Due to their capacity of multi-lineage differentiation and immunomodulation, MSCs have got great interest in various cell-based therapies, including the treatment or prevention of acute graft versus host disease, autoimmune diseases, and solid organ transplantation rejection. Multiple mechanisms underlying the MSC immunosuppressive effect have been elucidated, however, understanding of the underlying mechanisms remains far incomplete. As far as we know, to date, these MSC properties are largely due to the paracrine effects from the secretion of biologic factors.
Among the secreted factors, prostaglandins (PGs), a category of oxylipins, have been long associated with inflammation and targeted by cyclooxygenase (COX) inhibitors to treat inflammatory diseases. PGs are traditionally considered proinflammatory oxylipins, but a previous study (Tsoyi et al., 2013) showed that MSC, but not fibroblasts exposed to carbon monoxide, with docosahexaenoic acid substrate, produced proresolving oxylipins, particularly D-series resolvins, contributing to attenuating systemic inflammation and sepsis.
It is not the first time that oxylipins were reported to be involved in MSC's immuno-modulatory function. In another report, experiments provide evidence that human MSCs can promote the resolution of acute lung injury in mice, in part through the pro-resolving oxylipin, LXA4, and LXA4 itself could be considered as a therapeutic for acute respiratory distress syndrome (ARDS), mediating part of the potential therapeutic effects of MSCs (Fang et al., 2015). Furthermore, one of these MSC derived prostaglandins, prostaglandin E2 (PGE2), has been recognized as having anti-inflammatory and immunomodulatory effects. PGE2 initiates lipid mediator class switching, which results in a decrease in 5-lipoxygenase (LOX)-derived pro-inflammatory lipid mediators and an increase in 15-LOX-derived pro-resolving oxylipins (Levy et al., 2001).
It has been indicated in a previous review that inflammation and lipid signaling are intertwined modulators of homeostasis and immunity. Oxylipins and other lipid mediators’ function to regulate inflammatory responses both positively and negatively. Conversely, inflammatory signaling can also significantly affect the lipid oxidation process in MSCs. Furthermore, injurious stimuli (i.e., infection and inflammation) can induce the acute-phase response and lead to multiple alterations in the metabolism of lipids. Thus, it is critical to perform a detailed characterization of the altered oxylipin metabolism depending on inflammation in MSCs. And the findings may lay a great foundation for MSCs based therapies and further determine the ways to improve the therapeutic potential of MSCs.
A more detailed investigation of the downstream metabolic products of PGs and other polyunsaturated fatty acids (PUFAs) in ADSC is needed. To our knowledge, ADSCs derived oxylipins have never been comprehensively analyzed. Oxylipins are biosynthesized by enzymatic and non-enzymatic oxidation of polyunsaturated fatty acids (PUFAs), which form eicosanoids from the metabolism of 20-carbon arachidonic acid (AA) and eicosapentaenoic acid (EPA), 22-carbon docosanoids from docosahexaenoic acid (DHA), and 18-carbon oxidized metabolites of linoleic acid (LA) and linolenic acid (ALA). After decades of research, numerous biologic functions have been linked to them and many others are still being elucidated. Quantification of eicosanoids and oxylipins derived from other PUFAs in biological samples is crucial for a better understanding of the biology of these lipid mediators.
However, the analytical determination of these metabolites is challenging. Usually, the endogenous concentrations of these oxylipins are very low and they are instable in biological samples. The structural similarity between members of the oxylipin metabolome, particularly the isomers, adds difficulty in chromatographic separation of these compounds. The ideal analytical method should be able to simultaneously profile and determine all relevant species in a single analytical assay, because these compounds are produced within the same cascade and they all are part of a complex regulatory network. Therefore, a highly sensitive and selective analytical method is required for the comprehensive study of this class of lipids.
We utilized a sensitive and selective ultraperformance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method for the simultaneous profiling of 71 targeted oxylipins in ADSCs with or without proinflammatory stimulation. These quantified oxylipins are derived from six major n-6 and n-3 polyunsaturated fatty acids (PUFAs) which serve as oxylipin precursors, including arachidonic (AA), linoleic (LA), alpha-linolenic (ALA), (DGLA) eicosapentaenoic (EPA), and docosahexaenoic (DHA) acids. The targeted oxylipin panel provides a broad coverage of lipid mediators and pathway markers generated from cyclooxygenases, lipoxygenases, cytochrome P450 epoxygenases/hydroxylases, and non-enzymatic oxidation pathways. The limits of quantification (LOQ) and detection (LOD) were set at a signal-to-noise ratio (S/N) of 10 and 3, respectively.
Detection and quantification of critical oxylipins in inflammation are essential for immunological disease surveillance and therapeutic target discovery. As we know, MSCs express cyclooxygenase 1 and 2 and produce prostaglandin E2 (PGE2) constitutively. In accordance with our expectations, PGE2 was found highly produced in both stimulated or unstimulated cell samples (~5.88-fold overexpressed in stimulated cells), probably serving as the major precursor and switcher of many downstream oxylipins.
An eicosapentaenoic acid (EPA)-derived eicosanoid, 15-HEPE, which associates with anti-inflammatory properties, was found apparently upproduced (~2.2-fold) in inflammatory-stimulated ADSCs. Earlier studies have shown that 15-HEPE and 15-hydroxyeicosatetraenoic acid (15-HETE) inhibit 5-LOX activity and proinflammatory prostaglandin synthetase (Miller et al., 1988). The result in a decrease in 5-lipoxygenase (LOX)-derived proinflammatory lipid mediators and an increase in 15-LOX-derived proresolving oxylipins, which resembles the initiation of resolution of inflammation. Additionally, it was reported that 15-HETE and 15-HEPE are converted to lipoxins via 5-hydroperoxy, a 15-hydroxy acid derivative (Serhan, 2005). These lipid mediators, actively biosynthesized in the resolution phase of acute inflammation, control the duration and magnitude of inflammation. In a recent study, Kento Sawane and college identified 15-HEPE as a novel EPA-derived anti-inflammatory and/or proresolution lipid mediator in a PPARγ-dependent manner by 15-lipoxygenase activity in eosinophils (Sawane et al., 2019). Targeting the transcription factor peroxisome proliferator-activated receptor gamma (PPAR-γ), an important inflammation regulator, may serve as promising therapeutics to treat inflammatory disorders. Interestingly, a switch towards 15-lipoxygenase products, such as the pro-resolving lipid precursors 15-HEPE and 17-HDHA was observed after 24 h inflammatory stimulation in monocyte-derived macrophages.
Meanwhile, the levels of 7-HDHA, the pathway marker for D-series resolvins, were significantly higher in ADSCs subjects after stimulation. However, none of the D-series resolvins (RvD1, RvD5, RvD2, RvD3) were detected in all cell groups. Hence, we speculated that 7-HDHA might be the substrate for transcellular biosynthesis of D-series resolvins in other pro-solving immune cells such as neutrophils or macrophages, which needs further investigation.
In conclusion, we introduced a targeted UPLC- MS/MS lipid mediator metabolomics strategy for the detection, identification, and quantification of 71 oxylipins derived from LA (n=6), AA (n=31), ALA (n=3), EPA (n=15), DGLA (n=5), DHA (n=8), and others (n=3) in MSCs under pro-inflammatory stimulation.
The activation with proinflammatory cytokines of ADSCs induced the significant overexpression of nine oxylipins and the underexpression of none oxylipin, which induces profound changes in the downstream oxylipin functioning in the resolution of inflammation. These data indicate that MSCs might contribute to the resolution of inflammation in time by the production of proresolving lipids after an initial inflammatory stimulus. The results reported here make the first step towards a comprehensive characterization of MSCs derived oxylipins as potentially modulators of inflammation and immune.