MiR-155 Regulates PAD4-Dependent Formation of Neutrophil Extracellular Traps

Abstract

Accumulating data suggest that neutrophil extracellular traps (NETs) exert a key function in several diseases. Peptidylarginine deiminase 4 (PAD4) regulates NET formation via citrullination of histones. The aim of this study was to examine the role of miR-155 in controlling PAD4-dependent generation of NETs. Bone marrow neutrophils were stimulated with PMA and MIP-2. Pre-incubation of neutrophils with translational inhibitors (cycloheximide or puromycin) markedly decreased NET formation induced by PMA or MIP-2. Neutrophil transfection with a mimic miR-155 increased PMA-induced PAD4 mRNA expression and NET formation. In contrast, transfection with an antagomiR-155 decreased induction of PAD4 mRNA and NETs in response to PMA challenge. Bioinformatical examination of PAD4 revealed a potential binding site in AU-rich elements at the 3'-UTR region. MiR-155 binding to PAD4 was examined by use of target site blockers and RNA immunoprecipitation, revealing that miR-155 regulation of PAD4 mRNA is mediated via AU-rich elements in the 3'-UTR region. In conclusion, our findings demonstrate that miR-155 positively regulates neutrophil expression of PAD4 and expulsion of extracellular traps. Thus, our novel results indicate that targeting miR-155 might be useful to inhibit exaggerated NET generation in inflammatory diseases.

Keywords: NETosis; histone; inflammation; microRNA; neutrophils.

Figure 1

NET formation in neutrophils. (A) Neutrophils were preincubated with 10 μg/ml of cycloheximide or puromycin for 5–30 min and stimulated with PMA (500 nM) for 3 h at 37°C. (B) Neutrophils were preincubated with indicated concentrations of cycloheximide or puromycin for 30 min and stimulated with PMA (500 nM). DNA-histone complexes were quantified in supernatants by ELISA. (C) Levels of citrullinated Histone H3 and MPO in neutrophils (Ly6G+ cells) were determined by FACS. Neutrophils were preincubated with 10 μg/ml of cycloheximide or puromycin for 30 min and stimulated with PMA (500 nM). (D) Aggregate data of flow cytometry. Data represent mean ± SEM and n = 5. ^#P < 0.05 vs. control and *P < 0.05 vs. vehicle.

Figure 2

NETs were visualized by confocal immunofluorescence microscopy in isolated neutrophils. Cells were preincubated with or without 10 μg/ml cycloheximide and stimulated with PMA (500 nM) for 3 h at 37°C. (A–C) Neutrophils were immune-stained with antibodies to myeloperoxidase (MPO-green), citrullinated Histone H3 (citH3-red), hoechst-blue to counterstain DNA. Non-stimulated cells served as a control. Images are representative of four independent experiments. Scale bars = 10 μm. (D) NETs covered area per field of view were quantified using Fiji and expressed as percentage of H3cit covered area. Data represent mean ± SEM and n = 4. ^#P < 0.05 vs. control and *P < 0.05 vs. Vehicle.

Figure 3

NET formation in neutrophils. (A) Neutrophils were preincubated with 10 μg/ml of cycloheximide or puromycin for 5–30 min and stimulated with MIP-2 (100 ng/ml) for 3 h at 37°C. DNA-histone complexes were quantified in supernatants by ELISA. (B) Levels of citrullinated Histone H3 and MPO in neutrophils (Ly6G+ cells) were determined by FACS. Neutrophils were preincubated with 10 μg/ml of cycloheximide or puromycin for 30 min and stimulated with MIP-2 (100 ng/ml). (C) Aggregate data of flow cytometry. Data represent mean ± SEM and n = 5. ^#P < 0.05 vs. control and *P < 0.05 vs. vehicle.

Figure 4

NETs were visualized by confocal immunofluorescence microscopy in isolated neutrophils. Cells were preincubated with or without 10 μg/ml cycloheximide or puromycin and stimulated with MIP-2 (100 ng/ml) for 3 h at 37°C. (A–D) Neutrophils were immune-stained with antibodies to myeloperoxidase (MPO-green), citrullinated Histone H3 (citH3-red), hoechst-blue to counterstain DNA. Non-stimulated cells served as a control. Images are representative of four independent experiments. Scale bars = 10 μm. (E) NETs covered area per field of view were quantified using Fiji and expressed as percentage of H3cit covered area. Data represent mean ± SEM and n = 4. ^#P < 0.05 vs. control and *P < 0.05 vs. vehicle.

Figure 5

(A) miR-155-5p expression and (B) PAD4 mRNA expression. Neutrophils were stimulated with or without PMA (500 nM). Relative expression was determined by qRT-PCR. U6 and GAPDH were used as a house-keeping genes to normalize miR-155-5p and PAD4 expression, respectively. Relative expression was determined using 2^−ΔΔCT method. Data represent mean ± SEM and n = 5. *P < 0.05 vs. control.

Figure 6

Mimic miR-155 upregulates and AntagomiR-155 downregulates PAD4 mRNA expression in neutrophils. (A) miR-155-5p and (B) PAD4 mRNA expression in cells transfected with Ctrl-Mimic and miR-155 mimic. (C) miR-155-5p and (D) PAD4 mRNA expression in cells transfected with antagomiR-Ctrl and antagomiR-155-5p. Relative expression was determined by qRT-PCR. U6 and GAPDH were used as a house-keeping genes to normalize miR-155-5p and PAD4 expression, respectively. Relative expression was determined using 2^−ΔΔCT method. Data represent mean ± SEM and n = 5. *P < 0.05 vs. control.

Figure 7

Mimic miR-155 upregulates and AntagomiR-155 downregulates PAD4 mRNA expression in neutrophils stimulated with PMA (500 nM). (A) miR-155-5p and (B) PAD4 mRNA expression in cells transfected with Ctrl Mimic and mir-155 mimic. (C) miR-155-5p and (D) PAD4 mRNA expression in cells transfected with Ctrl-antagomiR and antagomiR-155-5p. Relative expression was determined by qRT-PCR. U6 and GAPDH were used as a house-keeping genes to normalize miR-155-5p and PAD4 expression, respectively. Relative expression was determined using 2^−ΔΔCT method. Data represent mean ± SEM and n = 5. ^#P < 0.05 vs. control and *P < 0.05 vs. vehicle.

Figure 8

Levels of DNA histone complexes in neutrophils transfected with (A) Ctrl Mimic or mir-155 mimic and (B) Ctrl-antagomiR or antagomiR-155-5p. Expression of (C) PAD4 and (D) citH3 protein as determined by western blot and aggregate data showing PAD4 and citH3 protein expression normalized to the total protein of the respective lane. Band intensity was quantified by using Image Lab™ software. Western blots are representative of 4 independent experiments. Data are expressed as mean ± SEM and n = 4–5 and represented as fold change. ^#P < 0.05 vs. control and *P < 0.05 vs. vehicle.

Figure 9

NETs were visualized by confocal immunofluorescence microscopy in neutrophils transfected with ctrl-antagomiR or antagomiR-155-5p and stimulated with PMA (500 nM). (A–D) Neutrophils were immune-stained with antibodies to myeloperoxidase (MPO-green), citrullinated Histone H3 (citH3-red), Hoechst-blue to counterstain DNA. Non-stimulated cells served as a control. Images are representative of four independent experiments. Scale bars = 10 μm. (E) NETs covered area per field of view were quantified using Fiji and expressed as percentage of H3cit covered area. Data represent mean ± SEM and n = 4. ^#P < 0.05 vs. control and *P < 0.05 vs. Ctrl-AntagomiR.

Figure 10

PAD4 is a direct target of miR-155-5p. (A) Predicted target sites of miR-155-5p in PAD4 mRNA 3-UTR sequence containing an ARE motifs (AAUUAAAAAU, AUUUA) are depicted in a shaded box. The seeding region of miR-155-5p complementary to ARE motifs was blocked using TSB, red sequence as described in section Materials and Methods. (B) TSB dose-dependently reversed the effect of miR-155-5p mimic on PAD4 mRNA expression in isolated neutrophils. GAPDH was used as a house-keeping genes to normalize PAD4 expression. Relative expression was determined using 2^−ΔΔCT method. Data represent mean ± SEM and n = 5. ^#P < 0.05 control-mimic and *P < 0.05 control TSB.

Figure 11

Neutrophils were transfected with ctrl-antagomiR, antagomiR-155-5p and then stimulated with or without PMA (500 nM). The amount of miR-155-5p and PAD4 mRNA were determined in input RNA by qRT-PCR in RIP assays. AntagomiR-155-5p decreased relative enrichment of (A) miR-155-5p and (B) PAD4 mRNA in Ago2 immunoprecipitates. Data are presented as fold change compared to anti-IgG ctrl. U6 and GAPDH were used as a house-keeping genes to normalize miR-155-5p and PAD4 expression, respectively. Relative expression was determined using 2^−ΔΔCT method. Data are expressed as mean ± SEM and n = 5. ^#P < 0.05 vs. control Ab and *P < 0.05 vs. anti-Ago2 Ab-Ctrl-antagomiR treated cells.