To better understand the function of a particular Tlr molecule, identification of its ligand(s) is crucial. Studies on mammalian Tlrs have identified a range of ligands for the different Tlrs, shed-ding light on their role in innate immunity. Although these studies may be helpful to direct studies on TLR orthologs in fish, true ligands of fish Tlrs have to be identified with functional studies. Of course, when studying non-mammalian Tlrs such as, for example, Tlr20 (chapter 4) there are no studies in mammalian vertebrates than can be referred to. Many conclusions on the putative func-tions of fish Tlrs and their potential ligands have been based on studies reporting upregulation of tlr gene expression by RT-qPCR. Of course, studying up-regulation of tlr gene expression can provide indirect evidence of their function (chapters 4, 6). However, many of these studies show a bias in their choice of ligands that are selected based on the assumption that fish equal mouse and/
or human with respect to ligand recognition by their TLR reportoire. More objective it may be to screen microarray or deep-sequencing datasets for changes in TLR gene expression after infection with whole pathogens.
One of the most common routes taken, and proven successful in the determination of ligands for several TLR [57, 58], is to overexpress the TLR of interest in a cell line that does not naturally express this receptor, for example a human cell line such as the HEK 293. Under the assumption that HEK 293 signalling pathway is conserved between human and fish. Following overexpression, activation by a ligand can be determined by reading, for example, induced levels of luciferase activ-ity using a reporter construct (chapters 4, 5). The reporter activactiv-ity is most often based on activation of NF-κB, a transcription factor highly conserved [59]. In our studies we did not manage to exploit NF-κB-based reporter assays in HEK 293 cells to unequivocally determine ligands for Tlr20 nor Tlr4. Hypothesizing that human cell lines such as HEK 293 may be too different from fish cells in crucial aspects to support ligand-determining studies, we also tried fish cell lines. The CLC (carp leukocyte culture) cell line originates from blood leukocytes of common carp [60], but may have become contaminated in many laboratories by cells from other, non-carp, cell lines (unpublished observations). The EPC (epithelioma papulosum cyprini) cell line originated from carp epidermal herpes virus-induced hyperplastic lesions with a reduced chromosome number (n = 94) compared with common carp [61]. Indeed, it was later discovered to be contaminated with cyprinid fathead minnow [62], but these cells are still considered close relatives of common carp. Thus, in theory, the EPC should support functional studies on carp molecules. Yet, NF-κB-based reporter assays using EPC cells did not unequivocally determine ligands for Tlr20 nor Tlr4. Most recently, we tried to re-fine our approach as to include a reporter construct based on activation of AP-1, another transcrip-tion factor highly conserved [63]. Again, we did not manage to unequivocally determine ligands for Tlr20 nor Tlr4. Future studies should focus on the use of other cell lines derived from carp.
Sub-cellular localization studies on Tlr20 and Tlr4 pointed at intracellular expression of these receptors in human (HEK 293) and fish cell lines. To date, the exact function of the
non-mammali-8
an Tlr20 is unknown (chapter 4) and the same is true for Tlr4 (chapter 5). Given these uncertain-ties, it may well be possible that these Tlrs are expressed intracellulary and not on the cell surface.
It may also be possible that overexpression of these molecules will lead to an aberrant expression inside the cell, for example, in the early secretory pathway, endoplasmic reticulum-associated deg-radation and autophagic pathways specialize in seeking misfolded polypeptides and mediate their degradation [64]. Where aberrant expression patterns of foreign molecules in human cell lines such as HEK 293 could be a true possibility, overexpression of carp Tlrs in EPC cells should lead to cor-rect expression patterns, however.
In this thesis we report the identification, in fish genomes, of a large number of molecules ac-cessory to the function of mammalian Tlrs (chapter 7). It could be a realistic option that human cell lines such as HEK 293 express accessory molecules that are (too) different from the fish orthologs to function optimally in conjunction with fish Tlrs, such as carp Tlr4 or Tlr20. At the same time, we can expect these accessory molecules in the fathead minnow cell line (EPC), to be most similar to those in carp. Yet, despite the genetic relation of the two fish species, the fact that these species have different number of chromosomes may result in their inability tot express all the molecules that other (leukocyte) cell types would express. In addition, it is not uncommon that cell lines grown for many years in vitro built up mistakes in their genomes [65]. Therefore, we examined the transcrip-tome of the EPC cell line (courtesy of H.P. Spaink and ZF Screens, Leiden, the Netherlands) for the presence of known accessory molecules (Table 1).
As can be concluded from the overview in Figure 4, not all accessory proteins previously thought to be involved in Tlr function were actually present in the transcriptome of the EPC cell line. For example, EPC cells do not express bpi/lbp, cd36, tril and unc93b1 that can be found in the carp genome. The apparent absence of these molecules in EPC could have affected the functional characterization of Tlrs overexpressed in this cell line. Of the missing accessory molecules, three (bpi/lbp, cd36 and tril) are mediators of ligand delivery and or recognition and one (unc93b1) is a trafficking factor. Trafficking factors are thought to help localize the Tlr into the correct sub-cellular compartment whereas mediators of ligand delivery and/or recognition may help sense and/or de-liver ligands. Future studies should focus on expressing one or several of the missing accessory mol-ecules in EPC to study their role in the functioning of fish Tlrs, among which are Tlr20 and Tlr4.
Overall, the molecular and functional characterization of a number of Tlrs and associated molecules considered important for the carp innate immune system, as described in this thesis, has allowed for much progress on the knowledge of fish Tlrs. Although the search for unique receptors on carp leukocytes sensing β-glucans may still be ongoing, the research described in this thesis has already contributed to the valorization and use of β-glucans as immunostimulants for sustainable aquaculture, potentially achieving a strategic improvement of fish health.
Table 1 Comparison of accessory molecules between carp genome and the EPC transcriptome. a bpi/lbp ancestral gene of LBP; b High mobility group-T protein; c unc93b1-like protein MFSD11; d cathepsins f.
Accessory molecules Carp genome EPC transcriptome
TLR processing factors cathepsind + +
aep + +
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