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

ABSTRACT: 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-?B 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-?, IL-1?, IL-6, and IL-8 as well as decreased p65 concentration, the subunit responsible for NF-?B activation and cytokine production and simultaneously stimulated release of anti-inflammatory mediators (IL-10 and TGF-?). 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.

SUBMITTER: Gierlikowska B

PROVIDER: S-EPMC7494907 | biostudies-literature | 2020 Jan

REPOSITORIES: biostudies-literature

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Project description:The receptors engaged during phagocytic particle uptake determine the signaling events that occur during phagosome formation and maturation. However, pathogens generally have multiple ligands, making it difficult to dissect the roles of individual receptors in these processes. Here, we used latex beads coupled to single ligands, focusing on IgG, mannan, LPS and avidin, and monitored phagocytic uptake rates, phago-lysosomal fusion events, macrophage gene expression and the proteomic composition of isolated phagosomes. The pattern of gene expression and the protein composition of isolated phagosomes showed that each bead ligand altered a distinct pattern of genes and led to a different composition of phagosomes. These data argue that activation of each receptor initiates a specific signature of signaling events that last many hours and influences several phagocytosis functions. All samples and controls were carried out in triplicates. J774.A1 cells (mouse macrophage-like cell line) were seeded onto 6-well plates one day before the experiment. Subsequently, cells were incubated with serum-free DME medium containing 0.01 % latex beads of 1 Âµm diameter coupled to the following ligands: avidin (Av), the Fc fragment of mouse IgG (Fc), lipopolysaccharides from Klebsiella pneumoniae (LPS) and mannan from Saccharomyces cerevisae (Man) for 30 and 60 minutes. After incubation, samples as well as untreated controls were washed twice in PBS and total RNA was extracted using RNeasy kit (Qiagen) following manufacturer's instructions. Each sample was hybridized to CodeLink Mouse whole genome bioarray slides (Amersham). This study was intended to analyze the role of receptor-ligand interactions on phagosome maturation and gene expression after receptor-mediated phagocytosis in macrophages. CodeLink EXP v4.0-processed data are represented in the Sample tables, GeneSpring-processed data are linked as a supplementary files to the matrix table, additional results available as a supplementary file on the Series record. http://www.biologie.uni-rostock.de/tierphysiologie/agkuznetsov.html

Project description:Background: The Janus kinase (JAK) 1/2 inhibitor ruxolitinib has been approved in an indication of myelofibrosis and is a candidate for the treatment of a number of inflammatory or autoimmune diseases. We assessed the effects of ruxolitinib on lipopolysaccharide (LPS)- and poly (I:C)-induced cytokine production by human lung macrophages (LMs) and on the LMs' phagocytic activity. Methods: Human LMs were isolated from patients operated on for lung carcinoma. The LMs were cultured with ruxolitinib (0.5 × 10-7 M to 10-5 M) or budesonide (10-11 to 10-8 M) and then stimulated with LPS (10 ng·ml-1) or poly (I:C) (10 μg·ml-1) for 24 h. Cytokines released by the LMs into the supernatants were measured using ELISAs. The phagocytosis of labelled bioparticles was assessed using flow cytometry. Results: Ruxolitinib inhibited both the LPS- and poly (I:C)-stimulated production of tumor necrosis factor alpha, interleukin (IL)-6, IL-10, chemokines CCL2, and CXCL10 in a concentration-dependent manner. Ruxolitinib also inhibited the poly (I:C)- induced (but not the LPS-induced) production of IL-1ß. Budesonide inhibited cytokine production more strongly than ruxolitinib but failed to mitigate the production of CXCL10. The LMs' phagocytic activity was not impaired by the highest tested concentration (10-5 M) of ruxolitinib. Conclusion: Clinically relevant concentrations of ruxolitinib inhibited the LPS- and poly (I:C)-stimulated production of cytokines by human LMs but did not impair their phagocytic activity. Overall, ruxolitinib's anti-inflammatory activities are less intense than (but somewhat different from) those of budesonide-particularly with regard to the production of the corticosteroid-resistant chemokine CXCL-10. Our results indicate that treatment with a JAK inhibitor might be a valuable anti-inflammatory strategy in chronic obstructive pulmonary disease, Th1-high asthma, and both viral and non-viral acute respiratory distress syndromes (including coronavirus disease 2019).

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

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