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

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

Hla induces programmed cell death in human monocytes, T cells and B cells during USA300 infection.

<p>Compiled flow cytometry analysis of human PMNs or PBMCs infected at a MOI of 10 with USA300, USA300Δ<i>hla</i>, USA300Δ<i>hla</i> Comp, USA300Δ<i>saeR/S</i>, or a DPBS control then examined using an Annexin V binding assay at 3 hours (A, C, E, and G) or ApoBrdU Tunel assay at 6 hours (B, D, F, and H). Figures represent 4 separate experiments using different blood donors with *P<0.05, **P<0.01, and ***P<0.001 relative to USA300 as determined by paired one-tailed t-test for A, C, E and G or one-way repeated measures ANOVA with Tukey’s post-test for B, D, F, and H.</p

Primer names and sequences.

<p>Primer names and sequences.</p

Hla-dependent human T cell and B cell plasma membrane permeablity occurs following 60 minutes exposure to USA300 supernatant.

<p>A) Representative flow cytometry dot plots of human PBMCs stained with propidium iodide following intoxication with filtered 5 hour supernatants from USA300 or USA300Δ<i>hla</i> diluted to a final concentration of 1∶6 for 30 minutes for CD14⁺ PBMCs or 180 minutes for CD3⁺ and CD19⁺ PBMCs. B) Representative histograms of data in part A with CD3⁺, CD14⁺, and CD19⁺ PBMCs exposed to supernatant from USA300 indicated by a dashed line, supernatant from USA300Δ<i>hla</i> indicated with grey shading, and TSB media alone indicated with black shading. Compiled results from at least 4 separate experiments shown in part A using different blood donors for C) CD3⁺, D) CD14⁺ and E) CD19⁺ PBMCs at indicated times following intoxication with *P<0.05, **P<0.01, and ***P<0.001 relative to USA300 as determined by paired one-tailed t-test.</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

Recombinant Hla alone induces programmed cell death in monocytes, T cells and B cells.

<p>Compiled flow cytometry analysis of human PMNs and CD3⁺, CD14⁺, or CD19⁺ PBMCs intoxicated for 60 minutes with increasing concentrations of recombinant Hla then examined using A) Annexin V binding or B) ApoBrdU Tunel assays. Figures represent 3 separate experiments using different blood donors with *P<0.05, **P<0.01, and ***P<0.001 relative to USA300 as determined by one-tailed t-test.</p