Role of vimentin in modulating immune cell apoptosis and inflammatory responses in sepsis

Abstract

New diagnostic biomarkers or therapeutic targets for sepsis have substantial significance for critical care medicine. In this study, 192 differentially expressed proteins were selected through iTRAQ. Based on cluster analysis of protein expression dynamics and protein-protein interactions, hemopexin, vimentin, and heat shock protein 90 were selected for further investigation. It was demonstrated that serum vimentin (VIM) levels were significantly increased in patients with sepsis and septic shock compared to controls and that VIM expression was significantly increased in lymphocytes isolated from septic shock and sepsis patients compared to controls. Moreover, a nonsurvivor group had higher serum VIM levels and VIM expression in lymphocytes. Caspase-3 was significantly upregulated in Jurkat T cells lacking VIM and when exposed to LPS compared to control cells. In contrast, caspase-3 was reduced nearly 40% in cells over-expressing VIM. IL-2, IL-10 and IFN-α levels were significantly decreased in cells lacking VIM compared to control cells, whereas they were not significantly altered in cells over-expressing VIM. These findings suggest that VIM modulates lymphocyte apoptosis and inflammatory responses and that VIM could be a new target for the diagnosis and prognostic prediction of patients with sepsis or septic shock.

Trial registration: ClinicalTrials.gov NCT01493466 NCT03253146.

Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1

Flowchart illustrating the clinical screening stage and clinical validation stage of the target proteins.

Figure 2

Protein expression patterns analyzed by series test of clusters. The expression patterns of 167 proteins were analyzed, and eight model profiles were used to summarize. Seven protein expression patterns showed significant p-values (p < 0.05). Inserts represent an identical expression trend for several column molecules. All of the molecules exhibiting this expression trend were displayed in different colored lines above the X-axis. Detailed information is shown in Supplemental Table 2.

Figure 3

Coexpression network of the proteins. Fifty-two proteins were analyzed and identified based on a protein coexpression network with k-core algorithm. Each circle represented one protein of potential interest. The size of each circle indicated the power of the interrelation among the proteins, and edges between two circles indicated interactions between proteins. The more edges of a given target, the more proteins connect to it and the more central role it plays within the network.

Figure 4

Comparison of serum vimentin concentrations and expression in lymphocytes. Serum vimentin (VIM) concentrations in sepsis patients and vimentin (VIM) expression in lymphocytes isolated from sepsis patients were quantified by ELISA and flow cytometry, respectively, as described in the Materials and Methods. Panel A,B: Comparison of serum vimentin (VIM) concentrations in the different groups by ELISA. Panel C,D: Representative dot plots and histograms of the flow cytometry analysis. Panel E,F: Comparison of vimentin (VIM) expression in lymphocytes isolated from control, sepsis and septic shock patients. *p < 0.05.

Figure 5

Vimentin expression in Jurkat cells and its effects on apoptosis. Jurkat cells were transfected with control siRNA, vimentin-specific siRNA or a plasmid expressing vimentin as described in the Materials and Methods. Cells were cultured in serum-free DMEM as indicated, followed by TUNEL assay analysis as described in the Materials and Methods. Panel A: Representative immunoblot indicating suppression of vimentin by siRNA (Vim-siRNA) or overexpression of vimentin (Vim-DNA). Panel B: Representative images of TUNEL assays in the absence of LPS. Green: TUNEL-positive staining; Red: cell nuclei.

Figure 6

Role of vimentin in modulating Jurkat cell apoptosis in response to LPS. Jurkat cells were transfected with control siRNA, vimentin-specific siRNA or a plasmid expressing vimentin as described in the Materials and Methods. Cells were then cultured for 6, 18 and 24 h in the presence or absence of LPS (10 µg/mL). Panel A: Representative images of TUNEL staining in the presence of LPS. Panel B: Quantitative comparison of TUNEL positivity in cells with or without LPS exposure. *p < 0.05 compared to control cells without LPS; #p < 0.05 compared to control cells with LPS. Data shown are from a single representative assessment; the experiments were repeated at least 3 times with similar results.

Figure 7

Expression of caspase-3 and Bcl-2 in Jurkat cells lacking vimentin or overexpressing vimentin. Jurkat cells were transfected with control siRNA, vimentin-specific siRNA or a plasmid expressing vimentin as described in the Materials and Methods. Cells were then cultured in serum-free DMEM in the presence or absence of LPS (10 µg/mL) for 24 h. Total cell lysates were analyzed via immunoblotting for caspase-3, Bcl-2 and ß-actin (as a loading control) as described in the Materials and Methods. Panel A: Representative immunoblots. Panel B: Semiquantitative comparison of target proteins with normalization. Vertical axes: protein level expressed as “Ratio versus control”, which was obtained as follows: (1). The densities of target protein versus internal control ß-actin were obtained. (2). The group of cells transfected with Con-siRNA and cultured in SF-DMEM was set as a reference (“Control = 1”), and the ratio of other groups versus this control group were obtained. Horizontal axes: cells transfected with control siRNA (Con-siRNA), vimentin-specific siRNA (Vim-siRNA) or vimentin expressing plasmid (Vim-vector). Open bar: cells were cultured in serum-free DMEM (SF-DMEM); closed bar: cells treated with 10 µg/mL LPS. #p < 0.05; *p < 0.05 compared to cells transfected with Con-siRNA and cultured in SF-DMEM. Data represent an average of 3 separate experiments.

Figure 8

Release of cytokines in the supernatants. Jurkat cells were transfected with control siRNA, vimentin-specific siRNA or a plasmid expressing vimentin as described in the Materials and Methods. Cells were then cultured in serum-free DMEM in the presence or absence of LPS (10 µg/mL) for 24 h. Supernatants were harvested and used for quantification of cytokines by ELISA as described in the Materials and Methods. Panel A: TNF-α. Panel B: IL-2. Panel C: IL-10. Panel D: IFN-γ. Vertical axes: cytokine level expressed as pg/day/105 cells; horizontal axes: cells transfected with control siRNA (Con-siRNA), vimentin-specific siRNA (Vim-siRNA) or the plasmid expressing vimentin (Vim-vector). Data are shown from one representative assessment; experiments were each repeated at least 3 times with similar results. Open bar: cells cultured in the serum-free DMEM (SF-DMEM); closed bar: cells treated with 10 µg/mL LPS. *p < 0.05 compared to cells cultured in SF-DMEM; #p < 0.05 compared to cells transfected with Con-siRNA and cultured in SF-DMEM.