A Novel Role for IκBζ in the Regulation of IFNγ Production

IκBζ is a novel member of the IκB family of NFκB regulators, which modulates NFκB activity in the nucleus, rather than controlling its nuclear translocation. IκBζ is specifically induced by IL-1β and several TLR ligands and positively regulates NFκB-mediated transcription of genes such as IL-6 and NGAL as an NFκB binding co-factor. We recently reported that the IL-1 family cytokines, IL-1β and IL-18, strongly synergize with TNFα for IFNγ production in KG-1 cells, whereas the same cytokines alone have minimal effects on IFNγ production. Given the striking similarities between the IL-1R and IL-18R signaling pathways we hypothesized that a common signaling event or gene product downstream of these receptors is responsible for the observed synergy. We investigated IκBζ protein expression in KG-1 cells upon stimulation with IL-1β, IL-18 and TNFα. Our results demonstrated that IL-18, as well as IL-1β, induced moderate IκBζ expression in KG-1 cells. However, TNFα synergized with IL-1β and IL-18, whereas by itself it had a minimal effect on IκBζ expression. NFκB inhibition resulted in decreased IL-1β/IL-18/TNFα-stimulated IFNγ release. Moreover, silencing of IκBζ expression led to a specific decrease in IFNγ production. Overall, our data suggests that IκBζ positively regulates NFκB-mediated IFNγ production in KG-1 cells

Effect of TNFα treatment on KG-1 IL-18R and IL-1R surface expression.

<p>KG-1 cells (10⁶/ml) were incubated with the indicated combinations of rIL-1β, rIL-18, rTNFα (10 ng/ml each) for 24 h. Cells were stained with IL-18R-PE and IL-1R-FITC followed by flow cytometry analysis. Results are representative of 3 separate experiments.</p

Silencing of IκBζ expression suppresses IFNγ and IL-6, not IL-8, mRNA and protein production.

<p>KG-1 cells (2×10⁶/ml) were nucleofected with a mixture of 3 different small interfering RNA (siRNA) oligonucleotides against IκBζ or 3 different scrambled siRNA oligonucleotides. After 2 h, cells were stimulated with a combination of rIL-1β, rIL-18 and rTNFα (10 ng/ml each) for 24 h. Cells were lysed for mRNA extraction. Messenger RNA (mRNA) was converted to cDNA, followed by quantitative PCR (qPCR) using primers specific for IFNγ (A), IL-6 (B) and IL-8 (C). Supernatants were harvested and analyzed for cytokine release by IFNγ (D), IL-6 (E) and IL-8 (F) ELISA. Results are shown as mean±S.E.M. *, <i>p</i><.05; **, <i>p</i><0.005 (A, B and C, n = 3) (D, E and F, n = 5).</p

The conditioned media from LPS/ATP-stimulated monocytes induces IκBζ expression in an IL-1β/TNFα-dependent, but IL-18-independent manner.

<p>KG-1 cells (10⁶/ml) were incubated with conditioned media from monocytes (10⁶/ml) that were stimulated with LPS (10 ng/ml, 4 h) and ATP (5 mM, last 15 min) for the indicated time points. For selected time points, the cells were incubated with the conditioned media in the presence of IL-1ra (100 µg/ml), IL-18R Ab (10 µg/ml), TNFα Ab (10 µg/ml), or different combinations of these neutralizing agents. Protein-matched total cell extracts were analyzed by Western blotting using anti-serum against IκBζ and actin Ab. Results are representative of 3 separate experiments.</p

IκBζ protein expression in KG-1 cells in response to IL-1β, IL-18 and TNFα stimulation.

<p>KG-1 cells (10⁶/ml) were stimulated with rIL-1β (A), rIL-18 (B), or rTNFα (C) (10 ng/ml each) for the indicated time points. At selected time points, the cells were incubated with rIL-1β and rIL-18 in the presence of IL-1ra (100 µg/ml) or IL-18 Ab (2 µg/ml), respectively. Protein-matched total cell extracts were analyzed by Western blotting using anti-serum against IκBζ, IkBα Ab and actin Ab. Results are representative of at least 3 separate experiments.</p

The synergistic effect of TNFα and IL-1β/IL-18 on IκBζ protein expression is partially suppressed with a TNFα-specific Ab, but completely blocked with IL-1β and/or IL-18 neutralization.

<p>KG-1 cells (10⁶/ml) were stimulated with rIL-1β, rIL-18, rTNFα (10 ng/ml each) (A), or the indicated combinations of these cytokines (B and C) for the indicated time points. At selected time points, the cells were incubated with the recombinant proteins in the presence of IL-1ra (100 µg/ml), IL-18R Ab (10 µg/ml), TNFα Ab (10 µg/ml), or different combinations of these neutralizing agents. Protein-matched total cell extracts were analyzed by Western blotting using anti-serum against IκBζ and actin Ab. Results are representative of at least 3 separate experiments.</p

Silencing of IκBζ expression.

<p>KG-1 cells (2×10⁶/ml) were nucleofected with a mixture of 3 different small interfering RNA (siRNA) oligonucleotides against IκBζ or 3 different scrambled siRNA oligonucleotides. After 2 h, cells were stimulated with a combination of rIL-1β, rIL-18 and rTNFα (10 ng/ml each) for the indicated time points. Protein-matched total cell extracts were analyzed by Western blotting using anti-serum against IκBζ and actin Ab. Results are representative of 5 separate experiments.</p

IκBζ mainly localizes to the nucleus.

<p>KG-1 cells (10⁶/ml) were stimulated with rIL-1β, rIL-18, rTNFα (10 ng/ml each), or different combinations of these cytokines for 8 h. Cells were harvested for subsequent cytosol and nuclear extraction. Protein-matched nuclear and cytosolic extracts were then analyzed by Western blotting with anti-serum against IκBζ, laminB1 Ab (nuclear marker) and IL-1β Ab (cytosolic marker). Results are representative of 3 separate experiments.</p