ABSTRACT
Objective:To investigate and analyze the effect of ubiquitin-editing protein A20 on monocytes activity in patients with ventilator-associated pneumonia (VAP).Methods:Twenty-four VAP patients (VAP group) and twelve healthy controls (control group) were included from the First Affiliated Hospital of Zhengzhou University between February 2019 and September 2019. Peripheral blood mononuclear cells (PBMCs) and bronchial alveolar lavage fluid (BALF) (both infection site and non-infection site) were collected from VAP patients, while PBMCs were collected from healthy controls. A20 level in CD14 + monocytes were measured. CD14 + monocytes and CD4 + T cells were purified from VAP patients. CD14 + monocytes were transfected by A20 siRNA. Transfected CD14 + monocytes were directly/indirectly co-cultured with autologous CD4 + T cells. The secretion of interferon-γ (IFN-γ) and interleukin-17 (IL-17) by CD4 + T cells was investigated. Transfected CD14 + monocytes were directly/indirectly co-cultured with NCI-H889 cells. Cytotoxicity, and cytokines/granzyme B level, and tumor necrosis factor-related apoptosis inducing ligand (TRAIL)/Fas ligand (FasL) level was assessed. Student t test or SNK-q test was used for comparison. Results:VAP group had elevated percentage of circulating CD14 +A20 + cells than control group [(66.14±19.62)% vs. (52.52±13.71)%, P<0.05], and also had increased A20 mean fluorescence intensity (MFI) than control group [(268.0±72.56) vs. (197.4±60.01), P<0.05]. The percentage of CD14 +A20 + cells in BALF from infection site was higher than from non-infection site in VAP group [(66.14±19.62)% vs. (52.52±13.71)%, P<0.05], while A20 MFI in infection site was also up-regulated compared with non-infection site [(268.0±72.56) vs. (197.4±60.01), P<0.05]. In direct contact co-culture, A20 siRNA transfected CD14 + monocytes, which were purified from peripheral blood and BALF of VAP patients, induced elevated percentage of IFN-γ and IL-17 secreting CD4 + T cells than un-transfection or control siRNA transfection ( P<0.05). However, there were no significant differences of CD4 +IFN-γ + or CD4 +IL-17 + percentages among un-transfection, control siRNA transfection, and A20 siRNA transfection ( P>0.05). A20 siRNA transfected CD14 + monocytes, which were purified from peripheral blood and BALF of VAP patients, induced increased target cell death in both direct and indirect contact co-culture than un-transfection or control siRNA transfection ( P<0.05). Tumor necrosis factor-α, IL-6, granzyme B level and TRAIL MFI was also up-regulated ( P<0.05). There was no remarkable difference of target cell death between direct and indirect contact co-culture ( P>0.05). Conclusions:A20 was increasingly expressed in monocytes of VAP patients, and might dampen the activity of monocytes.
ABSTRACT
Objective To investigate the changes in CD4+CD25+CD127 dim/- regulatory T cells ( Tregs) and interleukin ( IL)-35 in peripheral blood and bronchoalveolar lavage fluid ( BALF) samples from patients with ventilator-associated pneumonia ( VAP) . Methods A total of 23 patients with VAP and 11 normal controls ( NCs) were enrolled in this study. Peripheral blood mononuclear cells, serum and BALF samples were isolated and collected. Levels of IL-35 were measured by enzyme linked-immunosorbent assay. Percentages of CD4+CD25+CD127dim/-Tregs were measured by flow cytometry. CD4+CD25+CD127dim/-Tregs in BALF samples were isolated and purified, which were then stimulated with recombinant human IL-35 and co-cultured with autologous CD4+CD25-T cells. Cells and supernatants were harvested for analysis of cell proliferation and cytokine secretion. Results No significant difference in peripheral Tregs and serum IL-35 was found between patients with VAP and NCs. The percentages of Tregs and the levels of IL-35 in BALF samples collected from infectious sites were remarkably higher than those collected from non-infectious sites in patients with VAP. Moreover, there was a positive correlation between Tregs and IL-35 in BALF ( r=0. 441, P=0. 035). Tregs and IL-35 in BALF samples as well as Treg-secreted IL-35 were significantly re-duced in patients who had good response to therapy. However, no significant change in these parameters was observed in patients who had poor response to therapy. Besides, suppression of cell proliferation and IL-10 secretion that were related to Tregs were inhibited in patients whose condition was improved as compared with those in patients who had no response to therapy. Stimulation with recombinant human IL-35 enhanced the immunosuppressive function of purified Tregs that were separated from BALF of treatment-na?ve patients with VAP, which was mainly marked by suppressed cell proliferation, increased secretion of inhibitory cytokines (IL-35 and IL-10), and decreased secretion of proinflammatory cytokines (IFN-γ and TNF-α). However, IL-35 had little effect on the activities of Tregs that were separated from patients with VAP who responded to therapy. Conclusion Both IL-35 and Tregs are increased in BALF of patients with VAP and IL-35 en-hances the immunosuppressive function of Tregs, which indicates that IL-35-mediated modulation of Tregs might take part in the pathogenesis of VAP.
ABSTRACT
Objective To investigate the changes in CD4+CD25+CD127 dim/- regulatory T cells ( Tregs) and interleukin ( IL)-35 in peripheral blood and bronchoalveolar lavage fluid ( BALF) samples from patients with ventilator-associated pneumonia ( VAP) . Methods A total of 23 patients with VAP and 11 normal controls ( NCs) were enrolled in this study. Peripheral blood mononuclear cells, serum and BALF samples were isolated and collected. Levels of IL-35 were measured by enzyme linked-immunosorbent assay. Percentages of CD4+CD25+CD127dim/-Tregs were measured by flow cytometry. CD4+CD25+CD127dim/-Tregs in BALF samples were isolated and purified, which were then stimulated with recombinant human IL-35 and co-cultured with autologous CD4+CD25-T cells. Cells and supernatants were harvested for analysis of cell proliferation and cytokine secretion. Results No significant difference in peripheral Tregs and serum IL-35 was found between patients with VAP and NCs. The percentages of Tregs and the levels of IL-35 in BALF samples collected from infectious sites were remarkably higher than those collected from non-infectious sites in patients with VAP. Moreover, there was a positive correlation between Tregs and IL-35 in BALF ( r=0. 441, P=0. 035). Tregs and IL-35 in BALF samples as well as Treg-secreted IL-35 were significantly re-duced in patients who had good response to therapy. However, no significant change in these parameters was observed in patients who had poor response to therapy. Besides, suppression of cell proliferation and IL-10 secretion that were related to Tregs were inhibited in patients whose condition was improved as compared with those in patients who had no response to therapy. Stimulation with recombinant human IL-35 enhanced the immunosuppressive function of purified Tregs that were separated from BALF of treatment-na?ve patients with VAP, which was mainly marked by suppressed cell proliferation, increased secretion of inhibitory cytokines (IL-35 and IL-10), and decreased secretion of proinflammatory cytokines (IFN-γ and TNF-α). However, IL-35 had little effect on the activities of Tregs that were separated from patients with VAP who responded to therapy. Conclusion Both IL-35 and Tregs are increased in BALF of patients with VAP and IL-35 en-hances the immunosuppressive function of Tregs, which indicates that IL-35-mediated modulation of Tregs might take part in the pathogenesis of VAP.