Muramyl dipeptide synergizes with Staphylococcus aureus lipoteichoic acid to recruit neutrophils in the mammary gland and to stimulate mammary epithelial cells

Abstract

Staphylococcus aureus, a major pathogen for the mammary gland of dairy ruminants, elicits the recruitment of neutrophils into milk during mastitis, but the mechanisms are incompletely understood. We investigated the response of the bovine mammary gland to muramyl dipeptide (MDP), an elementary constituent of the bacterial peptidoglycan, alone or in combination with lipoteichoic acid (LTA), another staphylococcal microbial-associated molecular pattern (MAMP). MDP induced a prompt and marked influx of neutrophils in milk, and its combination with LTA elicited a more intense and prolonged influx than the responses to either stimulus alone. The concentrations of several chemoattractants for neutrophils (CXCL1, CXCL2, CXCL3, CXCL8, and C5a) increased in milk after challenge, and the highest increases followed challenge with the combination of MDP and LTA. MDP and LTA were also synergistic in inducing in vitro chemokine production by bovine mammary epithelial cells (bMEpC). Nucleotide-binding oligomerization domain 2 (NOD2), a major sensor of MDP, was expressed (mRNA) in bovine mammary tissue and by bMEpC in culture. The production of interleukin-8 (IL-8) following the stimulation of bMEpC by LTA and MDP was dependent on the activation of NF-κB. LTA-induced IL-8 production did not depend on platelet-activating factor receptor (PAFR), as the PAFR antagonist WEB2086 was without effect. In contrast, bMEpC and mammary tissue are known to express Toll-like receptor 2 (TLR2) and to respond to TLR2 agonists. Although the levels of expression of the inflammatory cytokines tumor necrosis factor alpha (TNF-α) and IL-1β were increased by LTA and MDP at the mRNA level, no protein could be detected in the bMEpC culture supernatant. The level of induction of IL-6 was low at both the mRNA and protein levels. These results indicate that MDP and LTA exert synergistic effects to induce neutrophilic inflammation in the mammary gland. These results also show that bMEpC could contribute to the inflammatory response by recognizing LTA and MDP and secreting chemokines but not proinflammatory cytokines. Overall, this study indicates that the TLR2 and NOD2 pathways could cooperate to trigger an innate immune response to S. aureus mastitis.

FIG. 1.

Cellular response induced by LTA and MDP in the mammary gland. Concentrations of cells in milk of mammary glands (quarters) infused at 0 h with different amounts of MDP or S. aureus LTA, alone or in combination, were monitored at 4, 8, 12, 24, 32, 48, 72, and 96 hpi. Data are median values and interquartile ranges. (A) Pilot experiment with three cows which received 1, 10, or 100 μg MDP in three different quarters. The fourth quarter served as a control. (B) Eleven cows received 20 μg MDP, 5 μg LTA, or the combination of 20 μg MDP and 5 μg LTA in three different quarters. *, statistical significance (P < 0.05, multiple comparisons) of values from challenged versus control quarters; $, significance (P < 0.05, multiple comparisons) of values from quarters challenged with MDP plus LTA versus either LTA or MDP. SCC, somatic cell count.

FIG. 2.

Concentrations of ELR+CXC neutrophil-oriented chemokines CXCL1 (A), CXCL2 (B), CXCL3 (C), and CXCL8 (D) in milk of quarters infused with either MDP, S. aureus LTA, or LTA plus MDP from the time of infusion (time zero) to 96 hpi. Data are from six cows (median values and interquartile ranges). *, significantly increased or decreased (CXCL3) concentrations relative to that at time zero (P < 0.05); $, significant difference between LTA plus MDP and LTA or MDP alone (P < 0.05).

FIG. 3.

Concentrations of the complement-derived chemoattractant C5a in milk of quarters infused with either MDP, S. aureus LTA, or LTA plus MDP from the time of infusion (time zero) to 96 hpi. Data are from six cows (median values and interquartile ranges). *, significantly increased concentration relative to that at time zero (P < 0.05); $, significant difference between LTA plus MDP and LTA or MDP alone (P < 0.05).

FIG. 4.

Concentrations of the proinflammatory cytokines IL-1β, IL-6, and TNF-α in milk of quarters infused with either MDP, S. aureus LTA, or LTA plus MDP from the time of infusion (time zero) to 96 hpi. Data are from 11 cows (median values and interquartile ranges). *, significantly increased concentration relative to that at time zero (P < 0.05); $, significant difference between LTA plus MDP and LTA or MDP alone (P < 0.05).

FIG. 5.

Secretion of the chemokines CXCL8 and CXCL3 by bMEpC stimulated with LTA and MDP alone or in combination. (a) Dose-response of bMEpC from one cow to LTA and MDP. (b) Susceptibility of LTA to PAF-AH or proteinase K. Purified LTA (200 ng/ml) was used to stimulate bMEpC; alternatively, LTA (200 ng/ml) was pretreated with the LTA-inactivating enzyme PAF-acetyl-hydrolase (LTA-PAF-AH) or with lipoproteins inactivating proteinase K (LTA-Prot K). LTA (200 ng/ml) was added to the treated LTA to check whether PAF-AH treatment had generated inhibitors of CXCL8 secretion (LTA-PAF-AH or LTA). (c) Secretion of CXCL8 by MEpC (median values from cells of five cows, first quartile [Q1] and Q3) in response to LTA and MDP at 8 h and 24 h poststimulation. Shown is the secretion of CXCL3 in response to LTA and MDP at 1 μg/ml. (d) Secretion of CXCL3 in response to LTA and MDP at 0.1 μg/ml. MDP-DD, inactive isoform of muramyl-dipeptide (MurNAc-d-Ala-d-iso-Gln); MDP, active isoform (MurNAc-l-Ala-d-iso-Gln).

FIG. 6.

Production of IL-6 by bMEpC stimulated with LTA or MDP at 1 μg/ml. (a) Production at the mRNA level at 2 h or 8 h poststimulation. (b) Production at the protein level at 24 h poststimulation. Shown are median values (Q1 and Q3) of bMEpC from five cows. There was a significant difference (P < 0.05 by Friedman test) only between medium only and LTA plus MDP at 8 h poststimulation for mRNA and at 24 h for protein secretion. RE/18S, expression relative to 18S rRNA.

FIG. 7.

Production of TNF-α and IL-1β by cells stimulated with LTA or MDP. (a) bMEpC stimulated with LTA (1 μg/ml), MDP (1 μg/ml), or a combination of LTA plus MDP (1 μg/ml) responded with an increased level of expression of TNF-α mRNA (P < 0.05 by Kruskal-Wallis test). Shown are median values (Q1 and Q3) from bMEpC of five cows. (b) TNF-α in cell culture supernatants after 16 h of culture; results are from a representative experiment with either bMEpC or leukocytes. (c) IL-1β mRNA levels in response to LTA or MDP. Shown are median values (Q1 an Q3) from bMEpC of five cows. (d) IL-1β in cell culture supernatants after 16 h of culture; results are from the bMEpC of five cows or a representative experiment with either bMEpC or leukocytes. RE/18S, expression relative to 18S rRNA; PMA, phorbol myristate acetate; Mono, monocytes; PBMC, peripheral blood mononuclear cells stimulated with PMA and ionomycin.

FIG. 8.

Expression of transcripts of genes encoding PRR (NOD2, TLR2, and TLR6), peptide transporter (PEPT1), or PAF receptor by MEpC or bovine mammary tissue. RNA was extracted from bMEpC of two cows (cows 2006 and 1018), which were left unstimulated or were stimulated with LTA plus MDP (1 μg/ml each) for 8 h. RNA was also obtained from the mammary tissue of uninfected uninflamed quarters of two lactating cows (cows 5011 and 4019). RT-PCR was performed as described in Materials and Methods. GAPDH expression was used as a positive control.

FIG. 9.

Effect of the PAFR inhibitor WEB2086 on the secretion of CXCL8 by bMEpC stimulated with LTA. Bovine MEpC were preincubated without (WEB 0) or with 10, 100, or 1,000 nM WEB2086 and then stimulated with 0.1 or 1 μg/ml LTA or not stimulated (medium). The cell culture supernatant was collected 16 h later, and CXCL8 concentrations were determined by ELISA. Results are median values (Q1 and Q3) from bMEpC of four cows. Reductions of CXCL8 concentrations by WEB2086 were not statistically significant (Friedman test).

FIG. 10.

Activation of NF-κB p65 in bMEpC after 2 h or 8 h of stimulation with LTA (1 μg/ml) or MDP (1 μg/ml) used alone or in combination. Activation was measured in nuclear cell extracts of bMEpC by ELISA. Results are median values (Q1 and Q3) from bMEpC of four cows. *, significant differences compared to the unstimulated (medium) bMEpC (P < 0.05 by Friedman test and Bonferroni's multiple comparisons).

FIG. 11.

Effect of a pharmacological inhibitor of NF-κB on CXCL8 secretion by bMEpC incubated with LTA or MDP. Bovine MEpC were preincubated for 1 h at 37°C with 15 μM NF-κB activation inhibitor (Calbiochem) before the addition of MAMPs (1 μg/ml). Cell culture supernatants were collected 8 h poststimulation, and CXCL8 concentrations were determined by ELISA. Results are median values (Q1 and Q3) from bMEpC of five cows *, reductions of CXCL8 concentrations by NF-κB inhibition are significant (P < 0.05 by Friedman test and Bonferroni's multiple comparisons).