resolvin D5

Abstract

One of the omega-3 essential fatty acids, docosahexaenoic acid (DHA), is a significant constituent of the cell membrane and the precursor of several potent lipid mediators. These mediators are considered to be important in preventing or treating several diseases. Resolvin D5, an oxidized lipid mediator derived from DHA, has been known to exert anti-inflammatory effects. However, the detailed mechanism underlying these effects has not yet been elucidated in human monocytic THP-1 cells. In the present study, we investigated the effects of resolvin D5 on inflammation-related signaling pathways, including the extracellular signal-regulated kinase (ERK)-nuclear factor (NF)-κB signaling pathway. Resolvin D5 downregulated the production of interleukin (IL)-6 and chemokine (C-C motif) ligand 5 (CCL5). Additionally, these inhibitory effects were found to be modulated by mitogen-activated protein kinase (MAPK) and NF-κB in lipopolysaccharide (LPS)-treated THP-1 cells. Resolvin D5 inhibited the LPS-stimulated phosphorylation of ERK and translocation of p65 and p50 into the nucleus, resulting in the inhibition of IL-6 and CCL5 production. These results revealed that resolvin D5 exerts anti-inflammatory effects in LPS-treated THP-1 cells by regulating the phosphorylation of ERK and nuclear translocation of NF-κB.

Effects of Resolvin D5 on IL-6 and CCL5 Production in LPS-Treated THP-1 Cells

To identify whether resolvin D5 has anti-inflammatory effects in LPS-stimulated THP-1 cells, the cells were pre-treated with resolvin D5 for 1 h, and then with LPS for 24 h. The RT-qPCR analyses revealed that at concentrations of 20–40 μM, resolvin D5 significantly inhibited the IL-6 and CCL5 mRNA levels following LPS treatment (). There was no contamination of LPS during resolvin D5 purification because resolvin D5 was extracted from the reaction solution not from the recombinant cells, and resolvin D5 itself did not induce IL-6 or CCL5 mRNA levels (data not shown), suggesting that there was no immunotoxicity in the resolvin D5-treated cells. Furthermore, ELISA results also showed that the secreted protein level of IL-6 in the THP-1 cell supernatants was reduced by resolvin D5. However, unlike the IL-6 level, the secreted CCL5 level was not inhibited notably (). Taking all results from and , it was revealed that resolvin D5 neither exerted cytotoxicity nor immunotoxicity and suppressed the expression or production of pro-inflammatory cytokine IL-6 and chemokine CCL5.

Effects of Resolvin D5 on MAPK Signaling in LPS-Treated THP-1 Cells

To identify whether resolvin D5 alters the LPS stimulation of MAPK signaling, several key MAPK members were examined including ERK, JNK, and p38. The phosphorylation of ERK was attenuated by resolvin D5 dose-dependently in THP-1 cells, while that of JNK and p38 was not altered (). Subsequently, the production of CCL5 and IL-6 was evaluated after the treatment with the ERK inhibitor, PD98059, to determine whether CCL5 and IL-6 expressions were modulated via ERK. Pre-treatment with PD98059 (20 μM) for 1 h before LPS treatment in the absence or presence of resolvin D5 (40 μM) attenuated the production of CCL5 and IL-6. These data indicated that LPS-induced CCL5 and IL-6 secretion was reduced by resolvin D5 via inhibition of the ERK/MAPK signaling pathway ().

Effects of Resolvin D5 on Transcription Factor Activities

Transcription factors are involved in the regulation of the expression of various genes via their phosphorylation and translocation. Therefore, the translocation of several transcription factors from the cytosol to the nucleus was investigated. NF-κB is a well-known transcription factor; the nuclear fractionation results revealed that the LPS-induced translocation of NF-κB was decreased by resolvin D5 ().

Discussion

The ubiquitous transcription factor NF-κB is important in inflammation, and responsible for the modulation of genes encoding proinflammatory cytokines such as TNF-α, IL-1, IL-6, IL-12, inducible enzymes such as cyclooxygenase (COX)-2, and inducible nitric oxide synthase (iNOS) [, ]. It is activated via signaling cascades triggered by extracellular stimuli such as LPS. Cell culture studies have shown that the ω-3 fatty acids EPA and DHA inhibit the LPS-induced production of TNF-α, IL-1, IL-6, COX-2, and iNOS in monocytes and macrophages. Additionally, ω-3 fatty acids inhibit the LPS-induced activation of NF-κB [-]. Resolvin D5, a lipid mediator that is biosynthesized from DHA, also inhibited the expression of inflammatory mediators via the NF-κB pathway in LPS-stimulated THP-1 cells. The nuclear translocation of the NF-κB subunits, p65 and p50, were upregulated after LPS stimulation; however, resolvin D5 treatment attenuated this translocation (). Other transcription factors involved in inflammation, such as activated protein-1 (AP-1) and STAT3, were also examined; resolvin D5 did not affect the translocation of these transcription factors. To determine the relationship between NF-κB and ERK, LPS-treated THP-1 cells were treated with the ERK inhibitor PD98059 in the presence or absence of resolvin D5. Indeed, the translocation of p65 was inhibited by PD98059, and it was revealed that NF-κB was regulated via the ERK signaling pathway (). To confirm the effects of resolvin D5 on NF-κB transcriptional activation, a transient transfection and luciferase assay were performed. The results revealed an effective inhibitory effect of resolvin D5 on the transcriptional level of NF-κB after LPS stimulation ().