Increased percentages of T helper cells producing IL-17 and monocytes expressing markers of alternative activation in patients with sepsis

Abstract

Background: A shift from Th1 to Th2 as well as an increase in Treg CD4+T cell subsets has been reported in septic patients (SP). Furthermore, these patients display modulation of monocyte function, with reduced production of pro-inflammatory cytokines upon LPS stimulus, which resembles the phenotype of alternatively activated macrophages. In this study, we evaluated the percentages of T cells differentiated into Th1, Th17 and Treg subsets, as well as the percentage of monocytes expressing markers of alternatively activated monocytes/macrophages (AAM) in SP.

Methodology/principal findings: Peripheral blood mononuclear cells (PBMC) were obtained from 32 healthy volunteers (HV) and from SP at admission (D0, n = 67) and after 7 days of therapy (D7, n = 33). Th1 and Th17 (CD3+CD8-) lymphocytes were identified by the intracellular detection of IFN-γ and IL-17, respectively, spontaneously and after PMA/Io stimulation, and Treg cells were identified by Foxp3+CD127- expression. Monocytes were evaluated for CD206 and CD163 expression. Absolute numbers of CD4+T lymphocytes were measured in whole blood samples by flow cytometry. The Mann-Whitney or Wilcoxon test was applied, as appropriate. The percentage of Th1 cells was lower in SP than in HV at admission after PMA/Io stimulation, whereas the percentage of Th17 cells was higher. In patients' follow-up samples, a higher percentage of Th1 cells and a lower percentage of Th17 cells were observed on D7 compared with the D0 samples. Treg cells remained unchanged. Septic patients showed a markedly increased proportion of monocytes expressing CD163 and CD206.

Conclusions/significance: Upon in vitro stimulus, the percentage of T helper lymphocytes producing IL-17 was higher in SP than in HV at admission, and the percentage producing IFN-γ was lower, a pattern that was reversed during follow-up. The increased expression of CD163 and CD206 indicates that monocytes may acquire the AAM phenotype during sepsis.

Figure 1. Strategy for the analysis of Th1 and Th17 lymphocytes.

Dot plots shown are representative of one healthy volunteer and one septic patient. T cells were identified based on CD3-APC-Cy7 staining and forward scatter versus side scatter parameters. For the analyses, T helper cells were gated as CD3+CD8−, and the percentages of cells producing IFN-γ or IL-17 were determined with the quadrants established based on the control samples.

Figure 2. Strategy for the analysis of T regulatory lymphocytes.

Dot plots shown are representative of one healthy volunteer and one septic patient. Fifty thousand events were acquired with CD3+ gating and forward scatter versus side scatter parameters. For the analyses, the first gate was determined based on low SSC and high CD45 APC-Cy7, and the second gate was based on CD4+CD3+ cells. The cells with the phenotype CD45+CD3+CD4+CD127-FOXP3+ were considered to be Treg lymphocytes. Quadrants or gates were established based on the isotype controls.

Figure 3. Strategy for the analysis of alternatively activated monocytes.

Dot plots shown are representative of one healthy volunteer and one septic patient. Twenty thousand events were acquired based on forward scatter versus side scatter parameters and CD14-positive stained cells. The percentages of cells expressing CD163 or CD206 were determined with quadrants based on the isotype controls.

Figure 4. Percentages of T helper (CD3+CD8−) producing IFN-γ (Th1 - A/D) and IL-17 (Th17 - B/E), and percentages of Treg (CD3+CD4+CD127-Foxp3+ - C) lymphocytes in healthy volunteers and septic patients with paired samples (D0 and D7). n  =  number of individuals in each group.

P values are shown. The Mann-Whitney test was used to compare healthy volunteer and patient samples (D0 and D7), and the Wilcoxon Signed-Rank Test was used to compare D0- and D7-related samples.

Figure 5. Percentages of T helper (CD3+CD8−) producing IFN-γ (Th1 - A/D) and IL-17 (Th17 - B/E), and percentages of Treg (CD3+CD4+CD127Foxp3+ - C) lymphocytes in non-survivors (n = 14) and survivors (n = 17) at admission (D0) and after 7 days (D7) in patients with follow-up samples. *P<0.05 (Wilcoxon Signed-Rank Test).

Figure 6. Percentages of AAM expressing CD163 (A) and CD206 (B) in healthy volunteers and septic patients with paired samples (D0 and D7).

n =  number of individuals in each group. P values are shown. The Mann-Whitney test was used to compare healthy volunteer and patient samples (D0 and D7), and the Wilcoxon Signed-Rank Test was applied to patients’ related samples.