Association between regulatory T cell activity and sepsis and outcome of severely burned patients: a prospective, observational study

Abstract

Introduction: To investigate the significance of changes in regulatory T cells (Tregs) activity and its relationship with sepsis, as well as outcome of patients with major burns.

Methods: The periphery blood samples of 106 patients were collected on post-burn days 1, 3, 7, 14, and 21. Tregs were isolated and their phenotypes (cytotoxic T-lymphocyte-associated antigen 4 and forkhead/winged helix transcription factor p3) were analyzed by flow cytometry, and the contents of cytokines (interleukin-10 and transforming growth factor-beta1) released into supernatants by Tregs were also determined by enzyme-linked immunosorbent assay kits. Gene expressions of cytokines were assessed by real-time quantitative polymerase chain reaction.

Results: Expressions of Tregs phenotypes and gene/protein expression of cytokines were all elevated after burn, and there were obvious differences among patients with various burn sizes. They were also higher in septic patients than those without sepsis. Among septic patients, the expressions of Tregs phenotypes and the levels of cytokines were markedly lower in the survival group than those in patients with fatal outcome.

Conclusions: Severe burn injury per se could lead to the changes in Tregs activities. Elevated levels of cytokines produced by Tregs and activation markers on Tregs surface might play an important role in the pathogenesis of sepsis and mortality in burned patients.

Figure 1

Isolation of CD4⁺CD25⁺ Tregs from the peripheral blood lymphocytes. CD4⁺CD25⁺regulatory T cells (Tregs) were isolated from the peripheral blood lymphocytes in two steps by magnetic cell sorting (MACS) system according to manufacturer's instructions. (a) T lymphocytes before isolation and (b) CD4⁺T cells are shown. (c) The purity of positively sorted CD4⁺CD25⁺ Tregs was 93.5 ± 1.7% with the survival rate of 96.2 ± 2.9%. (d) The purity of negatively sorted CD4⁺CD25-T cells was 89.6 ± 2.5%.

Figure 2

Flow cytometric analysis of phenotypes of Tregs. Increased expressions of CTLA-4 and FOXP3 on the surface of regulatory T cells (Tregs) from burned patients were found on postburn days (PBD) 1 to 21 compared with normal controls, and there were obvious differences among patients with various burn sizes ((a and b) a mean of all days). The expressions of CTLA-4 and FOXP3 were significantly higher in patients with serious burns during the whole observational period, and (c and d) they were much higher in septic patients than those without sepsis on PBD 3 to 21. (e and f) Among septic patients, the expressions of CTLA-4 and FOXP3 in the survival group were obviously lower than those in non-survival group on PBD 3 to 21. * P < 0.05, ** P < 0.01, Group I vs. normal group or sepsis group vs. non-sepsis group or non-survivors vs. survivors; #P < 0.05, ## P < 0.01, Group II vs. Group I; &P < 0.05, Group III vs. Group II.

Figure 3

ELISA analysis of IL-10 and TGF-β1 levels in Tregs supernatants. Elevated protein expressions of IL-10 and TGF-β1 in regulatory T cells (Tregs) from burned patients were detected on postburn days (PBD) 1 to 21 in comparison to normal controls, and there were obvious differences among patients with different extent of burn injury ((a and b) a mean of all days). Protein levels of (c) IL-10 and (d) TGF-β1 in Tregs were significantly higher in septic patients than those without sepsis on PBD 3 to 21. Among septic patients, (e) IL-10 and (f) TGF-β1 levels in the survivors were obviously lower than those with non-survivors on PBD 3 to 21. **P < 0.01, Group I vs. normal group, or sepsis group vs. non-sepsis group or non-survivors group vs. survivors group; ^##P < 0.01, Group II vs. Group I; ^&P < 0.05, ^&&P < 0.01, Group III vs. Group II.

Figure 4

SYBR green real-time RT-PCR analysis for mRNA expression of IL-10 and TGF-β1 in Tregs. Enhanced gene expressions of (a) IL-10 and (b) TGF-β1 in regulatory T cells (Tregs) from burned patients were detected on postburn days (PBD) 1 to 21 in comparison with normal controls, and there were obvious differences among patients with different extent of burn injury. mRNA expressions of (c) IL-10 and (d) TGF-β1 in Tregs were significantly higher in septic patients than those without sepsis on PBD 3 to 21. Among septic patients, (e) IL-10 and (f) TGF-β1 mRNA expressions in the survival group were markedly lower than those with fatal outcome on PBD 3 to 21. *P < 0.01, **P < 0.01, Group I vs. normal group or sepsis group vs. non-sepsis group, or non-survivors group vs. survivors group; ^#P < 0.01, ^##P < 0.01, Group II vs. Group I; ^&P < 0.05, ^&&P < 0.01, Group III vs. Group II.