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. 2018 Nov 13;8(4):144.
doi: 10.3390/biom8040144.

Furanoid F-Acid F6 Uniquely Induces NETosis Compared to C16 and C18 Fatty Acids in Human Neutrophils

Meraj A Khan  1 , Cecil Pace-Asciak  2   3 , Jassim M Al-Hassan  4 , Mohammad Afzal  5 , Yuan Fang Liu  6 , Sosamma Oommen  7 , Bincy M Paul  8 , Divya Nair  9 , Nades Palaniyar  10   11
Affiliations

Furanoid F-Acid F6 Uniquely Induces NETosis Compared to C16 and C18 Fatty Acids in Human Neutrophils

Meraj A Khan et al. Biomolecules. .

Abstract

Various biomolecules induce neutrophil extracellular trap (NET) formation or NETosis. However, the effect of fatty acids on NETosis has not been clearly established. In this study, we focused on the NETosis-inducing ability of several lipid molecules. We extracted the lipid molecules present in Arabian Gulf catfish (Arius bilineatus, Val) skin gel, which has multiple therapeutic activities. Gas chromatography⁻mass spectrometry (GC-MS) analysis of the lipid fraction-3 from the gel with NETosis-inducing activity contained fatty acids including a furanoid F-acid (F6; 12,15-epoxy-13,14-dimethyleicosa-12,14-dienoic acid) and common long-chain fatty acids such as palmitic acid (PA; C16:0), palmitoleic acid (PO; C16:1), stearic acid (SA; C18:0), and oleic acid (OA; C18:1). Using pure molecules, we show that all of these fatty acids induce NETosis to different degrees in a dose-dependent fashion. Notably, F6 induces a unique form of NETosis that is rapid and induces reactive oxygen species (ROS) production by both NADPH oxidase (NOX) and mitochondria. F6 also induces citrullination of histone. By contrast, the common fatty acids (PA, PO, SA, and OA) only induce NOX-dependent NETosis. The activation of the kinases such as ERK (extracellular signal-regulated kinase) and JNK (c-Jun N-terminal kinase) is important for long-chain fatty acid-induced NETosis, whereas, in F-acid-induced NETosis, Akt is additionally needed. Nevertheless, NETosis induced by all of these compounds requires the final chromatin decondensation step of transcriptional firing. These findings are useful for understanding F-acid- and other fatty acid-induced NETosis and to establish the active ingredients with therapeutic potential for regulating diseases involving NET formation.

Keywords: MAP kinases; NADPH oxidase; NETosis; ROS; catfish lipids; citrullination of histone; furanoid F-acids (F6); long-chain fatty acids; transcription.

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Conflict of interest statement

The authors declare no conflict of interest. J.M.A.-H. holds patents on the use of the Arabian Gulf catfish gel for treating various diseases. C.P.-A. holds patents on inflammatory/anti-inflammatory lipid compounds unrelated to this work. N.P. filed a patent on the use of compounds unrelated to this work to suppress NETosis.

Figures

Figure 1
Figure 1
Lipid fraction Ft-3 from total lipid extract of catfish epidermal gel secretion induces neutrophil extracellular trap (NET) formation (NETosis) in human neutrophils. (A) SytoxGreen-based NETosis kinetics suggest that lipid fraction 3 (Ft-3), isolated from the dermal gel secretion of the catfish, induces NETosis in a dose-dependent manner (n = 5, * p-value < 0.05 comparing with control; two-way analysis of variance (ANOVA) with Bonferroni’s multiple comparison post-test); (B) Confocal images of neutrophils treated with Ft-3 showing DNA-fibers (blue) colocalized with MPO (green), confirming the formation of NETs by this lipid fraction (n = 4; scale bar, 50 µm). See Supplementary Figure S1 for the control NETosis induced by typical agonists phorbol 12-myristate 13-acetate (PMA) and A23187, and confocal images; (C) Gas chromatography–mass spectrometry (GC-MS) profile of the methylated fraction present in Ft-3; (D) Relative amounts of the studied lipid compounds that were present in Ft-3 fraction (chemical structures shown as the free acids), as determined by GC-MS. See Supplementary Figure S2 for confirmation of the compounds listed in (D).
Figure 2
Figure 2
Furanoid acid (F-acid) F6 contributes to most of the rapid NETosis effect exerted by lipid fraction-3 (Ft-3). The SytoxGreen NETosis kinetics analysis was performed in the neutrophils treated either with −ve control (ethanol), or with different concentrations of palmitic acid (PA; 16:0), palmitoleic acid (PO; 16:1), stearic acid (SA; 18:0), oleic acid (OA; 18:1), or furanoid acid (F6). (A–D) The % DNA release data show slow kinetics and a lower % DNA release in neutrophils activated with PA, PO, SA, or OA. By contrast, F6-treated neutrophils (E) showed rapid kinetics and a greater % DNA release (n = 3, * p-value < 0.05 compared with the control; two-way ANOVA with Bonferroni’s multiple comparison post -test); (F) The comparative analyses of the % DNA release between F6 and other fatty acids showed up to 65% more NET release by F6 at 120 and 240 min (n = 3, * p-value < 0.05; One-way ANOVA with Dunnett’s post-test, compared to control.
Figure 3
Figure 3
Confocal images confirm that the fatty acids induce NETosis. (A) Neutrophils were treated either with −ve control (ethanol), +ve controls (PMA, A23187), 5 μg/0.1 mL of PA, PO, SA, OA, or F6 for 4 h. After 4 h the cells were fixed, immunostained, and imaged for myeloperoxidase (MPO) and DNA. MPO co-localizes to NET-DNA generated by PA, PO, SA, OA, or F6. Images show abundant NET-DNA and MPO colocalization in F6-treated cells, while other fatty acids show a lower amount of NET-DNA staining, confirming the SytoxGreen kinetics data (Blue, DAPI (4′,6-diamidino-2-phenylindole) staining for DNA; green, MPO; n = 3; scale bar 50 μm). See Supplementary Figures S1 and S2 for −ve control and +ve controls treated with PMA and A23187 images, and single channel images; (B) Neutrophils with NETs were quantified (n = 3; *, p < 0.05 compared to control; One-way ANOVA followed by the Dunnett’s post-test).
Figure 4
Figure 4
F6 modulates NETosis through both a NOX-dependent and NOX-independent pathway. (A) To confirm the involvement of NOX-mediated ROS production, a DHR123 assay was performed. Neutrophils were treated with cytosolic ROS indicator dye DHR123 and activated either with ethanol (−ve control), 5 μg/0.1 mL of PA, PO, SA, OA, or F6. ROS generation was assessed over 60 min post activation by a plate reader; (B) To examine the generation of mitochondrial ROS, MitoSox dye was used during the plate reader assay. Neutrophils were treated with MitoSox, a mitochondrial ROS indicative dye for 15 min, and activated either by ethanol (−ve control), 5 μg/0.1 mL of PA, PO, SA, OA, or F6 and the kinetics were assessed (n = 3, * p < 0.05 comparing with control; two-way ANOVA with Bonferroni’s multiple comparison post -test); (C,D) To test the involvement of the NETosis pathway, neutrophils were pre-incubated either with NOX-inhibitor (DPI; 1 µM) or mitochondrial ROS inhibitor (DNP; 25 µM) for 1 h, followed by activation either with ethanol (−ve control), 5 μg/0.1 ml of PA, PO, SA, OA, or F6. The % DNA release data at 4 h of activation, show NETosis suppression by diphenyl iodonium (DPI) in neutrophils activated with PA, PO, SA, OA, or F6 (5 µg/0.1 mL), while (D) 2,4-dinitrophenol (DNP) (ATP uncoupler; mitochondrial ROS inhibitor) only suppresses the NETosis induced by F6 (n = 4, * p < 0.05 comparing between compound with and without inhibitors; independent t-test).
Figure 5
Figure 5
F6 induces histone H3 citrullination (CitH3) during NETosis. (A) Neutrophils were incubated either with ethanol (−ve control), A23187 (+ve control), 5 μg/0.1 mL of PA, PO, SA, OA, or F6 for 120 min followed by the fixation and immunostaining of the CitH3. Cells were stained for CitH3 (red) and DNA (DAPI; blue). Confocal fluorescence images show the substantial CitH3 immunostaining in neutrophils treated with F6, while the control and PA-, PO-, SA-, or OA-treated neutrophils lack CitH3 immunostaining (scale bar, 50 μm; images are representative of three independent experiments). See the Supplementary Figure S4 for the CitH3 in the positive control A23187-treated neutrophils; (B) CitH3 was quantified from the images using Image J (n = 3; * p < 0.05 compared to control; One-way ANOVA followed by Dunnett’s post-test); (C) Histone H3 was quantified from the images using Image J (n = 3; One-way ANOVA; the differences in these controls are not significantly different; images not shown).
Figure 6
Figure 6
Different sets of kinases are involved in PA-, PO-, SA-, OA-, or F6-mediated NETosis. Neutrophils were pre-incubated for 1 h either with different kinase inhibitors Akt inhibitor XI, FR180204, SB202190, SP600125 for Akt, ERK, P38, and JNK, respectively, followed by incubation with ethanol (−ve control), PA, PO, SA, OA, or F6. (A–E) The % DNA release data at the final time point (240 min) show background NETosis suppressed by ERK inhibitor while ERK and JNK inhibitors suppress the NETosis induced by PA, PO, SA or OA; (F) By contrast, F6-mediated NETosis was suppressed by Akt, ERK, and JNK inhibitors (n = 3, * p < 0.05 comparing between compound with and without inhibitors; One-way ANOVA with Dunnett’s post-test).
Figure 7
Figure 7
Fatty acid-induced NETosis involves transcriptional firing. (A–F) Neutrophils were pre-incubated 1 h either with actinomycin-D followed by incubation with ethanol (−ve control), PA, PO, SA, OA, or F6. NETosis kinetics data indicate that the transcriptional firing is important for all the tested compounds as actinomycin-D suppresses NETosis released by these fatty acids (n = 3–4, * p-value < 0.05 comparing between compound with and without inhibitors; two-way ANOVA with Bonferroni’s multiple comparison post-test).
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