Tammar wallaby mammary cathelicidins are differentially expressed during lactation and exhibit antimicrobial and cell proliferative activity

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Abstract

Cathelicidins secreted in milk may be central to autocrine feedback in the mammary gland for optimal development in addition to conferring innate immunity to both the mammary gland and the neonate. This study exploits the unique reproductive strategy of the tammar wallaby (Macropus eugenii) model to analyse differential splicing of cathelicidin genes and to evaluate the bactericidal activity and effect of the protein on mammary epithelial cell proliferation. Two linear peptides, Con73 and Con218, derived from the heterogeneous carboxyl end of cathelicidin transcripts, MaeuCath1 and MaeuCath7 respectively, were evaluated for antimicrobial activity. Both Con73 and Con218 significantly inhibited the growth of Staphylococcus aureus, Pseudomonas aureginosa, Enterococcus faecalis and Salmonella enterica. In addition both MaeuCath1 and MaeuCath7 stimulated proliferation of primary tammar wallaby mammary epithelial cells (WallMEC). Lactation-phase specific alternate spliced transcripts were determined for MaeuCath1 showing utilisation of both antimicrobial and proliferative functions are required by the mammary gland and the suckled young. The study has shown for the first time that temporal regulation of milk cathelicidins may be crucial in antimicrobial protection of the mammary gland and suckled young and mammary cell proliferation.

Introduction

Evidence is now emerging to show that milk not only provides appropriate nutrition for the young but also plays a significant role in the development of the mammary gland (Nicholas et al., 1997). Major milk proteins, whey acidic protein (WAP) and alpha-lactalbumin for instance have been shown to enhance mammary epithelial cell proliferation (Topcic et al., 2009) and initiate apoptosis in mammary cells (Sharp et al., 2008) respectively. In this study we use the unique reproductive strategy of the marsupial, tammar wallaby (Macropus eugenii) to investigate the role of mammary-derived cathelicidins MaeuCath1 and MaeuCath7 and their derivative peptides Con73 and Con218, respectively in lactation.

Relative to eutherians, marsupials including wallabies have a short gestation period, leading to the birth of an altricial neonate. The tammar lactation cycle consists of one pre-parturition phase (phase 1) and three post-parturition phases (2A, 2B and 3) during which there is significant remodelling of the mother's mammary gland, changes in milk composition, and development of the neonate from a foetus-like form to adult-like anatomy (Nicholas, 1988, Bird et al., 1994, Nicholas et al., 1997, Brennan et al., 2007). During phase 2A which lasts up to 110 days post-partum, the neonate is permanently attached to the teat and feeds on milk which is low in protein and fat but high in carbohydrates (Nicholas et al., 1997). Phase 2B consists of days 100–200 post-partum when the pouch young detaches from the teat and only reattaches to feed. Phase 3 commences approximately 200 days post-partum and is characterised by secretion of high volumes of milk that is rich in protein and fat but low in carbohydrates (Nicholas, 1988, Nicholas et al., 1997). Development of the respiratory, cardio-vascular and adaptive immune systems in the tammar neonate occurs during lactation and therefore the milk must provide bioactives for immediate nutritional and immunological demands and to act as signals for development (Basden et al., 1997).

A number of genes, including cathelicidins, encode antimicrobial peptides that constitute a significant part of innate immunity in vertebrates (Yang et al., 2004). Cathelicidins belong to a family of proteins generally characterised by an N-terminal putative signal peptide, a conserved cathelin-like domain and a C-terminal heterogeneous antimicrobial domain (Zanetti et al., 1995). Their gene structure characteristically consists of four exons (Gudmundsson et al., 1995), the first three of which encode the pre-proregion (the putative signal peptide and Cathelin-like domain) and the last exon encodes the appropriate protease cleavage site and the C-terminal microbicidal domain. Cathelicidins usually exist in their inactive proform state stored in neutrophil granules. The mature peptide is formed by post-translational cleavage of the proform by different proteases depending on the species. Extrinsic factors, including LPS and LTA have been shown to stimulate mammary epithelial cells (Daly et al., 2009) and keratinocytes (Frohm et al., 1997) to express cathelicidin genes.

Numerous functions have been attributed to the two domains of the proform cathelicidin (Yang et al., 2004). The cathelin-like domain is thought to act as a buffer against possible intracellular cytotoxicity of the cationic C-terminal domain (Zanetti et al., 1995) in addition to antimicrobial activity (Li et al., 2000). The human cathelicidin peptide LL-37 has been shown to stimulate colonic mucus synthesis, induce mast cell chemotaxis , suppress neutrophil apoptosis, act as chemoattractant to neutrophils and CD4 T-lymphocytes and neutralise LPS (Yang et al., 2004). Several studies involving LL-37 in various human tissues have demonstrated that cathelicidins play a significant role in wound healing and tissue remodelling. LL-37 enhances re-epithelization of wounds in oral, intestinal, lung, and corneal epithelia implying a possible role in epithelial cell proliferation (Yang et al., 2004). Cathelicidins expressed in the mammary gland during lactation may therefore play a role in mammary tissue remodelling and epithelial cell proliferation.

The structure of a number of tammar mammary-derived cathelicidins has been elucidated and found to consist of typical cathelicidin features (Daly et al., 2008) but their function in lactation is not understood. MaeuCath1 and MaeuCath7 and their C-terminal peptides Con73 and Con218, respectively are for the first time characterised for bactericidal activity. The effect on the proliferation of tammar mammary epithelial cells (WallMEC) is examined and lactation-phase specific differential splicing investigated.

Section snippets

Experimentation with animals

Wallabies were kept in an open yard with adequate vegetation and water. Tissues were obtained following animal ethics approval by the University Animal Welfare Committee (AWC)

RNA purification and RT-PCR

Twenty-six mammary glands were excised for RNA isolation, including two each from non-pregnant (NP) and Phase 1, four from Phase 2A and six each from phases 2B, 3 and weaned (INV) tammar wallabies (Macropus eugenii). RNA was isolated in two lots of 13 using Tripure Isolation reagent (Roche Diagnostics, Castle Hill, NSW,

Alternate splicing of MaeuCath1

Differential splicing of the MaeuCath1 gene was observed during specific phases of the lactation cycle. Two different splice forms, MaeuCath1a and MaeuCath1b were confirmed by sequence analysis and their structure determined by comparison to the genomic MaeuCath1 gene sequence (Figs. 1A and 2). MaeuCath1a is 489 bp and comprises all four exons with no intron. MaeuCathlb is 697 bp and comprises exons 1 and 2, 256 bp of retained intron sequence and the 3′untranslated region. The translation stop

Discussion

Lactation in the tammar starts with birth of an altricial young and the immune-compromised neonate relies on antibodies and other immune factors secreted in the mother's milk for defence against microbes (Daly et al., 2007). The pouch of the tammar wallaby and other marsupials is rich in microflora, including potentially pathogenic bacteria (Yadav et al., 1972, Old and Deane, 1998). Thirty species of bacteria, including Staphylococcus aureus, Klebsiella pneumoniae, Pseudomonas aeruginosa and

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