Overview
Bioactive lipids are important molecular mediators in inflammatory settings. As inflammation, and particularly chronic inflammation, are important drivers of many chronic disorders, investigating the role of lipid mediators in inflammation could have a strong impact. Thus, our group aims to identify novel lipid mediators and lipid-related targets (i.e. receptors and enzymes) in inflammatory settings. Bioactive lipids are selected either based on reported effects or following their identification in lipidomics studies performed in our laboratory. The effects of these bioactive lipids are assessed, in vitro, ex vivo and in vivo, to determine their potential impact on inflammation (Figure 1). Once interesting lipids are selected and their effects identified, we turn to the identification of potential means to control their effects in vivo, for example by using agonists or antagonists of their receptors, or interfering with their metabolic pathways using pharmacological tools. A key aspect of our research strategy is to integrate the information gathered by quantifying the lipids and the information obtained by assessing their effects in our models. Over the years, this strategy allowed us to put forth several lipids and enzymes as important mediators of inflammation.
The biological activities of most lipid mediators are controlled by the balance between their production and degradation. Because most lipids have multiple metabolic pathways, measuring the expression or activities of the enzymes involved is often not enough to appreciate fully the overall activity of a lipid system. It is therefore crucial to quantify their endogenous levels. Thus, our group develops analytical methods to help understand the involvement of bioactive lipids in pathophysiological settings. We routinely use LC-MS methods allowing the relative quantification of a large number of lipids in a single run using an LTQ-Orbitrap (e.g. Guillemot-Legris et al. J. Neuroinfl., 2016). We also use validated methods for the absolute quantification of lipids of particular interest in our work (e.g. oxysterols, bile acids, endocannabinoids, …) (Mutemberezi et al. Anal Bioanal Chem, 2016; Masquelier et al. J Pharm Biomed Anal., 2016). Moreover, using a Xevo-TQS tandem quadrupole, in two ongoing theses, we are developing quantification methods for challenging lipids due to their low abundance (e.g. PGD2-G), as well as more general methods allowing the quantification of phospholipids and lysophospholipids (Pollet et al. Biomolecules, 2020). More recently, we developed a method allowing the quantification of linear and branched short chain fatty acids as well as TCA cycle metabolites. The method will be of large interest in the context of metabolic and microbiota studies (Paquot et al. in preparation).
Over the years, we have implemented in our laboratory several in vivo and in vitro models to study inflammation. We developed a recognized expertise in studying colon inflammation using models of inflammatory bowel diseases (IBD). These acute (e.g. DSS, TNBS, oxazolone) and chronic (e.g. cycles of DSS) models allow us to study the effect of modulating bioactive lipid levels on the evolution of colitis (e.g. Alhouayek et al. FASEB J 2015; Alhouayek et al. FASEB J., 2018; Guillemot-Legris et al. J. Crohns Colitis 2019). Other examples of models currently used in our research group are models of lung inflammation (LPS-induced inflammation, house dust mite-induced inflammation), of multiple sclerosis (EAE model in mice), and of inflammatory pain (carrageenan, LPS, capsaicin) (e.g. Bottemanne et al. FASEB J 2019; Mutemeberezi et al. J. Neuroinflammation 2018; Buisseret et al. BBA Mol. Cell Biol. Lipids, 2019; Orefice et al. Elife 2020, Buisseret et al. FASEB J. 2021; Bottemanne et al. Neurotherapeutics 2022). Besides the in vivo models, we rely also on in vitro models such as primary macrophages (alveolar and peritoneal) and neutrophils, primary glial cells, as well as tissue explants (e.g. colon and adipose tissue). In addition, our expanding network of clinical collaborations helps us improve the translational potential of our findings.
Research results
Endocannabinoids and related lipids
We and others have shown that several endocannabinoids and related lipids play an important role in inflammation.
We have shown that increasing 2-AG levels via MAGL inhibition reduces colitis in a partially CB1- and CB2-dependent manner (Alhouayek et al. FASEB J., 2011). We also showed that inhibition of ABHD6 increases 2-AG levels in some tissues, and has pronounced anti-inflammatory effects in vivo (Alhouayek et al. PNAS, 2013).
We showed that the ABHD6 inhibitor WWL70 strongly decreases all the hallmarks of lung inflammation (including neutrophil infiltration, cytokine secretion, and protein extravasation) induced by intratracheal administration of LPS, a model of acute lung injury. As macrophages and neutrophils are key cells in acute lung inflammation, we also studied ABHD6 inhibition on primary alveolar macrophages and neutrophils to explore their potential implication in the effects observed in vivo (Bottemanne et al. FASEB J, 2019).
We also demonstrated in vitro that ABHD6 inhibition in activated macrophages favors the production of PGD2-G, a bioactive lipid derived from 2-AG (Figure 2), that we found to have potent anti-inflammatory effects (Alhouayek et al. PNAS, 2013).
Demonstrating that PGD2-G has anti-inflammatory properties (Alhouayek et al. PNAS, 2013) opened several research projects in our research group. For instance, we demonstrated that administration of PGD2-G to mice having DSS-induced colitis allows for a strong reduction of the major hallmarks of the disease. Moreover, we could put forth the DP1 receptor as one of the receptors mediating the effects of PGD2-G in the colon (Alhouayek et al. FASEB J., 2018).
Additionally, in the context of a PhD thesis (supervisors Prof. des Rieux (ADDB) & Prof. Muccioli), we have developped an innovative formulation for PGD2-G, with the aim of increasing its delivery and efficacy in the CNS. Promising results obtained in a murine model of multiple sclerosis, the experimental autoimmune encephalomyelitis (EAE) model, support the feasibility and interest of this approach (Mwema A. et al. Nanomedicine. 2022).
Because inflammation can lead to painful sensations, we also asked whether PGD2-G could reduce inflammation-induced pain. We found that PGD2-G decreased hyperalgesia and edema in carrageenan-induced inflammatory pain, leading to a faster recovery. PGD2-G also decreased carrageenan-induced inflammatory markers in the paw as well as inflammatory cell recruitment. The effects of PGD2-G were independent from metabolite formation (PGD2 and 15d-PGJ2-G) or DP1 receptor activation in this model (Buisseret et al. BBA Mol. Cell Biol. Lipids, 2019). In a follow up study, we investigated the effects of the COX2-derived endocannabinoid metabolites (i.e. prostamides (PG-EAs) and prostaglandin glycerol esters (PG-Gs) on LPS-induced and carrageenan-induced hyperalgesia. Moreover, we compared in the same models the effect of the S- and R- enantiomers of flurbiprofen, the latter considered as a substrate selective COX inhibitor (Buisseret et al. FASEB J, 2021).
Besides these examples, our continuing efforts will contribute to highlight further the interest of modulating endocannabinoids and related lipids’ levels in inflammatory situations. For instance, we are actively collaborating with Dr Makriyannis and Dr Malamas (Northeastern University, Boston) on the characterization of the role of NAAA in inflammation (Alhouayek et al. FASEB J, 2015; Alhouayek et al. BBA Mol. Cell Biol. lipids, 2017).
Case in point, we assessed the effect of NAAA inhibition in the EAE mouse model of multiple sclerosis. Our results show that NAAA inhibition decreases inflammation and demyelination in this model (Figure 3). Interestingly, this was not the case with FAAH inhibition in the same setting. (Bottemanne et al. Neurotherapeutics 2021).
Oxysterols are considered as important lipid mediators, beyond their role in controlling lipid metabolism (Guillemot-Legris et al., Trends Mol. Med., 2016; Mutemberezi et al., Prog. Lipid Res. 2016).
We reported that colitis profoundly affects oxysterol levels, both in mice models and in human patients suffering from Crohn’s disease and ulcerative colitis. For some oxysterols we also found a link between the changes in oxysterol levels and alterations in the expression of key metabolic enzymes (e.g. cyp3A4) (Guillemot-Legris et al., J. Crohns Colitis, 2019). These compelling data were obtained thanks to a close collaboration with gastroenterologists from CHU UCL Namur and especially Dr Rahier. Because we are convinced that reporting lipid level alterations is a key step but hardly a goal per se, we are now investigating further the properties of oxysterols in colitis models. For instance, we reported already that the administration of 4β-hydroxycholesterol worsens the impact of DSS-induced colitis (Guillemot-Legris et al., J. Crohns Colitis, 2019).
In another series of experiments, using a mice model of diet-induced obesity, we found that obesity profoundly affects the levels of oxysterols in numerous tissues (Guillemot-Legris et al. Sci. Rep., 2016). We then investigated the consequences of these alterations on obesity in vivo and ex vivo on mice and human adipose tissue explants (Guillemot-Legris, Leloup et al., in preparation).
We also reported the effect of neuroinflammation on oxysterols and the potential effect of oxysterols on these models. Using in vitro models of primary glial cells, we found pronounced changes in oxysterol levels upon their activation with lipopolysaccharides (LPS). Moreover, several oxysterols were able to decrease LPS-induced activation of these primary glial cells (Mutemberezi et al. J. Neuroinfl., 2018).
Together these data are of interest when considering the phenomenon of obesity-induced neuroinflammation. Indeed, we (Guillemot-Legris et al. J. Neuroinfl., 2016) and others have shown that obesity leads to neuroinflammation (reviewed in Guillemot-Legris et al. Trends Neurosci., 2017). An exciting hypothesis is that changes in oxysterol levels might represent a potential explanation for the changes in inflammatory status found in the central and peripheral nervous system during obesity development.
Interestingly, the consequences of obesity on post-operative pain remain poorly explored. We showed that obesity affects the resolution of post-operative pain induced by hind paw incision and actually leads to a chronic pain state in mice. In this context, we found that following hind paw incision, high fat diet prolonged glial cell activation in the spinal cord. It also altered the expression of neurotrophins and increased inflammatory and endoplasmic reticulum stress markers in both central and peripheral nervous systems. Moreover, we show that a dietary intervention, leading to weight reduction and decreased inflammation, was able to restore normal pain sensitivity in mice suffering from chronic pain for more than 10 weeks. Thus, our data support the notion that obesity is responsible for pain chronicization (Guillemot-Legris et al. Brain Behav Immun, 2018). These findings are of clear importance in a clinical post-operative setting and we therefore aim to decipher further the underlying mechanisms, with several bioactive lipids as potential key mediators.
Conclusion
The examples of our current research described here clearly support the importance of increasing our understanding of bioactive lipid signaling in inflammation to put forth novel innovative therapeutic strategies.