INNATE IMMUNITY
2.1 Natural killer cells and receptors
Natural killer (NK) cells are involved in anticancer and antiviral immune defence, as they bind ligands presented on MHC I and II. NK cells are activated through cell surface receptors, which may be grouped into two superfamilies: C-type lectin superfamily (CLSF), which are encoded by the natural killer complex (NKC), and the immunoglobulin superfamily (IgSF), which are encoded by the leucocyte receptor complex (LRC) (Kelley et al.
2005).NKC gene structure and organisation is conserved among mammals (Kelley et al. 2005); however, gene number varies significantly between eutherians, mono- tremes and marsupials (Belov et al. 2007; Wong et al. 2009b; van der Kraan et al. 2013). All marsupials studied have a small NKC, with only two receptor orthologs characterised in the tammar wallaby, four in the brown antechinus (Antechinus stuartii) and five in the brush-tailed bettong (woylie) (Bettongia penicillata) and bare-nosed wombat (Vombatus ursinus) (Peel et al. 2022). Six receptor orthologs have been identified in the Tasmanian devil (Sarcophilus harrisii), grey short-tailed opossum (Mono- delphis domestica) and the koala (Phascolarctos cinereus) (Belov et al. 2007; van der Kraan et al. 2013; Morris et al. 2015; Johnson et al. 2018), which is fewer than in eutherians or the platypus (Kelley et al. 2005; Wong et al. 2009b). The platypus has the largest lineage-specific expansion of NKC genes in all mammals studied to date, with 213 receptors identified; however, only five show orthology to other mammalian NKC genes (Wong et al. 2009b).
The LRC has greatly expanded in marsupials since their evolutionary divergence from eutherian mammals and may have been driven by pathogen pressures to protect immunologically naive young (Belov et al. 2007). The opossum has a large lineage-specific expansion of IgSF genes, termed marsupial immunoglobulin-like receptors (MAIRs), which show a low level of orthology to eutherian LRC genes (Belov et al.
2007). Several MAIRs are conserved across all marsupial genomes studied to date (Peel et al. 2022). However, species-specific expansions of IgSF genes have also been identified in the koala (Morris et al. 2015), Tasmanian devil (van der Kraan et al. 2013), antechinus, numbat (Myrmecobius fasciatus), woylie and wombat (Peel et al. 2022). LRC genes could not be identified in the platypus and echidna genomes (Zhou et al. 2021), although genes within the extended LRC have been characterised (Wong et al. 2009b).NK cell receptors have only been investigated in one Australian bat species, the black flying-fox (Pteropus alecto) (Papenfuss et al. 2012; Zhang et al. 2013). Receptors belonging to the CLSF, IgSF and additional families such as the killer immunoglobulin-like receptors (KIRs) could not be identified in the genome or in various immune tissue transcriptomes. This may suggest that bat NK cells use a novel class of receptors for interaction with MHC-I molecules (Zhang et al. 2013). The only NK receptors characterised were the lectin-like CD94/ NKG2A inhibitory receptor, CD244 and co-receptors such as CD16 and CD56 (Papenfuss et al. 2012).
2.2 Toll-like receptors
The TLR are primarily found on the surface of antigenpresenting cells such as macrophages, where they recognise and bind pathogen-associated molecular patterns, leading to activation of the innate and adaptive immune responses. The full set of black flying-fox TLRs (TLR1-10 and 13) have been described, with TLR13 identified as a spliced and polyadenylated pseudogene (Cowled et al. 2011). In the Tasmanian devil (Cui et al. 2015a), koala (Cui et al. 2015b), antechinus, numbat, woylie, wombat (Peel et al. 2022) and opossum (Roach et al. 2005), clear eutherian orthologs of TLR2-5, TLR7-10 and TLR13 have been characterised. A marsupial-specific TLR, TLR1/6-like, has been identified in all marsupial species studied to date, but is absent in eutherian mammals. Phylogenetic analysis revealed that TLR1∕6-like is the ancestral gene, which duplicated to give rise to the TLR1 and TLR6 families present in eutherian mammals (Cui et al.
2015a; Cui et al. 2015b; Peel et al. 2022).Given their role in pathogen detection, the diversity of the TLR has been linked to the ability of individuals and populations to respond to new disease threats. The Tasmanian devil has critically low TLR diversity with 7 out of 10 TLR genes being monomorphic, which may contribute to disease vulnerability in this species (Cui et al. 2015a). This is not the case in the koala, as TLR diversity is at a similar level to other mammals (Cui et al. 2015b).
2.3 Cytokines
Marsupials and monotremes have a diverse repertoire of immune tissues and cells; however, the lack of in vivo research has led to questions regarding the functionality of their immune systems. Inconsistent reports describe delayed and/or dampened T and B lymphocyte responses and antibody production in these two mammalian lineages (Wilkinson et al. 1992; Wronski et al. 2003). The functionality of the bat immune system is also largely unknown and given their role as reservoirs for numerous pathogenic viruses (Wong et al. 2007), their immune system may possess unique characteristics and functions. Investigations into immune cell function in monotremes, marsupials and bats are hindered by the lack of speciesspecific immunological reagents. A small number of monotreme, marsupial and bat antibodies targeting key immune molecules such as immunoglobulins and CD8 are available and cross-reactivity among marsupial species is observed (Duncan et al. 2012). Cross-reactive antibodies raised against evolutionarily conserved components of human and mouse CD3, CD5, CD79a and CD79b successfully stained T and B lymphocytes in several marsupial species (Hemsley et al. 1995; Old and Deane 2002) as well as bats (Martinez Gomez et al. 2016). However, numerous immune cell populations are not captured by currently available antibodies. Table 7.1 outlines antibodies successfully used to stain monotreme, marsupial and bat immune cells.
Cytokines are small intracellular mediators that direct the immune response.
Understanding the cytokine network in monotremes and marsupials may reveal the complexity and functionality of their immune systems. Detection of proteins in marsupial immune cell cultures, which resemble eutherian cytokines such as interleukins (IL) and tumour necrosis factor (TNF), provides the first direct evidence of marsupial T lymphocyte functionality (Wilkinson et al. 1992). The emergence of PCR-based methods and the recent release of marsupial and monotreme genomes and transcriptomes have expanded our knowledge of cytokine families such as interleukins and interferons (IFN-α, -β and -γ), enabling comprehensive characterisation of cytokines and comparative analysis across all three mammalian lineages. Monotreme and marsupial cytokines are structurally similar to those of eutherians. All five major cytokine families have been identified, as well as cytokines associated with each of the four major T lymphocyte lineages (Th1, Th2, T17 and Treg) (Table 7.2). Given this, the complexity and functional capacity of the marsupial and monotreme immune system are similar to eutherian mammals.Of the major cytokine families, interferons (IFN) have mainly been characterised in bats because of their involvement in antiviral immunity. Bats have a contracted IFN genomic region, containing only 10 IFN genes, including three functional IFN-α loci. IFNα is constitutively and ubiquitously expressed across all tissues, thus providing a highly effective system for controlling viral replication (Zhou et al. 2016). Type III IFN (IFN-λ) was found to play an important role in the ability of bats to coexist with viruses through simultaneous suppression of type I IFN and induction of type III IFN after viral replication (Zhou et al. 2011).
2.4 Antimicrobial peptides
Antimicrobial peptides (AMPs) play an important role in rapid defence against invading pathogens (Kosciuczuk et al. 2012). There are two major families of AMPs in mammals: cathelicidins and defensins (Kosciuczuk et al.
2012). Both families have been identified in species from six bat families (Castellanos et al. 2023; Papenfuss et al. 2012). The function of bat AMPs has not been investigated but they may have novel functions given the coexistence of bats with pathogenic viruses and antiviral activity of other mammalian AMPs.The need for immunological protection during early pouch life has encouraged the expansion of cathelicidins in marsupials and monotremes, resulting in numerous diverse peptides. Although humans have only one cathelicidin gene (Kosciuczuk et al. 2012), opossums have 19 (Belov et al. 2007; Cho et al. 2020), Tasmanian devils have six (Peel et al. 2016), koalas have 10 (Peel et al. 2021) and the tammar wallaby has eight (Daly et al. 2008). Ten cathelicidin genes have been identified in the platypus genome (Warren et al. 2008; Zhou et al. 2021) and six have been identified in the echidna genome (Zhou et al. 2021). These small, cationic AMPs specifically target and kill a range of bacteria, fungi and enveloped viruses. Tasmanian devil and koala cathelicidins have broad-spectrum antibacterial activity and kill drug-resistant bacteria such as methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococcus (VRE) (Peel et al. 2016; Peel et al. 2021). Cathelicidins are expressed in the pouch and secreted into the milk of the tammar wallaby, as well as by the skin of the young from day 1 postpartum (Daly et al. 2008). Their presence within the pouch immediately after birth and their antimicrobial activity supports their functional role in protection of immunologically naive young.
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