The IgA receptor, FcαRI, may be dysfunctional in goats

Immunoglobulin A (IgA) is an antibody that plays a crucial role in the immune function of mucus membranes. TGF-β, together with IL-5, is responsible for class-switching of B lymphocytes into IgA-producing plasma cells (Coffman et al., Reference Coffman, Lebman and Shrader1989; Sonoda et al., Reference Sonoda, Matsumoto, Hitoshi, Ishii, Sugimoto, Araki, Tominaga, Yamaguchi and Takatsu1989). This local production of IgA is termed secretory IgA and is the predominant form of IgA in mucosal secretions (van Egmond et al., Reference van Egmond, Damen, van Spriel, Vidarsson, van Garderen and van de Winkel2001; Bakema and van Egmond, Reference Balic, Cunningham and Meeusen2011). Considering the importance of IgA in the protection of mucus membranes, it is unsurprising that studies investigating immune responses to parasitic infections have found associations between IgA and parasite-induced eosinophilia (Muraki et al., Reference Muraki, Gleich and Kita2011). IgA has been demonstrated to be associated with nematode fecundity and peripheral eosinophils, and therefore resistance to helminth infection in sheep (Gill et al., Reference Gill, Gray, Watson and Husband1993; Henderson and Stear, Reference Henderson and Stear2006; Halliday et al., Reference Halliday, Routledge, Smith, Matthews and Smmith2007; Hernández et al., Reference Hernández, Hernández, Stear, Conde-Felipe, Rodríguez, Piedrafita and González2016; Fairlie-Clarke et al., Reference Fairlie-Clarke, Kaseja, Sotomaior, Brady, Moore and Stear2019). The lack of an effective IgA response has also been implicated in increased susceptibility to parasite infection in goats (Basripuzi et al., Reference Basripuzi, Salisi, Isa, Busin, Cairns, Jenvey and Stear2018). Additionally, in vitro studies have indicated that parasite-induced eosinophil cytolysis may be dependent upon IgA binding to its receptor (Ueki et al., Reference Ueki, Melo, Ghiran, Spencer, Dvorak and Weller2013).

IgA exists as a monomer of two identical heavy chains and two identical light chains, but can form a dimer (secretory IgA) stabilized by disulphide bonds and a joining (J) chain (Woof and Russell, Reference Woof and Russell2011). In ruminants, most IgA in serum derives from mucosal surfaces and is largely dimeric (Scicchitano et al., Reference Scicchitano, Sheldrake and Husband1986). The FcαRI (CD89) is a transmembrane receptor and due to heavy glycosylation, eosinophil FcαRI is heavier (70–100 kDa) compared to macrophage and neutrophil FcαRI (55–75 kDa) (van Egmond et al., Reference van Egmond, Damen, van Spriel, Vidarsson, van Garderen and van de Winkel2001). FcαRI comprises a ligand-binding alpha chain, which consists of two extracellular Ig-like domains, a transmembrane domain, and a short cytoplasmic tail. Due to the lack of signalling motifs, FcαRI must associate with the FcRγ chain for signalling and function (van Egmond et al., Reference van Egmond, Damen, van Spriel, Vidarsson, van Garderen and van de Winkel2001).

Molecular modelling of the eosinophil IgA receptor indicates that it may be dysfunctional in goats (Basripuzi et al., Reference Basripuzi, Salisi, Isa, Busin, Cairns, Jenvey and Stear2018). Structural modelling of the IgA receptor indicated that the sheep and human receptors had similar structures. However, the goat receptor had a different conformation, with the C-terminus being bent away from the main body of the protein and lacked an alpha helix within the transmembrane domain (approximately 20 amino acids) (Fig. 1). Although the sheep and goat sequences showed high sequence identity (96%), the sheep and goat sequences showed only 55 and 54% sequence identity with the human protein. Additionally, a difference of only three amino acids was observed within the binding domain (EC1) and no differences were observed within the transmembrane domain. This seems to indicate that the loss of secondary structure within the transmembrane domain is most likely a consequence of sequence differences outside of this domain. As yet, no mouse homologue of FcαRI has been identified (van Egmond et al., Reference van Egmond, Damen, van Spriel, Vidarsson, van Garderen and van de Winkel2001; Decot et al., Reference Decot, Woerly, Loyens, Loiseau, Quatannens, Capron and Dombrowicz2005), thus if eosinophils and IgA interact to control nematodes through ADCC, mice may not be appropriate models for studying eosinophil activity against nematodes infecting humans and ruminants.

Fig. 1. The goat IgA receptor, FcαRI, may be dysfunctional in goats. Homology modelling of human, P24071.1 (A); sheep, XM_027976143.1 (B) and goat, XM_018059931.1 (C) IgA receptors revealed that human and sheep receptors have a similar conformation, however, the goat receptor has a C-terminus that is bent away from the main body of the protein and lacked an alpha helix within the transmembrane domain (blue).

Differences in eosinophil responsiveness are not due to IL-5 and IL-5Rα

IL-5 is a growth factor and chemoattractant of eosinophils and is involved in the recruitment, activation, degranulation and survival of eosinophils (Lopez et al., Reference Lopez, Sanderson, Gamble, Campbell, Young and Vadas1988; Horie et al., Reference Horie, Gleich and Kita1996; Fulkerson et al., Reference Fulkerson, Schollaert, Bouffi and Rothenberg2014; Sippel et al., Reference Sippel, Pierlot, Renault, Groenen and Strasser2018). IL-5 is an important cytokine in the differentiation and activation of anti-parasitic eosinophil responses and has been specifically targeted in mice to demonstrate the in vivo role of eosinophils in helminth infections, as discussed in previous reviews (Huang and Appleton, Reference Huang and Appleton2016; Meeusen and Balic, Reference Meeusen and Balic2000). However, the reagents and knock-out models used in these rodent studies are generally not available or suitable for large animal experimentation. Several studies have shown that mice vaccinated with recombinant cytokines can induce autoantibodies that specifically cancel out the activity of the native cytokine in vivo (Dalum et al., Reference Dalum, Butler, Jensen, Hindersson, Steinaa, Waterston, Grell, Feldmann, Elsner and Mouritsen1999; Hertz, et al., Reference Hertz, Mahalingam, Dalum, Klysner, Mattes, Neisig, Mouritsen, Foster and Gautam2001; Richard et al., Reference Richard, Grencis, Humphreys, Renauld and Van Snick2000). This approach may provide an alternative for the in vivo study of immune responses in large animals.

IL-5 acts on target cells by binding to its receptor, IL-5R, which consists of an α and a β subunit, however the α subunit is specific to IL-5R only (McBrien and Menzies-Gow, Reference McBrien and Menzies-Gow2017). IL-5 is a dimeric glycoprotein with a four-helix bundle motif. In complex, IL-5 forms a homodimer which is sandwiched by the IL-5Rα. Binding of IL-5 to the receptor alpha subunit results in recruitment of the β subunit to the receptor (Tavernier et al., Reference Tavernier, Devos, Cornelis, Tuypens, Van der Heyden, Fiers and Plaetinck1991; Kusano et al., Reference Kusano, Kukimoto-Niino, Hino, Ohsawa, Ikutani, Takaki, Sakamoto, Hara-Yokoyama, Shirouzu, Takatsu and Yokoyama2012). The human and mouse cDNA code for proteins of 134 and 133 amino acids in length, respectively, and have 70% amino acid sequence identity (Yamaguchi, Reference Yamaguchi1994).

Molecular modelling indicates that it is unlikely that IL-5 and its receptor are responsible for differences in eosinophil responses between sheep and goats. For IL-5, the predicted protein sequences were identical between sheep and goat, while for IL-5Rα, the predicted protein sequences differed in 6 out of the 432 amino acids. All of these differences occurred in the first 42 amino acids of the protein, which included within the 15–34 amino acids that aligned with the signal peptide for human IL-5Rα, as well as within a region lacking secondary structure. This indicates that the observed sequence differences are unlikely to result in a dysfunctional IL-5 and IL-5R.

The eotaxin receptor, CCR3, contains a frameshift mutation in goats

Eotaxin is a chemoattractant cytokine which is important for promoting eosinophil recruitment and degranulation (Garcia-Zepeda et al., Reference Garcia-Zepeda, Rothenberg, Ownbey, Celestin, Leder and Luster1996; Davoine and Lacy, Reference Davoine and Lacy2014), and has been shown to be important for eosinophil recruitment during helminth infections (Rothenberg et al., Reference Rothenberg, MacLean, Pearlman, Luster and Leder1997; Mochizuki et al., Reference Mochizuki, Bartels, Mallet, Christophers and Schröder1998; Ruth et al., Reference Ruth, Lukacs, Warmington, Polak, Burdick, Kunkel, Strieter and Chensue1998; Culley et al., Reference Culley, Brown, Conroy, Sabroe, Pritchard and Williams2000; Simons et al., Reference Simons, Rothenberg and Lawrence2005). Eotaxin belongs to the CC chemokine family, which is distinguished by two cysteines immediately adjacent to the N terminus. Eotaxin has been determined to be in equilibrium between a monomer and a dimer at near physiological pH, however, functional eotaxin is present as a monomer (Crump et al., Reference Crump, Rajarathnam, Kim, Clark-Lewis and Sykes1998). It exhibits a chemokine-like fold consisting of three anti-parallel β-strands with an overlying α-helix (Crump et al., Reference Crump, Rajarathnam, Kim, Clark-Lewis and Sykes1998). There are three molecules of Eotaxin, CCL11 (Eotaxin-1), CCL24 (Eotaxin-2) and CCL26 (Eotaxin-3); however, Eotaxin-1 is the dominant isoform. The highest levels of Eotaxin-1 are found in the GI system and can be produced by a variety of cells (Kitaura et al., Reference Kitaura, Nakajima, Imai, Harada, Combadiere, Tiffany, Murphy and Yoshie1996; Ying et al., Reference Ying, Robinson, Meng, Barata, McEuen, Buckley, Walls, Askenase and Kay1999). Eotaxin-1 is important for the release of eosinophil precursors from the bone marrow (Palframan et al., Reference Palframan, Collins, Williams and Rankin1998) and is activated by Th2 cytokines (Mochizuki et al., Reference Mochizuki, Bartels, Mallet, Christophers and Schröder1998) and inhibited by Th1 cytokines (Miyamasu et al., Reference Miyamasu, Yamaguchi, Nakajima, Misaki, Morita, Matsushima, Yamamoto and Hirai1999; Fukuda et al., Reference Fukuda, Yamada, Fujitsu, Kumagai and Nishida2002). Eotaxin-2 is synthesized and released by mucosal epithelial cells and macrophages, while Eotaxin-3 is produced by epithelial and endothelial cells (Kitaura et al., Reference Kitaura, Suzuki, Imai, Takagi, Suzuki, Nakajima, Hirai, Nomiyama and Yoshie1999; Shinkai et al., Reference Shinkai, Yoshisue, Koike, Shoji, Nakagawa, Saito, Takeda, Imabeppu, Kato, Hanai, Anazawa, Kuga and Nishi1999; Dulkys et al., Reference Dulkys, Schramm, Kimmig, Knöß, Weyergraf, Kapp and Elsner2001). Eotaxin-2 and Eotaxin-3 can also recruit eosinophils, but at later stages of infection (>24 h) (Ying et al., Reference Ying, Robinson, Meng, Barata, McEuen, Buckley, Walls, Askenase and Kay1999; Rosenwasser et al., Reference Rosenwasser, Zimmermann, Hershey, Foster and Rothenberg2003; Kalomenidis et al., Reference Kalomenidis, Stathopoulos, Barnette, Guo, Peebles, Blackwell and Light2005; Schratl et al., Reference Schratl, Sturm, Royer, Sturm, Lippe, Peskar and Heinemann2006).

All Eotaxin isoforms are associated with a single receptor, CCR3, however the receptor binds to the different isoforms with different affinities (Kitaura et al., Reference Kitaura, Suzuki, Imai, Takagi, Suzuki, Nakajima, Hirai, Nomiyama and Yoshie1999). CCR3 is very abundant in eosinophils (approximately 40–400 × 10³ receptors per cell), but is also expressed at lower levels in basophils, mast cells and a subset of Th2 lymphocytes (Sallusto et al., Reference Sallusto, Mackay and Lanzavecchia1997; Uguccioni et al., Reference Uguccioni, Mackay, Ochensberger, Loetscher, Rhis, LaRosa, Rao, Ponath, Baggiolini and Dahinden1997; Romagnani et al., Reference Romagnani, De Paulis, Beltrame, Annunziato, Dente, Maggi, Romagnani and Marone1999). CCR3 also binds to other non-eosinophil selective CC chemokines, but with lower affinity compared to Eotaxin-1 (Ponath et al., Reference Ponath, Qin, Post, Wang, Wu, Gerard, Newman, Gerard and Mackay1996; Baggiolini et al., Reference Baggiolini, Dewald and Moser1997; Sabroe et al., Reference Sabroe, Hartnell, Jopling, Bel, Ponath, Pease, Collins and Williams1999). CCR3 is a G-protein-coupled receptor of 335 amino acids in length and shares 63 and 51% sequence homology with CCR1 and CCR2, respectively (Daugherty et al., Reference Daugherty, Siciliano, DeMartino, Malkowitz, Sirotina and Springer1996). The CCR3 gene codes for four cysteine residues, one in each of the extracellular domains, and a serine/threonine-rich cytoplasmic tail, all of which are highly conserved features of chemokine receptors (Ponath et al., Reference Ponath, Qin, Post, Wang, Wu, Gerard, Newman, Gerard and Mackay1996). Uniquely, CCR3 contains a cluster of negatively charged amino acids distal to the transmembrane helix IV in the second extracellular loop (Daugherty et al., Reference Daugherty, Siciliano, DeMartino, Malkowitz, Sirotina and Springer1996).

Molecular modelling of the Eotaxin receptor, CCR3, indicates that it may be dysfunctional in goats. The sheep and goat CCR3 protein sequences differed by only two amino acids. The two sheep protein sequences were identical except for two substitutions. The two goat sequences were derived from the goat genome sequence and the other from mRNA extracted from the liver of an Osmanabadi goat. The goat genome sequence contained a frameshift deletion on chromosome 22 at 52,155,650, which corresponded to amino acid 330 of the CCR3 protein (Fig. 2). This deletion was not present in the mRNA; therefore, it is possible that this deletion is merely a sequencing artefact. Additionally, both goat sequences contain two amino acid substitutions in relation to the sheep sequences. However, it is unlikely that these substitutions would be the cause of any dysfunction, as the same amino acids are present in the same positions in the human CCR3 protein sequence. In any case, more research is necessary to establish if the potential frameshift mutation is polymorphic in goats and whether or not it may contribute to differences in eosinophil responsiveness between relatively resistant and susceptible goat breeds.

Fig. 2. Eotaxin receptor, CCR3, may be dysfunctional in goats. Homology modelling revealed that the two sheep sequences [Q9N0M0 (B), W5PXW1 (C)] were identical except for two substitutions and all substitutions were at the C-terminal end of the sequence. The two goat sequences [JO419941.2 (A), LOC5316646 (D)] contained two substitutions compared to the sheep sequences. In addition, the goat genome sequence contained a frameshift deletion, corresponding to amino acid 330 (pink). Substitutions are colour coded by the following: aa 354 (blue), aa 356 (red), aa 357 (green) and aa 358 (orange).

MBP-2 is the only MBP molecule present in ruminants

MBP-1 is an abundant granule protein of human eosinophils. Its homologue, MBP-2, is unique to eosinophils (Acharya and Ackerman, Reference Acharya and Ackerman2014). MBP is localized within the crystalline core of the eosinophil and is an important mediator of eosinophil function (Gleich and Adolphson, Reference Gleich, Adolphson and Dixon1986; Kita, Reference Kita2011). MBP is highly basic, which results in the binding of MBP to cell membranes. Cytotoxic mechanisms of MBP involve surface interchange to increase cell membrane permeability and interrupting tissue enzyme activity (Ackerman et al., Reference Ackerman, Gleich, Loegering, Richardson and Butterworth1985; Gleich and Adolphson, Reference Gleich, Adolphson and Dixon1986; Swaminathan et al., Reference Swaminathan, Weaver, Loegering, Checkel, Leonidas, Gleich and Acharya2001). MBP has been demonstrated to be toxic against Schistosoma mansoni by disrupting the cell membrane via the binding of heparin (Butterworth et al., Reference Butterworth, Wassom, Gleich, Loegering and David1979), as well as being important in the control of Litomosoides sigmodontis in mice (Specht et al., Reference Specht, Saeftel, Arndt, Endl, Dubben, Lee, Lee and Hoerauf2006). Structurally, MBP is most like C-type lectins, except that it lacks a calcium binding site, and instead binds selectively to heparin and heparin sulphate, glycosaminoglycans and chondroitin sulphate B (Swaminathan et al., Reference Swaminathan, Myszka, Katsamba, Ohnuki, Gleich and Acharya2005; Wagner et al., Reference Wagner, Ohnuki, Parsawar, Gleich and Nelson2007). Human MBP-1 is 222 amino acids long, consisting of a signalling peptide, pro-peptide and two chains (Swaminathan et al., Reference Swaminathan, Weaver, Loegering, Checkel, Leonidas, Gleich and Acharya2001). It has been suggested that the pro-peptide protects the eosinophil from MBP during transport from the Golgi apparatus to the crystalline core by masking the mature domain, as well as by blocking glycosylated binding sites to inactivate the protein (Swaminathan et al., Reference Swaminathan, Weaver, Loegering, Checkel, Leonidas, Gleich and Acharya2001). The mature domain is highly basic and is the region where carbohydrate recognition occurs. There is a 66% amino acid sequence identity between MBP-1 and MBP-2, with MBP-2 being less basic. MBP-2 contains 10 cysteine residues, eight of which are conserved in MBP-1, including those cysteines that are involved in disulphide bridges. The conservation of these disulphide bridges is consistent with other C-type lectins and is thought to be important for tertiary structure and function (Wagner et al., Reference Wagner, Ohnuki, Parsawar, Gleich and Nelson2007).

Sequence searches of MBP indicate that only MBP-2 is detectable in ruminant genomes; it may function similarly to MBP-1. A total of three sequences for sheep (XM_027979083.1, XM_027979084.1 and XM_02797908.1), two sequences for goats (XM_018058941.1 and XM_018058942) and one sequence for cows (NM_001098471.1) were retrieved. Of the two human MBP sequences, the ruminant sequences were most similar to MBP-2 (55–58% homology). Homology between the sheep sequences was between 72 and 79%, while homology between the goat sequences was 79%. The highest homology was observed between sheep sequence XM_027979084.1 and goat sequence XM_018058941.1, with 95%, which implies a recent divergence and that sheep and goats may have multiple MBP loci. Typical features of C-type lectins and MBP were conserved in the ruminant sequences, including the C-type lectin fold, disulphide-bonded cysteines, and the heparin binding sites. Despite higher homology between human MBP-2 and the ruminant sequences (Fig. 3), theoretical isoelectric point (pI) calculations for the ruminant sequences were most similar to the pI for human MBP-1, with the pI of all ruminant sequences being within 26% of the pI human MBP-1, compared to within 61% of the pI of MBP-2 (Table 1). It is possible that in the absence of MBP-1, ruminant MBP-2 may function as MBP-1, being more cytotoxic and abundant. Functional assays are required to determine if this hypothesis is in fact the case.

Fig. 3. Major basic protein 2 is the only MBP molecule in ruminants. Homology modelling of MBP-1 and MBP-2 C-type lectin fold revealed high structural similarities between human MBP-2 and ruminant MBP-2 molecules, however, theoretical isoelectric point (pI) computation suggests that ruminant MBP-2 molecules may function similarly to human MBP-1. Amino acids used to calculate theoretical pI are as follows: aspartic acid (green), glutamic acid (orange), histidine (yellow), cysteine (red), tyrosine (pink), lysine (aqua) and arginine (blue). Molecules are presented in the order of percentage of sequence identity to human MBP-1, CR450311.1 (A) (highest to lowest): human MBP-2, NM_006093.4 (B); sheep XM_027979083.1 (C); sheep XM_027979084.1 (D); goat XM_018058941.1 (E); cow NM_001098471.1 (F); sheep XM_027979081.1 (G) and goat XM_018058942 (H).

Table 1. Theoretical isoelectric point (pI) of sequences for MBP molecules from humans, cows, sheep and goats

Amino acid sequences were entered into the ExPASy Bioinformatics Resource Portal Compute pI/MW Tool (https://web.expasy.org/compute_pi/) for estimating average theoretical pI based upon the pK values of amino acids described in Bjellqvist et al. (Reference Bjellqvist, Hughes, Pasquali, Paquet, Ravier, Sanchez, Frutiger and Hochstrasser1993) and Bjellqvist et al. (Reference Bjellqvist, Basse, Olsen and Celis1994). The pK values in these studies were defined by examining polypeptide migration between pH 4.5 and 7.3; therefore, predictions for proteins outside of this pH range (cow NM_001098471.1, sheep XM_027979084.1, goat XM_018058941.1) may not be accurate.

Goat EPX lacks a nitrosylated tyrosine

Similar to MBP-2, eosinophil peroxidase (EPX) is unique to eosinophils and is the most abundant cationic protein within the matrix of the specific granule of the eosinophil (Acharya and Ackerman, Reference Acharya and Ackerman2014). Human EPX is 715 AA long, located on chromosome 17. It is structurally similar to myeloperoxidase (MPO), which is present in neutrophil-specific granules (Loughran et al., Reference Loughran, O'Connor, Ó’Fágáin and O'Connell2008). EPX uses hydrogen peroxide to produce toxic reactive oxygen species, such as hypohalous acids, and is capable of killing parasites, including S. mansoni (Auriault et al., Reference Auriault, Capron and Capron1982), Toxoplasma gondii (Locksley et al., Reference Locksley, Wilson and Klebanoff1982) and L. sigmodontis (Specht et al., Reference Specht, Saeftel, Arndt, Endl, Dubben, Lee, Lee and Hoerauf2006). In addition to eosinophils, mast cells also play a role in parasitic infections. High concentrations of EPX have been demonstrated to result in mast cell lysis. Mast cell lysis is followed by the binding of EPX to mast cell granules to form a complex, which results in the retention of secretory activity on mast cells (Henderson et al., Reference Henderson, Chi and Klebanoff1980). In addition, a study by Metzler et al. (Reference Metzler, Fuchs, Nauseef, Reumaux, Roesler, Schulze, Wahn, Papayannopoulos and Zychlinsky2011) demonstrated donors that were deficient in MPO failed to form neutrophil extracellular traps (NET), indicating that MPO is essential for NET formation. Based on this evidence, it is possible that EPX could be involved in eosinophil extracellular trap (EET) formation, which would implicate eosinophils in the direct control of parasitic infections.

EPX is structurally distinct from the other granule proteins, being a two-chain (55-kDa heavy chain and 12.5-kDa light chain) haemoprotein, although EPX is highly cationic, much like MBP-1 and eosinophil cationic protein. A feature of EPX is that it post-translationally modifies itself via the nitrosylation of a specific tyrosine residue (Tyr-488) during synthesis and packaging of the granule proteins into the developing eosinophil (Ulrich et al., Reference Ulrich, Petre, Youhnovski, Prömm, Schirle, Schumm, Pero, Doyle, Checkel, Kita, Thiyagarajan, Acharya, Schmid-Grendelmeier, Simon, Schwarz, Tsutsui, Shimokawa, Bellon, Lee, Przybylski and Döring2008). Tyr-488 has also been shown to be surface exposed, which may assist in the production of reactive oxygen species by EPX. Other granule proteins are also nitrosylated by EPX, but it is unclear whether this is important in protection of the host against helminths.

Molecular modelling of EPX indicates that this protein may be dysfunctional in goats. Based on searches using the human (NM_000502.6) and bovine (XM_024980582.1) EPX sequences, no annotated sequences were retrieved from either the sheep or goat genomes. However, short sequences that matched the reference, but not annotated with an associated gene, were retrieved and re-aligned to the reference. The sheep sequence contained one single-nucleotide polymorphism (SNP), while the goat sequence contained two SNP, one of which was in the same location as in the sheep sequence. The sheep and goat sequences were 90.3% homologous, with homology of sheep and goat to the cow sequence being 84.9 and 82.9%, respectively. The cow, sheep and goat sequences all contained MPO-like protein domains, all of which were conserved except for single-residue substitutions within each domain. In addition to the MPO-like domains, the cow and sheep sequences also contained a tryptic peptide fragment, which was conserved in all ruminant sequences except for a single substitution (alignment to human sequence; Arg⁴⁸³ to His⁴⁸³). Of note, the nitrosylated tyrosine was not conserved in the goat sequence and was substituted for a cysteine (Fig. 4). The nitrosylated tyrosine is important for EPX-mediated activities, including the post-translational nitration of eosinophil secondary granule proteins, which in turn, may influence inflammatory responses. The importance of a nitrosylated tyrosine in GIN infections of ruminants has not yet been established; however, the absence of this residue may be responsible for the relative susceptibility of goats to GIN infection. Functional studies are required to determine whether the SNP identified in the sheep and goat sequences may be sequencing error rather than true SNP, as well as to confirm the importance of the nitrosylated tyrosine in resistance and susceptibility to GIN infection.

Fig. 4. Eosinophil peroxidase may be dysfunctional in goats. Homology modelling EPX heavy chain revealed goat EPX does not contain a tyrosine involved which is involved in post-translational modification of eosinophil granule proteins during eosinophil maturation in human eosinophils. The nitrated tyrosine (green) in the human, NM_000502.6 (A); cow, XM_024980582.1 (B) and sheep, contig. AMGL01017333.1 (C) sequences have been replaced by a cysteine (red) in the goat, contig. LWLT01000022.1 (D) sequence.

Directions for future research

Phenotypic and bioinformatic analyses, in combination, are valuable in assessing differences in immune responses between species in order to better direct the design of functional studies. Some eosinophil proteins may be at least partly responsible for the susceptibility of certain ruminant species to GINs. In particular, sequence variation in the IgA receptor, FcαRI, the Eotaxin receptor, CCR3 and EPX may contribute to the susceptibility of goats to GIN infection. The different conformation of goat FcαRI, as compared to sheep and human, indicates that it is unlikely that it would be fully effective in antigen-dependent cellular cytotoxicity processes, which have been shown to be important for some helminth infections (Huang et al., Reference Huang, Gebreselassie, Gagliardo, Ruyechan, Luber, Lee, Lee and Appleton2015). In addition, goat CCR3 may contain a frameshift mutation, which would in turn affect the role of Eotaxin-1 in eosinophil recruitment and degranulation. Goat EPX lacks a nitrosylated tyrosine. In humans, the nitrosylated tyrosine in EPX is important for the post-translational modification of EDGPs during eosinophil maturation. Goat EPX may not be involved in this process. Finally, although MBP-1 is the most abundant and important cationic protein in human eosinophils, it has no orthologue in ruminant genomes. MBP-2 appears to be the only MBP molecule present in ruminants and it may function similarly to human MBP-1. Future research should focus on functional studies to confirm these findings and should involve the extraction of these proteins from the host of interest, followed by sequencing, crystallography and in vitro assays to assess the overall importance of these proteins to the susceptibility of ruminants to GIN.

Perhaps the clearest finding of this review is that bioinformatic analyses indicate that the function of specific eosinophil proteins may vary among host species. Therefore, functional studies of eosinophil activity should be performed in the target species. Extrapolations from one species to another need to be interpreted with great care.