IL-20 Signaling in Activated Human Neutrophils Inhibits Neutrophil Migration and Function

Abstract

Neutrophils possess multiple antimicrobial mechanisms that are critical for protection of the host against infection with extracellular microbes, such as the bacterial pathogen Staphylococcus aureus Recruitment and activation of neutrophils at sites of infection are driven by cytokine and chemokine signals that directly target neutrophils via specific cell surface receptors. The IL-20 subfamily of cytokines has been reported to act at epithelial sites and contribute to psoriasis, wound healing, and anti-inflammatory effects during S. aureus infection. However, the ability of these cytokines to directly affect neutrophil function remains incompletely understood. In this article, we show that human neutrophils altered their expression of IL-20R chains upon migration and activation in vivo and in vitro. Such activation of neutrophils under conditions mimicking infection with S. aureus conferred responsiveness to IL-20 that manifested as modification of actin polymerization and inhibition of a broad range of actin-dependent functions, including phagocytosis, granule exocytosis, and migration. Consistent with the previously described homeostatic and anti-inflammatory properties of IL-20 on epithelial cells, the current study provides evidence that IL-20 directly targets and inhibits key inflammatory functions of neutrophils during infection with S. aureus.

Figures

Figure 1. Human neutrophils express IL-20RB when activated

(A) Detection of indicated IL-20 receptor chains on live neutrophils isolated from peripheral whole blood. Flow cytometry histograms from a representative donor (left) and summary data from multiple donors of percentage of neutrophils staining positive for each receptor chain (right) are shown. (B) Detection of IL-20R receptor chains on live neutrophils that extravasated from circulation into suction blisters induced on healthy volunteers. The suction blisters contained saline or killed S. aureus (SA1 or SA2, two distinct clinical isolates). Flow cytometry dot plots from a representative blister subject (left) and summary data from multiple subjects (right, each line connects data from a unique subject, n=6) are shown. (C–E) Healthy donor-derived peripheral blood neutrophils were assessed for IL-20R chain expression after exposure to indicated conditions in vitro. In (C), neutrophils were stained directly after isolation (peripheral blood), after migration across transwell inserts to uninfected or S. aureus-infected BEAS2b cells, or after adherence and exposure to supernatant from S. aureus-infected BEAS2b cells (adherent+sup). In (D), neutrophils were stained directly after isolation (peripheral blood) or after migration across transwell inserts into wells containing IL-8 (100 ng/ml), S. aureus (2×106 CFU/well), or both. In (E), neutrophils were incubated in media (unprimed) or primed with TNFα (10 ng/ml, 30 minutes) and then incubated for 1 hour in the absence or presence of S. aureus (Sa, 1×106 CFU/well=MOI 1). Data show mean frequency of live CD66b+ cells that express indicated receptor chains from three healthy volunteers (C–D), or fold-change in frequency of receptor-positive cells relative to unprimed neutrophils in 6 healthy volunteers (E). Error bars reflect SEM. *p< 0.05, **p<0.01, ***p<0.001 compared to peripheral blood (C–D) or unprimed (E) neutrophils.

Figure 2. IL-20 inhibits killing of S. aureus by TNFα-primed neutrophils in vitro

(A) Bacterial CFU recovered from adherent peripheral blood neutrophils that were unprimed (left panel) or primed with TNFα (10 ng/mL, 30 min, right panel) and then incubated with S. aureus (MOI 1) in the presence or absence of IL-20 (50 ng/mL) for the indicated time. Data shown are the mean of results from seven donors with each donor assayed in quadruplicate. (B) ROS detected by luminescence of luminol (1 mM) added to wells with neutrophils that were treated as described in (A). Conditions were performed in triplicate and measurements were taken every 4 minutes; data shown as mean +/− SEM of results from four donors. (C) Neutrophils were incubated with S. aureus +/− IL-20 after priming with TNFα as described in (A). Gentamicin was added at the indicated time points after infection to kill extracellular bacteria and CFU were enumerated. Data shown as percent of S. aureus remaining compared to average of total S. aureus present in duplicate gentamicin-untreated wells and reflect mean +/− SEM of results from four donors. (D) Neutrophils primed with TNFα and then incubated with S. aureus (MOI 1) +/− IL-20 as described in (A), in the absence or presence of cytochalasin D (10 μg/mL) or DNase (10 U/mL). CFU was determined in quadruplicate for each sample at 4 hours post-infection. Data shown are mean +/− SEM of results from three donors. For (A) and (C–D): **p<0.01, ***p<0.001 compared to unprimed (A, left) or TNFα-primed (A, right; C–D) neutrophils.

Figure 3. IL-20 inhibits exocytosis of tertiary (gelatinase) granules

(A) Concentration of neutrophil elastase (primary/azurophilic granule marker) in supernatants from human neutrophils, incubated with S. aureus MOI 1 for indicated times, measured by reaction with colorimetric substrate, calculated using standard of known concentration. (B) Lactoferrin concentration, marker for secondary (specific) granules, measured by ELISA of neutrophil supernatants. Data shown in (A) and (B) as mean +/− SEM from three healthy donors. (C) Gelatin zymography of supernatants from neutrophils infected for indicated times with S. aureus (MOI 1). Results shown are representative of neutrophils from four independent healthy donors. (D) Concentration of MMP9, marker for tertiary (gelatinase) granule release, in supernatants of neutrophils infected for indicated times with S. aureus (MOI 1) +/− IL-20. Data shown as mean +/− SEM from four healthy donors. ***p<0.001 compared to TNFα+IL-20-treated neutrophils by Two-way ANOVA.

Figure 4. Inhibition of phagocytosis and tertiary granule exocytosis by IL-20 is mediated by ERK1/2

(A–B) Western blot of lysates of neutrophils that were treated with the indicated inhibitors and infected/stimulated with S. aureus and TNFα +/− IL-20 for 10 minutes. Results shown are representative of neutrophils from 3 healthy donors. (C) S. aureus CFU recovered from adherent peripheral blood neutrophils that were treated with the indicated inhibitors and then incubated with S. aureus (MOI 1) and TNFα (10 ng/mL) in the presence or absence of IL-20 (50 ng/mL) for four hours. (D) Neutrophils were treated with indicated inhibitors and incubated with S. aureus and TNFα +/− IL-20 as described in (B). Gentamicin was added at the indicated time points after infection to kill extracellular bacteria and CFU were enumerated. Data shown as percent of S. aureus remaining compared to average of total S. aureus present in duplicate gentamicin-untreated wells. (E) Gelatin zymography of cell free supernatants from neutrophils incubated with for one hour with S. aureus (MOI 1) and TNFα +/− IL-20 after treatment with indicated inhibitors. Results shown are representative of neutrophils from three healthy donors. (F) Concentration of MMP9 in supernatants of neutrophils infected for one hour with S. aureus (MOI 1) +/− IL-20 after priming under indicated conditions. For (C), (D), and (F), data shown reflect mean +/− SEM of results from three donors, and *p<0.05, **p<0.01, ***p<0.001 by t test.

Figure 5. IL-20 inhibits neutrophil migration in vitro

(A) 1.5×106 peripheral blood neutrophils were placed in the upper chamber of transwell plates. After 3 hours, the number of cells that migrated across the transwell insert to lower chambers was counted using a hemacytometer. The lower chambers contained media or supernatants from human bronchial epithelial (BEAS2b) cells that had been infected with S. aureus (MOI 2) for 4 hours, sterile-filtered, and then subjected to immunoprecipitation with isotype control or IL-20 antibody. Under some conditions (αIL-20+IL-20), recombinant IL-20 (10 ng/mL) was added back to supernatant after IL-20 immunoprecipitation. (B) Neutrophils were treated with indicated inhibitors and stimulated to migrate across a transwell chamber as in (A). (C) Neutrophils untreated or treated with indicated inhibitors and stimulated to migrate across transwell inserts for 1 hour to wells containing S. aureus (2×106 CFU/well) + IL-8 (100 ng/mL) alone or with IL-20 (10 ng/mL). (D) Western blot of lysates from neutrophils, untreated or treated with indicated inhibitors, infected with S. aureus (MOI 1) and stimulated with IL-8 alone or with IL-20 for 10 minutes. For A–C, data shown reflect mean +/− SEM of results from three donors, and *p<0.05, **p<0.01 by t test.

Figure 6. IL-20 modulates actin polymerization in neutrophils

(A) Representative confocal microscopy images of neutrophils incubated with indicated stimulation for 1 minute (Sa, S. aureus 2×106 CFU/well; IL-8, 100 ng/mL; TNFα, 10 ng/ml). Green, Alexa Fluor 488-phalloidin; blue, DAPI. (B) Quantification of Alexa Fluor 488-phalloidin fluorescence intensity per cell from analysis of 25 fields of view per condition that were generated by an automated tiling method and normalized to the total number of cells across all images with standard deviation. Data is representative of results from three different donors. ***p<0.001 by t test.