Squalene targets pro- and anti-inflammatory mediators and pathways to modulate over-activation of neutrophils, monocytes and macrophages

Highlights

• Squalene has anti-inflammatory activities on LPS-mediated inflammatory response.

• Squalene targets pro-inflammatory mediators such as TLR4, iNOS, COX-2 or MPO.

• Squalene enhanced expression of anti-inflammatory enzymes (HO-1).

• Squalene modulated Nrf2, NF-κB, MAPKs, MMPs and PPARγ signalling pathways.

• Squalene prevents the over-activation of neutrophils/monocytes/macrophages.

Abstract

Squalene is a natural triterpene consumed as an integral part of the human diet. Increasing evidence demonstrates that squalene has antioxidant, cardioprotective and anti-carcinogenic activities. Nevertheless, its anti-inflammatory properties remain unclear. The effects of squalene on lipopolysaccharide (LPS)-mediated inflammatory response in murine macrophages and human monocytes and neutrophils were investigated. Squalene reduced intracellular levels of ROS, nitrites, cytokines (TNF-α, IL-1β, IL-6 and IFN-γ) and pro-inflammatory enzymes (iNOS, COX-2 and MPO), including a decreased expression of TLR4 and key proteins for signalling pathways mediated by NF-κB (IκBα), MAPKs (JNK) and MMPs (1, 3 and 9). In addition, squalene enhanced expression levels of anti-inflammatory enzymes (HO-1) and transcription factors (Nrf2 and PPARγ). This study establishes that squalene has significant potential for management of inflammatory conditions characterized by an over-activation of neutrophils/monocytes/macrophages and thereby for the efficient termination of the inflammatory response.

Introduction

Squalene (2,6,10,15,19,23-hexamethyl tetracosahexaene) is a natural lipid belonging to the terpenoid family, containing six isoprene units, and a biochemical precursor of cholesterol biosynthesis and other biological substances. Besides, to be synthesized in the liver and the skin of humans, squalene is widely present in nature, especially in wheat germ, rice bran, shark liver and olive oils. Therefore, it is consumed as an integral part of the human diet. Up-to-date, anticancer, antioxidant, drug carrier, detoxifier, skin hydrating and emollient activities of squalene have been reported both in animal models and in vitro environments (Kim, Karadeniz, 2012, Reddy, Couvreur, 2009). In fact, squalene is a highly effective antioxidant acting as a direct reactive oxygen species (ROS) scavenging agent reducing intracellular oxidant stress and also protects human skin surfaces from lipid peroxidation as a quencher of singlet oxygen (Kabuto, Yamanushi, Janjua, Takayama, & Mankura, 2013). Moreover, this natural isoprenoid has been shown to ameliorate atherosclerotic lesions through the reduction of CD36 scavenger receptor expression in macrophages (Granados-Principal et al., 2012), in addition to important cardioprotective effects in several experimental models mainly by blocking lipid peroxidation (Lou-Bonafonte et al, 2012, Sabeena Farvin et al, 2004). However, although the importance as a powerful antioxidant agent of squalene has been largely described (Kim & Karadeniz, 2012), its anti-inflammatory properties still remain unclear.

Monocytes and macrophages are closely related to phagocytic cells that cooperate during the onset, progression and resolution of inflammation (Soehnlein & Lindbom, 2010). Pro-inflammatory, metabolic and immune stimuli increase recruitment of monocytes from blood vessels to peripheral sites where the differentiation into macrophages and dendritic cells occurs, contributing to host defence, tissue remodelling and repair (Gordon, Taylor, 2005, Shi, Pamer, 2011). Furthermore, neutrophils, as a key component of the inflammatory response, generate chemotactic signals that attract monocytes and dendritic cells, and influence whether macrophages differentiate to a predominantly pro- or anti-inflammatory state (Nathan, 2006). In these cell types, lipopolysaccharide (LPS) acts as an endotoxin by its binding to the CD14/TLR4/MD2 receptor complex, which promotes the secretion of pro-inflammatory cytokines such as tumour necrosis factor alpha (TNF-α), interleukin (IL)-8, IL-6 or interferon gamma (IFN)-γ among others (Chanput, Mes, Vreeburg, Savelkoul, & Wichers, 2010). Moreover, some transcription factors such as the peroxisome proliferator-activated receptor gamma (PPARγ), nuclear transcription factor (NF)-kB, nuclear factor-E2-related factor-2 (Nrf2) or mitogen-activated protein kinase (MAPK) family (Jung et al, 2010, Jung et al, 2010; Kang & Kim, 2013) have been shown to be major effectors in the LPS-induced inflammatory response through the induction of several pro-inflammatory enzymes including inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX)-2 (Takahashi, Kozaki, Yatabe, Achiwa, & Hida, 2002) or antioxidant enzymes, such as haem oxygenase 1 (HO-1) (Alexander, Mathie, & Peters, 2011). Importantly, matrix metalloproteinases (MMPs) regulate aspects of immune cell development, effector function, migration and ligand–receptor interactions and activate signal transduction pathways that control cytokine biosynthesis and direct systemic inflammation or barrier immunity. For this reason, the stimulation with LPS constitutes an excellent model for the screening and subsequent evaluation of the effects of candidate drugs on the inflammatory pathway (Sanchez Miranda, Perez Ramos, Fresan Orozco, Zavala Sanchez, & Perez Gutierrez, 2013).

Therefore, we investigated the anti-inflammatory activity of squalene on LPS-stimulated murine peritoneal macrophages and human blood monocytes and neutrophils. In this model, redox changes, myeloperoxidase (MPO) activity, pro-inflammatory (IL-1β, IL-6, TNF-α and IFN-γ) and anti-inflammatory (IL-10) cytokines production, gene/protein expression of pro-inflammatory (iNOS and COX-2) and anti-inflammatory (HO-1) enzymes were measured. NF-kB, Nrf2, MAPKs, MMPs (MMP-1, MMP-3 and MMP-9) and PPARγ signalling pathways were also explored to understand the underlying mechanisms by which squalene may prevent inflammation.