University of Veterinary Medicine Hannover
Institute for Immunology &
Research Center for Emerging Infections and Zoonoses
Veterinary Glycoimmunology:
Generation and in vitro application of a novel sheep C-type lectin receptor fusion protein library
THESIS
Submitted in partial fulfilment of the requirements for the degree
Doctor of Philosophy (PhD)
awarded by the University of Veterinary Medicine Hannover
by
Dimitri Leonid Lindenwald aus Bryansk
Hannover, Germany 2020
I Supervisor: Prof. Dr. Bernd Lepenies
Supervision Group:
Prof. Dr. Bernd Lepenies
Prof. Dr. Silke Rautenschlein, PhD Prof. Dr. Roland Lang
1st Evaluation:
Prof. Dr. Bernd Lepenies, Immunology Unit & Research Center for Emerging Infections and Zoonoses, University of Veterinary Medicine Hannover, Bünteweg 17, 30559 Hannover, Germany.
Prof. Dr. Silke Rautenschlein, PhD, Clinic for Poultry, University of Veterinary Medicine Hannover, Bünteweg 17, 30173 Hannover, Germany.
Prof. Dr. Roland Lang, Institute of Microbiology, Universitätsklinikum Erlangen, Wasserturmstraße 3, 91054 Erlangen, Germany.
2nd Evaluation:
Prof. Tina Sørensen Dalgaard, PhD, Department of Animal Science - ANIS Health, Aarhus University, Blichers Allé 20, P25, 3334, 8830 Tjele, Denmark
Date of final exam: 17.11.2020
Funding: This work was funded by the Nationale Forschungsplattform für Zoonosen (DLR/BMBF, Fkz. 01KI1724). I also acknowledge support from the Niedersachsen-Research Network on Neuroinfectiology (NRENNT-2), and thank Deutsche Forschungsgemeinschaft and the University of Veterinary Medicine Hannover, Foundation, for the support in publishing parts of this work within the funding programme Open Access Publishing.
II For my wife ❤
III Parts of the thesis have been previously published in peer-reviewed journals:
1. Lindenwald, D.L.; Monteiro, J.T.; Rautenschlein, S.; Meens, J.; Jung, K.; Becker, S.C.;
Lepenies, B. Ovine C-type lectin receptor hFc-fusion protein library - A novel platform to screen for host-pathogen interactions. Vet Immunol Immunopathol. 2020;224:
110047.
2. Lindenwald, D.L.; Lepenies, B. C-Type Lectins in Veterinary Species: Recent Advancements and Applications. Int J Mol Sci. 2020;21:5122.
Additional publications in peer-reviewed journals:
3. Waindok, P.; Janecek-Erfurth, E.; Lindenwald, D.L., Wilk, E.; Schughart, K.; Geffers, R.; Balas, L.; Durand, T.; Rund, K.M.; Schebb, N.H.; Strube, C. Multiplex profiling of inflammation-related bioactive lipid mediators in Toxocara canis- and Toxocara cati- induced neurotoxocarosis. PLoS Negl Trop Dis. 2019;13(9):e0007706.
4. Lindenwald, D.L.; Lepenies, B. [Immunological Tolerance and Nutraceuticals.]
Tierarztl Umsch. 2020;2020(03); pp. 78–82.
Book Chapter (submitted):
5. Ebbecke, T.; Diersing, C.; Lindenwald, D.L.; Stegmann, F.; Lepenies, B. C-type Lectins and their Roles in Disease and Immune Homeostasis. Comprehensive Glycoscience 2nd Edition.
IV I. Table of Contents
I. Table of Contents ... IV II. Abbreviation list ... VI
III. Abstract ... 1
IV. Deutsche Zusammenfassung ... 3
V. Introduction ... 5
1. The immune system ... 5
1.1 Innate and adaptive immunity ... 5
1.2 Pattern recognition receptors ... 8
1.3 Glycan recognition ... 9
1.4 PRR signaling ... 10
2. The C-type lectin receptor superfamily ... 12
2.1 Myeloid CTLs ... 12
3. Role of CTLs in veterinary species: Recent Advancements and Applications ... 15
1. Introduction ... 17
2. Protective Role of Veterinary Relevant CTLs ... 21
3. Detrimental Role of Veterinary Relevant CTLs ... 23
4. Harnessing the Power of CTLs ... 24
5. Conclusions ... 26
6. References ... 28
VI. Aims of the thesis ... 43
1. Aim: Generation of a sheep CTL hFc-fusion protein library ... 43
2. Aim: Functionality testing of the CTL hFc-fusion protein library ... 43
3. Aim: Pathogen screening with sheep CTL hFc-fusion protein library ... 43
4. Aim: Generation of further tools to characterize identified interactions ... 43
VII. Results ... 45
Ovine C-type lectin receptor hFc-fusion protein library – A novel platform to screen for host- pathogen interactions ... 45
1. Introduction ... 47
2. Materials and methods ... 49
2.1 Cloning and expression of the CTLR-hFc fusion protein library ... 49
2.2 Western Blot and silver staining ... 50
2.3 Bioinformatic analysis ... 51
V
2.4 ELISA-based binding studies ... 51
2.5 Flow cytometry-based binding study ... 52
2.6 Pathogen screening ... 53
3. Results... 54
3.1 Ovine CTLR hFc-fusion protein library ... 54
3.2 ELISA based binding studies ... 54
3.3 Flow cytometry based binding study ... 55
3.4 Pathogen screening ... 55
4. Discussion ... 56
5. Conclusion ... 57
6. Funding ... 58
7. Declaration of Competing Interest ... 58
8. Acknowledgement ... 58
9. Appendix A. Supplementary data ... 58
10. References ... 58
VIII. Discussion... 65
X. Concluding remarks ... 79
XI. References ... 81
XIII. Affidavit ... 97
XIV. Appendix ... 99
Supplemental figures ... 99
Generation of Aedes aegypti mosquito CTL hFc-fusion protein library. ... 101
Initial steps in the production of sheep FSHR-3 based reporter cell line. ... 102
VI II. Abbreviation list
Term Abbreviation
Absent in melanoma 2 AIM-2
Absent in melanoma 2-like receptors ALRs
Antibodies Abs
Antimicrobial peptides AMPs
Apoptosis-associated speck-like protein ASC
B-cell lymphoma protein 10 BCL10
Caprine arthritis encephalitis virus CAEV
Caprine nasal adenocarcinoma viruses CNAV
Cellular sarcoma Src
Chinese hamster ovary (cells) CHO
Clustered regularly interspaced short palindromic repeats CRISPR
Complement factor 3 convertase C3 convertase
CRISPR associated protein CAS
Carbohydrate recognition domain CRD
C-terminal domain CTD
C-type lectins CTLs
C-type lectin-like domain CTLD
C-type lectin galactose binding 6 CTLGA6
C-type lectin mannose binding 14 CTLMA14
CV-1 in origin, carrying SV40 COS7
Cyan florescent protein CFP
Damage associated molecular patterns DAMPs
Domain in apoptosis and interferon response DAPIN
Dendritic cells DCs
DC-specific intracellular adhesion molecule-3 grabbing non-integrin DC-SIGN
Double-stranded RNA dsRNA
Fc receptor signaling gamma chain FcRγ
Fetal bovine serum FBS
Follicle-stimulating hormone receptor 3 FSHR3
Foot-and-mouth disease FMD
Fragment of crystallization Fc
VII Follicle-stimulating hormone receptor isoform 3 FSHR3
Fucose Fuc
Galactose Gal
Glucose Glc
Goatpox GP
Green fluorescent protein GFP
Hematopoetic interferon-inducible nuclear domain HIN
Inflammatory bowel disease IBD
Immunoglobulin G1 IgG1
Immunoreceptor tyrosine-based activation motifs ITAMs Immunoreceptor tyrosine-based inhibitory motifs ITIMs
Interferon type I IFN-I
Interferon type II IFN-II
Invariant natural killer T cells iNKT
Jaagsiekte sheep retrovirus JSRV
Lipopolysaccharides LPS
LN-specific intracellular adhesion molecule-3 grabbing non-integrin L-SIGN
Lymph node LN
Lymphocyte antigen 75 Ly75
Macrophages Mϕ
Maedi-Visna virus MVV
Major histocompatibility complex I MHC-I
Major histocompatibility complex II MHC-II
Malignant catarrhal fever MCF
Mannose Man
Mannose binding lectin MBL
Mannose receptor MR
Mason-Pfizer monkey virus MPMV
MBL-associated serine proteases MASPs
Monoglucosyldiacylglycerol MGDG
Mitogen-activated protein MAPK
Monocytes Mo
Mosquito galactose-specific C-type lectin 1 mosGCTL-1 Mucosa-associated lymphoid tissue lymphoma translocation protein 1 MALT1
VIII
Multiple sclerosis MS
Mycoplasma mycoides subsp. capri Mmc
MyD88 adaptor-like MAL
Myeloid DAP12-associating lectin 1 MDL-1
Myeloid differentiation primary response 88 MyD88
N-Acetylgalactosamine GalNAc
N-Acetylglucosamine GlcNAc
N-Acetylneuraminic acid Neu5Ac
NACHT, LRR, FIIND, CARD and PYD domains-containing protein 1 NLRP1
Natural killer cells NK cells
N-Glycolylneuraminic Neu5Gc
NLR family CARD domain-containing protein 4 NLRC4
N-terminal caspase activation and recruitment domains CARDs Nucleotide-binding oligomerization domain-containing protein 1 NOD1 Nucleotide-binding oligomerization domain-like receptors NLRs
Ovine gammaherpesvirus 2 OHV-2
Ovine nasal adenocarcinoma viruses ONAV
Pathogen associated molecular patterns PAMPs
Phycoerythrin PE
Plasmocytoid dendritic cells pDC
Pattern recognition receptors PRRs
Rapidly accelerated fibrosarcoma 1 Raf-1
Red fluorescent protein RFP
Retinoic acid inducible gene 1 RIG-I
Retinoic acid inducible gene 1 like receptors RLRs Genetically encoded RFP based Ca2+ indicator for optical imaging 1 R-GECO1
Rift Valley Fever RVF
Sheeppox SP
Single domain antibody sdAB
Single nucleotide polymorphism SNP
Single-stranded DNA ssDNA
Spleen tyrosine kinase Syk
SRC homology region 2 domain-containing phosphatase-1/-2 SHP-1/SHP-2
Systemic sclerosis SS
IX
Tumor necrosis factor TNF
TIR-domain containing adaptor-inducing interferon-β TRIF
Toll like receptors TLRs
Toll/interleukin 1 receptor TIR
Trehalose-6,6’-dibehenate TDB
Trehalose-6,6’-dimycolate TDM
TRIF-related adaptor molecule TRAM
Tumor necrosis factor receptor superfamily member 8 TNFRSF8
UDP-di-N-acetylbacillosamine diNAcBac
West Nile Fever WNF
1 III. Abstract
Dimitri Leonid Lindenwald
Veterinary Glycoimmunology: Generation and in vitro application of a novel sheep C- type lectin receptor fusion protein library
C-type lectins (CTLs) are crucial innate pattern recognition receptors (PRRs) which appear throughout the animal kingdom, from nematodes and insects up to the higher animals and humans. They recognize various host and foreign carbohydrates, as well as further polypeptide and crystal ligands, evoke regulatory signals and participate in phagocytosis and priming of adaptive immune responses against viral, bacterial, fungal and parasitic infections.
In the first chapter of this thesis, general concepts regarding the immune system and specifically the role of relevant PRR families, such as the CTLs, Toll-like and NOD-like receptors, are introduced. Following, the state of research concerning CTLs in veterinary medicine is summarized. Though barely nascent, veterinary glycoimmunology already presents most promising concepts and methods, featuring the role of CTLs in controlling autoimmunity, host-pathogen interactions, and cancer. The detrimental effects of CTLs, such as their causative role in allergies and the immune compromising aspect of several CTLs posing as pathogen adhesion molecules, are also reviewed. Current problems and limitations of veterinary CTL- related research are discussed. Specifically, the limited applicability of mouse models in research on veterinary species, and deficits in species-specific characterization of CTL ligand preferences and the respective CTL-mediated signaling are addressed.
A novel sheep CTL fusion protein library providing a means to study sheep CTL/ligand interactions in vitro is introduced in the second chapter. It is comprised of eight distinct recombinant CTLs, all of which were produced in suspended Chinese hamster ovary (CHO) cells, expressed as homodimers fused to the human immunoglobulin G1 fragment of crystallization (IgG1 Fc).
The functionality of the sheep CTL hFc-fusion protein library was verified with known ligands of corresponding mouse CTL orthologues in ELISA- and flow cytometry-based binding studies. The obtained results indicate that similar (Dectin-1, Dectin-2, Mincle, DNGR-1) as well as differing (Langerin) ligand preferences of orthologous sheep and mouse CTLs exist.
Ensuing, the ovine CTL hFc-fusion protein library was applied in a proof-of-principle flow cytometry-based pathogen screening study with the model ruminant pathogen Mycoplasma
2 mycoides subsp. capri. Thereby, novel interactions of several sheep CTLs, such as MCL, MICL, and DCIR, were identified.
In addition, an Aedes aegypti mosquito CTL hFc-fusion protein library was generated for collaborative studies on zoonotic arboviruses, such as the Rift Valley Fever virus.
Finally, the generation of ovine CTL-expressing reporter cells was initiated to investigate the signal transducing potential of the respective CTL/ligand interactions. Results obtained with ovine follicle stimulating hormone isoform 3 (FSHR3) and derivative sheep Dectin-1 FSHR3 hybrid receptor reporter cell are, however, yet preliminary and require further studies.
3 IV. Deutsche Zusammenfassung
Dimitri Leonid Lindenwald
Veterinär-Glykoimmunologie: Etablierung und in vitro Studien mit einer neuen ovinen C-Typ Lektinrezeptor Fusionsprotein-Bibliothek
C-Typ Lektine (CTLs) sind wichtige Mustererkennungs-Rezeptoren (“pattern recognition receptors”, PRRs) auf Zellen des angeborenen Immunsystems. Sie kommen bei verschiedensten Vertretern des Tierreiches, von Nematoden über Insekten bis zu den höheren Tieren und dem Menschen, vor. Zu ihren Funktionen gehört die Erkennung von verschiedenen körpereigenen und körperfremden Kohlenhydraten sowie von weiteren Polypeptid- und Kristallliganden. In diesem Zusammenhang lösen sie regulatorische Signalkaskaden aus und beteiligen sich an der Phagozytose und der anschließenden Prägung („priming“) der adaptiven Immunität im Kampf gegen virale, bakterielle, mykotische und parasitäre Infektionen.
Im ersten Kapitel dieser Arbeit werden allgemeine Konzepte zum Immunsystem und insbesondere zur Rolle der bekanntesten PRR-Familien wie z.B. den CTLs, Toll-like- und NOD-like-Rezeptoren, vorgestellt. Im Folgenden wird der Forschungsstand zu CTLs in der Veterinärmedizin zusammengefasst. Obwohl erst im Entstehen begriffen, weist die Veterinär- Glykoimmunologie bereits jetzt vielversprechende Konzepte und Methoden auf, welche die Rolle von CTLs bei der Kontrolle von Autoimmunreaktionen, den Wechselwirkungen zwischen Wirt und Krankheitserreger und der Prävention von Krebs thematisieren. Die nachteiligen Wirkungen von manchen CTLs, wie beispielsweise eine Beteiligung an der Entstehung von Allergien und die Rolle von einigen CTLs als Pathogenadhäsionsmoleküle, werden ebenfalls besprochen. Daneben werden aktuelle Probleme und Limitationen der veterinärmedizinischen CTL-bezogenen Forschung ebenfalls diskutiert. Insbesondere werden die eingeschränkte Anwendbarkeit von Mausmodellen bei der Erforschung veterinärmedizinischer Spezies sowie die Defizite bei der artspezifischen Charakterisierung der CTL-Ligandenpräferenzen und der jeweiligen CTL-vermittelten Signalübertragung thematisiert.
Im zweiten Kapitel wird eine neue CTL-Fusionsproteinbibliothek aus Schaf vorgestellt, mit der CTL-Ligand-Interaktionen von Schaf-CTLs in vitro untersucht werden können. Sie besteht aus acht verschiedenen CTLs. Alle Fusionsproteine wurden in Ovarialzellen des chinesischen Hamsters (CHO) als Homodimere, fusioniert mit dem Fc-fragment des menschlichen Immunglobulin G1 (IgG1 Fc), hergestellt.
4 Die Funktionalität der Bibliothek wurde in ELISA- und Durchflusszytometrie-basierten Bindungsstudien mit entsprechenden Maus-CTL-Orthologen verglichen. Die erhaltenen Ergebnisse zeigen, dass sowohl vergleichbare (Dectin-1, Dectin-2, Mincle, DNGR-1) als auch unterschiedliche (Langerin) Ligandenpräferenzen von orthologen Schaf- und Maus-CTLs existieren.
Anschließend wurde die CTL-hFc-Fusionsproteinbibliothek aus Schaf in einer Durchflusszytometrie-basierten Pathogen-Bindungs-Studie mit dem Modell-Wiederkäuer- Pathogen Mycoplasma mycoides subsp. capri getestet. Dabei wurden neue Interaktionen mehrerer Schaf-CTLs wie MCL, MICL und DCIR gefunden. Zudem wurde eine CTL-hFc- Fusionsproteinbibliothek aus der Aedes aegypti Mücke für gemeinsame Studien zu zoonotischen Arboviren wie dem Rift Valley Fever-Virus erstellt.
Zum Schlusss wurden erste Arbeiten an CTL-exprimierenden Reporterzellen von Schafen durchgeführt, die zu Untersuchungen der Signaltransduktion der jeweiligen CTL- Ligandenbindungen genutzt werden sollen. Die Ergebnisse, die mit dem ovinen Follikel- stimulierenden Hormon Isoform 3 (FSHR3) und dem davon abgeleiteten ovinen Dectin-1- FSHR3-Hybridrezeptor erhalten wurden, sind jedoch noch präliminär und erfordern weitere grundlegende Anpassungen.
5 V. Introduction
1. The immune system
The inborn ability to distinguish between “self” and “non-self” on the molecular level is an indispensable requirement of life, which is already recognized in phylogenetically old prokaryotic organisms, such as bacteria [1] and archaea [2]. The respective self-nonself discrimination of these organisms encompasses clustered regularly interspaced short palindromic repeats and CRISPR associated protein (CRISPR-Cas) based antiviral resistance [3], protective biofilm formation [4] as well as the application of weaponized enzymes and phage-derived toxins [2, 5]. Inter- and intra-species communication systems, crucial for the synchronization and optimization of protective reactions [6], have independently evolved in different prokaryotes [7] and are considered precursors [8-10] of fungal [11, 12], plant [13, 14]
and animal [15] immune systems.
The immunity of higher animals including humans provides defense against various viral, microbial and multicellular pathogens [16-18], and contributes to the bidirectional host- microbiome [19, 20] and host-mycobiome [21] communication. Additionally, it maintains immune homeostasis by inducing tolerance to self-antigens [22], and by destroying cancerous cells [23]. This self-regulating system [24-26] incorporates both genetically fixed (innate) and acquired (adaptive) mechanisms [25]. Among them, specialized cell subsets represent cell- mediated immunity [27, 28], while secreted macromolecules such as the antimicrobial peptides (AMPs) and antibodies (Abs) are denoted as humoral immunity [29, 30] (Fig. 1).
1.1 Innate and adaptive immunity
Innate immunity combines passive barrier [31] and active pathogen destruction and uptake mechanisms provided by epithelial [32], endothelial [33], and circulating innate immune cells [34]. Structural keratins [35], complement factors and other AMPs produced by keratinocytes in both cornified [35-37] and mucosal skin [38] limit pathogen adhesion and entry, and demonstrate antibacterial properties in vitro [38-40] and in vivo [41]. Additionally, antimicrobial mucin glycosidase lysozyme and unspecific pathogen-binding natural antibodies (NAbs) [42], which are produced by naïve murine and human CD5+ or CD5- B-1 cells [42], contribute to innate humoral immunity and inactivate a broad range of bacterial, viral, fungal and parasitic pathogens [42-45].
Active phagocytosis-like pathogen uptake and subsequent lysis are mediated by epithelial [32] and endothelial [33] cells. For keratinocytes, on the other hand, apoptosis following
internalization of bacterial pathogens was described [46]. Albeit, major histocompatibility complex II (MHC-II) molecule expressing keratinocytes were also shown to upregulate antigen processing and presentation genes, and to interact with TH1 T-cells in the skin [47].
Further, the so called “tight junction” multiprotein cell membrane complexes prevent the uncontrolled paracellular transport of organisms, metabolites or antigens across skin and intestine basal membranes [31, 48]. Specialized innate immune cells, such as the dendritic cells
Figure 1.
The cellular and humoral components of the innate and adaptive immunity.
Soluble macromolecules, such as the antigen-specific antibodies (Abs) and their smaller variants, the monomeric single domain antibodies (sdAB) found in camelids and sharks, represent adaptive humoral immunity. Their unspecific counterparts, the natural Abs, constitute, along with the complement proteins and the antimicrobial peptides (AMPs), innate humoral immunity. Various cytokines and chemoattractants are utilized by both adaptive and innate immune cells, and therefore belong to both systems. Similarly, the natural killer (NK) cells, gamma delta T (γδT) cells and the invariant natural killer T cells (iNKT) can be placed between the innate and adaptive groups of the cell-mediated immunity [68]. In spite of shared ontogenetic origin with the adaptive immune cells, such as the classical B and T lymphocytes, these cells feature mainly innate immune functions, similar to those of the dendritic cells (DCs), macrophages (Mϕ), granulocytes, mast cells, epithelial and endothelial cells.
(DCs), monocytes (Mo), macrophages (Mϕ), granulocytes, natural killer (NK) cells and mast cells, destroy pathogens that manage to pass the barriers or have been transcytotically carried over by M-cells [49] in course of antigen sampling [50].
In some of these cells, for instance in Mϕ [51], DCs [52] and NK cells [53], immune memory functions [54, 55] were identified. Essentially, innate immune memory, also known as trained innate immunity, is based upon epigenetic effects: the expression of transcription factors and distinct immune receptors may be differentially regulated during the course of infection in innate immune cells [54]. This reprogramming, lasting from weeks to months, strengthens the unspecific immune response to following infections [56]. In this context, histone modifications were reported as key regulatory mechanisms of differential gene expression in innate immune cells and epithelia [54, 57]. Among them, acetylation, phosphorylation and methylation, which can modulate chromatin accessibility and compaction state within cells, thus affect the expression levels of specific genes [54].
The innate immune defense against pathogens is complemented by pathogen-specific adaptive immune cells, such as the B and T lymphocytes [58, 59]. Activated B cells, for instance, can differentiate into plasma cells to produce antigen-specific Abs [60] or smaller sdAbs, which were described in camelids [60] and sharks [61, 62]. Primed CD4+ and CD8+ T cells, on the other hand, can differentiate into cytokine producing effector cells, such as CD4+ regulatory and helper cells [59], and CD8+ cytotoxic T cells, respectively [63]. Further, memory B and T cells arise during the course of infection and persist for decades, ready to rapidly expand and resume protective functions upon reinfection [28, 64].
The respective activation and priming of adaptive immune cells against newly encountered infections is largely performed by professional antigen presenting cells (APCs), such as the Mo, Mϕ and DCs [24]. Although several lymphocyte subsets, such as the antigen specific B cells [65] and γδT cells [66], are also able to act as APCs in priming the adaptive immune response, their relative rareness [66-68] renders the innate APCs indispensable [68].
APCs commonly express phylogenetically conserved invariant intracellular and membrane bound immune receptors targeting molecules typical of various pathogens, such as bacterial cell wall glycoproteins and lipopolysaccharides (LPS) or fungal glycans. These are classified as pathogen associated molecular patterns (PAMPs) [69] (Tab. 1). The aforementioned receptors recognizing PAMPs, as well as damage associated molecular patterns (DAMPs) [70], are denominated as pattern recognition receptors (PRRs) [71].
1.2 Pattern recognition receptors
Among the distinct PRR groups, soluble and cell membrane bound PRRs exist [71]. The soluble nucleotide-binding oligomerization domain-like receptors (NLRs) [72], the absent in melanoma 2-like receptors (ALRs) [73] and the retinoic acid inducible gene 1 like receptors (RLRs) [74] represent cytosolic sensors. They are primarily characterized by the occurrence of respective family-specific functional domains – the oligomerization domain NACHT and, in case of NLR, the ligand-binding C-terminal leucine-rich repeat domain (LLR) [72]. ALRs are, on the other hand, defined by DNA-binding C-terminal hematopoietic interferon-inducible nuclear domain (HIN) and N-terminal domain in apoptosis and interferon response (DAPIN) domain [73], while RLRs feature a RNA-recognizing composite helicase and C-terminal domain (CTD) [74]. The likewise soluble S-type lectin receptors (galectins), on the other hand, occur in nucleus, cytosol, cell secrets and in the intracellular matrix and are characterized by a galactose-specific carbohydrate binding domain [75].
C-type lectin receptors (CTLs) are defined by their genetically conserved ligand binding C- type lectin-like domains [76, 77]. Toll like receptors (TLRs) are characterized by their respective N-terminal ligand binding LRRs and C-terminal Toll/IL-1 receptor (TIR) domains [78]. Although numerous members of these receptor groups are transmembrane proteins, soluble C-type lectins [79] and endosomal Toll-like receptors [80] also occur.
PAMP class Ligand (Examples) PRRs (Examples)
Nucleotides Bacterial and viral dsDNA/ssDNA/dsRNA
TLR (3, 7, 8, 9, 10) RLR (RIG-I) ALR (AIM2) NLR (NOD2)
Lipids Cell membrane TLR (1, 2, 4, 6)
Peptides
Flagella components TLR (5)
NLR (NLRC4)
Secreted toxins TLR (4)
NLR (NLRP1) Gram+/Gram- cell wall components NLR (NLRP7) Glycans Bacterial, viral, fungal and parasitic
glycoconjugates
NLR (NOD1) CTL (many) Galectins (many) Table 1. Overview of selected PAMPs and associated PRRs.
Distinct nucleic acids typical of viruses but absent in pro- and eukaryotes, for example single-stranded DNA (ssDNA, e.g. of parvoviruses) and double-stranded RNA (dsRNA, e.g. of Bluetongue virus), but also bacterial-specific sequences of double-stranded DNA (dsDNA) are recognized by various soluble and membrane-associated PRRs. These include endosomal TLRs [81, 82] and cytosolic RLR retinoic acid inducible gene 1 (RIG-I) [74] and ALR absent in melanoma 2 (AIM-2) receptors [83].
Di- and triacylated bacterial membrane lipoproteins are predominantly recognized by cell surface TLRs, namely the homodimerized TLR 2 or heterodimers thereof paired with TLR 1 or TLR 6 [84]. The lipopolysaccharides constituting cell walls of Gram negative bacteria are sensed by surface TLR 4 [84]. Further pathogen-associated structural and functional proteins, such as the flagellins and various peptide toxins are, on the other hand, recognized by the surface TLR 5 [84] and cytosolic NLR family CARD domain-containing protein 4 (NLRC4) [72].
1.3 Glycan recognition
In contrast to the genetically encoded amino acid sequences of proteins, the composite sugar structures of cell glycome, such as the N- and O-glycosylated proteins, glycosaminoglycans and glycosphingolipids, arise in the interplay of different sugar-transferring enzymes, the glycosyltransferases [85]. Both, the genetic repertoire and the activity of glycosyltransferases vary considerably between different species [86-88] (Fig. 2). Moreover, glycosylation patterns vary within an organism with respect to the cell and tissue type involved [89].
Vertebrate glycans have diverse functions in health and disease [85, 90]. Endothelial glycoproteins can, for instance, serve as tissue-specific adhesion factors and “guideposts” for lymphocyte migration [91] and homing [92]. Various leucocytes expressing specialized adhesion CTLs, named selectins, are able to bind them for blood vessel adherence and rolling prior to extravasation [93]. The sugar moieties of various cell receptors, for instance of B and T lymphocytes, also play a crucial role in cell-cell communication and differentiation processes [94, 95]. For instance, they can be bound and cross-linked by soluble galectins, which leads to an increase in relative receptor density and stabilizes cell-cell interactions [96]. Glycans are also important for intracellular sorting and transport of lysosomal and Golgi proteins [97].
Further, differential glycosylation of immune globulin hinge regions and Fc parts was shown to influence interactions with respective Fc-receptors expressed by immune cell subsets and determine the inflammatory response [98]. Next to that, histone glycosylation (O- GlcNAcylation) was, in addition to acetylation, methylation and phosphorylation [99], reported
to mediate repackaging of chromosomal DNA [100] and epigenetic modulation of cell metabolism and signaling [101]. Lastly, secreted and cell surface associated mucosal glycans are known to interact with CTLs and may induce a tolerogenic phenotype in DCs [102], preventing autoimmunity and further immune-mediated damage [103]. Some pathogens, however, evolved strategies to subvert these protective functions and use host glycans for adhesion, either via respective lectins or via direct glycan-glycan interactions [104].
Recognition of bacterial, viral, fungal and parasitic glycans is mediated by membrane-bound or secreted CTLs [105]. Several cytosolic PRRs, including NLR nucleotide-binding oligomerization domain-containing protein 1 (NOD1) [72] and various galectins [106], are also known to bind to diverse foreign glycans.
1.4 PRR signaling
Two main PRR signaling strategies either involving PRR associated effector domains, or those of recruited adapter proteins, exist [71]. Upon ligand binding, several inflammasome activating PRRs, such as the NLR family member NACHT, LRR, FIIND, CARD domain and PYD domains-containing protein 1 (NLRP1) and RIG-I signal via enzymatically active N- terminal caspase activation and recruitment domains (CARDs). NLRP3 as well as AIM2, on the other hand, lack intrinsic enzymatic activity and recruit CARD-bearing adapter apoptosis- associated speck-like protein (ASC) [107] instead. Conclusively, the CARD-activated effector
Figure 2.
Glycosylation patterns in different organisms.
The relatively simple cell wall glycans produced by prokaryotic microorganisms, such as bacteria and archea, differ from the heavily branched eukaryotic cell membrane associated glycans of yeasts, insects and mammals including humans. diNAcBac = UDP-di-N- acetylbacillosamine; Glc = glycose; GlcNAc = N-Acetylglucosamine; Gal = galactose; GalNAc
= N-Acetylgalactosamine; Fuc = fucose; Man = mannose; Neu5Gc = N-Glycolylneuraminic acid; Neu5Ac = N-Acetylneuraminic acid.
Caspase-1 can activate several proinflammatory cytokines, such as IL-1β and IL-18 [72] and induce either pyroptotic [74] or apoptotic [107] cell death.
In contrast, NLRs like NOD1/2 associate with receptor interacting protein 2 (RIP2). Thus, downstream transcription factors NF-κB and mitogen-activated protein kinases (MAPK) [108]
are mobilized and enhance the expression of proinflammatory cytokines, such as IL-1β [108].
Additionally, alternative RIG-I signaling mode involving the activation of the CARD9, mucosa-associated lymphoid tissue lymphoma translocation protein 1 (MALT1) and B-cell lymphoma protein 10 (BCL10) signalosome enhancing IL-1β production [109], was described.
Single-chain TLRs homo- and heterodimerize upon ligand binding and signal via their respective cytoplasmic TIR homology domains [110], which recruit adapter proteins like the myeloid differentiation primary response 88 (MyD88), MyD88 adaptor-like (MAL), TIR- domain containing adaptor-inducing interferon-β (TRIF) and TRIF-related adaptor molecule (TRAM) [84, 110].
Membrane-bound CTLs can signal via four amino acids long N-terminal immunoreceptor tyrosine-based activation motifs (ITAMs), which consist of a single tyrosine residue separated from leucine or isoleucine by two variable amino acids (YxxL/I) and are phosphorylated via spleen tyrosine kinase (Syk) [76]. Alternatively, some CTLs use immunoreceptor tyrosine- based inhibitory motifs (ITIMs), which characteristically feature an N-terminal serine, isoleucine, valine or leucine and a C-terminal isoleucine, valine or leucine. A single tyrosine within the ITIM sequence, which is phosphorylated upon activation, is preceded by one and followed by two more random amino acids (S/I/V/LxYxxI/V/L) [111]. The respective signal transduction occurs via the cellular sarcoma (Src) homology region 2 domain-containing phosphatase-1 and -2 (SHP-1 resp. SHP-2), which inhibit downstream signaling [111]. The general distinction between “activating” and “inhibiting” motifs is, however, inconsistent, since inhibiting effects following ITAM-mediated receptor signaling route were also described [112, 113]. In addition, flexible CTL signaling independent of said motifs using the rapidly accelerated fibrosarcoma 1 (Raf-1) adapter protein was shown [114] (Chapter 3, Fig. 1). For the humoral CTLs, a fundamentally different mode of action is described: the mannose binding lectin (MBL), for instance, binds to bacterial surface glycans via its CTL-like domains and recruits MBL-associated serine proteases (MASPs) [115]. The latter, in turn, cleave the inactive precursors of antimicrobial complement proteins, thus catalyze their activation and assembly, ultimately leading to the formation of the pathogen membrane attack complex [115].
Some PRRs feature overlapping ligand recognition, as seen in bacterial glycan binding by intracellular NLRs, membrane bound myeloid CTLs and the intra- and paracellular galectins
[106, 108]. Varying localization and downstream signaling pathways between distinct PRRs allow highly differentiated innate immune responses during the course of infection [26, 116, 117]. In addition, backup or secondary PAMP recognition systems may compensate for genetic variations rendering single host PRRs unresponsive to infections [118]. These variations, on the other hand, may also provide host populations with distinctive selectable PRR phenotypes needed to counter the high mutability rates of pathogens [119].
Downstream signaling pathways of distinct PRRs form an intricate net of self-regulated and cross-regulated connections, which encompasses multiple levels of synergistic and antagonistic effects [120]. Spontaneous [121-123] or pathogen-induced dysregulation [124-126] of the PRRs signaling networks can lead to immune failure or to immunopathology: The host either succumbs to facultative and obligate pathogens, unable to keep them in check [122, 124, 127], or loses self-tolerance, which may result in autoimmune diseases [128]. For instance, CTL- and TLR-mediated exacerbation was observed in neurodegenerative disorders [129], such as multiple sclerosis (MS) [130] and systemic sclerosis (SS) [131], respectively.
2. The C-type lectin receptor superfamily
Among the PRRs, the phylogenetically conserved CTLs are recognized as major orchestrators of the innate and adaptive immunity cross-talk and to maintain homeostasis [132, 133]. They are, however, also known to act as entry points of numerous pathogens [134, 135]
and are involved in the pathogenesis and progression of several immune-mediated diseases [130, 136, 137] and cancer [138]. Additionally, some CTLs engage in cross-talk with various other PRRs [120] and are known to assemble into homo- and hetero-multimers to increase ligand affinity and modulate signaling [76, 114].[139]
The CTL-like fold of ancestral CTL carbohydrate recognition domain (CRD) stayed highly conserved in different vertebrate CTL groups [140] (Fig. 3, Tab. 2). To date, 17 different types of CTLs are distinguished [141] (Tab. 2). In this thesis, however, a specific focus has been placed on the membrane-bound myeloid CTLs belonging to the groups II and V, which, along with the secreted collectins and ficolins [115], contribute to immune defense and immune homeostasis maintenance in veterinary species [105, 142].
2.1 Myeloid CTLs
The myeloid CTLs are predominantly expressed by myeloid progenitor cell line derived cells, such as the Mϕs, DCs and Kupffer cells [114, 143]. However, myeloid CTL expression by other cell types, such as the B lymphocytes [144] and enterocytes [145], is observed as well.
These CTLs generally feature a C-terminal carbohydrate recognition domain (CRD), a neck
13 Figure 3.
Amino acid sequence alignment and clustering of a myeloid CTL Dectin-1 from various animal species, performed with NGPhylogeny.fr suite [139].
region, and a transmembrane region connecting the CRD to the respective signaling domains and the associated cytosolic adapter proteins [114]. Predominantly recognized as major contributors in antifungal immunity [146], CTLs are also important in antiviral, antibacterial and antiparasitic immunity [77, 147]. They additionally control cancerogenesis [148], and are involved in various homeostatic regulatory processes [105, 111, 142].
Insights into CTL functions were mostly derived from studies performed with human patients and mouse models [142]. The transferability of the respective findings is also accepted for orthologues found in other species [149]. The little which is specifically known for CTLs of non-human and non-model species is mostly derived from population studies, which aim to associate varying farm [150, 151] and companion animal [152] CTL genotypes with respective phenotypes in health and disease. Recent advancements and example applications of CTL research in veterinary species, specifically focusing on pigs, cattle and sheep, are summarized in the ensuing review [142] and put this work into the broad context of veterinary glycoimmunologic research.
Group Name Example functions
I Lecticans Intracellular protein processing and trafficking II Asialoglycoprotein and DC
receptors DAMP and PAMP recognition
III Collectins PAMP binding and complement activation
IV Selectins Cell adhesion and homing
V NK-cell receptors Activating and inhibitory cell signaling, DAMP and PAMP recognition VI Multi-CTL-domain
endocytic receptors Recycling of endocytic receptors VII Reg group Assumed to be involved in physiological and
pathological processes in pancreas VIII Chondrolectin, Layilin Assumed to function as adhesion molecules
IX Tetranectin Tissue remodeling
X Polycystin Unknown, mutations in this CTL lead to autosomal dominant polycystic kidney disease (ADPKD)
XI Attractin Unknown
XII EMBP Eosinophil major basic protein. Formation of
eosinophilic granulas
XIII DGCR 2 Unknown, mutations in this CTL lead to DiGeorge syndrome
XIV Thrombomodulin Anticoagulation and cell adhesion
XV Bimlec Unknown
XVI SEEC SCP, EGF, EGF and CTL domain containing
protein. Function unknown
XVII CBCP Calx‐β and CTL domain containing protein.
Tissue remodeling
Table 2.
An overview of vertebrate CTL groups and their functions [141].
3. Role of CTLs in veterinary species: Recent Advancements and Applications
International Journal of
Molecular Sciences
Review
C-Type Lectins in Veterinary Species: Recent Advancements and Applications
Dimitri Leonid Lindenwald and Bernd Lepenies *
Immunology Unit & Research Center for Emerging Infections and Zoonoses (RIZ), University for Veterinary Medicine Hannover, Foundation, 30559 Hannover, Germany;
Dimitri.Leonid.Lindenwald@tiho-hannover.de
* Correspondence: bernd.lepenies@tiho-hannover.de; Tel.: +49-(0)5-11/953-6135 Received: 29 June 2020; Accepted: 17 July 2020; Published: 20 July 2020
Abstract: C-type lectins (CTLs), a superfamily of glycan-binding receptors, play a pivotal role in the host defense against pathogens and the maintenance of immune homeostasis of higher animals and humans. CTLs in innate immunity serve as pattern recognition receptors and often bind to glycan structures in damage- and pathogen-associated molecular patterns. While CTLs are found throughout the whole animal kingdom, their ligand specificities and downstream signaling have mainly been studied in humans and in model organisms such as mice. In this review, recent advancements in CTL research in veterinary species as well as potential applications of CTL targeting in veterinary medicine are outlined.
Keywords: C-type lectin; glycans; immune modulation; comparative immunology; veterinary immunology
Int. J. Mol. Sci. 2020, 21, 5122; doi:10.3390/ijms21145122 www.mdpi.com/journal/ijms
The extent of Dimitri L. Lindenwald contribution to the article is evaluated according to the following scale:
A. has contributed to collaboration (0-33%).
B. has contributed significantly (34-66%).
C. has essentially performed this study independently (67-100%).
1. Design of the project including design of individual experiments: - 2. Performing of the experimental part of the study: -
3. Analysis of the experiments: -
4. Presentation and discussion of the study in article form: B
17 1. Introduction
Glycans belong to the most abundant macromolecules constituting all living organisms. In multicellular animals, processes such as cell migration, homeostasis maintenance, and innate immune signaling rely on the ability of cells to recognize glycoconjugates, most often in the form of glycoproteins and glycolipids, via glycan binding proteins, the so-called lectins [1]. In the immune system, lectin receptors are either secreted or found on the cell surface of immune cells [2]. Three major receptor families that are involved in glycan recognition in the immune system include the galectins [3], siglecs [4], and C-type lectins (CTLs) [5]. Among these, the phylogenetically conserved CTLs proved to play a pivotal role in both host–pathogen interactions and homeostasis maintenance in vertebrate and in invertebrate species [5–7].
Myeloid CTLs are mainly expressed by antigen-presenting cells (APCs) and act as pattern recognition receptors (PRRs) that bind to pathogen and damage-associated molecular patterns (PAMPs and DAMPs) [5]. Most CTL receptors require Ca2+ ions for binding, hence the “C” in the name. However, some CTLs also bind carbohydrate, peptide, or crystalline ligands in a Ca2+-independent manner [5]. The importance of CTLs for antifungal immunity is well recognized in human medicine [8] (Table 1). For instance, an increased risk for candidiasis [9]
and a higher susceptibility to aspergillosis is associated with CTL polymorphisms in human patients [10]. However, CTLs are also chiefly important in the scope of immune homeostasis [11–14] and protection against bacteria, viruses, parasites, and cancer [15–19] (Fig. 1). They induce signal pathways leading to the expression of chemokines and cytokines, and they are involved in phagocytosis and antigen (cross-)presentation by molecules of the major histocompatibility complex (MHC) I or II to T-cells, thus bridging innate and adaptive immunity [20] (Fig. 1). CTLs associated with an immunoreceptor tyrosine-based activation motif (ITAM), such as the dendritic cell-associated lectin 1 (Dectin-1/Clec7a), and 2 (Dectin- 2/Clec6a) and the macrophage-inducible Ca2+-dependent lectin (Mincle/Clec4e), signal upon ligand binding via phosphorylation of the spleen tyrosine kinase (Syk). Syk activates further kinases such as the protein kinase C (PKC), which results in downstream activation and assembly of the caspase recruitment domain-containing protein 9 (CARD9), mucosa-associated lymphoid tissue lymphoma translocation protein 1 (MALT1), and B-cell lymphoma protein 10 (BCL10) complex. Finally, this leads to phosphorylation of IκB and translocation of the transcription factor NF-κB into the nucleus, where it enhances the transcription of numerous cytokine and chemokine genes [21]. This activation may be counteracted by CTLs such as the DC immunoreceptor (DCIR/Clec4a), which carry an immunoreceptor tyrosine-based inhibition
motif (ITIM) and engage the src homology domain-containing protein tyrosine phosphatases (SHP), thus restricting ITAM-mediated signals and limiting inflammation [22,23].
ITAM/ITIM-independent CTLs, such as the dendritic cell-specific ICAM-3-grabbing non- integrin (DC-SIGN/Clec4l/CD209) can also stimulate the activation of NF-κB via steroid receptor coactivator (SRC) and p21-activated kinase (PAK) or via the leukocyte-specific protein 1 (LSP-1), kinase suppressor of RAS 1 (KSR-1), and connector enhancer of kinase suppressor of RAS (CNK) rat sarcoma (RAS) signalosome [22]. However, CTLs were also shown to act as pathogen entry receptors and targets of immune escape [24] and may contribute to immune pathology in several infections [25–28], as well as in autoimmune diseases and cancer [20,29,30].
Table 1. Overview of selected human CTLs, including examples of respective ligands and functions.
C-Type
Lectin Main Expression Ligands Recognized Pathogens (Examples)
Dectin-1
Monocytes, Macrophages, Dendritic cells, NK cells,
(1→3)-β-D-glucans C. albicans, A.fumigatus, C. neoformans, Mycobacterium spp.
Dectin-2
Monocytes, Macrophages, Dendritic cells, NK cells,
Endothelial cells,
high-mannose oligosaccharides
C. albicans, A. fumigatus, M. tuberculosis, S.
mansoni
Mincle
Monocytes, Macrophages, Dendritic cells,
mycobacterial trehalose 6,6’-dimycolate (TDM), alpha-mannose residues, DAMPs
Mycobacterium spp., Malassezia spp.
DC-SIGN Dendritic cells
high-mannose and fucose-containing oligosaccharides
HIV-1, Dengue virus, Measles virus, SARS
coronavirus DCIR Monocytes,
Macrophages Mannose, fucose HIV-1
MICL
Macrophages, Monocytes, Granulocytes,
DAMPs, urate crystals,
hemozoin Unknown
MGL Monocytes,
Dendritic cells
terminal galactose and
N-acetylgalactosamine Influenza virus For further details, see contents of this review. For more detailed information on the role of CTLs in pathogen recognition, see review [5].
Figure 1. CTL functions and signaling pathways. CTLs recognize molecular patterns of fungal, parasitic, bacterial, and viral pathogens (so-called PAMPs) as well as those of dead and malignant cells (DAMPs). Upon pathogen binding, CTL–mediated signaling leads to cytokine and chemokine production and phagocytosis. The latter results in antigen (cross-)presentation and priming of T-cells. However, some viruses, such as the zoonotic Dengue fever virus, developed immune evasion mechanisms and may exploit CTLs such as DC-SIGN to promote viral transmission and dissemination.
Most insights into animal CTLs functions were gained in studies performed with model organisms, predominantly mice. In vitro CTL–ligand screenings using murine [31–33] or human [34, 35] recombinant CTL hFc-fusion protein libraries (Fig. 2) allowed for the identification of novel CTL/pathogen interactions and CTL ligands [34,36]. Further studies analyzed ligand binding and downstream signal transduction of mouse and human CTL using APCs [21,37–41] or CTL expressing reporter cell systems [42–45]. Data from human patients [46] and studies performed in CTL−/− mice or mice that were deficient for CTL-mediated signaling [47, 48] depict the effects of particular CTLs in vivo. However, ligand specificities of CTL orthologues, downstream signaling pathways, and effector functions may significantly vary among different species [44,49–59], thus emphasizing the need for CTL investigations performed in a species-specific manner (Fig. 3). In particular, there is a knowledge gap
regarding CTL function in veterinary species. In the following sections, we will discuss recent studies in this field and briefly highlight potential applications of CTL targeting in veterinary medicine.
Figure 2. Recombinant CTL libraries for in vitro screenings allow for the identification of CTL ligands. The murine [31] and ovine [60] CTL libraries were expressed as CTL-Fc fusion proteins. For the bovine [61] library, cow CTL and bacterial biotinylation site coding DNA fragments were fused and expressed in E. coli, yielding biotinylated fusion proteins that can be used for glycan array- and ELISA- based binding studies and high throughput pull-down assays.
Figure 3. Hierarchical clustering of amino acid sequences comprising Dectin-1 (Clec7a) CTLs of selected animal species, and humans. For the Atlantic salmon (Salmo salar), missing a corresponding ortholog Dectin-1 encoding gene, a functional ortholog C-type lectin receptor C was chosen. Remarkably, the degree of similarity in the Dectin-1 amino acid sequences mirrors the phylogenetic relationships between the respective species. Visualization and clustering were performed with NGPhylogeny.fr suite [62].
2. Protective Role of Veterinary Relevant CTLs
Most often, CTL functions in veterinary species were investigated in population screening studies. These studies correlated the course of infection- or general susceptibility-associated phenotypes with specific CTL genotypes, thus allowing for conclusions concerning the functions of individual CTLs in health and disease [63, 64]. By using such strategies, implications of CTLs in antimicrobial immunity were recently described. For instance, a link between different single nucleotide polymorphisms (SNPs) in the bovine Dectin-1 encoding gene and the susceptibility to Johne’s disease caused by Mycobacterium avium ssp.
paratuberculosis (MAP) was found in screening studies in Canadian [65] and in Indian cattle [66]. Similarly, multiple SNPs were described in the Dectin-1 encoding gene in pigs [67]. All SNPs discovered in commercial pig lines proved to be neutral when compared to the reference pig Dectin-1 in a NF-κB driven reporter system using the Dectin-1 ligand zymosan [67]. In contrast, the Dectin-1 isoform found exclusively in wild boars displayed a markedly enhanced activatory capability upon ligand stimulation. This augmented Dectin-1 signaling was suggested to negatively influence the overall fitness of its carriers, possibly leading to
overshooting immune responses to pathogenic and commensal fungi [67]. A SNP in the gene encoding for the humoral mannose binding lectin A (MBL1), on the other hand, was hypothesized to result in a loss-of-function type of mutation and in an increased shedding of Salmonella sp. in fattened pigs [68]. Consistently, a negative correlation between the concentration of the orthologous MBL and Salmonella susceptibility was observed in chicken [69]. However, multiple SNPs in the non-coding intron parts of the MBL gene were shown to correlate with varying serum MBL levels in Chinese Hu sheep [70], demonstrating that SNPs that do not directly affect the CTL protein sequence may nevertheless influence CTL levels in vivo.
The antimicrobial effects of several CTLs were recently shown for both sweet water [71]
and salt water [72] fish species. In carp, a number of CTLs were identified to be downregulated on macrophages upon stimulation with the ß-glucan curdlan [73], which is a well-known ligand of Dectin-1 in mammals [74]. This surprising effect may represent a negative feedback mechanism preventing an over-stimulation of the carp immune cells in the course of bacterial and fungal infections [73]. In contrast, several salmon genes encoding signaling molecules downstream of CTL receptors SCRLA, SCRLB, and SCRLC (Salmon C-type lectins A,B,C), such as the one encoding the fish analogue of the mammalian tyrosine kinase Syk, were significantly upregulated following ß-glucan stimulation. These findings suggest an involvement of CTLs in pathogen recognition and signal transduction in salmon [75].
A strong correlation between the protective Th1 response and Dectin-1 engagement was recently shown in mouse Leishmania spp. infections models [76, 77], demonstrating a crucial role of this specific CTL in anti-Leishmania immunity. The site-specific expansion of Dectin- 1 expressing DCs following intradermal injection of the specific Dectin-1 agonist curdlan sufficed to protect wild-type mice from illness following transdermal Leishmania infection, whereas Dectin-1−/− mice succumbed to the disease [76]. Leishmaniosis is an important and life-threatening disease in dogs; an insufficient Th1 response in favor of the detrimental Th2 response [78] in clinically affected canids renders vaccine development a significant challenge [79, 80]. However, the function of canine Dectin-1 during Leishmania spp. infection in dogs is yet unknown.
The influence of different CTL-associated alleles on anti-parasitic immunity was described in wild Soay sheep on the St Kilda archipelago, Scotland [81]. In this study, SNPs in the presumed cis-regulatory element of the clec16a gene, a CTL-encoding gene associated with immunoglobulin isotype deficiency disorders in humans and mice [82], strongly correlated with specific IgA levels against the intestinal roundworm Telodorsagia circumcincta in lambs as
well as in mature sheep [81]. In fish, CTLs may also contribute to protective immune responses against parasites as suggested by a positive correlation between macrophage mannose receptor 1 (MRC1/Clec13d) expression levels and the relative resistance of Atlantic [83] and pink salmon toward sea lice infestation [84]. These findings indicate that the selective breeding or genetic engineering introducing desirable CTL alleles into veterinary species might be a means to improve their performance and disease resistance in the future.
3. Detrimental Role of Veterinary Relevant CTLs 3.1. Pathological Inflammation
Dysregulation in CTL signaling can lead to sterile inflammation in the absence of any pathogen [29]. For instance, a possible involvement of Dectin-1 in sterile inflammation and postpartum placenta retention was suggested in cows, since higher numbers of Dectin-1- expressing uterine macrophages were detected in retention-affected cows compared to cows with a regular afterbirth [85]. Allergic hypersensitivity and immunopathology can also be mediated by CTLs: in horses, Dectin-1, Dectin-2, and macrophage lectin 2 (MGL/Clec10a) may contribute to severe allergic dermatitis following insect bites [86]. Similar findings were also obtained for mice and men, as Dectin-1−/− mice were largely protected against Aspergillus fumigatus-initiated corneal keratitis [87] and Dectin-1 blockade using the antagonist laminarin alleviated the severity of fungal keratitis in human patients [28]. Other CTLs may also be involved in immune pathology upon CTL engagement during infections.
For instance, the myeloid C-type lectin-like receptor (MICL/Clec12a) was shown to cross- prime CD8+ T-cells contributing to the development of experimental cerebral malaria [26] and to promote murine viral lymphocytic choriomeningitis virus (LCMV) infections by hampering pathogen clearance [88]. To date, there is a knowledge gap on how CTLs may contribute to immune pathology in veterinary species, thus highlighting the need for further research in this field.
3.2. Exploitation of CTLs by Pathogens
Numerous viral pathogens, among them arthropod-borne phleboviruses, such as Dengue virus and Rift Valley Fever virus, specifically target CTLs such as the human DC-SIGN to establish infections [89]. Similarly, the feline corona virus, a close relative of both the canine coronaviruses (CCoVs) and the porcine transmissible gastroenteritis virus (TGEV) [90], establish infection by exploiting the cat DC-SIGN [91]. Heterologous expression of human DC-
SIGN in otherwise resistant cells rendered them susceptible for infection with an avian corona virus, chicken Infectious Bronchitis virus (IBV) [92].
These studies on viral/DC-SIGN interactions indicate that CTLs represent relevant receptors for viral entry into host cells; thus, they may play a crucial role in the cross-species transmission of viruses.
However, not only viruses, but also bacteria and parasites were reported to highjack DC- SIGN or its orthologues, as recently demonstrated for the bacterium Yersinia pestis [93] and the apicomplexan parasite Toxoplasma gondii [94]. For the ruminant trematode parasite Fasciola hepatica, a strong downregulation of host DC effector functions via DC-SIGN was observed, finally leading to immune dysregulation and T-cell anergy [95]. The apicomplexan parasite Neospora caninum circulating between canine definitive hosts and bovine intermediate hosts causes large losses in dairy and beef production worldwide by inducing abortions [96]. In a murine model, N. caninum engaged Dectin-1 and thereby inhibited DC effector functions in wild-type mice compared to Dectin-1−/− mice [97]. Further helminths, such as the nematode Toxocara canis, were shown to synthesize a repertoire of mammalian-like CTLs [98,99] and unusual glycans [100], which might interfere with and subvert CTL-based glycan recognition in vivo [100]. In conclusion, the examples highlighted here demonstrate a variety of immune evasion strategies of parasites to interfere with mammalian CTL-mediated immunity [101].
4. Harnessing the Power of CTLs
CTLs represent attractive targets for immune modulation, not only in mice and humans, but also in veterinary species as they hold promise of novel and/or improved diagnostic, prophylactic, and therapeutic applications. In the following, we will briefly highlight some recent examples.
4.1. General Aspects
In veterinary research, cell-surface expressed CTLs can be used as cell-specific markers, thus allowing for the discrimination of immune cell subsets to elucidate specific functions. For instance, the analysis of antiviral effectorfunctions of porcine DCs was performed in Clec13B/LY75/CD205 positive DCs [102]. Accordingly, the in vitro characterization of rainbow trout immune cell subpopulations was performed by staining Clec4t1 positive monocyte-derived macrophage and DC precursor cells with CTL-specific antibodies [71].
Plate-bound or soluble recombinant CTL-based fusion proteins can in turn be used for binding studies with bacteria, viruses, fungi, and parasites in order to identify interactions of CTLs with
PAMPs [103]. In this regard, the lectin array technology offers excellent opportunities for the diagnosis of blood and urine infections [61] or protein glycosylation-associated disorders [104]
in animal or human patients.
4.2. Prophylaxis
CTL-targeting adjuvants, for instance the Mincle glycolipid ligand trehalose-6,6- dimycolate (TDM) derived from the M. tuberculosis cell wall and its synthetic analogue trehelose-6,6-dibehenate (TDB) [105], were recently evaluated for their immunogenic properties in mouse models [106,107]. In veterinary research, this approach was adopted for the development of a bovine tuberculosis vaccine [108]. In addition, the co-application of TDB and furfurman (targeting Dectin-2) in pigs as well as TDB and curdlan (targeting Dectin-1) along with further PRR targeting ligands in cattle markedly enhanced vaccination efficacy against Foot-and-Mouth Disease by providing robust and long-lasting effects in vivo [53].
Similarly, a TDB-based experimental liposomal vaccine adjuvant, CAF01, was shown to mediate long-lived M. tuberculosis-specific T-cell responses in humans [109] and enhanced the efficacy of a commercially available inactivated influenza vaccine in ferret models [110]. This adjuvant system was also used for rainbow trout immunization against Aeromonas salmonicida and induced enhanced cellular immunity in comparison to formulation with the standard adjuvant mineral oil [111]. Further trehalose-based CTL-targeting compounds, such as the 2- hydroxy benzoic acid coupled trehalose compound 6,6′-bis-(3,5-di-tert-butylsalisate)-α,α- trehalose (UM1024), demonstrated high Mincle targeting specificity and low cytotoxicity in mouse and human peripheral blood mononuclear cells (PBMCs) in vitro and robust immunogenicity in a mouse model [112]. In pigs, a recently characterized MICL ortholog was proposed as a selective antigen delivery target, since it mediated antigen uptake by pig dendritic cells in vitro [113]. In poultry, novel CTL-targeting compounds such as pustulan [114], as well as other promising CTL immunization targets, namely Clec13B [115,116], Clec17AL-A, and –B [117], the bird homologues of the mammalian Prolectin/Clec17A, were described. These studies indicate that CTLs in veterinary species are indeed promising targets to enhance vaccine efficacy; however, further research is needed to evaluate the potential of the respective adjuvant candidates in vivo.
4.3. Therapeutic Applications
CTL targeting can not only enhance the efficacy of vaccines, but it can also be adapted to metaphylaxis and therapy. As a potential treatment of parasitic infections, T-cell modulation
toward the favorable T helper type 1 response was achieved via the metaphylactic curdlan stimulation of Dectin-1 expressing DCs in the mouse model of cutaneous leishmaniosis. In this model, the co-injection of curdlan along with infectious L. major promastigotes resulted in a resistant phenotype observed in the otherwise highly susceptible BALB/c mouse strain [76].
This finding matches the protective properties of yeast glucans that had previously been described in murine leishmaniosis models [118,119]. The utility of this approach for leishmaniosis treatment in other species, such as the domestic and feral dogs [120], cats [121], and foxes [122] remains to be investigated.
DC-SIGN can serve as an adhesion and dissemination receptor for cat-born Toxoplasma gondii infection [94]. Therefore, the specific antibody- or glycan-mediated blocking of paralogues could possibly be applied to prevent toxoplasmosis in chicken [123], thus reducing the risk of alimentary infections in poultry meat consumers. Additionally, selective blocking of the corresponding DC-SIGN orthologue might be applied to limit IBV spread in chickens.
Although no chicken DC-SIGN orthologue has been identified yet [92], it is probable to exist due to the engagement of human DC-SIGN and the DC-SIGN-related protein L-SIGN (CD209L/Clec4m) by IBV to establish experimental infections in vitro [92].
Furthermore, CTLs represent important therapeutic targets in immune-mediated diseases.
As such, isolated helminth immunomodulatory compounds mimicking an infestation might be used to suppress autoimmunity in human patients [124]. Consistently, a desensitizing DC targeting construct composed of the mite allergoid and mannan, a ligand of the murine [125], ovine [60], and human [126] CTL Dectin-2, was described as a potential allergy treatment in dogs [127]. Another potential therapeutic CTL target is Mincle. The expression of Mincle along with Syk and CARD9 adapter proteins was described in cattle papillomavirus-associated urothelial tumor cells, suggesting their phagocytotic capacity and rendering Mincle a promising target in veterinary oncology [128].
5. Conclusions
Along with other PRRs, CTLs also are important constituents of the host–microbiome communication interface: symbiotic microbes interact with CTLs [129] and affect host cytokine production and CTL expression in trained innate immunity [130] and homeostasis [131] by epigenetic mechanisms. For the Dectin-1 targeting mushroom glucans lentinan [132] and proteo-β-glucan [133], a robust Dectin-1-mediated antidepressant-like effect was demonstrated in mouse models [133,134], illustrating the influence that CTLs may have upon animal and human cerebral functions via the microbiota–gut–brain signaling axis [135]. Many veterinary
and human nutraceuticals, or pharmacologically active nutrition additives [136,137], are also likely to exhibit their respective immune stimulating and/or modulatory functions via CTL- mediated signaling. Such an effect was also observed in a study performed in crayfish: crayfish susceptibility to the viral White-spot disease was reduced while the expression of hemocyte- associated crayfish CTL (X2C306-1) was simultaneously upregulated following the probiotic gavage of Bacillus amyloliquefaciens [138]. Further positive effects of carbohydrate supplements were demonstrated in lentinan-rich shiitake mushrooms gavage in a rat model of human dyslipidemia [139], probiotic glucan gavage in carp [140], and mannoprotein supplementation in adult and aging dogs [141].
Finally, functions of CTLs in intrauterine immunity and maternal–fetal tolerance [142], as well as in parturition [143], were shown in humans. Initial studies suggest a possible involvement of CTLs in veterinary species in these processes. For instance, a microarray-based differential gene expression investigation in pregnant sheep yielded several candidate CTLs, such as the DCAR/Clec4b, that were upregulated during the early gestation phase in the endometrium [144]. However, the impact of the respective CTLs on the placenta immunity in vivo is an open question for future research.
In conclusion, advancements in the understanding of CTL functions in veterinary species will open up new applications in veterinary medicine; yet, the current lack of knowledge clearly highlights the need for further research. To bridge this knowledge gap between model and target species, novel tools, such as recombinant bovine [61] and ovine [60] CTL receptor libraries, were recently generated. The role of the identified CTL interactions of veterinary relevant species with pathogens will be unravelled in further studies.
Author Contributions: Original draft preparation and visualization, D.L.L.; review and editing, B.L. All authors have read and agreed to the published version of the manuscript.
Acknowledgments: 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). This publication was supported by Deutsche Forschungsgemeinschaft and the University of Veterinary Medicine Hannover, Foundation, within the funding programme Open Access Publishing.
Conflicts of Interest: The authors declare no conflict of interest.
