Soluble CD14 produced by bovine mammary epithelial cells modulates their response to full length LPS.

Bovine mastitis remains a major disease in cattle world-wide. In the mammary gland, mammary epithelial cells (MEC) are sentinels equipped with receptors allowing them to detect and respond to the invasion by bacterial pathogens, in particular Escherichia coli. Lipopolysaccharide (LPS) is the major E. coli motif recognized by MEC through its interaction with the TLR4 receptor and the CD14 co-receptor. Previous studies have highlighted the role of soluble CD14 (sCD14) in the efficient recognition of LPS molecules possessing a full-length O-antigen (LPSS). We demonstrate here that MEC are able to secrete CD14 and are likely to contribute to the presence of sCD14 in milk. We then investigated how sCD14 modulates and is required for the response of MEC to LPSS. This study highlights the key role of sCD14 for the full activation of the Myd88-independent pathway by LPSS. We also identified several lncRNA that are activated in MEC in response to LPS, including one lncRNA showing homologies with the mir-99a-let-7c gene (MIR99AHG). Altogether, our results show that a full response to LPS by mammary epithelial cells requires sCD14 and provide detailed information on how milk sCD14 can contribute to an efficient recognition of LPS from coliform pathogens.

Invited review: A critical appraisal of mastitis vaccines for dairy cows.

Infections of the mammary gland remain a frequent disease of dairy ruminants that negatively affect animal welfare, milk quality, farmer serenity, and farming profitability and cause an increase in use of antimicrobials. There is a need for efficacious vaccines to alleviate the burden of mastitis in dairy farming, but this need has not been satisfactorily fulfilled despite decades of research. A careful appraisal of past and current research on mastitis vaccines reveals the peculiarities but also the commonalities among mammary gland infections associated with the major mastitis pathogens Escherichia coli, Staphylococcus aureus, Streptococcus uberis, Streptococcus agalactiae, or Streptococcus dysgalactiae. A major pitfall is that the immune mechanisms of effective protection have not been fully identified. Until now, vaccine development has been directed toward the generation of antibodies. In this review, we drew up an inventory of the main approaches used to design vaccines that aim at the major pathogens for the mammary gland, and we critically appraised the current and tentative vaccines. In particular, we sought to relate efficacy to vaccine-induced defense mechanisms to shed light on some possible reasons for current vaccine shortcomings. Based on the lessons learned from past attempts and the recent results of current research, the design of effective vaccines may take a new turn in the years to come.

Type 3 immunity: a perspective for the defense of the mammary gland against infections.

Type 3 immunity encompasses innate and adaptive immune responses mediated by cells that produce the signature cytokines IL-17A and IL-17F. This class of effector immunity is particularly adept at controlling infections by pyogenic extracellular bacteria at epithelial barriers. Since mastitis results from infections by bacteria such as streptococci, staphylococci and coliform bacteria that cause neutrophilic inflammation, type 3 immunity can be expected to be mobilized at the mammary gland. In effect, the main defenses of this organ are provided by epithelial cells and neutrophils, which are the main terminal effectors of type 3 immunity. In addition to theoretical grounds, there is observational and experimental evidence that supports a role for type 3 immunity in the mammary gland, such as the production of IL-17A, IL-17F, and IL-22 in milk and mammary tissue during infection, although their respective sources remain to be fully identified. Moreover, mouse mastitis models have shown a positive effect of IL-17A on the course of mastitis. A lot remains to be uncovered before we can safely harness type 3 immunity to reinforce mammary gland defenses through innate immune training or vaccination. However, this is a promising way to find new means of improving mammary gland defenses against infection.

Host factors determine the evolution of infection with Staphylococcus aureus to gangrenous mastitis in goats.

Staphylococcus aureus is the major cause of very severe mastitis of dairy goats. The initial objective of our study was to fine-tune an experimental model of infection of the goat mammary gland with two strains of S. aureus and two lines of goats (low and high somatic cell score lines). Following the challenge, the 10 infected goats divided in two clear-cut severity groups, independently of the S. aureus strain and the goat line. Five goats developed very severe mastitis (of which four were gangrenous) characterized by uncontrolled infection (UI group), whereas the other five kept the infection under control (CI group). The outcome of the infection was determined by 18 h post-infection (hpi), as heralded by the bacterial milk concentration at 18 hpi: more than 107/mL in the UI group, about 106/mL in the CI group. Leukocyte recruitment and composition did not differ between the groups, but the phagocytic killing at 18 hpi efficiency did. Contributing factors involved milk concentrations of α-toxin and LukMF' leukotoxin, but not early expression of the genes encoding the pentraxin PTX3, the cytokines IL-1α and IL-1ß, and the chemokines IL-8 and CCL5. Concentrations of TNF-α, IFN-γ, IL-17A, and IL-22 rose sharply in the milk of UI goats when infection was out of control. The results indicate that defenses mobilized by the mammary gland at an early stage of infection were essential to prevent staphylococci from reaching critical concentrations. Staphylococcal exotoxin production appeared to be a consequent event inducing the evolution to gangrenous mastitis.

IL-17A Is an Important Effector of the Immune Response of the Mammary Gland to Escherichia coli Infection.

The cytokine IL-17A has been shown to play critical roles in host defense against bacterial and fungal infections at different epithelial sites, but its role in the defense of the mammary gland (MG) has seldom been investigated, although infections of the MG constitute the main pathology afflicting dairy cows. In this study, we showed that IL-17A contributes to the defense of the MG against Escherichia coli infection by using a mouse mastitis model. After inoculation of the MG with a mastitis-causing E. coli strain, the bacterial load increased rapidly, triggering an intense influx of leukocytes into mammary tissue and increased concentrations of IL-6, IL-22, TNF-α, and IL-10. Neutrophils were the first cells that migrated intensely to the mammary tissue, in line with an early production of CXCL2. Depletion of neutrophils induced an increased mammary bacterial load. There was a significant increase of IL-17-containing CD4(+) αß T lymphocyte numbers in infected glands. Depletion of IL-17A correlated with an increased bacterial colonization and IL-10 production. Intramammary infusion of IL-17A at the onset of infection was associated with markedly decreased bacterial numbers, decreased IL-10 production, and increased neutrophil recruitment. Depletion of CD25(+) regulatory T cells correlated with a decreased production of IL-10 and a reduced bacterial load. These results indicate that IL-17A is an important effector of MG immunity to E. coli and suggest that an early increased local production of IL-17A would improve the outcome of infection. These findings point to a new lead to the development of vaccines against mastitis.

Mammary microbiota of dairy ruminants: fact or fiction?

Explorations of how the complex microbial communities that inhabit different body sites might contribute to health and disease have prompted research on the ways the harmonious relationship between a host and its microbiota could be used to keep animals healthy in their production conditions. In particular, there is a growing interest in the bacterial signatures that can be found in the milk of healthy or mastitic dairy cows. The concept of sterility of the healthy mammary gland of dairy ruminants has been challenged by the results of studies using bacterial DNA-based methodology. The newly obtained data have led to the concept of the intramammary microbiota composed of a complex community of diverse bacteria. Accordingly, mammary gland infections are not mere infections by a bacterial pathogen, but the consequence of mammary dysbiosis. This article develops the logical implications of this paradigm shift and shows how this concept is incompatible with current knowledge concerning the innate and adaptive immune system of the mammary gland of dairy ruminants. It also highlights how the concept of mammary microbiota clashes with results of experimental infections induced under controlled conditions or large field experiments that demonstrated the efficacy of the current mastitis control measures.

Permissiveness of bovine epithelial cells from lung, intestine, placenta and udder for infection with Coxiella burnetii.

Ruminants are the main source of human infections with the obligate intracellular bacterium Coxiella (C.) burnetii. Infected animals shed high numbers of C. burnetii by milk, feces, and birth products. In goats, shedding by the latter route coincides with C. burnetii replication in epithelial (trophoblast) cells of the placenta, which led us to hypothesize that epithelial cells are generally implicated in replication and shedding of C. burnetii. We therefore aimed at analyzing the interactions of C. burnetii with epithelial cells of the bovine host (1) at the entry site (lung epithelium) which govern host immune responses and (2) in epithelial cells of gut, udder and placenta decisive for the quantity of pathogen excretion. Epithelial cell lines [PS (udder), FKD-R 971 (small intestine), BCEC (maternal placenta), F3 (fetal placenta), BEL-26 (lung)] were inoculated with C. burnetii strains Nine Mile I (NMI) and NMII at different cultivation conditions. The cell lines exhibited different permissiveness for C. burnetii. While maintaining cell viability, udder cells allowed the highest replication rates with formation of large cell-filling Coxiella containing vacuoles. Intestinal cells showed an enhanced susceptibility to invasion but supported C. burnetii replication only at intermediate levels. Lung and placental cells also internalized the bacteria but in strikingly smaller numbers. In any of the epithelial cells, both Coxiella strains failed to trigger a substantial IL-1ß, IL-6 and TNF-α response. Epithelial cells, with mammary epithelial cells in particular, may therefore serve as a niche for C. burnetii replication in vivo without alerting the host's immune response.

Behavioral and patho-physiological response as possible signs of pain in dairy cows during Escherichia coli mastitis: A pilot study.

Bovine mastitis is one of the most common diseases in the dairy industry and it is a major welfare problem. Pain during mastitis is generally assessed through behavior but a combination of indicators would increase the chances of detecting pain and assessing its intensity. The aim of this study was to assess behavioral and patho-physiological responses as possible signs of pain experienced by cows after experimental intramammary challenge (mastitis) with Escherichia coli. Six Holstein-Friesian cows received an inoculation of E. coli P4 in one healthy quarter. Evolution of the disease was assessed using bacteriological growth and somatic cell counts (SCC). Cows' response to the challenge was monitored by direct behavioral and clinical observations, data loggers, rumen temperature sensors, and indicators of inflammation, stress, and oxidative status. From all data recorded, the variables that contributed most to the discrimination of mastitis phases were obtained by factorial discriminant analysis. Baseline levels of all indicators corresponded to values before challenge. Specifically, we weighted data relating to lying behavior by the observations at the same hour of the day before challenge to eliminate the circadian rhythm effect. We identified 3 phases that were discriminated by factorial discriminant analysis with good performance. Nine indicators varied according to the phase of the disease: cows' attitude toward their surroundings, tail position, clinical signs, ear position, variation of postural changes, concentrations of haptoglobin and serum amyloid A (SAA), cortisol blood levels, and rumen temperature (as a surrogate for body temperature). In phase 1 (4 to 8 h postinoculation), E. coli proliferated exponentially in milk but inflammation indicators remained at baseline levels. Cows were less attentive toward their surroundings (median score, 0.63), and postural changes (lying/standing) were less frequent (0.75 times from baseline). In phase 2 (12 to 24 h postinoculation), bacterial concentrations peaked around 12 h and then began to decrease concomitantly with a sharp SCC increase. Cows were less attentive toward their surroundings (score, 0.54), had high plasma cortisol (31.3 ng/mL) and SAA (100.3 µg/mL) concentrations, and rumen temperature was increased (40.3°C). In phase 3 (32 to 80 h postinoculation), bacterial concentrations decreased concomitantly with high SCC levels. Cows had high levels of haptoglobin (0.57 mg/mL) and SAA (269 µg/mL) but showed no behavioral changes. Dairy cows displayed changes of behavioral, inflammatory, and stress parameters after E. coli mammary inoculation. Our results suggest that cows may have experienced discomfort in the preclinical phase (phase 1) and pain in the acute phase (phase 2) but neither discomfort nor pain in the remission phase (phase 3). Although larger controlled studies are needed to confirm our findings, this knowledge could be useful for early detection of E. coli mastitis and for decision-making regarding the initiation of pain-relief treatment during mastitis in dairy cows. This would improve animal welfare and potentially faster disease remission.

Staphylococcus aureus Phenol-Soluble Modulins Impair Interleukin Expression in Bovine Mammary Epithelial Cells.

The role of the recently described interleukin-32 (IL-32) in Staphylococcus aureus-induced mastitis, an inflammation of the mammary gland, is unclear. We determined expression of IL-32, IL-6, and IL-8 in S. aureus- and Escherichia coli-infected bovine mammary gland epithelial cells. Using live bacteria, we found that in S. aureus-infected cells, induction of IL-6 and IL-8 expression was less pronounced than in E. coli-infected cells. Notably, IL-32 expression was decreased in S. aureus-infected cells, while it was increased in E. coli-infected cells. We identified the staphylococcal phenol-soluble modulin (PSM) peptides as key contributors to these effects, as IL-32, IL-6, and IL-8 expression by epithelial cells exposed to psm mutant strains was significantly increased compared to that in cells exposed to the isogenic S. aureus wild-type strain, indicating that PSMs inhibit the production of these interleukins. The use of genetically complemented strains confirmed this observation. Inasmuch as the decreased expression of IL-32, which is involved in dendritic cell maturation, impairs immune responses, our results support a PSM-dependent mechanism that allows for the development of chronic S. aureus-related mastitis.

Investigating the contribution of IL-17A and IL-17F to the host response during Escherichia coli mastitis.

Mastitis remains a major disease of cattle with a strong impact on the dairy industry. There is a growing interest in understanding how cell mediated immunity contributes to the defence of the mammary gland against invading mastitis causing bacteria. Cytokines belonging to the IL-17 family, and the cells that produce them, have been described as important modulators of the innate immunity, in particular that of epithelial cells. We report here that expression of IL-17A and IL-17F genes, encoding two members of the IL-17 family, are induced in udder tissues of cows experimentally infected with Escherichia coli. The impact of IL-17A on the innate response of bovine mammary epithelial cells was investigated using a newly isolated cell line, the PS cell line. We first showed that PS cells, similar to primary bovine mammary epithelial cells, were able to respond to agonists of TLR2 and to LPS, provided CD14 was added to the culture medium. We then showed that secretion of CXCL8 and transcription of innate immunity related-genes by PS cells were increased by IL-17A, in particular when these cells were stimulated with live E. coli bacteria. Together with data from the literature, these results support the hypothesis that IL-17A and IL-17 F could play an important role in mediating of host-pathogen interactions during mastitis.

Expression of the receptor for IgM (FcµR) by bovine neutrophils.

Bovine neutrophils possess a particular set of receptors for immunoglobulins. They have been shown to express a distinctive receptor for IgG2, and it has long been known that they interact poorly with IgG1 but that they can use IgM antibodies as opsonins. We show that the binding of labeled IgM was inhibited by unlabeled IgM but not by IgA, suggesting that bovine neutrophils express a specific IgM receptor. The binding of non-aggregated IgM is strong at 4 °C, but shedding occurs at 37 °C. We designed anti-peptide antibodies based on the sequence of the FcµR, the newly described receptor for IgM. These antibodies bound to bovine neutrophils at 4 °C. At 37 °C, labeling was lost, but the loss was inhibited by pretreatment with cytochalasin D, indicating internalization of the receptor after cross-linking by antibodies. Neutrophils that had internalized the receptor were no longer able to bind IgM. Eosinophils showed a low level of FcµR expression. FcµR expression by neutrophils was not increased by stimulation with Toll-like receptor agonists or the complement anaphylatoxin C5a, and decreased by TNF-α. Exposure of neutrophils to IFN-γ for 18 h increased FcµR expression without augmenting the binding of IgG1 or IgG2. We confirmed that bovine neutrophils can use IgM to phagocytose and kill bacteria without the help of Complement. Neutrophils that have migrated into the lumen of inflamed lactating mammary glands expressed the FcµR. These results indicate that bovine neutrophils express an IgM receptor, the FcµR, which is functional to contribute to the opsonophagocytosis of bacteria at inflammatory sites. Expression of the FcµR by neutrophils gives IgM a particular importance for the immune defense in the bovine species.

Saccharomyces cerevisiae as a platform for vaccination against bovine mastitis.

Mastitis is a major issue for the dairy industry. Despite multiple attempts, the efficacy of available mastitis vaccines is limited and this has been attributed to their incapacity to trigger robust cell-mediated immunity. Yeasts have recently been identified as promising antigen vectors capable of inducing T-cell responses, surpassing the antibody-biased mechanisms elicited by conventional adjuvanted vaccines. In this study, we combine in vitro, ex vivo, and in vivo approaches to evaluate the potential of the yeast Saccharomyces cerevisiae as a platform for novel vaccines against bovine mastitis. We demonstrate that S. cerevisiae is safe for intramuscular and intramammary immunisation in dairy cows. Vaccination resulted in a significant increase of IFNγ and IL-17 responses against the yeast platform but not against the vaccine antigen. These observations highlight that strategies to counterbalance the immunodominance of S. cerevisiae antigens are necessary for the development of successful vaccine candidates.

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-1ß 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.

Punch-excised explants of bovine mammary gland to model early immune response to infection.

BACKGROUND: Mammary gland (MG) infections (mastitis) are frequent diseases of dairy cows that affect milk quality, animal welfare and farming profitability. These infections are commonly associated with the bacteria Escherichia coli and Staphylococcus aureus. Different in vitro models have been used to investigate the early response of the MG to bacteria, but the role of the teat in mastitis pathogenesis has received less attention. In this study, we used punch-excised teat tissue as an ex vivo model to study the immune mechanisms that arise early during infection when bacteria have entered the MG. RESULTS: Cytotoxicity and microscopic analyses showed that bovine teat sinus explants have their morphology and viability preserved after 24 h of culture and respond to ex vivo stimulation with TLR-agonists and bacteria. LPS and E. coli trigger stronger inflammatory response in teat when compared to LTA and S. aureus, leading to a higher production of IL-6 and IL-8, as well as to an up-regulation of proinflammatory genes. We also demonstrated that our ex vivo model can be applied to frozen-stored explants. CONCLUSIONS: In compliance with the 3Rs principle (replacement, reduction and refinement) in animal experimentation, ex vivo explant analyses proved to be a simple and affordable approach to study MG immune response to infection. This model, which better reproduces organ complexity than epithelial cell cultures or tissue slices, lends itself particularly well to studying the early phases of the MG immune response to infection.

Bovine blood and milk T-cell subsets in distinct states of activation and differentiation during subclinical Staphylococcus aureus mastitis.

T-lymphocytes are key mediators of adaptive cellular immunity and knowledge about distinct subsets of these cells in healthy and infected mammary gland secretions remains limited. In this study, we used a multiplex cytometry panel to show that staphylococcal mastitis causes the activation of CD4+, CD8+ and γδ T-cells found in bovine milk. We also highlight remarkable differences in the proportions of naïve and memory T-cells subsets found in blood and milk. These observations will contribute to a better understanding of cell-mediated immune mechanisms in the udder and to the development of new therapeutic and preventive strategies targeting mastitis.

Genetic susceptibility to S. aureus mastitis in sheep: differential expression of mammary epithelial cells in response to live bacteria or supernatant.

Staphylococcus aureus is a prevalent pathogen for mastitis in dairy ruminants and is responsible for both clinical and subclinical mastitis. Mammary epithelial cells (MEC) represent not only a physical barrier against bacterial invasion but are also active players of the innate immune response permitting infection clearance. To decipher their functions in general and in animals showing different levels of genetic predisposition to Staphylococcus in particular, MEC from ewes undergoing a divergent selection on milk somatic cell count were stimulated by S. aureus. MEC response was also studied according to the stimulation condition with live bacteria or culture supernatant. The early MEC response was studied during a 5 h time course by microarray to identify differentially expressed genes with regard to the host genetic background and as a function of the conditions of stimulation. In both conditions of stimulation, metabolic processes were altered, the apoptosis-associated pathways were considerably modified, and inflammatory and immune responses were enhanced with the upregulation of il1a, il1b, and tnfa and several chemokines known to enhance neutrophil (cxcl8) or mononuclear leukocyte (ccl20) recruitment. Genes associated with oxidative stress were increased after live bacteria stimulation, whereas immune response-related genes were higher after supernatant stimulation in the early phase. Only 20 genes were differentially expressed between Staphylococcus spp-mastitis resistant and susceptible animals without any clearly defined role on the control of infection. To conclude, this suggests that MEC may not represent the cell type at the origin of the difference of mastitis susceptibility, at least as demonstrated in our genetic model. Supernatant or heat-killed S. aureus produce biological effects that are essentially different from those induced by live bacteria.

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.

Immune defenses of the mammary gland epithelium of dairy ruminants.

The epithelium of the mammary gland (MG) fulfills three major functions: nutrition of progeny, transfer of immunity from mother to newborn, and its own defense against infection. The defense function of the epithelium requires the cooperation of mammary epithelial cells (MECs) with intraepithelial leucocytes, macrophages, DCs, and resident lymphocytes. The MG is characterized by the secretion of a large amount of a nutrient liquid in which certain bacteria can proliferate and reach a considerable bacterial load, which has conditioned how the udder reacts against bacterial invasions. This review presents how the mammary epithelium perceives bacteria, and how it responds to the main bacterial genera associated with mastitis. MECs are able to detect the presence of actively multiplying bacteria in the lumen of the gland: they express pattern recognition receptors (PRRs) that recognize microbe-associated molecular patterns (MAMPs) released by the growing bacteria. Interactions with intraepithelial leucocytes fine-tune MECs responses. Following the onset of inflammation, new interactions are established with lymphocytes and neutrophils recruited from the blood. The mammary epithelium also identifies and responds to antigens, which supposes an antigen-presenting capacity. Its responses can be manipulated with drugs, plant extracts, probiotics, and immune modifiers, in order to increase its defense capacities or reduce the damage related to inflammation. Numerous studies have established that the mammary epithelium is a genuine effector of both innate and adaptive immunity. However, knowledge gaps remain and newly available tools offer the prospect of exciting research to unravel and exploit the multiple capacities of this particular epithelium.

Adaptive Cell-Mediated Immunity in the Mammary Gland of Dairy Ruminants.

Mastitis is one of the greatest issues for the global dairy industry and controlling these infections by vaccination is a long-sought ambition that has remained unfulfilled so far. In fact, gaps in knowledge of cell-mediated immunity in the mammary gland (MG) have hampered progress in the rational design of immunization strategies targeting this organ, as current mastitis vaccines are unable to elicit a strong protective immunity. The objectives of this article are, from a comprehensive and critical review of available literature, to identify what characterizes adaptive immunity in the MG of ruminants, and to derive from this analysis research directions for the design of an optimal vaccination strategy. A peculiarity of the MG of ruminants is that it does not belong to the common mucosal immune system that links the gut immune system to the MG of rodents, swine or humans. Indeed, the MG of ruminants is not seeded by lymphocytes educated in mucosal epithelia of the digestive or respiratory tracts, because the mammary tissue does not express the vascular addressins and chemokines that would allow the homing of memory T cells. However, it is possible to elicit an adaptive immune response in the MG of ruminants by local immunization because the mammary tissue is provided with antigen-presenting cells and is linked to systemic mechanisms. The optimal immune response is obtained by luminal exposure to antigens in a non-lactating MG. The mammary gland can be sensitized to antigens so that a local recall elicits neutrophilic inflammation and enhanced defenses locally, resulting from the activation of resident memory lymphocytes producing IFN-γ and/or IL-17 in the mammary tissue. The rational exploitation of this immunity by vaccination will need a better understanding of MG cell-mediated immunity. The phenotypic and functional characterization of mammary antigen-presenting cells and memory T cells are amongst research priorities. Based on current knowledge, rekindling research on the immune cells that populate the healthy, infected, or immunized MG appears to be a most promising approach to designing efficacious mastitis vaccines.