Placozoan fiber cells: mediators of innate immunity and participants in wound healing.

Placozoa is a phylum of non-bilaterian marine animals. These small, flat organisms adhere to the substrate via their densely ciliated ventral epithelium, which mediates mucociliary locomotion and nutrient uptake. They have only six morphological cell types, including one, fiber cells, for which functional data is lacking. Fiber cells are non-epithelial cells with multiple processes. We used electron and light microscopic approaches to unravel the roles of fiber cells in Trichoplax adhaerens, a representative member of the phylum. Three-dimensional reconstructions of serial sections of Trichoplax showed that each fiber cell is in contact with several other cells. Examination of fiber cells in thin sections and observations of live dissociated fiber cells demonstrated that they phagocytose cell debris and bacteria. In situ hybridization confirmed that fiber cells express genes involved in phagocytic activity. Fiber cells also are involved in wound healing as evidenced from microsurgery experiments. Based on these observations we conclude that fiber cells are multi-purpose macrophage-like cells. Macrophage-like cells have been described in Porifera, Ctenophora, and Cnidaria and are widespread among Bilateria, but our study is the first to show that Placozoa possesses this cell type. The phylogenetic distribution of macrophage-like cells suggests that they appeared early in metazoan evolution.

Transcriptomic Insights into Innate Immunity Responding to Red Rot Disease in Red Alga Pyropia yezoensis.

Pyropia yezoensis, one of the most economically important marine algae, suffers from the biotic stress of the oomycete necrotrophic pathogen Pythium porphyrae. However, little is known about the molecular defensive mechanisms employed by Pyr. yezoensis during the infection process. In the present study, we defined three stages of red rot disease based on histopathological features and photosynthetic physiology. Transcriptomic analysis was carried out at different stages of infection to identify the genes related to the innate immune system in Pyr. yezoensis. In total, 2139 up-regulated genes and 1672 down-regulated genes were identified from all the infected groups. Pathogen receptor genes, including three lectin genes (pattern recognition receptors (PRRs)) and five genes encoding typical plant R protein domains (leucine rich repeat (LRR), nucleotide binding site (NBS), or Toll/interleukin-1 receptor (TIR)), were found to be up-regulated after infection. Several defense mechanisms that were typically regarded as PAMP-triggered immunity (PTI) in plants were induced during the infection. These included defensive and protective enzymes, heat shock proteins, secondary metabolites, cellulase, and protease inhibitors. As a part of the effector-triggered immunity (ETI), the expression of genes related to the ubiquitin-proteasome system (UPS) and hypersensitive cell death response (HR) increased significantly during the infection. The current study suggests that, similar to plants, Pyr. yezoensis possesses a conserved innate immune system that counters the invasion of necrotrophic pathogen Pyt. porphyrae. However, the innate immunity genes of Pyr. yezoensis appear to be more ancient in origin compared to those in higher plants.

1-Octen-3-ol, a self-stimulating oxylipin messenger, can prime and induce defense of marine alga.

BACKGROUND: Short chain oxylipins in plants as the main volatile organic carbon have been speculated to playing an important role for plant innate immunity, however, not yet intensively studied and far away established as the fully recognized algae defense signals. RESULTS: The production of 1-octen-3-ol is self-amplified via the fatty acid-oxylipin metabolic cycle through positive feedback loop. Production of 1-octen-3-ol may act as a messenger that induces P. haitanensis to be in a "primed" state and ready for defense by upregulating the synthesis of methyl jasmonic acid, indole-3-acetic acid, and gibberellin A3. Production of these oxylipins also adjust the redox state in cells, resulting in host defense activation. CONCLUSIONS: We provide the first demonstration that 1-octen-3-ol from P. haitanensis, can act as a self-stimulating community messenger. The multiple effects of 1-octen-3-ol may explain why P. haitanensis, a very ancient lineage within plant kingdom, thrives in the niche of intertidal zones.

3­Bromo­5­(ethoxymethyl)­1,2­benzenediol inhibits LPS-induced pro-inflammatory responses by preventing ROS production and downregulating NF-κB in vitro and in a zebrafish model.

The anti-inflammatory effects of 3­bromo­5­(ethoxymethyl)­1,2­benzenediol (BEMB) from Polysiphonia morrowii were evaluated in lipopolysaccharide (LPS)-induced RAW264.7 cells and zebrafish embryo. BEMB showed anti-inflammatory effects by inhibiting the production of nitric oxide (NO) and reactive oxygen species (ROS), and the expression of inducible NO synthase (iNOS) and cyclooxygenase-2 (COX-2) in the LPS-activated RAW264.7 cells and zebrafish embryo without cytotoxicity. Moreover, BEMB suppressed the protein and mRNA expression levels of nuclear factor (NF)-κB (p65 and inhibitor of NF-κB [IκB]-A) in RAW264.7 cells and zebrafish embryo, respectively. Collectively, the results of this study indicate that BEMB suppressed the production of pro-inflammatory mediators such as NO, iNOS, and COX-2 as well as their regulation in LPS-induced RAW264.7 cells and zebrafish embryos by inhibiting ROS production and NF-κB expression. Therefore, this study suggests that BEMB could be a potential anti-inflammatory agent for the treatment of inflammatory diseases.

Lithothamnion muelleri controls inflammatory responses, target organ injury and lethality associated with graft-versus-host disease in mice.

Lithothamnion muelleri (Hapalidiaceae) is a marine red alga, which is a member of a group of algae with anti-inflammatory, antitumor, and immunomodulatory properties. The present study evaluated the effects of treatment with Lithothamnion muelleri extract (LM) in a model of acute graft-versus-host disease (GVHD), using a model of adoptive splenocyte transfer from C57BL/6 donors into B6D2F1 recipient mice. Mice treated with LM showed reduced clinical signs of disease and mortality when compared with untreated mice. LM-treated mice had reduced tissue injury, less bacterial translocation, and decreased levels of proinflammatory cytokines and chemokines (interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α), chemokine (C-C motif) ligand 2 (CCL2), chemokine (C-C motif) ligand 3 (CCL3) and chemokine (C-C motif) ligand 5 (CCL5)). The polysaccharide-rich fraction derived from LM could inhibit leukocyte rolling and adhesion in intestinal venules, as assessed by intravital microscopy. LM treatment did not impair the beneficial effects of graft-versus-leukaemia (GVL). Altogether, our studies suggest that treatment with Lithothamnion muelleri has a potential therapeutic application in GVHD treatment.

Costs and benefits of chemical defence in the Red Alga Bonnemaisonia hamifera.

A number of studies have shown that the production of chemical defences is costly in terrestrial vascular plants. However, these studies do not necessarily reflect the costs of defence production in macroalgae, due to structural and functional differences between vascular plants and macroalgae. Using a specific culturing technique, we experimentally manipulated the defence production in the red alga Bonnemaisonia hamifera to examine if the defence is costly in terms of growth. Furthermore, we tested if the defence provides fitness benefits by reducing harmful bacterial colonisation of the alga. Costly defences should provide benefits to the producer in order to be maintained in natural populations, but such benefits through protection against harmful bacterial colonisation have rarely been documented in macroalgae. We found that algae with experimentally impaired defence production, but with an externally controlled epibacterial load, grew significantly better than algae with normal defence production. We also found that undefended algae exposed to a natural epibacterial load experienced a substantial reduction in growth and a 6-fold increase in cell bleaching, compared to controls. Thus, this study provides experimental evidence that chemical defence production in macroalgae is costly, but that the cost is outweighed by fitness benefits provided through protection against harmful bacterial colonisation.

Production and characterization of a phage-display recombinant antibody against carrageenans: evidence for the recognition of a secondary structure of carrageenan chains present in red algae tissues.

We report the isolation, for the first time by phage display, of a scFv recombinant antibody called B3 directed against carrageenans, the major sulphated polysaccharides of red seaweeds. Immunoassays were used to characterize the binding of B3 antibodies toward the three main carrageenan forms (iota, kappa, and lambda) differing by their sulfonic ester content and the presence of 3,6-anhydrogalactose. In enzyme-linked immunoadsorbent assay (ELISA), B3 soluble scFv showed a high reactivity towards iota-carrageenan at any titer but, at high titer only, recognized also the highly sulfated lambda-form. Surface-adsorbed kappa-polymers were only recognized in presence of poly-L-lysine (PLL). The replacement of Na+ ions by K+ in the buffers had no effect on kappa-polymer detection but increased the binding of B3 antibodies toward both iota- and lambda-carrageenans, whereas addition of Ca2+ decreased sharply the recognition of the iota-form. In competitive assays, low titer B3 soluble scFv showed a iota>kappa>lambda selectivity and recognized a mixture of iota-oligomers with degrees of polymerization between 4 and 18 but not sub-fractions of 4 or 6 residues long. We suggest therefore that the B3 epitope could consist of a helical conformation of carrageenan chains. Immunofluorescence microscopy showed that, amongst other red algae, Chondrus gametophyte (containing iota-chains) was strongly recognized by B3 scFv whereas sporophytic tissues rich in lambda-carrageenans were not, assessing the preference of this probe for iota-carrageenans in situ. The high potential of the B3 recombinant probe is discussed.

The innate immunity of a marine red alga involves oxylipins from both the eicosanoid and octadecanoid pathways.

The oxygenated derivatives of fatty acids, known as oxylipins, are pivotal signaling molecules in animals and terrestrial plants. In animal systems, eicosanoids regulate cell differentiation, immune responses, and homeostasis. In contrast, terrestrial plants use derivatives of C18 and C16 fatty acids as developmental or defense hormones. Marine algae have emerged early in the evolution of eukaryotes as several distinct phyla, independent from the animal and green-plant lineages. The occurrence of oxylipins of the eicosanoid family is well documented in marine red algae, but their biological roles remain an enigma. Here we address the hypothesis that they are involved with the defense mechanisms of the red alga Chondrus crispus. By investigating its association with a green algal endophyte Acrochaete operculata, which becomes invasive in the diploid generation of this red alga, we showed that (1) when challenged by pathogen extracts, the resistant haploid phase of C. crispus produced both C20 and C18 oxylipins, (2) elicitation with pathogen extracts or methyl jasmonate activated the metabolism of C20 and C18 polyunsaturated fatty acids to generate hydroperoxides and cyclopentenones such as prostaglandins and jasmonates, and (3) C20 and C18 hydroperoxides as well as methyl jasmonate did induce shikimate dehydrogenase and Phe ammonialyase activities in C. crispus and conferred an induced resistance to the diploid phase, while inhibitors of fatty acid oxidation reduced the natural resistance of the haploid generation. The dual nature of oxylipin metabolism in this alga suggests that early eukaryotes featured both animal- (eicosanoids) and plant-like (octadecanoids) oxylipins as essential components of innate immunity mechanisms.

Macrophage stimulation activity of the polysaccharide fraction from a marine alga (Porphyra yezoensis): structure-function relationships and improved solubility.

The polysaccharide fraction from Porphyra yezoensis (PASF) has already been shown to stimulate murine phagocytic functions in vivo and in vitro [Y. Yoshizawa et al., Biosci. Biotech. Biochem., 57, 1862-1866 (1993)]. In this study, various treatments were applied to PASF to assess its structure-function relationships. Desulfation of PASF decreased in vitro macrophage-stimulation activity, while further sulfation of PASF did not change the activity. Among 7 fractions obtained by anion-exchange chromatography of PASF, stronger activity was found in the fractions having a lower or higher sulfate content than in those having a medium sulfate content. Digests of PASF with beta-agarase showed higher activity and solubility, and lower viscosity, than undigested PASF. These results indicate that the sulfate groups in PASF, probably porphyran, contributed to the macrophage stimulating activity, although a larger number of sulfate groups did not always cause stronger activity.

Carrageenans, highly sulfated polysaccharides and macrophage-toxic agents: newly found human T lymphocyte mitogens.

We obtained the following results, using carrageenans (CGNs) of three types (kappa, lambda and iota), macrophage-toxic agents. 1. CGNs were toxic to human monocytes, cytotoxicity varying from 40% to 60%, as judged by trypan blue dye exclusion test. 2. CGNs were proved to be newly found mitogens for human T cells, whereas CGNs are polyclonal B-cell activators in mice and induced DNA synthesis that peaked at days 6 or 7. In addition, iota CGN induced an early peak of 3H-thymidine uptake at day 1, which found out to be cytoplasmic uptake of 3H-thymidine. 3. Only lambda and iota CGN induced slightly higher DNA synthesis in purified B cells, but both failed to induce polyclonal antibody synthesis in B cells. 4. In spite of the fact the CGNs are macrophage-toxic agents, CGNs could induce Il-1 production by the surviving monocytes. Thus CGN-induced human T-cell activation requires a relatively smaller number of monocytes. 5. The mitogenic responses of T cells induced by kappa, lambda and iota CGN were significantly inhibited by cyclosporin A (CyA) treatment (250 ng/ml) and Con A and PHA responses were also inhibited by CyA addition. It was concluded that CGNs were newly found human T-cell mitogens, which is in sharp contrast with polyclonal B-cell activators in mice, and the mechanism of T-cell activation induced by CGNs was the same as that by Con A or PHA, though there may be another possibility of inhibition mechanism by CyA.

The immunochemistry of lambda-type carrageenans from certain red algae.

An antibody preparation directed against a structural feature associated with 6-sulphate groups was used to probe structural relations among certain lambda-type carrageenans. Immunochemical and chemical differences are described between the KC1-soluble carrageenans from tetrasporic algal plants of Gigartina corymbifera, Gigartina sp. from San Francisco Bay, Petrocelis middendorfii, Iridaea cordata, Rhodoglossum californicum, and Chondrus crispus. The differences in immunochemical reactivity of the Gigartina and Petrocelis carrageenans relative to the homologous antigen (Chondrus crispus lambda-carrageenan) are attributed to the lower content of 6-sulphate groups on the 4-linked residues in the former carrageenans. Both the immunochemical and chemical data suggest that the Gigartina and Petrocelis carrageenans are largely xi-like in structure but do contain lambda-like features. The i.r. spectrum of the Petrocelis carrageenan differs from that of the Gigartina carrageenans. The carrageenans from I. cordata and R. californicum differ to a lesser degree from Chondrus crispus lambda-type carrageenan. These differences cannot be accounted for by differences in the levels of 6-sulphate groups. Some other structural feature, as yet unidentified, is responsible for the discrepancy in the immunochemical reactivity of these carrageenans to the anti-lambda-carrageenan.

The chemistry and immunochemistry of carrageenans from Eucheuma and related algal species.

Carrageenans from several species of Eucheuma have been fractionated into KC1-soluble and KC1-insoluble fractions and analyzed by the usual chemical procedures. An anti-kappa-carrageenan, the reactivity of which is directed to kappa-structures (i.e., 3-linked galactose 4-sulphate, and 4-linked 3,6-anhydrogalactose) was used to analyze these carrageenans immunochemically. The antibody preparation shows only a small amount of cross-reactivity with iota-type carrageenans and thus could be used to distinguish kappa- and iota-type carrageenans, the latter having an index of homology of less than 0.2. A comparison of chemical and immunochemical data yielded further information as to the nature of the carrageenan-anti-carrageenan interaction, as well as elucidating the finer structure of carrageenans.

Comparative immunology of algal biliproteins.

The three spectroscopically distinct classes of phycobiliproteins characteristic of the Cyanophyta and Rhodophyta-phycocyanins, allophycocyanins, and phycoerythrins-share no common antigenic determinants detectable by the Ouchterlony double diffusion technique. Each class of phycobiliprotein, from both Cyanophyta and Rhodophyta, possesses a strong determinant common to all members of that class. With respect to an antiserum directed against a specific cyanophytan biliprotein, all heterologous biliproteins of the same class are immunologically identical, as shown by the fact that absorption with a given heterologous antigen simultaneously eliminates crossreactions with other heterologous antigens. A cryptophytan phycoerythrin was found to be immunologically unrelated to any of the cyanophytan or rhodophytan biliproteins examined.