The perilous scientific journey of a PhD project

Dec ,19 2016

As my PhD is nearly completed and we are at the moment writing up our results, I am realizing what a fascinating scientific journey these 3 years were, and nearing its completion, what unexpected shape the project took. Hence, I thought that sharing this odyssey would make an interesting blog.

When I began my INBIONET assignment in the group of Pavel Kovarik in April 2013 at the Max F Perutz Laboratories in Vienna, I was tasked with continuing a previous PhD student’s topic pertaining to the elucidation of novel pattern recognitions receptors, which unexpectedly recognized S. pyogenes nucleic acids to trigger both proinflammatory and type I interferon responses. The project was rather well advanced in that we had a wealth of very solid data showing that these responses to bacterial nucleic acids were dependent on a yet uncharacterized immune receptor other than the well-established TLR2, which was at that time widely believed to be the main receptor recognizing Gram positive bacteria via their cell wall components. Unfortunately, just as I was starting, some groups had been faster than us and had identified TLR13 as the receptor responsible for inducing the responses we were investigating.

Far from killing our project, the identification of TLR13 opened new questions. In fact this TLR had been previously largely neglected for the simple reason that it was only present in a few mammals like mice and rats but absent from the human repertoire of immune receptors. At the same time, our observation that human cells respond to S. pyogenes nucleic acids even when TLR2 is blocked, indicated that receptors existed within human immune cells, which had analogous functions to TLR13 in mice. Hence the hunt was on to identify these human receptors.

This was the beginning of the CRISPR/Cas9 era. In this context, we were luckily positioned, as next door to us, a student of Emmanuelle Charpentier was finishing his PhD and offered his help. Our strategy was to use haploid cell lines to perform small-scale genetic screens to identify our elusive receptor. Early on, we identified a major caveat: no matter what we tried, we could not get the haploid cells to behave like immune cells. Hence, we had to switch the strategy to standard diploid immune cell lines, which had the disadvantage of being harder to modify genetically. We were additionally slowed down by the fact that immune cells are very good at defending themselves from foreign genetic material and are hence notoriously difficult to transfect. Eventually, other groups were again faster than us and identified TLR8 in human cells to have analogous pattern recognition functions to TLR13. We none the less were successful in publishing our findings with the novelty that TLR2 and TLR13 are collaborative rather than redundant in triggering sufficiently protective immune responses to S. pyogenes.

Successful scientists never put all their eggs in the same basket. While we were exhausting all possible avenues on the topic of pattern recognition in triggering immune responses to S. pyogenes, a parallel project focused on elucidating the protective mechanisms engaged by type I interferon responses was beginning to fruition. My colleague Virginia Castiglia had established that type I interferons were modulating IL-1 responses to S. pyogenes and as such had the very important function of averting hyperinflammation resulting from infection. IL-1 cytokines being central and essential in triggering protective inflammation against bacterial pathogens, we now had evidence that loss of IL-1 regulation could also have devastating effects. Given these findings, it became very relevant to understand what IL-1-driven mechanism confer protection and how are adequate but moderated IL-1 responses triggered by S. pyogenes. In this manner, my PhD project took a very sudden in vivo turn in the second half. It was perfect timing, as this is around the time when we were completing the FELASA certificate with INBIONET.

Overall, despite the frustrating times and sinuous paths my projects took, pursuing this PhD has been an extremely rewarding experience. One important lesson to be drawn is that what may initially seem as a scientific dead-end is often only a catalyst for a new and more exciting research direction. In the end, the unexpected shifts in the focus of my project only left me with more technical experience, knowhow, co-authorships and tenacity.