[Thesis]. Manchester, UK: The University of Manchester; 2015.
Interleukin (IL)-1α and IL-1β are pivotal to the initiation and orchestration of inflammation.
Unlike most cytokines, IL-1 does not have a signal peptide and therefore secretion
requires 2 independent processes; an initial signal to induce the up-regulation of
the inactive precursor (pro-IL-1) and a second signal to drive cleavage and subsequent
secretion. Whereas many previous studies have focused on the mechanisms that drive
IL-1 secretion, the aims of this thesis were to investigate the processes that regulate
the intracellular precursors of IL-1 (pro-IL-1α and pro-IL-1β). The hypothesis here
was that regulation of these precursors may serve to control the vigour of IL-1 secretion
and, ultimately, may influence the potency of pro-inflammatory responses.Post-translational
modifications were of particular interest in this thesis, as these modifications are
becoming increasingly important to immune system function. Ubiquitination is an important
post-translational modification whereby ubiquitin, an 8.5kDa protein, is covalently
bound to lysine residues on substrate proteins. In chapter 2, evidence was provided
to show that in murine DC, IL-1α and IL-1β are polyubiquitinated and that, in both
DC and macrophages, this polyubiquitination drives the proteasomal degradation of
IL-1. In addition, these data demonstrated that in the presence of a second signal,
polyubiquitinated IL-1 is still available for secretion. Overall, these investigations
highlight that the polyubiquitination and proteasomal degradation of IL-1 serves as
an essential process in the regulation of IL-1 and, therefore, should be considered
as an extra dimension to the current two-signal paradigm of IL-1 release. To support
this work, an immortalized bone marrow derived murine macrophage cell line and a human
monocyte cell line that both stably express fluorescent IL-1β were employed to measure
the rate of IL-1β degradation. In these investigations, it was shown that fluorescence
is a reliable readout for measuring IL-1β degradation in these cell lines. In addition,
it was demonstrated that that TLR-stimulation leads to an inhibition in IL-1β ubiquitination
and degradation. Together, the work presented herein highlights that ubiquitination
actively regulates the vigour of IL-1β protein expression and thus may be an important
regulator of inflammation. To complement this work, a broader approach was taken,
whereby the interactome of pro-IL-1β was explored using a human protein microarray.
In these investigations, a human proteome microarray containing 19,951 unique proteins
was used to identify proteins that bind human recombinant pro-IL-1β. In these analyses,
calmodulin was identified as a particularly strong hit, with a SNR of ~11. Using an
ELISA-based protein-binding assay, the interaction of recombinant calmodulin with
pro-IL-1β, but not mature IL-1β, was confirmed and shown to be calcium dependent.
Finally, using small molecule inhibitors it was demonstrated that both calcium and
calmodulin were required for nigericin induced IL-1β secretion in human monocytes.
Collectively, the evidence presented in these investigations suggests that following
calcium influx, pro-IL-1β interacts with intracellular calmodulin and that this interaction
is central for IL-1β processing and release. In addition, a number of other potentially
important pro-IL-1β-interacting proteins were also identified in this work, including
IL22RA2 and PLCXD3. Overall, the work presented in this thesis serves to highlight
that IL-1 is regulated by a broad range of potentially important intracellular processes.
We postulate that these processes may be pivotal in the regulation of inflammation
and thus the maintenance of homeostasis.