Targeting Cytokine Release Through the Differential Modulation of Nrf2 and NF-κB Pathways by Electrophilic/Non-Electrophilic Compounds

Abstract

The transcription factor Nrf2 coordinates a multifaceted response to various forms of stress and to inflammatory processes, maintaining a homeostatic intracellular environment. Nrf2 anti-inflammatory activity has been related to the crosstalk with the transcription factor NF-κB, a pivotal mediator of inflammatory responses and of multiple aspects of innate and adaptative immune functions. However, the underlying molecular basis has not been completely clarified. By combining into new chemical entities, the hydroxycinnamoyl motif from curcumin and the allyl mercaptan moiety of garlic organosulfur compounds, we tested a set of molecules, carrying (pro)electrophilic features responsible for the activation of the Nrf2 pathway, as valuable pharmacologic tools to dissect the mechanistic connection between Nrf2 and NF-κB. We investigated whether the activation of the Nrf2 pathway by (pro)electrophilic compounds may interfere with the secretion of pro-inflammatory cytokines, during immune stimulation, in a human immortalized monocyte-like cell line (THP-1). The capability of compounds to affect the NF-κB pathway was also evaluated. We assessed the compounds-mediated regulation of cytokine and chemokine release by using Luminex X-MAP^® technology in human primary peripheral blood mononuclear cells (PBMCs) upon LPS stimulation. We found that all compounds, also in the absence of electrophilic moieties, significantly suppressed the LPS-evoked secretion of pro-inflammatory cytokines such as TNFα and IL-1β, but not of IL-8, in THP-1 cells. A reduction in the release of pro-inflammatory mediators similar to that induced by the compounds was also observed after siRNA mediated-Nrf2 knockdown, thus indicating that the attenuation of cytokine secretion cannot be directly ascribed to the activation of Nrf2 signaling pathway. Moreover, all compounds, with the exception of compound 1, attenuated the LPS-induced activation of the NF-κB pathway, by reducing the upstream phosphorylation of IκB, the NF-κB nuclear translocation, as well as the activation of NF-κB promoter. In human PBMCs, compound 4 and CURC attenuated TNFα release as observed in THP-1 cells, and all compounds acting as Nrf2 inducers significantly decreased the levels of MCP-1/CCL2, as well as the release of the pro-inflammatory cytokine IL-12. Altogether, the compounds induced a differential modulation of innate immune cytokine release, by differently regulating Nrf2 and NF-κB intracellular signaling pathways.

Keywords: MCP-1; NF-κB; Nrf2; TNFα; antioxidant; curcumin; cytokine release; inflammation.

Figure 1

Cell viability in undifferentiated THP-1 exposed to compounds and CURC. THP-1 cells were treated with compounds 1–4 and CURC at the indicated concentrations for 24 h. Cell viability was assessed by MTT assay. Data are expressed as means of percentage of cell viability ± SEM. Dunnett’s multiple comparison test; **p < 0.01; ***p < 0.001 and ****p < 0.0001 versus CTR; n = 4.

Figure 2

Nrf2 nuclear translocation and modulation of HO-1 protein content in THP-1 cells. (A) THP-1 cells were treated with compounds 1–4 and CURC at a concentration of 5 μM for 3 h. After isolation, nuclear extracts were examined by Western blot analysis and Nrf2 expression was determined using an anti-Nrf2 antibody. Anti-lamin A/C was used as protein loading control. Results are shown as means of Nrf2/lamin A/C ratio ± SEM. Dunnett’s multiple comparison test; **p < 0.01 and ****p < 0.0001 versus CTR; n = 5–7. (B) Total protein extracts of THP-1 cells, treated with compounds 1–4 and CURC at the concentration of 5 μM for 24 h, were analyzed for HO-1 protein content by Western blot analysis. Anti-tubulin was used as protein loading control. Results are shown as means of HO-1/Tubulin ratio ± SEM. Dunnett’s multiple comparison test; **p < 0.01 and ****p < 0.0001 versus CTR; n = 7.

Figure 3

Modulation of TNFα and IL-8 release in LPS-stimulated THP-1 cells exposed to compounds. THP-1 cells were treated with compounds 1–4 and CURC at a concentration of 5 μM for 24 h, and then stimulated with 10 ng/mL LPS for 3 h. TNFα (A) and IL-8 (B) protein release was measured in THP-1 supernatants by ELISA. Data are presented as means of stimulation index ± SEM. Dunnett’s multiple comparison test; ****p < 0.0001 versus CTR; n = 5.

Figure 4

Modulation of IL-1β release in LPS-stimulated THP-1 cells. (A) IL-1β protein secretion was measured in THP-1 cell supernatants stimulated with LPS at the indicated concentrations for 3 h. At the end of the treatment, IL-1β protein release was assessed by ELISA. Data are presented as means of released picograms per mL (pg/mL) ± SEM. Dunnett’s multiple comparison test; **p < 0.01 and ****p < 0.0001 versus CTR; n = 3. (B) IL-1β protein release was measured in THP-1 cells supernatants, treated for 24 h with compounds 1–4 and CURC at a concentration of 5 μM and then stimulated with 1 μg/mL LPS for 3 h. The level of IL-1β was assessed by ELISA. Data are presented as means of stimulation index ± SEM. Dunnett’s multiple comparison test; **p < 0.01 and ***p < 0.001 versus CTR; n = 3.

Figure 5

Optimization of Nfr2-silenced THP-1 model (A) and effect of Nrf2-knockdown on modulation of TNFα release by compounds 1, 3 and 4, upon LPS stimulation (B). (A) THP-1 cells were treated either with vehicle (WT), scrambled or siRNA_Nrf2 for 24 h. Where indicated MG132 was added 4 h before the end of the experiment to block the proteasomal degradation of Nrf2. After treatments, Nrf2 expression was determined in total protein extracts by Western blot analysis using an anti-Nrf2 antibody. Anti-α-tubulin was used as protein loading control. Results are shown as means of Nrf2/α-Tubulin ratio ± SEM. Unpaired Student t-test; **p < 0.01; n = 3. (B) TNFα amount was measured in the supernatants of THP-1 Nrf2-knockdown cells, treated with compounds 1, 3, and 4 at a concentration of 5 μM for 24 h and then stimulated with 10 ng/mL LPS for 3 h. The protein secretion of TNFα was determined by ELISA. Data are shown as means of stimulation index ± SEM. Dunnett’s multiple comparison test; ***p < 0.001 and ****p < 0.0001 versus WT LPS; ^##p < 0.01 and ^####p < 0.0001 versus siRNA_Nrf2 LPS; n = 3.

Figure 6

Modulation of NF-κB pathway by compounds and CURC in LPS-stimulated THP-1 cells. (A) THP-1 cells were treated with 5 μM compounds 1–4 and CURC for 24 h and then stimulated with 10 ng/mL LPS for 45 min. After stimulation, p-IκBα expression was determined in total protein extracts by Western blot analysis, using an anti-p-IκBα antibody. Anti-IκBα (total) was used to normalize the data. Results are shown as means of p-IκBα/IκBα ratio ± SEM. Dunnett’s multiple comparison test; ***p < 0.001 and ****p < 0.0001 versus LPS; n = 5. (B) THP-1 cells were treated for 24 h with compounds 1–4 and CURC at a concentration of 5 μM and then stimulated with 10 ng/mL LPS for 90 min. After isolation, nuclear extracts were examined by Western blot analysis and NF-κB expression was determined using an anti-NF-κB antibody. Anti-lamin A/C was used as protein loading control. Results are shown as means of NF-κB/Lamin A/C ratio ± SEM. Dunnett’s multiple comparison test; *p < 0.05, **p < 0.01 and ***p < 0.001 versus LPS; n = 5. (C, D) THP-1 cells were transiently transfected with pGL4.32 [luc2P/NF-κB-RE/Hygro] Vector reporter construct, and subsequently treated with compounds 1–4 and CURC at a concentration of 5 μM for 24 h. After treatments, the cells were stimulated (D) or not (C) with 10 ng/mL LPS for 6 h. For each condition, luciferase activity was expressed as RLU% and compared to CTR values assumed at 100%. Results are shown as means ± SEM. Dunnett’s multiple comparison test; ***p < 0.001 and ****p < 0.0001 versus LPS; n = 3.

Figure 7

Differential modulation of Nrf2 and NF-κB intracellular signaling pathways by compounds. Electrophile 1, carrying both the catechol moiety (red) and the α,β-unsaturated carbonyl group (blue), is the most active Nrf2 inducer, while being devoid of activity on NF-kB pathway. Conversely, the non-electrophilic compound 4, synthesized to exclude eventual oxidative transformation of the methoxyphenol ring (green) of 3 into reactive metabolites, is the most potent NF-kB inhibitor, with no impact on Nrf2 activation.

Figure 8

Schematic representation of the effects induced by compounds 1–4 on Nrf2 and NF-κB pathways.