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. 2017 Mar;104:10-19.
doi: 10.1016/j.freeradbiomed.2017.01.003. Epub 2017 Jan 4.

Nitro-oleic Acid Regulates Growth Factor-Induced Differentiation of Bone Marrow-Derived Macrophages

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Free PMC article

Nitro-oleic Acid Regulates Growth Factor-Induced Differentiation of Bone Marrow-Derived Macrophages

Hana Verescakova et al. Free Radic Biol Med. .
Free PMC article

Abstract

Many diseases accompanied by chronic inflammation are connected with dysregulated activation of macrophage subpopulations. Recently, we reported that nitro-fatty acids (NO2-FAs), products of metabolic and inflammatory reactions of nitric oxide and nitrite, modulate macrophage and other immune cell functions. Bone marrow cell suspensions were isolated from mice and supplemented with macrophage colony-stimulating factor (M-CSF) or granulocyte-macrophage colony-stimulating factor (GM-CSF) in combination with NO2-OA for different times. RAW 264.7 macrophages were used for short-term (1-5min) experiments. We discovered that NO2-OA reduces cell numbers, cell colony formation, and proliferation of macrophages differentiated with colony-stimulating factors (CSFs), all in the absence of toxicity. In a case of GM-CSF-induced bone marrow-derived macrophages (BMMs), NO2-OA acts via downregulation of signal transducer and activator of transcription 5 and extracellular signal-regulated kinase (ERK) activation. In the case of M-CSF-induced BMMs, NO2-OA decreases activation of M-CSFR and activation of related PI3K and ERK. Additionally, NO2-OA also attenuates activation of BMMs. In aggregate, we demonstrate that NO2-OA regulates the process of macrophage differentiation and that NO2-FAs represent a promising therapeutic tool in the treatment of inflammatory pathologies linked with increased accumulation of macrophages in inflamed tissues.

Keywords: Differentiation; Growth factors; Inflammation; Macrophages; Nitro-fatty acids; Nitro-oleic acid; Signaling pathways.

Conflict of interest statement

Conflict of Interest

Bruce A. Freeman and Steven R. Woodcock acknowledge an interest in Complexa, Inc., as scientific founder/shareholder (BAF) and consultant (SRW). All other authors confirm that there are no conflicts of interest in this manuscript.

Figures

Figure 1
Figure 1. Schematic representation of experimental setup
Upper parts briefly describe cell preparation, treatment, and cultivation time. Lower parts summarize days and types of measurements as well as cell type used. (A) BMC experiments. (B) RAW 264.7 experiments.
Figure 2
Figure 2. The effect of NO2-OA on BMM numbers, proliferation, and colony formation
Native BMCs were isolated and cultivated i) with M-CSF (20 ng/ml) or GM-CSF (20 ng/ml) and NO2-OA (3 μM) in DMEM for 7 days (A, B, and D) or ii) with M-CSF (20 ng/ml) or GM-CSF (20 ng/ml) and NO2-OA (3 μM) in methylcellulose solution for 15 days (C). For schematic representation of BMC experimental setup see Figure 1A. (A) Quantification of adherent BMM numbers; n=4. (B) Assessment of BMM proliferation; n=5. (C) Quantification of nonadherent BMM colonies; n=3. A p value of less than 0.05 was considered significant and statistical comparison is presented as follows: a, b, c, d, and e represents the group of data statistically significant when compared to the data in the first (a), second (b), third (c), fourth (d), and fifth (e) bar, respectively. (D) Representative images of adherent BMMs; magnification 40x.
Figure 3
Figure 3. The effect of NO2-OA on M-CSFR, PU.1, C/EBPβ, and c-Myc expression in CSF-induced BMMs differentiated for 7 days
Native BMCs were isolated and cultivated with M-CSF (20 ng/ml) or GM-CSF (20 ng/ml) and NO2-OA (3 μM) in DMEM for 7 days. Adherent BMMs were then activated with LPS (500 ng/ml) and IFN-γ (50 ng/ml) or with IL-4 (20 ng/ml) for 24 h. For schematic representation of BMC experimental setup see Figure 1A. (A) Expression of M-CSFR and β-actin in adherent BMMs; Western blot pictures represent one of three individual experiments. (B) Assessment of M-CSFR expression in adherent BMMs; n=3. A p value of less than 0.05 was considered significant and statistical comparison is presented as follows: a, b, c, d, and e represents the group of data statistically significant when compared to the data in the first (a), second (b), third (c), fourth (d), and fifth (e) bar, respectively. (C) Expression of PU.1, C/EBPβ, c-Myc, and β-actin in adherent BMMs; representative Western blot pictures.
Figure 4
Figure 4. The effect of NO2-OA on M-CSFR, PI3K, STAT5, ERK 1/2, PU.1, and β-actin expression in BMMs differentiated for 2 days
Native BMCs were isolated and cultivated with M-CSF (20 ng/ml) or GM-CSF (20 ng/ml) and NO2-OA (3 μM) for 2 days. For schematic representation of BMC experimental setup see Figure 1A. (A) Expression of M-CSFR, PI3K, STAT5, ERK 1/2, PU.1, and β-actin in adherent BMMs; Western blot pictures represent one of three or four individual experiments. (B, C) Assessment of M-CSFR and PU.1 expression in adherent BMMs; n=3–4. A p value of less than 0.05 was considered significant and statistical comparison is presented as follows: a, b, c, d, and e represents the group of data statistically significant when compared to the data in the first (a), second (b), third (c), fourth (d), and fifth (e) bar, respectively. (D, E) Pearson correlation between M-CSFR or PU.1 expression and β-actin expression in adherent BMMs; n=3. A p value of less than 0.05 was considered significant. (F) Assessment of BMM proliferation; n=3. A p value of less than 0.05 was considered significant and statistical comparison is presented as follows: a, b, c, d, and e represents the group of data statistically significant when compared to the data in the first (a), second (b), third (c), fourth (d), and fifth (e) bar, respectively.
Figure 5
Figure 5. The effect of NO2-OA on STAT5, ERK, M-CSFR, and PI3K activation during early phase of differentiation
Native BMCs were isolated and treated with M-CSF (20 ng/ml) or GM-CSF (20 ng/ml) and NO2-OA (3 μM) for 30 min (A–C). RAW 264.7 macrophages were treated with M-CSF (50 ng/ml) or GM-CSF (50 ng/ml) and NO2-OA (1 μM) for 5 min (D–H). For schematic representation of experimental setups see Figure 1. (A) Activation of STAT5 (Tyr694) and ERK 1/2 (Thr202/Tyr204) in adherent and nonadherent BMCs; Western blot pictures represent one of four individual experiments. (B, C) Assessment of STAT5 and ERK 1/2 phosphorylation in adherent and nonadherent BMCs; n=4. (D) Activation of STAT5 (Tyr694), ERK 1/2 (Thr202/Tyr204), M-CSFR (Tyr723), and PI3K (Tyr458) in RAW 264.7; Western blot pictures represent one of four individual experiments. (E–H) Assessment of STAT5, ERK 1/2, M-CSFR, and PI3K phosphorylation in RAW 264.7; n=4. A p value of less than 0.05 was considered significant and statistical comparison is presented as follows: a, b, c, d, and e represents the group of data statistically significant when compared to the data in the first (a), second (b), third (c), fourth (d), and fifth (e) bar, respectively.

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