The Impact of Cytokines on Neutrophils' Phagocytosis and NET Formation during Sepsis-A Review.

Sepsis is an overwhelming inflammatory response to infection, resulting in multiple-organ injury. Neutrophils are crucial immune cells involved in innate response to pathogens and their migration and effector functions, such as phagocytosis and neutrophil extracellular trap (NET) formation, are dependent on cytokine presence and their concentration. In the course of sepsis, recruitment and migration of neutrophils to infectious foci gradually becomes impaired, thus leading to loss of a crucial arm of the innate immune response to infection. Our review briefly describes the sepsis course, the importance of neutrophils during sepsis, and explains dependence between cytokines and their activation. Moreover, we, for the first time, summarize the impact of cytokines on phagocytosis and NET formation. We highlight and discuss the importance of cytokines in modulation of both processes and emphasize the direction of further investigations.

MicroRNAs as Regulators of Phagocytosis.

MicroRNAs (miRNAs) are short non-coding RNAs that regulate gene expression and thus act as important regulators of cellular phenotype and function. As their expression may be dysregulated in numerous diseases, they are of interest as biomarkers. What is more, attempts of modulation of some microRNAs for therapeutic reasons have been undertaken. In this review, we discuss the current knowledge regarding the influence of microRNAs on phagocytosis, which may be exerted on different levels, such as through macrophages polarization, phagosome maturation, reactive oxygen species production and cytokines synthesis. This phenomenon plays an important role in numerous pathological conditions.

Germacranolides from Carpesium divaricatum: Some New Data on Cytotoxic and Anti-Inflammatory Activity.

Carpesium divaricatum Sieb. & Zucc., a traditional medicinal plant used as an inflammation-relieving remedy, is a rich source of terpenoids. At least 40 germacrane-type sesquiterpene lactones, representatives of four different structural groups, were isolated from the plant. Cytotoxicity against cancer cells in vitro is the most frequently described biological activity of the compounds. However, little is known about the selectivity of the cytotoxic effect. The anti-inflammatory activity of the germacranolides is also poorly documented. The objective of the present study was to assess the cytotoxic activity of selected C. divaricatum germacranolides-derivatives of 4,5,8,9-tetrahydroxy-3-oxo-germacran-6,12-olide towards cancer and normal cell lines (including cells of different p53 status). Moreover, to assess the anti-inflammatory effect of the compounds, the release of four proinflammatory cytokines/chemokines (IL-1ß, IL-8, TNF-α and CCL2) by lipopolysaccharide-stimulated human neutrophils was measured by ELISA. The investigated sesquiterpene lactones demonstrated nonselective activity towards prostate cancer (Du145 and PC3) and normal prostate epithelial cells (PNT2) as well as against melanoma cells (A375 and HTB140) and keratinocytes (HaCaT). Cytotoxic activity against osteosarcoma cells was independent of their p53 status. In sub-cytotoxic concentrations (0.5-2.5 µM) the studied compounds significantly decreased cytokine/chemokine release by lipopolysaccharide-stimulated human leukocytes.

Phagocytosis, Degranulation and Extracellular Traps Release by Neutrophils-The Current Knowledge, Pharmacological Modulation and Future Prospects.

Neutrophils are crucial elements of innate immune system, which assure host defense via a range of effector functions, such as phagocytosis, degranulation, and NET formation. The latest literature clearly indicates that modulation of effector functions of neutrophils may affect the treatment efficacy. Pharmacological modulation may affect molecular mechanisms activating or suppressing phagocytosis, degranulation or NET formation. In this review, we describe the role of neutrophils in physiology and in the course of bacterial and viral infections, illustrating the versatility and plasticity of those cells. This review also focus on the action of plant extracts, plant-derived compounds and synthetic drugs on effector functions of neutrophils. These recent advances in the knowledge can help to devise novel therapeutic approaches via pharmacological modulation of the described processes.

Alantolactone Enhances the Phagocytic Properties of Human Macrophages and Modulates Their Proinflammatory Functions.

AIM OF THE STUDY: Phagocytosis is a crucial element of innate immune defense involved in bacterial killing. The aim of our study was to evaluate the influence of alantolactone on phagocytosis and cytokines release by THP1-derived macrophages. We assessed whether antimicrobial compound alantolactone (a sesquiterpene lactone present in Inula helenium L.) is able to stimulate immune functions of macrophages by increase of S. aureus uptake, phagosome acidification and further stimulation of phago-lysosomes formation. Simultaneously, we tested influence of alantolactone on cytokines/chemokines production and p65 NF-κB concentration. The activity of alantolactone was compared with clarithromycin at concentration 20 µM. METHODS: The cytotoxicity of alantolactone as well as S. aureus uptake, pH of phagosomes and phago-lysosomes fusion were analysed with flow cytometry. Reactive oxygen species and superoxide production were evaluated spectrophotometrically. The efficiency of phagocytosis was evaluated via quantifying viable bacteria (CFU). The effect on p65 protein concentration and cytokine production by macrophages were measured by enzyme-linked immunosorbent assay (ELISA). RESULTS: Alantolactone enhanced phagocytosis via increase of S. aureus uptake, acidification of phagosomes, and later stimulation of phago-lysosomes fusion. Alantolactone treatment resulted in ROS and superoxide production decrease. Furthermore, alantolactone inhibited production of pro-inflammatory cytokines TNF-α, IL-1ß, IL-6, and IL-8 as well as decreased p65 concentration, the subunit responsible for NF-κB activation and cytokine production and simultaneously stimulated release of anti-inflammatory mediators (IL-10 and TGF-ß). CONCLUSION: Results of our study indicate that alantolactone enhances clearance of S. aureus, and simultaneously modulates immune response, preventing collateral damage of the surrounding tissues. Considering the importance of phagocytosis in the pathogen killing, alantolactone may have a great potential as the supportive treatment of S. aureus infections. Further in vivo studies are warranted to confirm this hypothesis.

Constituents of Xerolekia speciosissima (L.) Anderb. (Inuleae), and Anti-Inflammatory Activity of 7,10-Diisobutyryloxy-8,9-epoxythymyl Isobutyrate.

Xerolekia speciosissima (L.) Anderb., a rare plant from the north of Italy, is a member of the Inuleae-Inulinae subtribe of the Asteraceae. Despite its close taxonomic relationship with many species possessing medicinal properties, the chemical composition of the plant has remained unknown until now. A hydroalcoholic extract from the aerial parts of X. speciosissima was analyzed by HPLC-DAD-MSn, revealing the presence of caffeic acid derivatives and flavonoids. In all, 19 compounds, including commonly found chlorogenic acids and less frequently occurring butyryl and methylbutyryl conjugates of dicaffeoylquinic and tricaffeoylhexaric acids, plus two flavonoids, were tentatively identified. Chromatographic separation of a hydroalcoholic extract from the capitula of the plant led to the isolation of (+)-dehydrodiconiferyl alcohol 4-O-ß-glucopyranoside, quercimeritrin, astragalin, isoquercitrin, 6-hydroxykaempferol-7-O-ß-glucoside, quercetagitrin, methyl caffeate, caffeic acid, protocatechuic acid, chlorogenic acid and 1,5-dicaffeoylquinic acid. Composition of a nonpolar extract from the aerial parts of the plant was analyzed by chromatographic methods supported with 1H-NMR spectroscopy. The analysis revealed the presence of loliolide, reynosin, samtamarine, 2,3-dihydroaromaticin, 2-deoxy-4-epi-pulchellin and thymol derivatives as terpenoid constituents of the plant. One of the latter compounds-7,10-diisobutyryloxy-8,9-epoxythymyl isobutyrate-at concentrations 0.5, 1.0 and 2.5 µM, significantly reduced IL-8, IL-1ß and CCL2 excretion by LPS-stimulated human neutrophils.

A New Diterpene and Anti-inflammatory Sesquiterpene Lactones from Sigesbeckia orientalis.

Sigesbeckia orientalis, more commonly referred to as Herba Sigesbeckiae or Xi Xian Cao in traditional Chinese medicine and hy thiêm in traditional Vietnamese medicine, is used in China and Vietnam to treat inflammatory diseases such as arthritis, rheumatism, and joint pain. In initial investigations, the dichloromethane extract from the aerial parts of S. orientalis showed distinct inhibitory effects on the release of interleukin-8 in human neutrophils. Therefore, the purpose of the present study was the phytochemical investigation of the bioactive dichloromethane extract and the in vitro analysis of the effects of the isolated compounds on interleukin-8, interleukin-1ß, tumor necrosis factor-α, and monocyte chemoattractant protein 1 release, and surface expression of adhesion molecules (CD11a, CD11b, and CD62L) in lipopolysaccharide-stimulated human neutrophils to identify the active principle(s). The separation of the bioactive dichloromethane extract using various chromatographic techniques led to the isolation of nine compounds. Their chemical structures were elucidated from nuclear magnetic resonance and mass spectrometry data. One diterpene, 17(13 → 14)-abeo-ent-3S*,13S*,16-trihydroxystrob-8(15)-ene, was identified as a new natural product. Three germacranolide sesquiterpene lactones inhibited interleukin-8 production with IC50 values between 1.6 and 6.3 µM, respectively, and tumor necrosis factor-α production with IC50 values between 0.9 and 3.3 µM, respectively. Furthermore, they significantly inhibited interleukin-1ß and monocyte chemoattractant protein 1 production and diminished the effects of lipopolysaccharide on the surface expression of the adhesion molecules CD11a, CD11b, and CD62L. These findings support the traditional use of S. orientalis in the treatment of inflammatory diseases.

Root Extracts From Ononis spinosa Inhibit IL-8 Release via Interactions With Toll-Like Receptor 4 and Lipopolysaccharide.

Extracts from the roots of Ononis spinosa L. (restharrow roots) are traditionally used for the treatment of patients with urinary tract infections due to its mild diuretic activity, caused by the inhibition of renal human hyaluronidase-1 by isoflavonoids. Preliminary studies also indicated anti-inflammatory effects. The following study aimed at investigating potential anti-inflammatory effects of restharrow extracts, prepared with solvents of different polarity. A dichloromethane extract (OS1), mainly composed of isoflavonoids and triterpenes as characterized by LC-MS, showed a concentration-dependent (25-100 µg/ml) inhibition of IL-8 and TNF-α release from LPS-stimulated human neutrophils. Significant inhibition was also found for the triterpene α-onocerin and the norneolignan clitorienolactone B, isolated from OS1. Further, OS1 and both compounds significantly decreased the expression of the adhesion molecules CD11b/CD18 and conversely increased the expression of CD62L in LPS-stimulated human neutrophils. This finding corresponds to a reduced inflammatory response by the inhibition of adhesion and migration of immune cells. As all of the observed effects are potentially mediated via Toll-like receptor 4 (TLR4) signaling, TLR4 transfected HEK293 cells were incubated with OS1. LPS-induced IL-8 secretion was significantly inhibited in a concentration-dependent manner, confirming TLR4 antagonism. This inhibition, however, was in part caused by an interaction of OS1 with LPS. In addition, also an aqueous extract containing high amounts of isoflavonoid glycosides and saponins from the roots of O. spinosa showed anti-inflammatory effects by interacting with the TLR4 signaling pathway. This study rationalizes the traditional use of extracts from O. spinosa for therapy of urinary tract infections, due to its potential anti-inflammatory effects that are mediated via TLR4 receptor antagonism.

Grindelia squarrosa Extract and Grindelic Acid Modulate Pro-inflammatory Functions of Respiratory Epithelium and Human Macrophages.

Aim of the study: Both nasal and bronchial epithelial cells have evolved sophisticated mechanisms involved in cellular response to bacterial infection. Recognition of pathogens by TLR receptors activate the NF-κB transcription factor, and lead to production of wide spectrum of cytokines (TNF-α, IL-1ß, IL-6 and IL-8). Released by epithelium proinflammatory cytokines intensify migration of macrophages to damaged tissues and modulate their pro-inflammatory functions. Based on traditional use of G. squarrosa aerial parts we hypothesized that successful treatment of cold-related diseases may arise from modulation of the pro-inflammatory functions of respiratory epithelium and human monocytes/macrophages. The biological activity of G. squarrosa extract and grindelic acid were compared with clarithromycin and budesonide used as positive controls. Methods: The expression of surface receptors (TLR-4, IL-10) and expression of adhesive molecules (ICAM-1, VCAM-1, E-selectin) was analyzed with flow cytometry. The macrophage attachment to the epithelial cells was assessed fluorimetrically. The p65 NF-κB concentration and cytokine production was measured spectrophotometrically using enzyme-linked immunosorbent assay. Antibacterial activity was examined by the standard disc-diffusion method and serial dilution method according to CLSI guidelines. Results: G. squarrosa extract and grindelic acid had no antimicrobial effect. However, we noticed significant modulation of pro-inflammatory functions of LPS-stimulated nasal and bronchial epithelium. G. squarrosa extract treatment resulted in decrease of TLR-4 expression and p65 NF-κB concentration and inhibition of cytokines synthesis (IL-8, TNF-α, IL-1ß and IL-6) in both cellular models. Additionally, G. squarrosa extract slightly modulated ICAM-1 expression affecting on attachment of macrophages to epithelium. Only G. squarrosa extract was able to stimulate the anti-inflammatory functions of macrophages by inducing TGF-ß release and IL-10 receptor surface expression. Grindelic acid, identified as a dominant compound in the plant extract, modulated pro-inflammatory functions of epithelium and macrophages slightly. Conclusion: The obtained results support traditional use of Grindelia squarrosa preparations for a treatment cold-associated diseases symptoms. In our opinion, the observed biological effect of extract may be a consequence of synergistic effect of all compounds present in the extract.

The bioactivity of flavonoid glucuronides and free aglycones in the context of their absorption, II phase metabolism and deconjugation at the inflammation site.

One of the most spread group of phenolics are flavonoids. Many studies focusing on the digestion and bioavailability of flavonoids have been carried out. Several possible directions of flavonoid metabolism are suspected and described in the literature. The aim of the present study was to evaluate the bioactivity of 8 flavonoid 3-O- and 7-O- glucuronides and 7 free aglycones on inflammatory response of PMNs and HUVECs in the context of their fate in humans after oral intake. The present study for the first time compared the activity of several most popular in plant flavonol and flavone aglycones and their beta-glucuronides. The results showed that in all in vitro experiments only aglycones have anti-inflammatory activity in PMNs and HUVECs models in the concentration range 1-50 µM. The most significant influence on the inflammatory response was observed in the case of HUVECs. Compounds were able to down-regulate levels of adhesion molecules (ICAM, VCAM and E-selectin). The possible deconjugation phenomenon at the inflammation site was evaluated using enzymes produces by stimulated PMNs. This is the first report suggesting the role of ß-glucuronidase in the inflammatory process taking place on the inflammation site. Additionally, the anti-inflammatory effect was significantly better for flavones.

Inula helenium and Grindelia squarrosa as a source of compounds with anti-inflammatory activity in human neutrophils and cultured human respiratory epithelium.

ETHNOPHARMACOLOGICAL RELEVANCE: During the epidemic season, over 90% of acute wheezing disease is associated with bronchial inflammation. Both neutrophil- and eosinophil-mediated inflammation have been involved in the pathophysiology of acute bronchitis, but neutrophil cell recruitment has been shown to be dominant. The ongoing inflammation increases the chemotaxis of neutrophils to inflamed site providing to their overaccumulation. The pharmacological reduction of neutrophil migration can be limited by suppression of major chemo-attractants and cytokines (IL-8, IL-1ß and TNF-α) release and downregulation of adhesive molecules. AIM OF THE STUDY: During a screening of plants traditionally used in respiratory tracts diseases (e.g. cough, rhinitis, bronchitis, throat infection, fever, influenza) in Europe, we have selected roots of Inula helenium and aerial parts of Grindelia squarrosa as a potential source of compounds limiting neutrophil migration. MATERIALS AND METHODS: The effect on IL-8, IL-1ß and TNF-α release by neutrophils and respiratory epithelium cell line (A549) was measured by enzyme-linked immunosorbent assay (ELISA). The surface expression of adhesive molecules was analyzed with flow cytometry, and the neutrophil attachment to the epithelial cells was assessed fluorimetrically. RESULTS: We confirmed the ability of selected extracts and compounds to suppress neutrophil binding to the epithelium surface via downregulation of ß2 integrin. Alantolactone and grindelic acid have shown significant suppression of IL-8, TNF-α and IL-1ß release comparable with budesonide, used as a positive control. CONCLUSIONS: The present study demonstrated that Inula helenium and Grindelia squarrosa, which have been traditionally used in Europe as medicinal plants, are a valuable source of active compounds with anti-inflammatory activity. Our observations justify the traditional use of I. helenium and G. squarrosa for a treatment of inflammation-based diseases in respiratory tract.