Antecedentes del problema

The NEMO network formed a nucleus of young scientists, of which one more PhD student (Inge Rosenbek Fink) and one post-doc fellow (Anders Østergaard) were appointed at Wageningen University, Cell Biology and Immunology group. Next to the work described in this thesis, they undertook a molecular and functional characterization of carp Tlr1 and Tlr2, in combination with the scavanger receptor Cd36 (Inge R. Fink). And, carp scavanger receptor ‘Scarf-1’ or C-type lectin receptors ‘Illrs’ (Anders Østergaard) as candidate receptors for recognition of β-glucans. The preliminary results of these studies in progress are relevant to the research described in this thesis and shortly discussed below.

Full-length cDNA sequences of carp Tlr1 and Tlr2 were obtained and synteny analyses support that these molecules are orthologs of mammalian TLR1 and TLR2. Real-time quantitative PCR analysis of basal gene expression revealed expression in immune relevant organs. Of interest, Tlr1 was found to be expressed mainly by B cells and neutrophils, and to a lower extent by macrophages, whereas Tlr2 was expressed by B cells and macrophages. The full-length sequences were used to create fluorescent protein-tagged receptors to visualize their sub-cellular localization. At this moment, transfection experiments are being carried out to determine binding of ligands, among which β-glucan but also previously identified ligands for TLR2 [29], in both human and fish cell lines. Based on conservation of amino acids known in mammals to be important for heterodimer formation, it can be hypothesized that heterodimer formation between Tlr1 and Tlr2 can also occur in common carp [30].

Two full-length scarf-1 genes have been obtained from the common carp genome, confirming

the hypothesis that carp has undergone an additional genome duplication event compared to zebrafish [31], where only one scarf-1 gene is found. Sequence analysis of Scarf-11 revealed a high level of amino acid conservation compared to the human orthologue, whereas synteny studies show conserved linkage with several neighbouring genes when comparing genomic regions from different species. Gene expression analysis of scarf-1 showed highest expression in endothelial cells, but macrophages, granulocytes, thrombocytes and thymocytes also showed scarf-1 gene expression.

Carp cd36, in contrast to scarf-1, was expressed at low levels only in carp leukocytes. Sub-cellular localization of Scarf-1 and Cd36 was studied by confocal microscopy of cell lines transfected with a fluorescently tagged receptor. These studies showed these receptors to be expressed at the cell surface. Experiments are being carried out to study the possible roles of Scarf-1 and Cd36 as internalizing receptors that could facilitate TLR activation upon phagocytosis of ligands, such as β-glucan [32].

There are more receptors that could be candidates for recognition of β-glucans in fish. In mammals, myeloid cells express several receptors capable of recognizing β-glucan, with the C-type lectin receptor (CLR) Dectin-1 in conjunction with TLR2, considered key receptors for recognition of β-glucan. In our studies we could clearly show that carp macrophages are less, but not unresponsive to selective Dectin-1 agonists, suggesting recognition of β-glucan by multiple pattern recognition receptors that could include TLR but also non-TLR receptors of the CLR superfamily (chapter 3). Within the CLR superfamily not only Dectin-1, but also Dectin-2 and Dectin-3 may have an active role in the recognition of β-glucan. Dectin-3 forms a heterodimer with Dectin-2 for recognizing infection with fungi and it is suggested that CLRs may in fact form a scala of different hetero- and homodimers providing host cells with receptors to detect a scala of microbial infections [33]. Although (all) dectin receptors may be absent from fish genomes [26], other CLRs may well play roles in the recognition of β-glucans in fish. Several CLRs of fish have been characterized that show characteristics of both group II and group V receptors. The family of immune-related, lectin-like receptors (Illrs), that are part of the group II CLRs and possess inhibiting and/or activating signalling motifs typical of group V receptors, could be candidate receptors for recognition of β-glucan. The illr genes are differentially expressed in the myeloid and lymphoid lineages in zebrafish [34] supporting the idea that illrs may function in natural killer (NK) as well as in myeloid cells. In mammals, the first line of defence is represented by macrophages and NK cells, both cell types well developed in teleosts. Two types of NK cell homologues have been described in fish: non-specific cytotoxic cells and NK-like cells [35]. In carp, illr genes have been identified, and multiple alignment have shown high identity between illr genes in zebrafish and carp suggesting that carp illrs are homolog of zebrafish illrs. The basal gene expression level of carp illrs show a similar expression pattern as observed in zebrafish. (Østergaard, unpublished data).

Besides Dectin-1-like receptors that can detect β-glucan and trigger antimicrobial activity such as phagocytosis and production of reactive oxygen species, the immunoglobulin (Ig) Fc receptor (FcγR) can also play a functional role in the activation of the phagocytosis process [36].

Likewise, the complement receptor 3 (CR3) is involved in the uptake of particulate β-glucan in mice; C3 enhances the uptake of particulate β-glucan and C3 “knockout” mice show reduced capacity to phagocytose [37]. Autophagy is an intracellular degradation process with a number of roles, one of which can be the protection of eukaryotic cells from invading microbes. Microtubule-associated protein light-chain 3 (LC3) is a key autophagy-related protein that is recruited to the double-membrane autophagosome responsible for sequestering material intended for delivery to lysosomes. LC3 can also be recruited to other membranes including single-membrane phagosomes,

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in a process termed LC3-associated phagocytosis (LAP). Dectin-1 mediates a phagocytic response to β-glucan via this mechanism [38]. Dectin-1 may be absent from fish genomes, but also TLRs can engage in autophagy through the activation of LC3, facilitating rapid recruitment to the phagosome [39].

Based on existing data in the literature, we hypothesize that recognition of β-glucan in fish could be by an interplay of phagocytic receptors and TLRs (Figure 2). Phagocytic receptors such as scavenger receptors, C-type lectin receptors (CLR) and/or FcγR receptors could bind β-glucan and activate phagocytosis, leading to a higher expression of TLRs in the phagosome that could sense β-glucan. The TIR domain of the TLRs could then activate the signal cascade that would lead to NF-κB activation and an upregulation of expression of cytokines such il-1β, il-6 and il-11, as well as production of reactive oxygen and nitrogen species that would help clear phagocytosed β-glucan.

Figure 2. Possible mechanism for recognition of particulate β-glucans in fish. Particulate β-glucans are recognizes by phagocytic receptors such C-type lectin (CLR), the immunoglobulin (Ig) Fc receptor (FCγR) or scavenger receptors (such Scarf-1 and Cd36) which will activate the phagocytosis process. Activation leads to stimulation of TLRs that sense β-glucan and leads to possible recruitment of (microtubule-associated protein light-chain 3) LC3. The activation of the TIR domain and the following intracellular signal cascade leads to the production of oxygen radicals (ROS) and nitrogen radicals (NO) and upregulation of cytokines such il-1β, il-6 and il-11.

Of interest also, are recent in vivo studies on whole fungal pathogens including Candida albicans. The innate immune system controls Candida infection in part through Dectin-1. C.

albicans masks the presence of β-glucan early during infection, but it becomes exposed later, allowing Dectin-1 to recognize Candida and mediate immunity [40]. In a recent (2013) paper,

microarray data from zebrafish infected with Candida albicans were used to describe an intercellular protein-protein interaction (PPI) network between host and pathogen. As part of this extensive work, important defense-related proteins in zebrafish were predicted [41] (see Figure 3). This dataset may be of particular interest for the identification of β-glucan receptors in fish, identifying in an unbiased manner candidate receptors for β-glucans for future research. This dataset may be of particular interest for the identification of β-glucan receptors in fish, identifying in an unbiased manner candidate receptors for β-glucans for future research. Important to understand which receptors sense the β-glucan it will be investigation of the The figure was taken and adapted from [41].

Figure 3. Protein-protein interaction (PPI) network during C. albicans infection of zebrafish. The figure shows protein-protein interaction (PPI) between the Candida albicans and Danio rerio. Microarray data from C. albicans and D.rerio were combined to show the proteins interaction between host and pathogen. The red box indicated the region were may be possible to identify the β-glucan receptors in fish.