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.

Detection of clonal antigen receptor gene rearrangement in dogs with lymphoma by real-time polymerase chain reaction and melting curve analysis.

BACKGROUND: Molecular techniques that detect canine lymphoma cells by their clonal antigen receptor gene rearrangement play an increasing role for diagnosis as well as for monitoring minimal residual disease during and after cytostatic therapy. However, the methods currently available are time-consuming and/or cost-intensive thus impeding the use in clinical routine. The aim of the present study was to develop and evaluate a real-time polymerase chain reaction (PCR) with subsequent melting curve analysis (MCA) for the detection of clonally rearranged antigen receptor genes in dogs with B and T cell lymphoma on non formalin-fixed and paraffin-embedded lymph node samples. RESULTS: In lymph node aspirates from 30 dogs with multicentric B cell lymphoma, real-time PCR with MCA detected clonal rearrangement in 100% and conventional PCR with polyacrylamide gel electrophoresis (PAGE) in 93% of samples. Both methods correctly identified clonality in 80% of lymph node aspirates of 10 dogs with T cell lymphoma. None of the two PCR systems detected clonal rearrangement in samples from 9 dogs with lymph node hyperplasia. Using a dilutional series with regular lymphoid desoxyribonucleic acid (DNA), detection limits of lymphoma DNA were as low as 0.8% and 6.25% for B and T cell clonal rearrangement with real-time PCR and MCA and at 3.13% and 12.5% with the conventional system. Median absolute detection limits of lymphoma DNA were shown to be at 0.1 ng and 1 ng for the B and T cell immunophenotype with the real-time PCR system and at 10 ng each with conventional PCR and PAGE. CONCLUSIONS: Real-time PCR with MCA is a convenient and reliable method with a good analytical sensitivity. Thus, the method may assist the detection of clonal antigen receptor gene rearrangement in canine lymphoma patients in a clinical setting also in the presence of small amounts of neoplastic cells.

Loss of connexin43 in murine Sertoli cells and its effect on blood-testis barrier formation and dynamics.

Connexin43 (Cx43) is the predominant testicular gap junction protein and in cases of impaired spermatogenesis, Cx43 expression has been shown to be altered in several mammals. Amongst other functions, Cx43 is supposed to regulate junction formation of the blood-testis barrier (BTB). The aim of the present study was to investigate the expression pattern of different tight junction (TJ) proteins of the murine BTB using SC-specific Cx43 knockout mice (SCCx43KO). Adult homozygous male SCCx43KO mice (SCCx43KO-/-) predominantly show an arrest of spermatogenesis and SC-only tubules that might have been caused by an altered BTB assembly, composition or regulation. TJ molecules claudin-3, -5 and -11 were examined in adult wild type (WT) and SCCx43KO-/- mice using immunohistochemistry (IHC) and quantitative real-time PCR (qRT-PCR). In this context, investigation of single tubules with residual spermatogenesis in SCCx43KO-/- mice was particularly interesting to identify a potential Cx43-independent influence of germ cells (GC) on BTB composition and dynamics. In tubules without residual spermatogenesis, a diffuse cytoplasmic distribution pattern for claudin-11 protein could be demonstrated in mutant mice. Nevertheless, claudin-11 seems to form functional TJ. Claudin-3 and -5 could not be detected immunohistochemically in the seminiferous epithelium of those tubules. Correspondingly, claudin-3 and -5 mRNA expression was decreased, providing evidence of generally impaired BTB dynamics in adult KO mice. Observations of tubules with residual spermatogenesis suggested a Cx43-independent regulation of TJ proteins by GC populations. To determine initial BTB formation in peripubertal SCCx43KO-/- mice, immunohistochemical staining and qRT-PCR of claudin-11 were carried out in adolescent SCCx43KO-/- and WT mice. Additionally, BTB integrity was functionally analysed using a hypertonic glucose fixative. These analyses revealed that SCCx43KO-/- mice formed an intact BTB during puberty in the same time period as WT mice, which however seemed to be accelerated.

Neutrophil degranulation differentially modulates phenotype and function of bovine monocyte subsets.

Monocytes and neutrophils are important players in the innate immune response and cooperate during infection and inflammation. In our study we analyzed the effects of neutrophil degranulation products (polymorphonuclear granulocytes degranulation products, PMN-DGP) on the activation, the adhesion and the migration of three bovine monocyte subsets, as well as their effects on monocyte-macrophage differentiation. Cross-linking of surface CD18 molecules on bovine PMN resulted in the release of primary, secondary and tertiary granules as well as of secretory vesicles. PMN-DGP induced a significant Ca2+-influx in classical (classical monocytes, cM) and intermediate monocytes (intermediate monocytes, intM) but not in non-classical monocytes (non-classical monocytes, ncM). A selective and up-regulated expression induced by PMN-DGP was only seen for CD11a and CD31 on intM. PMN-DGP induced a selective migration of intM in vitro. The presence of PMN-DGP during the differentiation of cM or intM into macrophages resulted in increased expression of membrane CD163 and reduced expression of MHC-II molecules. PMN-DGP-derived macrophages produced more IL-12 and IL-10 and showed enhanced phagocytosis and ROS production capacities. In conclusion, PMN-DGP selectively attract bovine intM and skew the functional maturation of cM and intM.

The chemokine CCL5 induces selective migration of bovine classical monocytes and drives their differentiation into LPS-hyporesponsive macrophages in vitro.

Human and mouse studies indicate distinct roles of selected chemokines for monocyte subset attraction. We therefore analyzed the still unknown sensitivity and response of bovine monocyte subsets toward two monocyte-attracting chemokines (CCL2, CCL5). Only CCL5 induced a significant Ca(2+)influx and migration response in bovine monocytes, with classical and intermediate monocytes being significantly stimulated and attracted compared to nonclassical monocytes. The presence of CCL5 during in vitro macrophage differentiation did not alter their capacity to phagocytize or to generate reactive oxygen species upon stimulation with E. coli. However, macrophages differentiated in the presence of CCL5 displayed an altered phenotype with significantly less expressed CD14 and MHC class II molecules, whereas CD16 was upregulated. Moreover, CCL5-differentiated macrophages displayed a reduced upregulation of CXCL8, ARG1, IL6 and IL10 mRNA. Taken together, CCL5 but not CCL2 mainly attract bovine classical monocytes and promote their differentiation into LPS-hypo-responsive macrophages.

Peripheral blood leukocytes of cows with subclinical endometritis show an altered cellular composition and gene expression.

Subclinical endometritis (SCE) is an important postpartum disease in dairy cows, but conventional cytobrush diagnosis often gives imprecise results. The aim of this study was to analyze disease-associated changes in peripheral blood as potential diagnostic parameters. Cellular subpopulations of blood leukocytes from cows with or without SCE (45-55 days postpartum) were flow-cytometrically quantified. Gene expression of whole blood leukocytes was assessed by PAXgene analysis. Subclinical endometritis cows showed significantly higher number of blood mononuclear cells and neutrophils. Among mononuclear cells, numbers of B-cells, NK-cells, and CD172a-positive monocytes were significantly elevated. Compared with non-SCE cows, blood leukocytes of SCE cows significantly expressed higher copy numbers of CXCL8, TNF, and IL12. To test whether circulating plasma factors are responsible for these changes, leukocytes, polymorphonuclear cells, and monocyte subpopulations (classical, intermediate, nonclassical) of healthy cows were stimulated with plasma of SCE and non-SCE cows. Although gene expression of whole leukocytes and polymorphonuclear cells remained unaltered, plasma from SCE animals significantly elevated expressed messenger RNA copy numbers of CXCL8, CXCL1, and IL1B in intermediate monocytes. In conclusion, elevated number of selected mononuclear subpopulations in peripheral blood and enhanced expression of distinct genes encoding for inflammatory mediators in blood leukocytes reflect the subclinical uterine inflammatory process in cows. Whether the observed changes in the periphery of SCE cows are the consequence of the uterine inflammatory process, or whether they affect the pathogenesis of the disease is currently unknown.

Recombinant bovine S100A8 and A9 enhance IL-1ß secretion of interferon-gamma primed monocytes.

Calgranulin A (S100A8) and B (S100A9) are found at high levels in inflamed tissue and have been associated with acute and chronic inflammatory disorders. Calgranulins are discussed as damage-associated molecular patterns (DAMPs). To analyze the role of calgranulins for inflammatory responses, bovine S100A8 and S100A9 were cloned, successfully expressed and FPLC-purified. Both molecules did not induce NF-κB activation in boTLR4-transfected HEK293 cells and stimulation of bovine monocytes with both proteins did not result in interleukin 1ß (IL-1ß) secretion or an upregulated mRNA expression of selected genes (IL1B, TNF, CXCL8, IL10, IL12). However, Interferon γ (IFN-γ) primed bovine monocytes released significantly higher amounts of IL-1ß after stimulation with S100A8, S100A9, and co-stimulation with adenosine triphosphate (ATP). In IL-4/IL-13-primed monocytes, the IL-1ß release was completely abrogated. The results imply that TLR4/MyD88/NF-κB-independent S100A8/A9-mediated activation of the inflammasome in cattle is favored in a Th1 environment and that S100A8 and S100A9 act as a DAMP in cattle.

Inflammasome activation in bovine monocytes by extracellular ATP does not require the purinergic receptor P2X7.

Extracellular adenosine triphosphate (ATP) is a second signal for the assembly of the NLR family, pyrin domain-containing 3 (NLRP3) inflammasome, which form a framework to activate caspase 1, leading to the processing and secretion of the pro-inflammatory cytokine interleukin-1ß (IL-1ß). The aim of the present study was to investigate the role of the ATP-gated ion channel subtype P2X7 receptor in the inflammasome activation of bovine monocytes. ATP-induced inflammasome assembly in bovine monocytes was shown by caspase-1 activation and the release of IL-1ß by LPS/ATP-stimulated bovine cells. The IL-1ß release depended on potassium efflux but was independent of reactive oxygen generation of bovine monocytes. Unlike in the human system, a P2X7 receptor antagonist did not block the ATP-induced release of IL-1ß of LPS-primed bovine cells. P2X7 mediated pore formation was observed in subsets of bovine T lymphocytes (CD4+>CD8+) but not in monocytes. In addition, ATP and 2-MeSATP but not the high affinity P2X7 agonist BzATP induced calcium influx in bovine monocytes. The data indicate that ROS generation plays no role in the ATP-induced activation of inflammasome in bovine monocytes and that P2X7-mediated pore formation is not necessary for the release of Interleukin-1ß.