Implications of ER stress, the unfolded protein response, and pro- and anti-apoptotic protein fingerprints in human monocyte-derived dendritic cells treated with alcohol

Abstract

Background: Dendritic cells (DCs) are responsible for the activation of T cells and B cells. There is accumulating evidence that psychoactive substances such as alcohol can affect immune responses. We hypothesize that this occurs by modulating changes in proteins triggering a process known as unfolded protein response (UPR). This process protects cells from the toxic effects of misfolded proteins responsible for causing endoplasmic reticulum (ER) stress. Although much is known about ER stress, little is understood about the consequences of ethanol use on DC's protein expression.

Methods: In this study, we investigated alterations in the proteins of human monocyte-derived dendritic cells (MDDC) treated with 0.1% of alcohol by two-dimensional (2D) gel electrophoresis followed by liquid chromatography-tandem mass spectrometry, protein identification, and confirmation at the gene expression level by qRT-PCR.

Results: Proteomes of related samples demonstrated 32 differentially expressed proteins that had a 2-fold or greater change in expression (18 spots were up-regulated and 14 were down-regulated), compared to the control cultures (untreated cells). Alcohol significantly changed the expression of several components of the UPR stress-induced pathways that include chaperones, ER stress, antioxidant enzymes, proteases, alcohol dehydrogenase, cytoskeletal and apoptosis-regulating proteins. qRT-PCR analyses highlighted the enhanced expression of UPR and antioxidant genes that increased (18 hours) with alcohol treatment.

Conclusion: Results of these analyses provide insights into alcohol mechanisms of regulating DC and suggest that alcohol induced specifically the UPR in DC. We speculate that activation of a UPR by alcohol may protect the DC from oxidant injury but may lead to the development of alcohol-related diseases.

Figure 1

Differentially expressed monocyte derived dendritic cell proteins identified by peptide mass fingerprinting. Down-regulated proteins are indicated with solid red squares in the DC control 2D gel image and up-regulated proteins are indicated with solid red squares in the DC+0.1% EtOH 2D gel image. The triangles represent less expression in the corresponding 2D gel. 2D spots are numbered as shown in table 1. The standard common proteins are indicated with black squares and letters.

Figure 2

Multichannel viewer of the overlapped images. Ethanol treatment (etOH) of monocytes derived dendritic cells treated with ethanol vs. control (non treated cells). Three-dimensional interpretation for six representative proteins identified by peptide mass finger printing and corresponding RT-PCR are shown around the 2D-GE images. Each pair of protein spots are 3D views of their relative peak volumes. The peak area comprising the entire volume represents the 2D-GE distributions of the protein spot in the gel, whereas the volume correlates to the protein concentration. The SSP # located in the respective 3D view images correspond to the protein volumes shown. Ethanol induced changes in gene expression. IDC (5 × 10⁵ cells/ml) were treated with ethanol (0.1% v/v) for 18h. Total RNA was extracted, reverse transcribed and subjected to Q-PCR. Data are presented as mean fold change in gene expression + SE of 3 independent experiments. Statistical significance was calculated by students “t” test. The blue circles and arrows indicate downregulated proteins and the black circle and arrow indicate an upregulated protein.

Figure 3

Proposed model highlighting UPR (unfolded protein response) pathway with pro-apoptotic and anti-apoptotic protein signatures triggered by ER stress in alcohol treated monocyte derived dendritic cell.