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. 2008 Jun 26;149(10):5209–5218. doi: 10.1210/en.2008-0476

Leptin Acts in the Periphery to Protect Thymocytes from Glucocorticoid-Mediated Apoptosis in the Absence of Weight Loss

Robert N Trotter-Mayo 1, Margo R Roberts 1
PMCID: PMC2582910  PMID: 18583419

Abstract

Leptin is a member of the IL-6 cytokine family and is primarily produced by adipose tissue. At high enough concentration, leptin engages leptin receptors expressed in the hypothalamus that regulate a variety of functions, including induction of weight loss. Mice deficient in leptin (ob/ob) or leptin receptor (db/db) function exhibit thymic atrophy associated with a reduction in double-positive (DP) thymocytes. However, the mediator of such thymic atrophy remains to be identified, and the extent to which leptin acts in the periphery vs. the hypothalamus to promote thymocyte cellularity is unknown. In the present study, we first demonstrate that thymic cellularity and composition is fully restored in ob/ob mice subjected to adrenalectomy. Second, we observe that ob/ob mice treated with low-dose leptin peripherally but not centrally exhibit increased thymocyte cellularity in the absence of any weight loss or significant reduction in systemic corticosterone levels. Third, we demonstrate that reconstitution of db/db mice with wild-type bone marrow augments thymocyte cellularity and restores DP cell frequency despite elevated corticosterone levels. These and additional data support a mode of action whereby leptin acts in the periphery to reduce the sensitivity of DP thymocytes to glucocorticoid-mediated apoptosis in vivo. Strikingly, our data reveal that leptin’s actions on thymic cellularity in the periphery can be uncoupled from its anorectic actions in the hypothalamus.

LEPTIN IS A 167-amino acid protein encoded by the obese (ob) gene and is now considered to be a member of the IL-6 cytokine superfamily (1,2,3). Leptin is produced principally by white adipose tissue and plasma levels are proportional to fat stores (1,4,5). When present at sufficient concentration, leptin gains access to the central nervous system (CNS) and activates leptin receptors expressed primarily in the hypothalamus that regulate food intake and energy expenditure. If present at adequate levels in the CNS, leptin communicates that body energy stores are satiated, resulting in suppression of food intake, sanctioning of energy expenditure, and reduction in body weight (1,2,3). Lack of leptin signaling caused by mutation of the leptin or leptin receptor gene in rodents and humans results in increased food intake and reduced energy expenditure resulting in marked obesity (1,2,4). Leptin’s central actions (i.e. within the CNS) are not limited to regulation of body weight but have pleiotropic effects on peripheral tissues and organs, resulting in increased thermogenesis, inhibition of bone formation via activation of the sympathetic nervous system, sanctioning of reproduction (6,7,8,9,10,11), and suppression of stressor-induced activation of the hypothalamus-pituitary-adrenal (HPA) axis and thus blunting of systemic glucocorticoid levels (12,13). In addition to obesity, mice lacking functional leptin (ob/ob) or leptin receptors (db/db) display decreased body temperature, increased bone density, infertility, and hypercorticosteronemia (14). Although the central actions of leptin are well described, ex vivo evidence suggests that leptin can also act directly on peripheral targets, including bone (15), adrenal cortex (16), pancreas, and muscle (17).

Accumulating evidence supports a role for leptin in the regulation of adaptive immunity, at least in part, by the direct action of leptin on T cells (18). Ob/ob and db/db mice exhibit increased susceptibility to infection and decreased Th1-type responses. Likewise, the reduction in plasma leptin levels observed for humans and rodents subjected to caloric restriction correlates with clinically significant T cell deficiency, impaired adaptive immune function, and markedly increased susceptibility to infection (19,20,21,22,23,24). One important factor contributing to the deficient adaptive immunity observed in the setting of deficient leptin function is thymic atrophy. Specifically, ob/ob and db/db mice exhibit a marked reduction in thymocyte cellularity, particularly of the CD4+CD8+ double-positive (DP) subset (25,26,27). Reduced thymocyte frequency correlates with markedly enhanced thymocyte apoptosis in ob/ob mice (25). Similarly, in malnourished humans and rodents with low leptin levels, the cellularity of the thymus is dramatically reduced principally due to significant loss of cortical (DP) thymocytes (25,28,29). Wild-type (WT) mice rendered leptin deficient due to acute or chronic depletion of fat mass also exhibit reduced thymocyte cellularity and increased thymocyte apoptosis (30). The reduced thymocyte cellularity observed in the setting of starvation is the direct result of reduced leptin levels because peripheral administration of leptin to calorically restricted WT mice protects them from starvation-induced thymic atrophy (25). Despite the progress that has been made in identifying some of the mechanisms by which leptin regulates functions such as body weight, comparatively little is known about the cellular basis of leptin’s actions on thymopoiesis, a process fundamental to effective adaptive immunity.

In the present report, we address two outstanding and highly interrelated questions regarding the underlying mechanism of leptin-mediated regulation of thymic cellularity. One important question to be resolved is the extent to which the thymic atrophy observed in the setting of leptin insufficiency in vivo is the direct consequence of deficient leptin activity or an indirect effect mediated by a secondary factor. With regard to the latter mechanism, the following circumstantial evidence suggests glucocorticoids as an attractive candidate. CD4+CD8+ DP thymocytes are among the few cell types that are exquisitely sensitive to the apoptotic actions of glucocorticoids, and ob/ob and db/db mice or rodents subjected to caloric restriction exhibit elevated levels of systemic corticosterone (the rodent glucocorticoid) accompanied by enhanced apoptosis of the DP thymocyte subset (25,31,32,33,34,35). The second related question we address is the extent to which leptin acts centrally vs. peripherally to promote thymic cellularity in vivo.

Sympathetic nerves innervate the thymus, and we recently identified neurons in the hypothalamus that regulate sympathetic outflow to the thymus (36). Leptin could therefore act centrally to promote thymic cellularity in a direct manner by activating neuronal pathways that regulate the activity of sympathetic nerves projecting to the thymus. Leptin could also act centrally in an indirect manner by suppressing levels of glucocorticoid levels for example. Previous studies have shown that delivery of exogenous leptin to the periphery of leptin-deficient (ob/ob) or fasted WT mice increases thymocyte cellularity and augments the DP to double-negative (DN) ratio (25,26). Similarly, transfer of wild-type white adipose tissue (WAT) to ob/ob mice has been shown to normalize thymic cellularity (37). However, the systemic leptin level achieved in each of these studies was high enough to activate leptin receptors expressed in the hypothalamus, as evidenced by the marked weight loss experienced by leptin-treated or WAT transplanted animals relative to controls. Therefore, the actions of leptin on thymic cellularity could have been due to its actions in the CNS, regardless of administration route. Support for a peripheral mechanism of leptin action is provided by a report in which thymocytes exposed to exogenous leptin exhibited decreased susceptibility to the apoptotic action of glucocorticoids in vitro (25). However, the physiological relevance of this observation and the extent to which such a mechanism operates in vivo remains to be determined.

In the present study, our major objectives were to determine the extent to which glucocorticoids mediate thymic hypocellularity in the setting of leptin deficiency and the relative contribution of central vs. peripheral actions of leptin to thymic cellularity. First, we examined thymocyte number and composition in ob/ob mice lacking systemic glucocorticoids due to adrenalectomy. Second, we delivered low-dose leptin continuously via osmotic pumps to the periphery or the CNS of ob/ob mice and compared the effects on plasma corticosterone levels and thymocyte number and composition. In addition, we monitored weight loss as a marker of central activation. Finally, we generated reciprocal bone marrow chimeras between WT and db/db mice. We then examined corticosterone levels and thymocyte cellularity and composition for chimeras in which functional leptin receptor expression was restricted to either hematopoietic cells (and therefore accompanied by elevated circulating corticosterone levels) or nonhematopoietic cells.

Materials and Methods

Mice

B6.V-Lep Ob/J (ob/ob C57BL/6), B6.Cg-m+/+ Leprdb (db/db C57BL/6), wild-type C57BL/6, and B6.SJL-Ptprca Pep3b/BoyJ (C57BL/6 mice carrying the CD45.1 antigen) were obtained from a commercial supplier (Jackson Laboratories, Bar Harbor, ME) and housed under controlled conditions (lights on 0700–1900 h). Animals were males in all the studies described and were 6–8 wk old at the time of experiment initiation, except where stated otherwise in the text. Animals were fed laboratory chow and water ad libitum. All experiments were performed in accordance with the National Institutes of Health and Institutional Animal Care and Use Guidelines. The Animal Research Committees at the University of Virginia approved all procedures and protocols.

Adrenalectomy

C57BL/6 and ob/ob C57BL/6 mice underwent bilateral adrenalectomy under anesthesia and were maintained on 0.9% saline drinking water to maintain electrolyte balance for 12 d after surgery until killed. A separate group of animals underwent sham adrenalectomy in which the adrenals were grasped with forceps but not removed.

Corticosterone and leptin determinations

For all experimental and control animals, blood was collected by cardiac puncture 12 d after leptin treatment. Animals were killed between 1000 and 1200 h and plasma levels of corticosterone and leptin measured by ELISA (no. DE3600 and MOB00, respectively; R&D Systems, Minneapolis, MN). The sensitivity of these assays was 27 pg/ml for corticosterone and 22 pg/ml for leptin.

Peripheral and central leptin infusion

We used miniosmotic pumps (Alzet model 1002, flow rate 0.25 μl/h; Durect Corp., Cupertino, CA) to continually administer leptin (Peprotech, Inc., Rock Hill, NJ) at 32 ng/μl or PBS to ob/ob animals, either peripherally (ip) or centrally (intracerebroventricularly). For continuous peripheral leptin infusion, a prefilled Alzet pump was inserted into a small incision in the peritoneum with the pump nozzle facing toward the chest cavity. For continuous central infusion, a cannula (Brain Infusion Kit 3; Durect) was implanted into the lateral cerebral ventricle with the coordinates 0.5 mm posterior to bregma, 1–1.6 mm lateral to the midline, and 2.5 mm below bregma. The cannula was attached to the Alzet pump via manufacturer-provided vinyl catheter tubing.

Bone marrow chimeras

Chimeric animals were created by bone marrow transplantation. Recipient mice (WT or db/db C57BL/6 expressing CD45.1 or B6.SJL mice expressing CD45.2) were irradiated with 1100 rad split dose with 3 h between doses. Then 2 × 105 bone marrow cells from donor mice were injected into lethally irradiated recipient mice. Thymi were removed from reconstituted animals at 7–8 wk after transplant and analyzed as described below. The extent of chimerism in reconstituted animals was determined by flow cytometric analysis to identify the relative percentage of CD45.1-negative vs. CD45.2-positive thymocytes. The mean reconstitution efficiency of db/db recipient mice by B6.SJL (WT) donor marrow was 96 ± 3%, and that of B6.SJL (WT) recipient mice by db/db donor marrow was 80 ± 8%.

Flow cytometry

Thymocytes were washed in cold PBS and erythrocytes were lysed by red cell lysis buffer (Sigma-Aldrich, St. Louis, MO) according to manufacturer instructions. Samples containing 1 × 106 cells were incubated with the appropriate antibodies for 45 min at 4 C. The antibodies used were CD8-FITC (no. 553031), CD4 APC (no. 553051), CD4-PE (no. 553730), CD45.1-PE (no. 553776), and CD45.2-PerCP-Cy5.5 (no. 552950; PharMingen, San Jose, CA). After staining, thymocytes were washed three times in ice-cold PBS and analyzed by flow cytometry using a FACS Calibur II (Becton Dickinson, San Jose, CA). In experiments involving bone marrow chimeras, analysis was always performed using a donor-specific (i.e. CD45.1 or CD45.2 positive) thymocyte gate.

Data analysis and statistical methods

For plasma leptin and corticosterone concentrations, analysis of raw ELISA data were performed as per manufacturer instructions. Determination of standard curves and unknown experimental values was performed using the graphing utility, Graph Pad Prism 4 (Graph Pad Software, San Diego, CA). P values were determined using a two-tailed student’s t test or ANOVA followed by Dunnett’s post hoc analysis where appropriate. All thymocyte data presented for bone marrow chimeras are based on donor-derived gates as described above.

Results

To investigate the role glucocorticoids play in the hypocellularity of the thymus in the setting of leptin deficiency, we ablated corticosterone production in ob/ob mice by surgical adrenalectomy. In parallel, a second group of ob/ob mice were subjected to sham adrenalectomy. WT mice were also subject to adrenalectomy and served as controls. Twelve days after surgery, mice were analyzed for plasma corticosterone levels (Fig. 1A) and thymocyte cellularity and composition by flow cytometric analysis (Fig. 1B). Plasma corticosterone levels were reduced in adrenalectomized WT (2.4 ng/ml) and ob/ob (13.3 ng/ml) mice relative to unmanipulated WT (54 ng/ml) and sham-ob/ob (141.6 ng/ml) controls (Fig. 1A). We observed that control ob/ob mice (sham-ob/ob) exhibited reduced total thymocyte cell number relative to WT mice, primarily due to loss of the DP (CD4+CD8+) subset (Fig. 1C). In addition, we noted that sham-ob/ob mice exhibited a small but statistically significant decrease (approximately one third) in the number of DN thymocytes relative to WT mice (Fig. 1C).

Figure 1.

Figure 1

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Impact of adrenalectomy on thymic cellularity and subset composition in ob/ob mice. Ob/ob (Ob) or C57BL/6 (WT) mice were subjected to adrenalectomy (AdX). In parallel, mice were subjected to sham surgery (Ob-Sh). Twelve days after surgery, plasma corticosterone levels and thymocyte number and composition were determined for WT (n = 6), WT-AdX (n = 6), Ob-Sh (n = 6), and Ob-AdX (n = 6) mice. A, Plasma corticosterone concentrations for each treatment group. *, P < 0.0001, compared with Ob-AdX mice; **, P < 0.0003, compared with WT-AdX group, and P < 0.03, compared with Ob-Sh group. B, Representative flow cytometric analysis of CD4/CD8 thymocyte subsets for each treatment group. C, CD4/CD8 thymocyte subset cell numbers for each treatment group. *, P < 0.0001, compared with DP subsets of the three other groups; **, P < 0.008, compared with DN subsets of the other three groups. D, Ratio of DP to DN thymocytes for each group. *, P < 0.0001, compared with each of the other three groups.

Examination of thymic cellularity and distribution of CD4/CD8 subsets revealed that adrenalectomy increased the total number of thymocytes in ob/ob mice by 8-fold relative to sham ob/ob controls, primarily due to the 10-fold increase in the DP (CD4+CD8+) subset (Fig. 1C). In addition, adrenalectomy of ob/ob mice increased the size of the DN (CD4−CD8−) subset by 2-fold. Consistent with previous reports, adrenalectomy of WT animals increased total thymocyte number by almost 3-fold, underscoring the sensitivity of DP thymocytes to low basal levels of systemic glucocorticoids (38). Although the total thymocyte number observed for adrenalectomized ob/ob mice exceeded that of unmanipulated WT animals, it was approximately 60% of that seen for adrenalectomized WT mice in this study. The slightly lower level may have been due to the less efficient ablation of corticosterone levels after adrenalectomy of ob/ob, compared with WT mice (Fig. 1A). Adrenalectomy of ob/ob mice resulted in complete restoration of the ratio of DP (CD4+CD8+) to DN (CD4−CD8−) cells, consistent with the marked recovery of the DP population (Fig. 1D). These data are consistent with endogenous glucocorticoids as the primary mediator of thymic atrophy in the setting of leptin deficiency in vivo.

We next designed a series of experiments to determine the relative contribution of central vs. peripheral actions of leptin to thymocyte cellularity and composition. To discriminate between the central and peripheral actions of leptin, we evaluated thymic cellularity and composition in 6- to 8-wk-old ob/ob mice that had received a constant rate of low dose leptin (8 ng/h) via intracerebroventricular (i.c.v.) or ip infusion over a 12-d period (Fig. 2A). This low dose of leptin has previously been shown to induce weight loss and bone loss when delivered centrally but not peripherally (7). We observed that ob/ob mice treated with leptin via either the i.c.v. or ip route exhibited increased total thymocyte number relative to PBS controls (5.3- and 4-fold, respectively, Fig. 2B) primarily due to an increase in the DP subset (6.1- and 4.7-fold, respectively) (Fig. 2C). Thymocyte numbers did not reach those of unmanipulated WT mice, however (Fig. 1C). Although i.c.v. leptin was slightly more effective than ip leptin at augmenting DP cell number and total thymocyte cellularity, these differences were not significant. DN thymocyte numbers were 2-fold higher for both leptin-treated groups relative to the PBS-treated control group. Both i.c.v. and ip leptin treatment increased the DP to DN thymocyte ratio relative to PBS controls (Fig. 2D). Consistent with central delivery, plasma leptin levels of i.c.v. leptin-treated animals were significantly lower than those of ip leptin-treated mice (Fig. 3A). Mice that had received i.c.v. leptin displayed a 2-fold reduction in plasma corticosterone levels relative to PBS controls (Fig. 3B). Although a slight reduction in corticosterone was noted after ip leptin treatment, it was not statistically significant. The most striking difference observed between i.c.v. and ip leptin-treated mice was the 16-fold reduction in body weight of the former but not the latter group relative to PBS controls (Fig. 3C). In summary, our data demonstrate that leptin can act in the periphery to augment thymocyte cellularity and DP to DN ratio at concentrations insufficient to induce weight loss.

Figure 2.

Figure 2

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Impact on thymocyte cellularity of central vs. peripheral administration of leptin to young mice. Six- to 8-wk-old ob/ob mice were treated with low-dose leptin at a constant rate (8 ng/h) via i.c.v. (ICV-Lep, n = 5) or ip (IP-Lep, n = 6) infusion for a period of 12 d. In parallel, a third group of mice received i.c.v. PBS (ICV-PBS, n = 5). After the 12-d treatment period, mice were analyzed for thymocyte cellularity and composition. A, Representative flow cytometric analysis of CD4/8 thymocyte subsets for each treatment group. B, Total number of thymocytes for each group. *, P < 0.01, compared with ICV-Lep and IP-Lep groups. C, CD4/CD8 thymocyte subset cell numbers for each treatment group. *, P < 0.001, compared with DP subsets of ICV-Lep and IP-Lep groups; **, P < 0.001, compared with DN subsets of ICV-Lep and IP-Lep groups. D, Ratio of DP to DN thymocytes for each treatment group. *, P < 0.001, compared with ICV-Lep and IP-Lep groups.

Figure 3.

Figure 3

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Impact on weight of central vs. peripheral administration of leptin to young ob/ob mice. The three groups of ob/ob mice described in Fig. 2 were weighed before and after the 12-d treatment period. In addition, plasma corticosterone and leptin levels were determined for each group. A, Plasma leptin concentrations for each treatment group. *, P < 0.02, compared with IP-Lep and ICV-PBS groups. B, Plasma corticosterone concentrations for each treatment group. *, P < 0.001, compared with IP-Lep and ICV-PBS groups. C, Total weight loss or gain in each treatment group. Initial body weights were not statistically different between any the three groups: 47.7 ± 4.3 g (ICV-Lep), 45.1 ± 1.1 g (IP-Lep), and 50.5 ± 2.9 g (ICV-PBS).

Because normal mice undergo thymic atrophy as they age, we next asked whether low-dose leptin could promote thymic cellularity in older mice lacking leptin function. Twenty-week-old ob/ob mice were infused with PBS or leptin (8 ng/h) via ip pump delivery as before and thymocyte subset numbers and plasma corticosterone levels determined after 12 d. Age- and sex-matched WT mice were used as controls (Fig. 4A). As expected for animals undergoing aging-related thymic involution, the total thymocyte count of 20-wk-old WT mice was approximately 2.0-fold lower than that of younger WT mice (6.3 ± 0.7 × 107). In contrast, there was no statistical difference in the total thymocyte number between 20-wk-old and young ob/ob mice. In older ob/ob mice, ip leptin increased total and DP thymocyte numbers by 3.7- and 4-fold, respectively (Fig. 4, B and C), slightly less than the increases observed with young ob/ob mice treated with ip leptin (4- and 4.7-fold, respectively). The DP to DN ratio increased in leptin-treated ob/ob mice as a result of increased DP cell numbers (Fig. 4D) but was slightly dampened by a concomitant 1.8-fold increase in the number of DN thymocytes (P = 0.06) as we observed for young leptin-treated ob/ob mice. Similar to our observations with young ob/ob mice treated in the same way, leptin administration did not reduce systemic corticosterone levels; indeed, plasma corticosterone levels appeared to be lower for PBS, relative to leptin-treated animals (Fig. 4E). Consistent with the data obtained for young mice treated with ip leptin, no change in body weight was observed (data not shown). These data support the conclusion that the capacity of leptin to promote thymocyte cellularity and DP numbers is retained in older animals, at least up to 20 wk of age.

Figure 4.

Figure 4

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Peripheral administration of low-dose leptin to older ob/ob mice. Twenty-week-old ob/ob mice were weighed and then treated for 12 d with ip leptin exactly as described for 6- to 8-wk-old mice (IP-Lep, n = 5) or with ip PBS (IP-PBS, n = 4). After the treatment period, mice were weighed again and analyzed for thymocyte cellularity/composition and plasma corticosterone levels. Sex- and age-matched C57BL/6 mice (WT, n = 5) were analyzed in parallel. A, Representative flow cytometric analysis of CD4/8 thymocyte subsets for each group. B, Total number of thymocytes for each group. *, P < 0.05, compared with IP-Lep and WT groups. C, CD4/CD8 thymocyte subset cell numbers for each group. *, P = < 0.002, compared with DP subset of IP-Lep and WT groups; **, P = 0.06 and 0.004, compared with DN subset of IP-Lep and WT groups, respectively. D, Ratio of DP to DN thymocytes for each group. *, P = < 0.003, compared with IP-Lep and WT. E, Plasma corticosterone concentrations for each group. *, P = < 0.03, compared with IP-Lep and WT groups.

Because it is formally possible that the protective effects of ip leptin were achieved via central activation of weight- and HPA-independent neural circuits, the peripheral actions of leptin were next explored in subsequent experiments involving bone marrow chimeras. We generated chimeric animals in which only radiosensitive hematopoietic cells expressed functional leptin receptors. Specifically, db/db mice were reconstituted with WT bone marrow harvested from B6.SJL (CD45.1+) mice to generate WT→db chimeras. In addition, we generated chimeric mice lacking functional leptin receptors on radiosensitive hematopoietic cells only. These db→WT chimeras were generated by reconstituting B6.SJL mice with db/db bone marrow. We then analyzed thymic cellularity and composition, using irradiated db/db mice reconstituted with db/db bone marrow (db→db mice) and WT mice as controls (Fig. 5A). This analysis revealed that WT→db chimeras exhibited a 4.7- and 5.6-fold increase in the number of total and DP thymocytes respectively, relative to db→db controls (Fig. 5, B and C), despite similarly elevated levels of corticosterone (Fig. 5D). In addition to the 4-fold elevation of corticosterone relative to WT animals, both db→db and WT→db mice exhibited markedly elevated plasma levels of leptin due to dysregulation of leptin receptor-mediated feedback inhibition (data not shown). The absolute number of DN cells was equivalent for both WT→db and db→db groups (Fig. 5C). These data demonstrate that leptin receptors expressed on radiosensitive hematopoietic cells augment thymic cellularity and normalize DP to DN ratios, despite the presence of elevated glucocorticoid levels and the altered milieu of the mouse. In db→WT chimeras, the total number and subset distribution of db/db thymocytes was comparable to that of thymocytes in untreated WT animals (Fig. 5, B and C). As expected, plasma corticosterone levels in db→WT chimeras did not vary significantly from WT levels (Fig. 5D). These data indicate that leptin receptors expressed on radiosensitive immune cells are dispensable for thymocyte cellularity under steady-state conditions associated with normal glucocorticoid levels.

Figure 5.

Figure 5

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Thymic cellularity and composition in bone marrow chimeras. Bone marrow chimeras between db/db (db) and C57BL/6 CD45.1+ (WT) mice were generated as described in Materials and Methods. A, Representative flow cytometric analysis of CD4/CD8 thymocyte subsets for db→db (n = 7), WT→db (n = 6), db→WT (n = 5), and unmanipulated WT (n = 3) mice. B, Total number of thymocytes for each group. *, P = < 0.001, compared with three other groups; **, P < 0.001, compared with three other groups. C, CD4/CD8 thymocyte subset cell numbers for each group. *, P < 0.05, compared with DN subset of db→WT and WT; **, P = < 0.001, compared with DP subset of WT→db, db→WT, and WT. D, Plasma corticosterone concentrations for each group. *, P < 0.005, compared with WT and db→WT groups only. E, Ratio of DP to DN thymocytes for each group. *, P < 0.05, compared with other three groups.

It should be noted that although WT thymocytes were present at markedly elevated numbers in the WT→db chimeras relative to db→db controls, they still did not reach the levels seen for unmanipulated WT mice or db/db thymocytes in db→WT chimeras7 (Fig. 5B). Examination of the DN subsets in the four experimental groups revealed that the number of DN thymocytes in WT→db chimeras and db→db animals was equivalent but significantly lower than that of db→WT chimeras and unmanipulated WT mice by a factor of 2.6 and 3.7, respectively (Fig. 5C). In contrast to DN thymocyte number, the DP to DN thymocyte ratios of WT→db chimeras, db→WT chimeras, and unmanipulated WT mice were equivalent (Fig. 5E).

Discussion

Leptin deficiency secondary to genetic mutation or restricted caloric intake is associated with reduced thymocyte numbers, particularly of the DP subset (25,26,27,30). Furthermore, ob/ob and db/db mice exhibit elevated levels of systemic glucocorticoids. Leptin appears to inhibit corticosterone secretion from the adrenal cortex, although additional mechanisms such as reduced corticosterone bioavailability (e.g. via alterations in the quantity of corticosterone binding globulin) may also play a role in the differential plasma glucocorticoid levels observed for wild-type vs. ob/ob and db/db mice. A direct role for glucocorticoids in the thymic atrophy observed in leptin deficiency was suggested by the observation that exogenous leptin appears to decrease dexamethasone-mediated apoptosis of thymocytes in vitro (25). However, evidence that glucocorticoids are the primary mediators of thymic atrophy in the setting of leptin insufficiency in vivo is lacking. In the present study, we have demonstrated that ablation of adrenally produced corticosterone via surgical removal of the adrenal glands restores both thymocyte number and DP to DN ratio in ob/ob mice to WT levels, supporting the conclusion that glucocorticoids are the primary mediators of thymic atrophy observed in the setting of leptin deficiency.

Exogenously supplied leptin has been reported to reverse thymic atrophy in starved WT animals and leptin-deficient ob/ob animals (25,26,27). Specifically, ip injection of exogenous leptin over several days enhanced thymocyte number and increased DP to DN ratios. Similarly, transfer of WAT to ob/ob mice rescues thymocyte cellularity (37). The systemic leptin levels achieved in these studies were high enough to activate leptin receptors expressed in the hypothalamus, as evidenced by the marked weight loss experienced by treated animals. Therefore, the extent to which leptin acts centrally vs. peripherally to promote thymic cellularity remains unresolved. To evaluate central and peripheral actions of leptin on thymic cellularity, we extended these earlier studies by comparing i.c.v. vs. ip delivery of leptin at a constant low dose over a period of 12 d. We observed that although both modes of leptin delivery augmented thymocyte cellularity, only ip administration did so in the absence of any weight loss. Indeed, ip leptin also augmented thymocyte cellularity and DP to DN ratios in older ob/ob mice in the absence of any weight loss. These data support the conclusion that leptin’s actions on thymic cellularity can be uncoupled from leptin-mediated activation of neural circuits regulating body weight. Presumably systemic leptin concentrations that are insufficient for activating hypothalamic leptin receptors involved in the feeding response are sufficient for promotion of thymic cellularity. Furthermore, ip leptin increased thymocyte numbers in the absence of a significant decrease in corticosterone levels, consistent with a previous report that exogenous leptin can decrease corticosterone-mediated apoptosis of thymocytes in vitro (25). Although central administration of leptin also increased thymocyte cellularity and DP to DN ratio, this may have been achieved, at least in part, by leptin-mediated suppression of the HPA axis, resulting in reduced corticosterone levels per se.

Normal mice exhibit loss of thymocyte cellularity during aging via a process thought to involve defects in thymic stroma and reduced numbers of thymocyte progenitors (39). Intriguingly, we observed that whereas 20-wk-old WT mice exhibited decreased thymic cellularity relative to young mice, age- and sex-matched ob/ob mice did not. Furthermore, we demonstrated that low dose leptin treatment augmented thymocyte cellularity and DP numbers in both young and 20-wk-old ob/ob animals with a similar efficiency. Although high-dose leptin treatment does not increase the thymic cellularity of young WT mice (26,40), it has been shown to partially attenuate the thymic atrophy of aged WT mice (40). These data suggest that leptin may play a role in age-dependent thymic atrophy.

Our data support a mechanism whereby leptin acts in the periphery to augment thymocyte cellularity, possibly by decreasing sensitivity to glucocorticoid-mediated apoptosis. However, the data do not rule out a mechanism whereby peripherally administered leptin is acting centrally to regulate thymocyte apoptosis because it is formally possible that the threshold concentration of leptin required for this activity in the hypothalamus is markedly lower than that for inducing weight loss. To further demonstrate the existence of a direct peripheral action of leptin on thymocytes, we therefore reconstituted db/db mice with WT bone marrow to generate chimeras in which functional leptin receptor expression was restricted to radiosensitive hematopoietic cells only. Such WT→db chimeras retained the metabolic alterations of db/db mice, such as obesity and elevated glucocorticoid levels, due to lack of leptin receptor function in the CNS and other nonhematopoietic tissues. These chimeras displayed increased numbers of total DP thymocytes relative to db→db controls and the ratio of DP to DN thymocytes was restored to the value seen for WT animals, despite retention of markedly elevated corticosterone levels. These data confirm and extend our previous results, demonstrating that leptin acts on immune cells in the periphery to augment thymic cellularity. These data, together with those of the adrenalectomy experiment, support a model in which activation of leptin receptors expressed on thymocytes serves to reduce their sensitivity to glucocorticoid-mediated apoptosis in vivo.

In striking contrast to db→db controls and WT→db chimeras, db→WT chimeras whose radiosensitive hematopoietic cells lacked functional leptin receptors exhibited normal thymocyte number and DP to DN ratios. As expected, such mice possessed basal levels of corticosterone indistinguishable from those of WT mice. These data support the conclusion that leptin receptors expressed on hematopoietic cells (presumably thymocytes) do not play a significant role in modulating thymocyte cell number and CD4/CD8 subset distribution under steady-state conditions associated with lack of HPA axis activation. Future studies will be required to determine whether leptin regulates other aspects of thymopoiesis under steady-state conditions.

In addition to leptin’s actions on DP thymocyte numbers, our studies with both ob/ob and db/db mice suggest that leptin also impacts thymocytes before the DP stage of development. We observed that ob/ob mice exhibit a modest but significant reduction in DN cell number relative to WT animals (2- to 3-fold), and this number was increased approximately 2-fold after ip or i.c.v. leptin treatment. Additionally, although the number of DN cells in db→db and WT→db mice was equivalent, it was significantly lower than that of db→WT and WT mice (3- to 4-fold). Consistent with the fact that DP cell number is known to be directly proportional to DN cell number (41,42), the total donor thymocyte number of WT→db chimeras was restrained at approximately 40% that of db→WT or WT mice, whereas the DP to DN ratio for WT thymocytes was completely normal. These observations suggest that the metabolic milieu of db/db and ob/ob mice suppresses DN cell number irrespective of whether these cells or their precursors express functional leptin receptors and does so even more markedly under conditions of bone marrow transplantation. Although functional leptin receptors are expressed on hematopoietic progenitors (43), our data suggest that leptin receptors expressed on radioresistant cells are responsible for the increased DN thymocyte frequency in WT relative to db/db mice. The increase in DN number could be achieved indirectly via normalization of the metabolic milieu or directly via leptin’s actions on stromal cells involved in early lymphocyte differentiation. With regard to the latter, it is intriguing that human bone marrow stromal cells express functional leptin receptors (44,45). Further studies are required to determine the factors that contribute to the reduced DN subset frequency observed in the setting of leptin deficiency.

The thymocyte phenotype we observed for ob/ob mice, namely markedly reduced thymocyte cellularity and DP to DN subset ratio relative to WT mice, has also been described for ob/ob mice in previous reports from three other groups (db/db mice were not evaluated) (25,26,30). We also found that ob/ob and db/db mice in a C57BL/6 background exhibit very similar thymocyte phenotypes. We are aware of only one report in which the thymocyte phenotype of db/db mice in a C57BL/6 background has been examined (46). That report, in striking contrast to the present study, describes reduced thymocyte cellularity (∼15% of WT levels) unaccompanied by any reduction in the DP to DN subset ratio (or any significant differences in relative frequencies of any of the four CD4/CD8 subsets) for male db/db mice at 10 wk of age. It is possible that environmental variation related to housing conditions for example, resulted in a decrease in the stress-mediated apoptotic response of DP thymocytes for the db/db mice described in the study by Palmer et al. (46). Of note, systemic corticosterone levels in the db/db mice described by Palmer et al. (46) were elevated to similar levels as those described in the present study, suggesting that an additional factor(s) regulating sensitivity of DP thymocytes to apoptosis may be involved. Despite the qualitatively different thymic phenotype of the db/db mice used in the present study to those described by Palmer et al. (46), the data in the latter study warrant further discussion. Consistent with our findings, the authors observed that total thymocyte number of db→WT bone marrow chimeras was equivalent to WT→WT controls. In addition, they compared WT→db bone marrow chimeras with WT→WT controls and observed that total thymocyte number of the former was approximately two thirds of WT control values. The authors interpreted these findings as supporting an indirect role for leptin in thymic cellularity whereby the db/db environment nonselectively suppresses thymocyte cellularity. Indeed, our data demonstrate that both the db/db and ob/ob milieu suppress DN cell number, as discussed above. However, because WT→db chimeras were not compared with age-matched db→db or db/db controls in the Palmer study (chimeras were ∼5 months old at time of analysis), a direct contribution of leptin receptor function to thymic cellularity cannot be definitively ruled in or out for the db/db mice used in their analysis.

For the last two centuries, the disproportionate atrophy of lymphoid tissues, particularly the thymus, has been a well-noted effect of starvation and is now appreciated to be the result of reduced leptin levels. In the present study, we have provided novel evidence that the loss of thymocyte cellularity observed in the setting of leptin deficiency is mediated primarily by glucocorticoids in vivo. We demonstrate for the first time that leptin receptors function in a direct manner to augment the DP thymocyte subset and thus promote thymocyte cellularity and do so without activating centralized leptin-sensitive circuits regulating food intake and body weight in the mouse. We propose that decreased plasma leptin levels may initiate food-seeking behaviors before the induction of thymic atrophy. As fat deposits and plasma leptin levels continue to drop and corticosterone levels rise during worsening starvation, elimination of immature thymocytes coupled with suppression of peripheral T cell function may conserve available energy stores for more urgent life-sustaining functions. In the context of caloric sufficiency, leptin may serve to protect immature thymocytes from glucocorticoids released in response to modest activation of the HPA axis occurring in response to a variety of stressors, including certain infections for example, and thus maintain de novo T cell production.

Acknowledgments

We are extremely grateful to Dr. Timothy Bender and Dr. Patrick Concannon for critical reading of this manuscript and Dr. James Mandell for his assistance in the intracerebroventricular administration studies.

Footnotes

This work was supported, in part, by Grant NS41213 from the National Institutes of Health (to M.R.R.) and funds from the Center for Public Health Genomics at the University of Virginia.

Disclosure Statement: R.T.-M. and M.R.R. have nothing to declare.

First Published Online June 26, 2008

Abbreviations: CNS, Central nervous system; DN, double negative; DP, double positive; HPA, hypothalamus-pituitary-adrenal; i.c.v., intracerebroventricular; WAT, white adipose tissue; WT, wild type.

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