Sex steroid blockade enhances thymopoiesis by modulating Notch signaling

Abstract

Paradoxical to its importance for generating a diverse T cell repertoire, thymic function progressively declines throughout life. This process has been at least partially attributed to the effects of sex steroids, and their removal promotes enhanced thymopoiesis and recovery from immune injury. We show that one mechanism by which sex steroids influence thymopoiesis is through direct inhibition in cortical thymic epithelial cells (cTECs) of Delta-like 4 (Dll4), a Notch ligand crucial for the commitment and differentiation of T cell progenitors in a dose-dependent manner. Consistent with this, sex steroid ablation (SSA) led to increased expression of Dll4 and its downstream targets. Importantly, SSA induced by luteinizing hormone-releasing hormone (LHRH) receptor antagonism bypassed the surge in sex steroids caused by LHRH agonists, the gold standard for clinical ablation of sex steroids, thereby facilitating increased Dll4 expression and more rapid promotion of thymopoiesis. Collectively, these findings not only reveal a novel mechanism underlying improved thymic regeneration upon SSA but also offer an improved clinical strategy for successfully boosting immune function.

© 2014 Velardi et al.

Figures

Figure 1.

AR negatively regulates Dll4 expression. (A) Molecular profiles of TSCs (CD45−) 4 d after testosterone treatment (n = 9). (B) Dll4 expression in sorted TSC populations (n = 12). (C) Dll4 expression in sorted cTECs and ECs 4 d after testosterone treatment (n = 12). (A–C, 3.5-wk-old mice; mRNA expression relative to vehicle control and normalized to β-actin.) (D) Schematic representation of Dll4 promoter with ARE regions shaded in yellow and regions of high conservation in exons (dark blue), UTRs (light blue), or noncoding (pink). (E) ARE matrix logo as annotated in the JASPAR database is represented (top) with the ARE sequences identified in the Dll4 promoter. (F) Dll4 expression in C9 cells 24 h after treatment with DHT or MDV3100 (MDV). mRNA expression relative to untreated control (n = 3). (G) AR binding at region C of the Dll4 promoter 2 h after DHT and AR inhibitor MDV3100 treatment (n = 4). (H) HEK293T cells were transfected with a Dll4 promoter-driven reporter plasmid and luciferase activity was measured 48 h after transfection (n = 4). Data represent the mean + SEM of two independent experiments unless otherwise specified. *, P ≤ 0.05; **, P ≤ 0.01, unpaired Mann-Whitney U test.

Figure 2.

Notch ligand DLL4 regulates thymopoiesis in a dosage-sensitive manner. (A and B) Lymphoid differentiation of sorted LSK after 12 d of culture with IL-7, FLT3-ligand, SCF, and scalar concentrations of rDLL4. (A) Absolute numbers of DN2 cells. (B) DN cell proportions, gated on Lin−CD45+. A and B represent the mean + SEM of one of two representative experiments performed in triplicate. (C) Dll4 expression in FACS-sorted cTEC from 6-wk-old Foxn1-cre::Dll4+/fl mice and Foxn1-cre::Dll4+/+ mice. (D) Total thymic cellularity of 5–6-wk-old Foxn1-cre::Dll4+/fl mice (n = 7) and Foxn1-cre::Dll4+/+ mice (n = 5). (E) Total thymic cellularity of K14-cre::Dll4+/fl mice (n = 7) and K14-cre::Dll4+/+ mice (n = 2). Data represents the mean + SEM of one experiment. ^, P = 0.0556. Data represent the mean + SEM of two independent experiments unless otherwise specified. *, P ≤ 0.05, unpaired Mann-Whitney U test.

Figure 3.

LHRH-Ant triggers thymic regeneration and increases Dll4 signaling within 7 d after treatment. (A) Testosterone levels in serum of 8–12-wk old male mice after treatment with LHRH-Ag (dotted line) or LHRH-Ant (solid line). (B) Total thymic cellularity 7, 14, and 28 d after treatment. (C) Absolute numbers of DN, DP, and CD4+ and CD8+ single-positive thymocytes. (A–C, n = 5–8 mice/group). (D) Lymphoid differentiation of sorted LSK cells after 12 d of culture with IL-7, FLT3-ligand, and concentrations of LHRH-Ant (mean + SEM from one of two representative experiments). (E–H) 7 d after treatment with LHRH-Ant. (E) Molecular analysis of CD45− TSCs (n = 8). (F) Dll4 expression in sorted cTECs and ECs (n = 12). (G) mRNA expression of Hes1 and Ptcra in sorted DN3 (CD44−CD25+) thymocytes (n = 8). (H) Mean fluorescence intensity (MFI) of CD25 in CD45+CD4−CD8−CD3−CD25+ thymocytes. mRNA expression relative to untreated control, A–C and E–H represent the mean + SEM of at least two independent experiments. *, P ≤ 0.05; **, P ≤ 0.01; ***/^^^, P ≤ 0.001, compared with vehicle (*) or LHRH-Ag–treated (^) mice. Statistical analysis between two groups was performed with the nonparametric, unpaired Mann-Whitney U test. ANOVA was used for comparisons between more than two groups.

Figure 4.

LHRH-Ant treatment restores thymopoiesis and accelerates peripheral reconstitution in immunocompromised recipients after SL-TBI. (A–E) Mice were pretreated 5 d before SL-TBI with vehicle or LHRH-Ant. (A) Total thymic cellularity. (B) Total number of splenocytes. (C) Absolute number of total and naive (N, CD62Lhi CD44lo) T cells. (D) CD5+ enriched splenocytes obtained from vehicle and LHRH-Ant treated mice 42 d after SL-TBI were assessed in vitro for proliferation. (E) Viral titer of LCMV in the spleen 8 d after infection (where mice were infected 14 d after SL-TBI). (F–H) Allo-HSCT was performed by lethally irradiating B6 mice and transplanting with 5 × 106 B10.BR TCD BM cells (mice were pretreated with vehicle or LHRH-Ant). (F) Total thymic cellularity. (G) Absolute number of total, effector memory, central memory, and naive T cells in the spleen 3 mo after transplant. (H) Allo-HSCT recipients were spiked with 2 × 106 B10.BR T cells to induce GVHD and median survival time measured. Survival data were analyzed with the Mantel-Cox log-rank test. (I) Total thymic cellularity of 9-mo-old male mice 28 d after treatment. (J) Total thymic cellularity of 8–12-wk-old or 9-mo-old female mice 28 d after treatment. (K) Total thymic cellularity at day 7 after SL-TBI of young C57BL/6 female mice pretreated with vehicle or LHRH-Ant 5 d before SL-TBI. Results are expressed as combined mean ± SEM of 8–15 mice for each group representing at least two independent experiments. */^, P ≤ 0.05; **/^^, P ≤ 0.01; ***/^^^, P ≤ 0.001, compared with untreated (*) and vehicle-treated (^) mice. Statistical analysis between two groups was performed with an unpaired Mann-Whitney U test. ANOVA was used for comparisons between more than two groups.