Pattern-recognizing Receptors Research Articles

Molecular mechanisms underlying the pathogenesis of septic multiple organ failure

Sepsis is defined as the body's overwhelming and life-threatening response to infection that can lead to tissue damage, organ failure, and death. Since bacterial infection is one of the main causes of sepsis, appropriate antimicrobial therapy remains the cornerstone of sepsis and septic shock management. However, since sepsis is a multifaceted chaos involving inflammation and anti-inflammation disbalance leading to the unregulated widespread release of inflammatory mediators, cytokines, and pathogen-related molecules leading to system-wide organ dysfunction, the whole body control to prevent the progression of organ dysfunction is needed. In sepsis and septic shock, pathogen-associated molecular patterns (PAMPs), such as bacterial exotoxins, cause direct cellular damage and/or trigger an immune response in the host. PAMPs are recognized by pattern recognizing receptors (PRRs) expressed on immune-reactive cells. PRRs are also activated by host nuclear, mitochondrial, and cytosolic proteins, known as damage-associated molecular patterns (DAMPs) that are released from cells during sepsis. Thus, most PRRs respond to PAMPs or DAMPs by triggering activation of transcriptional factors, NF-κB, AP1, and STAT-3. On the other hand, sepsis leads to immune (lymphocytes and macrophages) and nonimmune (endothelial and epithelial cells) cell death. Apoptosis has been the major focus of research on cell death in sepsis, but autophagy, necrosis, necroptosis, pyroptosis, NETosis, and ferroptosis may also play an important role in this critical situation. The recent development in our understanding regarding the cellular pathogenesis of sepsis will help in developing new treatment of sepsis.

Thrombin in the Crossroad Hemostasis and Inflammation

Hemostasis and immune responses are evolutionarily and functionally related systems on the coordinated work of which vital processes – protection from blood loss and pathogens, depend. Thrombin is the central enzyme of the coagulation system, which has pronounced pro–inflammatory activity and plays an important role in the pathogenesis of a wide range of infectious and non-infectious diseases. Many humoral immune factors regulating inflammation (IL-1α, C3 and C5 complement components) and cell migration to the lesion site (osteopontin, chimerin) are thrombin targets and become activated by proteolytic cleavage. The main thrombin receptors – protease-activating receptors (PARs), are expressed on many cells of the immune system and are considered as non–classical pattern-recognizing receptors (PRRs). The effect of thrombin on innate immune cells may not be related to its enzymatic effects. Thrombin action on adaptive immunity is just beginning to be studied. Recent studies show that thrombin can act as an alarmin, stimulate the maturation of dendritic cells and adaptive immune responses. The production of this factor also affects Th cell polarization, which determines immune response strategy. The study of the immune functions of the components of the coagulation system reveals new pathogenetic mechanisms of the development of sterile inflammation and expands existing possibilities of allergic, autoimmune and neuroinflammatory disease therapy.

Immunological Response to Respiratory Syncytial Virus Infection

Immunological response to Respiratory Syncytial Virus (RSV) involves multiple entities such as cellular, antibody, cytokine, chemokine, and Pattern Recognizing Receptors (PRRs) that are mobilized to subdue the airway and pulmonary effects of the virus. RSV predominantly involves the respiratory tract including the bronchioles and the lungs in addition to the upper respiratory passage. Although RSV afflicts all age groups predominantly in winter, severe infection is more common in the very young and in older individuals, i.e., 60-65 years of age. Recently, vaccines and monoclonal antibodies have been developed as a preventive strategy for the vulnerable.

The Cytoskeleton in Plant Immunity: Dynamics, Regulation, and Function.

The plant cytoskeleton, consisting of actin filaments and microtubules, is a highly dynamic filamentous framework involved in plant growth, development, and stress responses. Recently, research has demonstrated that the plant cytoskeleton undergoes rapid remodeling upon sensing pathogen attacks, coordinating the formation of microdomain immune complexes, the dynamic and turnover of pattern-recognizing receptors (PRRs), the movement and aggregation of organelles, and the transportation of defense compounds, thus serving as an important platform for responding to pathogen infections. Meanwhile, pathogens produce effectors targeting the cytoskeleton to achieve pathogenicity. Recent findings have uncovered several cytoskeleton-associated proteins mediating cytoskeletal remodeling and defense signaling. Furthermore, the reorganization of the actin cytoskeleton is revealed to further feedback-regulate reactive oxygen species (ROS) production and trigger salicylic acid (SA) signaling, suggesting an extremely complex role of the cytoskeleton in plant immunity. Here, we describe recent advances in understanding the host cytoskeleton dynamics upon sensing pathogens and summarize the effectors that target the cytoskeleton. We highlight advances in the regulation of cytoskeletal remodeling associated with the defense response and assess the important function of the rearrangement of the cytoskeleton in the immune response. Finally, we propose suggestions for future research in this area.

Modern view on the role of pattern-recognition receptors and signaling pathways in the development of innate immunity in viral infections

Features of organization and functioning of pattern-recognizing receptors and signaling pathways in induction of antiviral immune response are considered. The recognition of antigenic structures of the virus is carried out by pathogen-associated molecular patterns of innate immunity cells. These are Toll-like receptors, nucleotide-binding oligomerization domain-like receptors, lectin C-type receptors, and RIG-I-like receptors. The functioning of these receptor structures depends on protein molecules that provide activation signals. These are the adapter proteins of the primary response of myeloid differentiation 88, interleukin-1 receptor-associated kinase, nuclear factor-KB. Interactions of cellular proteins in the activation of signaling pathways are complex and receptor-ligand reactions can lead to different outcomes in a single cell, in most cases leading to a limitation of viral reproduction. An important obstacle to the effective recognition of viruses and the development of an adequate immune response is the close organization of viral antigens and receptor structures of the cell. The role of LGP2 protein molecules is associated with the peculiarities of the development of the immune response, which can be not only positive, but also negative regulators of the transmission of excitation signals from intracellular cytoplasmic helicase receptors. A number of viral proteins inhibit activation signals, which ultimately leads to various options for the development of the immune response. A special role belongs to the transmembrane protein of the endoplasmic reticulum, which increases the expression of interferon a stimulator of interferon genes that provide detection of deoxyribonucleic acid viruses. Maximum activation of this protein, ensures the effective development of a cellular antiviral immune response.

Proteins of the lectin pathway of the complement system activation: immunobiological functions, genetics and involvement in the pathogenesis of human diseases

The complement system is the most ancient components in the innate immunity, mainly functioning to primarily eliminate bacterial agents intravascularly. Moreover, the complement complex proteins play a role as a bridge between the systems of innate and adaptive immunity providing adequate conditions for maturation and differentiation of B- and T-lymphocytes. The complement system consists of plasma proteins and membrane receptors. Plasma proteins interact with each other via the three described cascade pathways lectin (which is most ancient phylogenetically), alternative and classical. Lectins are proteins comprising a separate superfamily of pattern-recognizing receptors able to sense molecules of oligo- and polysaccharide nature and induce their aggregation. Among all the lectins, ficolins (FCN) (common domain fibrinogen) and collectins (common domain collagen) mannose-binding lectin (MBL), hepatic and renal collectins have exert unique functions by complexing with carbohydrate components of microbial wall. Formation of a compound complex microbial wall polysaccharides + collectin/ficolin + specific mannose-binding lectin-associated serine proteases (MARP) results in the complement system activation, inflammatory reaction and bacterium elimination. Such scenario is proceeded along the lectin pathway compared to the two other pathways called classical and alternative. Examining a role of the complement system and congenital protein defects in the pathogenesis of various diseases is of topical interest because inborn deficiency of the complement components comprises at least 5% out of total primary immunodeficiency rate, whereas the aspects of their prevalence and pathogenesis remain unexplored. Relevance of investigating the complement system components for diverse populations is tremendous, taking into consideration accumulated evidence regarding an important role of the lectin pathway in viral infections. Lectins, the main proteins in the lectin pathway of the complement activation, are encoded by polymorphic genes, wherein single nucleotide polymorphisms (SNPs) result in altered protein conformation and expression, which, in turn, affects functionality and potential to respond to a pathogen. The distribution of the lectin polymorphic gene frequencies and their haplotypes displays extremely marked population differences. According to analyzing available data, population SNP frequencies including those associated with inborn deficiencies for components of the lectin pathway have been currently scarce or unexplored. hence, here we review major lectins and their functions, their functionally significant SNPs in diverse populations and their pathogenetic importance for host defense functions.

Toll-like Receptor 4, Osteoblasts and Leukemogenesis; the Lesson from Acute Myeloid Leukemia.

Toll-like receptor 4 (TLR4) is a pattern-recognizing receptor that can bind exogenous and endogenous ligands. It is expressed by acute myeloid leukemia (AML) cells, several bone marrow stromal cells, and nonleukemic cells involved in inflammation. TLR4 can bind a wide range of endogenous ligands that are present in the bone marrow microenvironment. Furthermore, the TLR4-expressing nonleukemic bone marrow cells include various mesenchymal cells, endothelial cells, differentiated myeloid cells, and inflammatory/immunocompetent cells. Osteoblasts are important stem cell supporting cells localized to the stem cell niches, and they support the proliferation and survival of primary AML cells. These supporting effects are mediated by the bidirectional crosstalk between AML cells and supportive osteoblasts through the local cytokine network. Finally, TLR4 is also important for the defense against complicating infections in neutropenic patients, and it seems to be involved in the regulation of inflammatory and immunological reactions in patients treated with allogeneic stem cell transplantation. Thus, TLR4 has direct effects on primary AML cells, and it has indirect effects on the leukemic cells through modulation of their supporting neighboring bone marrow stromal cells (i.e., modulation of stem cell niches, regulation of angiogenesis). Furthermore, in allotransplant recipients TLR4 can modulate inflammatory and potentially antileukemic immune reactivity. The use of TLR4 targeting as an antileukemic treatment will therefore depend both on the biology of the AML cells, the biological context of the AML cells, aging effects reflected both in the AML and the stromal cells and the additional antileukemic treatment combined with HSP90 inhibition.

CD36 expression intensity on different subpopulations of circulating monocytes depending on the extent of subclinical atherosclerosis

It is known that chronic activation of innate immunity and persistent low-intensity inflammation play a crucial role in the initiation and progression of atherosclerosis. It was found that atherogenic lipoproteins can act as inducers of the inflammatory response through ligand-receptor interaction with pattern-recognizing receptors of immunocompetent cells, such as CD36 (SR-B2) and Toll-like receptors. It is suggested that expression of CD36 on circulating monocytes may represent the burden of systemic atherosclerosis and, therefore, act as its diagnostic marker. The aim of the present study was to assess the intensity of CD36 expression on circulating monocytes of different subpopulations in patients without established cardiovascular disease (CVD) depending on the extent of subclinical atherosclerosis of peripheral arteries. One hundred patients without established atherosclerotic CVD, 49 (49.0%) men and 51 (51.0%) women, were included in the study. Monoclonal antibody conjugates were used to phenotype monocyte subpopulations. The expression of CD36 on CD14++CD16- monocytes (classical monocytes), CD14+CD16+ monocytes (intermediate monocytes), CD14+CD16++ monocytes (non-classical monocytes) was determined by the average fluorescence intensity. There was a statistically significant decrease in CD36 expression intensity on classical and non-classical monocytes with increasing number of vascular basins affected by atherosclerosis. A statistically significant decrease in CD36 expression intensity on classical and non-classical monocytes was found in the patients with two vascular beds lesions in comparison with patients with a single vascular bed lesion upon pairwise comparisons.

The Role of Siglec-G on Immune Cells in Sepsis.

Sepsis is a life-threatening clinical syndrome that results from an overwhelming immune response to infection. During sepsis, immune cells are activated by sensing pathogen-associated molecular patterns and damage-associated molecular patterns (DAMPs) through pattern recognizing receptors (PRRs). Regulation of the immune response is essential to preventing or managing sepsis. Sialic acid-binding immunoglobulin-type lectin-G (Siglec-G), a CD33 group of Siglec expressed in B-1a cells and other hematopoietic cells, plays an important immunoregulatory role. B-1a cells, a subtype of B lymphocytes, spontaneously produce natural IgM which confers protection against infection. B-1a cells also produce IL-10, GM-CSF, and IL-35 to control inflammation. Sialic acids are present on cell membranes, receptors, and glycoproteins. Siglec-G binds to the sialic acid residues on the B cell receptor (BCR) and controls BCR-mediated signal transduction, thereby maintaining homeostasis of Ca++ influx and NFATc1 expression. Siglec-G inhibits NF-κB activation in B-1a cells and regulates B-1a cell proliferation. In myeloid cells, Siglec-G inhibits DAMP-mediated inflammation by forming a ternary complex with DAMP and CD24. Thus, preserving Siglec-G’s function could be a novel therapeutic approach in sepsis. Here, we review the immunoregulatory functions of Siglec-G in B-1a cells and myeloid cells in sepsis. A clear understanding of Siglec-G is important to developing novel therapeutics in treating sepsis.

Physiological and pathogenic role of scavenger receptors in humans

The scavenger receptors (SRs)) include > 30 different molecules structurally classified into 11 classes (A to L). They are expressed mostly on stromal macrophages, and their expression may be augmented in direct dependence with concentrations of their ligands. The SRs are heterogenous by their structure, however, being common in their functional potential. E.g., different SR classes may participate in absorption of modified low-density lipoproteins and glycated proteins, apoptotic and ageing cells, altered erythrocytes and platelets, like as a big variety of other endogenous ligands from metabolic and cellular “trash”. A common property of SRs is their participation in removal of small pathogen amounts from blood circulation, regulation of cell and tissue stress responses, ability to form complicated receptor complexes with other receptor types including integrins and toll-like receptors. Opposite to classic pattern-recognizing receptors, the SR involvement does not always elicit a pronounced cellular activation and development of pro-inflammatory cellular stress. The SR functional effects provide interactions between different physiological events and immune system, including the processes of neuroendocrine and metabolic regulation. These mechanisms provide both homeostatic stability and, likewise, act at the border of normal and pathological conditions, i.e., participating in pathogenesis of transitional processes, e.g., physiological ageing. Moreover, the SR-associated processes represent a key pathogenetic factor in different somatic diseases, e.g., those associated with low-intensity chronic inflammation, including obesity, type 2 diabetes, atherosclerosis, arterial hypertension, various neurodegenerative disorders. Similarly, the SRs are involved into the processes of cancer transformation and antitumor response, different processes of classical inflammation, from antigen presentation to the morphofunctional T cell and macrophage polarization in the inflammation foci and immunocompetent organs. SR are playing a controversial role in development of acute systemic inflammation, the main reason for lethal outcomes in the intensive care wards. Targeted effects upon the SRs represent a promising approach when treating a broad variety of diseases, whereas detection of membrane-bound and soluble SR forms could be performed by means of diagnostic and monitoring techniques in many human disorders.

ANALYSIS OF CYTOARCHITECTONICS OF TLR2<sup>+</sup> AND TLR4<sup>+</sup> LYMPHOCYTES AND TRANSCRIPTIONAL ACTIVITY OF THE GENES <i>Gp2, Spi-B, Nf-kB1, с-REL, TNFα</i> AND <i>TNFr</i> IN GALT OF RATS IN EXPERIMENTAL DIABETES MELLITUS AND AFTER PENTOXIFYLLINE ADMINISTRATION

Summary.Changes in the state of gut-associated lymphoid tissue (GALT) and the composition of the intestinal microbiome, both in experimental STZ-induced diabetes and in development of type 1 diabetes in humans as well as chronic inflammation due to stimulation of innate immunity are crucially important in the development of type 1 diabetes mellitus. One of the most important mediators for interactions between the intestinal microbiome and GALT are specialized M cells of the follicle-associated epithelium, providing transcytotic delivery of antigens to the underlying lymphoid structures. TNFα-signaling also plays a supporting role in the formation of M cells. Therefore, the aim of our work was to study some features of TLRs expression and transcriptional activity of the Gp2, Spi-B, Nf-kB1, c-Rel, TNFα and TNFr genes in GALT in experimental diabetes mellitus (EDM), and after pentoxifylline administration. To identify TLR2+ cells and TLR4+ cells, an immunofluorescence method was used with monoclonal antibodies to corresponding pattern-recognizing receptors. To study the transcriptional activity of genes, the method of real-time reverse transcription polymerase chain reaction (RT-PCR) was used. In the course of developing experimental pathology, at the terms of 2 and 4 weeks, a decrease in the total density of TLR2+ and TLR4+ lymphocytes was observed in lamina propria of villus (villus) and subepithelial zone isolated lymphoid follicles (ILF) of rat ileum. At the same time, the density of TLR2 on the membrane of immunopositive cells was increased for small lymphocytes, and TLR4 density has became higher in medium and small lymphocytes. The pentoxifylline administration to diabetic rats resulted in a decrease in the total density of TLR2+ cells at the 2nd week of development of the pathology, and an increase in this index at the 4th week. The total density of TLR4+ cells showed changing growth rates only in villus at the 2nd week of EDM development in the presence of pentoxifylline. Changes in the density of TLR2 and TLR4 on the surface of lymphocytes were multidirectional. The development of diabetes is also reflected in the transcriptional induction of genes of the key transcription factors NF-kB1 and c-Rel in GALT cells at both the 2nd and 4th week of the development of EDM. Meanwhile, administration of pentoxifylline resulted in a significantly reduced level of normalized expression of NF-kB1 mRNA during the entire observation period and increased this indicator for c-Rel mRNA at the 2nd week. The growth of normalized expression of markers of M cells Gp2 and Spi-B was observed both on the 2nd and on the 4th week of the development of experimental pathology. Administration of pentoxifylline to diabetic animals was largely reflected in the change in the intensity of mRNA expression of the mature M cell Gp2 marker. This parameter was increased during the 2nd week of developing pathology, and on the 4th week, a downward trend was shown. The development of EDM led to a significantly increased level of near-normalized expression of proinflammatory TNFα cytokine and its receptor TNFr, and demonstrated a trend towards their decrease following pentoxifylline administration in diabetic animals.

Preterm Life in Sterile Conditions: A Study on Preterm, Germ-Free Piglets.

Preterm infants born with immature organ systems, which can impede normal development, can also be highly sensitive to different biological and/or environmental factors. Animal models could aid in investigating and understanding the effects of different conditions on the health of these immunocompromised infants. The epitheliochorial placentation of the pig prevents the prenatal transfer of protective colostral immunoglobulins. Surgical colostrum-deprived piglets are free of maternal immunoglobulins, and the cells that are normally provided via colostrum. We bred preterm germ-free piglets in sterile conditions and compared them with their term counterparts. Enterocyte development and intestinal morphology, tight junction proteins claudin-1 and occludin, pattern-recognizing receptors, adaptor molecules and coreceptors (RAGE, TLR2, TLR4, TLR9, MyD88, TRIF, MD2, and CD14), and inflammasome NLRP3 transcription were all evaluated. The production of inflammatory mediators IFN-α, IL-4, IL-6, IL-8, IL-10, IL-12/23 p40, TNF-α, IFN-γ, and high mobility group box 1 (HMGB1) in the intestine of germ-free piglets was also assessed. In the preterm germ-free piglets, the ileum showed decreased lamina propria cellularity, reduced villous height, and thinner and less distinct stratification – especially muscle layer, in comparison with their term counterparts. Claudin-1 transcription increased in the intestine of the preterm piglets. The transcription levels of pattern-recognizing receptors and adaptor molecules showed ambiguous trends between the groups. The levels of IL-6, IL-8, IL-10, and TNF-α were increased in the preterm ileum numerically (though not significantly), with statistically significant increases in the colon. Additionally, IL-12/23 p40 and IFN-γ were statistically significantly higher in the preterm colon. Both blood plasma and intestinal HMGB1 levels were nonsignificantly higher in the preterm group. We propose that the intestine of the preterm germ-free piglets showed “mild inflammation in sterile conditions.” This model, which establishes preterm, hysterectomy-derived germ-free piglets, without protective maternal immunoglobulins, can be used to study influences of microbiota, nutrition, and therapeutic interventions on the development and health of vulnerable immunocompromised preterm infants.

Lectins and glycoconjugates in presentation of antigens and protection against patogens.

The current data on using lectin receptors and glycoconjugates (GC) in study of pathways of intercellular communications within innate immunity network coupled to adaptive immunity wеre overviewed and systematized. A superfamily of GC-specific C-type lectin receptors (CLR), as a constituent of pattern recognizing receptors (PRR), involves in immunity to produce redistribution of groups of cytokines, chemokines and specific antibodies. CLR on surface of multiple subpopulations of antigen-presenting cells (APC) and other cells of immunity are under consideration (monocyte derived macrophages, macrophages, immature and mature dendritic cells, other myeloid cells, neutrophils, basophils, lymphocytes, etc.). CLR cofunction both each other and/or together with other PRR and immune receptors. CLR are favourable for quicker and GC-specific innate type presentation of important danger antigenic patterns (PAMP, MAMP, DAMP, SAMP, TAMP) using their specific targeting through currently/ adaptively optimal pathways within intercellular branched network of activated/ polarized subpopulations of cells mentioned above. CLR agonist GC (recombinant, synthetic, conjugates to carriers (adaptive proteins, etc.), assembled into nanoparticles and liposomes) can serve successful and specific adjuvants in vaccine strategies. CLR and GC pairs participate in on duty biocontrol of immunobiological supervising in organism, modulate/ switch recognition and protection cell pathways of immune responses (presentation, reciprocally organized protective attacks, rates and amplitudes of activation and inhibition of cell communicative chains). Lectins characterized both cell and cell communicative chain current functional biomarkering that is of importance for clinical diagnostics. The knowledge on CLR-GC pattern relationships is perspective for study and influence fine mechanisms of cell GC-targeting and immunity, improvement of traditional vaccines, development of GC-specific and highly effective pattern vaccines against different infections or tumors. New specific vaccine strategies are possible towards different biotopes in organism (also for mucosal opened cavities).

PHARMACOLOGICAL CHARACTERIZATION OF BAN2401-MEDIATED Aβ PROTOFIBRIL CLEARANCE BY MICROGLIA

Amyloid β (Aβ) peptides aggregate into soluble, large molecular weight species (protofibrils) that eventually form the insoluble fibrils deposited in plaques. Aβ protofibrils are the dominant form of soluble Aβ in Alzheimer disease (AD) brains and are thought to play a role in the pathogenesis of AD through their deleterious effects on a number of physiological processes. BAN2401 is a humanized monoclonal IgG1 antibody with high selectivity for Aβ protofibrils that is currently in clinical development for early AD. mAb158, the mouse precursor, has previously been shown to remove insoluble amyloid plaque in mice, neutralize large soluble Aβ aggregates in vivo/in vitro and facilitate their clearance via a shift toward FcγR-dependent microglial phagocytosis. The present work aims to further characterize the mechanism of action of BAN2401-mediated Aβ protofibril clearance. Protofibril uptake was assessed in cultured human primary microglia prepared from AD brain by visualizing fluorescent tagged antibody under high power microscopy. Protofibril uptake was also assessed in an EOC-20 microglia cell antibody dependent cellular phagocytosis (ADCP) assay by measuring intracellular Aβ via ELISA in the absence or presence of FcγR blocker or F(ab’)2 fragments. Aβ protofibrils were readily taken up in human AD-derived microglia in an FcγR-independent manner, while BAN2401 facilitated further uptake of protofibrils in FcγR-mediated fashion. BAN2401 facilitated protofibril uptake in EOC-20 microglia cells was blocked with an FcγR blocker or F(ab’)2 fragments (EC50=257±61 ng/mL; corrected for background uptake). Aβ can be taken up by microglia via a non-specific pattern-recognizing receptor pathway or through an FcγR-mediated process. Previous work has demonstrated that BAN2401 facilitates protofibril clearance, and this clearance was inhibited by an FcγR blocker to levels lower than those seen in the absence of BAN2401, suggesting a shift from non-specific uptake toward microglial activation and FcγR-dependent microglial phagocytosis. The present studies isolate the FcγR-dependent component of BAN2401-mediated protofibril uptake and provide further support for antibody dependent cellular phagocytosis of Aβ protofibrils as the primary mechanism of action for BAN2401.

Activation of toll-like receptor signaling pathways leading to nitricoxide-mediated antiviral responses.

Toll-like receptors (TLRs), well-characterized pattern-recognizing receptors of the innate arm of the immune system, are vital in detecting pathogen-associated molecular patterns (PAMPs). The TLR-PAMP interaction initiates an intracellular signaling cascade, predominantly culminating in upregulation of antiviral components, including inducible nitric oxide synthase (iNOS). After activation, various TLR pathways can promote iNOS production via the myeloid differentiation primary response-88 (MyD-88) adapter protein. Subsequently, iNOS facilitates production of nitric oxide (NO), a highly reactive and potent antiviral molecule that can inhibit replication of RNA and DNA viruses. Furthermore, NO can diffuse freely across cell membranes and elicit antiviral mechanisms in various ways, including direct and indirect damage to viral genomes. This review emphasizes current knowledge of NO-mediated antiviral responses elicited after activation of TLR signaling pathways.

P133: DNA inhibits dsRNA-activated NF-kB-based inflammation in tumour cells: The role of Ku protein

The strong connection between cancer-related inflammation and tumour development with pattern – recognizing receptors (PRRs) activation results in identification of new target molecules that could lead to improved cancer diagnosis and treatment. TLR3, RIG1 and MDA5 synthetic ligand poly(I:C) was shown to trigger apoptosis in cancer cells. However, TLR3 signaling also includes NF-kB transcription factor which has emerged as endogenous tumour promoter via stimulation of pro-inflammatory tumour microenvironment, enhancement of angiogenesis, tumour cell proliferation and metastasis. Thus down regulation of NF-kB-mediated effects after TLR3 activation is needed for implementation of TLR3 ligand-based therapy into clinical trials. We have earlier demonstrated that DNA and sequence specific ODNs inhibit poly(I:C)-induced production of pro-inflammatory cytokines in human primary fibroblasts and endothelial cells (Cherepanova et al., Immunobiology, 2013). Using these specific ODNs and affinity modification/isolation approach combined with subsequent MALDI-TOF the main cellular targets for these ODNs were identified as Ku protein – heterodimer of KU70 and KU80 (Cherepanova et al., Exp. Opin. Biol. Ther., 2012). The goals of this study are to reveal whether the ODNs target poly(I:C)-induced activation in tumour cells: cervical carcinoma (Hela) and epidermoid carcinoma (A431) and to confirm Ku protein involvement in the observed effect. Poly(I:C) was shown to activate IL-6 production in Hela and A431 cells, and specific ODNs inhibit this activation with comparable efficacy as in primary cells. ODNs decrease both IL-6 concentration and mRNA expression rate in poly(I:C)-activated cells, indicating that NF-kB activation is also impaired. To reveal whether Ku protein is involved in the inhibiting effect of ODNs, Ku70 level in Hela cells was reduced by siRNA, which were shown to inhibit the expression of Ku70 by up to 70%. Inhibiting efficacy of ODN is almost twice lower in Ku70 knock-down cells as compared to control siRNA, indicating that ODN binding to Ku protein is important for the inhibition. Whether Ku is directly involved in nucleic acid pattern recognition or have auxiliary functions, mediating ODN localization or poly(I:C)-proteins interactions, remains to be investigated. Nevertheless, efficient NF-kB pathway targeting by specific ODNs may improve dsRNA-based cancer therapy.

Involvement of γδ T cells and scavenger receptors in the immunopathogenesis of Pemphigus Vulgaris (BA11P.129)

Abstract Pemphigus Vulgaris (PV) is an auto-immune blistering disease mostly due to formation of auto-antibodies against desmoglein3 (Dsg3) and aberrant T cell function. γδ T cells play significant role in the immune surveillance at the epithelial surface and modulate other immune cells. Scavenger receptors (CD36 and CD163) are Pattern recognizing receptor (PRR) molecules and key players in several inflammatory diseases. γδ T cells and its signature molecular markers has found to be involved in several autoimmune diseases. However the role of γδ T cells and associated Scavenger receptor molecules are not characterized in the Immunopathogenesis of PV. In our study we observed significantly higher frequency of γδ T cells (6.7% vs. 3.8%) and IL17 producing γδ T cells in circulation of PV patients (n=25) vs. controls (n=15) respectively along with Th1 polarization (p<0.010) suggesting the potential role of γδ T cells and IL17 secreting γδ T cells in the pathogenesis of PV. Cytotoxic activity of γδ T cells from PV patients (26±11%) was observed to be higher as compare to control (12±7%). The molecular expression (mRNA) of scavenger receptors CD36 and CD163 were elevated in patients as compared to controls indicating the possible involvement of these PRR along with γδ T cells. These findings may help to understand the pathogenesis of PV and find better approach for novel therapeutics.

TLR3 Plays Significant Roles against HBV-Associated HCC.

Toll-like receptor 3 (TLR3) is a pattern-recognizing receptor that is involved in immune signaling and plays a crucial role in survival by being able to recognize various viral components including double-stranded RNA (dsRNA). The role of TLR3 in hepatocellular carcinoma (HCC) with hepatitis B virus (HBV) infections is not well understood. To investigate the ability of TLR3 in regulating HBV replication in HCC, 80 cases of human HCC were collected and their tissue microarray was made. In HCC cells, the expression and location of TLR3, hepatitis-associated virus, and interstitial immunoreactive cells were assayed with immunohistochemical staining. The apoptosis of tumor cells was also detected by TUNEL stain. Correlations between TLR3 expression and HBV infection, interstitial immunoreactive cells, and cells apoptosis in HCC were investigated. In addition, we explored whether TLR3 agonist dsRNA can inhibit HepG2.2.15 cells secreting HBV. We found that the cytoplasmic expression of TLR3 in HCC is positively related to HBsAg infection and HCC with cirrhosis and promotes interstitial immunoreactive cells infiltration and cancer cells apoptosis. In HepG2.2.15 cells, dsRNA inhibited the secretion of HBV and induced apoptosis. These results indicate that TLR3 signaling activity may be involved in immune responses against HBV in HCC.

Different Toll-Like Receptor Expression Patterns in Progression toward Cancer.

Pattern recognition forms the basis ofthe innate immune system, allowing it tomaintain systemic homeostasis by reject-ing harmful molecular structures. Immunecells as well as epithelial cells located nearthe host–environment boundary, expresspattern-recognizing receptors, the activa-tion of which launches cascades lead-ing to immune response and apopto-sis (1,2). Human toll-like receptors(TLRs) 1–10, a family of trans-membranereceptor proteins comprising one typeof pattern-recognizing receptors, partic-ipate in these immunological processes.In healthy immune cells and epithelialcells, TLR 1, 2, 4, 5, 6, and 10 are usu-ally expressed on the cell surface, whereasTLR 3, 7, 8, and 9 are mainly expressedon the surfaces of endosomes, lysosomes,and endoplasmic reticulum (3). Microbialmolecular structures activating pattern-recognizing receptors are called pathogen-associated molecular patterns (PAMPs)(1,2). Besides exogenous structures, alsoendogenous molecules, released, for exam-ple, from inflamed or damaged tissues, canactivate pattern-recognizing receptors andare called danger- or damage-associatedmolecular patterns (DAMPs) (4, 5). Lig-ands binding to TLR lead to the activa-tion of several intracellular signaling path-ways, activating, for instance, nuclear tran-scription factors that initiate host defensefunctions via secretion of co-stimulatoryfactors and cytokines (6). TLR activationleads also to regulation of gene expression,cell proliferation, differentiation, mitosis,cell-cycle regulation, and apoptosis (7).

Vaccine Adjuvants in Fish Vaccines Make a Difference: Comparing Three Adjuvants (Montanide ISA763A Oil, CpG/Poly I:C Combo and VHSV Glycoprotein) Alone or in Combination Formulated with an Inactivated Whole Salmonid Alphavirus Antigen

Most commercial vaccines offered to the aquaculture industry include inactivated antigens (Ag) formulated in oil adjuvants. Safety concerns are related to the use of oil adjuvants in multivalent vaccines for fish, since adverse side effects (e.g., adhesions) can appear. Therefore, there is a request for vaccine formulations for which protection will be maintained or improved, while the risk of side effects is reduced. Here, by using an inactivated salmonid alphavirus (SAV) as the test Ag, the combined use of two Toll-like receptor (TLR) ligand adjuvants, CpG oligonucleotides (ODNs) and poly I:C, as well as a genetic adjuvant consisting of a DNA plasmid vector expressing the viral haemorrhagic septicaemia virus (VHSV) glycoprotein (G) was explored. VHSV-G DNA vaccine was intramuscularly injected in combination with intraperitoneal injection of either SAV Ag alone or combined with the oil adjuvant, Montanide ISA763, or the CpG/polyI:C combo. Adjuvant formulations were evaluated for their ability to boost immune responses and induce protection against SAV in Atlantic salmon, following cohabitation challenge. It was observed that CpG/polyI:C-based formulations generated the highest neutralizing antibody titres (nAbs) before challenge, which endured post challenge. nAb responses for VHSV G-DNA- and oil-adjuvanted formulations were marginal compared to the CpG/poly I:C treatment. Interestingly, heat-inactivated sera showed reduced nAb titres compared to their non-heated counterparts, which suggests a role of complement-mediated neutralization against SAV. Consistently elevated levels of innate antiviral immune genes in the CpG/polyI:C injected groups suggested a role of IFN-mediated responses. Co-delivery of the VHSV-G DNA construct with either CpG/polyI:C or oil-adjuvanted SAV vaccine generated higher CD4 responses in head kidney at 48 h compared to injection of this vector or SAV Ag alone. The results demonstrate that a combination of pattern recognizing receptor (PRR) ligands, such as CpG/polyI:C, increases both adaptive and innate responses and represents a promising adjuvant strategy for enhancing the protection of future viral vaccines.