VII. Results
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
Mus musculus 264 99% 49,43% NP_001192292.1
MICL Ovis aries 265 Querry Querry XP_004006929.2
Dectin-2 Ovis aries 237 Querry Querry XP_012030843.2
Homo sapiens 237 100% 59,34% NP_057268.1
Langerin Ovis aries 329 Querry Querry XP_004006101.2
Homo sapiens 328 98% 70,46% NP_056532.4 C
Mus musculus 330 96% 62,26% XP_006506202.1
Table 3.
Pairwise nucleotide sequence alignments of sheep and human resp. mouse CTLs performed with NCBI BLAST blastp suite [161] provide comparissons of length, querry coverage and sequence identity of orthologuous CTLs in corresponding species.
[161]
The novel ovine CTL hFc-fusion protein library generated within the scope of this thesis [153] aims to provide a tool for research on CTL-ligand recognition in the context of host- microbiome [19] as well as host-pathogen [162] interactions in sheep. The adjunct hFc-tag
allows its use in various scenarios (chapter I.3 Fig. 2), such as ELISA-based ligand screenings [154] with various glycan [163, 164] or glycomimetic [165] compounds. The applicability of the presented library for this use was verified in comparative experiments with corresponding mouse CTL orthologues, revealing overlapping ligand preferences for ovine and murine Dectin-1, Dectin-2, DNGR-1 and Mincle [153]. Alongside, pronounced differences were recognized between murine and ovine Langerin [153], which is, however, well in line with the previously recognized species-specific ligand preferences of this CTL observed in humans and mice [166]. In addition, hitherto unknown interactions of the ovine DCIR, MCL and MICL with the model ruminant pathogen Mmc were observed in a proof-of-principle flow cytometry-based binding study [153]. Thus, the generated ovine CTL hFc-fusion protein library represents a valuable extension of the previously generated comprehensive murine CTL hFc-fusion protein library, which served as a starting point of this PhD thesis [154].
Initially comprised of 11 myeloid murine CTLs [154], the murine hFc-fusion protein library was later extended with mouse myeloid DAP12-associating lectin 1 (MDL-1), and human DC-specific- resp. human lymph node (LN)-specific intracellular adhesion molecule-3 grabbing non-integrin (DC-SIGN resp. L-SIGN) [167]. The recombinant proteins expressed in suspended CHO cells, feature mammalian-type glycosylation and are routinely purified using Protein G columns [154]. The library, applied in ELISA- and flow cytometry-based binding studies, provide numerous clues for further pathogen-related research. For instance, its unbiased application revealed the recognition of zoonotic La Crosse virus by mouse Mincle [167]. In addition, novel mouse Dectin-1 interaction with the intestinal pathogen Campylobacter jejuni [168] and Mincle interaction with group A Streptococci (GAS) were detected [169]. The GAS cell wall lipoteichonic acid anchoring molecule monoglucosyldiacylglycerol (MGDG) was identified as a ligand of murine Mincle, thus contributing to the murine anti-GAS immune response [169]. Similarly, selective binding of the murine MICL to plasmodial hemozoin, initially observed in a binding study [170], led to the characterization of the role of MICL in cerebral malaria pathogenesis in a mouse model [170]. Multiple mouse CTL hFc-fusion proteins, such as Mincle, Dectin-2 and MCL, as well as the human DC-SIGN, were also shown to bind to the intact and homogenized facultative pathogenic fungus Pneumocystis carinii, as well as to its isolated major surface glycoprotein antigen [171, 172]. The role of these CTLs during infection needs to be further clarified in future studies.
Numerous studies performed with human CTL hFc-fusion proteins, which were mainly produced in human embryonic kidney 293 (HEK293) cells [173], are also reported. In particular, the recognition of mycobacterial cord factor Trehalose-6,6’-dimycolate (TDM) resp.
its synthetic analogue Trehalose-6,6’-dibehenate (TDB) by human Mincle hFc-fusion protein, observed in ELISA-based binding studies [173, 174], allowed to subsequently characterize the role of Mincle in innate responses of human and mouse APCs [173, 175] in vitro, and in vivo [174]. Following research revealed that mouse macrophage expression of Mincle and other Dectin-2 family members was upregulated by TDB via Mincle-induced tumor necrosis factor (TNF) signaling [175], while Mincle-independent [176] TDM signaling resulted in cell cycle related, antimicrobial and antigen presentation pathway related molecules being downregulated [176, 177].
An alternative approach using truncated human and mouse Strep-tag II-fused CTLs expressed in BL21(DE3) E. coli was also reported [166, 178]. In these studies, pronounced differences in ligand recognition between human and mouse Langerin [166] were revealed. In addition, distinct single nucleotide polymorphisms (SNP) in human Langerin were shown to significantly compromise Langerin-mediated uptake of Staphylococcus aureus [178] in vitro.
The bovine CTL library, which was already introduced in chapter I, was produced recombinantly in bacteria, namely also in BL21(DE3) E. coli; the respective CTLs were, however, biotinylated instead [179]. The bovine library was used in unbiased screenings with autofluorescent or fluorophore-conjugated bacteria, such as various E. coli strains, Mycobacterium bovis and Staphylococcus aureus, revealing distinct CTL-specific binding profiles [179].
The utility of each library was demonstrated in corresponding binding studies. However, only few comparisons between different recombinant CTL libraries, specifically between mammalian cell-expressed and the E. coli-expressed receptor libraries, were performed [180].
Production costs of E. coli-expressed CTLs are generally lower compared to the ones expressed in mammalian expression systems; especially so, should up-scaling of the process be considered [181, 182]. The latter, on the other hand, might be a better model in the context of receptor glycosylation-dependent interactions: mammalian-type glycosylation featured by many CTLs cannot be achieved in currently available E. coli expression systems [183]. For several CTLs, differential receptor glycosylation was shown to influence receptor functions in vitro [184-186]. For instance, deglycosylation of the macrophage mannose receptor (MMR) CTL domain abrogated its ability to bind mannose [185]. For, DCIR, on the other hand, an increased binding affinity following deglycosylation was reported [186]. Further, a positive correlation between the CRD stability and glycosylation status was reported for DC-SIGN, thus demonstrating a glycoform-dependent variance in CRD properties, which might impact CTL binding in vitro [187]. Moreover, the hFc-tag-mediated dimeric structure of the recombinant
Fc-fusion proteins mirrors the close proximity spatial distribution and rearrangement of CTLs observed on immune cells [188]. CTL avidity was shown to increase in multivalent receptor systems [180], possibly rendering hFc-fusion CTL proteins more ligand sensitive and the respective interactions more stable when compared to monomeric counterparts [189, 190].
However, further comparisons are needed. As an initial step, comparative binding studies with orthologous members of the biotinylated cattle and hFc-fusion sheep CTL libraries might be performed.
With regard to other ruminants, such as cattle, antelopes and cervides [191], a certain degree of concordance between orthologous CTLs might well be expected. The applicability of sheep CTLs to model CTL interactions of more distantly related species, such as pigs, is, however, questionable. Despite both pigs and sheep belong to the even-toed ungulates, their CTL ligand specificities might significantly differ due to the respective immunological adaptations in terms of pathogen recognition [192] and microbiome maintenance [193]. Accordingly, extension of the current CTL hFc-fusion protein library with CTLs of pigs and further veterinary species, allowing the dissection of respective host CTL and microorganisms co-evolution [194] and cross species pathogen transmission [195], is intended and shall be performed in the future.In this regard, CTLs of arthropods like mosquitos, which serve as transmission vectors for many veterinary and zoonotic diseases [196, 197], represent an important research field. While being known to participate in gut microbiome homeostasis and in host-pathogen interaction within the mosquito vectors [198], the involvement of arthropod CTLs in pathogen transmission is not yet understood. In particular, their role for the transmission of arboviruses, such as the West Nile virus [199] and the Rift Valley Fever (RVF) virus, which use sheep and other ruminants as amplification vectors before reaching humans population [200], requires further investigation. In humans, DC-SIGN [201] was reported to act as an adhesion molecule in RVF and other arboviral infections [202, 203]. However, no direct orthologue of this CTL has yet been identified neither in mosquitos, nor in sheep. Nevertheless, multiple mosquito CTLs were shown to play important roles in arboviral infections [198]. To this end, an Aedes aegypti mosquito CTL hFc-fusion protein library encompassing five distinct mosquito CTLs was generated within the scope of this thesis (Fig. 4, Tab. 4). Among these, some promising candidates, such as the mosquito galactose-specific C-type lectin 1 (mosGCTL-1), which was identified as an important mosquito susceptibility factor to West Nile Fever (WNF) virus infection [204], are presented. Since little is known about the remaining library members, namely the C-type lectin galactose binding 6 (CTLGA6) and 14 (CTLMA14), and C-type lectin 23 (CTL23), they represent interesting candidates for binding studies. In particular, experiments
involving native and fluorescently labelled viruses and recombinant viral
involving native and fluorescently labelled viruses and recombinant viral