Cellular Model of Endotoxin Tolerance in Astrocytes: Role of Interleukin 10 and Oxylipins

Abstract

A phenomenon of endotoxin tolerance where prior exposure of cells to minute amounts of lipopolysaccharide (LPS) causes them to become refractory to a subsequent high-amount endotoxin challenge is well described for innate immune cells such as monocytes/macrophages, but it is still obscure for brain cells. We exposed primary rat cortical astrocytes to a long-term low-grade concentration of LPS, followed by stimulation with a middle-grade concentration of LPS. Inflammatory markers, i.e., pro-inflammatory cytokine TNFα, inducible enzymes COX-2 and iNOS, anti-inflammatory cytokine interleukin 10 (IL-10) detected at the mRNA and protein levels reveal similarities between astrocytes and macrophages in the model, i.e., tolerance in pro-inflammatory markers and priming in IL-10. Long-term or short-term treatment with IL-10 does not change cell sensitivity for LPS, which makes doubtful its involvement in the mechanisms of cell tolerance development. Significant changes occur in the oxylipin profiles measured by UPLC-MS/MS analysis. The priming occurs in the following compounds: 11-HETE, PGD 2 , PGE 2 , cyclopentenone prostaglandins, and TXB 2 . Tolerance is observed for 12-HHT, PGF 2α , and 6-keto-PGF 1α . As far as we know, this is the first report on changes in oxylipin profiles in the endotoxin tolerance model. The data can greatly improve the understanding of oxylipins' role in inflammatory and resolution processes in the brain and mechanisms of astrocyte involvement in neuroinflammation.

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

“…For ET-treated cells, there was a decrease in PGF 2α , 6-keto-PGF 1α , 12-HHT, 14,15-DHET, 12-HETE, and 5-HETE and an increase in PGA 2 +PGJ 2 , PGE 2 , PGD 2 , TXB 2 , 11-HETE, and 13-HDoHE (quantitative data presented in Figure S2). The data coincide with previously published findings [26]. For IL-4-treated cells, there was a decrease in PGF 2α 16-HDoHE and 20-HDOHE, and an increase in 12-HHT, PGA 2 +PGJ 2 , PGD 2 , TXB 2 , 11-HETE, 14,15-DHET.…”

“…There was no influence on the alternative activation-related genes IL-10, MRC1, FIZZ1, and Ym1 (Figure 1). Levels of iNOS, TNFα and IL-10 mRNA expression for all treatments were comparable with previously published data [26].…”

“…ET treatment resulted in tolerance for acute LPS stimulation, i.e., a decrease in GBP2, iNOS, TNFα, and CXCL10 mRNA expression. Such changes for TNFα and iNOS were shown previously [26]. The mechanism of these changes and the functional significance of such transcriptional adaptations still need to be investigated further, but such differences in the responses of cells adapted to anti-inflammatory stimuli upon the activation of innate immunity signaling pathways should be taken into account during a study of the mechanisms of inflammatory responses in CNS.…”

“…Mechanisms of these changes and the possibilities of directed regulation are unexplored. Although we characterized astrocyte oxylipin synthesis for pro-inflammatory stimuli [20,26,27], the data concerning oxylipin synthesis in various astrocyte polarization states are missing. Therefore, we stimulated astrocytes with interleukin 4 (IL-4), interleukin 10 (IL-10), and LPS for 24 h (h) and subdivided their phenotypic polarization into A1-like (pro-inflammatory, classical) and A2-like (anti-inflammatory, alternative) using the previously suggested markers [23,28].…”

“…This prompts an additional question concerning the endotoxin tolerance phenomenon in relation to astrocytes. Recently, we have shown that the phenomenon exists in astrocytes [26]. Accordingly, we included the endotoxin tolerance model in a comparison series of various treatments leading to the adaptation of astrocytes in their ability to respond to acute LPS stimulation.…”