Sex-Mediated Differences in LPS Induced Alterations of TNFα, IL-10 Expression, and Prostaglandin Synthesis in Primary Astrocytes

Abstract

Although many neurological and psychiatric disorders reveal clear sex-dependent variations, the molecular mechanism of this process is not clear enough. Astrocytes are involved in the response of neural tissue to injury and inflammation, produce steroid hormones, and sense steroid presence. To explore the hypothesis that astrocytes may participate in sex-mediated differences of inflammatory responses, we have examined whether male and female primary rat astrocytes show different responses to lipopolysaccharide (LPS) as a toll-like receptor 4 (TLR4) agonist. Levels of mRNA and proteins of tumor necrosis factor alpha (TNFα), interleukin-10 (IL-10), and cyclooxygenase (COX)-2 were assessed using qPCR, immunoblotting, and ELISA. UPLC-MS/MS was used to detect prostaglandins (PGs). LPS stimulation resulted in different levels of cytokine production; more TNFα and less IL-10 were produced in female cells compared with male astrocytes. Although the levels of the COX-2 expression were not altered, LPS significantly induced the synthesis of PGs with notable sex-related differences. PGE₂ and PGD₂ were less and 6-keto-PGF_1α was more upregulated in female astrocytes, and TXB₂ had similar levels in cells obtained from males and females. Trilostane, an inhibitor of 3β-Hydroxysteroid dehydrogenase (3β-HSD), inhibited the LPS-induced TNFα production and the release of PGE₂, PGD₂, and 6-keto-PGF_1α in female astrocytes. Thus, male and female astrocytes differentially respond to inflammatory challenges on the level of production of cytokines and steroid hormones. Sex-mediated differences in pro- and anti-inflammatory responses should be taken into consideration for the effective treatment of disorders with neuroinflammation.

Keywords: 3β-HSD; COX-2; IL-10; LPS; TLR4; TNFα; astrocytes; neuroinflammation; sex difference; trilostane.

Figure 1

Comparison of astrocyte cultures obtained from male and female rats. (a) Representative immunofluorescence images showing female (F) and male (M) astrocytes cell culture morphology and purity. The cultures were fixed with 3% paraformaldehyde and incubated with DAPI (4′,6-diamidino-2-phenylindole, blue), OX-42 (Anti-CD11b/c antibody OX-42, red), and GFAP (glial fibrillary acidic protein, green). The last panels are the merged images; (b) An example of agarose (2%) electrophoresis of PCR products. Samples obtained from male tails have two products (margin lanes), and from the female tails—one product (central lanes). All of the products have length between 250 and 500 bp. The molecular weight marker ladder is not shown; (c) Comparison of basal levels of released mediators. TNFα (left scale) and IL-10 (right scale) concentrations were measured by ELISA in supernatant samples of male (white bars) and female (black bars). Values represent mean ± SEM from three independent experiments performed in triplicate.

Figure 2

Sex differences in tumor necrosis factor alpha (TNFα), cyclooxygenase (COX)-2, and interleukin-10 (IL-10) expression during acute inflammation. Male (white) and female (black) astrocytes cultures were kept for 4 h with lipopolysaccharide (LPS) (100 ng/mL), then the concentrations of IL-10 (a) and TNFα (b) were measured by ELISA in the supernatants samples. The results are represented as mean ± SEM from three independent experiments performed in triplicate. (c) COX-2 protein levels were measured by Western blotting. Equal protein loading was confirmed using a β-tubulin antibody. The blot is representative of three independent experiments. * p < 0.05 compared with unstimulated cells, # p < 0.05 compared with indicated bars (sex difference), ^ p < 0.05 compared with male COX-2 basal protein levels.

Figure 3

Sex differences in PGE₂, PGD₂, TXB₂, and 6-keto-PGF_1α releases after LPS-induced inflammatory responses. Purified cultures of male and female astrocytes were treated with LPS (100 ng/mL) and concentrations of prostaglandins in culture media were analyzed by Ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). Samples were collected 4 h after LPS. The results are represented as a scheme of the metabolic pathway with intermediate mediators (blue frame) and enzymes (above lines, representing chemical reactions). Values are represented mean ± SEM from three independent experiments performed in triplicate. * p < 0.05 compared with unstimaluted cells, # p < 0.05 compared with indicated bars (sex difference). Abbreviations: COX—cyclooxygenase; TXAS—thromboxane A synthase; PGES—prostaglandin E synthase; PGDS—prostaglandin D synthase; PGIS—prostaglandin I synthase.

Figure 4

Trilostane differentially affects acute inflammatory responses in male and female astrocytes. Astrocytes were pretreated for 0.5 h with trilostane (TR; 25 μM) and then stimulated with LPS for 4 h. (a–d) Concentrations of prostaglandins in the supernatants were measured using UPLC-MS/MS; (e,f) Changes in the TNFα and IL-10 release levels in cell supernatants were measured using ELISA; (g) A scheme summarizing the differences in synthesis of prostaglandins, and the release of TNFα and IL-10 in male and female astrocytes upon LPS challenges (↑: increased release, ↑↑: strongly increased release, ↓: decreased release, –: no effect). All of the data are represented as ratios to LPS treatment (LPS treatment was accepted as 100%). White bars indicate the male culture, black bars indicate the female culture. Values are represented as mean ± SEM from three independent experiments performed in triplicate. * p < 0.05 compared with unstimulated cells, # p < 0.05 compared with indicated bars (sex difference).

Figure 5

Metabolism of neuroactive steroids in astrocytes and role of trilostane as a competitive inhibitor of 3β-hydroxysteroid dehydrogenase (3β-HSD), the key enzyme of steroid transformations. “Sulf” means the sulfate metabolites of substances, “X” means the blocking effect of trilostane, straight arrows connect substrates and metabolites, and dotted arrows indicate participation of several enzymatic transformations (adapted from [49]).