N. J. Galbraith1, S. P. Walker1, C. Bishop1, J. V. Carter1, M. Cahill1, S. A. Gardner1, H. C. Polk1 1University Of Louisville,Department Of Surgery,Louisville, KY, USA
Introduction:
The magnitude of the immune response following major trauma is predictive of mortality. IκK-16, a selective inhibitor of IκB kinase (IκK), has shown promising results by limiting end-organ damage in experimental models of sepsis and hemorrhagic shock. The purpose of this study was to determine the influence of IκK-16 on miRNA-155 expression and the human monocyte inflammatory response.
Methods:
Primary human monocytes were freshly isolated from healthy donors using CD14 magnetic beads and cultured in standard medium. Purity of ≥95% was confirmed by flow cytometry. Cells were treated for 1h with either IκK-16 (100nM unless otherwise specified) or diluted DMSO as a control. Monocytes were then stimulated with lipopolysaccharide (LPS) 100ng/mL for 16 h. Cell viability was determined by Trypan Blue staining and manual counting by standard microscopy. The influence of IκK-16 on IκK phosphorylation was assessed by Western Blot. Intracellular expression of miRNA-155 was measured at qRT-PCR. TNF-α and IL-10 supernatant protein concentrations were measured by ELISA. Cell surface protein expression of CD14 and HLA-DR were measured by flow cytometry. Paired T-test or Wilcoxon Rank Sign Test was used where appropriate, with signficance set at 0.05.
Results:
IκK-16 did not influence monocyte cell viability. IκK-16 treatment suppressed IκK phosphorylation. IκK-16 treatment lead to a downregulation of miRNA-155 expression compared with control (p<0.05). Levels of both TNF-α and IL-10 were suppressed in a dose-dependent fashion with IκK-16 treatment (p<0.05). However, levels of monocyte HLA-DR expression were not significantly altered by IκK-16 treatment, and monocyte surface CD14 expression was augmented by IκK-16 treatment.
Conclusion:
IκK-16 treatment suppresses activation of the IκK signaling pathway in the human monocyte, and does not appear to be toxic to cells. Both pro-inflammatory and anti-inflammatory cytokine responses are also suppressed, which may occur due to modulation of MiRNA-155 expression. The antigen presenting capacity of monocytes was not impaired by IκK-16 treatment. These results are promising for future studies examining the role of IκK-16 as a potential immunomodulatory therapy.