Common mechanisms cannot explain time- and dose-dependent DNA methylation changes in earthworms exposed to cadmium.

DNA hypermethylation caused by environmental pollutants like cadmium (Cd) has already been demonstrated in many invertebrates, including earthworms. However, the exact epigenetic mechanisms that drive this hypermethylation are largely unknown and even basic DNA methylation and demethylation processes are hardly characterized. Therefore, we used an important bioindicator, the earthworm Lumbricus terrestris, as a model organism to determine time- and dose-dependent effects of Cd on global and gene-specific DNA methylation and its underlying mechanisms. We revealed Cd-induced adenine and cytosine hypermethylation using specific antibodies in dot blots and found that the methylation level of adenine compared to cytosine changed even to a bigger extent. However, the levels of hydroxymethylated cytosine did not differ between treatment groups. General methylation and demethylation components like methyltransferases (DNMT1 and 3), and ten-eleven translocation (TET) genes were confirmed in L. terrestris by quantitative RealTime PCR. However, neither gene expression, nor DNMT and TET enzyme activity showed significant differences in the Cd exposure groups. Using bisulfite conversion and sequencing, gene body methylation (gbm) of metallothionein 2 (MT2), one of the most important detoxification proteins, was characterized. Cd-dependent changes in MT2 gbm could, however, not be correlated to MT2 gene activity evaluated by quantitative RealTime PCR. Future directions as well as missing links are discussed in the present study hinting towards the importance of studying epigenetic marks and mechanistic insights in a broad variety of species to deepen our knowledge on the effects of changing environmental conditions.

ß-1,3-glucan-lacking Aspergillus fumigatus mediates an efficient antifungal immune response by activating complement and dendritic cells.

Complement system and dendritic cells (DCs) form - beside neutrophils and macrophages - the first line of defense to combat fungal infections. Therefore, we here studied interactions of these first immune elements with Aspergillus fumigatus lacking ß-1,3-glucans (fks1tetOnrep under repressed conditions) to mechanistically explain the mode of action of echinocandins in more detail. Echinocandins are cell wall active agents blocking ß-glucan synthase, making the A. fumigatus fks1tetOn mutant a good model to study immune-modulatory actions of these drugs. We now demonstrate herein, that complement was activated to significantly higher levels by the fks1-deficient strain compared to its respective wild type. This enhanced covalent linking of complement fragments to the A. fumigatus fks1tetOnrep mutant further resulted in enhanced DC binding and internalization of the fungus. Additionally, we found that fks1tetOnrep induced a Th1-/Th17-polarizing cytokine profile program in DCs. The effect was essentially dependent on massive galactomannan shedding, since blocking of DC-SIGN significantly reduced the fks1tetOnrep-mediated induction of an inflammatory cytokine profile.Our data demonstrate that lack of ß-1,3-glucan, also found under echinocandin therapy, results in improved recognition of Aspergillus fumigatus by complement and DCs and therefore not only directly affects the fungus by its fungistatic actions, but also is likely to exert indirect antifungal mechanisms by strengthening innate host immune mechanisms.Abbreviations: C: complement; CR:complement receptor; DC: dendritic cell; iDC: immature dendritic cell; DC-SIGN: Dendritic Cell-Specific Intercellular adhesion molecule-3-Grabbing Non-integrin; ERK: extracellular signal-regulated kinases; JNK : c-Jun N-terminal kinases; MAPK: mitogen-activated protein kinase; NHS: normal human serum; PRR: pattern recognition receptor; Th :T helper; TLR :Toll-like receptor; WT: wild type.