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. 2008 Sep;32(9):1565-73.
doi: 10.1111/j.1530-0277.2008.00726.x. Epub 2008 Jul 9.

Acute alcohol intake induces SOCS1 and SOCS3 and inhibits cytokine-induced STAT1 and STAT3 signaling in human monocytes

Oxana Norkina  1 , Angela Dolganiuc, Donna Catalano, Karen Kodys, Pranoti Mandrekar, Amber Syed, Marian Efros, Gyongyi Szabo
Affiliations

Acute alcohol intake induces SOCS1 and SOCS3 and inhibits cytokine-induced STAT1 and STAT3 signaling in human monocytes

Oxana Norkina et al. Alcohol Clin Exp Res. 2008 Sep.

Abstract

Background: Acute alcohol consumption is associated with induction of immuno-inhibitory cytokines and down-regulation of pro-inflammatory responses to various pathogens. We previously reported that alcohol activates janus kinase/signal transducer and activator of transcription (JAK/STAT) signaling leading to IL-10 induction. The JAK-STAT pathway also activates its own negative regulators, suppressors of cytokine signaling (SOCS) 1 and SOCS3. SOCS proteins are inducible inhibitors that negatively regulate STAT3/STAT1 signaling pathways induced by cytokines, IL-6 or IFNs. Here we aimed to explore the effect of acute alcohol on induction of SOCS1/SOCS3 and regulation of STAT3/STAT1 pathways induced by IL-6 or IFNs in human monocytes.

Methods: Blood samples from normal volunteers were collected before and 24 hours after consumption of 2 ml vodka/kg body weight. For in vitro experiments human monocytes were pretreated with ethanol (EtOH) followed by stimulation with cytokines; proteins were analyzed by Western blot, nuclear protein binding to DNA by EMSA, and RNA by real time PCR.

Results: Acute in vivo or in vitro alcohol treatment increased both SOCS1 and SOCS3 RNA expression in monocytes. Alcohol treatment resulted in increased STAT3 and STAT1 DNA binding capacity. Activation of both STAT1 and STAT3 has been shown to induce SOCS1/3. We hypothesized that induction of SOCS proteins by alcohol in turn may lead to modulation of cytokine signaling through STAT1 and STAT3. Indeed, we observed significant down-regulation of IL-6-, IFNalpha- and IFNgamma-induced STAT1 DNA binding as well as inhibition of IL-6- and IFNgamma-induced STAT3 when alcohol was added to monocytes 3 hours prior to the cytokine stimulation. Consistent with inhibition of IL-6-induced STAT3 DNA binding in alcohol-pretreated cells, the levels of IL-6-dependent genes, MCP-1 and ICAM-1, was reduced after IL-6 stimulation. Similar to EtOH alone, combined EtOH+IL-6 simulation resulted in increased expression of both SOCS3 and SOCS1 genes.

Conclusion: While acute alcohol treatment alone activates STAT1/3 signaling pathways and induces SOCS3 and SOCS1 levels in monocytes, alcohol also leads to down-regulation of IL-6-, IFNalpha-, and IFNgamma-induced signaling via STAT1/STAT3 pathways, likely through excessive SOCS activation.

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Figures

Fig. 1
Fig. 1
Acute alcohol exposure induces SOCS1 and SOCS3 expression in human monocytes in vivo and in vitro. (A, B) Human monocytes were collected before and 24 hours after in vivo alcohol exposure. The mRNA expression of SOCS3 (A) and SOCS1 (B) was quantified using specific primers in real-time PCR and normalized for housekeeping gene 18S. The results are shown as mean ± SE from n = 4. (C, D) Monocytes from normal alcohol-naïve individuals were stimulated in vitro with EtOH (25 mM) for 5 hours. The mRNA of SOCS3 (C) and SOCS1 (D) was quantified as above and is shown as fold change compared to control cells from n = 4.
Fig. 2
Fig. 2
Acute alcohol induces STAT3 DNA binding in vivo. Monocytes were separated from donors before and after in vivo alcohol consumption. Some cells were stimulated ex vivo, as indicated, with LPS (1 µg/ml) for 45 minutes. Five µg of the nuclear extract was subjected to the conventional EMSA with radioactive-labeled consensus STAT3 oligonucleotide. The protein-STAT3 oligonucleotide complexes were separated on polyacrylamide gel, dried and exposed to autoradiography. One representative gel (A) and densitometric analysis from n = 3 is shown.
Fig. 3
Fig. 3
Ethanol attenuates cytokine-induced STAT3 DNA binding in human monocytes in vitro. Human monocytes were stimulated with IL-6 (5 ng/ml) or IFNα (10 U/ml) for 45 minutes without or with ethanol (50 mM) pretreatment for 3 hours prior to the cytokine stimulation, as indicated. Five µg of nuclear protein were analyzed for STAT3 DNA binding capacity using a consensus specific radioactive labeled oligonucleotide. One representative gel (A) and the densitometric analysis (B) from n = 5 are shown. To confirm the reagent specificity (C), nuclear extracts were incubated with a consensus specific radioactive labeled STAT3 oligonucleotide (line S-sample) in the presence of mutant STAT3 (line M-mutant) or cold consensus STAT3 (line C-cold competition) oligonucleotide.
Fig. 4
Fig. 4
Ethanol induces STAT1 DNA binding and attenuates cytokine-elicited STAT1 DNA binding in human monocytes. Human monocytes were stimulated with IL-6 (5 ng/ml), IFNγ (0.1 ng/ml) or IFNα (10 U/ml) for 45 minutes without or with ethanol (50 mM) pretreatment for 3 hours prior to cytokine stimulation, as indicated. Five µg of nuclear protein were analyzed for STAT1 DNA binding capacity using a specific radioactive labeled oligonucleotide. As a specificity control, IL-6-stimulated sample was preincubated with 100x cold STAT1 oligonucloitide (competition) prior to radioactive STAT1 oligonucleotide. One representative gel (A) and the densitometric analysis (B) from n = 5 are shown. To confirm the reagent specificity (C), nuclear extracts were incubated with a consensus specific radioactive labeled STAT1 oligonucleotide (line S-sample) in the presence of mutant STAT1 (line M-mutant) or cold consensus STAT1 (line C-cold competition) oligonucleotide.
Fig. 5
Fig. 5
Ethanol induces mRNA expression of ICAM-1 and MCP-1, but inhibits IL-6-induced mRNA expression of these genes. Monocytes were analyzed untreated (control) or after pretreatment with ethanol (50 mM) for 5 hours, for ICAM-1 (A) or MCP-1 (B) mRNA expression using real time PCR. Gene expression was normalized to 18S housekeeping control and the data presented as fold change compared to control (n = 4). (C, D) Monocytes were treated with IL-6 (5 ng/nm) for 5 hours without or with ethanol (50 mM) pretreatment prior to stimulation (for 3 hours) with IL-6 (as indicated). ICAM-1 (C) or MCP-1 (D) gene expression was quantified using real time PCR, as above. The data are presented as percent of IL-6-induced level of ICAM-1 or MCP-1 expression shown as 100% (n = 4). (E, F) The samples from panels A–D were analyzed for SOCS3 (E) and SOCS1 (F) expression, as above. Gene expression was normalized to 18S housekeeping control and the data presented as fold change compared to control (n = 4).
Fig. 6
Fig. 6
The model of ethanol-induces modulation of STAT/SOCS signaling pathways. Ethanol alone induces activation of STAT3/STAT1, which triggers transcription of STAT-dependent genes, including SOCS1 and SOCS3. Pretreatment with ethanol prior to stimulation with cytokines leads to down-regulation of IL-6-, IFNα-, and IFNγ-induced signaling via STAT1/-STAT3 pathways, likely through excessive SOCS activation. The arrows indicate the effect of ethanol.
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