Interplay between NOD1 and TLR4 Receptors in Macrophages: Nonsynergistic Activation of Signaling Pathways Results in Synergistic Induction of Proinflammatory Gene Expression.

Interactions between pattern-recognition receptors shape innate immune responses to pathogens. NOD1 and TLR4 are synergistically interacting receptors playing a pivotal role in the recognition of Gram-negative bacteria. However, mechanisms of their cooperation are poorly understood. It is unclear whether synergy is produced at the level of signaling pathways downstream of NOD1 and TLR4 or at more distal levels such as gene transcription. We analyzed sequential stages of human macrophage activation by a combination of NOD1 and TLR4 agonists (N-acetyl-d-muramyl-l-alanyl-d-isoglutamyl-meso-diaminopimelic acid [M-triDAP] and LPS, respectively). We show that events preceding or not requiring activation of transcription, such as activation of signaling kinases, rapid boost of glycolysis, and most importantly, nuclear translocation of NF-κB, are regulated nonsynergistically. However, at the output of the nucleus, the combination of M-triDAP and LPS synergistically induces expression of a subset of M-triDAP- and LPS-inducible genes, particularly those encoding proinflammatory cytokines (TNF, IL1B, IL6, IL12B, and IL23A). This synergistic response develops between 1 and 4 h of agonist treatment and requires continuous signaling through NOD1. The synergistically regulated genes have a lower basal expression and higher inducibility at 4 h than those regulated nonsynergistically. Both gene subsets include NF-κB-inducible genes. Therefore, activation of the NF-κB pathway does not explain synergistic gene induction, implying involvement of other transcription factors. Inhibition of IKKß or p38 MAPK lowers agonist-induced TNF mRNA expression but does not abolish synergy. Thus, nonsynergistic activation of NOD1- and TLR4-dependent signaling pathways results in the synergistic induction of a proinflammatory transcriptional program.

Glycolytic reprogramming of macrophages activated by NOD1 and TLR4 agonists: No association with proinflammatory cytokine production in normoxia.

Upon activation with pathogen-associated molecular patterns, metabolism of macrophages and dendritic cells is shifted from oxidative phosphorylation to aerobic glycolysis, which is considered important for proinflammatory cytokine production. Fragments of bacterial peptidoglycan (muramyl peptides) activate innate immune cells through nucleotide-binding oligomerization domain (NOD) 1 and/or NOD2 receptors. Here, we show that NOD1 and NOD2 agonists induce early glycolytic reprogramming of human monocyte-derived macrophages (MDM), which is similar to that induced by the Toll-like receptor 4 (TLR4) agonist lipopolysaccharide. This glycolytic reprogramming depends on Akt kinases, independent of mTOR complex 1 and is efficiently inhibited by 2-deoxy-d-glucose (2-DG) or by glucose starvation. 2-DG inhibits proinflammatory cytokine production by MDM and monocyte-derived dendritic cells activated by NOD1 or TLR4 agonists, except for tumor necrosis factor production by MDM, which is inhibited initially, but augmented 4 h after addition of agonists and later. However, 2-DG exerts these effects by inducing unfolded protein response rather than by inhibiting glycolysis. By contrast, glucose starvation does not cause unfolded protein response and, in normoxic conditions, only marginally affects proinflammatory cytokine production triggered through NOD1 or TLR4. In hypoxia mimicked by treating MDM with oligomycin (a mitochondrial ATP synthase inhibitor), both 2-DG and glucose starvation strongly suppress tumor necrosis factor and interleukin-6 production and compromise cell viability. In summary, the requirement of glycolytic reprogramming for proinflammatory cytokine production in normoxia is not obvious, and effects of 2-DG on cytokine responses should be interpreted cautiously. In hypoxia, however, glycolysis becomes critical for cytokine production and cell survival.

Synergistic interactions between NOD receptors and TLRs: Mechanisms and clinical implications.

Interactions between pattern recognition receptors (PRRs) shape innate immune responses to particular classes of pathogens. Here, we review interactions between TLRs and nucleotide-binding oligomerization domain 1 and 2 (NOD1 and NOD2) receptors, two major groups of PRRs involved in innate recognition of bacteria. Most of experimental data both in vitro and in vivo suggest that NODs and TLRs synergize with each other at inducing the production of cytokines and antimicrobial peptides. Molecular mechanisms of this synergy remain poorly understood, although several scenarios can be proposed: (i) direct interactions of signaling pathways downstream of NODs and TLRs; (ii) mutual transcriptional regulation of unique components of NOD-dependent and TLR-dependent signaling pathways; and (iii) interactions at the post-transcriptional level. Potential practical implications of NOD-TLR synergy are dual. In sepsis, where synergistic effects probably contribute to excessive proinflammatory cytokine production, blockade of NOD1, and/or NOD2 in addition to TLR4 blockade may be required to achieve therapeutic benefit. On the other hand, synergistic combinations of relatively small doses of NOD and TLR agonists administered before infection could be used to boost innate resistance against bacterial pathogens.

A cell-based test system for the assessment of pharmacokinetics of NOD1 and NOD2 receptor agonists.

Agonists of nucleotide oligomerization domain (NOD) 1 and NOD2 receptors represent a promising class of immunostimulants and immunological adjuvants. Here, we describe a cell-based test system to assess their pharmacokinetics. In this system, NOD1 and NOD2 agonist concentrations in sera are determined using a reporter cell line, 293Luc, which contains an NF-κB-inducible luciferase reporter construct and naturally expresses NOD1 and NOD2. The 293Luc cells dose-dependently respond to different NOD1 and NOD2 agonists in the nanomolar to low-micromolar concentration range. To verify that the NF-κB-inducing activity of serum samples is due to the administered agonist and not to secondarily induced endogenous molecules, a 293Luc-derived NOD1/NOD2 double-knockout clone is used. Within-run and between-run precisions of the system are <15% and <20%, respectively. Applicability of the novel assay is illustrated by studying pharmacokinetics of two specific NOD2 agonists (N­acetyl­d­glucosaminyl­N­acetyl­d­muramyl­l­alanyl­d­isoglutamine and N­glycolyl­d­muramyl­l­alanyl­d­isoglutamine) and a specific NOD1 agonist (N­acetyl­d­glucosaminyl­N­acetyl­d­sorbitolamine­d­lactoyl­l­alanyl­d­isoglutamyl­meso­diaminopimelic acid). In summary, the test system described here can potentially be used to assess pharmacokinetics of NOD1 and NOD2 agonists in different animal species.

NOD1 and NOD2: Molecular targets in prevention and treatment of infectious diseases.

Nucleotide-binding oligomerization domain (NOD) 1 and NOD2 are pattern-recognition receptors responsible for sensing fragments of bacterial peptidoglycan known as muropeptides. Stimulation of innate immunity by systemic or local administration of NOD1 and NOD2 agonists is an attractive means to prevent and treat infectious diseases. In this review, we discuss novel data concerning structural features of selective and non-selective (dual) NOD1 and NOD2 agonists, main signaling pathways and biological effects induced by NOD1 and NOD2 stimulation, including induction of pro-inflammatory cytokines, type I interferons and antimicrobial peptides, induction of autophagy, alterations of metabolism. We also discuss interactions between NOD1/NOD2 and Toll-like receptor agonists in terms of synergy and cross-tolerance. Finally, we review available animal data on the role of NOD1 and NOD2 in protection against infections, and discuss how these data could be applied in human infectious diseases.

The Role of the p38-MNK-eIF4E Signaling Axis in TNF Production Downstream of the NOD1 Receptor.

Activation of nucleotide-binding oligomerization domain (NOD) 1 and NOD2 by muropeptides triggers a complex transcriptional program in innate immune cells. However, little is known about posttranscriptional regulation of NOD1- and NOD2-dependent responses. When stimulated with a prototypic NOD1 agonist, N-acetylglucosaminyl-N-acetylmuramyl-l-alanyl-d-isoglutamyl-meso-diaminopimelic acid (GM-triDAP), human monocyte-derived macrophages (MDM) produced an order of magnitude more TNF, IL-6, and pro-IL-1ß than did monocyte-derived dendritic cells (MDDC), despite similar NOD1 expression, similar cytokine mRNA kinetics, and comparable responses to LPS. TNF production by GM-triDAP-activated MDM was independent of autocrine IL-1. However, GM-triDAP-activated MDM translated TNF mRNA more efficiently than did MDDC. As an underlying mechanism, NOD1 triggering in MDM caused a more potent and long-lasting activation of the signaling axis involving p38 MAPK, MAPK-interacting kinase (MNK), and eukaryotic translation initiation factor 4E, which is a critical regulator of translation. Furthermore, MNK controlled TNF mRNA abundance in MDDC and MDM upon NOD1 triggering. NOD1-dependent responses were more sensitive to MNK inhibition than were TLR4-dependent responses. These results demonstrate the importance of the p38-MNK-eukaryotic translation initiation factor 4E axis in TNF production downstream of NOD1.

Effects of the antioxidants Trolox, Tiron and Tempol on neutrophil extracellular trap formation.

Neutrophils can entrap and kill pathogens by releasing of neutrophil extracellular traps (NETs), in addition to their routine functions such as phagocytosis and degranulation. NETs consist of a DNA backbone supplemented by multiple bactericidal proteins from the nucleus, the cytoplasm and the granules. Neutrophils release NETs after their activation by a number of physiological and pharmacological stimuli. In addition to the antimicrobial function, NETs are involved in the pathogenesis of various autoimmune and inflammatory diseases. Since NET formation predominantly depends on the generation of reactive oxygen species (ROS), all substances that are capable of scavenging ROS or inhibiting the enzymes responsible for their synthesis should prevent ROS-associated NET release. The aim of this study was to test substances with an antioxidant activity, such as Trolox, Tiron, and Tempol, for their capacity to inhibit NET formation by primary human neutrophils in vitro. We revealed for the first time an inhibitory effect of Trolox on ROS-dependent NET release. We also established a suppressive effect of Tempol on NET formation that manifested itself in a wide range of concentrations. In this study, no inhibitory influence of Tiron on NET release was revealed. All tested substances exerted a significant dose-dependent antioxidative effect on ROS generation induced by phorbol 12-myristate 13-acetate (PMA). We suggest that the antioxidants Trolox and Tempol should be recommended for treating autoimmune and inflammatory diseases that implicate ROS-dependent NET release.

Complexes of DNA with the Antimicrobial Peptide LL37 Augment NK Cell Functions by Inducing Type I Interferon Production from Circulating Monocytes and Plasmacytoid Predendritic Cells.

The cationic antimicrobial peptide, LL37, forms electrostatic complexes with DNA (LL37-DNA), which are potent activators of circulating plasmacytoid predendritic cells (ppDCs) and monocytes. However, the effects of LL37-DNA on other immune cell types, such as NK cells, are poorly characterized. In this study, we show that complexes of human genomic DNA (hgDNA) or synthetic double-stranded oligodeoxynucleotides with LL37 strongly enhance natural cytotoxicity of human peripheral blood mononuclear cells (PBMCs) upon an overnight culture, whereas hgDNA alone has no effect, and LL37 alone is moderately active. LL37-DNA complexes potentiate degranulation of, and interferon (IFN)-γ production by, NK cells upon subsequent encounter of K562 target cells. The complexes do not influence percentages of NK cells among PBMCs or the expression of cytotoxic proteins by NK cells. Using neutralizing anticytokine antibodies and immunomagnetic depletion of different subpopulations of PBMCs, we found that the effect of LL37-DNA on NK cells is indirect and mediated by type I IFNs produced by monocytes and, to a lesser extent, by ppDCs. We discuss possible roles of LL37-DNA complexes in the regulation of NK cell functions and in the treatment of cancer.

Reduced degranulation of NK cells in patients with frequently recurring herpes.

NK cells lyse virus-infected cells by degranulation; however, alterations in NK cell degranulation in persistent viral infections have not been directly studied. Earlier reports have documented a decrease in NK activity in patients with frequently recurring herpes (FRH). We corroborate these findings by showing that the degranulation responses of blood NK cells from patients with FRH, both during relapse and during remission, are significantly lower than those in healthy donors. The impaired degranulation was probably not caused by defective target cell recognition, since it was observed upon stimulation both with K562 cells and with a receptor-independent stimulus (phorbol 12-myristate 13-acetate plus ionomycin). We also show that the intracellular expression of perforin and CD107a by NK cells from patients with FRH is not different from that in healthy donors, thus excluding that the low NK cell degranulation in FRH is caused by a smaller size of the lytic granule compartment. We confirm previous reports on lowered NK activity in FRH patients and show that NK activity is significantly impaired only during remission, but not relapse; the causes for the discrepancy between the low degranulation and "normal" NK cell activity during relapse are discussed. In all, these data point at the deficit of NK cell degranulation in FRH. Whether this is a predisposing factor or a consequence of herpes simplex virus infection requires further investigation.

Muropeptides trigger distinct activation profiles in macrophages and dendritic cells.

Bacterial peptidoglycan and its muropeptide derivatives potently activate mammalian innate immune system and are promising immunomodulators and vaccine adjuvants. However, their effects on human antigen-presenting cells, such as dendritic cells (DCs) and Mphi, are not fully understood. Lysozyme treatment of PG from Salmonella typhi yielded three muropeptides, GlcNAc-MurNAc-L-Ala-D-isoGlu-meso-DAP (GM-3P), GlcNAc-MurNAc-L-Ala-D-isoGlu-meso-DAP-D-Ala (GM-4P), and a dimer (GM-4P)(2), in which two GM-4P monomers are linked through their peptidic moieties. All three muropeptides induced TNF-alpha and IL-6 production by Mphi (GM-3P>GM-4P>>(GM-4P)(2)), but failed to trigger TNF-alpha, IL-6 and IL-12p70 production by immature DCs. At the same time, muropeptide-stimulated DCs abundantly produced inflammatory chemokines IL-8, MIP-1 alpha and MIP-1 beta, as well as displayed signs of phenotypic and functional maturation. Thus, muropeptide-dependent pro-inflammatory cytokine production is repressed in DCs. While this defect may be partly compensated in vivo by muropeptide-activated Mphi, neither Mphi nor DCs produce Th1- or Th17-polarizing cytokines upon muropeptide stimulation, which may contribute to the preferential induction of Th2 responses by muropeptides and should be taken into account when designing muropeptide-based immunomodulators and adjuvants.

Study of interaction between the polyoxidonium immunomodulator and the human immune system cells.

Polyoxidonium (PO) is a high-molecular weight physiologically active compound with pronounced immunomodulating activity, an N-oxidized polyethylene-piperazine derivative. The aim of our work was to study cellular and molecular mechanisms of the action of PO on the human peripheral blood leukocytes. By means of flow cytometry it was established that the binding of fluorescein-isothiocyanate-labeled PO (FITC-labeled PO) occurs more rapidly with monocytes and neutrophils than with lymphocytes (7- to 8-fold weaker as compared with monocytes). Using colloidal gold-labeled PO and electron microscopy it was shown with that the preparation penetrates into leukocytes by endocytosis. PO is localized in endoplasmic vesicles of cellular cytosol. Analysis of one of the crucial signal transducer, the intracellular Ca(2+), performed with the Fluo-3 fluorescent dye, showed that PO does not induce Ca(2+) mobilization from the intracellular calcium stores and influx of extracellular Ca(2+). The study of the intracellular hydrogen peroxide (H(2)O(2)) production with the 2',7'-dichlorfluorescein indicator demonstrated that PO significantly increases the level of intracellular H(2)O(2) in monocytes and neutrophils, however, this increase is much less as compared with phorbol myristate acetate stimulation. The analysis of immunomodulating effect produced by PO proved its stimulating activity on some cytokines production in vitro, e.g. interleukin 1beta (IL-1beta), tumor necrosis factor (TNF)-alpha and IL-6. A dose-dependent increase in the intracellular killing by blood phagocytes was established under the action of PO.

Effect of polyoxidonium on the phagocytic activity of human peripheral blood leukocytes.

The effect of polyoxidonium on the functional activity of human peripheral blood phagocytes was studied in vitro. Polyoxidonium is an N-oxidized polyethylene-piperazine derivative, a water-soluble high-molecular synthetic immunomodulator. It was established that a one-hour incubation of leukocytes with polyoxidonium increases the ability of leukocytes to kill the ingested Staphylococcus aureus in a dose-dependent manner. This increase was observed in leukocytes obtained from healthy individuals and from patients with chronic granulomatous disease. The study of phagocyte spontaneous and stimulated chemiluminescence showed a significant decrease in the quantity of chemiluminescent impulses in the extracellular space in the presence of polyoxidonium both in luminol- and lucigenin-dependent chemiluminescence assays. Polyoxidonium proved to have an antioxidant activity at all doses tested (100, 250, and 500 &mgr;g/ml). Evaluation of the intracellular hydrogen peroxide (H(2)O(2)) level with a fluorescent indicator dichlorofluorescein showed that incubation with polyoxidonium leads to a higher luminescence intensity of dichlorofluorescein, thus indicating an increase in the intracellular H(2)O(2) level. This increase was not as substantial as in the case of stimulation with phorbol myristate acetate. When polyoxidonium was used at a dose of 500 &mgr;g/ml, the difference with the control was significant for neutrophils and monocytes. Polyoxidonium can be used as adjuvant in combined treatment of acute and chronic infections of any etiology, and in the treatment of chronic granulomatous disease and secondary immunodeficiences along with etiotropic drugs.

Current Features of Secondary (Acquired) Types of Immune Deficiency.

Secondary (acquired) types of immune deficiencies (SID) take a leading place in practice of modern clinical immunology. The causes for SID development are extremely variable. Special attention is concerned with accumulating facts about target action of microorganisms, and first of all viruses, on certain processes in immune system. Damageable action of HIV-1 on cell elements expressing CD4 molecules is known in most precise manner. It is noteworthy that the search of real molecular defects, induced by microorganisms in immune system is required. It is not to be ruled out that the increased level of apoptosis of immune system cells is one of the causes of SID. The basis of it is disbalance between positive and negative activation processes of immunocompetent cells. Multiple factors may serve as apoptogens, including drugs (glucocorticoids etc.), xenobiotics, physical factors (radiation) and many others. In practice of clinical laboratories a certain spectrum of immunological investigations is recommended that allows to diagnose the degree of immunopathology. At present, in clinical practice these methods are focused around flow cytometry (immunophenotyping), immunodiffusion and immunoenzyme tests (determination of immunoglobulins, cytokines, other soluble components of immune system), tests of estimation of immunocompetent cell activation, proliferation and differentiation. As a prospective, some methods, based on identification of molecular defects in cells and soluble factors of immune system, may be taken into consideration.

Some Peculiarities of Humoral Antibacterial Immunity in HIV-infected and AIDS Patients.

The investigation was outlined to study antibodies against some antigens of extracellular microbes associated with inflammation in broncho-pulmonary system and accessory nasal sinus - Staphylococcus aureus, Streptococcus pneumoniae, Branhamella catarrhalis - in individuals (18 patients) with different stages of HIV-infection. The level of antibodies was measured by ELISA and their Ab affinity was assessed by sodium thiocyanate-induced alteration of antibody-antigen interaction. To determine interrelations between antibody production and CD4(+) T lymphocyte number flow cytometry was employed. At the early stages of HIV-infection the levels of antibodies against Streptococcus pneumoniae and GMGM decreased, in comparison with HIV-negative donors. During HIV-infection course levels of antibodies against Staphylococcus aureus peptidoglycan, its antigen determinants and Streptococcus pneumoniae somatic antigen increased. Time affinity of antibodies against these antigens decreased. At all stages of HIV-infection and at all forms of its complications, we observed an increase of titer of antibodies to GMDP, antigenic determinant of peptidoglycan, which carried immunostimulating and adjuvant activities. HIV patients with CD4(+) T lymphocyte number <200 cells/&mgr;l displayed higher level of antibodies to bacterial antigens than that in patients with CD4(+) T lymphocyte number 200-400 per ml. The development of humoral immune response against some of extracellular bacteria is characterized, on the one hand, by their increased levels, and on the other hand, decreased affinity.

Competitive Specificity Analysis of Natural Antibodies against Epitope of Bacterial Cell Wall Peptidoglycoan: Glucosaminylmuramyl Dipeptide Carrying Adjuvant Activity.

The natural antibodies against glucosaminylmuramyl dipeptide (GMDP), the epitope of peptidoglycan of bacterial cell wall, isolated from human serum by thermal extraction possess a capability to cross-react with determinant of glycan chain - tetrasaccharide consisting of N-acetylglucosamine and N-acetylmuramic acid. The intensity of interaction of natural anti-GMDP-antibodies with specific ligand is significantly higher than with tetrasaccharide. The natural antibodies against tetrasaccharide carry properties of heteroclitic antibodies, i.e. the intensity of their interaction with heterologous ligand, GMDP, is significantly higher than with homologous one, tetrasaccharide GMGM. GMDP is supposed to be specific antigenic peptidoglycan determinant against which the antibodies reacting with various intensity to homologous (GMDP) and relative (tetrasaccharide) hapten are formed in the process of natural immunization.

Affinity of Serum Natural Antibodies against Peptidoglycan Bacterial Cell Component with Adjuvant Activity.

We studied affinity of natural antibodies of human serum against glucosaminylmuramyl dipeptide (GMDP), the epitope peptidoglycan bacterial cell wall component which carries adjuvant activity. Antibodies against GMDP were isolated from the blood sera of healthy donors using thermal extraction of antibodies from specific ligand on plastic. Determination of the dissociation constant (K(d)) showed equal K(d) in the serum and affinity-purified anti-GMDP-antibodies, i.e. extraction by this method led to the isolation of all subpopulations of antibodies in spite of their affinity. K(d) of serum, affinity-purified and monoclonal anti-GMDP-antibodies proved of low value - 10(-6) M, and according to this index anti-GMDP-antibodies may be classified between anti-protein and anti-carbohydrate antibodies.