like receptor (TLR)-3, but, the status of retinoic acid-inducible gene I (RIG-I), a sensor of cytosolic nucleic acids, is unknown. Primary endometrial epithelial and stromal cells had low expression of RIG-I, which was increased in stromal cells after 12 h transfection with the TLR3 ligand Poly(I:C), a synthetic analog of double-stranded RNA. Furthermore, short interfering RNA targeting TLR3, or interferon (IFN) regulatory transcription factor 3, an inducer of type I IFN transcription, reduced Poly(I:C)-induced RIG-I protein expression and reduced inflammatory mediator secretion from stromal cells. We conclude that antiviral defense of endometrial stromal cells requires coordinated recognition of PAMPs, initially via TLR3 and later via inducible RIG-I.

Maturation-dependent expression of AIM2 in human B-cells.

Intracellular DNA- and RNA-sensing receptors, such as the IFN-inducible protein Absent in Melanoma 2 (AIM2), serve as host sensors against a wide range of infections. Immune sensing and inflammasome activation by AIM2 has been implicated in innate antiviral recognition in many experimental systems using cell-lines and animal models. However, little is known about the expression and function of AIM2 in freshly isolated human cells. In this study we investigated the expression of AIM2 in different cell types derived from human cord and adult peripheral blood, in steady state and following in vitro-activation. Adult but not cord blood B-cells expressed high levels of AIM2 mRNA at steady state. In adults, AIM2 was primarily expressed in mature memory CD27+ B-cells. Both adult and cord blood derived B-cells could induce their transcription of AIM2 mRNA in response to type II IFN but not type I IFN or the AIM2 ligand poly dA:dT. Upon B-cell receptor stimulation, B-cells from adult blood expressed reduced levels of AIM2 mRNA. In addition, we show that adult B-cells were able to release IL-1β upon stimulation with synthetic DNA. We conclude that functional AIM2 is preferentially expressed in adult human CD27+ B-cells, but is absent in cord blood mononuclear cells.

CD4+ T Cell and NK Cell Interplay Key to Regression of MHC Class Ilow Tumors upon TLR7/8 Agonist Therapy.

One of the next challenges in cancer immunotherapy is the resistance of tumors to T-cell-based treatments through loss of MHC class I. Here, we show that under these circumstances, the Toll-like receptor (TLR)-7/8 ligand imiquimod, but not the TLR3 ligand poly I:C or TLR9 ligand CpG, mediated an effective antitumor response. The rejection of these immune-escaped cancers was mediated by NK cells and CD4+ T cells, whereas activated CD8+ T cells were dispensable. Application of the innate immune stimulator at a distant site activated NK cells and thereby elicited tumor-specific T-cell responses in tumor-bearing mice. Mechanistically, imiquimod activated NK cells to kill tumor cells, resulting in release of tumor antigens and induction of tumor-specific CD4+ T cells. These T helper cells provoked a strong induction of CXCL9 and CXCL10 in the tumor environment. Simultaneously, imiquimod induced the expression of the cognate chemokine receptor CXCR3 on peripheral lymphocytes. This ignited intratumoral CD4+ T-cell infiltration and accumulation, which was critical for tumor rejection; CXCR3 blocking antibodies mitigated the clinical response. In the effector phase, NK cell recruitment to tumors and their activation depended on CD4+ T cells. Together, we have uncovered a potent immune axis of tumor-specific CD4+ T cells and NK cells that eliminates escaped MHC-Ilow tumors. Cancer Immunol Res; 5(8); 642-53. ©2017 AACR.

Synergistic Role of Endothelial ERG and FLI1 in Mediating Pulmonary Vascular Homeostasis.

Endothelial cell (EC) activation underlies many vascular diseases, including pulmonary arterial hypertension (PAH). Several members of the E-twenty six (ETS) family of transcription factors are important regulators of the gene network governing endothelial homeostasis, and their aberrant expression is associated with pathological angiogenesis. The goal of this study was to determine whether deficiencies of the ETS family member, Friend leukemia integration 1 transcription factor (FLI1), and its closest homolog, ETS-related gene (ERG), are associated with PAH. We found that endothelial ERG was significantly reduced in the lung samples from patients with PAH, as well as in chronically hypoxic mice. Functional studies revealed that depletion of ERG or FLI1 in human pulmonary ECs led to increased expression of inflammatory genes, including IFN genes, whereas genes regulating endothelial homeostasis and cell-cell adhesion were down-regulated. Simultaneous knockdown of both ERG and FLI1 had synergistic or additive effects on the expression of these genes, suggesting that ERG and FLI1 coregulate at least a subset of their target genes. Functionally, knockdown of ERG and FLI1 induced cell monolayer permeability with a potency similar to that of vascular endothelial growth factor. Notably, stimulation of ECs with Toll-like receptor 3 ligand poly(I:C) suppressed ERG expression and induced ERG dissociation from the IFNB1 promoter, while promoting signal transducers and activators of transcription 1 (STAT1) recruitment. Consistent with the up-regulation of inflammatory genes seen in vitro, Erg and Fli1 double-heterozygote mice showed increased immune cell infiltration and expression of cytokines in the lung. In conclusion, loss of ERG and FLI1 might contribute to the pathogenesis of vascular lung complications through the induction of inflammation.

Abstract 2313: Promoting caspase-8-dependent apoptosis signaling using 17-beta-hydroxywithanolides

Abstract We have previously reported that withanolide E (WE), a steroidal lactone from Physalis peruviana, was highly active in sensitizing various human carcinoma cell lines to TRAIL-mediated apoptosis. Therefore, over 100 natural and semi-synthetic withanolides were evaluated for their ability to promote caspase-8-dependent cancer cell death. Our studies identified several withanolides that were 4-8 fold more potent than WE in sensitizing the renal carcinoma cells and melanoma cells to caspase-8-dependent apoptosis in response to either TRAIL or the TLR3 ligand poly (I:C). All active withanolides were 17-beta-hydroxywithanolides (17-BHW). The highly active 17-BHWs were more efficient than withanolide E at reducing cellular levels of both cFLIPL and cFLIPS and enhancing caspase-8 activation. Furthermore, immunoprecipitation of the TRAIL death-inducing signaling complex (DISC), or the related ripoptosome, demonstrated enhanced levels of both FADD and RIP1 in these macromolecular apoptosis signaling complexes following treatment with active 17-BHWs. The 17-BHWs used in this work were obtained by the application of an efficient method of plant biomass production involving our innovative and patented soil-less aeroponic cultivation of P. crassifolia and P. peruviana and by chemical modification of natural withanolides produced by these plants. Preliminary structure activity relationship (SAR) studies suggested that the enone moiety in ring A was essential for activity. In addition, acetoxylation at C-18, an alpha orientation of the side-chain lactone group and the double bond at C-24(25) of the lactone ring played important roles in determining the activity of 17-BHWs as apoptosis sensitizers. This suggests that the 17-BHW scaffold is amenable to optimization by a medicinal chemistry approach, which could lead to the identification of highly active natural product-based sensitizers of cancer cells to caspase-8-dependent apoptosis. The cellular molecular target(s) of active 17-BHWs are currently under further investigation. Funded by FNLCR Contract HHSN261200800001E. Citation Format: Alan D. Brooks, Ya-ming Xu, E. M. Kithsiri Wijeratne, Curtis J. Henrich, Poonam Tewary, Leslie Gunatilaka, Thomas J. Sayers. Promoting caspase-8-dependent apoptosis signaling using 17-beta-hydroxywithanolides [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2313. doi:10.1158/1538-7445.AM2017-2313

INCREASED EXPRESSION OF TOLL-LIKE RECEPTOR (TLR)-3 IN ALZHEIMER’S DISEASE BRAINS: A PROTECTIVE OR PATHOGENIC RESPONSE?

Toll-like receptors (TLR) are pattern recognition receptors involved in immune responses to microbial organisms. Studies have also shown their involvement in inflammatory responses to amyloid beta (Aβ) and aggregated α-synuclein. Activation of these receptors can amplify inflammation, but also promote phagocytosis of Aβ. Activation of TLR-9 resulted in significant reduction of Aβ in animal AD models. Activation of TLR-3 could have therapeutic potential due to its induction of beta-interferon responses. The premise of this study was observation of increased numbers of TLR-3 immunoreactive cells in AD affected brains. This study had two components; firstly staged series of human brain samples from low plaque (LP) and high plaque (HP) non-demented (ND) cases and AD cases were used to identify TLR-3 cellular localization using immunohistochemistry, and to quantify changes of expression of TLR-3 mRNA with disease by quantitative polymerase chain reaction (PCR). The second component involved in vitro studies of TLR-3 expression and activation using human postmortem brain-derived microglia and vascular endothelial cells. Quantification of TLR-3 immunoreactivity in middle temporal gyrus using Image J software to measure area occupied showed increase of 101.8% in AD cases compared to LPND (p=0.0036), and 16.5% increase between LPND and HPND (NS). TLR-3 immunoreactivity showed significant correlation with scores for brain plaque loads (Spearman r=0.464, p<0.01) and tangle loads (r=0.561, p<0.001). TLR-3 immunoreactivity was primarily in microglia, but also visible in subsets of astrocytes and vascular endothelial cells. Increased TLR-3 immunoreactivity was noticeable in microglia associated with Aβ plaques. Quantitative PCR showed significant increase in TLR-3 mRNA between AD and ND cases (p=0.043), interferon-β (p=0.036) and interferon regulatory factor-3 (p=0.013). Significant increase in TLR-9 mRNA expression in AD cases was also detected in these samples (p=0.0019). In vitro experiments showed that treatment with TLR-3 ligand poly I:C did not induce significant increased expression of TLR-3 mRNA in microglia, but a tenfold increase in brain-derived endothelial cells.

Comparison and immunobiological characterization of retinoic acid inducible gene-I-like receptor expression in mesenchymal stromal cells

Due to their immunomodulatory and regenerative properties, Mesenchymal stromal cells (MSC) have generated major interests in several clinical settings including transplantation and inflammatory diseases. MSC functions can be influenced by their tissue origin. Their microenvironment strongly affects their biology notably through TLR sensing. In this study, we show that MSC isolated from four different sources express another type of cytosolic pathogen recognition receptors known as retinoic acid inducible gene-I (RIG-I)-like receptors (RLR). RLR activation in MSC induces the production of Type I IFN (IFN-β) and Type III IFN (IFN-λ1). The highest producers are adipose tissue(AT)-MSC. We further show that Interferon production is induced through TBK1/IKK-ε signaling and IRF7 phosphorylation. Depending on MSC source, the knockdown of TLR3 and/or RIG-I decreases the MSC response to RLR ligand poly(I:C)/Lyovec. Among the different MSC types, AT-MSCs display the highest sensitivity to viral stimuli as shown by the alteration of their viability after prolonged stimulation. Our work indicates that this could be linked to an increase of pro-apoptotic Noxa expression. Finally, the expression of IDO1 and LIF upon RLR activation indicate the increase of MSC immunomodulatory potential, especially in AT-MSCs. Altogether, these data should be considered when designing MSC-based therapy in clinical settings where inflammation or infection are present.

Modulation of human umbilical cord‐derived mesenchymal stromal cells immunophenotype through cellular priming

The in vitro immunomodulation effects of bone marrow‐derived mesenchymal stromal cells (BM‐MSCs) can be drastically altered by stimulation of toll‐like receptor (TLR) 3 and 4 into diametrically different immunomodulatory phenotypes similar to activated macrophage type 1 (M1) and macrophage type 2 (M2) polarization. BM‐MSCs activated by TLR4 ligand lipopolysaccharide (LPS) display an enhanced pro‐inflammatory phenotype (MSC1). In contrast, BM‐MSCs stimulated by Poly I:C, a TLR3 ligand that simulates a viral infection, display an anti‐inflammatory phenotype, called MSC2. This phenomenon has been termed “MSC priming”. The objective of this study was to determine whether human umbilical cord‐derived (HUC) MSCs display enhanced immune suppression after MSC1 versus MSC2 priming. Four different media conditions: 1) the TLR4 ligand LPS, 2) the TLR3 ligand Poly I:C, 3) interferon gamma (IFN‐γ) which upregulates TLR4 receptors and 4) human platelet lysate (HPL) versus control (multipotent adult progenitor cell (MAPC) medium. Five independent MSC lines were tested. Immune properties were assayed by examining the cell's suppression on the proliferation of stimulated murine splenocytes and 2) cytokine production: PGE2 and indoleamine 2,3‐dioxygenase (IDO) via ELISA. Cells cultured in IFN‐γ, LPS and Poly I:C media showed significant upregulation of IDO compared to MAPC medium. The expression of PGE2 was not significantly altered. Compared with MAPC medium, HPL medium inhibited the immunosuppressive effects of MSCs. In contrast, IFN‐γ and MAPC medium had the greatest immunosuppressive effects. Umbilical cord‐derived MSCs did not undergo MSC1 priming by TLR3 ligand stimulation like bone marrow‐derived MSCs.

Epigenetic modifications influence TLR3 expression and activity

BackgroundToll‐like receptors (TLRs) are a family of pattern recognition receptors (PRRs) which recognise conserved pathogen associated molecular patterns (PAMPs) present in bacteria and viruses e.g. TLR3 recognises dsRNA from viruses. TLRs are expressed on a variety of cells including intestinal epithelial cells and play an important role in the immune response by stimulating cytokine release via phosphorylation of signalling proteins, but it is not known how their expression is regulated. We hypothesised that epigenetic mechanisms such as DNA methylation and histone deacetylation may play a role. In order to investigate this, we studied the effect of inhibition and/or genetic knockout of the enzymes responsible for these events (DNA methyltransferases (DNMT) or histone deacetylase complexes (HDAC)) on TLR3 expression and signalling in HCT‐116 intestinal epithelial cells.MethodsHCT‐116 (both wild‐type (WT) and DNMT 1/3B double knockout (DKO) cells) cells were either untreated or treated with the pan‐HDAC inhibitor suberanilohydroxamic acid (SAHA, 10μM, 48hrs), or the DNMT inhibitor 5‐aza‐2‐deoxycytidine (500nM, 72hrs), followed by stimulation with the TLR3 ligand Poly I:C (10μg/ml, 6 or 24 hrs). Cytokine mRNA expression was measured using quantitative polymerase chain reaction (QPCR) while protein release was measured using ELISA of the cell culture supernatant. Intracellular signalling protein phosphorylation was measured using Western blotting. Statistical analyses was performed in SPSS and in all cases n ≥ 3.ResultsTreatment of HCT‐116 WT cells with Poly I:C resulted in a significant increase in TLR3 expression (p&lt;0.001) and in the mRNA expression of cytokines (p&lt;0.001) and also the release of cytokines (p&lt;0.001). Poly I:C treatment also resulted in increased phosphorylation of signalling proteins (NF‐κB, IκBα, JNK, p38, IRF3). Pre‐treatment with SAHA, 5‐aza‐2‐deoxycytidine and genetic knockout of DNTM1/3b prevented this Poly I:C induced increase in TLR3 expression and cytokine mRNA expression, cytokine release and signalling protein phosphorylation.ConclusionInhibition of HDACs or DNMTs by pharmacological means or genetic knockout prevents TLR3 signalling and the activation of the TLR3 inflammatory pathway. This suggests that viral induced inflammation may be controlled using epigenetic modifying drugs.Support or Funding InformationCollege of Science Scholarship, NUI Galway

Elevated Expression of Chemokine CXCL13 in Chronic Hepatitis B Patients Links to Immune Control during Antiviral Therapy.

C–X–C-chemokine ligand 13 (CXCL13), the ligand for C–X–C chemokine receptor type 5 (CXCR5), is a major regulator of B-cell trafficking and plays an integral role in age-dependent clearance of hepatitis B virus (HBV) in the mouse model. However, the expression and function of CXCL13 in patients with chronic hepatitis B (CHB) remain unknown. By use of liver cell subpopulations isolated from CHB patients, we found that CXCL13 mRNA was abundantly expressed in Kupffer cells (KCs), but not in primary hepatocytes, liver sinusoidal endothelial cells, and hepatic stellate cells. Interestingly, KC isolated from HBV-positive liver had much higher level of CXCL13 expression than non-HBV-infected controls. And its expression was induced by toll-like receptor 3 ligand poly I:C stimulation. Moreover, intense expression of CXCL13 protein and accumulation of CD4+ T and B cells were evident in follicular-like structures in the liver tissue of CHB patients, which indicated its chemotactic effect on CXCR5+ CD4+ cells and B cells. Consistently, the levels of serum CXCL13 were significantly higher in the CHB patients than in healthy controls. Furthermore, CXCL13 concentration was increased in the complete response (CR) group during weeks 0–12 and did not change significantly during the course of telbivudine treatment, compared with the patients who didn’t achieve CR. In conclusion, the HBV-related increase of CXCL13 production in KC and serum CXCL13 level during telbivudine treatment might be associated with immune control of chronic HBV infection.

Porcine Dendritic Cells as an In Vitro Model to Assess the Immunological Behaviour of Streptococcus suis Subunit Vaccine Formulations and the Polarizing Effect of Adjuvants.

An in vitro porcine bone marrow-derived dendritic cell (DC) culture was developed as a model for evaluating immune polarization induced by adjuvants when administered with immunogens that may become vaccine candidates if appropriately formulated. The swine pathogen Streptococcus suis was chosen as a prototype to evaluate proposed S. suis vaccine candidates in combination with the adjuvants Poly I:C, Quil A ®, Alhydrogel ®, TiterMax Gold ® and Stimune ®. The toll-like receptor ligand Poly I:C and the saponin Quil A ® polarized swine DC cytokines towards a type 1 phenotype, with preferential production of IL-12 and TNF-α. The water-in-oil adjuvants TiterMax Gold ® and Stimune ® favoured a type 2 profile as suggested by a marked IL-6 release. In contrast, Alhydrogel ® induced a type 1/type 2 mixed cytokine profile. The antigen type differently modified the magnitude of the adjuvant effect, but overall polarization was preserved. This is the first comparative report on swine DC immune activation by different adjuvants. Although further swine immunization studies would be required to better characterize the induced responses, the herein proposed in vitro model is a promising approach that helps assessing behaviour of the vaccine formulation rapidly at the pre-screening stage and will certainly reduce numbers of animals used while advancing vaccinology science.

The antiviral restriction factor IFN-induced transmembrane protein 3 prevents cytokine-driven CMV pathogenesis.

The antiviral restriction factor IFN-induced transmembrane protein 3 (IFITM3) inhibits cell entry of a number of viruses, and genetic diversity within IFITM3 determines susceptibility to viral disease in humans. Here, we used the murine CMV (MCMV) model of infection to determine that IFITM3 limits herpesvirus-associated pathogenesis without directly preventing virus replication. Instead, IFITM3 promoted antiviral cellular immunity through the restriction of virus-induced lymphopenia, apoptosis-independent NK cell death, and loss of T cells. Viral disease in Ifitm3–/– mice was accompanied by elevated production of cytokines, most notably IL-6. IFITM3 inhibited IL-6 production by myeloid cells in response to replicating and nonreplicating virus as well as following stimulation with the TLR ligands Poly(I:C) and CpG. Although IL-6 promoted virus-specific T cell responses, uncontrolled IL-6 expression in Ifitm3–/– mice triggered the loss of NK cells and subsequently impaired control of MCMV replication. Thus, IFITM3 represents a checkpoint regulator of antiviral immunity that controls cytokine production to restrict viral pathogenesis. These data suggest the utility of cytokine-targeting strategies in the treatment of virus-infected individuals with impaired IFITM3 activity.

Bovine Endometrial Cells Mount Innate Immune Response to the Intracellular Ligands CL097 and Poly(dA:dT) Indicating Roles against Uterine Viruses

Uterine infection and endometritis cause infertility and economic losses in the cattle industry. The innate immune response of the endometrium is critical in the elimination of pathogenic organisms that invade the uterus in postpartum cows. This study investigated the response of bovine endometrium to synthetic intracellular ligands which activate innate immunity by stimulating similar receptors to those used to recognise the presence of some viruses. Mixed primary epithelial and stromal cell cultures were treated with 5 μg/ml of CL097 (a TLR7/8 ligand) or 2 μg/ml of poly(dA:dT) (a DNA analogue) for either 6 h or 24 h. Cellular responses were assessed by the mRNA expression of 18 immune-related genes and 3 endogenous reference genes by conventional PCR followed by qRT-PCR from four replicate experiments. Bovine endometrial cells expressed the cytosolic pattern recognition receptors (PRRs) DDX58 (RIG-I), IFIH1 (MDA5) and LRRFIP1 which act as intracellular nucleic acid sensors. Neither ligand altered the expression of the extra-cytosolic pattern recognition receptors (PRRs) TLR3, TLR4, TLR7 or TLR8 whereas poly(dA:dT) treatment increased the expression of IFIH1 and DDX58. Treated cells also responded to CL097 or poly(dA:dT) with a differential up-regulation of genes involved in innate immune response including type I interferon/antiviral response (MX1, IFNAR1), antimicrobial activity (MUC1, SLPI) and cytokine activity (TNF, IL1B, IL8). Bovine endometrial cells therefore express both cytosolic and extra-cytosolic intracellular PRRs and are able to mount an innate immune response upon stimulation with intracellular ligands. This suggests an important role for these cells in the defence against viruses that may be present in the uterus in postpartum cows.

Abstract A029: A vaccine-induced, antigen-specific TH9 immune response blocks tumor cell engraftment

Abstract Metastatic cancer remains the main cause of cancer-related mortality. Therapies are needed to address this significant unmet medical need. The implantation of cancer cells resulting in metastatic foci is the consequence of a multistep process that is seldom diagnosed or targeted for therapeutic intervention. In light of its adhesive properties and deregulated overexpression by cancer cells, the carcinoembryonic antigen (CEA) has been identified as a molecular determinant linked to cellular events defining the metastatic cascade including their attachment to the ECM and to the formation of micrometastases. Herein, we report that a vaccination strategy yielding an antigen-specific TH9 response induces long term host surveillance and prevents the engraftment of circulating cancer cells. Specifically, we show that vaccination with a recombinant CEA IgV-like N domain, formulated with the TLR3 ligand poly I:C, elicits a CEA-specific TH9 response, wherein IL-9 secreting TH cells act in concert with CEA N domain-specific antibodies as well as activated mast cells in preventing tumor cell engraftment. Vaccine-imparted protection was reversed upon depleting CD3+ T cells, neutralizing serum IL-9, pharmacologically stabilizing or by depleting of CD117+ mast cells, in vivo. The development of this immune response was dependent on TLR3, since interference with the TLR3-dsRNA complex formation led to a reduction in vaccine-imparted protection and a shift in the resulting immune response toward a TH2 response. These results highlight the first instance of a vaccine induced tumor antigen-specific TH9 immune response and points to the existence of an alternate tumor eradication mechanism that may be exploited for developing anti-metastasic immunotherapies. (Supported by OCIR-IPDC,CBCF and CIHR) Citation Format: Aws Abdul-Wahid, Aaron Prodeus, Marzena Cydzik, Mays Alwash, Jean Gariepy. A vaccine-induced, antigen-specific TH9 immune response blocks tumor cell engraftment [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr A029.