Sustained thymopoiesis and improvement in functional immunity induced by exogenous KGF administration in murine models of aging

Abstract

Age-related thymopoietic insufficiency has been proposed to be related to either defects in lymphohematopoietic progenitors or the thymic microenvironment. In this study, we examined whether keratinocyte growth factor (KGF), an epithelial cell-specific growth factor, could increase thymopoietic capacity in aged mice by restoration of the function of thymic epithelial cells (TECs). The thymic cellularity in KGF-treated aged mice increased about 4-fold compared to placebo-treated mice, resulting in an equivalent thymic cellularity to young mice. Enhanced thymopoiesis was maintained for about 2 months after a single course of KGF, and sustained improvement was achieved by administration of monthly courses of KGF. With the enhanced thymopoiesis after KGF treatment, the number of naive CD4 T cells in the periphery and T-cell-dependent antibody production improved in aged mice. KGF induced increased numbers of TECs and intrathymic interleukin-7 (IL-7) production and reorganization of cortical and medullary architecture. Furthermore, KGF enhanced thymopoiesis and normalized TEC organization in klotho (kl/kl) mice, a model of premature degeneration and aging, which displays thymopoietic defects. The result suggests that TEC damage is pathophysiologically important in thymic aging, and KGF therapy may be clinically useful in improving thymopoiesis and immune function in the elderly.

Figure 1

KGF treatment restores thymopoiesis in aged mice. The thymic cellularity of young (6 weeks old, n = 5) and aged mice (15 months old, n = 5-11) was analyzed 1 to 3 months after a single 3-day course of PBS or KGF treatment. (A) Mean absolute thymocyte numbers ± SD from either PBS- or KGF-treated young and aged mice. *P < .05, PBS versus KGF; †P < .05, 1 month versus 2 months; ‡P < .05, 2 months versus 3 months; NS indicates not significant. ▩ indicates PBS-treated mice; □, KGF-treated mice. (B) Increases in the number of TN, DP, SP CD4, and SP CD8 thymic subsets 1 month after KGF treatment. The fold increase of the number of thymocyte subsets is shown in parenthesis. *P < .05, PBS versus KGF.

Figure 2

KGF treatment increases the number of T cells and T-cell–dependent antibody production in the periphery. (A) Blood was drawn from young or aged mice (n = 5-7 per group) 1 month after PBS or KGF treatment, and analyzed for mature CD4⁺ and CD8⁺ T cells. ▩ indicates PBS-treated aged mice; □, KGF-treated aged mice. *P < .05, PBS versus KGF. (B) The mean absolute cell number ± SD of naive CD4⁺ T cells that were CD45RB^high and CD44^low in the blood from PBS- or KGF-treated young or aged mice (n = 5-7 per group). *P < .05, PBS versus KGF. (C) KLH-specific IgM and IgG1 titer in young and aged mice. Mice were immunized with KLH at day 31 and day 45 after initiation of PBS or KGF treatment. Anti-KLH titer (mean ± SD shown) was measured 2 weeks after each immunization (n = 7 per group). *P < .05, PBS-treated aged mice versus KGF-treated aged mice; NS indicates not significant.

Figure 3

Repeated KGF treatment (3 monthly courses of 3 days each) maintained enhanced thymopoiesis in aged mice. The thymic cellularity was analyzed after a single course or multiple courses of KGF treatment to aged mice. Mean absolute thymocyte numbers ± SD from PBS-treated young mice (▥); aged mice evaluated 3 months after PBS treatment (▩); aged mice 3 months after a single course of KGF treatment (□); or aged mice 3 months after initiation of monthly courses (3) of KGF treatment (▧) (n = 5 in each group). *P < .05; NS indicates not significant.

Figure 4

KGF treatment normalized intrathymic IL-7 expression, thymic architecture, and TEC organization in aged mice. TEC organization in aged mice was compared 8 days or 1 month after initiation of a single 3-day course of PBS or KGF treatment. (A) Total RNA from whole thymus on day 8 after initiation of PBS or KGF treatment was analyzed for the epithelium-specific transcript EVA and IL-7 by quantitative RT-PCR (n = 5 per group). Each transcript level was normalized to β-actin expression. *P < .05; NS indicates not significant. (B) H&E staining of thymic sections from normal young PBS- or KGF-treated aged mice. (C) Immunofluorescence staining for K5 and K8, and DAPI nuclear staining. The thymus in aged mice displayed indistinct corticomedullary junctions and decreased TEC populations with areas that were completely devoid of keratin-positive TECs (asterisk). KGF treatment normalized TEC organization in aged mice. The magnification was 200 ×.

Figure 5

KGF treatment increases thymic cellularity and intrathymic IL-7 expression in kl/kl mice. wt littermates (2 weeks old, n = 5) or kl/kl mice (2 weeks old, n = 12) were treated with PBS or KGF for 3 days, and the thymic cellularity was analyzed on day 31. (A) The total thymic cellularity and (B) the absolute numbers of TN, DP, CD4 SP, and CD8 SP thymic subpopulations are shown; results are ± SD. The fold increase of the number of thymocyte subsets is shown in parenthesis. *P < .05, PBS versus KGF.

Figure 6

Premature thymic involution in kl/kl mice is related to disruption of thymic microenvironmental organization, which is restored by KGF. (A) The intrathymic levels of EVA, an epithelial cell–specific marker, and IL-7 transcripts in wt littermates or kl/kl mice (2 weeks old, n = 5 per group) were analyzed by quantitative RT-PCR on day 8 after initiation of PBS or KGF treatment. Each transcript level was normalized to β-actin expression. *P < .05; NS indicates not significant. (B)Thymic architecture of wt and kl/kl mice was compared 1 month after PBS or KGF treatment by H&E staining and immunofluorescence staining of K8, K5, and DAPI for nuclear staining. Arrows indicate cysts observed in the kl/kl thymus. The magnification was 100 × for H&E staining and 200 × for immunofluorescence staining.