Identification of monoclonal antibodies cross-reactive with bottlenose dolphin orthologues of the major histocompatibility complex and leukocyte differentiation molecules

Highlights

• Cross-reactive monoclonal antibodies that recognize bottlenose dolphin major histocompatibility class I and II, CD11a, CD18, CD14, CD16, CD163 and CD172a were identified.

• Bottlenose dolphin monocytes co-express CD14, CD16 and CD163.

• Major histocompatibility class II is expressed on bottlenose dolphin resting and activated lymphocytes in addition to monocytes.

Abstract

The slow progress in understanding immunotoxic effects of environmental contaminants and their influence on disease susceptibility in whales is largely due to the limited information available on the immune systems and immune function of species included in the Cetancodontamorpha clade. Studies in species in the other major clades included in the Artiodactylamorpha, Ruminantiamorpha, Suinamorpha, and Camelidamorpha have revealed the immune systems are similar, but not identical. The present study was undertaken to expand the available monoclonal antibody reagents needed to gain insight into the composition, function, and evolution of the immune system in Cetancodontamorpha, using the dolphin (Tursiops truncatus) as a model cetacean species. Screening of a set of mAbs that recognize highly conserved epitopes expressed on the major histocompatibility complex (MHC) and leukocyte differentiation molecules (LDMs) in cattle by flow cytometry revealed some of the mAbs recognize epitopes conserved on dolphin orthologues of MHC class I, MHC class II, CD11a, CD14, CD16, CD18, CD163 and CD172a. Comparison of the amino acid sequences of dolphin and bovine orthologues revealed limited changes in sequence have occurred during speciation, suggesting an approach for developing cross-reactive mAbs for use in cetacean research.

Introduction

Interest has continued to grow over the past few years in determining the effect of environmental contaminants on immune system function in marine mammals. This includes xenobiotics, pathogens unique to the marine environment, and pathogens of land vertebrates that find their way into the marine environment [reviewed in (Beineke et al., 2010, Venn-Watson et al., 2008, Waltzek et al., 2012)]. Interest has also increased in metabolic conditions that occur in aging bottlenose dolphins that parallel those in humans. Bottlenose dolphins can develop a subclinical metabolic syndrome, with elevated insulin, triglycerides, glucose, and ferritin, accompanied by fatty liver disease (Venn-Watson et al., 2012, Venn-Watson et al., 2011, Venn-Watson et al., 2013). Further, current models, based on data derived from studies in other species, suggest that metabolic perturbations arise from disrupted immune homeostasis in sites such as adipose tissue and liver, with activation status of leukocytes in these sites contributing to the disease pathology (Olefsky and Glass, 2010, Osborn and Olefsky, 2012). Thus, studies of metabolic perturbations in dolphins would also greatly benefit from having reagents available to define the contribution of specific leukocyte subsets to these pathogeneses. The reagents would also facilitate the use of dolphins as a model to study similar metabolic disorders in humans.

At this juncture, however, there has been limited progress in using dolphins as sentinels to detect changes harmful to inhabitants of the marine ecosystem or as an animal model for diseases that occur in dolphins and humans. This is in part because of a limited understanding of how the immune systems have evolved in marine mammals and, in part, the availability of reagents needed to characterize the respective immune systems for functional studies (Beineke et al., 2010, De Guise et al., 1997, De Guise et al., 1998, De Guise et al., 2004, De Guise et al., 2002, Jaber et al., 2003, Jaber et al., 2010).

The rationale for undertaking the present study was twofold. The first was to expand our ability to study host-pathogen interactions in the dolphin that might be influenced by contaminants in the environment. The second was a broader interest in understanding how immune systems have evolved in different mammalian species, especially in the evolution of immune systems in members of the Artiodactylamorpha which includes the clades Cetaceamorpha, Ruminantiamorpha, Suinamorpha, and Camelidamorpha (Spaulding et al., 2009). Studies of species in these clades have shown the immune systems have evolved from common ancestral genes, some of which have undergone changes affecting the function of the immune systems not seen in other clades of mammals. One unique feature of the evolution of Ruminantiamorpha, Suinamorpha, and Camelidamorpha not seen in other clades of mammals is the evolution of a protein containing the scavenger receptor cysteine rich (SRCR) domain, workshop cluster one (WC1), which was first described in cattle (Morrison and Davis, 1991). WC1 orthologues have been identified in Ruminantiamorpha (ruminants), Suinamorpha (pigs), and Camelidamorpha (camels, llamas, alpacas) (Davis et al., 2000, Mosaad et al., 2006). The WC1 proteins are encoded by a common ancestral gene that has undergone different evolutionary histories in the different clades. The WC1 proteins are only expressed on a subset of γδ T cells, and appear to play a unique role in the function of this subset of γδ T cells (Telfer and Baldwin, 2015). The WC1 SRCR containing genes are included in a larger group of genes encoding paralogues containing the SRCR domain, the CD163 family of proteins, Spα, CD5 and CD6 (Herzig et al., 2010). The CD163 gene family is closely related to the WC1 gene family suggesting a common phylogenetic origin. In contrast to WC1, the CD163 gene family is present in Artiodactylamorpha and other distantly related clades of mammalian species (Herzig et al., 2010). No species in Cetaceamorpha have been examined for presence of the orthologues of WC1.

Efforts to screen mAbs developed against leukocyte differentiation molecules (LDMs), made in different species of land mammals, have shown some mAbs recognize highly conserved epitopes expressed on orthologous molecules, providing a start for assembling sets of reagents for immunological investigations (Davis et al., 1995, Davis et al., 2007, Davis and Hamilton, 1998, Davis et al., 2000, Davis et al., 2001, Saalmuller et al., 2005). These studies have shown the more closely related the species are, the greater the probability that a mAb, developed in any of the respective species, will recognize an epitope conserved on orthologues in the other species e.g. goats, sheep, water buffalo, llama/alpaca, rabbits, and hamster (Davis et al., 2007, Davis and Hamilton, 2008, Davis et al., 2001, Davis et al., 1987, Grandoni et al., 2017, Rees et al., 2017, Saalmuller et al., 2005). These studies also showed that a more direct approach is needed for distantly related species for the development of mAbs needed for the species of interest (Davis and Hamilton, 2008, Davis et al., 2000). Both approaches have been used to identify sets of mAbs for use in the study of cetaceans (De Guise et al., 1997, De Guise et al., 1998, De Guise et al., 2002, Jaber et al., 2003, Jaber et al., 2010, Romano et al., 1992). Thus far, mAbs specific for MHC class I and II molecules, CD2, CD3 and CD4, developed against bovine and human MHC and LDMs, were shown to recognize epitopes conserved on orthologues in some cetacean species (De Guise et al., 1997, Jaber et al., 2003, Romano et al., 1992). In addition, mAbs developed against CD2, CD19, CD21 and CD45R in bottlenose dolphin (Tursiops truncatus) were shown to identify orthologues in other species of cetaceans (wild common dolphin, striped dolphin, killer whale and beluga) (De Guise et al., 1998, De Guise et al., 2002). Most recently, an extensive set of mAbs developed against human, rat and mouse LDM were screened for potential cross-reactivity with dolphin LDM by Nouri-Shirazi et al. (Nouri-Shirazi et al., 2017). They identified 11 of 56 commercially available mAbs with potential cross-reactivity with dolphin LDM, two of which yielded patterns of labeling consistent with MHC II and CD11b (Nouri-Shirazi et al., 2017). Although this is important progress, considerably more reagents are needed to fully characterize the immune system in the Cetaceamorpha clade and meet the challenges of studying the impact of environmental contaminants and pathogens present in the marine ecosystem [reviewed in (Beineke et al., 2010, Venn-Watson et al., 2008, Waltzek et al., 2012)]. The present study was conducted as part of an ongoing effort to expand reagents available for studying the evolution and function of the cetacean immune system and factors affecting the immune response to pathogens and parasites, using the dolphin as the model cetacean. Flow cytometry (FC) and mAbs known to recognize conserved epitopes expressed on MHC and LDMs in cattle were used to initiate the studies.