Abstract
Interleukin 10 (IL-10) is a dynamic cytokine produced by most adaptive and innate immune cells. Though known as a potent anti-inflammatory cytokine, IL-10 has been shown to promote the immune stimulatory activity of B cells, cytotoxic T cells, natural killer cells and mast cells. This has hindered the use of IL-10 as a therapeutic in immune mediated diseases. It is hypothesised that IL-10 induces its immune suppressive activity through the JAK-STAT3 pathway and immune stimulatory activity through the P13K-AKT1 pathway. We hypothesized that using structurally guided human IL-10 mutants with altered receptor affinity, can potentially bias IL-10 signalling towards therapeutic immune suppressive pathways.
Peripheral blood mononuclear cells (PBMC) from four healthy donors were stimulated with LPS and wild-type human (h)IL-10 or receptor-selective mutants IL-10s. The supernatant was collected and the levels of IL-1β measured using an enzyme linked immunosorbent assay (ELISA). To detect pSTAT3 and pAKT1 signalling, PBMC derived from two healthy donors were stimulated with hIL-10 or receptor-selective mutant IL-10s. PBMC were stained for CD3, CD8, CD19 and CD14 cell surface markers as well as intracellular phosphorylated (p) proteins pAKT1 and pSTAT3. Phospho-flow cytometry was used to detect phosphorylated proteins in specific cells, with changes in the geometric mean fluorescence intensity (gMFI) measured.
One mutant was found to have slightly greater anti-inflammatory activity than hIL-10 as it induced lower levels of IL-1β. The rest of the mutants did not inhibit the production of IL-1β to the same extent as hIL-10. Phosphorylation of STAT3 was induced to similar levels by hL- 10 and the IL-10 mutants across the CD3+ T cells, CD8+ T cells, CD19+ B cells and CD14+ monocytes. However, stimulation with hIL-10 induced substantially more phosphorylation of AKT1 across the immune cells compared to the mutant IL-10s.
These results suggest that changes to receptor affinity may not affect the JAK-STAT3 pathway however P13K-AKT1 signalling may be subject to manipulation. This could provide a mechanism of decoupling the immune stimulatory and immune suppressive activity of IL-10 which would contribute to improved IL-10 therapies for immune mediated disease.