| dc.description.abstract | The innate immune system functions as the first line of defence against pathogen infection. Innate immune systems are functionally present across the animal kingdom and mediate host defence against infection via a myriad of complex pathways. One such pathway involvesrapid induction of gene expression of innate immune signalling molecules such as cytokines and chemokines, to mount and co-ordinate appropriate responses. In particular, detection of pathogens, for example by Toll-like receptor 4 (TLR4) sensing LPS, leads to gene induction of type I interferons (IFN-I), and IFN-Is subsequently stimulated a transcriptional programme of IFN-stimulated genes (ISGs). Intense investigation has been performed into the underlying mechanisms of transcriptional regulation of innate immune gene expression. Contrastingly, the mechanisms by which such gene expression is controlled at the level of translation is less well studied and understood.
The Elongator complex is an evolutionarily conserved multiprotein complex that is crucial in modifying Uridines at the wobble position of tRNA molecules, to ensure efficient and proper decoding of mRNA codons during translation. Elongator is known to regulate the translation of proteins involved in processes ranging from cell cycle and DNA damage response, to nutrient starvation and CNS development. However, the contribution of the Elongator complex to innate immunity is poorly understood. Therefore, the aim of this project was to assess the role of the Elongator complex in innate immune responses in macrophages.
To do this, we used macrophages lacking ELP3, the catalytic subunit of the Elongator complex. Quantitative proteomics analysis of Elp3-/- macrophages following LPS treatment, showed a strong impairment in the expression of proteins involved in IFN-I-mediated signalling. We demonstrated that LPS-mediated gene induction of IFN-I and ISGs was impaired in Elp3-/- cells. Furthermore, ELP3 was necessary for ISG induction mediated directly by IFN-I stimulation. TYK2 is essential for IFN-I signalling and ELP3 was likely required for TYK2 activation, as gene induction downstream of other cytokine cascades which utilise TYK2, were also impaired in the absence of ELP3. As regards the requirement of ELP3 for LPS-stimulated IFN-I induction, we found that ELP3 was necessary for TLR4-mediated IRF3 activation. Thus we demonstrated a two-fold requirement for ELP3 in TLR4-mediated IFN-I induction and signalling.
Interestingly, although many pathogen sensing pathways utilise IRF3, ELP3 was only necessary for gene induction downstream of TLRs and the RNA sensor RIG-I, and not DNA sensing via STING-mediated signalling pathways. Supporting this, innate immune gene induction in response to RNA virus infection was abrogated in Elp3-/- cells. Interestingly however, ELP3 was also required for Influenza A virus replication, suggesting a bi-directional role for ELP3 in IFN-I induction and RNA virus replication. We also established that the PYHIN family of proteins were enriched in Elongator-dependent codons, and that ELP3 was required for p205 protein expression, but not mRNA induction following IFN? treatment.
In summary, this work clarifies and reveals a pivotal role for Elongator in IFN-I gene induction and signalling in macrophages, as well as demonstrating a dual role for Elongator in innate responses to viruses and viral replication. | en |
