Exogenous Ubiquitin Differentially Modulates the Phenotype and Function of M1 and M2 Macrophages

Figures

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  Figure 1. Exogenous UB decreases secretion of inflammatory cytokines in the conditioned media. Peritoneal macrophages, pretreated with UB (20 µg/mL) for 30 min, were treated with IFN-γ (100U/mL; M1 polarization) or IL-4 (20 ng/mL; M2 polarization) for 72 h. Levels of cytokines/chemokines were measured in the conditioned media using a Bio-Plex pro Mouse Cytokine 8-plex kit. Cytokines/chemokines are: (A) IFN-γ; (B) IL-4; (C) TNF-α; (D) IL-1β; (E) IL-2; (F) IL-5; (G) IL-10; (H) GM-CSF. Data were analyzed using one-way ANOVA followed by a post hoc Tukey’s test (* p < 0.05 vs. CTL, # p < 0.05 vs. IFN-γ; $ p < 0.05 vs. IL-4;) and 2-tailed Student’s t-test (@ p < 0.05 vs. CTL); n = 4–5. The comparison groups were CTL, IFN-γ, UB+IFN-γ, and UB; and CTL, IL-4, UB+IL-4, and UB.

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  Figure 2. Exogenous UB modulates the phenotype and cell surface area of macrophages. Peritoneal macrophages, pretreated with UB (20 µg/mL) for 30 min, were treated with IFN-γ (100 U/mL; M1 polarization) or IL-4 (20 ng/mL; M2 polarization) for 72 h. Macrophages were stained by Phalloidin-iFluor 555 (F-actin staining; red) and Hoechst 33258 (nuclear staining; blue). Images were acquired using the EVOS m7000 microscope (A) and cell surface area was quantified using NIS-Elements software (B). Data were analyzed using a 2-tailed Student’s t-test. # p < 0.05 vs. IFN-γ; n = 3.

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  Figure 3. Exogenous UB affects efferocytosis of macrophages. Peritoneal macrophages, pretreated with UB (20 µg/mL) for 30 min, were treated with IFN-γ (100U/mL; M1 polarization) or IL-4 (20 ng/mL; M2 polarization) for 72 h. Cells were then incubated overnight with PKH67-labeled apoptotic neutrophils (green) and then immune-stained with anti-F4/80 antibodies (red). Nuclei were stained using Hoechst (blue). Images, captured using EVOS m7000 microscope, were analyzed using Nikon-NIS Elements software. White arrows indicate macrophages with engulfed apoptotic neutrophils. Data were analyzed using one-way ANOVA followed by a post hoc Tukey’s test. * p < 0.05 vs. CTL; $ p < 0.05 vs. IL-4; n = 3. The comparison groups were CTL, IFN-γ, UB+IFN-γ, and UB; and CTL, IL-4, UB+IL-4, and UB.

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  Figure 4. Exogenous UB modulates migration of M1- and M2-polarized macrophages. Peritoneal macrophages were treated with UB (20 µg/mL) for 30 min. The cells were then exposed to IFN-γ (100U/mL; M1 polarization) or IL-4 (20 ng/mL; M2 polarization) for 72 h, followed by creation of a wound using a sterile plastic pipette tip in the cell monolayer. Cell migration into the wound area was assessed 4 h after wound creation. To achieve this, images of the wound area were acquired using EVOS m7000 microscope. The captured images were analyzed with Nikon-NIS Elements software. The upper panels demonstrate the wound area at 0 and 4 h after wound creation. The lower panel shows the mean data as distance in µm traveled since 0 h timepoint. Data were analyzed using one-way ANOVA followed by a post hoc Tukey’s test (* p < 0.05 vs. CTL, # p < 0.05 vs. IFN-γ) and 2-tailed Student’s t-test ($ p < 0.05 vs. IL-4); n = 5. The comparison groups were CTL, IFN-γ, UB+IFN-γ, and UB; and CTL, IL-4, UB+IL-4, and UB.

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  Figure 5. Exogenous UB decreases STAT1 and FAK activation in M1-polarized macrophages. Peritoneal macrophages, pretreated with UB (20 µg/mL) for 30 min, were treated with IFN-γ (100U/mL; M1 polarization) for 72 h. Cell lysates were analyzed by Western blot using anti-p-STAT1, STAT1, p-FAK, FAK, and GAPDH antibodies. After protein transfer, the membrane was cut into two portions. The upper portion, above the ~70 kDa marker, was probed for p-STAT1 and p-FAK, while the lower portion was probed for GAPDH. After stripping the upper portion, the membrane was probed for STAT1 and FAK. Thus, normalization for STAT1 and FAK was performed using the same GAPDH signal. (A) The upper panel exhibits immunoblotting for p-STAT1, STAT1 and GAPDH. (B) The upper panel exhibits immunoblotting for p-FAK, FAK and GAPDH. The lower panels (A,B) exhibit quantitative analyses normalized to STAT1, FAK or GAPDH. Data were analyzed using a 2-tailed Student’s t test or one-way ANOVA followed by a post hoc Tukey’s test. * p < 0.05 vs. CTL, # p < 0.05 vs. IFN-γ; n = 3.

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  Figure 6. Exogenous UB affects STAT6 and FAK activation in M2-polarized macrophages. Peritoneal macrophages, pretreated with UB (20 µg/mL) for 30 min, were treated with IL-4 (20 ng/mL; M2 polarization) for 72 h. Cell lysates were analyzed by Western blot using anti-p-STAT6, STAT6, p-FAK, FAK, and GAPDH antibodies. After protein transfer, the membrane was cut into two portions. The upper portion, above the ~70 kDa marker, was probed for p-STAT6 or p-FAK, while the lower portion was probed for GAPDH. After stripping the upper portion, the membrane was probed for STAT6 or FAK. (A) The upper panel exhibits immunoblotting for p-STAT6, STAT6, and GAPDH. (B) The upper panel exhibits immunoblotting for p-FAK, FAK, and GAPDH. The lower panels (A,B) exhibit quantitative analyses normalized to STAT6, FAK, or GAPDH. Data were analyzed using a two-tailed Student’s t test or one-way ANOVA followed by a post hoc Tukey’s test. $ p < 0.05 vs. IL-4; n = 3–6.

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  Figure 7. Exogenous UB had no effect on ubiquitination of cellular proteins. Peritoneal macrophages, pretreated with UB (20 µg/mL) for 30 min, were treated with IFN-γ (100 U/mL; M1 polarization) or IL-4 (20 ng/mL; M2 polarization) for 72 h. Cell lysates were analyzed by Western blot using anti-UB antibodies. (A) UB immunostaining. (B) Total protein stain. (C) Quantitative data for the ubiquitinated proteins normalized to total protein staining (entire lane). Data were analyzed using one-way ANOVA followed by a post hoc Tukey’s test. n = 3–4; Std, standard. The comparison groups were CTL, IFN-γ, UB+IFN-γ, and UB; and CTL, IL-4, UB+IL-4, and UB.