Role of Neutrophil NETS in pregnancy and auto-inflammatory conditions (rheumatoid arthritis)
The previous focus of our lab was on the analysis of cell-free DNA, liberated by dying cells into the circulation. Our specific aim was to use this for the development of non-invasive tests for prenatal diagnosis, by exploiting the finding that during pregnancy, the placenta releases DNA of fetal origin into the maternal circulation. By the use of sophisticated genome sequencing approaches this approach has successfully made the transition from bench to clinic, and is being offered commercially by a number of providers e.g., Sequenom, Verinata, Natera etc .
For this reason we sought a new research focus and have turned our attention to a rather pivotal question that still remained, namely, what was the source of this maternal cell-free DNA and why was it elevated in certain conditions like preeclampsia?
A hint of what the source of the maternal cell-free DNA could be was provid- ed by a fascinating discovery in 2004, which described that upon activation circulatory neutrophils could expel their nuclear DNA into the extracellular environment, where it could serve as a sticky trap to ensnare and kill microorganisms. Consequently these structures were called NETS or neutrophil extracellular traps.
Prompted by these findings we investigated whether such events occurred in preeclampsia, and were startled to determine that NETs could be triggered in isolated neutrophils by treatment with inflammatory placental debris as found in preeclampsia. We also detected large numbers of NETs directly in the intervillous space in preeclamptic placentae .
Since preeclampsia is a highly inflammatory condition, we were curious whether such events occurred in other auto-inflammatory disorders e.g. rheumatoid arthritis (RA). In collaboration with Prof. P. Hasler, KSA, Aarau, we observed that the levels of cell-free DNA were much higher in serum samples than plasma, and that this was significantly different to the same analysis performed on healthy control samples . As this difference was not evident in rapidly processed EDTA plasma samples, it implied that during the clotting process during serum generation, a population of cells was liberating their DNA into the extracellular milieu in an increased amount. We postulated that these could be neutrophils undergoing NETosis.
Recently we were able to confirm that this suspicion via the detection of NETs associated neutrophil granular components in serum samples, as well as in culture supernatants of isolated neutrophils (Chowdhury et al., submitted). These studies furthermore implicated enhanced NETosis in the underlying aetiology of RA, in that the presence of the deiminating enzymes PAD2 and 4 on these extracellular structures may drive the generation of auto-antibodies against citrullinated peptides (ACPA).
We are now focusing our attention on the regulation of NETosis in normal pregnancy, and how this is dysregulated under a number of different complications (Giaglis et al., in preparation).
In a collaborative study with Prof. M. Bühler, FMI, we concluded our analysis of the role of miRNA in regulating trophoblast differentiation. This study indicated that the expression of miR-455 and miR-210 was sensitive to hypoxic conditions and their abundance was enhanced in preeclamptic placentae. Bioinformatic analysis of predicted targets suggested that MUC1 could be regulated by miR-455-3P, a feature we confirmed by gene transfections as- says. These experiments also linked HIF2A in this regulatory pathway (Lale- vee et al., in preparation).
Development of new markers to detect preeclampsia
A major obstetrical concern is that no reliable diagnostic aids exist to identify pregnancies at risk for preeclampsia, as well as a number of other complications. In past experiments we examined whether the maternal plasma proteome can be examined by quantitative isobaric labeling (iTRAQ) mass spectrometric approaches. In order to evaluate the suitability of such an approach we first examined pregnancies bearing a fetus with trisomy 21, as this involved the sole addition of a single chromosome, and as such was not as complex as multifactorial conditions such as preeclampsia. This study indicated that this approach was feasible, as we detected quantitative changes in concentration of known screening markers such as betaHCG.
In a subsequent study we extended this study and examined 1st trimester maternal plasma samples from pregnancies at risk for preeclampsia, in which we also detected potentially new screening biomarkers, as well as confirming changes in currently used ones. In order to extend the scope and variety of potential biomarkers, we examined for changes in gene expression in placental samples obtained from normal healthy deliveries and those affected by preeclampsia. It is now our intention to establish a large biobank to test the efficacy of our newly found biomarkers more rigorously. In addition, we will determine whether we can enhance the specificity and sensitivity of current screening tests, for the detection of late onset preeclampsia. We will also determine whether proteomic approaches can be used to develop new biomarkers for preterm labour or gestational diabetes.