Mast cell-derived interleukin-4 mediates activation of dendritic cell during toll-like receptor 2-mediated inflammation.

Introduction: During an innate inflammation, immune cells form distinct pro- and anti-inflammatory regions around pathogen-containing core-regions. Mast cells are localized in an anti-inflammatory microenvironment during the resolution of an innate inflammation, suggesting antiinflammatory roles of these cells. Methods: High-content imaging was used to investigated mast cell-dependent changes in the regional distribution of immune cells during an inflammation, induced by the toll-like receptor (TLR)-2 agonist zymosan. Results: The distance between the zymosan-containing core-region and the anti-inflammatory region, described by M2-like macrophages, increased in mast cell-deficient mice. Absence of mast cells abolished dendritic cell (DC) activation, as determined by CD86-expression and localized the DCs in greater distance to zymosan particles. The CD86- DCs had a higher expression of the pro-inflammatory interleukins (IL)-1ß and IL-12/23p40 as compared to activated CD86+ DCs. IL-4 administration restored CD86 expression, cytokine expression profile and localization of the DCs in mast cell-deficient mice. The IL-4 effects were mast cell-specific, since IL-4 reduction by eosinophil depletion did not affect activation of DCs. Discussion: We found that mast cells induce DC activation selectively at the site of inflammation and thereby determine their localization within the inflammation. Overall, mast cells have antiinflammatory functions in this inflammation model and limit the size of the pro-inflammatory region surrounding the zymosan-containing core region.

Central carbon metabolism exhibits unique characteristics during the handling of fungal patterns by monocyte-derived dendritic cells.

Monocyte-derived dendritic cells (MDDCs) are key players in the defense against fungal infection because of their outstanding capacity for non-opsonic phagocytosis and phenotypic plasticity. Accordingly, MDDCs rewire metabolism to meet the energetic demands for microbial killing and biomass synthesis required to restore homeostasis. It has been commonplace considering the metabolic reprogramming a mimicry of the Warburg effect observed in tumor cells. However, this may be an oversimplification since the offshoots of glycolysis and the tricarboxylic acid (TCA) cycle are connected in central carbon metabolism. Zymosan, the external wall of Saccharomyces cerevisiae, contains ß-glucan and α-mannan chains that engage the C-type lectin receptors dectin-1/2 and Toll-like receptors. This makes it an optimal fungal surrogate for experimental research. Using real-time bioenergetic assays and [U-13C]glucose labeling, central hubs connected to cytokine expression were identified. The pentose phosphate pathway (PPP) exhibited a more relevant capacity to yield ribose-5-phosphate than reducing equivalents of NADPH, as judged from the high levels of isotopologues showing 13C-labeling in the ribose moiety and the limited contribution of the oxidative arm of the PPP to the production of ROS by NADPH oxidases (NOX). The finding of 13C-label in the purine ring and in glutathione unveiled the contribution of serine-derived glycine to purine ring and glutathione synthesis. Serine synthesis also supported the TCA cycle. Zymosan exhausted NAD+ and ATP, consistent with intracellular consumption and/or extracellular export. Poly-ADP-ribosylated proteins detected in the nuclear fractions of MDDCs did not show major changes upon zymosan stimulation, which suggests its dependence on constitutive Fe(II)/2-oxoglutarate-dependent demethylation of 5-methylcytosine by TET translocases and/or demethylation of histone H3 lysine 27 by JMJD demethylases rather than on NOX activities. These results disclose a unique pattern of central carbon metabolism following fungal challenge, characterized by the leverage of glycolysis offshoots and an extensive recycling of NAD+ and poly(ADP-ribose).

Role of nitric oxide and signaling pathways modulating the stimulatory effect of snake venom secretory PLA2S on non-opsonized zymosan phagocytosis by macrophages.

The phagocytic activity of macrophages activated with MT-II, a Lys-49 PLA2 homolog, and MT-III, an Asp-49 PLA2, from Bothrops asper snake venom, was investigated in this study using a pharmacological approach. Stimulating thioglycollate-elicited macrophages with both venom components enhanced their ability to phagocytose non-opsonized zymosan particles. MT-II and MT-III-induced phagocytosis was drastically inhibited by pretreating cells with L-NAME, aminoguanidine or L-NIL, cNOS or iNOS inhibitors, or with ODQ (sGC inhibitor) or Rp-cGMPS (PKG inhibitor). These results indicate that the NO/sGC/GMP/PKG pathway plays an essential role in the ß-glucan-mediated phagocytosis induced in macrophages by these venom-secretory PLA2s.

Immunization with a low dose of zymosan A confers resistance to depression-like behavior and neuroinflammatory responses in chronically stressed mice.

Stimulation of the innate immune system prior to stress exposure is a possible strategy to prevent depression under stressful conditions. Based on the innate immune system stimulating activities of zymosan A, we hypothesize that zymosan A may prevent the development of chronic stress-induced depression-like behavior. Our results showed that a single injection of zymosan A 1 day before stress exposure at a dose of 2 or 4 mg/kg, but not at a dose of 1 mg/kg, prevented the development of depression-like behaviors in mice treated with chronic social defeat stress (CSDS). The prophylactic effect of a single zymosan A injection (2 mg/kg) on CSDS-induced depression-like behaviors disappeared when the time interval between zymosan A and stress exposure was extended from 1 day or 5 days to 10 days, which was rescued by a second zymosan A injection 10 days after the first zymosan A injection and 4 days (4×, once daily) of zymosan A injections 10 days before stress exposure. Further analysis showed that a single zymosan A injection (2 mg/kg) 1 day before stress exposure could prevent the CSDS-induced increase in pro-inflammatory cytokines in the hippocampus and prefrontal cortex. Inhibition of the innate immune system by pretreatment with minocycline (40 mg/kg) abolished the preventive effect of zymosan A on CSDS-induced depression-like behaviors and CSDS-induced increase in pro-inflammatory cytokines in the brain. These results suggest that activation of the innate immune system triggered by zymosan A prevents the depression-like behaviors and neuroinflammatory responses in the brain induced by chronic stress.

IL-23 regulation of myeloid cell biology during inflammation.

Interleukin (IL)-23 is implicated in the pathogenesis of several inflammatory diseases and is usually linked with helper T cell (Th17) biology. However, there is some data linking IL-23 with innate immune biology in such diseases. We therefore examined the effects of IL-23p19 genetic deletion and/or neutralization on in vitro macrophage activation and in an innate immune-driven peritonitis model. We report that endogenous IL-23 was required for maximal macrophage activation by zymosan as determined by pro-inflammatory cytokine production, including a dramatic upregulation of granulocyte-colony stimulating factor (G-CSF). Furthermore, both IL-23p19 genetic deletion and neutralization in zymosan-induced peritonitis (ZIP) led to a specific reduction in the neutrophil numbers, as well as a reduction in the G-CSF levels in exudate fluids. We conclude that endogenous IL-23 can contribute significantly to macrophage activation during an inflammatory response, mostly likely via an autocrine/paracrine mechanism; of note, endogenous IL-23 can directly up-regulate macrophage G-CSF expression, which in turn is likely to contribute to the regulation of IL-23-dependent neutrophil number and function during an inflammatory response, with potential significance for IL-23 targeting particularly in neutrophil-associated inflammatory diseases.

Dectin-1/SYK Activation Induces Antimicrobial Peptide and Negative Regulator of NF-κB Signaling in Human Oral Epithelial Cells.

BACKGROUND/AIM: Oral epithelial cells serve as the primary defense against microbial exposure in the oral cavity, including the fungus Candida albicans. Dectin-1 is crucial for recognition of ß-glucan in fungi. However, expression and function of Dectin-1 in oral epithelial cells remain unclear. MATERIALS AND METHODS: We assessed Dectin-1 expression in Ca9-22 (gingiva), HSC-2 (mouth), HSC-3 (tongue), and HSC-4 (tongue) human oral epithelial cells using flow cytometry and real-time polymerase chain reaction. Cell treated with ß-glucan-rich zymosan were evaluated using real-time polymerase chain reaction. Phosphorylation of spleen-associated tyrosine kinase (SYK) was analyzed by western blotting. RESULTS: Dectin-1 was expressed in all four cell types, with high expression in Ca9-22 and HSC-2. In Ca9-22 cells, exposure to ß-glucan-rich zymosan did not alter the mRNA expression of chemokines nor of interleukin (IL)6, IL8, IL1ß, IL17A, and IL17F. Zymosan induced the expression of antimicrobial peptides ß-defensin-1 and LL-37, but not S100 calcium-binding protein A8 (S100A8) and S100A9. Furthermore, the expression of cylindromatosis (CYLD), a negative regulator of nuclear factor kappa B (NF-κB) signaling, was induced. In HSC-2 cells, zymosan induced the expression of IL17A. The expression of tumor necrosis factor alpha-induced protein 3 (TNFAIP3), a negative regulator of NF-κB signaling, was also induced. Expression of other cytokines and antimicrobial peptides remained unchanged. Zymosan induced phosphorylation of SYK in Ca9-22 cells, as well as NF-κB. CONCLUSION: Oral epithelial cells express Dectin-1 and recognize ß-glucan, which activates SYK and induces the expression of antimicrobial peptides and negative regulators of NF-κB, potentially maintaining oral homeostasis.

Zymosan and lipopolysaccharide decrease gene expression of neuronal nitric oxide synthase in peripheral organs in chicks.

Nitric oxide (NO) is gaseous bioactive molecule that is synthesized by NO synthase (NOS). Inducible NOS (iNOS) expression occurs in response to pathogenic challenges, resulting in the production of large amounts of NO. However, there is a lack of knowledge regarding neuronal NOS (nNOS) and endothelial NOS (eNOS) in birds during pathogenic challenge. Therefore, the present study was conducted to determine the influence of intraperitoneal (IP) injection of zymosan (cell wall component of yeast) and lipopolysaccharide (LPS, a cell wall component of gram-negative bacteria) on NOS expression in chicks (Gallus gallus). Furthermore, the effect of NOS inhibitors on the corresponding behavioral and physiological parameters was investigated. Zymosan and LPS injections induced iNOS mRNA expression in several organs. Zymosan had no effect on eNOS mRNA expression in the organs investigated, whereas LPS increased its expression in the pancreas. Zymosan and LPS decreased nNOS mRNA expression in the lung, heart, kidney, and pancreas. The decreased nNOS mRNA expression in pancreas was probably associated with the NO from iNOS provided that such effect was reproduced by IP injection of sodium nitroprusside, which is a NO donor. Furthermore, pancreatic nNOS mRNA expression decreased following subcutaneous injection of corticosterone. Furthermore, IP injections of a nonspecific NOS inhibitor, NG-nitro-L-arginine methyl ester, and an nNOS-specific inhibitor, 7-nitroindazole, resulted in the significant decreases in food intake, cloacal temperature, and feed passage via the digestive tract in chicks. Collectively, the current findings imply the decreased nNOS expression because of fungal and bacterial infections, which affects food intake, body temperature, and the digestive function in birds.

Changed regulation of granulocyte NADPH oxidase activity in the mouse model of obesity-induced type 2 diabetes mellitus.

NADPH oxidase is a target of hyperglycemia in type 2 diabetes mellitus (T2DM), which causes dysregulation of enzyme. Alterations in regulation of NADPH oxidase activity mediated receptor and non-receptor signaling in bone marrow granulocytes of mice with obesity-induced T2DM were studied. The animals fed high fat diet (516 kcal/100 g) for 16 weeks. NADPH oxidase-related generation of reactive species (RS) at normo- and hyperthermia was estimated using chemiluminescent analysis. The redox status of the cells was assessed by Redox Sensor Red CC-1. Baseline biochemical indicators in blood (glucose, cholesterol, HDL and LDL levels) were significant higher in T2DM mice versus controls. Using specific inhibitors, signaling mediated by formyl peptide receptors (FPRs) to NADPH oxidase was shown to involve PLC, PKC, cytochrome p450 in both control and T2DM groups and PLA2 in controls. In T2DM regulation of NADPH oxidase activity via mFpr1, a high-affinity receptors, occurred with a significant increase of the role of PKC isoforms and suppression of PLA2 participation. Significant differences between this regulation via mFpr2, low-affinity receptors, were not found. Non-receptor activation of NADPH oxidase with ionomycin (Ca2+ ionophore) or phorbol ester (direct activator of PKC isoforms) did not revealed differences in the kinetic parameters between groups at 37 °C and 40 °C. When these agents were used together (synergistic effect), lower sensitivity of cells to ionophore was observed in T2DM at both temperatures. Redox status in responses to opsonized zymosan was higher in T2DM mice at 37 °C and similar to control levels at 40 °C. ROC-analysis identified Tmax, RS production and effect of opsonized zymosan as the most significant predictors for discriminating between groups. It was concluded that Ca2+-dependent/PKC-mediated regulation of NADPH oxidase activity was altered in BM granulocytes from diabetic mice.

Transcriptomic analysis of turbot (Scophthalmus maximus) treated with zymosan a reveals that lncRNAs and inflammation-related genes mediate the protection conferred against Aeromonas salmonicida.

Aeromonas salmonicida is one of the most harmful pathogens in finfish aquaculture worldwide. Immunostimulants such as ß-glucans are used to enhance the immunity of cultured fish. However, their effects on fish physiology are not completely understood. In the present work, we evaluated the effect of a single intraperitoneal (ip) injection of zymosan A on fish survival against A. salmonicida infection. A single administration of this compound protected fish against A. salmonicida challenge and reduce the bacterial load in the head kidney one week after its administration. Transcriptome analyses of head kidney samples revealed several molecular mechanisms involved in the protection conferred by zymosan A and their regulation by long noncoding RNAs. The transcriptome profile of turbot exposed only to zymosan A was practically unaltered one week after ip injection. However, the administration of this immunostimulant induced significant transcriptomic changes once the fish were in contact with the bacteria and increased the survival of the infected turbot. Our results suggest that the restraint of the infection-induced inflammatory response, the management of apoptotic cell death, cell plasticity and cellular processes involving cytoskeleton dynamics support the protective effects of zymosan A. All this information provides insights on the cellular and molecular mechanisms involved in the protective effects of this widely used immunostimulant.

Dectin-1 ligands produce distinct training phenotypes in human monocytes through differential activation of signaling networks.

Cells of the innate immune system retain memory of prior exposures through a process known as innate immune training. ß-glucan, a Dectin-1 ligand purified from the Candida albicans cell wall, has been one of the most widely utilized ligands for inducing innate immune training. However, many Dectin-1 ligands exist, and it is not known whether these all produce the same phenotype. Using a well-established in vitro model of innate immune training, we compared two commercially available Dectin-1 agonists, zymosan and depleted zymosan, with the gold standard ß-glucan in the literature. We found that depleted zymosan, a ß-glucan purified from Saccharomyces cerevisiae cell wall through alkali treatment, produced near identical effects as C. albicans ß-glucan. However, untreated zymosan produced a distinct training effect from ß-glucans at both the transcript and cytokine level. Training with zymosan diminished, rather than potentiated, induction of cytokines such as TNF and IL-6. Zymosan activated NFκB and AP-1 transcription factors more strongly than ß-glucans. The addition of the toll-like receptor (TLR) ligand Pam3CSK4 was sufficient to convert the training effect of ß-glucans to a phenotype resembling zymosan. We conclude that differential activation of TLR signaling pathways determines the phenotype of innate immune training induced by Dectin-1 ligands.

Sex differences in zymosan-induced behavioral visceral hypersensitivity and colorectal afferent sensitization.

Sex differences in visceral nociception have been reported in clinical and preclinical studies, but the potential differences in sensory neural encoding of the colorectum between males and females are not well understood. In this study, we systematically assessed sex differences in colorectal neural encoding by conducting high-throughput optical recordings in intact dorsal root ganglia (DRGs) from control and visceral hypersensitive mice. We found an apparent sex difference in zymosan-induced behavioral visceral hypersensitivity: enhanced visceromotor responses to colorectal distension were observed only in male mice, not in female mice. In addition, a higher number of mechanosensitive colorectal afferents were identified per mouse in the zymosan-treated male group than in the saline-treated male group, whereas the mechanosensitive afferents identified per mouse were comparable between the zymosan- and saline-treated female groups. The increased number of identified afferents in zymosan-treated male mice was predominantly from thoracolumbar (TL) innervation, which agrees with the significant increase in the TL afferent proportion in the zymosan group as compared with the control group in male mice. In contrast, female mice showed no difference in the proportion of colorectal neurons between saline- and zymosan-treated groups. Our results revealed a significant sex difference in colorectal afferent innervation and sensitization in the context of behavioral visceral hypersensitivity, which could drive differential clinical symptoms in male and female patients.NEW & NOTEWORTHY We used high-throughput GCaMP6f recordings to study 2,275 mechanosensitive colorectal afferents in mice. Our results revealed significant sex differences in the zymosan-induced behavioral visceral hypersensitivity, which were present in male but not female mice. Male mice also showed sensitization of colorectal afferents in the thoracolumbar pathway, whereas female mice did not. These findings highlight sex differences in sensory neural anatomy and function of the colorectum, with implications for sex-specific therapies for treating visceral pain.

Meloxicam treatment disrupts the regional structure of innate inflammation sites by targeting the pro-inflammatory effects of prostanoids.

BACKGROUND AND PURPOSE: Non-steroidal anti-inflammatory drugs (NSAIDs) are the most widely prescribed drugs in the world due to their analgesic, antipyretic and anti-inflammatory effects. However, NSAIDs inhibit prostanoid synthesis, interfering with their pro-inflammatory and anti-inflammatory functions and potentially prolonging acute inflammation. EXPERIMENTAL APPROACH: We used high-content immunohistochemistry to define the impact of meloxicam treatment on spatially separated pro-inflammatory and anti-inflammatory processes during innate inflammation in mice induced by zymosan. This allowed us to determine the effect of meloxicam treatment on the organization of pro-inflammatory and anti-inflammatory microenvironments, thereby identifying relevant changes in immune cell localization, recruitment and activation. KEY RESULTS: Meloxicam treatment reduced zymosan-induced thermal hypersensitivity at early time points but delayed its resolution. High-content immunohistochemistry revealed that the pro-inflammatory area was smaller after treatment, diminishing neutrophil recruitment, M1-like macrophage polarization, and especially phagocytosis by neutrophils and macrophages. The polarization of macrophages towards the M2-like anti-inflammatory phenotype was unaffected, and the number of anti-inflammatory eosinophils actually increased. CONCLUSION AND IMPLICATIONS: High-content immunohistochemistry was able to identify relevant meloxicam-mediated effects on inflammatory processes based on alterations in the regional structure of inflammation sites. Meloxicam delayed the clearance of pathogens by inhibiting pro-inflammatory processes, causing prolonged inflammation. Our data suggest that the prescription of NSAIDs as a treatment during an acute pathogen-driven inflammation should be reconsidered in patients with compromised immune systems.

Doxorubicin-loaded zymosan nanoparticles: Synergistic cytotoxicity and modulation of apoptosis and Wnt/ß-catenin signaling pathway in C26 colorectal cancer cells.

Zymosan is a ß-glucan isolated from Saccharomyces cerevisiae that could be employed for drug delivery. We synthesized zymosan nanoparticles and measured their structural and morphological properties using XRD, UV-Vis spectroscopy, TEM and AFM. The loading of doxorubicin (DOX) onto the nanoparticles was confirmed by FT-IR, and the DOX release was shown to be pH-dependent. The effect of these agents on C26 cell viability was evaluated by MTT tests and the expression of genes connected with the Wnt/ß-catenin pathway and apoptosis were analyzed by RT-qPCR and Western blotting. Treatments were able to suppress the proliferation of C26 cells, and the zymosan nanocarriers loaded with DOX enhanced the anti-proliferative effect of DOX in a synergistic manner. Zymosan nanoparticles were able to suppress the expression of cyclin D1, VEGF, ZEB1, and Twist mRNAs. Treatment groups upregulated the expression of caspase-8, while reducing the Bax/Bcl-2 ratio, thus promoting apoptosis. In conclusion, zymosan nanoparticles as DOX nanocarriers could provide a more targeted drug delivery through pH-responsiveness, and showed synergistic cytotoxicity by modifying Wnt/ß-catenin signaling and apoptosis.

Possible role of neuropeptide Y on zymosan- and lipopolysaccharide-induced change in gastrointestinal feed passage via the medulla oblongata in chicks.

Zymosan is a fungi-derived pathogen-associated molecular pattern. It activates the immune system and induces the reduction of feed passage rate in the gastrointestinal tract of vertebrates including birds. However, the mechanism mediating the zymosan-induced inhibition of feed passage in the gastrointestinal tract remains unknown. Since the medulla oblongata regulates the digestive function, it is plausible that the medulla oblongata is involved in the zymosan-induced inhibition of feed passage. The present study was performed to identify the genes that were affected by zymosan within the medulla oblongata of chicks (Gallus gallus) using an RNA sequencing approach. We found that mRNAs of several bioactive molecules including neuropeptide Y (NPY) were increased with an intraperitoneal (IP) injection of zymosan. The increase of mRNA expression of NPY in the medulla oblongata was also observed after the IP injection of lipopolysaccharide, derived from gram-negative bacteria. These results suggest that medullary NPY is associated with physiological changes during fungal and bacterial infection. Furthermore, we found that intracerebroventricular injection of NPY and its receptor agonists reduced the feed passage from the crop. Additionally, the injection of NPY reduced the feed passage from the proventriculus to lower digestive tract. NPY also suppressed the activity of duodenal activities of amylase and trypsin. The present study suggests that fungi- and bacteria-induced activation of the immune system may activate the NPY neurons in the medulla oblongata and thereby reduce the digestive function in chicks.

A new acyl derivative of sulfadimethoxine inhibits phagocyte oxidative burst and ameliorates inflammation in a mice model of zymosan-induced generalised inflammation.

Chronic inflammation and oxidative stress play a pivotal role in the pathophysiology of most challenging illnesses, including cancer, Alzheimer's, cardiovascular and autoimmune diseases. The present study aimed to investigate the anti-inflammatory potential of a new sulfadimethoxine derivative N-(4-(N-(2,6-dimethoxypyrimidin-4-yl) sulfamoyl) phenyl) dodecanamide (MHH-II-32). The compound was characterised by applying 1H-, 13C-NMR, EI-MS and HRFAB-MS spectroscopic techniques. The compound inhibited zymosan-induced oxidative bursts from whole blood phagocytes and isolated polymorphonuclear cells with an IC50 value of (2.5 ± 0.4 and 3.4 ± 0.3 µg/mL), respectively. Furthermore, the inhibition of nitric oxide with an IC50 (3.6 ± 2.2 µg/mL) from lipopolysaccharide-induced J774.2 macrophages indicates its in vitro anti-inflammatory efficacy. The compound did not show toxicity towards normal fibroblast cells. The observational findings, gross anatomical analysis of visceral organs and serological tests revealed the non-toxicity of the compound at the highest tested intraperitoneal (IP) dose of 100 mg/kg in acute toxicological studies in Balb/c mice. The compound treatment (100 mg/kg) (SC) significantly (P < 0.001) downregulated the mRNA expression of inflammatory markers TNF-α, IL-1ß, IL-2, IL-13, and NF-κB, which were elevated in zymosan-induced generalised inflammation (IP) in Balb/c mice while upregulated the expression of anti-inflammatory cytokine IL-10, which was reduced in zymosan-treated mice. No suppressive effect was observed at the dose of 25 mg/kg. Ibuprofen was taken as a standard drug. The results revealed that the new acyl derivative of sulfadimethoxine has an immunomodulatory effect against generalised inflammatory response with non-toxicity both in vitro and in vivo, and has therapeutic potential for various chronic inflammatory illnesses.

Interception Proximity Labeling for Interrogating Cell Efflux Microenvironment.

The difficulty in elucidating the microenvironment of extracellular H2O2 efflux has led to the lack of a critical extracellular link in studies of the mechanisms of redox signaling pathways. Herein, we mounted horseradish peroxidase (HRP) to glycans expressed globally on the living cell surface and constructed an interception proximity labeling (IPL) platform for H2O2 efflux. The release of endogenous H2O2 is used as a "physiological switch" for HRP to enable proximity labeling. Using this platform, we visualize the oxidative stress state of tumor cells under the condition of nutrient withdrawal, as well as that of macrophages exposed to nonparticulate stimuli. Furthermore, in combination with a proteomics technique, we identify candidate proteins at the invasion interface between fungal mimics (zymosan) and macrophages by interception labeling of locally accumulated H2O2 and confirm that Toll-like receptor 2 binds zymosan in a glycan-dependent manner. The IPL platform has great potential to elucidate the mechanisms underlying biological processes involving redox pathways.

Anti-Inflammatory Activity of Fucan from Spatoglossum schröederi in a Murine Model of Generalized Inflammation Induced by Zymosan.

Fucans from marine algae have been the object of many studies that demonstrated a broad spectrum of biological activities, including anti-inflammatory effects. The aim of this study was to verify the protective effects of a fucan extracted from the brown algae Spatoglossum schröederi in animals submitted to a generalized inflammation model induced by zymosan (ZIGI). BALB/c mice were first submitted to zymosan-induced peritonitis to evaluate the treatment dose capable of inhibiting the induced cellular migration in a simple model of inflammation. Mice were treated by the intravenous route with three doses (20, 10, and 5 mg/kg) of our fucan and, 1 h later, were inoculated with an intraperitoneal dose of zymosan (40 mg/kg). Peritoneal exudate was collected 24 h later for the evaluation of leukocyte migration. Doses of the fucan of Spatoglossum schröederi at 20 and 10 mg/kg reduced peritoneal cellular migration and were selected to perform ZIGI experiments. In the ZIGI model, treatment was administered 1 h before and 6 h after the zymosan inoculation (500 mg/kg). Treatments and challenges were administered via intravenous and intraperitoneal routes, respectively. Systemic toxicity was assessed 6 h after inoculation, based on three clinical signs (bristly hair, prostration, and diarrhea). The peritoneal exudate was collected to assess cellular migration and IL-6 levels, while blood samples were collected to determine IL-6, ALT, and AST levels. Liver tissue was collected for histopathological analysis. In another experimental series, weight loss was evaluated for 15 days after zymosan inoculation and fucan treatment. The fucan treatment did not present any effect on ZIGI systemic toxicity; however, a fucan dose of 20 mg/kg was capable of reducing the weight loss in treated mice. The treatment with both doses also reduced the cellular migration and reduced IL-6 levels in peritoneal exudate and serum in doses of 20 and 10 mg/kg, respectively. They also presented a protective effect in the liver, with a reduction in hepatic transaminase levels in both doses of treatment and attenuated histological damage in the liver at a dose of 10 mg/kg. Fucan from S. schröederi presented a promising pharmacological activity upon the murine model of ZIGI, with potential anti-inflammatory and hepatic protective effects, and should be the target of profound and elucidative studies.

Dynamics of Docosahexaenoic Acid Utilization by Mouse Peritoneal Macrophages.

In this work, the incorporation of docosahexaenoic acid (DHA) in mouse resident peritoneal macrophages and its redistribution within the various phospholipid classes were investigated. Choline glycerophospholipids (PC) behaved as the major initial acceptors of DHA. Prolonged incubation with the fatty acid resulted in the transfer of DHA from PC to ethanolamine glycerophospholipids (PE), reflecting phospholipid remodeling. This process resulted in the cells containing similar amounts of DHA in PC and PE in the resting state. Mass spectrometry-based lipidomic analyses of phospholipid molecular species indicated a marked abundance of DHA in ether phospholipids. Stimulation of the macrophages with yeast-derived zymosan resulted in significant decreases in the levels of all DHA-containing PC and PI species; however, no PE or PS molecular species were found to decrease. In contrast, the levels of an unusual DHA-containing species, namely PI(20:4/22:6), which was barely present in resting cells, were found to markedly increase under zymosan stimulation. The levels of this phospholipid also significantly increased when the calcium-ionophore A23187 or platelet-activating factor were used instead of zymosan to stimulate the macrophages. The study of the route involved in the synthesis of PI(20:4/22:6) suggested that this species is produced through deacylation/reacylation reactions. These results define the increases in PI(20:4/22:6) as a novel lipid metabolic marker of mouse macrophage activation, and provide novel information to understand the regulation of phospholipid fatty acid turnover in activated macrophages.

Modeling complement activation on human glomerular microvascular endothelial cells.

Introduction: Atypical hemolytic uremic syndrome (aHUS) is a rare kidney disease caused by dysregulation of the complement alternative pathway. The complement dysregulation specifically leads to damage to the glomerular endothelium. To further understand aHUS pathophysiology, we validated an ex vivo model for measuring complement deposition on both control and patient human glomerular microvascular endothelial cells (GMVECs). Methods: Endothelial cells were incubated with human test sera and stained with an anti-C5b-9 antibody to visualize and quantify complement depositions on the cells with immunofluorescence microscopy. Results: First, we showed that zymosan-activated sera resulted in increased endothelial C5b-9 depositions compared to normal human serum (NHS). The levels of C5b-9 depositions were similar between conditionally immortalized (ci)GMVECs and primary control GMVECs. The protocol with ciGMVECs was further validated and we additionally generated ciGMVECs from an aHUS patient. The increased C5b-9 deposition on control ciGMVECs by zymosan-activated serum could be dose-dependently inhibited by adding the C5 inhibitor eculizumab. Next, sera from five aHUS patients were tested on control ciGMVECs. Sera from acute disease phases of all patients showed increased endothelial C5b-9 deposition levels compared to NHS. The remission samples showed normalized C5b-9 depositions, whether remission was reached with or without complement blockage by eculizumab. We also monitored the glomerular endothelial complement deposition of an aHUS patient with a hybrid complement factor H (CFH)/CFH-related 1 gene during follow-up. This patient had already chronic kidney failure and an ongoing deterioration of kidney function despite absence of markers indicating an aHUS flare. Increased C5b-9 depositions on ciGMVECs were observed in all samples obtained throughout different diseases phases, except for the samples with eculizumab levels above target. We then tested the samples on the patient's own ciGMVECs. The C5b-9 deposition pattern was comparable and these aHUS patient ciGMVECs also responded similar to NHS as control ciGMVECs. Discussion: In conclusion, we demonstrate a robust and reliable model to adequately measure C5b-9-based complement deposition on human control and patient ciGMVECs. This model can be used to study the pathophysiological mechanisms of aHUS or other diseases associated with endothelial complement activation ex vivo.