Hepatic Transcriptome Analysis Identifies Divergent Pathogen-Specific Targeting-Strategies to Modulate the Innate Immune System in Response to Intramammary Infection.

Mastitis is one of the major risks for public health and animal welfare in the dairy industry. Two of the most important pathogens to cause mastitis in dairy cattle are Staphylococcus aureus (S. aureus) and Escherichia coli (E. coli). While S. aureus generally induces a chronic and subclinical mastitis, E. coli is an important etiological pathogen resulting in an acute and clinical mastitis. The liver plays a central role in both, the metabolic and inflammatory physiology of the dairy cow, which is particularly challenged in the early lactation due to high metabolic and immunological demands. In the current study, we challenged the mammary glands of Holstein cows with S. aureus or E. coli, respectively, mimicking an early lactation infection. We compared the animals' liver transcriptomes with those of untreated controls to investigate the hepatic response of the individuals. Both, S. aureus and E. coli elicited systemic effects on the host after intramammary challenge and seemed to use pathogen-specific targeting strategies to bypass the innate immune system. The most striking result of our study is that we demonstrate for the first time that S. aureus intramammary challenge causes an immune response beyond the original local site of the mastitis. We found that in the peripheral liver tissue defined biological pathways are switched on in a coordinated manner to balance the immune response in the entire organism. TGFB1 signaling plays a crucial role in this context. Important pathways involving actin and integrin, key components of the cytoskeleton, were downregulated in the liver of S. aureus infected cows. In the hepatic transcriptome of E. coli infected cows, important components of the complement system were significantly lower expressed compared to the control cows. Notably, while S. aureus inhibits the cell signaling by Rho GTPases in the liver, E. coli switches the complement system off. Also, metabolic hepatic pathways (e.g., lipid metabolism) are affected after mammary gland challenge, demonstrating that the liver restricts metabolic tasks in favor of the predominant immune response after infection. Our results provide new insights for the infection-induced modifications of the dairy cow's hepatic transcriptome following mastitis.

At Least Two Genes Encode Many Variants of Irak3 in Rainbow Trout, but Neither the Full-Length Factor Nor Its Variants Interfere Directly With the TLR-Mediated Stimulation of Inflammation.

The interleukin-1-receptor-associated kinase 3 (IRAK3) is known in mammals as a negative feedback regulator of NF-κB-mediated innate-immune mechanisms. Our RNA-seq experiments revealed a prototypic 1920-nt sequence encoding irak3 from rainbow trout (Oncorhynchus mykiss), as well as 20 variants that vary in length and nucleotide composition. Based on the DNA-sequence information from two closely related irak3 genes from rainbow trout and an irak3-sequence fragment from Atlantic salmon retrieved from public databases, we elucidated the underlying genetic causes for this striking irak3 diversity. Infecting rainbow trout with a lethal dose of Aeromonas salmonicida enhanced the expression of all variants in the liver, head kidney, and peripheral blood leucocytes. We analyzed the functional impact of the full-length factor and selected structural variants by overexpressing them in mammalian HEK-293 cells. The full-length factor enhanced the basal activity of NF-κB, but did not dampen the TLR2-signaling-induced levels of NF-κB activation. Increasing the basal NF-κB-activity through Irak3 apparently does not involve its C-terminal domain. However, more severely truncated factors had only a minor impact on the activity of NF-κB. The TLR2-mediated stimulation did not alter the spatial distribution of Irak3 inside the cells. In salmonid CHSE-214 cells, we observed that the Irak3-splice variant that prominently expresses the C-terminal domain significantly quenched the stimulation-dependent production of interleukin-1ß and interleukin-8, but not the production of other immune regulators. We conclude that the different gene and splice variants of Irak3 from trout play distinct roles in the activation of immune-regulatory mechanisms.

The first line of defence: insights into mechanisms and relevance of phagocytosis in epithelial cells.

Epithelial tissues cover most of the external and internal surfaces of the body and its organs. Inevitably, these tissues serve as first line of defence against inorganic, organic, and microbial intruders. Epithelial cells are the main cell type of these tissues. Besides their function as cellular barrier, there is growing evidence that epithelial cells are of particular relevance as initial sensors of danger and also as executers of adequate defence responses. These cells feature various essential functions to maintain tissue integrity in health and disease. In this review, we survey some of the different innate immune functions of epithelial cells in mucosal tissues being constantly exposed to a plethora of harmless contaminants but also of pathogens. We discuss how epithelial cells avoid inadequate immune responses in such conditions. In particular, we will focus on the diverse types and mechanisms of phagocytosis used by epithelial cells to not only maintain homeostasis but to also harness the host response against invading pathogens.

Pathogen-specific responses in the bovine udder. Models and immunoprophylactic concepts.

Bovine mastitis is a disease of major economic effects on the dairy industry worldwide. Experimental in vivo infection models have been widely proven as an effective tool for the investigation of pathogen-specific host immune responses. Staphylococcus aureus (S. aureus) and Escherichia coli (E. coli) are two common mastitis pathogens with an opposite clinical outcome of the disease. E. coli and S. aureus have proven to be valid surrogates to model clinical and subclinical mastitis respectively. Contemporary transcriptome profiling studies demonstrated that the transcriptomic response in the teat reflects the course of pathogen-specific mastitis, being ultimately determined by the immune response of the mammary epithelial cells. After an experimental in vivo challenge, E. coli induces a vigorous early transcriptional response in udder tissue being quantitatively and - notably - qualitatively distinct from the much weaker response against an S. aureus infection. E. coli mastitis models proved that the local response in the infected udder quarters is accompanied by a response in non-infected neighbouring udder quarters modulating systemically their immune responsiveness. Immunomodulation of the udder was investigated in animal models. Pathophysiological consequences were studied after intramammary administration of cytokines, chemokines, growth factors, steroidal anti-inflammatory drugs, or priming of tissue resident cells with pathogen-derived molecules. The latter approaches resulted only in a temporal protection of the udder, reducing transiently the risk of infection but sustained lowering of the severity of an eventually occurring mastitis. They offer an alternative to vaccination trials, which over decades also did not yield protection against new infections.

Differentiating Staphylococcus aureus from Escherichia coli mastitis: S. aureus triggers unbalanced immune-dampening and host cell invasion immediately after udder infection.

The etiology determines quality and extent of the immune response after udder infection (mastitis). Infections with Gram negative bacteria (e.g. Escherichia coli) will quickly elicit strong inflammation of the udder, fully activate its immune defence via pathogen receptor driven activation of IκB/NF-κB signaling. This often eradicates the pathogen. In contrast, Gram-positive bacteria (e.g. Staphylococcus aureus) will slowly elicit a much weaker inflammation and immune response, frequently resulting in chronic infections. However, it was unclear which immune regulatory pathways are specifically triggered by S. aureus causing this partial immune subversion. We therefore compared in first lactating cows the earliest (1-3 h) udder responses against infection with mastitis causing pathogens of either species. Global transcriptome profiling, bioinformatics analysis and experimental validation of key aspects revealed as S. aureus infection specific features the (i) failure to activating IκB/NF-κB signaling; (ii) activation of the wnt/ß-catenin cascade resulting in active suppression of NF-κB signaling and (iii) rearrangement of the actin-cytoskeleton through modulating Rho GTPase regulated pathways. This facilitates invasion of pathogens into host cells. Hence, S. aureus mastitis is characterized by eliciting unbalanced immune suppression rather than inflammation and invasion of S. aureus into the epithelial cells of the host causing sustained infection.

ST2 from rainbow trout quenches TLR signalling, localises at the nuclear membrane and allows the nuclear translocation of MYD88.

The mammalian interleukin 1 receptor-like 1 receptor (IL1RL1), commonly known as ST2, is thought to downregulate TLR signalling by sequestering the signalling adapter MYD88 (myeloid differentiation primary response protein 88). ST2 sequences are known in several fish species, but none of them have functionally been examined. We characterised ST2 from rainbow trout (Oncorhynchus mykiss) and the structure of its encoding gene. The primary sequence of ST2 is only weakly conserved from fish to human. However, the amino acid sequences forming the interfaces for ST2 and MYD88 interaction are well conserved throughout evolution. High similarity of the gene segmentation unambiguously proves the common ancestry of fish and mammalian ST2. Trout ST2 and trout MYD88 genes were constitutively expressed in embryonic, larval and adult trout. In vivo infection with Aeromonas salmonicida did not modulate the mRNA levels of both factors. Overexpressing trout ST2 in the mammalian HEK-293 reconstitution system of TLR2 signalling quenched the Escherichia coli-induced activation of NF-κB and SAA promoters in a dose-dependent fashion. The expression of GFP-tagged trout ST2 in human HEK-293 or trout CHSE-214 cells surprisingly revealed that (i) ST2 localised abundantly at the nuclear membrane rather than at the cell membrane and (ii) the coexpression of both ST2 and MYD88 allowed the translocation of trout MYD88 from cytoplasm to nucleus, as assessed using confocal microscopy and Western blotting. Hence, we validated that trout ST2 is a dampener of TLR signalling and interacts with MYD88. The spatial distribution of these factors raises questions about how this repressive mechanism functions.

TLR ligands, but not modulators of histone modifiers, can induce the complex immune response pattern of endotoxin tolerance in mammary epithelial cells.

Excessive stimulation of the TLR4 axis through LPS reduces the expression of some cytokine genes in immune cells, while stimulating the expression of immune defense genes during a subsequent bacterial infection. This endotoxin tolerance (ET) is mediated via epigenetic mechanisms. Priming the udder of cows with LPS was shown to induce ET in mammary epithelial cells (MEC), thereby protecting the udder against reinfection for some time. Seeking alternatives to LPS priming we tried to elicit ET by priming MEC with either lipopeptide (Pam2CSK4) via the TLR2/6 axis or inhibitors of histone-modifying enzymes. Pre-incubation of MEC with Pam2CSK4 enhanced baseline and induced expression of bactericidal (ß-defensin; SLPI) and membrane protecting factors ( SAA3, TGM3), while reducing the expression of cytokine- and chemokine-encoding genes ( TNF, IL1ß) after a subsequent pathogen challenge, the latter, however, not as efficiently as after LPS priming. Pre-treating MEC with various inhibitors of histone H3 modifiers (for demethylation, acetylation or deacetylation) all failed to induce any of the protective factors and only resulted in some dampening of cytokine gene expression after the re-challenge. Hence, triggering immune functions via the TLR axis, but not through those histone modifiers, induced the beneficial phenomenon of ET in MEC.

Bovine milk RNases modulate pro-inflammatory responses induced by nucleic acids in cultured immune and epithelial cells.

Activation of innate immune receptors by exogenous substances is crucial for the detection of microbial pathogens and a subsequent inflammatory response. The inflammatory response to microbial lipopolysaccharide via Toll-like receptor 4 (TLR4) is facilitated by soluble accessory proteins, but the role of such proteins in the activation of other pathogen recognition receptors for microbial nucleic acid is not well understood. Here we demonstrate that RNase4 and RNase5 purified from bovine milk bind to Salmonella typhimurium DNA and stimulate pro-inflammatory responses induced by nucleic acid mimetics and S. typhimurium DNA in an established mouse macrophage cell culture model, RAW264.7, as well as in primary bovine mammary epithelial cells. RNase4 and 5 also modulated pro-inflammatory signalling in response to nucleic acids in bovine peripheral blood mononuclear cells, although producing a distinct response. These results support a role for RNase4 and RNase5 in mediating inflammatory signals in both immune and epithelial cells, involving mechanisms that are cell-type specific.

Stearoyl-CoA desaturase 1 expression is downregulated in liver and udder during E. coli mastitis through enhanced expression of repressive C/EBP factors and reduced expression of the inducer SREBP1A.

BACKGROUND: Stearoyl-CoA desaturase 1 (SCD1) desaturates long chain fatty acids and is therefore a key enzyme in fat catabolism. Its synthesis is downregulated in liver during illnesses caused by high levels of circulating lipopolysaccharide (LPS). SCD1 expression is known to be stimulated under adipogenic conditions through a variety of transcription factors, notably SREBP1 and C/EBPα and -ß. However, mechanisms downregulating SCD1 expression during illness related reprograming of the metabolism were unknown. Escherichia coli elicited mastitis is an example of such a condition and was found to downregulates milk and milk fat synthesis. This is in part mediated through epigenetic mechanisms. We analyzed here mechanism controlling SCD1 expression in livers and udders from cows suffering from experimentally induced E. coli mastitis. RESULTS: We validated with RT-qPCR that SCD1 expression was reduced in these organs of the experimental cows. They also featured decreased levels of mRNAs encoding SREBP1a but increased levels for C/EBP α and -ß. Chromatin accessibility PCR (CHART) revealed that downregulation of SCD1 expression in liver was not caused by tighter chromatin compaction of the SCD1 promoter. Reporter gene analyses showed in liver (HepG2) and mammary epithelial (MAC-T) model cells that overexpression of SREBP1a expectedly activated the promoter, while unexpectedly C/EBPα and -ß strongly quenched the promoter activity. Abrogation of two from among of the three C/EBP DNA-binding motifs of the promoter revealed that C/EBPα acts in cis but C/EBPß in trans. Overexpressing truncated C/EBPα or -ß factors lacking their repressive domains confirmed in both model cells the direct action of C/EBPα, but not of C/EBPß on the promoter. CONCLUSIONS: We found no evidence that epigenetic mechanism remodeling the chromatin compaction of the SCD1 promoter would contribute to downregulate SCD1 expression during infection. Instead, our data show for the first time that C/EBP factors may repress SCD1 expression in liver and udder rather than stimulating as it was previously shown in adipocytes. This cell type specific dual and opposite function of C/EBP factors for regulating SCD1 expression was previously unknown. Infection related activation of their expression combined with downregulated expression of SREBP1a explains reduced SCD1 expression in liver and udder during acute mastitis.

Early transcriptional events in the udder and teat after intra-mammary Escherichia coli and Staphylococcus aureus challenge.

Intra-mammary bacterial infections can result in harmful clinical mastitis or subclinical mastitis with persistent infections. Research during the last decades closely examined the pathophysiology of inflamed udders. Initial events after pathogen perception but before the onset of mastitis have not been examined in vivo The objective of this study was to develop a mastitis model in cows by monitoring initial transcriptional pathogen-specific host response before clinical signs occur. We applied a short-term infection model to analyse transcripts encoding chemokines, cytokines and antimicrobial molecules in the teat cistern (TC) and lobulo-alveolar parenchyma (LP) up to 3 h after challenge with E and Staphylococcus aureus Both pathogens elicited an immune reaction by 1 h after challenge. Escherichia coli induced all analysed factors (CCL20, CXCL8, TNF, IL6, IL12B, IL10, LAP, S100A9); however, S. aureus failed to induce IL12B, IL10, LAP and S100A9 expression. The E. coli-induced up-regulation was 25-105 times greater than that after S. aureus challenge. Almost all the responses were restricted to the TC. The short-term mastitis model demonstrates that a divergent pathogen-specific response is generated during the first h. It confirms that the first transcripts are generated in the TC prior to a response in the LP.

Comparison of the pathogen species-specific immune response in udder derived cell types and their models.

The outcome of an udder infection (mastitis) largely depends on the species of the invading pathogen. Gram-negative pathogens, such as Escherichia coli often elicit acute clinical mastitis while Gram-positive pathogens, such as Staphylococcus aureus tend to cause milder subclinical inflammations. It is unclear which type of the immune competent cells residing in the udder governs the pathogen species-specific physiology of mastitis and which established cell lines might provide suitable models. We therefore profiled the pathogen species-specific immune response of different cell types derived from udder and blood. Primary cultures of bovine mammary epithelial cells (pbMEC), mammary derived fibroblasts (pbMFC), and bovine monocyte-derived macrophages (boMdM) were challenged with heat-killed E. coli, S. aureus and S. uberis mastitis pathogens and their immune response was scaled against the response of established models for MEC (bovine MAC-T) and macrophages (murine RAW 264.7). Only E. coli provoked a full scale immune reaction in pbMEC, fibroblasts and MAC-T cells, as indicated by induced cytokine and chemokine expression and NF-κB activation. Weak reactions were induced by S. aureus and none by S. uberis challenges. In contrast, both models for macrophages (boMdM and RAW 264.7) reacted strongly against all the three pathogens accompanied by strong activation of NF-κB factors. Hence, the established cell models MAC-T and RAW 264.7 properly reflected key aspects of the pathogen species-specific immune response of the respective parental cell type. Our data imply that the pathogen species-specific physiology of mastitis likely relates to the respective response of MEC rather to that of professional immune cells.

Streptococcus uberis strains isolated from the bovine mammary gland evade immune recognition by mammary epithelial cells, but not of macrophages.

Streptococcus uberis is frequently isolated from the mammary gland of dairy cattle. Infection with some strains can induce mild subclinical inflammation whilst others induce severe inflammation and clinical mastitis. We compared here the inflammatory response of primary cultures of bovine mammary epithelial cells (pbMEC) towards S. uberis strains collected from clinical or subclinical cases (seven strains each) of mastitis with the strong response elicited by Escherichia coli. Neither heat inactivated nor live S. uberis induced the expression of 10 key immune genes (including TNF, IL1B, IL6). The widely used virulent strain 0140J and the avirulent strain, EF20 elicited similar responses; as did mutants defective in capsule (hasA) or biofilm formation (sub0538 and sub0539). Streptococcus uberis failed to activate NF-κB in pbMEC or TLR2 in HEK293 cells, indicating that S. uberis particles did not induce any TLR-signaling in MEC. However, preparations of lipoteichoic acid (LTA) from two strains strongly induced immune gene expression and activated NF-κB in pbMEC, without the involvement of TLR2. The immune-stimulatory LTA must be arranged in the intact S. uberis such that it is unrecognizable by the relevant pathogen receptors of the MEC. The absence of immune recognition is specific for MEC, since the same S. uberis preparations strongly induced immune gene expression and NF-κB activity in the murine macrophage model cell RAW264.7. Hence, the sluggish immune response of MEC and not of professional immune cells to this pathogen may aid establishment of the often encountered belated and subclinical phenotype of S. uberis mastitis.

Structurally diverse genes encode Tlr2 in rainbow trout: The conserved receptor cannot be stimulated by classical ligands to activate NF-κB in vitro.

The mammalian toll-like receptor 2 (TLR2) is a dominant receptor for the recognition of Gram-positive bacteria. Its structure and functional properties were unknown in salmonid fish. In RT-PCR and RACE experiments, we obtained the full-length cDNA sequence encoding Tlr2 from rainbow trout (Oncorhynchus mykiss) as well as a copy of an unspliced nonsense message from a highly segmented gene. The primary structure of the encoded receptor complies with the domain structure and ligand-binding sites known from mammals and other fish species and sorts well into the evolutionary tree of teleostean Tlr2s. We retrieved a gene version encoding the receptor on a single exon (tlr2a) and also a partial sequence of a second gene variant being segmented into multiple exons (tlr2b). Surprisingly, the abundances of both transcript variants accounted only for ∼10% of all Tlr2-encoding transcripts in various tissues and cell types of healthy fish. This suggests the expression of several distinct tlr2 gene variants in rainbow trout. We expressed tlr2a in HEK-293 cells, but were unable to demonstrate its functionality through NF-κB activation. Neither synthetic lipopeptides known to stimulate mammalian TLR2 nor different bacterial challenges induced OmTLR2-mediated NF-κB activation, not in HEK-293 or in salmon CHSE-214 cells. Positive demonstration of TLR2-MYD88 interaction excluded that its functional impairment caused the failure of NF-κB activation. We discuss impaired heterodimerization with a necessary Tlr partner as one from among several alternatives to explain the dysfunction of Tlr2a in the interspecies reconstitution system of TLR signaling.

Hepatic TLR4 signaling is activated by LPS from digestive tract during SARA, and epigenetic mechanisms contribute to enforced TLR4 expression.

Subacute ruminal acidosis (SARA) is known to trigger a systemic inflammatory response that is possibly caused by the translocation of lipopolysaccharides (LPS) from the gastrointestinal tract into the bloodstream. The aim of this study is to investigate this causal relationship between the increases of circulating LPS and liver inflammation. Here we found that SARA goats exhibited significantly increased LPS concentrations in both the rumen and portal vein. The livers of these goats exhibited increased mRNA concentrations of pro-inflammatory genes that indicated inflammation. Meanwhile, the occurrence of liver inflammation was further validated by the enhanced protein expression of those cytokines in the livers of SARA goats. These increased expressions of detected pro-inflammatory genes were likely mediated by enforced TLR4 signaling because SARA increased the concentrations of TLR4 mRNA and protein in the liver and the abundance of both the NF-kB-p65 factor and its active phosphorylated variant. We also verified that the enhanced TLR4 expression was accompanied by chromatin decompaction and demethylation of the proximal TLR4 promoter. Hence, epigenetic mechanisms are involved in the enforced expression of immune genes during SARA, and these findings open innovative routes for interventions via the modulation of these epigenetic mechanisms.

Three promoters with different tissue specificity and pathogen inducibility express the toll-like-receptor 2 (TLR2)-encoding gene in cattle.

Toll-like-receptor 2 (TLR2) is a dominant receptor for perceiving presence of bacterial pathogens. The promoter controlling its tissue specific and infection induced expression in cattle was unknown. We structurally defined with 5'-RACE experiments three promoters (P1-3) controlling TLR2 expression in udder, liver and other tissues of cows suffering from E. coli mastitis. P1 is 5'-adjacent to exon 1 as defined by the prototypical TLR2 cDNA sequence. Exon 1 is spliced to the protein-encoding exon 2. P2 and P3 reside in intron 1, express exon 1A and exon 1B, respectively which are each spliced to exon 2. Infection induced massively (>30-fold) activity of P1 and P2, but not of P3 in udders and also somewhat in liver. However, the GC-rich housekeeping promoter P3 expressed exon1B in many tissues providing the wealth of TLR2-encoding transcripts. Similar induction data were obtained after challenging primary cultures of mammary epithelial cells (pbMEC) with E. coli. Reporter gene analyses in pbMEC and the liver cell line HepG2 collectively validated that P1 and constructs containing segments from P2/P3 are in principle capable to drive gene expression. Our structural data provide the basis for more detailed molecular analyses of the infection and tissue specific regulation of TLR2 expression.

Aeromonas salmonicida Infection Only Moderately Regulates Expression of Factors Contributing to Toll-Like Receptor Signaling but Massively Activates the Cellular and Humoral Branches of Innate Immunity in Rainbow Trout (Oncorhynchus mykiss).

Toll-like receptors (TLRs) are known to detect a defined spectrum of microbial structures. However, the knowledge about the specificity of teleost Tlr factors for distinct pathogens is limited so far. We measured baseline expression profiles of 18 tlr genes and associated signaling factors in four immune-relevant tissues of rainbow trout Oncorhynchus mykiss. Intraperitoneal injection of a lethal dose of Aeromonas salmonicida subsp. salmonicida induced highly increased levels of cytokine mRNAs during a 72-hour postinfection (hpi) period. In contrast, only the fish-specific tlr22a2 and the downstream factor irak1 featured clearly increased transcript levels, while the mRNA concentrations of many other tlr genes decreased. Flow cytometry quantified cell trafficking after infection indicating a dramatic influx of myeloid cells into the peritoneum and a belated low level immigration of lymphoid cells. T and B lymphocytes were differentiated with RT-qPCR revealing that B lymphocytes emigrated from and T lymphocytes immigrated into head kidney. In conclusion, no specific TLR can be singled out as a dominant receptor for A. salmonicida. The recruitment of cellular factors of innate immunity rather than induced expression of pathogen receptors is hence of key importance for mounting a first immune defense against invading A. salmonicida.

Extracellular milieu grossly alters pathogen-specific immune response of mammary epithelial cells.

BACKGROUND: Considerably divergent data have been published from attempts to model the E. coli vs. S. aureus specific immune reaction of the udder using primary cultures of bovine mammary epithelial cells from cows (pbMEC). Some groups reported a swift, strong and transient inflammatory response against challenges with E. coli and only a weak and retarded response against S. aureus, in agreement with the respective reaction of the udder. Others found almost the reverse. Presence or absence of fetal calf serum distinguished the experimental setting between both groups. We examined here if this causes the divergent reaction of the pbMEC towards both pathogen species. We challenged pbMEC with proteins from heat killed E. coli or S. aureus pathogens or purified TLR2 and TLR4 ligands. The stimuli were applied in normal growth medium with (SM10) or without (SM0) 10% fetal calf serum, or in the basal medium supplemented with 10 mg/ml milk proteins (SM Milk). RESULTS: Withdrawal of FCS slowed down and decreased the extent by which E. coli or LPS enhanced the expression of cyto- and chemokine encoding genes through impaired TLR4 signalling but enforced their expression during stimulation with S. aureus. SM Milk strongly quenched the induction of those genes. S. aureus strain specific differences in the reaction of the pbMEC could only be recorded in SM0. NF-κB factors were activated by E. coli in all stimulation media, but only to a small extent by S. aureus, solely in SM0. Purified ligands for TLR2 stimulated expression of those genes and activated NF-κB equally well in SM10 and SM0. The mRNA destabilizing factor tristetraproline was only induced by E. coli in SM10 and by purified PAMPs. CONCLUSIONS: Our data cross validate the correctness of previously published divergent data on the pathogen-specific induction of key immune genes in pbMEC. The differences are due to the presence of FCS, modulating signalling through TLR4 and TLR-unrelated pathogen receptors. S. aureus does not substantially activate any TLR signalling in MEC. Rather, receptors distinct from TLRs perceive the presence of S. aureus and control the immune response against this pathogen in MEC.

Feeding a high-grain diet reduces the percentage of LPS clearance and enhances immune gene expression in goat liver.

BACKGROUND: The effects of feeding a high-grain (HG) diet on lipopolysaccharide (LPS) clearance and innate immune defence responses in the liver remain unclear. Therefore, we conducted the present study in which twelve female goats were randomly assigned to either a treatment group fed a HG diet (60% grain, n = 6) or a control group fed a low grain diet (LG; 40% grain, n = 6) for 6 weeks. Catheters were installed in the mesenteric, portal and hepatic veins, as well as one femoral artery of the goats, for determining blood flow and net clearance rate of LPS in the liver. Plasma and tissue samples were collected in the week 6 for analyzing pro-inflammatory cytokines, acute phase protein and biochemical parameters, as well as expression of genes involved in immune response. RESULT: HG diet feeding increased blood flow and LPS concentration in the portal vein, hepatic vein and artery. Hepatic net LPS clearance showed that HG diet feeding elevated the rate of hepatic LPS clearance, but decreased the percentage of removed LPS accounting for the total entry of LPS into the liver. Our results demonstrated that the feeding of HG diet increased plasma concentrations of pro-inflammatory cytokines and acute phase proteins and triggered a systemic inflammatory response. In addition, peripheral blood plasma concentrations of alanine aminotransferase, alkaline phosphatase and total bilirubin were increased in the HG group compared to the LG group. This indicated that the impairment of hepatocytes occurred after 6 weeks of HG diet feeding. The expression of genes involved in immune response and Toll-like receptor (TLR)4 protein in the liver was up-regulated in the HG group compared to the LG group, indicating that increased entry of LPS enhanced hepatic immune defence responses and contributed to hepatic inflammatory responses. CONCLUSION: These results provide insight into the capacity of the liver to clear LPS. The increased entry of LPS into liver enhanced hepatic immune defence responses, thereby elevated the rate of LPS clearance. However, the reduction of the percentage of hepatic LPS clearance could be due to the formation of hepatocyte lesion during HG diet feeding.

Epigenetic mechanisms contribute to enhanced expression of immune response genes in the liver of cows after experimentally induced Escherichia coli mastitis.

Endotoxins, such as lipopolysaccharide (LPS), are released during infection with Gram-negative bacteria, which can result in excessive activation of toll-like receptor (TLR) signalling. The aim of the present study was to investigate whether epigenetic mechanisms are involved in controlling the onset and progression of the systemic inflammatory response. Using chromatin accessibility by real-time (CHART) PCR to assess livers from cows with experimentally induced Escherichia coli mastitis, this study demonstrated that the chromatin at the site of the promoters of the genes encoding TLR2, TLR4, lipopolysaccharide binding protein (LBP) and haptoglobin (HP) was opened up 24 h after infection, accompanied by enhanced mRNA expression by these genes. Such modulation did not occur in the same samples for the αS1-casein promoter, which served as a negative control. Demethylation of the TLR4 promoter accompanied opening up of chromatin. These data suggest that modulation of epigenetic factors might offer a novel approach to treating adverse systemic reactions elicited in cows with E. coli mastitis.

Two TIR-like domain containing proteins in a newly emerging zoonotic Staphylococcus aureus strain sequence type 398 are potential virulence factors by impacting on the host innate immune response.

Staphylococcus aureus, sequence type (ST) 398, is an emerging pathogen and the leading cause of livestock-associated methicillin-resistant S. aureus infections in Europe and North America. This strain is characterized by high promiscuity in terms of host-species and also lacks several traditional S. aureus virulence factors. This does not, however, explain the apparent ease with which it crosses species-barriers. Recently, TIR-domain containing proteins (Tcps) which inhibit the innate immune response were identified in some Gram-negative bacteria. Here we report the presence of two proteins, S. aureus TIR-like Protein 1 (SaTlp1) and S. aureus TIR-like Protein 2 (SaTlp2), expressed by ST398 which contain domain of unknown function 1863 (DUF1863), similar to the Toll/IL-1 receptor (TIR) domain. In contrast to the Tcps in Gram-negative bacteria, our data suggest that SaTlp1 and SaTlp2 increase activation of the transcription factor NF-κB as well as downstream pro-inflammatory cytokines and immune effectors. To assess the role of both proteins as potential virulence factors knock-out mutants were created. These showed a slightly enhanced survival rate in a murine infectious model compared to the wild-type strain at one dose. Our data suggest that both proteins may act as factors contributing to the enhanced ability of ST398 to cross species-barriers.