Blunted Expansion of Regulatory T Lymphocytes Is Associated With Increased Bacterial Translocation in Patients With Major Depressive Disorder

Abstract

Background: Major Depressive Disorder (MDD) is associated with both proinflammatory and adaptive immune response abnormalities. Regulatory T lymphocytes (Tregs), a subtype of CD4+ T cells, are relevant for maintaining immune-inflammatory system homeostasis and control of inflammation such as the kind potentially induced by the interactions between the intestinal microbiome and gut mucosa. We investigated the Treg population and its distribution along their stages of differentiation/activation, as well as its function in MDD patients without concomitant diseases. We also studied the potential association between Treg alterations, intestinal barrier damage, and bacterial translocation. Methods: 30 MDD patients and 20 healthy controls were studied. The levels of circulating CD25FoxP3⁺ Tregs and their distribution on the naïve (T_N), effector (T_E), central (T_CM), and effector memory(T_EM) differentiation/activation stages were analyzed using polychromatic flow cytometry. Chemokine receptors (CCR) 2, 5, and 6, and the intracytoplasmic IL-10 expression by the Tregs were also analyzed. The serum IL-10 was measured using Luminex. The serum levels of zonulin and the intestinal fatty acid-binding protein (I-FABP), both markers of gut barrier function, and the LPS-binding protein (LBP), a marker of bacterial translocation, were measured using an enzyme-linked immunosorbent assay. Results: MDD patients had increased number of circulating Tregs cells with enhanced number of Tregs at the T_N, T_E, T_CM, and T_EM stages. The percentage of Tregs cells at T_N stage was significantly higher in MDD patients. The percentage of Tregs that expressed CCR2 and CCR6 was increased as well as those expressing IL-10. MDD patients had significantly increased levels of circulating I-FABP and LBP. MDD patients with high LBP levels had a significant reduction in the number of circulating Tregs compared to normal-LBP MDD patients. Conclusions: MDD patients showed an expansion of circulating Tregs and their CD25^highFoxP3⁺ and CD25^lowFoxP3⁺ subsets throughout the different stages of CD4+ T lymphocyte differentiation/activation. Tregs also showed an increased frequency of cells expressing CCR6 and CCR2. IL-10 Treg production was also enhanced in MDD patients that concurrently had increased serum IL-10 levels. However, this Treg expansion was blunted in MDD patients with gut barrier damage and increased bacterial translocation.

Keywords: CD4+ lymphocytes; LPS-binding protein; chemorreceptors; gut barrier; interleukin 10 (IL10); major depressive disorder; regulatory T lymphocytes (Treg).

Figure 1

Dot plots represent the flow cytometry gating strategy and histograms of the circulating total Tregs and their CD25^highFoxP3⁺ and CD25^lowFoxP3⁺ subsets in a representative case of MDD. The first row dot plots represent the selected gates and percentages to obtain the total Tregs (A), the second those of the individualized total Tregs and their CD25^highFoxP3⁺ and CD25^lowFoxP3⁺ subsets (B, respectively). The (C) shows dot plots of the selected gates and percentages of the CCR2 (superior line), CCR5 (medium line), and CCR6 (lower line) expression by total Tregs and their CD25^highFoxP3⁺ and CD25^lowFoxP3⁺ subsets.

Figure 2

Absolute number and frequency of total circulating Tregs and their CD25^hiFoxP3⁺ and CD25^lowFoxP3⁺ subsets in MDD patients and healthy controls. Absolute number (cells/μl) (y axis) of CD25⁺FoxP3⁺ lymphocytes and their CD25^hiFoxP3⁺ and CD25^lowFoxP3⁺ subsets in MDD patients (gray box plots) and healthy controls (white box plots) are shown in panel (A). Frequency of the total Tregs and their CD25^hiFoxP3⁺ and CD25^lowFoxP3⁺ subsets in the circulating CD4+ T lymphocytes in MDD patients (gray box plots) and controls (white box plots) are shown in (B). The top of the rectangle indicates the third quartile, the horizontal line near the middle of the rectangle indicates the median, and the bottom of the rectangle indicates the first quartile. A vertical line extends from the top of the rectangle to indicate the maximum value, and another vertical line extends from the bottom of the rectangle to indicate the minimum value. *Significant difference between MDD and healthy controls for the indicated variable.

Figure 3

Absolute number and frequency of circulating total Tregs and their CD25^hiFoxP3⁺ and CD25^lowFoxP3⁺ subsets at the T_N, T_CM T_EM, and T_E stages of CD4+ T lymphocyte differentiation/activation in MDD patients and healthy controls. Absolute number (cells/μl) (y axis) of Tregs lymphocytes and their CD25^hiFoxP3⁺ and CD25^lowFoxP3⁺ subsets at the T_N, T_CM T_EM, and T_E stages of CD4+ T lymphocyte differentiation/activation in MDD patients (gray box plots) and healthy controls (white box plots) are shown in (A–C), respectively. Frequency of the total Tregs and their CD25^hiFoxP3⁺ and CD25^lowFoxP3⁺ subsets at the T_N, T_CM T_EM, and T_E stages of differentiation/activation in MDD patients (gray box plots) and controls (white box plots) are shown in (D–F), respectively. The top of the rectangle indicates the third quartile, the horizontal line near the middle of the rectangle indicates the median, and the bottom of the rectangle indicates the first quartile. A vertical line extends from the top of the rectangle to indicate the maximum value, and another vertical line extends from the bottom of the rectangle to indicate the minimum value. *Significant difference between MDD and healthy controls for the indicated variable.

Figure 4

Frequency of CCR2, CCR5 and CCR6 expression by Tregs and their CD25^hiFoxP3⁺ and CD25^lowFoxP3⁺ subsets in MDD patients and healthy controls, Frequencies of total Tregs and their CD25^hiFoxP3⁺ and CD25^lowFoxP3⁺ subsets (y axis) in MDD patients (gray box plots) and healthy controls (white box plots) that express the CCR2 (A), CCR5 (B), and CCR6 (C). The top of the rectangle indicates the third quartile, the horizontal line near the middle of the rectangle indicates the median, and the bottom of the rectangle indicates the first quartile. A vertical line extends from the top of the rectangle to indicate the maximum value, and another vertical line extends from the bottom of the rectangle to indicate the minimum value. *Significant difference between MDD and healthy controls for the indicated variable.

Figure 5

Frequency of IL-10 expression and number of circulating total Tregs and of those at the T_N, T_CM T_EM, and T_E stages of differentiation/activation able to produce this cytokine in MDD patients and healthy controls. Frequencies of total Tregs and of those at the T_N, T_CM T_EM, and T_E stages of differentiation/activation (y axis) in MDD patients (gray box plots) and healthy controls (white box plots) that express IL10 (x axis) after in vitro PMA (50 ng/ml) stimulation for 4 h in total (A). Absolute number (cells/μl) (y axis) of circulating total Tregs and of those at the T_N, T_CM T_EM, and T_E stages of differentiation/activation in MDD patients (gray box plots) and healthy controls (white box plots) that are able to express the indicated cytokine (x axis) after in vitro PMA stimulation for 4 h (B). Serum concentrations (pg/μl) (y axis) of IL-10 MDD patients (gray box plots) and healthy controls (white box plots) (C). The top of the rectangle indicates the third quartile, the horizontal line near the middle of the rectangle indicates the median, and the bottom of the rectangle indicates the first quartile. A vertical line extends from the top of the rectangle to indicate the maximum value, and another vertical line extends from the bottom of the rectangle to indicate the minimum value. *Significant difference between MDD and healthy controls for the indicated variable.

Figure 6

Serum levels of LBP, zonulin and I-FABP in MDD patients and absolute number of circulating Tregs and their CD25^hiFoxP3⁺ and CD25^lowFoxP3⁺ subsets in high-LBP and normal-LBP MDD patients. Serum concentrations (y axis) of LBP (ng/ml), zonulin (mg/ml) and I-FABP (pg/ml) in MDD patients (gray box plots) and healthy controls (white box plots) (A). Absolute number of circulating Tregs and their CD25^hiFoxP3⁺ and CD25^lowFoxP3⁺ subsets (y axis) in high-LBP (ascending lined box plots) and normal-LBP (descending lined box plots) MDD patients subsets and healthy controls (white box plots) (B). The top of the rectangle indicates the third quartile, the horizontal line near the middle of the rectangle indicates the median, and the bottom of the rectangle indicates the first quartile. A vertical line extends from the top of the rectangle to indicate the maximum value, and another vertical line extends from the bottom of the rectangle to indicate the minimum value. *Significant difference between MDD and healthy controls for the indicated variable.