Low-dose Drosera rotundifolia induces gene expression changes in 16HBE human bronchial epithelial cells.

Drosera rotundifolia has been traditionally used for the treatment of respiratory diseases in phytotherapy and homeopathy. The mechanisms of action recognized so far are linked to the known effects of specific components, such as flavonoids, but are not completely understood. In this study, the biological functions of D. rotundifolia were explored in vitro following the treatment of bronchial epithelial cells, which are the potential targets of the pharmacological effects of the herbal medicine. To do so, the whole plant ethanolic extract was 1000-fold diluted in water (D. rotundifolia 3×) and added to a 16HBE human cell line culture for 3 h or 6 h. The effects on gene expression of the treatments and corresponding controls were then investigated by RNA sequencing. The differentially expressed genes were validated through RT-qPCR, and the enriched biological functions involved in the effects of treatment were investigated. D. rotundifolia 3× did not impair cell viability and was shown to be a stimulant of cell functions by regulating the expression of dozens of genes after 3 h, and the effects were amplified after 6 h of treatment. The main differentially expressed genes encoded ligands of epithelial growth factor receptor, proteins involved in xenobiotic detoxification and cytokines, suggesting that D. rotundifolia 3× could stimulate self-repair systems, which are impaired in airway diseases. Furthermore, D. rotundifolia 3× acts on a complex and multifaceted set of genes and may potentially affect different layers of the bronchial mucosa.

Human neutrophils activated by TLR8 agonists, with or without IFNγ, synthesize and release EBI3, but not IL-12, IL-27, IL-35, or IL-39.

The IL-12 family of cytokines plays crucial functions in innate and adaptive immunity. These cytokines include heterodimers sharing distinct α (IL-12A, IL-23A, and IL-27A) with two ß (IL-12B and Epstein-Barr virus induced gene 3 [EBI3]) chains, respectively, IL-12 (IL-12B plus IL-12A) and IL-23 (IL-12B plus IL-23A) sharing IL-12B, IL-27 (EBI3 plus IL-27A), IL-35 (EBI3 plus IL-12A), and IL-39 (EBI3 plus IL-23A) sharing EBI3. In this context, we have recently reported that highly pure neutrophils incubated with TLR8 agonists produce functional IL-23. Previously, we showed that neutrophils incubated with LPS plus IFNγ for 20 h produce IL-12. Herein, we investigated whether highly pure, TLR8-activated, neutrophils produce EBI3, and in turn IL-27, IL-35, and IL-39, the IL-12 members containing it. We report that neutrophils incubated with TLR8 ligands, TNFα and, to a lesser extent, LPS, produce and release remarkable amounts of EBI3, but not IL-27A, consequently excluding the possibility for an IL-27 production. We also report a series of unsuccessful experiments performed to investigate whether neutrophil-derived EBI3 associates with IL-23A to form IL-39. Furthermore, we show that neutrophils incubated with IFNγ in combination with either TLR8 or TLR4 ligands express/produce neither IL-12, nor IL-35, due to the inability of IFNγ, contrary to previous findings, to activate IL12A transcription. Even IL-27 was undetectable in supernatants harvested from IFNγ plus R848-treated neutrophils, although they were found to accumulate IL27A transcripts. Finally, by immunohistochemistry experiments, EBI3-positive neutrophils were found in discrete pathologies only, including diverticulitis, cholecystitis, Gorham disease, and Bartonella Henselae infection, implying a specific role of neutrophil-derived EBI3 in vivo.

Fibronectin Gene Up-regulation by Arnica montana in Human Macrophages: Validation by Real-Time Polymerase Chain Reaction Assay.

BACKGROUND AND AIM: Arnica montana L. (Arnica m.) is a popular traditional medicine, used for its therapeutic properties in healing traumas, but little is known about its biological action on tissue formation and repair. This new work tested the effects of Arnica m. homeopathic dilutions on human macrophages, key cells in tissue defence and repair. MATERIALS AND METHODS: Macrophages derived from the THP-1 cell line were differentiated with interleukin-4 to induce a 'wound-healing'-like phenotype, and treated with various dilutions of Arnica m. centesimal (100 times) dilutions (2c, 3c, 5c, 9c, and 15c) or control solvent for 24 hours. RNA samples from cultured cells were analysed by real-time quantitative polymerase chain reaction in five separate experiments. RESULTS: Arnica montana at the 2c dilution (final concentration of sesquiterpene lactones in cell culture = 10-8 mol/L) significantly stimulated the expression of three genes which code for regulatory proteins of the extracellular matrix, namely FN1 (fibronectin 1, % increase of 21.8 ± standard error of the mean 4.6), low-density lipoprotein-receptor-related protein 1 (% increase of 33.4 ± 6.1) and heparan sulphate proteoglycan 2 (% increase of 21.6 ± 9.1). Among these genes, the most quantitatively expressed was FN1. In addition, FN1, unlike other candidate genes, was upregulated in cells treated with higher dilutions/dynamisations (3c, 5c, and 15c) of Arnica m. CONCLUSION: The results support evidence that the extracellular matrix is a potential therapeutic target of Arnica m., with positive effects on cell adhesion and migration during tissue development and healing.

Human neutrophils activated via TLR8 promote Th17 polarization through IL-23.

Human neutrophils contribute to the regulation of inflammation via the generation of a range of cytokines that affect all elements of the immune system. Here, we investigated their ability to express some of the members of the IL-12 family after incubation with TLR8 agonists. Highly pure human neutrophils were thus incubated for up to 48 h with or without R848, or other TLR8 agonists, to then measure the expression levels of transcripts and proteins for IL-12 family member subunits by RNA-seq, reverse transcription quantitative PCR, and ELISA. We show a TLR8-mediated inducible expression of IL-12B and IL-23A, but not IL-12A, mRNA, which occurs via chromatin remodeling (as assessed by ChIP-seq), and subsequent production of IL-23 and IL-12B, but no IL-12, proteins. Induction of IL-23 requires endogenous TNF-α, as both mRNA and protein levels were blocked in TLR8-activated neutrophils via a TNF-α-neutralizing Ab. We also show that supernatants from TLR8-activated neutrophils, but not autologous monocytes, induce the differentiation of Th17 cells from naïve T cells in an IL-23-dependent fashion. This study unequivocally demonstrates that highly pure human neutrophils express and produce IL-23, further supporting the key roles played by these cells in the important IL-17/IL-23 network and Th17 responses.

Cytokine production by human neutrophils: Revisiting the "dark side of the moon".

Polymorphonuclear neutrophils are the most numerous leucocytes present in human blood, and function as crucial players in innate immune responses. Neutrophils are indispensable for the defence towards microbes, as they effectively counter them by releasing toxic enzymes, by synthetizing reactive oxygen species and by producing inflammatory mediators. Interestingly, recent findings have highlighted an important role of neutrophils also as promoters of the resolution of inflammation process, indicating that their biological functions go well beyond simple pathogen killing. Consistently, data from the last decades have highlighted that neutrophils may even contribute to the development of adaptive immunity by performing previously unanticipated functions, including the capacity to extend their survival, directly interact with other leucocytes or cell types, and produce and release a variety of cytokines. In this article, we will summarize the main features of, as well as emphasize some important concepts on, the production of cytokines by human neutrophils.

A Reappraisal on the Potential Ability of Human Neutrophils to Express and Produce IL-17 Family Members In Vitro: Failure to Reproducibly Detect It.

Neutrophils are known to perform a series of effector functions that are crucial for the innate and adaptive responses, including the synthesis and secretion of a variety of cytokines. In light of the controversial data in the literature, the main objective of this study was to more in-depth reevaluate the capacity of human neutrophils to express and produce cytokines of the IL-17 family in vitro. By reverse transcription quantitative real-time PCR, protein measurement via commercial ELISA, immunohistochemistry (IHC) and immunofluorescence (IF), flow cytometry, immunoblotting, chromatin immunoprecipitation (ChIP), and ChIP-seq experiments, we found that highly pure (>99.7%) populations of human neutrophils do not express/produce IL-17A, IL-17F, IL-17AF, or IL-17B mRNA/protein upon incubation with a variety of agonists. Similar findings were observed by analyzing neutrophils isolated from active psoriatic patients. In contrast with published studies, IL-17A and IL-17F mRNA expression/production was not even found when neutrophils were incubated with extremely high concentrations of IL-6 plus IL-23, regardless of their combination with inactivated hyphae or conidia from Aspergillus fumigatus. Consistently, no deposition of histone marks for active (H3K27Ac) and poised (H3K4me1) genomic regulatory elements was detected at the IL-17A and IL-17F locus of resting and IL-6 plus IL-23-stimulated neutrophils, indicating a closed chromatin conformation. Concurrent experiments revealed that some commercial anti-IL-17A and anti-IL-17B antibodies (Abs), although staining neutrophils either spotted on cytospin slides or present in inflamed tissue samples by IHC/IF, do not recognize intracellular protein having the molecular weight corresponding to IL-17A or IL-17B, respectively, in immunoblotting experiments of whole neutrophil lysates. By contrast, the same Abs were found to more specifically recognize other intracellular proteins of neutrophils, suggesting that their ability to positively stain neutrophils in cytospin preparations and, eventually, tissue samples derives from IL-17A- or IL-17B-independent detections. In sum, our data confirm and extend, also at epigenetic level, previous findings on the inability of highly purified populations of human neutrophils to express/produce IL-17A, IL-17B, and IL-17F mRNAs/proteins in vitro, at least under the experimental conditions herein tested. Data also provide a number of justifications explaining, in part, why it is possible to false positively detect IL-17A+-neutrophils.

Human Neutrophils Produce CCL23 in Response to Various TLR-Agonists and TNFα.

CCL23, also known as myeloid progenitor inhibitory factor (MPIF)-1, macrophage inflammatory protein (MIP)-3, or CKß8, is a member of the CC chemokine subfamily exerting its effects via CCR1 binding. By doing so, CCL23 selectively recruits resting T lymphocytes and monocytes, inhibits proliferation of myeloid progenitor cells and promotes angiogenesis. Previously, we and other groups have reported that human neutrophils are able to produce chemokines upon appropriate activation, including CCR1-binding CCL2, CCL3, and CCL4. Herein, we demonstrate that human neutrophils display the capacity to also express and release CCL23 when stimulated by R848 and, to a lesser extent, by other pro-inflammatory agonists, including LPS, Pam3CSK4, and TNFα. Notably, we show that, on a per cell basis, R848-activated neutrophils produce higher levels of CCL23 than autologous CD14+-monocytes activated under similar experimental conditions. By contrast, we found that, unlike CD14+-monocytes, neutrophils do not produce CCL23 in response to IL-4, thus indicating that they express CCL23 in a stimulus-specific fashion. Finally, we show that the production of CCL23 by R848-stimulated neutrophils is negatively modulated by IFNα, which instead enhances that of CCL2. Together, data extend our knowledge on the chemokines potentially produced by neutrophils. The ability of human neutrophils to produce CCL23 further supports the notion on the neutrophil capacity of orchestrating the recruitment of different cell types to the inflamed sites, in turn contributing to the control of the immune response.

Inhibition of the NF-κB and p38 MAPK pathways by scopoletin reduce the inflammation caused by carrageenan in the mouse model of pleurisy.

Natural products have long been used worldwide as therapeutic agents, but it is only recently, in response to the new challenges posed by global population aging, that interest in research into potentially therapeutic natural products has reemerged. In this context, coumarins, chemical compounds found in plants that have known anti-inflammatory activity, are promising candidates for the development of new drugs. In this study we test the effect of scopoletin, a coumarin found in several plant species, on carrageenan-induced inflammation in the mouse model of pleurisy. Initially, the effects of scopoletin on leukocyte migration and exudate concentrations were evaluated at three different doses (0.1, 1 and 5 mg/kg) and time (0.5-4 h before pleurisy). In the next step, we chose the lowest dose capable of inhibiting the inflammatory parameters (1 mg/kg), in order to analyze the myeloperoxidase and adenosine deaminase activities, the nitric oxide, tumor necrosis factor-α, and interleukin 1ß levels in the fluid leakage, and the p65 subunit of NF-κB and p38 MAPK phosphorylation. Scopoletin at a dose of 1 mg/kg was able to significantly reduce cell migration and exudation to the pleural fluid (p < 0.01). Scopoletin at the same dose also decreased the myeloperoxidase and adenosine-deaminase activities and nitric oxide, tumor necrosis factor-α, and interleukin-1ß levels (p < 0.01). In addition, it significantly reduced p65 and p38 phosphorylation in the mouse lungs (p < 0.01). Our results reinforce that scopoletin has important anti-inflammatory activity, and shows, that this effect can be attributed to the ability of this compound to inhibit the phosphorylation of NF-κB and p38 MAPK.

IFNα enhances the production of IL-6 by human neutrophils activated via TLR8.

Recently, we reported that human neutrophils produce biologically active amounts of IL-6 when incubated with agonists activating TLR8, a receptor recognizing viral single strand RNA. In this study, we demonstrate that IFNα, a cytokine that modulates the early innate immune responses toward viral and bacterial infections, potently enhances the production of IL-6 in neutrophils stimulated with R848, a TLR8 agonist. We also show that such an effect is not caused by an IFNα-dependent induction of TLR7 and its consequent co-activation with TLR8 in response to R848, but, rather, it is substantially mediated by an increased production and release of endogenous TNFα. The latter cytokine, in an autocrine manner, leads to an augmented synthesis of the IkBζ co-activator and an enhanced recruitment of the C/EBPß transcription factor to the IL-6 promoter. Moreover, we show that neutrophils from SLE patients with active disease state, hence displaying an IFN-induced gene expression signature, produce increased amounts of both IL-6 and TNFα in response to R848 as compared to healthy donors. Altogether, data uncover novel effects that type I IFN exerts in TLR8-activated neutrophils, which therefore enlarge our knowledge on the various biological actions which type I IFN orchestrates during infectious and autoimmune diseases.

7-prenyloxi-6-methoxycoumarin from Polygala sabulosa A.W. Bennett Regulates p38 MAPK and NF-kB Pathways Inhibiting the Inflammation Induced by Carrageenan in the Mouse Model of Pleurisy.

CONTEXT: Polygala sabulosa, popularly known as "timutu-pinheirinho," has been used in Brazilian folk medicine for the treatment of bowel and kidney disorders and as an expectorant. OBJECTIVE: Evaluate the anti-inflammatory effects of the crude extract (CE), acetonic fraction (Ac), and the main compound, 7-prenyloxi-6-methoxycoumarin (PC) on a mouse model of carrageenan-induced pleurisy. MATERIALS AND METHODS: A mouse model of carrageenan-induced pleurisy was used to investigate the effects of P. sabulosa CE, Ac and PC on leukocyte migration, exudate formation, activities of myeloperoxidase (MPO), and adenosine-deaminase (ADA), levels of tumor necrosis factor-α (TNF-α), interleukin 1ß (IL-1ß) and nitric oxide (NO). In addition, the effect of the plant material on lung histology was also evaluated. The effects of PC on the TNF-α, IL-1ß and NO synthase 2 (NOS2) mRNA expression, were also investigated. Finally, the effect of PC on the nuclear factor-kappa B (NF-κB) and p38 mitogen-activated protein kinase (p38 MAPK) was also evaluated. RESULTS: CE, Ac and PC reduced inflammation in the pleural cavity and lungs. This effect was evidenced by reduction on all inflammatory parameters evaluated; the exception being the inability of the CE to inhibit exudate formation. In isolation, PC showed reduction on mRNA levels of TNF-α, IL-1ß and NOS2, and on activation of the NF-κB and p38 MAPK pathways. CONCLUSION: The presented results show that P. sabulosa has significant anti-inflammatory activity, as does its main compound, PC. Moreover, the results suggest that PC exerts its effects mainly by inhibited the NF-κB and p38 MAPK pathways.

Polygala molluginifolia A. St.-Hil. and Moq. prevent inflammation in the mouse pleurisy model by inhibiting NF-κB activation.

UNLABELLED: This study was conducted to investigate the anti-inflammatory activity of Polygala molluginifolia (Polygalaceae) on the mouse pleurisy model induced by carrageenan. P. molluginifolia is a plant native to southern Brazil that is popularly called "canfora". The Polygala genus is used to treat different pathologies, including inflammatory diseases, in traditional medicine. MATERIAL AND METHODS: The whole P. molluginifolia plant material was extracted by maceration with 96% ethanol. The crude hydroalcoholic extract (CE) was subjected to chromatographic procedures to produce various derivate fractions, including its aqueous (Aq), ethyl acetate (EtOAc), and hexane (Hex) fractions. Compound 1 (5,3',4'-trihydroxy-6″,6″-dimethylpyrano [2″,3″:7,6] isoflavone) (Iso), which was isolated from the EtOAc fraction, and Compound 2 (rutin) (Rut), which was isolated from the Aq fraction, were identified using ¹H and ¹³C NMR spectroscopy and quantified using an HPLC apparatus. RESULTS: The CE, the Aq, EtOAc, and Hex fractions, and the isolated compounds Iso and Rut were able to reduce cell migration and exudation. Furthermore, the plant material also decreased the myeloperoxidase (MPO) and adenosine-deaminase (ADA) activities and the nitric oxide (NO(x)), tumor necrosis factor-alpha (TNF-α), and interleukin-1 beta (IL-1ß) levels. In addition, Iso and Rut reduced the TNF-α and IL-1ß mRNA expression levels and significantly decreased NF-κB p65 phosphorylation. CONCLUSION: The results show that P. molluginifolia has a significant anti-inflammatory action and that this effect is due, at least in part, to the presence of Iso and Rut in large amounts. Moreover, this effect was found to be closely related to the inhibitory effects of the isolated compounds on the NF-κB pathway.