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

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

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

<i>Map3k8</i>^{<i>–/–</i>}mice are resistant to <i>H</i>. <i>polygyrus</i> infection.

<p>A) WT and <i>Map3k8</i>^{<i>–/–</i>}mice were infected with 200 L3 stage <i>H</i>. <i>polygyrus</i> larvae. Adult luminal worms from the intestinal tissue were counted on days 14 (D14) and 28 (D28). B) Fecal egg burden in <i>H</i>. <i>polygyrus</i> infected WT and <i>Map3k8</i>^{<i>–/–</i>}mice at D14 and D28. C) ATP levels of adult <i>H</i>. <i>polygyrus</i> worms harvested from the duodenal tissue of WT and <i>Map3k8</i>^{<i>–/–</i>}mice at day 14 post infection. D) Representative FACS plots from the mesenteric lymph nodes (mLNs) and frequency of CD4⁺ <i>Il4</i>^GFP+ and CD4⁺ <i>FoxP3</i>^RFP+ T cells from the spleen, mLNs and Peyer’s patches (PP) of D14 <i>H</i>. <i>polygyrus</i> infected WT and <i>Map3k8</i>^{<i>–/–</i>}mice. E) Total number of cells from the spleen, mLNs and PP of D14 <i>H</i>. <i>polygyrus</i> infected WT and <i>Map3k8</i>^{<i>–/–</i>}mice. F) Arbitrary units (a.u.) of <i>H</i>. <i>polygyrus</i> adult worm extract (HEX)-specific IgG1 in the serum of D14 <i>H</i>. <i>polygyrus</i> infected WT and <i>Map3k8</i>^{<i>–/–</i>}mice. G) HEX-specific IL-5 and IL-13 in mLN cell culture supernatants of D14 <i>H</i>. <i>polygyrus</i> infected WT and <i>Map3k8</i>^{<i>–/–</i>}mice. Data from readouts in A) and B) represent 7–8 mice/genotype pooled from 2 independent experiments. Data from C)—G) is representative of 2–3 independent experiments with 3–4 mice/genotype and 4 adult worms/mouse. * and ** denote p≤0.05 using a two-tailed Mann-Whitney test.</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

Ccl24 neutralization reduced expression of early type-2 memory responses, correlating with a loss of resistance to <i>H</i>. <i>polygyrus</i> in <i>Map3k8</i>^{<i>–/–</i>}mice.

<p>A) <i>Arg1</i> expression; B) <i>Retnla</i> expression; C) <i>Chil3</i> expression; D) <i>Ear11</i> expression from small intestinal duodenal tissue of D5 <i>H</i>. <i>polygyrus</i> infected WT and <i>Map3k8</i>^{<i>–/–</i>}mice treated with Ccl24 neutralizing antibody or isotype control antibody. E) Adult luminal worms in the D21 <i>H</i>. <i>polygyrus</i> infected WT and <i>Map3k8</i>^{<i>–/–</i>}mice treated with Ccl24 neutralizing antibody or isotype control. Data represents 8–11 mice/genotype/group, pooled from 2 independent experiments. * denotes p≤0.05 using Mann-Whitney test.</p

Enhanced <i>Ccl24</i>, myeloid cells and <i>Il4</i>^<i>GFP+</i> Th2 cell infiltration in <i>Map3k8</i>^{<i>–/–</i>}mice.

<p>A) Representative FACS profile for CD11c⁺ CD11b⁺ cells from the intestinal LP of D5 <i>H</i>. <i>polygyrus</i> infected WT and <i>Map3k8</i>^{<i>–/–</i>}mice. B) <i>Ccl24</i> and <i>Ear11</i> expression from CD11c⁺ CD11b⁺ cells D5 <i>H</i>. <i>polygyrus</i> infected WT and <i>Map3k8</i>^{<i>–/–</i>}mice. C-H) Frequency and number of (C) eosinophils (live/CD45⁺/SiglecF⁺/SSChi); (D) neutrophils (live/CD45⁺/SiglecF^-/CD11b⁺/Ly6G⁺); (E) monocytes (live/CD45⁺/SiglecF^-/CD11b⁺/Ly6G^-/Ly6C⁺); (F) <i>Il4</i>^<i>GFP+</i> Th2 cells (live/CD45⁺/CD4⁺/CD3⁺/TCRβ⁺/<i>Il4</i>^<i>GFP+</i>) in the intestinal LP of D5 <i>H</i>. <i>polygyrus</i> infected WT and <i>Map3k8</i>^{<i>–/–</i>}mice. Data from B) is representative of 2 independent experiments with 3 biological replicates per group. Data from C-H) is representative of 2 independent experiments with 5–6 mice/group. * and ** denotes p≤0.05 using Mann-Whitney test.</p