Inositol hexakisphosphate kinase 1 regulates neutrophil function in innate immunity by inhibiting phosphatidylinositol-(3,4,5)-trisphosphate signaling

Abstract

Inositol phosphates are widely produced throughout animal and plant tissues. Diphosphoinositol pentakisphosphate (InsP7) contains an energetic pyrophosphate bond. Here we demonstrate that disruption of inositol hexakisphosphate kinase 1 (InsP6K1), one of the three mammalian inositol hexakisphosphate kinases (InsP6Ks) that convert inositol hexakisphosphate (InsP6) to InsP7, conferred enhanced phosphatidylinositol-(3,4,5)-trisphosphate (PtdIns(3,4,5)P3)-mediated membrane translocation of the pleckstrin homology domain of the kinase Akt and thus augmented downstream PtdIns(3,4,5)P3 signaling in mouse neutrophils. Consequently, these neutrophils had greater phagocytic and bactericidal ability and amplified NADPH oxidase–mediated production of superoxide. These phenotypes were replicated in human primary neutrophils with pharmacologically inhibited InsP6Ks. In contrast, an increase in intracellular InsP7 blocked chemoattractant-elicited translocation of the pleckstrin homology domain to the membrane and substantially suppressed PtdIns(3,4,5)P3-mediated cellular events in neutrophils. Our findings establish a role for InsP7 in signal transduction and provide a mechanism for modulating PtdIns(3,4,5)P3 signaling in neutrophils.

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Figure 1: Disruption of InsP6K1 in mouse neutrophils augments PtdIns(3,4,5)P3 signaling.
Figure 2: Disruption of InsP6K1 leads to enhanced chemoattractant-elicited intracellular and extracellular superoxide production in mouse neutrophils.
Figure 3: Pharmacological inhibition of InsP6K activity augments PtdIns(3,4,5)P3 signaling and NADPH oxidase–mediated production of superoxide in human primary neutrophils.
Figure 4: InsP6K1 disruption does not alter the amount of PtdIns(3,4,5)P3 in neutrophils.
Figure 5: Overexpression of InsP6Ks suppresses PtdIns(3,4,5)P3 signaling in dHL60 cells.
Figure 6: Inhibition of superoxide production by InsP7 in a cell-free reconstitution assay.
Figure 7: Chemoattractant stimulation rapidly diminishes InsP7 in neutrophils.
Figure 8: Enhanced in vivo bacteria-killing ability of Ip6k1−/− mice.
Figure 9: Augmented in vitro bacteria-killing ability of Ip6k1−/− neutrophils, with enhanced phagocytosis and ROS production.

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Acknowledgements

We thank L. Silberstein, J. Manis and L. Chai for discussions. Supported by the US National Institutes of Health (HL085100, AI076471, HL092020 and GM076084 to H.R.L.).

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Authors

Contributions

A.P., Y.J., A.C., S.H.S. and H.R.L. designed the experiments; A.P., Y.J., A.C., Y.L., S.K.J., J.Z., S.G.R., F.L., S.M., J.S. and C.B. did the experiments; A.P., Y.J., A.C., S.H.S. and H.R.L. analyzed data; and A.P., Y.J., S.H.S. and H.R.L. wrote the manuscript.

Corresponding author

Correspondence to Hongbo R Luo.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–10 and Supplementary Methods (PDF 1117 kb)

41590_2011_BFni2052_MOESM6_ESM.mov

Chemoattractant-elicited polarization of WT neutrophils (MOV 2006 kb)

Supplementary Movie 1

Chemoattractant-elicited polarization of WT neutrophils (MOV 2006 kb)

41590_2011_BFni2052_MOESM7_ESM.mov

Chemoattractant-elicited polarization of InsP6K−/− neutrophils (MOV 1696 kb)

Supplementary Movie 2

Chemoattractant-elicited polarization of InsP6K−/− neutrophils (MOV 1696 kb)

41590_2011_BFni2052_MOESM8_ESM.mov

Adhesion of WT (left) and InsP6K−/− (right) neutrophils under shear flow (MOV 799 kb)

Supplementary Movie 3

Adhesion of WT (left) and InsP6K−/− (right) neutrophils under shear flow (MOV 799 kb)

41590_2011_BFni2052_MOESM9_ESM.mov

Detachment of adhered WT (left) and InsP6K−/− (right) neutrophils under shear flow (MOV 1119 kb)

Supplementary Movie 4

Detachment of adhered WT (left) and InsP6K−/− (right) neutrophils under shear flow (MOV 1119 kb)

41590_2011_BFni2052_MOESM10_ESM.mov

Chemotaxis of WT neutrophils (bottom) towards 1 AM of fMLP (top) in EZ-TAXIScan chamber (MOV 882 kb)

Supplementary Movie 5

Chemotaxis of WT neutrophils (bottom) towards 1 AM of fMLP (top) in EZ-TAXIScan chamber (MOV 882 kb)

41590_2011_BFni2052_MOESM11_ESM.mov

Chemotaxis of InsP6K1−/− neutrophils (bottom) towards 1 AM of fMLP (top) in EZ-TAXIScan chamber (MOV 862 kb)

Supplementary Movie 6

Chemotaxis of InsP6K1−/− neutrophils (bottom) towards 1 AM of fMLP (top) in EZ-TAXIScan chamber (MOV 862 kb)

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Prasad, A., Jia, Y., Chakraborty, A. et al. Inositol hexakisphosphate kinase 1 regulates neutrophil function in innate immunity by inhibiting phosphatidylinositol-(3,4,5)-trisphosphate signaling. Nat Immunol 12, 752–760 (2011). https://doi.org/10.1038/ni.2052

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