The regulatory activity of interleukin-35 to CD4+ and CD8+ T cells in sepsis patients

Objective To investigate plasma interleukin-35 (IL-35) expression in sepsis patients, and to assess the regulatory activity of IL-35 on CD4+ and CD8+ T cells in sepsis patients. Methods A prospective study was conducted. Forty-one sepsis patients and nineteen healthy controls were enrolled in this study. According to the survival outcome on 28 day after admission, sepsis patients were further divided into the survival group (n=30) and death group (n=11). Peripheral blood samples were collected within one hour of visit. Plasma IL-35 level was measured by enzyme linked immunosorbent assay (ELISA), while CD3+, CD4+, and CD8+ T cell counts were measured by flow cytometry. The correlation between IL-35 expression/T cell counts and sequential organ failure assessment (SOFA) score was also assessed. Peripheral CD4+ and CD8+ T cells from sepsis patients were purified and stimulated with recombinant human IL-35 for 48 h. mRNA relative levels of T-bet, GATA-3, FoxP3, and RORγt in CD4+ T cells and perforin, granzyme B, and FasL in CD8+ T cells were measured by real-time PCR. Interferon-γ (IFN-γ), IL-4, IL-10, IL-17, and tumor necrosis factor-α (TNF-α) levels in the cultured supernatants were measured by ELISA. Transcription factor level and cytokine expression was compared prior to and post IL-35 stimulation. Results Plasma IL-35 was significantly elevated in sepsis patients when compared with controls [(76.76±10.33) pg/mL vs (27.53±8.31) pg/mL, P 0.05). Moreover, T cell counts were negatively correlated with SOFA score (P<0.01). IL-35 stimulation resulted in the reduction of mRNA relative level of Th1 transcription factor T-bet and Th17 transcription factor RORγt in CD4+ T cells from sepsis patients (P<0.01), while elevation in regulatory T cell transcription factor FoxP3 mRNA (P<0.01). However, IL-35 stimulation did not affect Th2 transcription factor GATA-3 mRNA in CD4+ T cells (P=0.745). IFN-γ and IL-17 production by CD4+ T cells was significantly decreased in response to IL-35 stimulation (P<0.05), while IL-10 was increased (P<0.01) and IL-4 was comparable (P=0.536) between CD4+ T cells with and without IL-35 stimulation. IL-35 stimulation led to the down-regulation of mRNA relative level of perforin and granzyme B (P<0.01). However, FasL mRNA was comparable between CD8+ T cells with and without IL-35 stimulation (P=0.795). IFN-γ and TNF-α secretion was also reduced in response to IL-35 stimulation (P<0.05). Conclusion Elevated plasma IL-35 level is an important indicator for poor prognosis of sepsis patients. IL-35 inhibits the activity of CD4+ and CD8+ T cells in sepsis patients, and might take part in the pathogenesis of sepsis. Key words: Sepsis; Interleukin-35; Cytokine; T lymphocytes; Immune