Bovine CCL28 Mediates Chemotaxis via CCR10 and Demonstrates Direct Antimicrobial Activity against Mastitis Causing Bacteria

<div><p>In addition to the well characterized function of chemokines in mediating the homing and accumulation of leukocytes to tissues, some chemokines also exhibit potent antimicrobial activity. Little is known of the potential role of chemokines in bovine mammary gland health and disease. The chemokine CCL28 has previously been shown to play a key role in the homing and accumulation of IgA antibody secreting cells to the lactating murine mammary gland. CCL28 has also been shown to act as an antimicrobial peptide with activity demonstrated against a wide range of pathogens including bacteria, fungi and protozoans. Here we describe the cloning and function of bovine CCL28 and document the concentration of this chemokine in bovine milk. Bovine CCL28 was shown to mediate cellular chemotaxis via the CCR10 chemokine receptor and exhibited antimicrobial activity against a variety of bovine mastitis causing organisms. The concentration of bovine CCL28 in milk was found to be highly correlated with the lactation cycle. Highest concentrations of CCL28 were observed soon after parturition, with levels decreasing over time. These results suggest a potential role for CCL28 in the prevention/resolution of bovine mastitis.</p></div

Primer names and sequences.

<p>Primer names and sequences.</p

Bovine CCL28 mediates migration of CCR10 transfected cells Supernatant fluids from Cos-7 cells transfected with mCCL28, bCCL28, or empty vector controls were placed in the bottom well of a Transwell migration chamber.

<p>Cells expressing mCCR10 were placed in the upper chamber and allowed to migrate for 1.5hrs. Both murine and bovine CCL28 mediated migration of CCR10 transfectants significantly better than empty vector controls *p<0.05, as determined by Mann Whitney <i>U</i> test. Results are from four separate experiments. Average migration of cells migrating to mCCL28 was 18.4% (SE 3.71), migration to bCCL28 9.4% (SE 2.12), and migration to empty vector 0.1% (SE 0.01). Error bars represent standard error of the mean. NS = Not Significant.</p

Recombinant bCCL28 has potent broad spectrum antimicrobial activity against mastitis causing bacteria.

<p>Bovine CCL28 demonstrates dose dependent antimicrobial activity against <i>Pseudomonas aeruginosa</i> (<i>A)</i>, methicillin-resistant <i>Staphylococcus aureus</i> (MRSA) <i>(B)</i>, <i>Streptococcus uberis (C)</i>, and <i>Streptococcus agalactiae (D)</i>. Bacteria were incubated with varied doses of bCCL28 or bCCL27 for 1 hour. Percent survival was calculated using the following equation: (CFU_{pathogen + chemokine}/CFU_{pathogen only}) x 100. *p<0.05, **p<0.01 as determined by Mann Whitney <i>U</i> test. Results are from four or more separate experiments. Error bars represent standard error of the mean. Details on percent survival, standard error, and the number of times each experiment was performed are available in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0138084#pone.0138084.s001" target="_blank">S1 Fig</a>.</p

Recombinant bCCL28 exhibits antimicrobial activity <i>in vitro</i>.

<p><i>E</i>. <i>coli</i> was incubated with 0.25 μM of mouse or bovine CCL28 or CCL27 for 2 hours. Percent survival was calculated using the following equation: (CFU_{pathogen + chemokine}/CFU_{pathogen only}) x 100. **p<0.01 as determined by Mann Whitney <i>U</i> test. Results are from three separate experiments, each performed in duplicate. Survival of bacteria in the CCL27 treated groups was scaled to 100% and the amount of killing in the CCL28 treated groups is shown as percent survival compared to the CCL27 treated group. Average bacterial survival seen in mCCL28 treated bacteria 28.3% (SE 1.35), survival observed in bCCL28 treated bacteria 30.3% (SE 1.29). Error bars represent standard error of the mean.</p

CCL28 levels in bovine milk are highest soon after parturition and are not correlated with somatic cell count.

<p>Milk CCL28 levels were determined by ELISA using antibodies generated against human CCL28. No correlation was seen between somatic cell counts and CCL28 protein levels in bovine milk (A). A strong correlation was seen (R² = 0.35) between higher levels of CCL28 at the beginning of lactation (B). Diamonds represent individual milk samples. X’s represent the average CCL28 level for each time point indicated in the x-axis.</p

Alignment of bovine and murine CCL28.

<p>Alignment of bovine, pig, murine and human CCL28 sequences demonstrates high amino acid homology at the N-terminus of the protein and lower homology at the C-terminus. Asterisks (*) denote amino acid changes between murine, pig, human and bovine CCL28 sequences. The RKDRK sequence, which has previously been shown to be essential for optimal antimicrobial function of mCCL28 and corresponding bovine sequence, is boxed.</p