Increased systemic and brain cytokine production and neuroinflammation by endotoxin following ethanol treatment

Abstract

Background: Cytokines and alcohol share a common modulation of inflammation and hormones as well as being implicated in multiple diseases, but the mechanisms are poorly understood. The purpose of this study was to investigate the interaction of liver, serum and brain cytokines as well as whether ethanol would potentiate endotoxin (Lipopolysaccharide, LPS) responses once ethanol had cleared.

Methods: Male C57BL/6J mice were treated intragastrically with water (control) or ethanol (5 g/kg, i.g., 25% ethanol, w/v), with volumes matched, for 1 day or daily for 10 days. Mice were then injected intraperitoneally with saline (control) or LPS (3 mg/kg, i.p.) in saline 24 hrs after the last dose of ethanol. Gene expression and protein synthesis of proinflammatory cytokines and anti-inflammatory cytokine, oxidative enzymes, microglial activation and inhibition of neurogenesis were examined using real-time PCR, ELISA, and immunohistochemistry.

Results: LPS increased proinflammatory cytokines (TNFalpha, MCP-1, IL-1beta) several fold in liver, brain and serum at 1 hr. Ethanol is known to increase liver cytokines and alter the risk of multiple chronic diseases. Ten daily doses of ethanol increased brain and liver TNFalpha, and pretreatment with ethanol potentiated LPS-induced increases in TNFalpha, MCP-1, IL-1beta in liver, serum and brain. Proinflammatory cytokine levels in liver and serum returned to basal levels within a day, whereas brain proinflammatory cytokines remained elevated for long periods. IL-10, an anti-inflammatory cytokine, is reduced in brain by ethanol and LPS, while brain proinflammatory cytokines remain increased, whereas liver IL-10 is increased when proinflammatory cytokines have returned to control levels. Activation of brain microglia indicated by morphological changes, reduced neurogenesis and increased brain expression of COX-2 and gp91phox NADPH oxidase subunit mRNA were found in the 10 daily doses of ethanol-pretreated LPS group.

Conclusion: Acute increases in serum cytokines induce long lasting increases in brain proinflammatory cytokines. Ten daily doses of ethanol exposure results in persistent alterations of cytokines and significantly increases the magnitude and duration of central and peripheral proinflammatory cytokines and microglial activation. Ethanol induced differential anti-inflammatory cytokine IL-10 responses in liver and brain could cause long lasting disruption of cytokine cascades that could contribute to protection or increased risk of multiple chronic diseases.

Figure 1

Effects of a single dose of ethanol exposure on LPS-induced liver, serum and brain TNFα production. Male C57BL/6J mice were treated intragastrically with ethanol (5 g/kg, i.g.) for one day. Mice were injected intraperitoneally (i.p.) with LPS (3 mg/kg) 24 hrs after ethanol treatment. Liver, serum and brain samples were collected at 1 hr post LPS treatment. Analysis of TNFα and MCP-1 protein was conducted by ELISA. (A) LPS and ETOH-LPS rapidly increased liver, serum and brain TNFα protein. (B) MCP-1 protein in liver, serum and brain was increased after LPS treatment alone or combined LPS and ethanol treatments. The results are the means ± SEM (n = 6 per group). * P < 0.05, ** P < 0.01, compared with the saline controls.

Figure 2

A single dose of ethanol treatment enhanced LPS-induced IL-1β protein in brain and serum, but not in liver. Male C57BL/6J mice were treated intragastrically with ethanol (5 g/kg, i.g.) for one day. Mice were injected intraperitoneally (i.p.) with LPS (3 mg/kg) 24 hrs after ethanol treatment. Liver, serum and brain samples were collected at 1 hr after LPS injection. Liver, serum and brain IL-1β protein was measured by ELISA. (A) A single ethanol dose increased LPS-induced brain IL-1β production. (B) A single dose of ethanol increased LPS-induced IL-1β protein in serum. (C) A single dose of ethanol did not show potentiation to LPS-stimulated increase in IL-1β protein in liver. The results are the means ± SEM (n = 6 per group). * P < 0.05, ** P < 0.01, compared with the saline controls.^# P < 0.05, ^## P < 0.01, compared with LPS-treated group.

Figure 3

Effects of 10 daily doses of ethanol exposure on LPS-induced liver, serum and brain TNFα and MCP-1 production. Male C57BL/6J mice were treated intragastrically with ethanol (5 g/kg, i.g.) daily for 10 days and injected intraperitoneally (i.p.) with LPS (3 mg/kg) 24 hrs after ethanol treatment. Liver, serum and brain samples were collected 1 hr post LPS treatment. TNFα and MCP-1 protein was determined by ELISA. (A) In 10-day ethanol pre-treated group, LPS significantly increased liver, serum and brain TNFα protein compared with LPS alone group. (B) Exposure to 10 daily doses of ethanol resulted in a significant increase in MCP-1 protein in liver, serum and brain. The results are the means ± SEM of two experiments performed with 6 mice each group. * P < 0.05, ** P < 0.01, compared with the saline controls. ^# P < 0.05, ^## P < 0.01, compared with LPS-treated group.

Figure 4

Effects of 10 daily doses of ethanol exposure on LPS-induced liver, serum and brain IL-1β production. Male C57BL/6J mice were treated intragastrically with ethanol (5 g/kg, i.g.) for 10 days and injected intraperitoneally (i.p.) with LPS (3 mg/kg) 24 hrs after ethanol treatment. Liver, serum and brain samples were collected 1 hr after LPS injection. LPS significantly increased IL-1β protein in liver, serum and brain after 10 days of ethanol administration compared with LPS alone treatment. The results are the means ± SEM of two experiments performed with 6 mice each group. * P < 0.05, ** P < 0.01, compared with the saline control mice. ^# P < 0.05, ^## P < 0.01, compared with LPS-treated mice.

Figure 5

Immunocytochemical analysis of microglia. Male C57BL/6J mice were treated intragastrically with ethanol (5 g/kg, i.g.) for 10 days and injected intraperitoneally (i.p.) with LPS (3 mg/kg, i.p.) 24 hrs after ethanol treatment. Mice were sacrificed 1 hr following saline or LPS injection. Brain sections were fixed and stained with Iba1 antibody. Ten daily doses of ethanol exposure potentiated LPS-induced microglial activation. In either LPS or ethanol treated groups, most of the microglia were in a resting morphological shape. However, in the ethanol pre-treated group, LPS increased Iba1 staining in some brain regions such as cortex, hippocampus and substantia nigra. Iba1-immunoreactive (IR) cells showed an increased cell size, irregular shape consistent with morphological changes in activated microglia.

Figure 6

Ten daily doses of ethanol exposure reduced neurogenesis in hippocampal dentate gyrus during combined ethanol and LPS treatments. Male C57BL/6J mice were treated intragastrically with ethanol (5 g/kg, i.g.) for either 1 day or 10 days and injected intraperitoneally (i.p.) with LPS (3 mg/kg, i.p.) 24 hrs after ethanol treatment. Mice were sacrificed 1 hr following saline or LPS administration. Brain sections were fixed and stained with anti-mouse PCNA (a marker for proliferation of neural progenitor cells) and anti-goat doublecortin (a marker for immature or newly born neurons) antibodies. (A) Ethanol (5 g/kg, i.g., 1 day) pre-treated group, LPS did not show a decrease in the number of PCNA and doublecortin-IR cells. (B) Ethanol (5 g/kg, i.g., 10 days) pre-treated mice, LPS significantly decreased PCNA and doublecortin-IR cells, suggesting that 10 daily doses of ethanol inhibits neurogenesis during combined ethanol and LPS treatments. (C) The pictures represent PCNA-IR cells in control (upper panel) and ETOH-LPS treated (lower panel) dentate gyri of the hippocampus. (D) Representative pictures of doublecortin (DCX) immunoreactivity. DCX expression was shown in control brain (upper panel) and ETOH-LPS treated brain (lower panel).

Figure 7

Effects of 10 daily doses of ethanol on IL-10 in brain and liver at 1 week after LPS treatment. Male C57BL/6J mice were treated intragastrically with ethanol (5 g/kg, i.g.) for 10 days and were injected intraperitoneally (i.p.) with LPS at the indicated doses 24 hrs after ethanol treatment. Brains and livers were collected at 1 week after LPS injection. The level of IL-10 protein was measured by ELISA. (A) Ten doses of ethanol decreased brain IL-10 and enhanced LPS-induced decrease in IL-10 in a LPS dose-dependent manner at 1 week after LPS treatment. (B) There was a significant increase in liver IL-10 protein in 10 daily doses of ethanol, LPS (0.3 and 3 mg/kg) and ETOH-LPS groups. P < 0.05, ** P < 0.01, compared with the saline controls.^# P < 0.05, ^## P < 0.01, compared with the corresponding LPS-treated group.

Figure 8

Ten daily doses of ethanol administration potentiated LPS-induced brain TNFα, MCP-1 and IL-1β production that remained elevated at 1 week. Male C57BL/6J mice were treated intragastrically with ethanol (5 g/kg, i.g.) for 10 days and were injected intraperitoneally (i.p.) with LPS at indicated doses 24 hrs after ethanol treatment. Brains were collected at 1 week after LPS injection. The levels of TNFα, MCP-1 and IL-1βprotein were measured by ELISA. (A) In the10-dose ethanol pre-treated group, brain TNFβ protein significantly increased in a LPS dose-dependent manner. (B) Exposure to 10 daily doses of ethanol resulted in a significant increase in brain MCP-1 in a LPS dose-dependent manner. (C) Brain IL-1βprotein synthesis was also enhanced by ethanol pre-treatment. The results are the means ± SEM (n = 6 per group). * P < 0.05, ** P < 0.01, compared with the saline controls.^#P < 0.05, ^## P < 0.01, compared with the corresponding LPS-treated group.