Cytokine-Glycosaminoglycan Interactions and the Role of Dermatan Sulphate-PG and TGF-β1 in the Regulation of Interferon-γ Production by Murine NK Cells

Enhancing the understanding of the biological and functional role of specific cytokines in the regulation of Natural Killer (NK) cell activity is vital to cell-mediated immunity against infections and tumour development. Binding of cytokines with glycosaminoglycans (GAGs) and proteoglycans (PGs) have been known as a potential mechanism for regulating biological activity of many cytokines. In this thesis work, the binding of interleukins IL-11, IL-18 and IL-22 to heparin/HS was investigated using an ELISA approach, alongwith examination of primary sequences, 3-D protein structures and molecular docking calculations. Our results demonstrate that these interleukins do not bind to heparin/HS, which implies that the heparin-binding property should not be considered a generic characteristic for any class of cytokines. Further research focussed on examining the functional role of NK cell-surface PG, particularly dermatan sulphate (DS), in IL-12 signalled IFN-γ production using a PG synthesis inhibitor, p-nitrophenyl-β-xyloside. Our findings show partial inhibition of IFN-γ due to disrupted GAG/PG metabolism. Mechanistically, DS-PG interference was found independent of STAT-4 phosphorylation but suggestive of involvement essentially at the transcriptional or post-transcriptional level. In addition, our data implies that p-nitrophenyl-β-xyloside exerts cellular effects attenuating NK cell response to IL-12, independent of GAG-PGs. Finally, an interplay of molecular mechanisms between immunoregulatory factors, TGF-β1 and IL-12, in suppressing IFN-γ expression in mNK cells is studied. A clear evidence of partial inhibition of IL-12-induced IFN-γ production independent of NK cell proliferation is presented that does not directly involve key transcription factors of IFN-γ expression, STAT-4 and T-bet. However, promoter and early inhibition (3-6 hrs) studies suggested a possible role of TGF-β1 in chromatin remodelling of Ifng locus that regulates IFN-γ expression. Overall, identifying IL-heparin/HS interactions within different cytokine families and dissecting molecular events of cytokine signalling in NK cells are therapeutically important in recognising potential targets to manage cell-mediated immune responses.