Abstract
Interleukin-10 (IL-10) is a multifunctional cytokine that regulates both innate and adaptive immune responses but is best known for its anti-inflammatory properties. Active IL-10 functions as a dimer that binds to a heterotetrameric receptor composed of two high affinity IL-10R1 subunits and two lower affinity IL-10R2 subunits. When bound, it predominantly activates the JAK1-STAT3 signalling pathway, inducing the transcription of anti-inflammatory genes.
Despite its potent anti-inflammatory activity, IL-10 can occasionally elicit pro-inflammatory responses instead. Recent findings suggest that IL-10R2 may play a role in this response as high affinity hIL-10 variants caused pro-inflammatory responses, whereas low affinity variants resulted in anti-inflammatory activity. These findings highlight the need for a deeper understanding of IL-10 interactions under physiological conditions. However, current IL-10 receptor binding assays are often conducted under artificial conditions, limiting their ability to reflect the complexity of IL-10 signalling on intact cells.
NanoLuc luciferase (NLuc) is a bioluminescent enzyme engineered for high stability and luminescent signal output. Previously it has been used to label proteins and measure their receptor binding to cells via bioluminescence with great success. This project aimed to utilise this technique to develop a more physiologically relevant approach to study IL-10 binding to its receptor. Human IL-10 (hIL-10)–NLuc fusion proteins, containing either one or two NLuc enzymes per dimer, were produced from HEK293 cells and purified via affinity chromatography. Their binding was tested by an in vitro competitive displacement hIL-10R1 ELISA, where both proteins could displace wild-type hIL-10 from its receptor, with the single NLuc variant exhibiting greater binding. Anti-inflammatory activity was assessed by measuring each fusion protein’s ability to suppress lipopolysaccharide-induced IL-1β production, where only the variant containing a single NLuc enzyme provided anti-inflammatory activity.
To measure fusion proteins binding to the hIL-10 receptor expressed by human cell lines, the proteins were incubated with either THP-1 cells (which natively express the hIL-10 receptor) or HEK293 cells (transfected to express hIL-10R1). Following incubation, addition of the NLuc substrate produced bioluminescence, allowing direct measurement of hIL-10 binding. However, only background levels of bioluminescence were detected after washing, likely due to low surface receptor expression. To increase receptor availability, vesicles were prepared from membranes of hIL-10R1-expressing HEK293 cells. When these vesicles were incubated with hIL-10–NLuc fusion proteins, bioluminescence was detected for both fusion proteins.
Overall, this study successfully produced novel hIL-10–NLuc fusion proteins that allowed hIL-10 binding to hIL-10R1 to be measured in vesicles. The development of this new IL-10 receptor binding assay provides a more native approach to study its receptor interactions, and with further assay optimisation, these results could be potentially replicated in cells.