Inflammatory mechanisms in bacterial infections: focus on mast cells and mastitis

Inflammation is an unspecific response of the immune system to pathogens, for example, invasion by bacteria. This thesis focuses on two aspects of inflammation in the context of bacterial infections: (1) mast cells and (2) mastitis. Mast cells are potent proinflammatory leucocytes that have been implicated in the defence against bacterial infections. Mastitis is an inflammation of the mammary tissue and is one of the most economically destructive disease in the dairy industry worldwide. Here, mast cell synthesis of the potent pro-angiogenic vascular endothelial growth factor (VEGF) in response to stimuli with Staphylococcus aureus (S. aureus) was studied using an in vitro model of primary mouse mast cells. VEGF synthesis was found to be dependent on the presence of live whole bacteria. Previous in vivo investigations of the roles of mast cells in bacterial infections have been conducted using c-Kit-dependent mast cell-deficient mice. These mice suffer from numerous abnormalities in addition to the lack of mast cells. Instead, we used newer, cKit-independent mast cell-deficient mice (Mcpt5-Cre), which have fewer non-mast cell related abnormalities. We found no impact of the mast cell deficiency on the course of intraperitoneal S. aureus infection (e.g., bacterial clearance and cytokine production). We differentiated the virulence of, and response to, a set of clinical bacterial strains of bovine mastitis origin. Escherichia coli (E. coli) and S. aureus strains were injected intraperitoneally into mice. One E. coli strain(strain 127) was found to consistently cause more severe infection (judged by a clinical score) and induce a distinct profile of cytokines (CXCL1, G-CSF, CCL2). The concentrations of these cytokines correlated with both the clinical score and bacterial burden. The kinetics of the clinical and molecular changes that occurred during acute bovine mastitis were studied using a bovine in vivo model in which mastitis was induced by an intramammary infusion of E. coli lipopolysaccharide. Changes in clinical parameters (clinical score, milk changes, rectal temperature) as well as in milk and plasma cytokine concentrations and changes in the metabolome were registered. The progression of these changes occurred in the following order: (1) signs of inflammation in the udder and an increase in milk cytokine concentrations (after/at two hours), (2) visible changes in the milk and an increase in milk somatic cell counts (SCCs) (four hours), (3) changes in the plasma metabolome(four hours) and (4) changes in the milk metabolome (24 hours)

Troubleshooting the GFP-tagging gene knockout (GGKO) method for the Leptosphaeria maculans effectors AvrLm6 and AvrLm4-7

An attempt was made to GFP-tag the effector proteins of AvrLm6 and AvrLm4-7 using the GFP-tagging gene knockout method (GGKO) developed by Saitoh et al. (2008) in order to determine whether or not they are secreted. Successful pETHG-(target)KO vectors were not generated. The protocol was examined for potential errors. Fatal errors were pinpointed to the ligation reaction and the transformation required to generate and propagate the desired vector pETGH-(target)KO. The Downstreams Flanking Region inserts were evidently successfully ligated into the pETHG vector but for the Upstreams Flanking Region inserts the results were highly ambiguous. Due to a mistake, too high vector:insert ratios were used; altering them to the recommended 1:1 – 1:5 is hence expedient. It was reasoned that the bacteria possibly absorbed empty pETHG vectors instead of putative insert-carrying pETHG-(target)KO vectors during the transformation. The procedure could be improved by digesting pETHG twice prior to ligation as well by separating linearised and uncleaved pETHG by gel extraction. The latter could also be performed on the ligation product. Suggested general improvements include: Sequencing the PCR product and purifying it of potential restriction enzyme inhibitors, use the maximal incubation time for the restriction enzymes, expand the colony screening, increase the spectinomycin concentration and test different bacterial strains in the transformation.Ett försök gjordes att GFP-markera AvrLm6 och AvrLm4-7 proteinerna via ”GFP-tagging gene knockout” (GGKO) vektor systemet som utvecklats av Saitoh et al. (2008). Syftet var att utröna om genprodukterna från dessa gener återfinns inne i värdväxten under infektionens gång. Inga pETHG-(target)KO vektorer genererades under projektets gång. Via analys av resultat från rutinmässiga kontroller i experimentet samt tester av vissa steg begränsades de potentiella kloningsproblemen till transformeringsteget med viss tvekan med avseende på om uppströmsflankerande regionen ligerades till pETHG eller ej. På grund av ett misstag användes fel vektor:insert förhållande i ligeringsreaktionen. De rekommenderade 1:1 – 1:5 förhållandena bör således användas. För att förhindra den potentiella upptagningen av tomma pETHG under transformeringen, kan pETHG behandlas två gånger med restriktionsenzymen därtill kan linjära vektorer separeras från obehandlade vektorer via gel extraktion. Det senare kan även utföras på ligeringsprodukten. Allmänna förbättringar innefattar: Sekvensera PCR produkten samt avlägsna potentiella restriktionsenzym inhibitorer, använda den maximala inkubationstiden för restriktionsenzym, utöka koloni screeningen, höja spektinomycin koncentrationen samt testa olika bakteriestammar för transformeringen

Absence of changes in the milk microbiota during Escherichia coli endotoxin induced experimental bovine mastitis

Changes in the milk microbiota during the course of mastitis are due to the nature of a sporadic occurring disease difficult to study. In this study we experimentally induced mastitis by infusion of Escherichia coli endotoxins in one udder quarter each of nine healthy lactating dairy cows and assessed the bacteriological dynamics and the milk microbiota at four time points before and eight time points after infusion. As control, saline was infused in one udder quarter each of additionally nine healthy cows that followed the same sampling protocol. The milk microbiota was assessed by sequencing of the 16 S rRNA gene and a range of positive and negative controls were included for methodological evaluation. Two different data filtration models were used to identify and cure data from contaminating taxa. Endotoxin infused quarters responded with transient clinical signs of inflammation and increased SCC while no response was observed in the control cows. In the milk microbiota data no response to inflammation was identified. The data analysis of the milk microbiota was largely hampered by laboratory and reagent contamination. Application of the filtration models caused a marked reduction in data but did not reveal any associations with the inflammatory reaction. Our results indicate that the microbiota in milk from healthy cows is unaffected by inflammation

Mastitis Pathogens with high Virulence in a Mouse Model Produce a Distinct cytokine Profile In Vivo

Mastitis is a serious medical condition of dairy cattle. Here, we evaluated whether the degree of virulence of mastitis pathogens in a mouse model can be linked to the inflammatory response that they provoke. Clinical isolates of Staphylococcus aureus (S. aureus) (strain 556 and 392) and Escherichia coli (E. coli) (676 and 127), and laboratory control strains [8325-4 (S. aureus) and MG1655 (E. coli)], were injected i.p. into mice, followed by the assessment of clinical scores and inflammatory parameters. As judged by clinical scoring, E. coli 127 exhibited the largest degree of virulence among the strains. All bacterial strains induced neutrophil recruitment. However, whereas E. coli 127 induced high peritoneal levels of CXCL1, G-CSF, and CCL2, strikingly lower levels of these were induced by the less virulent bacterial strains. High concentrations of these compounds were also seen in blood samples taken from animals infected with E. coli 127, suggesting systemic inflammation. Moreover, the levels of CXCL1 and G-CSF, both in the peritoneal fluid and in plasma, correlated with clinical score. Together, these findings suggest that highly virulent clinical mastitis isolates produce a distinct cytokine profile that shows a close correlation with the severity of the bacterial infection

Absence of changes in the milk microbiota during Escherichia coli endotoxin induced experimental bovine mastitis

Changes in the milk microbiota during the course of mastitis are due to the nature of a sporadic occurring disease difficult to study. In this study we experimentally induced mastitis by infusion of Escherichia coli endotoxins in one udder quarter each of nine healthy lactating dairy cows and assessed the bacteriological dynamics and the milk microbiota at four time points before and eight time points after infusion. As control, saline was infused in one udder quarter each of additionally nine healthy cows that followed the same sampling protocol. The milk microbiota was assessed by sequencing of the 16 S rRNA gene and a range of positive and negative controls were included for methodological evaluation. Two different data filtration models were used to identify and cure data from contaminating taxa. Endotoxin infused quarters responded with transient clinical signs of inflammation and increased SCC while no response was observed in the control cows. In the milk microbiota data no response to inflammation was identified. The data analysis of the milk microbiota was largely hampered by laboratory and reagent contamination. Application of the filtration models caused a marked reduction in data but did not reveal any associations with the inflammatory reaction. Our results indicate that the microbiota in milk from healthy cows is unaffected by inflammation

The Effect of Lipopolysaccharide-Induced Experimental Bovine Mastitis on Clinical Parameters, Inflammatory Markers, and the Metabolome : A Kinetic Approach

Mastitis is an inflammatory condition of the mammary tissue and represents a major problem for the dairy industry worldwide. The present study was undertaken to study how experimentally induced acute bovine mastitis affects inflammatory parameters and changes in the metabolome. To this end, we induced experimental mastitis in nine cows by intramammary infusion of 100 pg purified Escherichia call lipopolysaccharide (LPS) followed by kinetic assessments of cytokine responses (by enzyme-linked immunosorbent assay), changes in the metabolome (assessed by nuclear magnetic resonance), clinical parameters (heat, local pain perception, redness, swelling, rectal temperature, clot formation, and color changes in the milk), and milk somatic cell counts, at several time points post LPS infusion. Intramammary LPS infusion induced clinical signs of mastitis, which started from 2 h post infusion and had returned to normal levels within 24-72 h. Milk changes were seen with a delay compared with the clinical signs and persisted for a longer time. In parallel, induction of IL-6 and TNF-alpha were seen in milk, and there was also a transient elevation of plasma IL-6 whereas plasma TNF-alpha was not significantly elevated. In addition, a robust increase in CCL2 was seen in the milk of LPS-infused cows, whereas G-CSF, CXCL1, and histamine in milk were unaffected. By using a metabolomics approach, a transient increase of plasma lactose was seen in LPS-induced cows. In plasma, significant reductions in ketone bodies (3-hydroxybutyrate and acetoacetate) and decreased levels of short-chain fatty acids, known to be major products released from the gut microbiota, were observed after LPS infusion; a profound reduction of plasma citrate was also seen. Intramammary LPS infusion also caused major changes in the milk metabolome, although with a delay in comparison with plasma, including a reduction of lactose. We conclude that the LPS-induced acute mastitis rapidly affects the plasma metabolome and cytokine induction with similar kinetics as the development of the clinical signs, whereas the corresponding effects in milk occurred with a delay

Mast cells are activated by Staphylococcus aureus in vitro but do not influence the outcome of intraperitoneal S. aureus infection in vivo

Staphylococcus aureus is a major pathogen that can cause a broad spectrum of serious infections including skin infections, pneumonia and sepsis. Peritoneal mast cells have been implicated in the host response towards various bacterial insults and to provide mechanistic insight into the role of mast cells in intraperitoneal bacterial infection we here studied the global effects of S. aureus on mast cell gene expression. After co-culture of peritoneal mast cells with live S. aureus we found by gene array analysis that they up-regulate a number of genes. Many of these corresponded to pro-inflammatory cytokines, including interleukin-3, interleukin-13 and tumour necrosis factor-α. The cytokine induction in response to S. aureus was confirmed by ELISA. To study the role of peritoneal mast cells during in vivo infection with S. aureus we used newly developed Mcpt5-Cre(+) × R-DTA mice in which mast cell deficiency is independent of c-Kit. This is in contrast to previous studies in which an impact of mast cells on bacterial infection has been proposed based on the use of mice whose mast cell deficiency is a consequence of defective c-Kit signalling. Staphylococcus aureus was injected intraperitoneally into mast-cell-deficient Mcpt5-Cre(+) × R-DTA mice using littermate mast-cell-sufficient mice as controls. We did not observe any difference between mast-cell-deficient and control mice with regard to weight loss, bacterial clearance, inflammation or cytokine production. We conclude that, despite peritoneal mast cells being activated by S. aureus in vitro, they do not influence the in vivo manifestations of intraperitoneal S. aureus infection

Table_1_The Effect of Lipopolysaccharide-Induced Experimental Bovine Mastitis on Clinical Parameters, Inflammatory Markers, and the Metabolome: A Kinetic Approach.DOCX

<p>Mastitis is an inflammatory condition of the mammary tissue and represents a major problem for the dairy industry worldwide. The present study was undertaken to study how experimentally induced acute bovine mastitis affects inflammatory parameters and changes in the metabolome. To this end, we induced experimental mastitis in nine cows by intramammary infusion of 100 µg purified Escherichia coli lipopolysaccharide (LPS) followed by kinetic assessments of cytokine responses (by enzyme-linked immunosorbent assay), changes in the metabolome (assessed by nuclear magnetic resonance), clinical parameters (heat, local pain perception, redness, swelling, rectal temperature, clot formation, and color changes in the milk), and milk somatic cell counts, at several time points post LPS infusion. Intramammary LPS infusion induced clinical signs of mastitis, which started from 2 h post infusion and had returned to normal levels within 24–72 h. Milk changes were seen with a delay compared with the clinical signs and persisted for a longer time. In parallel, induction of IL-6 and TNF-α were seen in milk, and there was also a transient elevation of plasma IL-6 whereas plasma TNF-α was not significantly elevated. In addition, a robust increase in CCL2 was seen in the milk of LPS-infused cows, whereas G-CSF, CXCL1, and histamine in milk were unaffected. By using a metabolomics approach, a transient increase of plasma lactose was seen in LPS-induced cows. In plasma, significant reductions in ketone bodies (3-hydroxybutyrate and acetoacetate) and decreased levels of short-chain fatty acids, known to be major products released from the gut microbiota, were observed after LPS infusion; a profound reduction of plasma citrate was also seen. Intramammary LPS infusion also caused major changes in the milk metabolome, although with a delay in comparison with plasma, including a reduction of lactose. We conclude that the LPS-induced acute mastitis rapidly affects the plasma metabolome and cytokine induction with similar kinetics as the development of the clinical signs, whereas the corresponding effects in milk occurred with a delay.</p