Formyl Peptide Receptors in Mice and Men: Similarities and Differences in Recognition of Conventional Ligands and Modulating Lipopeptides.

The pattern recognition formyl peptide receptors (FPRs) belong to the class of G-protein-coupled receptors (GPCRs), the largest group of cell surface receptors involved in a range of physiological processes and pathologies. The FPRs have regulatory function in the initiation as well as resolution of inflammatory reactions, making them highly interesting as targets for drug development. Recent research in the GPCR/FPR fields has uncovered novel receptor biology concepts, including biased signalling/functional selectivity, allosteric modulation, receptor reactivation and receptor cross-talk. When it comes to allosteric modulators, 'tailor-made' lipopeptides (pepducins and lipopeptoids) represent a novel concept of GPCR/FPR regulation. This MiniReview is focused on the basis for recognition of conventional ligands and immunomodulating lipopeptides, novel allosteric modulators for the FPRs, receptors that are highly expressed by both human and mouse neutrophils. The FPRs play key roles in host defence against microbial infections, tissue homeostasis and the initiation as well as resolution of inflammation but there are both similarities and differences in ligand recognition between mice and men. Thus, identification and functional characterization of activating and inhibiting ligands should provide insights into future design of FPR-based animal models of human diseases and development of therapeutics for treating inflammatory diseases.

2'Fluoro Modification Differentially Modulates the Ability of RNAs to Activate Pattern Recognition Receptors.

Although the use of RNAs has enormous therapeutic potential, these RNA-based therapies can trigger unwanted inflammatory responses by the activation of pattern recognition receptors (PRRs) and cause harmful side effects. In contrast, the immune activation by therapeutic RNAs can be advantageous for treating cancers. Thus, the immunogenicity of therapeutic RNAs should be deliberately controlled depending on the therapeutic applications of RNAs. In this study, we demonstrated that RNAs containing 2'fluoro (2'F) pyrimidines differentially controlled the activation of PRRs. The activity of RNAs that stimulate toll-like receptors 3 and 7 was abrogated by the incorporation of 2'F pyrimidine. By contrast, incorporation of 2'F pyrimidines enhanced the activity of retinoic acid-inducible gene 1-stimulating RNAs. Furthermore, we found that transfection with RNAs containing 2'F pyrimidine and 5' triphosphate (5'ppp) increased cell death and interferon-ß expression in human cancer cells compared with transfection with 2'hydroxyl 5'ppp RNAs, whereas RNAs containing 2'O-methyl pyrimidine and 5'ppp completely abolished the induction of cell death and cytokine expression in the cells. Our findings suggest that incorporation of 2'F and 2'O-methyl nucleosides is a facile approach to differentially control the ability of therapeutic RNAs to activate or limit immune and inflammatory responses depending on therapeutic applications.

Mosla scabra flavonoids ameliorate the influenza A virus-induced lung injury and water transport abnormality via the inhibition of PRR and AQP signaling pathways in mice.

ETHNOPHARMACROLOGICAL RELEVANCE: Mosla scabra (Thunb.) C.Y. Wu and H.W. Li has been used as a traditional medicinal herb for centuries in East Asian countries. It has antibacterial, antiviral, antioxidant, anti-inflammatory and immunomodulatory effects. In folk medicine, it is used as a remedy for the treatment of pulmonary diseases, such as fever, cold, cough, pulmonary edema and emphysema. AIM OF THE STUDY: This study was to investigate the protective mechanism of total flavonoids from M. scabra (MF) in influenza A virus (IAV)-infected mice. MATERIALS AND METHODS: The mice were infected with IAV and then were treated daily with MF for five days. At the end of the experiment, the levels of inflammatory-related cytokines (IFN-α, IL-6, TNF-α and IL-1ß) were determined by ELISA. Pathological changes of lung tissue were examined by H&E staining. The protein expressions of AQP5, p-p38, caspase-3 and NF-κB p65 were detected by western blot analysis while the gene expressions of key effectors in AQP5 and PRRs signaling pathways were detected by real-time Fluorescence Quantitative Polymerase Chain Reaction (RFQ-PCR) analysis. RESULTS: The results showed that treatment with MF at doses of 120-360mg/kg for five days to IAV-infected mice significantly attenuated IAV-induced pulmonary injury and decreased the serum levels of IL-6, TNF-α and IL-1ß, but increased IFN-α levels. MF treatment could up-regulate the mRNA expressions of TLR-7, RIG-1, TRAF6, Bcl-2, Bax, VIPR1, PKCα and AQP5 and down-regulate caspase-3 and NF-κB p65 protein expression. CONCLUSION: Treatment with MF could significantly alleviate IAV-induced pulmonary inflammation, apoptosis and water transport abnormality, which was probably through the regulation of TLR7, RIG-1 and AQP5 signaling pathway.

PtLGBP, a pattern recognition receptor in Portunus trituberculatus involved in the immune response against different challenges.

Lipopolysaccharide and b-1,3-glucan binding protein (LGBP) is a pattern recognition receptor that can recognize and bind LPS and b-1,3-glucan. LGBP has crucial roles in innate immune defense against Gram-negative bacteria and fungi. In this study, LGBP functions in Portunus trituberculatus innate immunity were analyzed. First, the mRNA expression of PtLGBP in hemocytes, hepatopancreas, and muscle toward three typical pathogen-associated molecular patterns (PAMPs) stimulations were examined using real-time PCR. Results show that the overall trend of relative expressions of the LGBP gene in three tissues is consistent, showing up-down trend. In each group, the highest expression of the LGBP gene was at 3 and 12 h post-injection. The LGBP gene is also expressed significantly higher in the hemocytes and hepatopancreas than in the muscle. The highest level of LGBP was in the lipopolysaccharides (LPS) and glucan-injected group, whereas the lowest level was in the PGN-injected group. Furthermore, bacterial agglutination assay with polyclonal antibody specifically for PtLGBP proved that the recombinant PtLGBP (designated as rPtLGBP) could exhibit obvious agglutination activity toward Gram-negative bacteria Escherichia coli, Vibrio parahaemolyticus, and V. alginolyticus; Gram-positive bacteria Bacillus subtilis; and fungi Saccharomyces cerevisiae. LGBP in Portunus trituberculatus possibly served as a multi-functional PRR. In addition, LGBP is not only involved in the immune response against Gram-negative and fungi, as manifested in other invertebrates, but also has a significant role in anti-Gram-positive bacteria infection.

Programmed necrosis, not apoptosis, is a key mediator of cell loss and DAMP-mediated inflammation in dsRNA-induced retinal degeneration.

There is no known treatment for the dry form of an age-related macular degeneration (AMD). Cell death and inflammation are important biological processes thought to have central role in AMD. Here we show that receptor-interacting protein (RIP) kinase mediates necrosis and enhances inflammation in a mouse model of retinal degeneration induced by dsRNA, a component of drusen in AMD. In contrast to photoreceptor-induced apoptosis, subretinal injection of the dsRNA analog poly(I : C) caused necrosis of the retinal pigment epithelium (RPE), as well as macrophage infiltration into the outer retinas. In Rip3(-/-) mice, both necrosis and inflammation were prevented, providing substantial protection against poly(I : C)-induced retinal degeneration. Moreover, after poly(I : C) injection, Rip3(-/-) mice displayed decreased levels of pro-inflammatory cytokines (such as TNF-α and IL-6) in the retina, and attenuated intravitreal release of high-mobility group box-1 (HMGB1), a major damage-associated molecular pattern (DAMP). In vitro, poly(I : C)-induced necrosis were inhibited in Rip3-deficient RPE cells, which in turn suppressed HMGB1 release and dampened TNF-α and IL-6 induction evoked by necrotic supernatants. On the other hand, Rip3 deficiency did not modulate directly TNF-α and IL-6 production after poly(I : C) stimulation in RPE cells or macrophages. Therefore, programmed necrosis is crucial in dsRNA-induced retinal degeneration and may promote inflammation by regulating the release of intracellular DAMPs, suggesting novel therapeutic targets for diseases such as AMD.

The therapeutic potential of modifying inflammasomes and NOD-like receptors.

Inflammasomes are the central processing units (CPUs) responsible for decoding and integrating signals of foreignness, damage, danger, and distress released by pathogens, cells, and tissues. It was initially thought that the inflammasomes participated only in pathogen recognition and in the pathogenesis of a few, rare, hereditary inflammatory disorders. On the contrary, it is now clear that they have a central role in the pathogenesis of basically all types of chronic inflammation, in metabolic diseases and cancer. So far, six or possibly eight inflammasome subtypes have been identified. Their main, but by no means exclusive, function is to catalyze conversion of pro-IL-1ß and pro-IL-18 into their respective mature forms. However, the different inflammasome subtypes may also participate in additional responses, e.g., proliferation, regulation of glycolytic metabolism, or cell activation, albeit it is not clear whether these effects are still mediated through IL-1ß release or via modulation of other caspase-1-dependent or -independent pathways. Central to inflammasome organization and activity are proteins belonging to the nucleotide binding domain, leucine-rich repeat, or NOD-like receptor family. One relevant exception is the AIM2 inflammasome. NOD-like receptors belong to the superfamily of pattern recognition receptors, a group of highly conserved molecules specialized in the recognition of invariant molecular patterns diffused across species. Given their potent proinflammatory activity, it is anticipated that inflammasome activation is tightly controlled. In this review, I will summarize essential features of the known NOD-like receptors, the basic molecular structure of inflammasomes, their participation in pathophysiological responses, and their possible exploitation for therapy.

Matrix metalloproteinase 13 expression in response to double-stranded RNA in human chondrocytes.

OBJECTIVE: To investigate the mechanism of matrix metalloproteinase 13 (MMP-13) expression in chondrocytes via pattern-recognition receptors (PRRs) for double-stranded RNA (dsRNA). METHODS: Differential expression of PRRs was determined by real-time reverse transcription-polymerase chain reaction (RT-PCR) of RNA from patients with osteoarthritis (OA) and patients with femoral neck fracture (as normal control). Isolated human articular chondrocytes and the chondrosarcoma cell line SW-1353 were activated with poly(I-C) of different molecular weights as a dsRNA mimic, and changes in gene and protein expression were monitored by real-time RT-PCR and immunoblotting, respectively. RESULTS: The dsRNA signaling moieties Toll-like receptor 3 (TLR-3), retinoic acid-inducible gene 1 (RIG-1), and nucleotide-binding oligomerization domain-like receptor X1 were all differentially expressed in OA cartilage compared to normal cartilage, as determined by gene expression screening. Depletion of the dsRNA-sensing receptors TLR-3, RIG-1, or melanoma differentiation-associated gene 5 (MDA-5) suppressed the induction of MMP13 messenger RNA (mRNA) expression by poly(I-C), regardless of its mode of delivery. In addition, depletion of the downstream transcription factor interferon regulatory factor 3 resulted in reduced induction of MMP13 mRNA expression by poly(I-C). CONCLUSION: Signaling by dsRNA in chondrocytes requires a range of PRRs, including TLR-3, RIG-1, and MDA-5, for the full-induction of MMP13, thus providing tight regulation of a gene critical for maintenance of cartilage integrity. Our data add to the understanding of MMP13 regulation, which is essential before such mechanisms can be exploited to alleviate the cartilage destruction associated with OA.

Blueprints of signaling interactions between pattern recognition receptors: implications for the design of vaccine adjuvants.

Innate immunity activation largely depends on recognition of microorganism structures by Pattern Recognition Receptors (PRRs). PRR downstream signaling results in production of pro- and anti-inflammatory cytokines and other mediators. Moreover, PRR engagement in antigen-presenting cells initiates the activation of adaptive immunity. Recent reports suggest that for the activation of innate immune responses and initiation of adaptive immunity, synergistic effects between two or more PRRs are necessary. No systematic analysis of the interaction between the major PRR pathways were performed to date. In this study, a systematical analysis of the interactions between PRR signaling pathways was performed. PBMCs derived from 10 healthy volunteers were stimulated with either a single PRR ligand or a combination of two PRR ligands. Known ligands for the major PRR families were used: Toll-like receptors (TLRs), C-type lectin receptors (CLRs), NOD-like receptors (NLRs), and RigI-helicases. After 24 h of incubation, production of tumor necrosis factor alpha (TNF-α), interleukin-1 beta (IL-1ß), IL-6, and IL-10 was measured in supernatants by enzyme-linked immunosorbent assay (ELISA). The consistency of the PRR interactions (both inhibitory and synergistic) between the various individuals was assessed. A number of PRR-dependent signaling interactions were found to be consistent, both between individuals and with regard to multiple cytokines. The combinations of TLR2 and NOD2, TLR5 and NOD2, TLR5 and TLR3, and TLR5 and TLR9 acted as synergistic combinations. Surprisingly, inhibitory interactions between TLR4 and TLR2, TLR4 and Dectin-1, and TLR2 and TLR9 as well as TLR3 and TLR2 were observed. These consistent signaling interactions between PRR combinations may represent promising targets for immunomodulation and vaccine adjuvant development.

Immunomodulatory properties of antifungal agents on phagocytic cells.

Phagocytic cells, particularly neutrophils and monocytes/macrophages, are the first line and the most effective form of innate host defence against pathogenic fungi. During antifungal therapy these phagocytic cells are also exposed to antifungal agents. In the phagocyte-fungus-antifungal agent interplay, drugs may directly interact with phagocytes through specific pattern recognition receptors, leading to altered antifungal activities. Antifungal agents, through modulation of fungal virulence, may initiate different immune response programs in the phagocytes, leading to antifungal synergism/antagonism or up-regulation of gene expression for a pro-inflammatory response. Additionally, indirect modulation of phagocyte behavior by pretreatment of neutrophils, monocytes, and macrophages with cytokines and exposure to antifungal agents have shown promising findings for combined drug-cytokine therapy that may improve treatment of life-threatening fungal diseases. In this review, we discuss the main in vitro and in vivo immunomodulatory effects of antifungal agents on phagocytes in response to pathogenic fungi, as well as we address underlying immunopharmacologic mechanisms and their potential impact on clinical outcome.

Pathogen-associated molecular patterns on biomaterials: a paradigm for engineering new vaccines.

Vaccine development has progressed significantly and has moved from whole microorganisms to subunit vaccines that contain only their antigenic proteins. Subunit vaccines are often less immunogenic than whole pathogens; therefore, adjuvants must amplify the immune response, ideally establishing both innate and adaptive immunity. Incorporation of antigens into biomaterials, such as liposomes and polymers, can achieve a desired vaccine response. The physical properties of these platforms can be easily manipulated, thus allowing for controlled delivery of immunostimulatory factors and presentation of pathogen-associated molecular patterns (PAMPs) that are targeted to specific immune cells. Targeting antigen to immune cells via PAMP-modified biomaterials is a new strategy to control the subsequent development of immunity and, in turn, effective vaccination. Here, we review the recent advances in both immunology and biomaterial engineering that have brought particulate-based vaccines to reality.

Src kinase-targeted anti-inflammatory activity of davallialactone from Inonotus xeranticus in lipopolysaccharide-activated RAW264.7 cells.

BACKGROUND AND PURPOSE: Mushrooms are popular both as food and as a source of natural compounds of biopharmaceutical interest. Some mushroom-derived compounds such as beta-glucan have been shown to be immunostimulatory; this study explores the anti-inflammatory properties of hispidin analogues derived from the mushroom, Inonotus xeranticus. We sought to identify the molecular mechanism of action of these hispidin analogues by determining their effects on lipopolysaccharide (LPS)-mediated inflammatory responses in a macrophage cell line. EXPERIMENTAL APPROACH: The production of inflammatory mediators was determined by Griess assay, reverse transcription-PCR and ELISA. The inhibitory effect of davalliactone on LPS-induced activation of signalling cascades was assessed by western blotting, immunoprecipitation and direct kinase assay. KEY RESULTS: In activated RAW264.7 cells, davallialactone strongly downregulated LPS-mediated inflammatory responses, including NO production, prostaglandin E2 release, expression of proinflammatory cytokine genes and cell surface expression of co-stimulatory molecules. Davallialactone treatment did not alter cell viability or morphology. Davallialactone was found to exert its anti-inflammatory effects by inhibiting a signalling cascade that activates nuclear factor kappa B via PI3K, Akt and IKK, but not mitogen-activated protein kinases. Treatment with davallialactone affected the phosphorylation of these signalling proteins, but not their level of expression. These inhibitory effects were not due to the interruption of toll-like receptor 4 binding to CD14. In particular, davallialactone strongly inhibited the LPS-induced phosphorylation and kinase activity of Src, implying that Src may be a potential pharmacological target of davallialactone. CONCLUSIONS AND IMPLICATIONS: Our data suggest that davallialactone, a small molecule found in edible mushrooms, has anti-inflammatory activity. Davallialactone can be developed as a pharmaceutically valuable anti-Src kinase agent.

Immune activation by a sterile aqueous extract of Cordyceps sinensis: mechanism of action.

Cordyceps sinensis is a fungus that has been used for over 2,000 years in China as a treatment for a variety of conditions including infectious diseases. The available evidence suggests a hypothesis that any efficacy of C. sinensis as an anti-infective therapeutic would be related to a role as an activator of innate immune responses. The objectives of this study were first to investigate the ability of C. sinensis to activate pro-inflammatory responses in macrophages in vitro and induce protective responses against intracellular pathogens in vivo, and second to characterize a method of action. We found that C. sinensis activates murine macrophages to produce a variety of pro-inflammatory cytokines. IFN-gamma synergizes with C. sinensis to amplify this response. Bacterial endotoxin contamination was ruled out as a potential artefact. The evidence presented in this study supports a hypothesis that C. sinensis activates macrophages by engaging Toll-like receptors and inducing mitogen-activated protein kinase (MAPK) pathways characteristic of inflammatory stimuli.

A modeling comparison of projection neuron- and neuromodulator-elicited oscillations in a central pattern generating network.

Many central pattern generating networks are influenced by synaptic input from modulatory projection neurons. The network response to a projection neuron is sometimes mimicked by bath applying the neuronally-released modulator, despite the absence of network interactions with the projection neuron. One interesting example occurs in the crab stomatogastric ganglion (STG), where bath applying the neuropeptide pyrokinin (PK) elicits a gastric mill rhythm which is similar to that elicited by the projection neuron modulatory commissural neuron 1 (MCN1), despite the absence of PK in MCN1 and the fact that MCN1 is not active during the PK-elicited rhythm. MCN1 terminals have fast and slow synaptic actions on the gastric mill network and are presynaptically inhibited by this network in the STG. These local connections are inactive in the PK-elicited rhythm, and the mechanism underlying this rhythm is unknown. We use mathematical and biophysically-realistic modeling to propose potential mechanisms by which PK can elicit a gastric mill rhythm that is similar to the MCN1-elicited rhythm. We analyze slow-wave network oscillations using simplified mathematical models and, in parallel, develop biophysically-realistic models that account for fast, action potential-driven oscillations and some spatial structure of the network neurons. Our results illustrate how the actions of bath-applied neuromodulators can mimic those of descending projection neurons through mathematically similar but physiologically distinct mechanisms.

Targeting intracellular mediators of pattern-recognition receptor signalling to adjuvant vaccination.

PRR (pattern-recognition receptor) signalling is involved early in the immune response and therefore would be attractive to target during vaccination. The use of PRR ligands has shown some success; however, toxicity and non-specificity are issues with this strategy. The targeting of PRR intracellular signalling networks would allow for greater specificity and reduced systemic toxicity. The present review examines the successes seen with overexpression or repression of PRR signalling molecules.