Oxidative burst and nitric oxide responses in carp macrophages induced by zymosan, MacroGard(®) and selective dectin-1 agonists suggest recognition by multiple pattern recognition receptors.

ß-Glucans are glucose polymers that are found in the cell walls of plants, bacteria, certain fungi, mushrooms and the cell wall of baker's yeast. In mammals, myeloid cells express several receptors capable of recognizing ß-glucans, with the C-type lectin receptor dectin-1 in conjunction with Toll-like receptor 2 (TLR2), considered key receptors for recognition of ß-glucan. In our studies to determine the possible involvement of these receptors on carp macrophages a range of sources of ß-glucans were utilized including particulate ß-glucan preparations of baker's yeast such as zymosan, which is composed of insoluble ß-glucan and mannan, and MacroGard(®), a ß-glucan-based feed ingredient for farmed animals including several fish species. Both preparations were confirmed TLR2 ligands by measuring activation of HEK293 cells transfected with human TLR2 and CD14, co-transfected with a secreted embryonic alkaline phosphatase (SEAP) reporter gene. In addition, dectin-1-specific ligands in mammals i.e. zymosan treated to deplete the TLR-stimulating properties and curdlan, were monitored for their effects on carp macrophages by measuring reactive oxygen and nitrogen radicals production, as well as cytokine gene expression by real-time PCR. Results clearly show the ability of carp macrophages to strongly react to particulate ß-glucans with an increase in the production of reactive oxygen and nitrogen radicals and an increase in cytokine gene expression, in particular il-1ß, il-6 and il-11. We identified carp il-6, that was previously unknown. In addition, carp macrophages are less, but not unresponsive to selective dectin-1 agonists, suggesting recognition of ß-glucans by multiple pattern recognition receptors that could include TLR but also non-TLR receptors. Candidate receptors for recognition of ß-glucans are discussed.

ß-glucan enriched bath directly stimulates the wound healing process in common carp (Cyprinus carpio L.).

Wound healing is a complex and well-organized process in which physiological factors and immune mechanisms are involved. A number of different immune modulators have been found to enhance the non-specific defence system in vertebrates, among which ß-glucans are the most powerful and extensively investigated. The aim of the present study was to investigate the biological impact of two different commercially available ß glucan containing products on the wound healing process in carp. Throughout a two week experiment fish were kept either untreated (control), or in water supplemented with the two different types of ß-glucans. The wound healing process was monitored using a multispectral visualisation system. The correlation between wound closure and immune response was investigated by measuring the gene expression patterns of IL-1ß, IL-6 family member M17, IL-8 and Muc5b, and measurement of production of radical oxygen species. PAMPs/DAMPs stimulation caused by the wounding and or ß-glucans resulted in an inflammatory response by activating IL-1ß, IL-6 family member M17 and IL-8 and differences in the expression pattern were seen depending on stimuli. IL-1ß, IL-6 family member M17 and IL-8 were activated in all wounds regardless of treatment. Expression of all three interleukins was highly up regulated in control wounded muscle already at day 1 post-wounding and decreased at subsequent time-points. The reverse was the case with control wounded skin, where expression increased from day 1 through day 14. The results for the ß-glucan treated wounds were more complex. The images showed significantly faster wound contraction in both treated groups compared to the control. The obtained results clearly demonstrated that a ß glucan enriched bath promotes the closure of wounds in common carp and induce a local change in cytokine expression.

Comparative study of ß-glucan induced respiratory burst measured by nitroblue tetrazolium assay and real-time luminol-enhanced chemiluminescence assay in common carp (Cyprinus carpio L.).

The respiratory burst is an important feature of the immune system. The increase in cellular oxygen uptake that marks the initiation of the respiratory burst is followed by the production of reactive oxygen species (ROS) such as superoxide anion and hydrogen peroxide which plays a role in the clearance of pathogens and tissue regeneration processes. Therefore, the respiratory burst and associated ROS constitute important indicators of fish health status. This paper compares two methods for quantitation of ROS produced during the respiratory burst in common carp: the widely used, single-point measurement based on the intracellular reduction of nitroblue tetrazolium (NBT) and a real-time luminol-enhanced assay based on the detection of native chemiluminescence. Both assays allowed for detection of dose-dependent changes in magnitude of the respiratory burst response induced by ß-glucans in head kidney cells of carp. However, whereas the NBT assay was shown to detect the production of only superoxide anions, the real-time luminol-enhanced assay could detect the production of both superoxide anions and hydrogen peroxide. Only the chemiluminescence assay could reliably record the production of ROS on a real-time scale at frequent and continual time intervals for time course experiments, providing more detailed information on the respiratory burst response. The real-time chemiluminescence assay was used to measure respiratory burst activity in macrophage and neutrophilic granulocyte-enriched head kidney cell fractions and total head kidney cell suspensions and proved to be a fast, reliable, automated multiwell microplate assay to quantitate fish health status modulated by ß-glucans.

Carp head kidney leukocytes display different patterns of oxygen radical production after stimulation with PAMPs and DAMPs.

Wound healing and tissue regeneration are essential mechanisms to ensure the survival and health of any organism. Despite this, only a few studies have been devoted to study tissue regeneration during wound healing in fish. Reactive oxygen species (ROS), in particular hydrogen peroxide, play an important dual role both for promoting tissue repair, but also for eradication of pathogens. This study aims at dissecting the contribution of PAMPs (using ß-glucan) and DAMPs in the respiratory burst response of carp head kidney-derived leukocytes, and address their contribution to wound healing processes. Consistent with a pathogen eradication strategy, ROS responses to PAMP stimulation (ß-glucan) was fast, vigorous and highly dominated by production of superoxide anion. In contrast, stimulation with DAMPs led to a slow, subtle but long-lasting production of oxygen radicals dominated by hydrogen peroxide. Using an in vitro model of scratch-wounded CCB fibroblast cell cultures and a novel PhotoID proliferation assay, stimulation with low and continuous levels of hydrogen peroxide (5 µM) led to a slight increase in the percentage of wound recovery and thus promoted wound closure. In contrast, high doses of hydrogen peroxide (300 µM) impaired fibroblast scratch-wound recovery and caused cell death. These results elucidate the capacity of hydrogen peroxide to influence the fate of tissue regeneration through the establishment of environments suitable for promoting either tissue regeneration or oxidative stress and thereby potential tissue damage. Direct in vitro stimulation with ß-glucans did not impact fibroblast scratch-wound recovery, which further suggests that interaction with tissue-resident leukocytes or other components of the fish immune system are required to induce fibroblast proliferation and thus for the accelerated wound healing promoted by ß-glucan stimulation.

Cell cultures from the symbiotic soft coral Sinularia flexibilis.

The symbiotic octocoral Sinularia flexibilis is a producer of potential pharmaceuticals. Sustainable mass production of these corals as a source of such compounds demands innovative approaches, including coral cell culture. We studied various cell dissociation methodologies and the feasibility of cultivation of S. flexibilis cells on different media and cell dissociation methodologies. Mechanical dissociation of coral tissue always yielded the highest number of cells and allowed subsequent cellular growth in all treatments. The best results from chemical dissociation reagents were found with trypsin-ethylene diamine tetraacetic acid. Coral cells obtained from spontaneous dissociation did not grow. Light intensity was found to be important for coral cell culture showing an enduring symbiosis between the cultured cells and their intracellular algae. The Grace's insect medium and Grace's modified insect medium were found to be superior substrates. To confirm the similarity of the cultured cells and those in the coral tissue, a molecular test with Internal Transcribed Spacer primers was performed. Thereby, the presence of similar cells of both the coral cells and zooxanthella in different culture media was confirmed.