Thymic indoleamine 2,3-dioxygenase-positive eosinophils in young children: potential role in maturation of the naive immune system

Abstract

Eosinophils expressing indoleamine 2, 3-dioxygenase (IDO) may contribute to T-helper cell (Th)2 predominance. To characterize human thymus IDO+ eosinophil ontogeny relative to Th2 regulatory gene expression, we processed surgically obtained thymi from 22 children (age: 7 days to 12 years) for immunohistochemistry and molecular analysis, and measured cytokine and kynurenine levels in tissue homogenates. Luna+ eosinophils ( approximately 2% of total thymic cells) decreased in number with age (P = 0.02) and were IDO+. Thymic IDO immunoreactivity (P = 0.01) and kynurenine concentration (P = 0.01) decreased with age as well. In addition, constitutively-expressed interleukin (IL)-5 and IL-13 in thymus supernatants was highest in youngest children. Eosinophil numbers correlated positively with expression of the Th2 cytokines IL-5, IL-13 (r = 0.44, P = 0.002), and IL-4 (r = 0.46, P = 0.005), transcription factor signal transducer and activator of transcription-6 (r = 0.68, P = 0.001), and the chemokine receptor, CCR3 (r = 0.17, P = 0.04), but negatively with IL-17 mRNA (r = -0.57, P = 0.02) and toll-like receptor 4 expression (r = -0.74, P = 0.002). Taken together, these results suggest that functional thymic IDO+ eosinophils during human infant life may have an immunomodulatory role in Th2 immune responses.

Figure 1

Presence of eosinophils and l-kynurenine in the thymus of young children. Representative histological sections of Luna-positive eosinophils in the thymus of children at 1 week (A), 1 year (B), and 12 (C) years of age. We also detected eosinophils within Hassall’s corpuscles in the early perinatal period, at 2 weeks (D) and 4 weeks (E) after birth. Many of these eosinophils were degranulated (F, age 4 weeks). In the same 4-week-old neonate, kynurenine-immunoreactive cells were detected throughout the thymus (G) including Hassall corpuscles (H). Isotype control staining for kynurenine antibody is shown in (I).

Figure 2

Age-dependent decrease in the number of eosinophils in the thymus. A: Number of Luna-positive eosinophils in the thymus of children <6 months of age (n = 7), 6 to 12 months of age (n = 5), 1 to 5 years (n = 7), and older than 5 (n = 3). The bar represents group means. *P = 0.02 between <6-month and >5-year age groups. B: Representative flow cytometry density plot illustrating the presence of eosinophils (CCR3⁺ and MBP⁺) in the thymus of a 2-week-old infant, which make up approximately 2% of the total cell population.

Figure 3

Thymic eosinophils contain IDO and their numbers decrease with age. A: Immunodetection of Luna-positive eosinophils (red) and IDO-positive cells (brown) showing the majority of eosinophils to contain IDO (co-localization shown by arrows). B: Confirmatory immunofluorescent staining using BMK-13 monoclonal antibody (directed against human eosinophil major basic protein) conjugated to BODIPY FL (green, C) and IDO human antibody conjugated to rhodamine tetramethylrhodamine B isothiocyanate (red, D) demonstrating co-localization of the two markers (E). Inserts illustrate lack of staining with isotype controls for BMK-13 (IgG1 conjugated to BODIPY FL in C) or IDO (IgG1, IgG2a, IgG2b, and IgG3 conjugated to tetramethylrhodamine B isothiocyanate in D). The number of IDO-positive eosinophils in the thymus decreased with age (F). *P = 0.01 between <6-month and >5-year-age groups.

Figure 4

The ontogeny of global expression of Th1/Th2 genes in the thymus. High (A), moderate (B), and low (C) baseline expression of Th1 and Th2 genes in the thymus obtained from children <6 months of age (black squares, n = 7), 6 to 12 months of age (black circles, n = 5), 1 to 5 years (black diamond, n = 7), and children >5 years of age (black triangle, n = 3). Targets were examined using real-time PCR and results are represented as means ± SEM. IL-5 mRNA and IL-17 mRNA were measured but values were below level of detection. *P < 0.05 versus <6-month-old age group.

Figure 5

Correlations between Luna-positive eosinophils and Th1/Th2 markers. Correlation between individual subjects’ Luna-positive eosinophil count and IDO (A), STAT-6 (B), FOXP3 (C), IL-4 (D), CCR3 (E), IL-17 (F), GATA-3 (G), TLR-4 (H), or T-bet (I) mRNA gene expression. R values are the correlation coefficients between the two variables. All correlations are linear except for IL-17 (F) and TLR-4 (H).

Figure 6

Correlations between IDO-positive cells and Th1/Th2 markers. Correlation between individual subjects’ IDO-positive cells and STAT-6 (A), IL-4 (B), FOXP3 (C), GATA-3 (D), and TLR-4 (E) mRNA expression. R values are the correlation coefficients between the two variables.

Figure 7

Baseline cytokine and innate gene expression in the human thymus. A: Baseline cytokine protein production in the supernatants from homogenized thymic tissue and B: mRNA expression of innate (TLR) genes in unstimulated cells from neonates <6 months old (white bars, n = 7), infants between 6 and 12 months of age (gray bars, n = 5), 1- to 5-year-old children (black bars, n = 7), and children older than 5 years (striped bars, n = 3). Results are represented as means ± SE. In (B), RNA results are normalized to the reference housekeeping gene for ubiquitin-conjugating enzyme E2D2 and represented as means ± SE. There was no significant differences in constitutive (or basal) cytokine levels or expression of innate TLRs in the thymus between age groups although a trend for increased IFNγ (P = 0.071) and decreased IL-5 (P = 0.062) protein is seen in children >5 years of age compared with <6-month-old infants.

Figure 8

Age-dependent decrease in l-kynurenine activity in the human thymus. Age-dependent decrease in l-kynurenine concentration in the supernatants from thymus of neonates <6 months of age (n = 7), infants between 6 and 12 months of age (n = 5), 1- to 5-year-old children (n = 7), and children older than 5 years of age (n = 3). The bar represents group means. *P = 0.01 between <6-month and >5-year age groups. Concentration is given in μM/L.