Priming of udders with LPS protects for a limited time against manifestation of experimentally induced mastitis in mid-lactating cows [10, 23]. Mild endotoxin stimulation might thus possibly be developed as a means to reduce both, incidence and severity of mastitis in critical periods of the lactation cycle. We have undertaken the current study to learn more about the broader implications of a mild endotoxin pre-stimulation (priming) of primary MEC cultures upon their subsequent reaction towards a challenge with heat inactivated E. coli particles. We have previously reported on relevance and some limitations of the MEC model to study mastitis related immune regulations, on the E. coli challenge as also applied here and on the general techniques of our microarray analysis [17, 18, 22, 27, 28]. We based the current study on using primary MEC cultures from three different cows. Using this small number of biological replica might limit the statistical power. However, we have previously documented that the intra-animal variability of pathogen induced gene expression between those cultures is much less than observed between cows . We discuss our key observations in terms of priming (i) enforced sentinel functions of the MEC, (ii) improved protection against pathogens and tissue damage and (iii) reduced expression of master cytokines and potentially harmful factors limiting the risk of induced immune-pathology.
Priming enforces sentinel functions of the MEC
A mild LPS priming (1 μg/udder quarter, for 12 h) was found to significantly increase in vivo the number of somatic cells in udders . Thus, it was not surprising to find in our in vitro pbMEC model that LPS priming enhanced quite strongly the level of mRNAs encoding the chemokines CCL5 (also known as RANTES) and CXCL8. Both factors are known to trigger diapedesis of mononuclear cells, T-cells and macrophages (CCL5 ) and polymorph nuclear granulocytes (PMN; CXCL8 ) through the endothelia of the blood vessels recruiting them into the inflamed sites. Elevated levels of both factors enhance the abundance of professional sentinel cells (e.g. macrophages) in the udder and conceivably improve the competence of the gland to counter act recurrent infections. It is known from macrophages that LPS priming enhances CCL5 expression in response to a second LPS stimulus .
In contrast to observations made on endotoxin tolerant macrophages [14, 15], we found that priming increased the abundances of a variety of mRNA moieties encoding peptidases for antigen processing (ERAP2, CTTS) and MHC-II factors (bovine orthologs of HLA-DQA1, -DRA, -DQB1) in MEC. While MEC are generally not considered as professional antigen-presenting cells (APCs) it is known that alveolar epithelial cells express MHC-II factors and are relevant APCs in the lung . Thus, enhanced expression of these factors in LPS-primed MEC suggests an improved readiness of the primed cells for antigen presentation and thus improving their sentinel competence.
Priming improves protection against pathogens and tissue damage
Priming increased the levels of mRNAs encoding the bactericidal β-defensin lingual antimicrobial peptide (LAP). It was shown in macrophages that ET remodels the chromatin at the promoters of such "not LPS tolerant" effector genes of immune defence through differential histone modification making them more quickly responsive to a second stimulus . Epigenetic mechanism might also operate during ET at the LAP promoter of the MEC, since mastitis induced expression of the LAP gene requires chromatin decompaction at the promoter .
Focussing on LAP as an example for a β-defensin encoding gene serves only as a paradigm for the regulation of this class of molecules, which are so abundantly encoded in the bovine genome. More than 100 highly related copies of β-defensin-encoding genes were uncovered in the analysis of the entire bovine genome . The infection induced expression of LAP and other β-defensins in the udder has previously been reported [18, 19, 34]. The wealth of these β-defensin-encoding genes could not be analysed with the tools applied in this study, but LPS priming-enhanced abundance of the LAP-encoding RNA might be indicative for the regulation of other members of this gene family. Hence, LPS priming supports protection against colonization of the udder by bacterial pathogens. This was indeed found to be the case in vivo [10, 23].
Priming increased also the mRNA level of the defensin like factor secretory leukocyte peptidase inhibitor (SLPI), known as contributing to the defence against bacteria, fungi and retroviruses in epithelial tissues . This multifunctional serine protease inhibitor protects epithelial tissue during inflammation from the attack by endogenous proteolytic enzymes. Moreover, SLPI is known to bind and synergize with proepithelin. This growth factor promotes proliferation of epithelial cells and suppresses activation of neutrophils. SLPI abrogates proteolytic cleavage of proepithelin into the inflammation sustaining factor epithelin . Lactotransferrin (LTF) is another multifunctional factor revealing LPS priming-related upregulated mRNA levels. This factor has bacteriostatic, bactericidal and some immune modulating properties, but also the capability to bind LPS (reviewed in ). It was shown that LTF pre-treatment protects mice from pathophysiological effects of LPS and enhances the survival after an LPS injection . Priming enhanced the levels of the mRNA encoding the complement factor CFB. This protein activates the alternative complement pathway thereby strengthening unspecific immune defence mechanisms. Expression of this factor in the udder is also strongly induced during E. coli-elicited mastitis .
Priming increased the mRNA abundances of several factors protecting against membrane damage associated with the so called "bystander killing" effect of activated immune defence. These include the decay accelerating factor for complement CD55 (also known as DAF) protecting membranes of the host cells against misguided attacks through complement factors and also the glycoprotein mucin 1 (MUC1). This mucin is a key component of the apical (luminal) membrane of the MEC  forming a physical barrier against microbial attacks . Increased expression of the transglutaminases TGM1 and TGM3 indicates stabilization of tissues and cells by protein cross-linking . These factors contribute also to the formation of tight junctions between mammary epithelial cells and are known to promote cross linking of proteins in the extracellular matrix thereby initiating wound healing.
Priming reduces expression of master cytokines and of potentially harmful factors
It is known from macrophages that endotoxin tolerance (ET) down-regulates the expression of the inflammatory master cytokines tumor necrosis factor α (TNF-α), interleukin 1 (IL-1) and IL-6 (reviewed in ). They all belong to the class of "LPS-tolerant" genes which become refractory in macrophages to repeated LPS stimulation . We now made a similar observation in the MEC. LPS priming moderately, yet significantly reduces their mRNA levels after a subsequent E. coli challenge in these cells. These three factors together orchestrate and dominate many aspects of the local as well as systemic immune response [42, 43]. While their adequate induction through invading pathogens is indispensable for mounting a productive immune defence in the host, their overshooting expression may be detrimental. Confining their expression is therefore an overarching beneficial effect of ET and contributes to prevent immune-pathology. Our data suggest that this principle applies also to the regulation of the immune defence in the udder mediated through MEC. Moreover, we validated reduced and confined expression for some of their secondary response factors during the subsequent pathogen challenge. These factors include the nitric oxide synthase NOS2. This enzyme produces nitric oxide radicals, which are bactericidal but also harmful to the host cells and tissues as well. Similarly, expression of the metallopeptidases (MMP9, MMP13) was found to be confined by LPS priming. These proteases may disintegrate the extra-cellular matrix and connectives. LPS priming appears to prohibit their exuberant production and thus reduces activity of these potentially harmful factors.
Expression of many factors associated with cell death was found to be limited by LPS priming. These factors include the caspases CASP4 and -7, DAXX, CFLAR to name only some of them. Reduced and limited induction of these factors is conceivably a consequence of reduced TNF-α induction and indicates improved preservation of cell- and tissue-integrity.
Reduced and dampened induction of the TLR/NF-κB axis of pathogen signal transduction appears to be crucial for the ET mediated modulation of the immune response in the MEC. Not only was the pathogen stimulated expression of several TLRs reduced (TLR2, -3,-4) but also were factors induced by the LPS priming which are known to interfere with NF-κB activation, including the coronin CORO1A and SPLI. CORO1A is known to suppress TLR2, TLR3, TLR4 and TNF-α mediated NF-κB activation as well as IFN-β promoter activation . Indeed, we found that the mRNA expression of various type I interferon response factors was decreased by priming (e.g. myxovirus resistance 2 [MX2], receptor transporter protein 4 [RTP4], 2'-5'-oligoadenylate synthetase 1 [OAS1], interferon stimulated exonuclease gene 20 kDa [ISG20]). The factor SLPI has not only a broad spectrum of antimicrobial activities but also anti-inflammatory/immunomodulatory functions . SLPI interferes also with TLR-4 mediated NF-κB activation by inhibiting the interaction between CD14 and LPS  thus quenching the production of inflammatory factors like TNF-α, NOS2, COX2, and MMPs [47, 48].