Untersuchungen in vitro zu epigenetisch wirksamen Immunmodulatoren bei der Pathogen-spezifischen Mastitis des Rindes

Mastitis is among the most important diseases in dairy cows. It accounts for large parts of antimicrobial drug use worldwide. Two of the most important mastitis pathogens are Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus). Due to the imminent normative to reduce the use of antimicrobial drugs in livestock, new ways for therapy and prophylaxis of mastitis are needed. Recently epigenetic regulation of inflammation by chromatin modifications has increasingly drawn attention. Currently assorted epigenetic modifiers have already been approved for the use in humans, however little is known about their actions in the bovine system. The aim of this study was to investigate whether three selected epigenetic modifiers (ViD3, SAHA and S2101) influence the initial immune response towards mastitis pathogens in bovine udder tissue in vitro. Additionally, the epigenetic signature of polymorphonuclear neutrophils (PMN) should be identified for the better understanding of their role during pathogen control and abatement. Tissue explants of the teat cistern and udder parenchyma were collected from 21 cows and were incubated for 36 hours in the absence and presence of epigenetic modifiers. Additionally, the tissue was stimulated with heat-inactivated particles of E. coli and S. aureus. After incubation, the explants were tested by RT-qPCR for transcript abundances of immune related candidate genes. Gene expression was validated in culture supernatants by an AlphaLISA® assay. Furthermore, the culture supernatants were analyzed for their chemotactic capacity through a chemotaxis assay and their ability to activate bovine PMN by measuring Ca++-influx. Statistical analysis of data was performed with the program “R” version 3.2.3. For the epigenetic profiling of PMN, blood and milk samples of three cows with mastitis were collected. After isolation of PMN from blood and milk, the PMN were fixated and stained by intracellular immunofluorescence of specific histone regions. After incubation and stimulation, the explants showed a comparably high metabolic activity in the teat cistern (76 %) and the udder parenchyma (50 %). The addition of pathogens (E. coli and S. aureus) or epigenetic modifiers (VitD3, SAHA and S2101) did not affect to the metabolic activity of the explants. The epigenetic modifier S2101 significantly inhibited the pathogen induced upregulation of transcripts for the chemokine CXCL8, the cytokine TNF (P < 0,05) and the antimicrobial peptides S100A9 and LAP. The epigenetic modifier SAHA inhibited the upregulation of S100A9 and LAP (P < 0,01) but did not inhibit the expression of CXCL8 and TNF. The regulation of the cytokine IL 10 was neither affected by SAHA nor S2101. The transcriptional regulation of CXCL8, TNF and IL 10 was validated through the product analysis by AlphaLISA® and functionally by a chemotaxis assay and the intracellular Ca2+-influx. VitD3 had no effect on the immune response of udder tissue in vitro after stimulation with mastitis pathogens. The epigenetic signature of PMN showed just minor differences. The PMN from milk revealed a lower fluorescence intensity for H3K9ac than the PMN from blood. Regarding methylation there were no differences found between blood and milk PMN. In conclusion these data show the potential of epigenetic modifiers (SAHA and S2101) to block overshooting inflammation in the udder. Thus, epigenetic modifiers may serve in future as immune modulators for the treatment and/or prophylaxis of clinical mastitis