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. 2014 Aug 15;193(4):1975-87.
doi: 10.4049/jimmunol.1303468. Epub 2014 Jul 14.

Moderate alcohol induces stress proteins HSF1 and hsp70 and inhibits proinflammatory cytokines resulting in endotoxin tolerance

Sujatha Muralidharan  1 , Aditya Ambade  1 , Melissa A Fulham  1 , Janhavee Deshpande  1 , Donna Catalano  1 , Pranoti Mandrekar  2
Affiliations

Moderate alcohol induces stress proteins HSF1 and hsp70 and inhibits proinflammatory cytokines resulting in endotoxin tolerance

Sujatha Muralidharan et al. J Immunol. .

Abstract

Binge or moderate alcohol exposure impairs host defense and increases susceptibility to infection because of compromised innate immune responses. However, there is a lack of consensus on the molecular mechanism by which alcohol mediates this immunosuppression. In this study, we show that cellular stress proteins HSF1 and hsp70 play a mechanistic role in alcohol-mediated inhibition of the TLR4/MyD88 pathway. Alcohol exposure induced transcription factor HSF1 mRNA expression and DNA binding activity in primary human monocytes and murine macrophages. Furthermore, HSF1 target gene hsp70 mRNA and protein are upregulated by alcohol in monocytes. In vitro pre-exposure to moderate alcohol reduced subsequent LPS-induced NF-κB promoter activity and downstream TNF-α, IL-6 and IL-1β production in monocytes and macrophages, exhibiting endotoxin tolerance. Mechanistic analysis demonstrates that alcohol-induced HSF1 binds to the TNF-α promoter in macrophages at early time points, exerting transrepression and decreased TNF-α expression. Furthermore, association of hsp70 with NF-κB subunit p50 in alcohol-treated macrophages correlates with reduced NF-κB activation at later time points. Hsp70 overexpression in macrophages was sufficient to block LPS-induced NF-κB promoter activity, suggesting alcohol-mediated immunosuppression by hsp70. The direct crosstalk of hsp70 and HSF1 was further confirmed by the loss of alcohol-mediated endotoxin tolerance in hsp70- and HSF1-silenced macrophages. Our data suggest that alcohol-mediated activation of HSF1 and induction of hsp70 inhibit TLR4-MyD88 signaling and are required for alcohol-induced endotoxin tolerance. Using stress proteins as direct drug targets would be clinically relevant in alcohol abuse treatment and may serve to provide a better understanding of alcohol-mediated immunosuppression.

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Conflict of interest statement

Conflict of Interest

The authors have no competing financial interest.

Figures

Figure 1
Figure 1. Pre-exposure of human monocytes to alcohol results in decreased levels of TLR4- induced pro-inflammatory cytokine mRNA
(A-C) Adherence isolated human monocytes were treated with 100 ng/ml LPS or 25 mM alcohol (Et) alone for 2 hours or pre-exposed to 25 mM alcohol for 1, 3, 5, 7 or 24 hours followed by LPS stimulation for 2 hours. Levels of TNFα (A), IL-1β (B) and IL-6 (C) mRNA were analyzed by qPCR. Fold change in expression of genes was calculated with respect to untreated. LPS tolerance control: 10 ng/ml LPS for 24 hrs followed by LPS (100 ng/ml). Data summarize mean ± SD of 13 independent experiments. (* p<0.05, ** p<0.01, *** p<0.005, ns not significant)
Figure 2
Figure 2. Pre-exposure of human monocytes to alcohol inhibits TLR4-induced pro-inflammatory cytokine protein levels
(A-C) Adherence isolated human monocytes were treated with 100 ng/ml LPS or 25 mM alcohol (Et) alone or pre-exposed to 25 mM alcohol for 1, 3, 5, 7 or 24 hours followed by LPS stimulation for 18-22 hours (overnight). Secreted TNFα (A), IL-1β (B) and IL-6 (C) protein in overnight culture supernatants was analyzed by ELISA. LPS tolerance control: 10 ng/ml LPS for 24 hrs followed by LPS (100 ng/ml). Data summarize mean ± SD of 13 independent experiments. (* p<0.05, ** p<0.01, *** p<0.005, ns not significant)
Figure 3
Figure 3. Alcohol induces HSF1 expression and activation in human monocytes and RAW macrophages
(A) Human monocytes isolated by adherence were exposed to 25 mM alcohol (Et) for 2-24 hours and total RNA collected at different timepoints were analyzed for HSF1 mRNA by qPCR. Graph depicts mean ± SD of 7 independent experiments. (B) RAW macrophages were exposed to 25 or 50 mM alcohol (Et) for 2-24 hours and total RNA collected at different timepoints were analyzed for HSF1 mRNA by qPCR. Data summarize mean ± SD of 3 independent experiments. (C) Adherence isolated human monocytes were treated with 100 ng/ml LPS, 25 mM alcohol (Et) alone or pre-exposed to 25 mM alcohol followed by LPS stimulation for indicated timepoints. HS denotes heat shocked control: 42°C for 45 minutes. Total RNA was subjected to HSF1 mRNA determination by qPCR. Data summarizes mean ± SD of 7 independent experiments. (D-F) RAW macrophages were stimulated with 100 ng/ml LPS for 30 minutes, 25 or 50 mM alcohol (Et) alone for 1 or 24 hours or pre-exposed to 25 or 50 mM alcohol for 1 or 24 hours followed by LPS stimulation for 30 minutes. HS denotes heat shocked control: 42°C for 45 minutes. (D) HSF1 DNA-binding activity was detected in nuclear extracts by EMSA using a 32P-labeled, double-stranded HSE oligonucleotide. A representative experiment is depicted and graph summarizes mean ± SD of 4 independent experiments. A 20-fold excess of unlabeled oligonucleotide added to the heat shocked sample to confirm specificity of HSF1 binding was included as competition control (Comp). (E) Expression of hsp90β in the cytoplasmic extracts was assayed by western blotting. A representative experiment is depicted and graph summarizes mean ± SD of 4 independent experiments. (F) Phosphorylated HSF1 (phosphoserine 326) was detected in nuclear lysates by ELISA and normalized to total HSF1. Graph depicts mean ± SD of 4 independent experiments. (* p<0.05, ** p<0.01, ns not significant)
Figure 4
Figure 4. Alcohol induces HSF1-mediated hsp70 expression in human monocytes and RAW macrophages
(A) Human monocytes isolated by adherence were exposed to 25 mM alcohol (Et) for 2-24 hours and total RNA collected at different timepoints were analyzed for hsp70 mRNA by qPCR. Graph depicts mean ± SD of 7 independent experiments. (B and C) Adherence isolated human monocytes were treated with 100 ng/ml LPS, 25 mM alcohol (Et) alone or pre-exposed to 25 mM alcohol followed by LPS stimulation for indicated timepoints. HS denotes heat shocked control: 42°C for 45 minutes. (B) Total RNA was subjected to hsp70 mRNA determination by qPCR. Data summarizes mean ± SD of 7 independent experiments. (C) hsp70 protein was detected in whole cell lysates by western blotting. The densitometry graph represents quantitation of bands seen in the gel and depicts mean ± SD (n=9). Representative gels are shown with hsp70 (top) and loading control, β-actin (bottom). (D) RAW macrophages were stimulated with 100 ng/ml LPS or 50 mM alcohol (Et) for 1 hour or pre-exposed to alcohol for 1 hour followed by LPS stimulation for 1 hour. HS denotes heat shocked control: 42°C for 45 minutes. Chromatin immunoprecipitation assay was performed using anti-HSF1 antibody and semi-quantitative PCR was carried out using hsp70 promoter specific primers. A representative gel picture is shown above the densitometry graph, which represents quantitation of bands seen in the gel (n=4). Input DNA is shown to ensure equal amount of the sheared DNA. (* p<0.05, ** p<0.01, *** p<0.005, ns not significant)
Figure 5
Figure 5. Alcohol inhibits TLR4-stimulated NF-κB activation via hsp70 and HSF1
(A-B) RAW macrophages were stimulated with 100 ng/ml LPS for 30 minutes, 50mM alcohol (Et) alone for 1 or 24 hours or pre-exposed to alcohol followed by LPS stimulation for 30 minutes. NF-κB subunits p65 (A) and p50 (B) were detected in nuclear lysates by western blotting. The densitometry graph represents quantitation of bands seen in the gel and depicts mean ± SD of 4 independent experiments. Representative gels are shown with p65 (top) or p50 (top) and loading control TATA-binding protein 1 (TBP-1) (bottom). (C) RAW macrophages were transiently transfected with pNF-κBluc at 1:3 DNA to lipofectamine ratio. 24 hours after transfection, macrophages were treated with 100 ng/ml LPS for 6 hours, 50 mM alcohol (Et) alone for 24 hours or pre-exposed to alcohol for 1 or 24 hours followed by LPS stimulation for 6 hours and luciferase activity was measured in the cell lysates. Graph depicts mean ± SD (n=3). (D-E) RAW macrophages were stimulated with 100 ng/ml LPS for 1 hour or 50mM alcohol (Et) for 1 or 24 hours or pre-exposed to alcohol for 1 or 24 hours followed by LPS stimulation for 1 hr. HS denotes heat shocked control: 42°C for 45 minutes. (D) Whole cell lysates were used for immunoprecipitation with anti-p50 antibody and levels of hsp70 and p50 in immunoprecipitated samples were analyzed by immunoblotting. The densitometry graph represents quantitation of bands seen in the gel and depicts mean ± SD of 3 independent experiments. Representative gels are shown with hsp70 (top) and p50 (bottom). (E) Chromatin immunoprecipitation assay was performed using anti-HSF1 antibody and semi-quantitative PCR was carried out using TNFα promoter specific primers. A representative gel picture is shown above. The densitometry graph represents quantitation of bands seen in the gel (n=4). Input DNA is shown to ensure equal amount of the sheared DNA. (* p<0.05, ** p<0.01, *** p<0.005)
Figure 6
Figure 6. Overexpression of hsp70 is sufficient to mimic alcohol-mediated induction of TLR4 tolerance
(A) RAW macrophages were transiently transfected with pCMV5-hsp70 using 1:1, 1:2 and 1:3 ratios of 2 μg DNA to lipofectamine. 24 hours after transfection, whole cell lysate was collected and analyzed for hsp70 protein by western blotting. The densitometry graph represents quantitation of bands seen in the gel and depicts mean ± SD (n=5). Representative gels are shown with hsp70 (top) and loading control, β-actin (bottom). UT denotes untransfected and HS denotes heat shocked control: 42°C for 45 minutes. (B) RAW macrophages were transiently transfected with 0.5, 1 or 1.5 μg pCMV5-hsp70 in combination with 0.5 μg p(κB)4-luc at 1:3 DNA to lipofectamine ratio, maintaining total plasmid DNA at 2 μg. 24 hours after transfection, macrophages were stimulated with 100 ng/ml LPS for 6 hours and luciferase activity was measured in the cell lysates. Graph depicts mean ± SD (n=3). (C-E) RAW macrophages were transiently transfected with 2 μg pCMV5-hsp70 using 1:3 ratio of DNA to lipofectamine. 24 hours after transfection, macrophages were stimulated with 100 ng/ml LPS for 6 hours and culture supernatants were assayed for secreted TNFα (C), IL-1β (D) and IL-6 (E) by ELISA. Graph depicts mean ± SD (n=4). (* p<0.05, ** p<0.01, *** p<0.005)
Figure 7
Figure 7. HSF1 and hsp70 are required for alcohol-mediated TLR4 tolerance
(A and C) RAW macrophages were transfected with 10 nM siRNA targeting HSF1 (A) or hsp70 (B) or negative control (Neg ctrl) siRNA. Total RNA was subjected to HSF1 or hsp70 mRNA determination by qPCR and percent knockdown in unstimulated and heat shocked cells (42°C for 45 min) was calculated with respect to untransfected for each condition. Graph depicts mean ± SD (n=3). (B and D) RAW macrophages were transfected with 10 nM siRNA targeting HSF1 (B) or hsp70 (D) or negative control (Neg ctrl) siRNA. 24 hours after transfection, macrophages were stimulated with 100 ng/ml LPS for 2 hours, 50 mM alcohol (Et) alone for 24 hours or pre-exposed to alcohol followed by LPS stimulation for indicated times. TNFα mRNA was analyzed by qPCR. Graph depicts mean ± SD of 3 independent experiments. (* p<0.05, ns not significant)
Figure 8
Figure 8. Alcohol-induced stress proteins HSF1 and hsp70 play an important role in alcohol-induced TLR4-MyD88 tolerance
LPS stimulation induces downstream TLR4-MyD88 signaling resulting in NF-κB activation and production of pro-inflammatory cytokines. However, alcohol pre-exposure induced HSF1 and hsp70 (highlighted in gray) directly interact with subsequent LPS stimulated immune signaling molecules resulting in inhibition of NF-κB activation and pro-inflammatory cytokine production causing endotoxin tolerance.
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