The cytokine mRNA increase induced by withdrawal from chronic ethanol in the sterile environment of brain is mediated by CRF and HMGB1 release - PubMed (original) (raw)

The cytokine mRNA increase induced by withdrawal from chronic ethanol in the sterile environment of brain is mediated by CRF and HMGB1 release

Buddy A Whitman et al. Alcohol Clin Exp Res. 2013 Dec.

Abstract

Background: Many neurobiological factors may initiate and sustain alcoholism. Recently, dysregulation of the neuroimmune system by chronic ethanol (CE) has implicated Toll-like receptor 4 (TLR4) activation. Even though TLR4s are linked to CE initiation of brain cytokine mRNAs, the means by which CE influences neuroimmune signaling in brain in the absence of infection remains uncertain. Therefore, the hypothesis is tested that release of an endogenous TLR4 agonist, high-mobility group box 1 (HMGB1) and/or corticotropin-releasing factor (CRF) during CE withdrawal are responsible for CE protocols increasing cytokine mRNAs.

Methods: Acute ethanol (EtOH; 2.75 g/kg) and acute lipopolysaccharide (LPS; 250 μg/kg) dosing on cytokine mRNAs are first compared. Then, the effects of chronic LPS exposure (250 μg/kg for 10 days) on cytokine mRNAs are compared with changes induced by CE protocols (15 days of continuous 7% EtOH diet [CE protocol] or 3 intermittent 5-day cycles of 7% EtOH diet [CIE protocol]). Additionally, TLR4, HMGB1, and downstream effector mRNAs are assessed after CE, CIE, and chronic LPS. To test whether HMGB1 and/or CRF support the CE withdrawal increase in cytokine mRNAs, the HMGB1 antagonists, glycyrrhizin and ethyl pyruvate, and a CRF1 receptor antagonist (CRF1RA) are administered during 24 hours of CE withdrawal.

Results: While cytokine mRNAs were not increased following acute EtOH, acute LPS increased all cytokine mRNAs 4 hours after injection. CE produced no change in cytokine mRNAs prior to CE removal; however, the CE and CIE protocols increased cytokine mRNAs by 24 hours after withdrawal. In contrast, chronic LPS produced no cytokine mRNA changes 24 hours after LPS dosing. TLR4 mRNA was elevated 24 hours following both CE protocols and chronic LPS exposure. While chronic LPS had no effect on HMGB1 mRNA, withdrawal from CE protocols significantly elevated HMGB1 mRNA. Systemic administration of HMGB1 antagonists or a CRF1RA significantly reduced the cytokine mRNA increase following CE withdrawal. The CRF1RA and the HMGB1 antagonist, ethyl pyruvate, also reduced the HMGB1 mRNA increase that followed CE withdrawal.

Conclusions: By blocking HMGB1 or CRF action during CE withdrawal, evidence is provided that HMGB1 and CRF release are critical for the CE withdrawal induction of selected brain cytokine mRNAs. Consequently, these results clarify a means by which withdrawal from CE exposure activates neuroimmune function in the sterile milieu of brain.

Keywords: CRF1 Receptor Antagonist; Chronic Ethanol Withdrawal; Cytokines; HMGB1 Antagonists; Lipopolysaccharide.

Copyright © 2013 by the Research Society on Alcoholism.

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Figures

Figure 1

Figure 1

Protocols for the experimental treatments described in this paper.

Figure 2

Figure 2. Cytokine-mRNA and TLR4-mRNA expression following acute treatment with ethanol (2.75 g/kg) and LPS (250 μg/kg)

(A) The acute single dose of 2.75 g/kg of ethanol I.P resulted in no significant increase in cytokine-mRNAs for CCL2, IL1-β, or TNFα (all _p_>0.05). (B) The acute single 250 μg/kg dose of LPS IP increased cytokine-mRNAs for CCL2, IL1-β, and TNFα four hours following injection [(CCL2: t(17)=6.07, p<0.0001; IL1-β: _t_(16)=6.31, _p_<0.0001; TNFα: _t_(13)=4.97, _p_=0.0003). Twenty-four hours after the acute dose of ethanol the levels of cytokine-mRNAs were not significantly different from control P>0.05. Acute-ethanol (C) and Acute LPS (D) treatments did not alter TLR4 mRNA expression either at four hours (Fig C and D) or 24-hrs after their acute dosing (_p_>0.05). Thus, the acute ethanol exposure did not recapitulate the increase in cytokine-mRNAs induced by acute LPS administration. * Significantly different from control (p<0.05).

Figure 3

Figure 3. Cytokine-mRNA expression following a chronic-ethanol-protocol (Fig 1.C) and a chronic-LPS protocol (Fig 1D)

Twenty-four-hours after withdrawal from 15 days of 7% ethanol-diet (Chronic ethanol), cytokine-mRNAs for CCL2, IL1-β, and TNFα are significantly increased compared to non-treated controls (CD) (t(15)=4.53, _p_=0.0004; t(20)=3.42, _p_=0.0027; t(21)=5.443, p<0.0001, respectively. In contrast, 24-hours after the chronic-LPS exposure cytokine-mRNAs for CCL2, IL1-β, and TNFα were not significant increased (all cytokines = _p_>0.05). Again, the CE- and chronic-LPS protocols differed in their expression of cytokine-mRNAs. * Significantly different from Control (CD) (p<0.05).

Figure 4

Figure 4. Effect of the chronic-ethanol protocol and chronic-LPS protocol on mRNAs for TLR4, HMGB1, My88, and NFκB

(A). Chronic-ethanol significantly increased TLR4 mRNA expression [t(23)=2.669, _p_=0.0137]. (B) Chronic-LPS (250 μg/kg/day for 10 days; Fig 1D) also significantly increased TLR4 [t(18)=2.83, _p_=0.0112]. (C) Chronic-Ethanol significantly increased HMGB1 mRNA exposure [t(16)=3.618, _p_=0.0023]. (D) Chronic-LPS was without effect on mRNA for HMGB1 (p>0.05). (E-H) Chronic-ethanol and chronic-LPS did not significantly affect either My88 (EF) or NF-κB (GH) mRNA levels (p>0.05). * Significantly different from Control (p<0.05).

Figure 5

Figure 5. Time course of changes for cytokine-mRNAs (top) and TLR4- and HMGB1-mRNAs (bottom) while on (o-hr) and 24-hours after withdrawal from the CE-protocol

For cytokine-mRNAs (top), CCL2, IL1-β, and TNFα mRNAs significantly peaked 24-hours after withdrawal from the CE-protocol compared to control [CCL2-mRNA = Control 100 ± 31.1 vs. CCL2 peak = 202.0 ± 33.3, _p_=0.025 (F(4,25)= 3.06, _p_=0.035]. [IL1-β-mRNA = Control 100 ± 11.0 vs. IL1-β peak = 193.8 ± 38.9, _p_=0.0039) (F(4,27)= 5.05, _p_=0.036]; TNFα = Control 100 ± 9.3 vs. TNFα-Peak= 207.3 ± 38.7, _p_=0.0006) (F(4,27)=7.30; _p_=0.0004]. At time t=0, cytokine-mRNAs were not significantly increased (p>0.05). The peak expression of TLR4-mRNA 24-hours following withdrawal from the CE-protocol (Bottom) was 218.7 ± 19.4 for TLR4 vs 100 ± 13.9 for control (F(4,27)=8.64, _p_=0.0001) and 164.5 ± 15.6 for HMGB1 vs 100 ± 21.9 for the Control (F(2,26)=3.51, _p_=0.02). At T=0 (0 hr) TLR4 was not significantly different from control (p>0.05). However, while the HMGB1-mRNA elevation at T=0 (0 hr) is significant, but only to a minor degree (p<0.05), an additional investigation was unable to duplicate this significant increase [Control = 100 ± 16.4% vs HMGB1-mRNA = 120 ± 13.5%; p>0.05]. See text for descriptions of changes for cytokine-mRNAs and the TLR4 and HMGB1-mRNAs 7 days after withdrawal from the CE-protocol.

Figure 6

Figure 6. Comparison of CE-protocol (Chronic ETOH; see Fig 1C) and Chronic-Intermittent Ethanol (Cycled ETOH; see Fig 1F)

Twenty-four hours after removal from ethanol diet, both the Chronic Ethanol- and Cycled Ethanol-treatment groups showed significant increases in CCL2, IL1-β, and TNFα-mRNA [F(2,18)= 5.211, _p_=0.018; F(2,24)=9.718, _p_=0.0009; F(2,25)=6.475, _p_=0.006, respectively]. However, a statistical difference did not occur between the cytokine-mRNAs for the Chronic ETOH and Cycled ETOH-treatment groups (p>0.05). * Significantly different from Control (p<0.05).

Figure 7

Figure 7. Comparison of CE- and cycled-ethanol treatment groups on mRNAs for TLR4, HMGB1, MyD88 and NF-κB

(A) TLR4 was significantly elevated in both the chronic and cycled ethanol groups (F(2,23)=8.744, _p_=0.0017); However, a difference between the chronic and cycled groups was not observed (_p_>0.05). (B) Both the chronic and cycled ethanol protocol groups significantly elevated HMBG1-mRNA (F(2,24)=3.787, _p_=0.0466), but no difference between the chronic- and cycled-ethanol protocols was noted (_p_>0.05). (C&D) Neither the chronic nor the cycled ethanol protocol groups caused a discernible effect on levels of MyD88 or NF-κB _p_>0.05). * Significantly different from CD (p<0.05).

Figure 8

Figure 8. Testing of a CRF1RA [CP154,526 (CP: 10 mg/kg)] or the HMGB1 antagonists, glycyrrhizin (Gly: 50 mg/kg) and ethyl-pyruvate (EP: 75 mg/kg), on the cytokine-mRNA increases induced by 24-hrs of withdrawal (WD) from CE-exposure. Each drug was administered at 3 and 12 hours into the withdrawal period after cessation of ethanol diet exposure

There were significant group effects for each mRNA [TNFα = F(4, 37) = 6.16, p<0.05; IL1β = F(4,37) = 3.46, p<0.05; and CCL2 = F(4,42) = 4.28, p<0.05]. TNFα, CCL2, & IL1β-mRNAs were significantly elevated 24-hours after withdrawal from the CE-24h-WD protocol (P<0.05), in accord with results in Fig 3. The CRF1 receptor antagonist CP154,526 (CP: 10 mg/kg) and ethyl-pyruvate (EP: 75 mg/kg) significantly reduced the cytokine mRNA increases for TNFα and IL1β 24-hours after removal from the CE-protocol (p<0.05). After the HMGB1 inhibitor, glycyrrhizin (Gly: 50 mg/kg), the suppression of CCL2-mRNA from the CE-24-WE-veh did not reach statistical significance (p=0.059). *Significantly different from CE-24h-WD (p<0.05).

Figure 9

Figure 9. Testing of the CRF1-receptor antagonist CP154,526 (CP: 10 mg/kg) or the HMGB1-antagonists, glycyrrhizin (Gly: 50 mg/kg) and ethyl-pyruvate (EP: 75 mg/kg), on the HMGB1-mRNA increase induced by withdrawal (WD) from chronic ethanol (CE)-exposure

See Figure 8 for further details. Overall effect of groups was significant, [F(4,41) = 3.96, p<0.05]. The CRF1 receptor antagonist [CP154,526 (CP: 10 mg/kg)] and ethyl-pyruvate (EP: 75 mg/kg) significantly reduced the HMGB1-mRNA increase after removal from the CE-24-WD-protocol (p<0.05). The trend toward suppression of HMGB1 with glycyrrhizin after the CE-24-WD protocol did not reach statistical significance (p=0.094). * Significantly different from CE-24h-WD (p<0.05).

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