Activation of Innate Immunity by Therapeutic Nucleic Acids

Nucleic acid-based therapeutics have gained increased attention during recent decades because of their wide range of application prospects. Immunostimulatory nucleic acids represent a promising class of potential drugs for the treatment of tumoral and viral diseases due to their low toxicity and stimulation of the body’s own innate immunity by acting on the natural mechanisms of its activation. The repertoire of nucleic acids that directly interact with the components of the immune system is expanding with the improvement of both analytical methods and methods for the synthesis of nucleic acids and their derivatives. Despite the obvious progress in this area, the problem of delivering therapeutic acids to target cells as well as the unresolved issue of achieving a specific therapeutic effect based on activating the mechanism of interferon and anti-inflammatory cytokine synthesis. Minimizing the undesirable effects of excessive secretion of inflammatory cytokines remains an unsolved task. This review examines recent data on the types of immunostimulatory nucleic acids, the receptors interacting with them, and the mechanisms of immunity activation under the action of these molecules. Finally, data on immunostimulatory nucleic acids in ongoing and completed clinical trials will be summarized.

Toll-like receptor 4-mediated cytokine synthesis and post-stroke depressive symptoms

Altered cytokine synthesis thought to contribute to the pathophysiology of post-stroke depression (PSD). Toll-like receptor 4 (TLR4) is a master regulator of innate immunity. The aim of this study was to explore the putative association between TLR4-mediated cytokine synthesis and subsequent symptoms of PSD. In total, 262 patients with ischemic stroke and without a history of PSD were included. Depressive symptoms were assessed using the Patient Health Questionnaire-9 in 170 patients on Day 8 and in 146 at 3 months after stroke. Blood samples taken on Day 3 after stroke were stimulated ex vivo with lipopolysaccharide (LPS). Ex vivo synthesized cytokines (TNFα, IP-10, IL-1β, IL-6, IL-8, IL-10, and IL-12p70) and circulating cytokines (TNFα, IL-6, sIL-6R, and IL-1ra) were measured using the enzyme-linked immunoassay or cytometric method. RNA sequencing was used to determine the gene expression profile of LPS-induced cytokines and chemokines. LPS-induced cytokine synthesis and the gene expression of TLR4-dependent cytokines and chemokines did not differ between patients with and without greater depressive symptoms. The plasma level of IL-6, but not TNFα, sIL-6R, and IL-1ra, was higher in patients who developed depressive symptoms at 3 months after stroke (median: 4.7 vs 3.4 pg/mL, P = 0.06). Plasma IL-6 predicted the severity of depressive symptoms at 3 months after stroke (β = 0.42, P = 0.03). In conclusion, TLR4-dependent cytokine synthesis was not associated with greater post-stroke depressive symptoms in this study. Circulating IL-6 might be associated with depressive symptoms occurring at 3 months after stroke.

Anandamide Influences Interleukin-1β Synthesis and IL-1 System Gene Expressions in the Ovine Hypothalamus during Endo-Toxin-Induced Inflammation

This study evaluated the effect of anandamide (AEA) on interleukin (IL)-1β synthesis and gene expression of IL-1β, its type I (IL-1R1) and II (IL-1R2) receptors, and IL-1 receptor antagonist (IL-1RN) in the hypothalamic structures, involved in the central control of reproduction, during inflammation. Animals were intravenously (i.v.) injected with bacterial endotoxin-lipopolysaccharide (LPS) (400 ng/kg) or saline, and two hours after LPS administration., a third group received i.v. injection of AEA (10 μg/kg). Ewes were euthanized one hour later. AEA injection (p < 0.05) suppressed LPS-induced expression of IL-1β protein in the hypothalamus. The gene expression of IL-1β, IL-1RN, and IL-1R2 in the hypothalamic structures was higher (p < 0.05) in animals treated with both LPS and AEA in comparison to other experimental groups. AEA administration did not influence LPS-stimulated IL-1R1 gene expression. Our study shows that AEA suppressed IL-1β synthesis in the hypothalamus, likely affecting posttranscriptional levels of this cytokine synthesis. However, anti-inflammatory effect of AEA might also result from its stimulating action on IL-1RN and IL-1R2 gene expression. These results indicate the potential of endocannabinoids and/or their metabolites in the inhibition of inflammatory process at the level of central nervous system, and therefore their usefulness in the therapy of inflammation-induced neuroendocrine disorders.

The glutaminase (CgGLS-1) mediates anti-bacterial immunity by prompting cytokine synthesis and hemocyte apoptosis in Pacific oyster Crassostrea gigas

Glutaminase, an amidohydrolase enzyme that hydrolyzes glutamine to glutamate, plays crucial roles in various immunomodulatory processes such as cell apoptosis, proliferation, migration, and secretion of cytokines. In the present study, a glutaminase homologue (designated as CgGLS-1) was identified from Pacific oyster Crassostrea gigas, whose open reading frame was of 1836 bp. CgGLS-1 exhibited high sequence identity with vertebrate kidney-type GLS, and closely clustered with their homologues from mollusc C. virginica. The enzyme activity of recombinant CgGLS-1 protein (rCgGLS-1) was estimated to be 1.705 U/mg. CgGLS-1 mRNA was constitutively expressed in all the tested tissues of oysters, with the highest expression level in hemocytes. CgGLS-1 mRNA expression in hemocytes was significantly up-regulated and peaked at 6 h (2.07-fold, p < 0.01) after lipopolysaccharide (LPS) stimulation. The CgGLS-1 protein was mainly distributed in the cytoplasm with a significant co-location with mitochondria in oyster hemocytes. The content of Glu in the oyster serum was significantly decreased after the inhibition of CgGLS-1 using specific inhibitor Bis-2- [5-(phenyl acetamido)-1,3,4-thiadiazol-2-yl] ethyl sulfide (BPTES), and the expression levels of CgmGluR6, CgAP-1, cytokines CgIL17-5 and CgTNF-1 were significantly decreased after BPTES and LPS stimulation. The transcripts of CgCaspase3 as well as the apoptosis index of hemocytes were also decreased. These results collectively suggest that CgGLS-1 is the enzyme to synthesize Glu in oyster, which can modulate anti-bacterial immunity by regulating the secretion of pro-inflammatory cytokines CgIL17-5 and CgTNF-1, as well as hemocyte apoptosis.

TLR4-associated IRF-7 and NFĸB signaling acts as a molecular link between androgen and metformin activities and cytokine synthesis in the PCOS endometrium

Background Low-grade chronic inflammation is commonly seen in polycystic ovary syndrome (PCOS) patients with elevated levels of inflammatory cytokines in the endometrium. However, our understanding of the mechanisms underlying cytokine synthesis and increased endometrial inflammation in PCOS patients remains limited. Methods Endometrial biopsy samples were collected from non-PCOS (n = 17) and PCOS (n = 22) patients either during the proliferative phase of the menstrual cycle or with hyperplasia. Endometrial explants were prepared from PCOS patients and subjected to pharmacological manipulation in vitro. The expression and localization of TLR2/4, key elements of innate immune signal transduction and NFκB signaling pathways, and multiple cytokines were comprehensively evaluated by Western blotting, immunohistochemistry, and immunofluorescence in endometrial tissues. Results We demonstrated the distribution of protein expression and localization associated with the significantly increased androgen receptor, TLR2, and TLR4-mediated activation of IRF-7 and NFkB signaling, cytokine production, and endometrial inflammation in PCOS patients compared to non-PCOS patients with and without endometrial hyperplasia. In vitro experiments showed that 5α-dihydrotestosterone (DHT) enhanced androgen receptor, TLR4, IRF-7, and p-NFκB p65 protein expression along with increased IFNα and IFNɣ abundance. The effects of DHT on IRF-7, p-NFκB p65, and IFN abundance were abolished by flutamide, an anti-androgen. Although 17β-estradiol (E2) decreased p-IRF-7 expression with little effect on TLR-mediated IRF7 and NFκB signaling or on cytokine protein levels, exposure to metformin alone or in combination with E2 suppressed IRAK4, p-IRF-7, IRF-7, IKKα, p-NFκB p65, IFNɣ, and TNFα protein expression. Conclusion Cytokine synthesis and increased endometrial inflammation in PCOS patients is coupled to androgen-induced TLR4/IRF-7/NFkB signaling, which is be inhibited by metformin treatment.

MicroRNA regulatory mechanisms in atherosclerosis

Atherosclerosis is a chronic inflammatory disease of the arterial wall, characterized by a change in the intima of the arteries in the form of focal lipid deposits and the formation of a fibrous lid. Atherosclerosis is considered as the main cause of myocardial infarction, ischemic stroke and chronic lower limb ischemia. The pathogenesis of atherosclerosis is complex, and genetic mechanisms of atherosclerosis have not been fully investigated. The recent studies have shown that microRNAs (miRNAs) can play a role in the development of atherosclerosis. MiRNAs are short, non-coding RNA molecules 1822 nucleotides in length that suppress gene expression at the post-transcriptional level by binding to the 3ʹ-untranslated region of mRNA targets. MiRNAs are involved in virtually all biological processes, including cell proliferation, apoptosis, and cell differentiation. MiRNAs control aging and dysfunction of endothelial cells, proliferation and migration of vascular smooth muscle cells, as well as cytokine synthesis and polarization of macrophages. In this paper, we focus on how miRNAs can influence the pathogenesis of atherosclerosis.

IMMUNE RESPONS OF CYTOKINES IN DIFFERENT COURSE OF VIRAL ENCEPHALITIS IN CHILDREN

105 children aged 1-17 years with viral encephalitis (EF) were examined. Determined cytokines (TNF-α, IL-4, IL-10, IFN-α and IFN-γ) in serum and CSF. In acute course (n = 50) dominated (74%) Th1 type of immune response (IR) with a high average IFN-γ in the blood and CSF and IFN-γ / IL-4 = 8.3 and 11.9. With a prolonged flow (n = 25) - Th2 with indices IFN-γ / IL-4 = 0.75 and 0.79. In chronic (n = 30) - suppressive type of IR, in which the blood cytokines did not exceed the norm, and CSF were lower than in acute and prolonged course. Cytokines in acute and prolonged course of EF were higher in serum, and in chronic ~ 1.3-1.7 times higher in CSF than in blood, indicating their intrathecal synthesis in the long-term course of neu-roinfection. Types of IR and the intensity of cytokine synthesis were interrelated with the nature of the course of EF in children and the severity of infectious and cerebral symptoms.

LIPOPOLYSACCHARIDE-INDUCED IMMUNOLOGICAL STRESS AT EARLY STAGES OF PREGNANCY AFFECTS THE DEVELOPMENT OF GONADOTROPIN RELEASING-HORMONE (GNRH)-PRODUCING SYSTEM

The aim of the present work was to study the development of afferent bonds between GnRH- and monoaminergic neurons in rat fetuses and to identify possible targets affected by LPS-induced inflammation. The innervation was analyzed using retrograde tracing method with DiI dye. At ED17 and ED21 olfactory bulbs (the area of GnRH migration) are innervated with monoaminergic neurons of septum and in lateral hypothalamus. The GnRH- and monoaminergic neuron interaction zones are sensitive to LPS (E. coli) prenatal exposure, which induces pro-inflammatory cytokine synthesis. We suppose that the olfactory bulbs of fetal forebrain can be a possible area of cytokine influence on GnRH- and monoaminergic neuron interaction.