Correlation of the expression of YY1 and Fas cell surface death receptor with apoptosis of peripheral blood mononuclear cells, and the development of multiple organ dysfunction in children with sepsis

Abstract

Multiple organ dysfunction (MOD) is a lethal complication in children with sepsis. Apoptosis of several cell types is involved in this process, and it is associated with increased Fas cell surface death receptor (Fas) expression. As YY1 transcription factor (YY1) negatively regulates the expression of Fas in cancer models, and is associated with the clinical outcome, it may be important in MOD. The present study aimed to determine the association between the expression of Fas, YY1 and apoptosis in children with sepsis, and its association with MOD, these factors were analyzed in 30 pediatric patients that had been diagnosed with sepsis. Peripheral blood mononuclear cells were purified from patients, and YY1 and Fas protein expression was assessed by immunocytochemistry. Apoptosis was determined by terminal deoxynucleotidyl transferase dUTP nick‑end labeling. Sepsis was monitored using clinical parameters, pediatric logistic organ dysfunction (PELOD) score and the pediatric mortality index. The results demonstrated that Fas expression was directly correlated with apoptosis levels and the expression of YY1 was inversely correlated with apoptosis levels. Patients with high levels of apoptosis exhibited increased disease severity and poor clinical outcome. Notably, the findings of the present study demonstrated that there were higher survival rates in patients with high YY1 expression, compared with those with low YY1 expression. Additionally, patients with MOD exhibited lower proportions of apoptotic cells compared with sepsis patients without MOD. Furthermore, the PELOD score was positively correlated with Fas and inversely correlated with YY1 expression. Finally, high apoptosis and low YY1 expression were prognostic factors associated with poor survival rates. These data suggested that YY1 may be important for apoptosis induction via the regulation of Fas during sepsis. Therefore, Fas may be a potential therapeutic target to prevent MOD through regulation of YY1 expression. Furthermore, YY1 and Fas expression in PBMCs may be used to as prognostic markers.

Figure 1.

Septic pediatric patients exhibit increased levels of apoptosis. (A) Apoptosis was evaluated using an in situ TUNEL assay in PBMCs from pediatrics patients with sepsis. Upper panels: Negative control assay with no enzyme, healthy control PBMCs and PBMCs from pediatric patients with sepsis. Magnification at ×40, and ×100 in lower right square. Bottom panel: Quantification of TUNEL-positive cells (%) in healthy controls and patients with sepsis (*P=0.0007 vs. healthy controls; Student's t test). (B) Fas expression was determined by immunocytochemistry on PBMCs from pediatric patients with sepsis. Upper panels: Isotype control, healthy control and patient with sepsis. Magnification at ×40, and ×100 in lower right square. Bottom panel: Quantification of Fas-positive cells (%) in healthy controls and patients with sepsis (*P=0.022 vs. healthy controls; Student's t test). (C) YY1 expression was determined by immunohistochemistry in PBMCs from pediatric patients with sepsis. Upper panels: Isotype control, healthy control and patient with sepsis. Magnification at ×40, and ×100 in lower right square. Bottom panel: Quantification of YY1-positive cells (%) in healthy controls and patients with sepsis (*P<0.05 vs. healthy controls; Student's t test). Correlation analysis between apoptosis and (D) Fas and (E) YY1 expression in PBMCs from pediatric patients with sepsis. (*P<0.05; r=0.5013 and 0.4003, respectively; Spearman's correlation analysis). (F) FasL expression was determined by immunocytochemistry in PBMCs from pediatric patients with sepsis. Upper panel: Isotype control, healthy control and patient with sepsis. Magnification. ×40, and ×100 on lower right square. Bottom panel: Quantification of FasL-positive cells (%) in healthy controls and patients with sepsis (*P=0.033 vs. healthy controls; Student's t test). Data are presented as the mean ± standard deviation of three independent experiments. TUNEL, terminal deoxynucleotidyl-transferase-mediated dUTP nick end labeling; PBMCs, peripheral blood mononuclear cells; Fas, Fas cell surface death receptor; YY1, YY1 transcription factor; FasL, Fas ligand.

Figure 2.

Percentage of TUNEL-positive (apoptotic) cells in PBMCs from pediatric patients with sepsis. (A) Percentage of apoptotic cells was compared between the severities of sepsis. (P=0.001, sepsis vs. MOD). (B) Correlation analysis between levels of apoptosis and outcome of patients with sepsis (P=0.0344; Student's t-test; alive vs. did not survive). TUNEL, terminal deoxynucleotidyl transferase dUTP nick-end labeling; PBMC, peripheral blood mononuclear cell; MOD, multiple organ dysfunction.

Figure 3.

Correlation analysis between the PELOD index and apoptosis, Fas expression and YY1 expression. (A) Correlation analysis between apoptosis levels and PELOD index in PBMCs from pediatric patients with sepsis. There was a positive correlation between the PELOD index and apoptosis. (Spearman test, P=0.01, r=0.413). (B) Correlation analysis of Fas expression and PELOD index in PBMCs derived from pediatric patients with sepsis. There was a positive correlation between the PELOD index and Fas expression (Spearman test, P=0.024, r=0.2). (C) Correlation analysis between YY1 expression and PELOD Index in PBMCs derived from pediatric patients with sepsis. There is no significant correlation between YY1 expression and the PELOD index. (Spearman test, P=0.21, r=0.29). PELOD, pediatric logistic organ dysfunction; PBMC, peripheral blood mononuclear cell; Fas, Fas cell surface death receptor; YY1, YY1 transcription factor.

Figure 4.

Survival curves for pediatric patients with sepsis with different levels of apoptosis or YY1 expression. (A) Percentage overall survival curve of patients with high/low apoptosis in peripheral blood mononuclear cells analyzed by terminal deoxynucleotidyl transferase dUTP nick-end labeling. High, 48% survival (n=18); low, 83% survival (n=12). P=0.044. (B) Percentage survival curve for pediatric patients with sepsis with high/low YY1 expression. High, 90% survival (n=16); low, 42% survival (n=14). P=0.012. YY1, YY1 transcription factor.

Figure 5.

Frequency of PELOD index in PBMCs from pediatric patients with sepsis. PELOD index was compared with outcome of patients with sepsis. (Student's t-test, P=0.021, alive vs. did not survive). PELOD, pediatric logistic organ dysfunction; PBMC, peripheral blood mononuclear cell.