Pascal Rainard

Differential induction of complement fragment C5a and inflammatory cytokines during intramammary infections with Escherichia coli and Staphylococcus aureus.

ABSTRACT The prompt recruitment of neutrophils to the site of infection is essential for the defense of the bovine mammary gland against invading pathogens and is determinant for the outcome of the infection.Escherichia coli is known to induce clinical mastitis, characterized by an intense neutrophil recruitment leading to the eradication of the bacteria, whereas Staphylococcus aureusinduces subclinical mastitis accompanied by a moderate neutrophil recruitment and the establishment of chronic mastitis. To elicit the neutrophil recruitment into the udder, inflammatory mediators must be produced after recognition of the invading pathogen. To our knowledge, those mediators have never been studied during S. aureusmastitis, although understanding of the neutrophil recruitment mechanisms could allow a better understanding of the differences in the pathogeneses elicited by E. coli and S. aureus. Therefore, we studied, at several time points, the accumulation of neutrophils and the presence of the chemoattractant complement fragment C5a and of the cytokines interleukin-1β (IL-1β), tumor necrosis factor alpha, and IL-8 in milk after inoculation of E. colior S. aureus in lactating bovine udders. The low levels of C5a and the absence of cytokines in milk from S. aureus-infected cows, compared to the high levels found in milk from E. coli-infected animals, mirror the differences in the severities of the two inflammatory reactions. The cytokine deficit in milk after S. aureus inoculation in the lactating bovine mammary gland could contribute to the establishment of chronic mastitis. This result could help in the design of preventive or curative strategies against chronic mastitis.

Cells and Cytokines in Inflammatory Secretions of Bovine Mammary Gland

In response to invading bacteria, the mammary gland is protected by a variety of defence mechanisms, which can be separated into two distinct categories: innate immunity and specific immunity. Milk somatic cells consist of several cell types, including neutrophils, macrophages, lymphocytes and a smaller percentage of epithelial cells. In the healthy lactating mammary gland, macrophages are the predominant cell type whereas neutrophils are the major cell population during early inflammation. Following a bacteria invasion, neutrophil recruitment is elicited by inflammatory mediators that are produced in the infected gland by cells, possibly macrophages, activated by bacteria phagocytosis or responding to bacterial toxins or metabolites. Several cytokines, including interleukin- (IL-) 1 beta, IL-6, IL-8, tumour necrosis factor- (TNF-) alpha and interferon- (IFN-) gamma are known to be important to elicit the acute phase response and allow the accumulation of leukocytes at the site of infection. In addition to their role in early non-specific defences, macrophages also play a key role in the specific immune system, as antigen processing and presenting cells for the T cells. Few lymphocytes are found in milk of healthy glands where the predominant phenotype is CD8+ T cells. During the inflammatory reaction, T cells are recruited in milk and CD4+ cells become the predominant phenotype. The understanding of the specific and nonspecific immune mechanisms involved in the mammary gland defence against invading bacteria may lead to the development of new vaccines and to the use of cytokines to design immunomodulatory strategies for the control of bovine mastitis.

Leucotoxic Activities of Staphylococcus aureus Strains Isolated from Cows, Ewes, and Goats with Mastitis: Importance of LukM/LukF′-PV Leukotoxin

ABSTRACT Among the toxins that Staphylococcus aureus is able to secrete, bi-component toxins named leukotoxins target specifically leukocytes, mainly phagocytic cells. Isolates from cows, goats and ewes with mastitis were selected on the basis of the presence or not of the genes encoding the recently described LukM/LukF′-PV leukotoxin. Of the 128 isolates tested, 126 had moderate to high leukotoxic activity to bovine polymorphonuclear cells (PMN). The supernatants of lukM-positive isolates were much more leukotoxic than the supernatants of lukM-negative isolates: mean leukotoxic titers were 122 versus 20 and 581 versus 26 for isolates of bovine and caprine origin, respectively. Among lukM/lukF′-PV positive isolates, those of caprine and ovine origins were more leukotoxic than were isolates of bovine origin (P < 0.01). The two most abundant proteins in the culture supernatant of a highly toxic isolate were purified and identified as the two components of LukM (LukM and LukF′-PV) on the basis of their molecular mass, N-terminal amino acid sequence, and high synergistic activity. LukM/LukF′-PV induced the flattening of bovine PMN at a concentration as low as 3.6 ng/ml (0.1 nM). A higher concentration (18 ng/ml) was necessary to produce the same effect on caprine or ovine PMN. Affinity-purified antibodies to LukM or to LukF′-PV neutralized the leukotoxic effect of all the culture supernatants. They neutralized with the same efficiency the toxic activity of supernatants from lukM/lukF′-PV positive or negative isolates. These results establish that LukM/LukF′-PV is very active on PMN of ruminants and suggest that this leukotoxin could be the most active leukotoxin produced by mastitis isolates. They prompt further studies to delineate the contribution of LukM/LukF′-PV to the pathogenesis of mastitis in ruminants and the protective effect of antibodies to this leukotoxin.

Differential response of bovine mammary epithelial cells to Staphylococcus aureus or Escherichia coli agonists of the innate immune system

Mastitis caused by Escherichia coli and Staphylococcus aureus is a major pathology of dairy cows. To better understand the differential response of the mammary gland to these two pathogens, we stimulated bovine mammary epithelial cells (bMEC) with either E. coli crude lipopolysaccharide (LPS) or with S. aureus culture supernatant (SaS) to compare the transcriptomic profiles of the initial bMEC response. By using HEK 293 reporter cells for pattern recognition receptors, the LPS preparation was found to stimulate TLR2 and TLR4 but not TLR5, Nod1 or Nod2, whereas SaS stimulated TLR2. Biochemical analysis revealed that lipoteichoic acid, protein A and α-hemolysin were all present in SaS, and bMEC were found to be responsive to each of these molecules. Transcriptome profiling revealed a core innate immune response partly shared by LPS and SaS. However, LPS induced expression of a significant higher number of genes and the fold changes were of greater magnitude than those induced by SaS. Microarray data analysis suggests that the activation pathways and the early chemokine and cytokine production preceded the defense and stress responses. A major differential response was the activation of the type I IFN pathway by LPS but not by SaS. The higher upregulation of chemokines (Cxcl10, Ccl2, Ccl5 and Ccl20) that target mononuclear leucocytes by LPS than by SaS is likely to be related to the differential activation of the type I IFN pathway, and could induce a different profile of the initial recruitment of leucocytes. The MEC responses to the two stimuli were different, as LPS was associated with NF-κB and Fas signaling pathways, whereas SaS was associated with AP-1 and IL-17A signaling pathways. It is noteworthy that at the protein level secretion of TNF-α and IL-1β was not induced by either stimulus. These results suggest that the response of MEC to diffusible stimuli from E. coli and S. aureus contributes to the onset of the response with differential leucocyte recruitment and distinct inflammatory and innate immune reactions of the mammary gland to infection.

Kinetics of cells and cytokines during immune-mediated inflammation in the mammary gland of cows systemically immunized with Staphylococcus aureus α-toxin

Abstract:Objective: To examine changes in inflammatory mediators, lymphocyte subpopulations and neutrophil activation that occur during an immune-mediated recruitment of neutrophils in the mammary gland.¶Subjects: 11 clinically healthy cows.¶Treatment: 5 cows received 2 subcutaneous injections of 30 μg of α-toxin of Staphylococcus aureus, two months apart. Three months after the last immunization, 5 μg of α-toxin were injected, via the teat end, in one randomly selected quarter of the 5 immunized cows and of the 6 unimmunized cows (control group).¶Methods: Blood and milk samples were collected at several times during 4 days post-challenge. Blood and milk cells were purified to be stained with specific mAbs and analysed by flow cytometry, or to be used for cytokine RT-PCR. Bovine serum albumin, haptoglobin, cytokines and C5a were also analysed in milk or plasma samples using radial immunodiffusion assay or ELISA.¶Results: Large amounts of cells (> 1 million/ml of milk) were recruited in the quarters of the immunized cows, whereas no recruitment occurred in the control group. In blood of immunized animals, haptoglobin was present and expression of surface adhesion molecules on neutrophils was modified whereas no change was observed concerning the lymphocyte subpopulations. On milk-derived neutrophils, the expression of CD11b and CD18 was upregulated compared to blood, in contrast to CD62L that was downregulated. The CD8+ cells were recruited as soon as 12 h post-challenge, in contrast to the CD4+ cells, 96 h post-challenge. No IL-1β, TNF-α, IL-8 and C5a were detected using ELISA. mRNA of IL-1α, IL-1β, IL-6, TNF-α, IL-8 and IL-12 were found in almost all the samples.¶Conclusions: The immunization triggered an early and massive neutrophil recruitment from the blood into the milk compartment as well as the recruitment of a cytotoxic/suppressor lymphocyte population during the early acute phase response. These results could help to devise new vaccinal strategies to fight against staphylococcal mastitis.¶

Repertoire of Escherichia coli agonists sensed by innate immunity receptors of the bovine udder and mammary epithelial cells

Escherichia coli is a frequent cause of clinical mastitis in dairy cows. It has been shown that a prompt response of the mammary gland after E. coli entry into the lumen of the gland is required to control the infection, which means that the early detection of bacteria is of prime importance. Yet, apart from lipopolysaccharide (LPS), little is known of the bacterial components which are detected by the mammary innate immune system. We investigated the repertoire of potential bacterial agonists sensed by the udder and bovine mammary epithelial cells (bMEC) during E. coli mastitis by using purified or synthetic molecular surrogates of bacterial agonists of identified pattern-recognition receptors (PRRs). The production of CXCL8 and the influx of leucocytes in milk were the readouts of reactivity of stimulated cultured bMEC and challenged udders, respectively. Quantitative PCR revealed that bMEC in culture expressed the nucleotide oligomerization domain receptors NOD1 and NOD2, along with the Toll-like receptors TLR1, TLR2, TLR4, and TLR6, but hardly TLR5. In line with expression data, bMEC proved to react to the cognate agonists C12-iE-DAP (NOD1), Pam3CSK4 (TLR1/2), Pam2CSK4 (TLR2/6), pure LPS (TLR4), but not to flagellin (TLR5). As the udder reactivity to NOD1 and TLR5 agonists has never been reported, we tested whether the mammary gland reacted to intramammary infusion of C12-iE-DAP or flagellin. The udder reacted to C12-iE-DAP, but not to flagellin, in line with the reactivity of bMEC. These results extend our knowledge of the reactivity of the bovine mammary gland to bacterial agonists of the innate immune system, and suggest that E. coli can be recognized by several PRRs including NOD1, but unexpectedly not by TLR5. The way the mammary gland senses E. coli is likely to shape the innate immune response and finally the outcome of E. coli mastitis.

The chemokine CXCL3 is responsible for the constitutive chemotactic activity of bovine milk for neutrophils.

Bovine milk is known to exert a potent chemotactic activity on neutrophils, but the responsible agent has not been identified. The objective of the study was to characterize the main biochemical component responsible for this chemotactic activity. A neutrophil shape change assay was used to locate active milk fractions separated by chromatography. A single protein was isolated and identified by amino acid sequencing and mass spectrometry as CXCL3. Recombinant bovine chemokines and specific antibodies were used to show that normal milk contains active concentrations of CXCL1 (1-5ng/ml) and CXCL3 (100-500ng/ml), whereas CXCL2 and CXCL8/IL-8 were not detected. Depletion experiments with antibodies showed that CXCL3 was the main chemotaxin for neutrophils in normal (non-mastitic) milk. The chemokine CXCL3 was located by immunohistochemistry in mammary epithelial cells, and abundant mRNA was found in uninflamed mammary tissue, suggesting constitutive secretion by the lactating mammary epithelium. These results indicate that CXCL3/GRO-gamma is the major chemotactic factor for neutrophils in bovine milk in the absence of inflammation, and that it is secreted constitutively in milk by mammary epithelial cells. This finding prompts the question of the biological significance of permanent high concentrations of a CXC chemokine in milk.

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.

Staphylococcal-associated molecular patterns enhance expression of immune defense genes induced by IL-17 in mammary epithelial cells

Interleukin-17A (IL-17A) and IL-17F have been shown to mediate a crucial crosstalk between the immune system and various epithelial tissues, stimulating various defensive mechanisms to bacterial infections. A number of studies have characterized the response to IL-17A and IL-17F of epithelial cells from airways, intestine, and skin, but not from the mammary gland. To evaluate the potential contribution of IL-17 to the immune defense of the mammary gland, we analyzed the effects of recombinant bovine IL-17A and IL-17F on primary bovine mammary epithelial cells (MEC) by quantitative PCR and ELISA. We found expression (mRNA) of the two components of the IL-17 receptor complex, IL-17RA and IL-17RC, in mammary tissue and MEC in vitro. The expression of a number of genes encoding cytokines, chemokines and proteins endowed with antibacterial activities was increased by IL-17A, and to a lesser extent by IL-17F, but the magnitude of responses was modest. As expected, responses were augmented by the combination of IL-17A or IL-17F with TNF-α. Interestingly, responses of a few of the tested genes, such as IL8, CCL20, iNOS, and CfB, were augmented by the combination of IL-17A with staphylococcal lipoteichoic acid or muramyl dipeptide, bacterial agonists of the innate immune system. This can be interpreted as indicating that IL-17A and IL-17F are tailored to exert their full potential in a septic environment. MEC responses were characterized by the expression of chemokines targeting not only neutrophils (CXCL3 and CXCL8) but also mononuclear leucocytes (CCL2, CCL20). Production of IL-6 was low and the inflammatory cytokines TNF-α and IL-1β were expressed (mRNA) but proteins were not secreted. Altogether, our results suggest that IL-17A and IL-17F have a potential to modulate the mammary gland immune response to mastitis-causing pathogens.

A colorimetric microassay for opsonins by reduction of NBT in phagocytosing bovine polymorphs

The adaptation of the reduction of nitro blue tetrazolium (NBT) by phagocytes to the measurement of the opsonising capacity of serum or milk whey is described. A dose-response curve of the absorbance of solubilised reduced NBT to the concentration of opsonins, along with a close correlation (r = 0.94) between the colorimetric NBT assay and ingestion of group B streptococci (as measured by reduction of [3H]thymidine incorporation by extracellular bacteria), was established. This NBT reduction test, employing 96-well flat-bottom microtitre plates, is simple, semiautomated, requires a low number of PMN, and is applicable to a large number of samples, which renders it useful as a screening test for the opsonising capacity of sera or other biologic fluids.