The opposite effects of acute and chronic alcohol on lipopolysaccharide-induced inflammation are linked to IRAK-M in human monocytes.

Acute alcohol decreases but chronic alcohol treatment of human monocytes increases LPS-induced TNF-α production. A, Human monocytes were exposed to 25 mM alcohol (Et) from 1 to 7 days followed by LPS (100 ng/ml) treatment for 18 h and detection of TNF-α in the cell-free supernatants by ELISA. Mean values of TNF-α (pg/ml/106 cells) ± SE from a total of ten individuals are shown (compared with LPS; *, p < 0.001). B, LPS (100 ng/ml) treatment for 3 h and TNF-α mRNA determination by real-time PCR using specific TNF-α primers as described in Materials and Methods. The bar graph represents fold induction of mRNA ± SE (compared with LPS; *, p < 0.01; **, p < 0.001; n = 6).

Alcohol alters IRAK-M and IRAK-1 levels without any effect on CD14 and TLR4 expression. A, Human monocytes were either exposed to 25 mM alcohol (Et) for 1 day (acute) or 7 days (chronic), stained with CD14 and TLR4 Abs, and analyzed by flow cytometry. Iso-type IgG Abs were used as negative controls. B, Human monocytes were exposed to 25 mM alcohol for 7 days (Chr) followed by LPS (100 ng/ml) for 15 min. Immunoprecipitation (IP) of the cytoplasmic extracts with IRAK-1 Ab in lysis buffer was conducted as described in Materials and Methods. The kinase assay was performed with MBP (myelin basic protein) as substrate. The proteins were separated on SDS-PAGE and the gel depicts the 32P-phosphorylated MBP. Equal protein in the IP samples was determined by immunoblotting (WB) with IRAK-1 Abs. The bar graph below represents the cpm incorporated in the substrate as measured by scintillation counter. (compared with unst; *, p < 0.01; compared with LPS; **, p < 0.05; n = 4). C, Human monocytes were exposed to 25 mM alcohol (Et) for 1 day (Ac) or 7 days (Chr) followed by LPS (100 ng/ml) treatment for 6 h and IRAK-M mRNA determination by real-time PCR using specific IRAK-M primers as described in Materials and Methods. The bar graph represents fold induction of mRNA ± SE (compared with unst; *, p < 0.01; compared with LPS; **, p < 0.02; n = 4). D, Monocytes were exposed to 25 mM alcohol (Et) for 1 day (Ac) or 7 days (Chr) followed by LPS (100 ng/ml) treatment for 24 h and IRAK-M protein levels determined by immunoblotting using anti-IRAK-M Abs as described in Materials and Methods. Equal loading of protein is demonstrated using an internal control anti-β-actin Ab.

IRAK-M expression regulates TNF-α during alcohol exposure. A, Raw macrophages (control cells) were transfected with control siRNA (scr) and IRAK-M-specific siRNA and after 48 h exposed to 25 mM alcohol followed by LPS for 6 h. Total RNA extracts were analyzed for IRAK-M mRNA by real time PCR and bar graph (upper panel) represented as percentage of IRAK-M gene expression (compared with control; *, p < 0.009). Whole cell lysates in the presence or absence of LPS for 6 h were analyzed for IRAK-M protein by Western blot analysis (middle panel) and supernatants analyzed for TNF-α production by ELISA (lower panel). Mean values of TNF-α (pg/ml/106 cells) ± SE from three experiments are shown (compared with control siRNA; #, p < 0.007; **, p < 0.01). B, Raw 264.7 macrophages were exposed to chronic alcohol for 7 days, transfected with either control vector or IRAK-M overexpression vector (Origene) for 36 h and treated without (inset graph) or with LPS for 6 h. Whole cell lysates were subject to Western blotting using anti- IRAK-M (upper panel) Ab and supernatents were analyzed for TNF-α by ELISA (lower panel). Mean values of TNF-α (pg/ml/106 cells) ± SE from a three experiments are shown (compared with vector control; *, p < 0.01; **, p < 0.001).

Acute alcohol decreases whereas chronic alcohol increases LPS-induced IKK kinase activity in human monocytes. Human monocytes were exposed to alcohol (Et) for 1 day (Ac) or 7 days (Chr) followed by stimulation with LPS (100 ng/ml) for 15 min. IP of the cytoplasmic extracts with IKKβ Abs in lysis buffer was followed by kinase assays using the GST-IκBα as substrate as described in Materials and Methods. The proteins were separated on SDS-PAGE and the gels depict the 32P-phosphorylated GST-IκBα. Equal protein loading in the input samples was determined by immunoblotting with β-actin Ab. The bar graph represents the cpm incorporated in the substrate as measured by scintillation counter. (compared with unst; *, p < 0.05; compared with LPS; #, p < 0.05; **, p < 0.01; n = 3).

Inhibition of MAPK-ERK activation by acute alcohol whereas chronic alcohol increases phospho-ERK levels in monocytes. Monocytes were exposed to 25 mM alcohol (Et) for 1 day (Ac) or 7 days (Chr) followed by LPS (100 ng/ml) treatment for 15 min. A, Total and phospho-ERK, phospho-p38 and phospho-JNK protein levels determined by immunoblotting using anti-specific phospho-antibodies as described in Materials and Methods. B, Mean density units ± SE of the phospho-ERK bands from a total of six individuals is shown. (compared with LPS; *, p < 0.05; #, p < 0.02, n = 6). C, Monocytes were exposed to 25 mM alcohol (Et) for 7 days (Chr) followed by incubation with ERK inhibitor (PD98059, 50 μM) for 4 h and stimulation with LPS (100 ng/ml) for 18 h. Cell-free supernatants were collected and assayed for TNF-α in an ELISA. The data are represented as mean ± SE (compared with LPS; **, p < 0.001; compared with Chr Et plus LPS; *, p < 0.02, n = 6).

Alcohol exposure, acute and chronic, promotes LPS-induced IκBα degradation in monocytes. Monocytes were exposed to alcohol for 1 (Ac), 4, and 7 days followed by simulation with LPS (100 ng/ml) for 60 min. Cytoplasmic extracts were prepared and immunoblotted with total IκBα Ab. The bar graph represents mean density units ± SE from a total of six individuals (compared with LPS; *, p < 0.02, n = 6).

Opposite effects of acute and chronic alcohol exposure on NFκB binding activity is independent of alcohol metabolism in human monocytes. A, Monocytes were exposed to alcohol for 1 (Ac), 4, and 7 days followed by simulation with LPS (100 ng/ml) for 60 min. NFκB was detected in the nuclear extracts by EMSA using a 32P-labeled double-stranded NFκB oli-gonucleotide. The bar graph shows mean density ± SE of a total of six individuals (compared with LPS; *, p < 0.01). B, RAW 264.7 macrophages were exposed to alcohol (Et) for 1 (ac) or 4 days (chr) and then transiently transfected with NFκB reporter gene constructs carrying five tandem copies of the NFκB binding site in front of the firefly luciferase gene and the Renilla luciferase construct. At 24 h after transfection, cells were treated with LPS (100 ng/ml) for 8 h. Cells were then lysed to determine firefly luciferase activity and normalized to the Renilla luciferase activity. The bar graph represents fold activation of the luciferase gene as compared with the unstimulated control of a total of three experiments. (compared with LPS; *, p < 0.01; **, p < 0.02, n = 3). C, Monocytes were exposed to 25 mM alcohol (Et) for 1 to 7 days and CYP2E1 protein levels determined by immunoblotting using anti-CYP2E1 Abs as described in Materials and Methods. The bar graph represents mean density ± SE of a total of six individuals. D, Monocytes were exposed to alcohol for 7 days along with 4-methyl-prazole or cyanamide followed by stimulation with LPS (100 ng/ml) for 60 min. NFκB was detected in the nuclear extracts by EMSA using a 32P-labeled double-stranded NFκB oligonucleotide. The bar graph shows mean density ± SE of a total of six individuals (compared with unst; *, p < 0.001; compared with LPS; **, p < 0.001). E, Monocytes were exposed to 25 mM alcohol (Et) for 7 days (Chr) along with 4-methyl-prazole or cyanamide followed by stimulation with LPS (100 ng/ml) for 18 h. Cell-free supernatants were collected and assayed for TNF-α in an ELISA. The data are represented as mean ± SE (compared with LPS; *, p < 0.001, n = 6).

Alcohol, based on the length of exposure, alters IRAK-M to differently regulate LPS signaling in human monocytes. Acute alcohol exposure induces whereas chronic alcohol decreases IRAK-M in human monocytes to either reduce or augment activity of down-stream LPS signaling molecules IRAK-1, IKK, ERK, and NFκB, and finally TNF-α production, respectively.