In situ generation, metabolism and immunomodulatory signaling actions of nitro-conjugated linoleic acid in a murine model of inflammation

Abstract

Conjugated linoleic acid (CLA) is a prime substrate for intra-gastric nitration giving rise to the formation of nitro-conjugated linoleic acid (NO₂-CLA). Herein, NO₂-CLA generation is demonstrated within the context of acute inflammatory responses both in vitro and in vivo. Macrophage activation resulted in dose- and time-dependent CLA nitration and also in the production of secondary electrophilic and non-electrophilic derivatives. Both exogenous NO₂-CLA as well as that generated in situ, attenuated NF-κB-dependent gene expression, decreased pro-inflammatory cytokine production and up-regulated Nrf2-regulated proteins. Importantly, both CLA nitration and the corresponding downstream anti-inflammatory actions of NO₂-CLA were recapitulated in a mouse peritonitis model where NO₂-CLA administration decreased pro-inflammatory cytokines and inhibited leukocyte recruitment. Taken together, our results demonstrate that the formation of NO₂-CLA has the potential to function as an adaptive response capable of not only modulating inflammation amplitude but also protecting neighboring tissues via the expression of Nrf2-dependent genes.

Keywords: Electrophile; Inflammation; Macrophage; NF-κB; Nitration; Nitro-fatty acid; Nrf2.

Graphical abstract

Fig. 1

LPS/IFN-γ activated macrophages generate NO₂-CLA. RAW264.7 were activated in the presence of 50 µM CLA for 24 h and NO₂-FA formation measured in media. A) MRM analysis for synthetic mixed ¹⁵NO₂-CLA (top), synthetic 9-¹⁵NO₂-CLA (middle) and RAW264.7-generated NO₂-CLA (bottom). B) Detection of dihydro-NO₂-CLA by MRM analysis. Panels show mixed dihydro-¹⁵NO₂-CLA (top), dihydro-9-¹⁵NO₂-CLA (middle) and RAW264.7-derived dihydro-NO₂-CLA (bottom). Peaks 1 and 2 indicate the corresponding 12-NO₂ and 9-NO₂ positional isomers. C-D) Dose- and time-dependence of NO₂-FA formation by activated RAW264.7 cells. Data are represented as mean ± SEM (n = 3–6). E) Dose-response for NO₂-FA formation by M1, M2 and M0 polarized BMDM. F) NO₂-FA formation in wild-type (WT) BMDM, WT in the presence of the iNOS inhibitor 1400 W (100 µM) or in iNOS^-/- BMDM. Dose responses were established at 20 h and time courses were generated using 50 µM CLA. Data are represented as mean ± SEM (n = 4). ND: Not detected. LOQ: Limit of quantification.

Fig. 2

In situgenerated NO₂-CLA is metabolized by macrophages. A) Representative LC-MS/MS traces obtained after 24 h incubation of LPS/IFN-γ activated RAW264 cells with synthetic NO₂-CLA (5 µM, top) or in situ formed NO₂-CLA (from 50 µM CLA, middle). Blue traces correspond to unsaturated nitroalkene species and red traces correspond to saturated dihydro metabolites. Unsaturated metabolites retain electrophilic reactivity as indicated by their complete consumption upon incubation with 500 mM BME for 2 h before extraction as opposed to dihydro-derivatives which are not affected by BME treatment (bottom). Dose and time-dependence for the generation of one- (B, D) and two-round (C, E) β-oxidation metabolites of NO₂-CLA and dihydro-NO₂-CLA derivatives by LPS/IFN-γ activated RAW264.7 cells. Data are means ± SEM (n = 3–6).

Fig. 3

Differential accumulation and metabolism of NO₂-CLA positional isomers. Time-dependent generation of 9- and 12-NO₂-CLA (A) and their corresponding reduction metabolites (B) by LPS/IFNγ activated RAW264.7 cells. Data are means ± SD (n = 3–6). Synthetic 12-NO₂-CLA is more efficiently metabolized by RAW264.7 cells (C) leading to a more prominent accumulation of dihydro-12-NO₂-CLA (D). Data are means ± SD (n = 3) * p < 0.05 as determined by two-way ANOVA.

Fig. 4

NO₂-CLA inhibits NF-κB signaling. A) Dose-dependent inhibition of iNOS expression in LPS/IFN-γ activated RAW264.7 cells by synthetic 9- and 12-NO₂-CLA. B-C) Inhibition of IL-6 and MCP-1 secretion by activated RAW264.7 cells after 24 h co-treatment with CLA (10 µM) or the indicated NO₂-CLA doses. Data are shown as means ± SEM. * p < 0.05 vs. no NO₂-CLA controls as determined by one-way ANOVA and Dunnett's multiple comparisons test (n = 3–4). D-E) Dose-dependent NO₂-CLA inhibition of IL-6 and iNOS gene expression in LPS/IFN-γ activated BMDM. Data are means ± SEM. * p < 0.05 vs. no NO₂-CLA controls as determined by one-way ANOVA and Dunnett's multiple comparisons test (n = 4). F-G) CLA inhibition of IL-6 and iNOS expression in LPS/IFN-γ activated BMDM is dependent on iNOS activity as demonstrated by the use of iNOS^-/- BMDM and the iNOS inhibitor 1400 W (100 µM). Data is presented as mean ± SEM. * p < 0.05 as determined by one-way ANOVA and Dunnett's multiple comparisons test vs. corresponding no CLA controls (n = 4).

Fig. 5

NO₂-CLA activates Nrf2 signaling. A) Dose-dependent induction of HO-1 and NQO-1 expression by 9- and 12-NO₂-CLA in RAW264.7 cells. M: Methanol vehicle control. C: CLA (10 µM). B-C) NO₂-CLA induces HO-1 and NQO-1 expression in LPS/IFN-γ activated BMDM. Data are means ± SEM. * p < 0.05 as determined by one-way ANOVA and Dunnett's multiple comparisons test vs. no NO₂-CLA controls (n =4). CLA treatment (10 µM) had no effect on mRNA levels. D) HO-1 and NQO-1 protein induction by NO₂-CLA or CLA (10 µM) treatment in in LPS/IFN-γ activated BMDM.

Fig. 6

Zymosan-A injection induces CLA nitrationin vivo. A) Representative MRM traces showing total (MRM: 324/46, top); and specific 9- (324/168 dark grey, middle), 12-NO₂-CLA (324/157, light grey, middle) formation. Synthetic ¹⁵N-labeled NO₂-CLA standard (bottom) showing chromatographic co-elution with the endogenous products in top and middle panels. B) Time course of peritoneal NO₂-CLA formation. Box and whiskers plot indicate median, 25th to 75th percentiles and range. * p < 0.05 versus 1 h time-point as determined by one-way ANOVA and Dunnett's multiple comparisons test (n = 6 per time point).

Fig. 7

NO₂-CLA modulates acute inflammatory responsesin vivo. A) Representative flow cytometry dot plot of exudate cells from vehicle (PEG), CLA (2.5 mg/mouse) or NO₂-CLA (2.5 mg/kg). The dynamics of PMN infiltration are indicated in the gated population 12 h and 24 h post zymosan-A i.p. injection. B) Exudate PMN numbers were calculated. Results are mean ± SEM (n = 6 per time point). NO₂-CLA reduced PMN numbers as determined by two-way ANOVA and Holm-Sidak multiple comparisons test, * p < 0.01 and ** p < 0.0001 vs. PEG; # p < 0.001 and ## p < 0.0001 vs. CLA. C) Pro-inflammatory cytokines IL-6 and D) MCP-1 levels at 12 h. Results are mean ± SEM, (n= 6 per time point). * p < 0.05 as determined by one-way ANOVA.

Scheme 1

Proposed model for NO₂-CLA formation and signaling actions under inflammatory conditions. Macrophage activation leads to iNOS expression, ^•NO-derived species formation and in situ NO₂-CLA generation. The electrophilic reactivity of the nitroalkene moiety in NO₂-CLA results in Nrf2 activation and in the inhibition of NF-κB dependent gene expression, thus inducing antioxidant/cytoprotective responses and limiting both reactive species generation and leukocyte recruitment to sites of inflammation. In addition to mediating CLA nitration, macrophages also regulate NO₂-CLA levels by β-oxidation and nitroalkene reduction to non-electrophilic nitroalkane derivatives.