Oxylipin Profiles as Functional Characteristics of Acute Inflammatory Responses in Astrocytes Pre-Treated with IL-4, IL-10, or LPS

Abstract

Functional phenotypes, which cells can acquire depending on the microenvironment, are currently the focus of investigations into new anti-inflammatory therapeutic approaches. Glial cells, microglia, and astrocytes are major participants in neuroinflammation, but their roles differ, as microglia are cells of mesodermal origin, while astrocytes are cells of ectodermal origin. The inflammatory phenotype of cells can be modulated by ω-6- and ω-3-polyunsaturated fatty acid-derived oxylipins, although data on changes in oxylipin profiles in different cell adaptations to pro- and anti-inflammatory stimuli are scarce. Our study aimed to compare UPLC-MS/MS-measured oxylipin profiles in various rat astrocyte adaptation states. We used cells treated for 24 h with lipopolysaccharide (LPS) for classical pro-inflammatory adaptation and with interleukin 4 (IL-4) or 10 (IL-10) for alternative anti-inflammatory adaptation, with the resulting phenotypes characterized by quantitative real-time PCR (RT-PCR). We also tested long-term, low-concentration LPS treatment (endotoxin treatment) as a model of astrocyte adaptations. The functional response of astrocytes was estimated by acute (4 h) LPS-induced cell reactivity, measured by gene expression markers and oxylipin synthesis. We discovered that, as well as gene markers, oxylipin profiles can serve as markers of pro- (A1-like) or anti-inflammatory (A2-like) adaptations. We observed predominant involvement of ω-6 polyunsaturated fatty acid (PUFA) and the cyclooxygenase branch for classical (LPS) pro-inflammatory adaptations and ω-3 PUFA and the lipoxygenase branch for alternative (IL-4) anti-inflammatory adaptations. Treatment with IL-4, but not IL-10, primes the ability of astrocytes to activate the innate immunity signaling pathways in response to LPS. Endotoxin-treated astrocytes provide an alternative anti-inflammatory adaptation, which makes cells less sensitive to acute LPS stimulation than the IL-4 induced adaptation. Taken together, the data reveal that oxylipin profiles associate with different states of polarization to generate a pro-inflammatory or anti-inflammatory phenotype. This association manifests itself both in native cells and in their responses to a pro-inflammatory stimulus.

Keywords: IL-10; LPS; eicosanoids; endotoxin tolerance; inflammation; interleukins IL-4; oxylipins; polarization; rat astrocytes.

Figure 1

Identification of astrocytes polarization state in primary rat astrocyte cultures based on gene-expression profiles. (A) Heat map representation; (B) quantitative analysis of expression data. The primary rat astrocyte cultures were treated with lipopolysaccharide (LPS, 100 ng/mL), interleukin 4 (IL-4, 10 ng/mL) or interleukin10 (IL-10, 20 ng/mL) for 24 h or adapted to endotoxin in the tolerance model (ET, LPS 10 ng/mL, 48h), and the total RNAs were isolated. The mRNA levels of polarization state markers (C3, GBP2, IL-1β, iNOS, TNFα, CXCL10, IL-10, MRC1, FIZZ1 and Ym1) were determined by quantitative real-time PCR (RT-PCR). The values are normalized to β-actin mRNA levels. The results are expressed as fold-changes, relative to untreated cells. The values represent a mean ± SEM from three independent experiments. * p < 0.05, compared with the unstimulated cells.

Figure 2

Effects of polarized astrocytes on the inflammatory response. The primary rat astrocyte cultures were pretreated with IL-4 (10 ng/mL) or IL-10 (20 ng/mL) for 24 h or adapted to endotoxin in the tolerance model (LPS 10 ng/mL, 48h, ET) and then stimulated with LPS (100 ng/mL) for 4h (the level of gene expression under LPS stimulation is shown by the dotted line). (A), (C): the mRNA levels were determined by real-time RT-PCR. The values are normalized to β-actin mRNA levels. (B): the TNFα and IL-1β protein release measured by ELISA in supernatant samples. The results are expressed as fold-changes, relative to the LPS-stimulated cells. (C): the results are represented as lg, relative to the control cells. The values represent a mean ± SEM from three independent experiments. * p < 0.05, compared with the LPS-stimulated cells, # p < 0.05, compared with the unstimulated cells.

Figure 3

Effect of astrocyte polarization on the oxylipins release. A heat map representation of oxylipin production of n-6 and n-3 fatty acid-derived lipid mediators. (A) The primary rat astrocytes were treated with IL-4 (10 ng/mL), IL-10 (20 ng/mL), or LPS (100 ng/mL) for 24 h or adapted to endotoxin in the tolerance model (ET). (B) Primary rat astrocytes were pretreated with IL-4 (10 ng/mL), IL-10 (20 ng/mL), or adapted to endotoxin (ET, LPS 10 ng/mL for 48 h) and were then stimulated with LPS (100 ng/mL) for 4 h. Concentrations of oxylipins in supernatants were measured using UPLC-MS/MS. The heat map shows relative amounts of each lipid mediator compared to the control. The horizontal axis indicates the stimuli, while the vertical axis indicates the relative amount (log2) of each lipid mediator. Metabolites were divided into: Lipoxygenase (LOX), cyclooxygenase (COX), and cytochrome (CYP) pathways involved in their synthesis.

Figure 4

Schema of oxylipins’ profiles for astrocytes classical (LPS) and alternative (IL-4) polarization states. Tested oxylipins, synthesized via lipoxygenase (LOX), cyclooxygenase (COX), or cytochrome (CYP) metabolic branches, are marked with a red frame for classical and a blue frame for alternative polarizations. An arrow of the corresponding color means a decrease (↓) or an increase (↑) in the synthesis of a metabolite.

Figure 5

General scheme of treatment.