VII. Results
2. Materials and methods
3.2 ELISA based binding studies
In order to verify the functionality of the newly produced ovine CTLR hFc-fusion proteins, ELISA-based binding studies were performed. Given the observed amino acid sequence similarity of CTLRs in different species (Fig. 1), we hypothesized that known ligands of murine and human CTLRs might be recognized by ovine CTLRs as well. For Dectin-1 testing, a β-glucan zymosan (Saijo and Iwakura, 2011) derived from Saccharomyces cerevisiae yeast, was selected. Zymosan is a common fungal and yeast cell wall constituent. For Dectin-2 and Langerin testing, α-linked mannose polysaccharide mannan (Hanske et al., 2017; Saijo and Iwakura, 2011) prepared from Saccharomyces cerevisiae cell wall via alkaline extraction was selected. For Mincle testing, a synthetic mycobacterial cord factor analog Trehalose-6,6-dibehenate (TDB) (Ishikawa et al., 2009) was applied. Indeed, substantial binding to the respective ligands could be observed for ovine and murine Dectin-1, Dectin-2 and Mincle, (Fig.
3A, B, D). In contrast, only marginal binding of ovine Langerin to mannan was detected (Fig.
3C), thus suggesting a potentially different ligand preference of ovine Langerin compared to its murine orthologue.
55 3.3 Flow cytometry based binding study
For the comparison of binding preferences between ovine and murine DNGR-1, a flow cytometry-based approach was chosen. To this end, CHO-cells were subjected to two freeze-thaw cycles in order to expose intracellular F-actin filaments, which act as potent DAMPs and are a known ligand of the murine DNGR-1 (Ahrens et al., 2012). Hierarchical gating strategy was applied to discriminate live cells from dead cells
and debris (Suppl. Fig. 1). The F-actin filaments were stained by murine as well as ovine DNGR-1 hFc-fusion proteins. The intact cells on the other hand did not reveal any substantial binding to murine or ovine DNGR-1 (Fig. 3E).
3.4 Pathogen screening
The applicability of the ovine CTLR hFc-fusion protein library for a flow-cytometry based pathogen screening was verified with Mycoplasma mycoides subsp. capri (Mmc). In the flow-cytometric analysis, DNA-positive cells were considered intact and gated accordingly for CTLR binding assessment (Fig. 4A).
Remarkably, selective binding of ovine DCIR, MCL and MICL hFc-fusion proteins to Mmc was observed (Fig. 4B) which calls for further examination. In contrast, no substantial binding was detected for Dectin-1, Dectin-2, DNGR-1, Langerin or Mincle, which might indicate a limited role of these CTLRs in Mmc recognition. However, we cannot formally exclude that certain binding epitopes on the Mmc surface may have been masked by the fixation procedure.
Fig. 4. Ovine CTLR hFc-fusion protein library application to screen for pathogen/CTLR interactions. Flow cytometry-based binding study with the ovine CTLR hFc-fusion protein library and Mycoplasma mycoides subsp. capri (Mmc) GM12 was performed. Mmc was gated for DNA-positive events to distinguish intact Mmc from debris (A). Binding of ovine CTLR hFc-fusion proteins to DNA-positive Mmc was investigated (B). Two independent experiments were performed in duplicates each.
4. Discussion
To screen for pathogen-host interactions, a comprehensive library of murine CTLR hFc-fusion protein receptors was previously established (Maglinao et al., 2014; Mayer et al., 2018;
Monteiro et al., 2019). In order to meet the need for comparable research tools aiming at the elucidation of pathogen-host interactions in large animals, in this study sheep CTLRs were selected. The importance of sheep farming for developing and developed countries as well as sheep’s role as vectors of multiple zoonotic diseases (Ganter, 2015), such as Rift Valley Fever, Tuberculosis and Brucellosis, highlights the relevance of small ruminant immunology research.
Chimeric CTLRs produced as dimeric human hFc-fusion proteins in a mammalian cell system exploits the advances of multivalent ligand recognition while at the same time mimicking the multimerization or high density clustering observed for some CTLRs in vivo (Dam and Brewer, 2010; Richardson and Williams, 2014). The versatile hFc-tag supports standard methods of fusion protein purification and detection while being compatible with CHO-cell protein expression system which provides a mammalian-type glycosylation. It has indeed been shown that glycosylation may impact CTLR binding capacity in vitro (Bloem et al., 2013; Su et al., 2005). The occurrence of recombinant proteins of different sizes, as observed for ovine Langerin and MICL, suggests the presence of alternative glycoforms, which may impact the functionality of the respective CTLR (Su et al., 2005) and which has been observed for human MICL in vivo (Marshall et al., 2006). Different CTLR variants characterized by SNPs in the respective CTLDs might feature alternative ligand preferences or affected binding capacities (Kumar et al., 2019). The capability of ovine Dectin-2, Mincle and DNGR-1 (one SNP each) to bind distinct ligands as well as the pathogen binding capacity of the ovine MICL (three distinct SNPs), all discussed in detail below, however, suggest a limited role of these point mutations for the function of respective CTLRs. The occurrence of CTLR polymorphisms in distinctive sheep populations (Zhou et al., 2010) still remains largely unknown. Furthermore, the sheep genome assembly Oar_v4.0 referenced in our study, which is built upon the genome sequencing data of a female and a male Texel sheep (Archibald et al., 2010), does not necessarily feature the most common CTLR variants in different sheep races.
The higher degree of morphological similarity of ruminant CTLRs to the human rather than to the murine orthologs (Fig. 1) allude that ligand preferences of ovine CTLRs might be even more similar to human CTLRs compared to murine ones and should be investigated in the future. The functional evaluation of the selected CTLR-hFc fusion protein library members nevertheless revealed comparable ligand binding preferences for most tested ovine CTLRs and their murine orthologs. The barely detectable binding of the ovine Langerin to mannan in
contrast to the substantial binding of the murine Langerin is in accordance with the recently discovered species-specific differences in murine and human Langerin ligand preferences (Hanske et al., 2017). This finding, as well as the newly-discovered species-specific variety in signal transduction and immune stimulation shown for murine and human Dectin-1 (Takano et al., 2017), and differences seen in Mincledependent TDB-mediated immune stimulation in humans, mice and cattle (Thakur et al., 2018) highlight the need for further studies on target species’ CTLRs and respective ligand interactions.
In our proof-of-principle flow cytometry-based pathogen binding study with the ovine CTLR hFc-fusion protein library, binding of ovine DCIR, MCL and MICL to Mmc was observed.
Being primarily associated with DAMP recognition and downregulation of the immune response (Fujikado et al., 2008; Redelinghuys et al., 2016), these CTLRs have also been shown to play an important role in PAMP recognition and related immune signaling (Bloem et al., 2014; Raulf et al., 2019; Wang et al., 2015). The elucidation of associated molecular mechanisms alongside with the investigation of these CTLRs involvement in Mmc infections in vivo thus can become the scope of further studies.
Neither Mmc, nor other mycoplasmas have yet been reported to interact with transmembrane CTLRs of any species. However, for human and rat lung surfactant protein D (SP-D), a soluble C-type lectin, specific binding to Mycoplasma pneumoniae has been described (Chiba et al., 2002). Binding studies with CTLRs of further species as well as the application of the ovine CTLR hFc-fusion protein library for screenings with additional ruminant and non-ruminant Mycoplasma species might provide useful insights into veterinary relevant mycoplasmoses.
5. Conclusion
The here generated ovine CTLR hFc-fusion protein library is a versatile tool allowing for the identification of pathogen interactions with ovine CTLRs. However, it may not only provide insights into ligand preferences of single ovine CTLRs, but it also enables a comparison with CTLRs across different species. Further screenings with bacterial and viral pathogens of sheep and other species as well as the extension of the library with additional ovine CTLRs such as MDL-1, MGL-1, Clec2L and Clec15B, and comparisons between different CTLR isoforms are conceived.
6. Funding
This work was supported by the “Nationale Forschungsplattform für Zoonosen”
(DLR/BMBF, Fkz. 01KI1724). We also acknowledge support from the Niedersachsen-Research Network on Neuroinfectiology (NRENNT-2).
7. Declaration of Competing Interest
No author has any competing interests or conflicts of interests.
8. Acknowledgement
We thank Julian Glanz and Anna Sternberg for their assistance in bioinformatic analysis and Silke Schöneberg for technical assistance. We thank the Clinic for Swine, Small Ruminants and Forensic Medicine and the Institute for Pathology, University for Veterinary Medicine Hannover, Foundation, for the provided biological materials.
9. Appendix A. Supplementary data
Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.vetimm.2020.110047.
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VIII. Discussion
This thesis addresses one of the limitations of current research on CTLs in veterinary glycoimmunology, namely the lack of reliable tools to investigate CTL/ligand interactions of veterinary species in vitro. The need for specific tools in immunologic research is apparent, since major differences in PRRs ligand preferences have recently been revealed among phylogenetically distinct animals [142, 155], questioning the applicability of results obtained in model species [156]. Lacking more suitable alternatives, current studies in veterinary immunology are yet often compelled to using recombinant human PRRs [157] or mouse models [158-160] (Tab. 3).
Name Species Length Coverage Identity Accession Nr.
Dectin-1 Ovis aries 246 Querry Querry XP_027823677.1
Homo sapiens 247 100% 71,26% NP_922938.1
Mus musculus 244 99% 55,51% NP_064392.2
DNGR-1 Ovis aries 241 Querry Querry XP_004006925.3
Homo sapiens 241 100% 68,46% NP_997228.1
Homo sapiens 241 100% 68,46% NP_997228.1