Innate lymphoid cell type 3-derived interleukin-22 boosts lipocalin-2 production in intestinal epithelial cells via synergy between STAT3 and NF-κB - PubMed (original) (raw)

Innate lymphoid cell type 3-derived interleukin-22 boosts lipocalin-2 production in intestinal epithelial cells via synergy between STAT3 and NF-κB

Maarten Coorens et al. J Biol Chem. 2019.

Abstract

Escherichia coli and Klebsiella pneumoniae are opportunistic pathogens that are commonly associated with infections at mucosal surfaces, such as the lung or the gut. The host response against these types of infections includes the release of epithelial-derived antimicrobial factors such as lipocalin-2 (LCN-2), a protein that specifically inhibits the iron acquisition of Enterobacteriaceae by binding and neutralizing the bacterial iron-scavenging molecule enterobactin. Regulation of epithelial antimicrobial responses, including the release of LCN-2, has previously been shown to depend on IL-22, a cytokine produced by innate lymphoid cells type 3 (ILC3) during Enterobacteriaceae infections. However, much remains unknown about the extent to which antimicrobial responses are regulated by IL-22 and how IL-22 regulates the expression and production of LCN-2 in intestinal epithelial cells (IECs). Our study demonstrates how IL-22-induced activation of STAT3 synergizes with NF-κB-activating cytokines to enhance LCN-2 expression in human IECs and elucidates how ILC3 are involved in LCN-2-mediated host defense against Enterobacteriaceae. Together, these results provide new insight into the role of ILC3 in regulating LCN-2 expression in human IECs and could prove useful in future studies aimed at understanding the host response against Enterobacteriaceae as well as for the development of antimicrobial therapies against Enterobacteriaceae-related infections.

Keywords: STAT3; antimicrobial peptide (AMP); innate immunity; interleukin-22; microbiology; mucosal immunology; signal transduction.

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Conflict of interest statement

The authors declare that they have no conflicts of interest with the contents of this article

Figures

Figure 1.

Cytokine-mediated antimicrobial gene and protein expression in human IECs. A, HT-29 cells were stimulated for 24 h with IL-17A (50 ng/ml), IL-22 (50 ng/ml), TNF (20 ng/ml), IFN-γ (10 ng/ml), or a combination of these cytokines after which gene expression for the indicated genes was determined by qPCR. Subsequent hierarchical clustering was performed based on the calculated Pearson distance between genes. Colors represent average fold change on log scale compared with untreated controls based on three independent experiments. B, HT-29 cells and primary human CD45-depleted intestinal cells were stimulated for 24 h with IL-17A (50 ng/ml), IL-22 (50 ng/ml), TNF (20 ng/ml), and IFN-γ (10 ng/ml) after which gene expression for 19 antimicrobial genes was determined by qPCR. For HT-29 cells, values represent average fold change compared with no stimulation based on three independent experiments. For primary IECs, values indicate average fold change compared with no stimulation for three individual patient samples. C, HT-29, SW480, Caco-2, T84, and HCT116 cells were stimulated for 24 h with IL-17A (50 ng/ml), IL-22 (50 ng/ml), TNF (20 ng/ml), and IFN-γ (10 ng/ml) after which gene expression for the indicated genes was determined by qPCR. Values indicate the mean with S.E. of three independent experiments. Statistical analysis was performed with two-way ANOVA followed by Dunnett's post hoc test as compared with the unstimulated cells (expression value of 1). D–F, HT-29 and T84 cells were stimulated for 24 h with IL-17A (50 ng/ml), IL-22 (50 ng/ml), TNF (20 ng/ml), and IFN-γ (10 ng/ml) after which hBD-2 and LCN-2 concentrations in supernatant were determined by ELISA, and nitrite concentrations were determined by Griess assay. Values indicate the mean with S.E. of three independent experiments. Statistical analysis performed with two-way ANOVA followed by Šidák's post hoc test. p < 0.005 = **, and p < 0.0005 = ***.

Figure 2.

Specific cytokine synergy regulates the expression and release of LCN-2, NO, and hBD-2. A–F, HT-29 cells were stimulated for 24 h with all possible combinations of IL-22 (50 ng/ml), IL-17A (50 ng/ml), TNF (20 ng/ml), and IFN-γ (10 ng/ml) after which qPCR was performed to determine LCN2 (A) DEFB4 (B), and NOS2 (C) expression. Protein levels for LCN-2 (D) and hBD-2 (E) were determined in supernatants by ELISA, whereas nitrite levels (F) were determined by Griess assay. Values indicate the mean with S.E. of three independent experiments. Statistical analysis was performed with one-way ANOVA followed by Dunnett's post hoc test as compared with unstimulated cells. G, Venn diagrams representing gene expression levels (left) and protein/nitrite levels (right) for different cytokine combinations. Color intensity corresponds to the values in A–F redistributed on a gray scale from 0 to 255. p < 0.05 = *, p < 0.005 = **, and p < 0.0005 = ***.

Figure 3.

Involvement of STAT3 in IL-22, TNF, and IL-17A–mediated LCN-2 production. A and B, HT-29 cells were stimulated for 30 min with the indicated cytokines after which protein lysate was used for Western blot analysis to determine levels of pSTAT3–Tyr-705, pSTAT3–Ser-727, ac-STAT3–Lys-685, STAT3, and total protein. A, images of Western blottings representative of three independent experiments. B, band intensity quantification. Values indicate the mean with S.E. of three independent experiments. Statistical analysis was performed with two-way ANOVA followed by Tukey's post hoc test. C and D, HT-29 cells were stimulated for 24 h with IL-22 (50 ng/ml), IL-17A (50 ng/ml), TNF (20 ng/ml), and IFN-γ (10 ng/ml) in combination with indicated concentrations of STATTIC or DMSO control after which LCN-2 concentrations were determined by ELISA (C) and HT-29 viability was determined by WST-1 assay (D). Values indicate the mean with S.E. of three independent experiments. Statistical analysis was performed with two-way ANOVA followed by Dunnett's post hoc test. E, HT-29 cells were stimulated with indicated cytokine combinations for 30 min after which ChIP-qPCR was performed using anti-STAT3 or control IgG. Fold-increase in enrichment is shown compared with unstimulated cells. Values indicate the mean with S.E. of three independent experiments. Statistical analysis was performed with two-way ANOVA followed by Tukey's post hoc test. p < 0.05 = *, p < 0.005 = **, and p < 0.0005 = ***. Molecular masses indicate the location of the closest protein ladder marker on the blot.

Figure 4.

Involvement of NF-κB in IL-22, TNF, and IL-17A-mediated LCN-2 production. A and B, HT-29 cells were stimulated for 30 min with the indicated cytokines after which protein lysate was used for Western blot analysis to determine levels of pNF-κB–Ser-536, ac-NF-κB–Lys-310, IκB, NF-κB, and total protein. A, images of Western blottings representative of three independent experiments. B, band intensity quantification. Values indicate the mean with S.E. of three independent experiments. Statistical analysis was performed with two-way ANOVA followed by Tukey's post hoc test. C and D, HT-29 cells were stimulated for 24 h with IL-22 (50 ng/ml), IL-17A (50 ng/ml), TNF (20 ng/ml), and IFN-γ (10 ng/ml) in combination with indicated concentrations of BMS-345541 or DMSO control after which LCN-2 concentrations were determined by ELISA (C) and HT-29 viability was determined by WST-1 assay (D). Values indicate the mean with S.E. of three independent experiments. Statistical analysis was performed with two-way ANOVA followed by Dunnett's post hoc test. E, HT-29 cells were stimulated with indicated cytokine combinations for 30 min after which ChIP-qPCR was performed using anti-NF-κB or control IgG. Fold-increase in enrichment was compared with unstimulated cells. Values indicate the mean with S.E. of three independent experiments. Statistical analysis was performed with two-way ANOVA followed by Tukey's post hoc test. p < 0.05 = *, p < 0.005 = **, p < 0.0005 = ***. Molecular masses indicate the location of the closest protein ladder marker on the blot.

Figure 5.

Synergy between STAT3 and NF-κB inducers regulates LCN-2 production. A and B, HT-29 cells were stimulated for 30 min with the indicated cytokines after which protein lysate was used for Western blot analysis to determine levels of pNF-κB–Ser-536, ac-NF-κB–Lys-310, IκB, NF-κB, and total protein. A, images of Western blottings representative of three independent experiments. B, band intensity quantification. Values indicate the mean with S.E. of three independent experiments. Statistical analysis was performed with two-way ANOVA followed by Tukey's post hoc test. C and D, HT-29 cells were stimulated for 30 min with the indicated cytokines after which protein lysate was used for Western blot analysis to determine total levels of pSTAT3–Tyr-705, pSTAT3–Ser-727, ac-STAT3–Lys-685, STAT3, and total protein. C, images of Western blottings representative of two independent experiments. D, band intensity quantification. Values indicate the mean with S.E. of two independent experiments. Statistical analysis was performed with two-way ANOVA followed by Tukey's post hoc test. E, HT-29 cells were stimulated for 24 h with the indicated combinations of IL-22 (50 ng/ml), IL-17A (50 ng/ml) + TNF (20 ng/ml), IL-6 (50 ng/ml), and IL-1β (50 ng/ml), after which LCN-2 concentrations were determined in the cell supernatant by ELISA. Values indicate the mean with S.E. of three independent experiments. Statistical analysis was performed with one-way ANOVA followed by Šidák's post hoc test. F, HT-29 cells were stimulated for 24 h with all indicated combinations of IL-22 (50 ng/ml), IL-17A (50 ng/ml) + TNF (20 ng/ml), IL-6 (50 ng/ml), and IL-1β (50 ng/ml) in combination with 5 μ

BMS-345541, 2.5 μ

STATTIC, or DMSO control, after which LCN-2 concentrations were determined in cell supernatant by ELISA. Values indicate the mean with S.E. of three independent experiments. Statistical analysis was performed with two-way ANOVA followed by Dunnett's post hoc test as compared with unstimulated cells. p < 0.05 = *, p < 0.005 = **, p < 0.0005 = ***. Molecular masses indicate the location of the closest protein ladder marker on the blot.

Figure 6.

ILC3 regulates LCN-2 production in human IECs through IL-22 release. A, from patient samples (n = 5), isolated NKp44+ and NKp44− ILC3 were stimulated with 50 ng/ml IL-2, 50 ng/ml IL-1β, and 50 ng/ml IL-23 for 2 days, after which supernatant was collected separately for each patient sample to stimulate HT-29 cells for 24 h. RNA was isolated to determine LCN2 expression by qPCR. Graph shows the LCN2 response of HT-29 cells to individual supernatant samples as well as the mean with S.E. over all samples. Statistical analysis was performed with two-way ANOVA followed by Tukey's post hoc test. B and C, from patient samples (n = 5), isolated ILC3 (combined NKp44+ and NKp44−) was stimulated with 50 ng/ml IL-2, 50 ng/ml IL-1β, and 50 ng/ml IL-23 for 2 days, after which supernatant was collected separately for each patient sample. HT-29 cells were stimulated for 24 h with indicated cytokines or ILC3 supernatant with added anti-IL-22 (25 μg/ml), control IgG (25 μg/ml), or PBS. B, RNA was isolated to determine LCN2 expression by qPCR, and C, supernatant was collected to determine LCN-2 concentrations in cell supernatant. Graphs show the LCN-2 response of HT-29 cells to individual supernatant samples as well as the mean with S.E. over all samples. Values indicate the mean with S.E. of three independent experiments. n = 3–5, except for IL-23 + IL-1β + IL-2, which is n = 2. Statistical analysis was performed with two-way ANOVA followed by Tukey's post hoc test. p < 0.05 = *, p < 0.005 = **, and p < 0.0005 = ***.

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Innate lymphoid cell type 3-derived interleukin-22 boosts lipocalin-2 production in intestinal epithelial cells via synergy between STAT3 and NF-κB - PubMed (original) (raw)