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. 2013 Jul;94(1):171-82.
doi: 10.1189/jlb.1212659. Epub 2013 Apr 26.

Alcohol-induced IL-1β in the brain is mediated by NLRP3/ASC inflammasome activation that amplifies neuroinflammation

Dora Lippai  1 , Shashi Bala, Jan Petrasek, Timea Csak, Ivan Levin, Evelyn A Kurt-Jones, Gyongyi Szabo
Affiliations

Alcohol-induced IL-1β in the brain is mediated by NLRP3/ASC inflammasome activation that amplifies neuroinflammation

Dora Lippai et al. J Leukoc Biol. 2013 Jul.

Abstract

Alcohol-induced neuroinflammation is mediated by proinflammatory cytokines, including IL-1β. IL-1β production requires caspase-1 activation by inflammasomes-multiprotein complexes that are assembled in response to danger signals. We hypothesized that alcohol-induced inflammasome activation contributes to increased IL-1β in the brain. WT and TLR4-, NLRP3-, and ASC-deficient (KO) mice received an ethanol-containing or isocaloric control diet for 5 weeks, and some received the rIL-1ra, anakinra, or saline treatment. Inflammasome activation, proinflammatory cytokines, endotoxin, and HMGB1 were measured in the cerebellum. Expression of inflammasome components (NLRP1, NLRP3, ASC) and proinflammatory cytokines (TNF-α, MCP-1) was increased in brains of alcohol-fed compared with control mice. Increased caspase-1 activity and IL-1β protein in ethanol-fed mice indicated inflammasome activation. TLR4 deficiency protected from TNF-α, MCP-1, and attenuated alcohol-induced IL-1β increases. The TLR4 ligand, LPS, was not increased in the cerebellum. However, we found up-regulation of acetylated and phosphorylated HMGB1 and increased expression of the HMGB1 receptors (TLR2, TLR4, TLR9, RAGE) in alcohol-fed mice. NLRP3- or ASC-deficient mice were protected from caspase-1 activation and alcohol-induced IL-1β increase in the brain. Furthermore, in vivo treatment with rIL-1ra prevented alcohol-induced inflammasome activation and IL-1β, TNF-α, and acetylated HMGB1 increases in the cerebellum. Conversely, intracranial IL-1β administration induced TNF-α and MCP-1 in the cerebellum. In conclusion, alcohol up-regulates and activates the NLRP3/ASC inflammasome, leading to caspase-1 activation and IL-1β increase in the cerebellum. IL-1β amplifies neuroinflammation, and disruption of IL-1/IL-1R signaling prevents alcohol-induced inflammasome activation and neuroinflammation. Increased levels of acetylated and phosphorylated HMGB1 may contribute to alcoholic neuroinflammation.

Keywords: CNS; HMGB1; MCP-1; TNF-α; cerebellum.

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Figures

Figure 1.
Figure 1.. Proinflammatory cytokines are increased in alcohol-induced brain injury.
WT mice were fed with an EtOH (n=6) or a control (PF; n=8) diet for 5 weeks. Astrocyte (GFAP) and microglia (Iba1) markers (A), as well as IL-1ra (E) were assessed by real-time PCR from whole cerebellar RNA extract, normalized to 18S. Inflammatory cytokines, TNF-α (B) and MCP-1 (C) of whole cerebellar lysates, and IL-1β (D and F) of cerebellar and cerebral lysates were measured by specific ELISAs. Mature IL-1β of whole cerebellar lysates were assessed by Western blot using β-actin as a loading control (G) and quantified further by densitometry (H). Bars represent mean ± sem (*P<0.05, relative to appropriate PF controls by the Kruskal-Wallis nonparametric test).
Figure 2.
Figure 2.. Cerebella of TLR4-deficient, alcohol-fed mice are partially protected against inflammatory cytokine production.
WT (n=8 or 7) and TLR4-KO (n=8 or 13) mice were fed with a control (PF) or EtOH diet for 5 weeks, respectively. Inflammatory cytokines, TNF-α (A), MCP-1 (B), and IL-1β (C) of whole cerebellar lysates, were measured by specific ELISAs. Endotoxin measurement was executed in serum and whole cerebellar lysates of WT mice by LAL assay (D). Bars represent mean ± sem (*P<0.05, relative to appropriate PF or WT-EtOH controls; #P<0.05, relative to appropriate WT-PF controls by the Kruskal-Wallis nonparametric test).
Figure 3.
Figure 3.. Active endogenous danger molecule, HMGB1, and its receptors are elevated in alcohol-fed mice in the cerebellum.
WT mice were fed with an EtOH (n=6) or a control (PF; n=8) diet for 5 weeks. TLR2, TLR4, TLR9, and RAGE receptors and HMGB1 (A) were assessed by real-time PCR from whole cerebellar RNA extracts, normalized to 18S. Acetyl (Ac)- and phospho (P)-HMGB1 of whole cerebellar lysates were analyzed by IP (B) and assessed further by densitometry, using β-actin Western blot for loading control on a separate gel (C). Cerebellar lysate of one representative ethanol-fed mouse was applied for IgG control (ctr), using the same amount of protein. Total HMGB1 of whole cerebellar lysates was analyzed by Western blot using β-actin as a loading control (D) and quantified further by densitometry (E). Bars represent mean ± sem (*P<0.05, relative to appropriate PF controls by the Kruskal-Wallis nonparametric test).
Figure 4.
Figure 4.. Inflammasome complex is up-regulated and activated in alcohol-fed mice in the cerebellum.
WT mice were fed with an EtOH (n=6) or a control (PF; n=8) diet for 5 weeks. Various inflammasome sensors (NLRP1, NLRP3, NLRC4, AIM2) and Pannexin-1 (A) and the inflammasome adaptor (ASC), the inflammasome effector (procaspase-1), and pro-IL-1β (B) were assessed by real-time PCR from whole cerebellar RNA extracts, normalized to 18S. Inflammasome activity was measured by a caspase-1 colorimetric assay (C) from whole cerebellar lysates. The caspase-1 p10 level (D) was visualized on a Western blot, using β-actin as a loading control, and quantified by densitometry (E). NLRP1, NLRP3, NLRC4, AIM2, and Pannexin-1 (F) and ASC, procaspase-1, and pro-IL-1β (G) were assessed by real-time PCR from whole cortical RNA extracts. Inflammasome activity was measured by a caspase-1 colorimetric assay (H) from whole cortical lysates. Bars represent mean ± sem (*P<0.05, relative to appropriate PF controls by the Kruskal-Wallis nonparametric test).
Figure 5.
Figure 5.. NLRP3 or ASC deficiency prevents caspase-1 activity and IL-1β production in alcohol-fed mice in the cerebellum.
WT (n=7 or 14), NLRP3 (n=8 or 10), and ASC-KO (n=9 or 12) mice were fed with a control (PF) or an EtOH diet for 5 weeks, respectively. IL-1β protein level of whole cerebellar lysates was measured by specific ELISA (A). Mature IL-1β of whole cerebellar lysates was assessed by Western blot using β-actin as a loading control (B) and quantified further by densitometry (C). Inflammasome activity was evaluated by a caspase-1 colorimetric assay from whole cerebellar lysates (D). Bars represent mean ± sem (*P<0.05, relative to appropriate PF or WT-EtOH controls; #P<0.05, relative to appropriate WT-PF controls by the Kruskal-Wallis nonparametric test).
Figure 6.
Figure 6.. TLR4 deficiency does not decrease caspase-1 activity in alcohol-fed mice in the cerebellum.
WT (n=8 or 7) and TLR4-KO (n=8 or 13) mice were fed with a control (PF) or an EtOH diet for 5 weeks, respectively. TLR4, various inflammasome receptors (NLRP1, NLRP3, NLRC4), and Pannexin-1 (A) and the inflammasome adaptor ASC, the inflammasome effector procaspase-1, and pro-IL-1β (B) were assessed by real-time PCR from whole cerebellar RNA extracts, normalized to 18S. Inflammasome activity was evaluated by the caspase-1 colorimetric assay from whole cerebellar lysates (C). Bars represent mean ± sem (*P<0.05, relative to appropriate PF or WT-EtOH controls; #P<0.05, relative to appropriate WT-PF controls by the Kruskal-Wallis nonparametric test).
Figure 7.
Figure 7.. IL-1ra, anakinra, treatment attenuates the effect of alcohol-feeding on caspase-1 activity and IL-1β production in murine cerebella.
WT mice were fed with a PF or an EtOH diet for 5 weeks and received daily i.p. IL-1ra (anakinra: 25 mg/kg) or an equal amount of saline injections. Inflammatory cytokines (TNF-α, MCP-1, and pro-IL-1β) and procaspase-1 (A) were assessed by real-time PCR from whole cerebellar RNA extract, normalized to 18S. Proinflammatory cytokines, TNF-α (B), MCP-1 (C), and IL-1β (D) of whole cerebellar lysates were measured by specific ELISAs. Inflammasome activity was measured by the caspase-1 colorimetric assay from whole cerebellar lysates (E). Acetyl-HMGB1 of whole cerebellar lysates were analyzed by IP (F) and assessed by densitometry, using β-actin Western blot for loading control on a separate gel (G). The cerebellar lysate of one representative ethanol-fed, IL-1ra-treated mouse was applied for IgG control, using the same amount of protein. Bars represent mean ± sem (*P<0.05, relative to appropriate PF or saline treated-EtOH controls; #P<0.05, relative to appropriate saline-treated PF controls by the Kruskal-Wallis nonparametric test. PF-saline, n = 8; PF-IL-1ra, n = 5; EtOH-saline, n = 7; EtOH-IL-1ra, n=9).
Figure 8.
Figure 8.. IL-1β induces TNF-α and MCP-1 in the cerebellum.
WT mice received 100 ng (30 μl) intracranial mouse rIL-1β or an equal amount of saline solution (n=7 or 8). Inflammatory cytokines (TNF-α, MCP-1, and pro-IL-1β) were assessed by real-time PCR from whole cerebellar RNA extract, normalized to 18S (A). Proinflammatory cytokines, TNF-α (B) and MCP-1 (C), of whole cerebellar lysates were measured by specific ELISAs. Bars represent mean ± sem (*P<0.05, relative to appropriate saline-treated controls by the Kruskal-Wallis nonparametric test).
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References

    1. Holley M. M., Kielian T. (2012) Th1 and Th17 cells regulate innate immune responses and bacterial clearance during central nervous system infection. J. Immunol. 188, 1360–1370 - PMC - PubMed
    1. Glass C. K., Saijo K., Winner B., Marchetto M. C., Gage F. H. (2010) Mechanisms underlying inflammation in neurodegeneration. Cell 140, 918–934 - PMC - PubMed
    1. Tracey K. J. (2010) Understanding immunity requires more than immunology. Nat. Immunol. 11, 561–564 - PMC - PubMed
    1. Alfonso-Loeches S., Pascual-Lucas M., Blanco A. M., Sanchez-Vera I., Guerri C. (2010) Pivotal role of TLR4 receptors in alcohol-induced neuroinflammation and brain damage. J. Neurosci. 30, 8285–8295 - PMC - PubMed
    1. Szabo G., Mandrekar P., Petrasek J., Catalano D. (2011) The unfolding web of innate immune dysregulation in alcoholic liver injury. Alcohol Clin. Exp. Res. 35, 782–786 - PMC - PubMed

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