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Declining expression of a single epithelial cell-autonomous gene accelerates age-related thymic involution

Declining expression of a single epithelial cell-autonomous gene accelerates age-related thymic... Introduction Aging process is gradual and complex involved in degeneration in many physiological systems and organs. The rate of degeneration varies among individual organs, tissue types, and cells. The primary organ responsible for acquired immunity, thymus gland, ages much earlier and more rapidly than most other organs. In human beings the onset of degeneration begins at puberty. Typical phenotypes of thymic aging are the thymic involution ( Lynch et al. , 2009 ), thymic adiposity ( Youm et al. , 2009 ), and decline of T-lymphopoiesis, i.e. reduction in thymic niches and gradual loss of newly produced naïve T cells ( Bodey et al. , 1997 ; Petrie, 2002 ; Gruver & Sempowski, 2008 ), which results in a restricted T-cell receptor repertoire and an expansion of preexisting memory T cell pool. It therefore compromises T-cell-mediated immunity because of the inability to generate new immune responses. The aged thymus also shows a disproportionate loss of thymic epithelial cells (TECs) ( Gray et al. , 2006 ) and disrupted thymic architecture ( Gui et al. , 2007 ), which results in an increased risk of autoimmunity through the escape of potentially self-reactive T cells from the disrupted thymic microenvironment. Therefore, age-related immune deficiency impacts elderly individuals encountering new and chronic infections, as well as those undergoing radio-chemo-therapeutics for cancer or other ailments. Age-related thymic involution is generally believed to result from deterioration of the interactions between lymphohematopoietic progenitor cells (LPCs) and nonhematopoietic thymic stromal cells (TSCs), primarily composed of thymic epithelial cells (TECs), in the thymus ( Taub & Longo, 2005 ; Ladi et al. , 2006 ). These interactions encompass many genetic pathways or programs in either LPCs of hematopoietic origin or TSCs of nonhematopoietic origin. We ( Gui et al. , 2007 ; Zhu et al. , 2007 ) and others ( Aspinall & Andrew, 2000 ) have hypothesized that age-related thymic involution is triggered primarily by deterioration of the thymic microenvironment, because of TEC dysfunction. In support of this concept, improving TEC function by infusing keratinocyte growth factor ( Min et al. , 2007 ; Rossi et al. , 2007 ), or directly supplying TEC-derived interleukin-7 ( Henson et al. , 2005 ), which is required for early T-cell development in the thymus, ameliorated deficient thymopoiesis owing to aging. Therefore, changes in gene expression related to TEC function may primarily regulate age-related thymic involution. FoxN1 is an epithelial cell-autonomous gene, which is expressed in epithelia of the thymus, skin, and mammary gland, and regulates thymic organogenesis in the thymus ( Nehls et al. , 1994, 1996 ) and is involved in successful generation of the T-cell immune system ( Frank et al. , 1999 ; Adriani et al. , 2004 ; Coffer & Burgering, 2004 ; Cunningham-Rundles & Ponda, 2005 ; Amorosi et al. , 2008 ). The mechanism by which FoxN1 regulates thymic organogenesis and T-cell development in the fetal murine thymus has been extensively studied ( Nehls et al. , 1996 ; Su et al. , 2003 ; Bleul et al. , 2006 ). However, the function of FoxN1 in the postnatal thymus and during thymic aging has not been fully addressed, and remains uncertain, because of the lack of a temporally controlled and tissue-specific loss-of-function model. Although FoxN1 mRNA expression in the thymus declines with age in wild-type (WT) mice ( Ortman et al. , 2002 ), changes in FoxN1 protein levels have not been detected. Furthermore, it is unclear whether reduced FoxN1 mRNA expression is the cause or the effect of thymic aging, because aging-induced TEC deterioration may affect gene expression in TECs. To address this question, we developed a loxP -floxed- FoxN1 mouse model, denoted as ‘fx’ ( Cheng et al. , 2010 ). Crossbreeding these mice with ubiquitously expressed inducible CreER T (uCreER T ) transgenic mice, we attained a temporally controlled FoxN1 knockout mouse allele (denoted as ‘uCreERT-fx’ mouse). Conditional ubiquitous deletion of FoxN1 in the young adult thymus of uCreERT-fx/fx mouse by tamoxifen (TM)-induction caused acute thymic atrophy within 5 days, whereas gradual deletion of FoxN1 in these mice through spontaneous uCreER T activation accelerated thymic involution. The thymic aging phenotype in the uCreERT-fx/fx mice could be observed at a much earlier age (∼ 3–6 months old), which mimicked the naturally aged (∼ 18–22 months old) thymus. Thymic involution was accompanied by changes in thymic architecture, and a sharp decrease in the numbers of CD4 + 8 + double-positive (DP) and CD4 + single-positive (SP) thymocytes, and MHC-II hi UEA-1 hi medullary TECs (mTECs). By intrathymic administration of exogenous FoxN1 -cDNA in middle-aged and aged WT mice, a rapid test of gain-of-function approach, thymic involution, and declining thymic function could be partially rescued, resulting in increased thymic size, thymocyte numbers, and functional CD4 + T cells. Thus, we for the first time provided the empirical evidence supporting that the role of FoxN1 is a potential cause to be related to age-related thymic involution. Results Spontaneous FoxN1 deletion and decline of FoxN1 + TECs with age in uCreER T -fx/fx mice Homozygous FoxN1 fx (fx/fx) mice carrying the uCreER T transgene ( Hayashi & McMahon, 2002 ) were intraperitoneally injected with ≥ 3 doses of TM, which caused deletion of exons 5 and 6 (denoted as ‘खE5&6’) of the FoxN1 locus ( Fig. 1A ) mediated by Cre-recombinase, and resulted in significant thymic atrophic phenotype, as described in our parallel study ( Cheng et al. , 2010 ). However, the uCreER T transgene has a low level of spontaneous activation even without TM-induction ( Bleul et al. , 2006 ; Matsuda & Cepko, 2007 ), causing gradual excision of the loxP -floxed FoxN1 gene with time, resulting in progressive loss of FoxN1 with age in uCreER T -fx/fx mice. The spontaneous deletion of FoxN1 was evaluated in the different-aged thymi of the uCreER T -fx/+ and uCreER T -fx/fx mice by PCR assay ( Fig. 1B ). The FoxN1 खE5&6 deletion was increased with age (arrows in Fig. 1B ) and was seen in both uCreER T -fx/+ heterozygous and uCreER T -fx/fx homozygous mice. To determine whether spontaneous FoxN1 recombination causes a decrease in FoxN1 expression at the protein level, we carried out immunofluorescent staining with FoxN1 antibody on cryosections. As shown in Figure 2 , we demonstrated, for the first time, that the FoxN1 positive (FoxN1 + ) TECs (at protein level) were reduced with age in the naturally aged thymus ( Fig. 2A,D ), although the FoxN1 mRNA was reported to be reduced with age in these mice ( Ortman et al. , 2002 ). Importantly, the reduction in FoxN1 + TECs occurred much earlier in the uCreER T -fx/fx mice ( Fig. 2B,D ), which could be seen as an overt decrease starting at the age of 3 months. By 6–9 months of age, uCreER T -fx/fx mice expressed FoxN1 + TECs at a level equivalent to a 22-month-old WT mouse ( Fig. 2B,D ). Distribution of FoxN1 + TECs in cortex and medulla in 1-month-old uCreER T -fx/fx thymus were almost the same as that in young WT thymus [ Fig. 2C and our previous article ( Cheng et al. , 2010 )]. The results suggest that loss of FoxN1 + TECs is accelerated with age in the uCreER T -fx/fx mice compared with the naturally aged mice, as a result of spontaneous uCreER T activation. Accelerated decreases in thymus size, and TEC and thymocyte numbers in uCreER T -fx/fx mice correlated with age To answer whether progressive loss of FoxN1 + TECs with age accelerates thymic involution, we observed thymic size changes in uCreER T -fx/fx mice and found that the size was gradually reduced at an accelerated rate, showing significant differences from 3 months of age when compared to fx/fx-only (without Cre-recombinase) and uCreER T -fx/+ littermates ( Fig. 3B and data not shown). The 3-month-old uCreER T -fx/fx mice showed a thymic size equivalent to that of ∼ 14-month-old WT mice ( Fig. 3A and data not shown). By 5–6 months old, the uCreER T -fx/fx mouse thymus was as small as the thymus of a 22-month-old WT mouse ( Fig. 3A,B ). The absolute number of total CD45 − TSCs, total thymocytes, particularly DP and CD4 + SP thymocytes ( Fig. 3C,D ), was found to negatively correlate with ages and showed an accelerated decline in the uCreER T -fx/fx mice. The age-related decline of DP and CD4 + SP absolute cell numbers were steeper in uCreER T -fx/fx mice providing proof that loss of FoxN1 + TECs accelerated thymic aging. However, the proportions of thymocytes expressing CD4 and CD8 in all groups of mice were less affected (data not shown). This is consistent with the changes in the naturally aged thymus, where there is a reduction in absolute thymocyte number, but no alterations in the proportions of major thymocyte subpopulations ( Thoman, 1995 ; Aspinall, 1997 ). Because in the uCreER T - fx/+ mice, one copy of floxed- FoxN1 gene undergoes a spontaneous gradual deletion over time ( Fig. 1B ), this also produces thymic involution with age. This is consistent with prior findings of mildly reduced thymic size in FoxN1 +/nu heterozygous nude mice with a congenital loss of one FoxN1 copy, despite normal thymic structure ( Scheiff et al. , 1978 ; Kojima et al. , 1984 ), and reflects a haploinsufficient feature of the FoxN1 gene. But the involution in uCreER T - fx/+ heterozygous mice did not occur as early or to as severe a degree as was seen in uCreER T - fx/fx homozygous mice (samples in Fig. 3B middle column, and Fig. 3C,D ), which suggests that the thymic phenotype is sensitive to FoxN1 gene dosage ( Chen et al. , 2009 ). Fx/fx-only mice that lacked Cre-recombinase showed normal thymic size, similar to WT mice ( Fig. 3A,B ), indicating that the phenotype was attributed to FoxN1 gene recombination and not to the loxP -floxed (fx) allele. Accelerated changes in thymic microarchitecture in uCreER T -fx/fx mice resembled that of the naturally aged thymus In our previous report ( Gui et al. , 2007 ), we analyzed the architecture of the naturally aged thymus with antibodies to Keratin-5 (K5) and K8 and found that the aged thymus (> 18 months old) showed a disorganized cortex and medulla, with poorly defined boundaries between cortical and medullary regions (corticomedullary junction, CMJ) and a mosaic pattern of intermingled medullary TECs (mTECs, presenting K5 + ) and cortical TECs (cTECs, presenting K8 + ). The uCreER T - fx/fx mice also showed these phenotypes with age, but it significantly occurred as early as at age of 3–6 months of age in these mice ( Fig. 4 ), including the changes in gross and microarchitectures observed by H&E staining ( Fig. 4A ), and sparse K5 + mTECs and mingled K5 and K8 staining with an indistinct CMJ ( Fig. 4B ). The results enhanced the fact that the uCreER T -fx/fx mice undergo accelerated thymic aging because of the loss of FoxN1 + TECs with age. Reduced MHC-II hi UEA-1 hi mTECs in uCreER T -fx/fx mice resembled those found in the naturally aged thymus The thymus in C57BL/6 WT mice normally undergoes initial involution from ∼ 3 months of age, but the observable shrinkage is pronounced from ∼ 12 months of age and is characterized by reduced thymic size and decreased cellularity of thymocytes and TECs. Detailed information on specific thymic phenotypes during aging is limited, because in the aged thymus, except for decreased cellularity, all T-cell subsets are present and the proportions of T-cell subsets do not change ( Thoman, 1995 ; Aspinall, 1997 ; Chidgey et al. , 2007 ). Furthermore, although the percentage of double negative 1 thymocytes has been reported to be relatively increased by some investigators ( Thoman, 1995 ; Aspinall, 1997 ), it is not a specific thymic aging phenotype. Thymic aging has also been associated with development of a poorly defined CMJ ( Takeoka et al. , 1996 ), but evaluation of this feature is somewhat various. As we found that the percentage of MHC-II hi UEA-1 hi mature mTECs was reduced in the conditional FoxN1 knockout mice in our parallel study ( Cheng et al. , 2010 ), and this subpopulation was reported to have a high FoxN1 expression in WT mice ( Chen et al. , 2009 ), as well as aged mice are known to have gradually reduced FoxN1 expression ( Ortman et al. , 2002 ), we hypothesized that reduction in MHC-II hi UEA-1 hi subpopulation may be related to natural thymic aging and should show in the naturally aged thymus. Indeed, flow cytometry analysis showed that the percentage of MHC-II hi UEA-1 hi mature mTECs did decline with age in WT mice ( Fig. 5A,B ). Coinciding with the flow cytometry results, our immunohistology results showed a similar reduction in UEA-1 + TECs during the thymic aging process ( Fig. 5C ). These changes paralleled the steady reduction in thymic size with age in WT mice ( Fig. 3A ). Mice of the fx/fx-only, without Cre-recombinase, had thymic size and MHC-II hi UEA-1 hi mTEC numbers that were similar to those of WT mice ( Fig. 5C,D ), indicating again that the loxP -floxed allele will not induce phenotype changes, and the fx/fx-only mice can be used as controls in place of WT mice. To determine whether the decline of MHC-II hi UEA-1 hi mTECs in naturally aged mice is more closely related to the gradual reduction in FoxN1 expression, we observed the changes in MHC-II hi UEA-1 hi mTECs in uCreER T -fx/fx mice, which have a spontaneous FoxN1 deletion. We found that the loss of MHC-II hi UEA-1 hi subset in uCreER T -fx/fx mice was accelerated, which was significantly reduced at 3–6 months of age ( Fig. 6A,C ). The slope of decline was steeper than those observed in fx/fx-only and uCreER T -fx/+ mice, ( Fig. 6B ). We noticed that onset was various in individual mice and complete penetrance of this phenotype needs a span of several months, but this is a valuable phenotype related to thymic aging and FoxN1 + TEC reduction. Exogenous FoxN1 could partially, but significantly, rescue natural aging-related thymic involution, increase thymocyte number and elevate peripheral CD4 + T-cell function If gradual decline of FoxN1 expression is a potential cause of age-related thymic involution, then, enhancement of FoxN1 expression in the middle-aged and/or aged thymus should retard further thymic involution and/or rejuvenate thymic function. For a rapid test of this hypothesis, we performed intrathymic injection of either the FoxN1 -cDNA–bearing vector or the empty vector into middle-aged (9–12 months old) and aged (18 months old) age-matched paired WT mice. The results showed the following: (i) Nonviral polyethylenimine (PEI)-mediated FoxN1 -cDNA plasmid delivery ( Boussif et al. , 1995 ) can transform ∼ 20–30% of thymic cells based on GFP expression (data not shown). Although GFP + cells included both thymocytes and TECs, FoxN1 only provides functionality in TECs, but not in thymocytes. (ii) Injection of FoxN1 -cDNA plasmid into thymic anlage and/or peri-thymus of nude mice, which have a germline mutation of FoxN1 with athymia phenotype, could also partially rescue the thymic phenotype. Number of thymocytes was significantly increased, and mature CD4 + SP and CD8 + SP T cells could be found in the FoxN1 -cDNA-infused nude mice ( Fig. 7A ). The results suggest that a FoxN1 -cDNA plasmid, when injected using a PEI in vivo delivery approach, can work well as an intrathymic injection system. (iii) Comparing the age-matched paired FoxN1 -cDNA plasmid- and empty vector-infused groups, the thymic size and number of thymocytes in the FoxN1 -cDNA plasmid-infused group were always larger or more than that in the empty vector-infused age-matched mice for both ages tested (two examples shown in Fig. 7B ). (iv) Total thymocyte number either in middle-aged or in aged mice was significantly increased after infusion of FoxN1 -cDNA plasmid ( Fig. 7C,D ). (v) The percentage of intracellular IL-2 + peripheral CD4 + T-cell population cannot be elevated in response to costimulation of CD3 and CD28 antibodies in the aged mice ( Zhu et al. , 2007 ) ( Fig. 8 A, the second row), whereas it was significantly elevated in aged mice following two infusions of the FoxN1 -cDNA plasmid, given every 3 weeks, for a total incubation of 6 weeks, compared with the group infused with empty vector. The partial rescue of natural aging-related thymic involution and improved thymic function, thereby partially rejuvenating peripheral CD4 + T-cell function in the aged mice resulting from supplying aged WT mice with exogenous FoxN1 confirms that shortage of FoxN1 in the thymus is a key issue related to T-cell immune system aging. Discussion Under the physiological condition, thymic aging is regulated by changes in gene expression. These changes should be accounted for as epigenetic changes including chromatin remodeling and DNA modifications, rather than genetic changes, because there is no DNA sequence change. However, by studying which genes are expressed and how changes in expression affect rates of thymic aging by using a genetic model should provide insights into single gene function in aging. A conventional gene knockout model cannot be used in this type of aging study because suddenly shutting down a gene does not mimic a natural aging scenario, whereas the conditional spontaneous gene deletion model can be used for this study because, like natural aging, this deletion model generates a process with progressive/gradual declining gene expression. We, herein, established such a genetic model by using a loxP -uCreER T approach to study changes in expression of the FoxN1 gene with thymic aging, and found that loss of epithelial cell-autonomous gene FoxN1 is a key to induction and acceleration of age-related thymic involution. This was confirmed not only by spontaneously deleting FoxN1 with age to accelerate thymic aging phenotypes via the loss-of-function approach, but also by supplying exogenous FoxN1 cDNA to partially, but significantly, rescue naturally aging-related thymic involution and improve naturally aged thymic function, through a rapid test of the gain-of-function approach. Although FoxN1 mRNA has been reported to decrease with age ( Ortman et al. , 2002 ), it has long been speculated whether decline of FoxN1 expression is a cause of age-related thymic involution or an effect arising from thymic epithelial cell deterioration during the thymic aging process. The results in this study demonstrate that gradual loss of FoxN1 through uCreER T spontaneous activation caused accelerated thymic aging, including atrophic thymic size, disrupted thymic microarchitecture – especially an indistinct CMJ, accelerated reduction in number of TECs – prominent decline in MHC-II hi UEA-1 hi mTEC subset in percentage and numbers, and sharply reduced number of thymocytes – particular in DP and CD4 + SP subsets. All these phenotypes resemble those of the naturally aged thymus. On the other hand, input of exogenous FoxN1 cDNA locally rescued the naturally aged thymic phenotype, including reversed thymic involution, increased number of total thymocytes, and rescued peripheral CD4 + T-cell function in response to costimulation by CD3 and CD28 antibodies. All these results provided evidence that loss of FoxN1 is a potential cause of age-related thymic involution in mice. Age-related thymic involution may be through several potential mechanisms, in which there is defect in interactions between TECs and LPCs. Thymic atrophy/involution can be triggered by defects in hematopoietic cell function, such as knockout of T-cell receptor rearrangement gene and depletion of T-cell progenitors ( Gruver et al. , 2007 ), or defects in nonhematopoietic cell function, such as alteration of cytokines produced by TECs of the stroma ( Lynch et al. , 2009 ) and knockout of FgfR2-IIIb gene ( Revest et al. , 2001 ). There are two opposing views of the mechanisms that mediate the initial changes in age-related thymic involution. One holds that LPCs, including hematopoietic stem cells (HSCs), and their downstream multipotent progenitors (MPPs) and early thymic progenitors (ETPs), in aged animals develop cumulative intrinsic defects with age to trigger thymic involution ( Min et al. , 2004 ). The other maintains that aging causes dysfunction of basic thymic microenvironmental cells, composed primarily of TECs, causing secondary changes in T-cell precursors – thymocytes, thereby triggering thymic involution ( Min et al. , 2007 ; Zhu et al. , 2007 ). Based on our studies in this report, we favor the hypothesis that the dominant defect in age-related thymic involution is primarily derived from TEC dysfunction in the thymus, which in turn cause age-related thymic lymphopoietic insufficiency, but we do not rule out that LPCs will eventually develop an irreversible intrinsic defect during aging. Indeed, we found that aged (> 22 months old) murine bone marrow cannot efficiently compete with its young counterpart to develop equivalent mature T cells under the same microenvironment in an in vivo competitive model (data unpublished), which consists with other group’s experiment ( Zediak et al. , 2007 ), although it has the capacity to do so in a young microenvironment with a noncompetitive style ( Zhu et al. , 2007 ). However, the intrinsic defect in bone marrow occurs chronologically later than the thymic involution. We believe that aging causes TEC dysfunction at first, and then additionally causes the LPC intrinsic defect. Both TEC and LPC defects amplify the age-related thymic involution. Generally, there is not one specific phenotype which can be used to define thymic aging except for thymic involution. In our studies, we found that decreases in one of the mTEC subsets – MHC-II hi UEA-1 hi population and FoxN1 + TECs can be used to define thymic aging, because both TEC subsets were found to decrease in inverse proportional to an increase in the age of the thymus. The MHC-II hi UEA-1 hi TECs are a mature mTEC subset. The loss of this subset may be because these cells being more sensitive to loss of FoxN1 and/or they fail to be continuously replaced by TEC progenitors in situ . This is related to FoxN1 function in the postnatal thymus, which we reported in our parallel study ( Cheng et al. , 2010 ). Recently, there has been an increased interest in rejuvenation strategies for the immune system ( Chidgey & Boyd, 2006 ; Lynch et al. , 2009 ). Restoration of dysfunctional thymic epithelial cells is an effective strategy for rejuvenation of the T-cell immune system. Administration of keratinocyte growth factor can promote mitosis in differentiated TECs; administration of a nonredundant cytokine Interleukin-7, mainly produced by TSCs in the thymus, can augment thymocyte proliferation, whereas administration of FoxN1 cDNA may have potential benefits for promoting TEC progenitor differentiation and maintaining mature mTEC survival. Incorporation of multiple strategies may succeed in creating rejuvenating conditions that more closely resemble conditions found in the natural physiological state, resulting in a more effective strategy. In summary, age-related thymic involution may occur and be regulated through several potential mechanisms including defects in hematopoietic and nonhematopoietic cells. However, alteration of epithelial cell-autonomous gene expression, such as expression of FoxN1 , in TECs may be considered as a key factor of age-related thymic involution for several reasons: (i) decrease in FoxN1 expression with age causes, while providing exogenous FoxN1 prevents progressive TEC impairment and thymic involution (ii), TEC defect occurs earlier than hematopoietic stem cell intrinsic deterioration (iii), and infusion of young LPCs into the aged thymus cannot reverse aged TEC defects, while replacement of aged TEC meshwork with young one can promote aged LPCs to produce normal levels of T cells ( Zhu et al. , 2007 ). However, it is unlikely that changes in only one or two genes can fully explain the complex process of age-related thymic involution, in which there may be multiple defects in the interactions between TECs and LPCs. Therefore, we will continue to decipher these mechanisms from understanding how aging impairs epithelial cell-autonomous gene expression and determining what are the downstream genes targeted by FoxN1. Experimental procedures Mice and genotyping The FoxN1 fx (fx) mice were generated as described in our parallel study ( Cheng et al. , 2010 ), in which exons 5 and 6 (DNA binding domain) of the FoxN1 locus are flanked by two loxP sites. We crossbred fx/fx homozygous mice with pCAGG-CreER™ mice (Jackson Lab, Bar Harbor, ME, USA; #004682) ( Hayashi & McMahon, 2002 ), carrying a tamoxifen(TM)-inducible ubiquitous Cre (which we refer to here as uCreER T ), then backcrossed them with fx/fx mice to obtain fx/fx and fx/+ carrying uCreER T mice. All young mice were genotyped by PCR at 3–4 weeks of age, using DNA obtained from the tails. Multiple-primer PCR (PCR was performed with three primers of A, B, and C, in one reaction) was used for genotyping and to check fx locus deletion ( Fig. 1 ). Primer locations are shown in Figure 1A (arrows): 5′-primer-A: 5′-cca acc tcc tgg gga cat ga-3′ and 3′-primer-B: 5′-tag gag gag ggg agc gcc ta-3′, which produce 566-bp and 648-bp amplicons for the WT and floxed- FoxN1 (fx) loci, respectively, as well as 5′-primer-C: 5′-gtg ggc ttt tca cca tcc ta-3′, which produces a ∼ 444-bp amplicon in the mutant locus with deleted exons 5 and 6 of FoxN1 (denoted as ‘खE5&6′), with primer-B. Aged (≥ 18 months old) WT mice were purchased from National Institutes for Aging (Bethesda, MD, USA). All animal experiments were performed according to the protocols approved by the Institutional Animal Care and Use Committee of the University of Texas Health Science Center at Tyler, in accordance with guidelines of the National Institution of Health, USA. FoxN1 + TECs and microarchitecture determined by histological and immunofluorescent analysis For immunofluorescent staining, cryosections (6 ॖm) were fixed in cold acetone and blocked with donkey serum in TBS containing 0.5% BSA or with an endogenous biotin-blocking kit (Invitrogen, Carlsbad, CA, USA) if a biotinylated antibody was used. Primary antibodies were rabbit anti-mouse FoxN1 ( Itoi et al. , 2007 ) or rabbit anti-mouse K5 (Covance, Berkeley, CA, USA), and rat anti-mouse K8 (Troma-1 supernatant) or biotinylated-anti-UEA-1 (Vector Labs, Burlingame, CA, USA). Secondary reagents were Cy3-donkey anti-rabbit IgG, FITC-donkey anti-rat IgG, Cy3-streptavidin (all from Jackson Immunoresearch), or AlexaFluor-488-anti-rabbit IgG (Invitrogen), respectively. For hematoxylin and eosin (H&E) staining, thymi were fixed, cut into 5-ॖm-thick paraffin sections, and stained with H&E. Number and proportions of subpopulations of thymocytes and TECs analyzed by flow cytometry Single-cell suspension of thymocytes, prepared with cell strainers ( Zhu et al. , 2007 ), was stained with combinations of either fluorochrome-conjugated anti-mouse-CD3, -CD4, and -CD8 ( Zhu et al. , 2007 ). Enzymatically (Collagenase-V/DNase-I) dissociated thymic cells ( Gui et al. , 2007 ) were stained with combinations of fluorochrome-conjugated anti-mouse-CD45, -MHC-II (M5/114), -Ly51 (6C3), and either FITC-anti-mouse-EpCam (G8.8) (BioLegend, San Diego, CA, USA) or FITC-UEA-1 (Vector Labs). Data were acquired with a dual-laser FACS Calibur system and analyzed using CellQuest (BD Biosciences, San Jose, CA, USA) and FlowJo (Tree Star Inc., Ashland, OR, USA) software. Intrathymic transformation of FoxN1 cDNA FoxN1 -cDNA, preceded by a Kozak sequence to enhance expression and followed by the internal ribosome entry site (IRES) and the green fluorescent protein (GFP) reporter gene, is driven by the CMV promoter in pADTrack vector [kindly provided by Dr. Brissette ( Prowse et al. , 1999 ; Weiner et al. , 2007 )]. The control vector is an empty pADTrack plasmid. FoxN1 -cDNA plasmid was delivered in vivo by a nonviral PEI-mediated method ( Boussif et al. , 1995 ; Neu et al. , 2005 ). A mixture of 40-ॖg plasmid and 6.4 ॖL of PEI (Genesee Scientific Corp., San Diego, CA, USA) at ionic balance N/P ratio = 8, in ∼ 25-ॖl volume was intrathymically injected into each middle-age or aged mouse at three sites of the thymic lobes under anesthesia and suprasternal notch surgery ( Zhu et al. , 2007 ). The middle-aged mice were injected once, and 1 month after injection the thymi were analyzed by flow cytometry, whereas the aged mice were injected twice at 3-week intervals, and 3 weeks after the follow-up injection the thymi were analyzed by flow cytometry. Analysis of intracellular IL-2 in peripheral CD4 + T cells in response to costimulation of CD3 and CD28 antibodies Spleen cells were freshly isolated from either FoxN1 -cDNA plasmid- or empty vector-infused 18-month-old mice, which were injected intrathymically every 3 weeks over a 6-week period for a total of two injections. The erythrocytes were depleted with ACK (Ammonium-Chloride-Potassum) lysis buffer (pH7.2, 0.15 m m NH 4 Cl/1.0 m m KHCO 3 /0.1 m m Na 2 EDTA). The spleen cells (2 × 10 6 /well) were cultured with anti-mouse CD3ॉ and CD28 antibodies (2 ॖg/mL each) supplemented with GolgiStop (4 ॖg/mL) for 5 h in a 48-well plate. The harvested cells were stained for surface CD4, then fixed with 1% PFA (Paraformaldehyde)/PBS overnight, permeabilized with 0.1% Triton-X100 in 0.1% sodium citrate, pH 7.2, then stained with IL-2 antibody intracellularly. The results were analyzed by a FACS Calibur. All Antibodies and GolgiStop were purchased from BD Pharmingen (San Diego, CA, USA). Statistics Comparisons were made by the paired Student’s t -test. P < 0.05 was considered statistically significant. Correlations were analyzed by linear regression using Prism-4 software (GraphPad). http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Aging Cell Wiley

Declining expression of a single epithelial cell-autonomous gene accelerates age-related thymic involution

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Wiley
Copyright
Journal compilation © 2010 Blackwell Publishing Ltd/The Anatomical Society of Great Britain and Ireland
ISSN
1474-9718
eISSN
1474-9726
DOI
10.1111/j.1474-9726.2010.00559.x
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20156205
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Abstract

Introduction Aging process is gradual and complex involved in degeneration in many physiological systems and organs. The rate of degeneration varies among individual organs, tissue types, and cells. The primary organ responsible for acquired immunity, thymus gland, ages much earlier and more rapidly than most other organs. In human beings the onset of degeneration begins at puberty. Typical phenotypes of thymic aging are the thymic involution ( Lynch et al. , 2009 ), thymic adiposity ( Youm et al. , 2009 ), and decline of T-lymphopoiesis, i.e. reduction in thymic niches and gradual loss of newly produced naïve T cells ( Bodey et al. , 1997 ; Petrie, 2002 ; Gruver & Sempowski, 2008 ), which results in a restricted T-cell receptor repertoire and an expansion of preexisting memory T cell pool. It therefore compromises T-cell-mediated immunity because of the inability to generate new immune responses. The aged thymus also shows a disproportionate loss of thymic epithelial cells (TECs) ( Gray et al. , 2006 ) and disrupted thymic architecture ( Gui et al. , 2007 ), which results in an increased risk of autoimmunity through the escape of potentially self-reactive T cells from the disrupted thymic microenvironment. Therefore, age-related immune deficiency impacts elderly individuals encountering new and chronic infections, as well as those undergoing radio-chemo-therapeutics for cancer or other ailments. Age-related thymic involution is generally believed to result from deterioration of the interactions between lymphohematopoietic progenitor cells (LPCs) and nonhematopoietic thymic stromal cells (TSCs), primarily composed of thymic epithelial cells (TECs), in the thymus ( Taub & Longo, 2005 ; Ladi et al. , 2006 ). These interactions encompass many genetic pathways or programs in either LPCs of hematopoietic origin or TSCs of nonhematopoietic origin. We ( Gui et al. , 2007 ; Zhu et al. , 2007 ) and others ( Aspinall & Andrew, 2000 ) have hypothesized that age-related thymic involution is triggered primarily by deterioration of the thymic microenvironment, because of TEC dysfunction. In support of this concept, improving TEC function by infusing keratinocyte growth factor ( Min et al. , 2007 ; Rossi et al. , 2007 ), or directly supplying TEC-derived interleukin-7 ( Henson et al. , 2005 ), which is required for early T-cell development in the thymus, ameliorated deficient thymopoiesis owing to aging. Therefore, changes in gene expression related to TEC function may primarily regulate age-related thymic involution. FoxN1 is an epithelial cell-autonomous gene, which is expressed in epithelia of the thymus, skin, and mammary gland, and regulates thymic organogenesis in the thymus ( Nehls et al. , 1994, 1996 ) and is involved in successful generation of the T-cell immune system ( Frank et al. , 1999 ; Adriani et al. , 2004 ; Coffer & Burgering, 2004 ; Cunningham-Rundles & Ponda, 2005 ; Amorosi et al. , 2008 ). The mechanism by which FoxN1 regulates thymic organogenesis and T-cell development in the fetal murine thymus has been extensively studied ( Nehls et al. , 1996 ; Su et al. , 2003 ; Bleul et al. , 2006 ). However, the function of FoxN1 in the postnatal thymus and during thymic aging has not been fully addressed, and remains uncertain, because of the lack of a temporally controlled and tissue-specific loss-of-function model. Although FoxN1 mRNA expression in the thymus declines with age in wild-type (WT) mice ( Ortman et al. , 2002 ), changes in FoxN1 protein levels have not been detected. Furthermore, it is unclear whether reduced FoxN1 mRNA expression is the cause or the effect of thymic aging, because aging-induced TEC deterioration may affect gene expression in TECs. To address this question, we developed a loxP -floxed- FoxN1 mouse model, denoted as ‘fx’ ( Cheng et al. , 2010 ). Crossbreeding these mice with ubiquitously expressed inducible CreER T (uCreER T ) transgenic mice, we attained a temporally controlled FoxN1 knockout mouse allele (denoted as ‘uCreERT-fx’ mouse). Conditional ubiquitous deletion of FoxN1 in the young adult thymus of uCreERT-fx/fx mouse by tamoxifen (TM)-induction caused acute thymic atrophy within 5 days, whereas gradual deletion of FoxN1 in these mice through spontaneous uCreER T activation accelerated thymic involution. The thymic aging phenotype in the uCreERT-fx/fx mice could be observed at a much earlier age (∼ 3–6 months old), which mimicked the naturally aged (∼ 18–22 months old) thymus. Thymic involution was accompanied by changes in thymic architecture, and a sharp decrease in the numbers of CD4 + 8 + double-positive (DP) and CD4 + single-positive (SP) thymocytes, and MHC-II hi UEA-1 hi medullary TECs (mTECs). By intrathymic administration of exogenous FoxN1 -cDNA in middle-aged and aged WT mice, a rapid test of gain-of-function approach, thymic involution, and declining thymic function could be partially rescued, resulting in increased thymic size, thymocyte numbers, and functional CD4 + T cells. Thus, we for the first time provided the empirical evidence supporting that the role of FoxN1 is a potential cause to be related to age-related thymic involution. Results Spontaneous FoxN1 deletion and decline of FoxN1 + TECs with age in uCreER T -fx/fx mice Homozygous FoxN1 fx (fx/fx) mice carrying the uCreER T transgene ( Hayashi & McMahon, 2002 ) were intraperitoneally injected with ≥ 3 doses of TM, which caused deletion of exons 5 and 6 (denoted as ‘खE5&6’) of the FoxN1 locus ( Fig. 1A ) mediated by Cre-recombinase, and resulted in significant thymic atrophic phenotype, as described in our parallel study ( Cheng et al. , 2010 ). However, the uCreER T transgene has a low level of spontaneous activation even without TM-induction ( Bleul et al. , 2006 ; Matsuda & Cepko, 2007 ), causing gradual excision of the loxP -floxed FoxN1 gene with time, resulting in progressive loss of FoxN1 with age in uCreER T -fx/fx mice. The spontaneous deletion of FoxN1 was evaluated in the different-aged thymi of the uCreER T -fx/+ and uCreER T -fx/fx mice by PCR assay ( Fig. 1B ). The FoxN1 खE5&6 deletion was increased with age (arrows in Fig. 1B ) and was seen in both uCreER T -fx/+ heterozygous and uCreER T -fx/fx homozygous mice. To determine whether spontaneous FoxN1 recombination causes a decrease in FoxN1 expression at the protein level, we carried out immunofluorescent staining with FoxN1 antibody on cryosections. As shown in Figure 2 , we demonstrated, for the first time, that the FoxN1 positive (FoxN1 + ) TECs (at protein level) were reduced with age in the naturally aged thymus ( Fig. 2A,D ), although the FoxN1 mRNA was reported to be reduced with age in these mice ( Ortman et al. , 2002 ). Importantly, the reduction in FoxN1 + TECs occurred much earlier in the uCreER T -fx/fx mice ( Fig. 2B,D ), which could be seen as an overt decrease starting at the age of 3 months. By 6–9 months of age, uCreER T -fx/fx mice expressed FoxN1 + TECs at a level equivalent to a 22-month-old WT mouse ( Fig. 2B,D ). Distribution of FoxN1 + TECs in cortex and medulla in 1-month-old uCreER T -fx/fx thymus were almost the same as that in young WT thymus [ Fig. 2C and our previous article ( Cheng et al. , 2010 )]. The results suggest that loss of FoxN1 + TECs is accelerated with age in the uCreER T -fx/fx mice compared with the naturally aged mice, as a result of spontaneous uCreER T activation. Accelerated decreases in thymus size, and TEC and thymocyte numbers in uCreER T -fx/fx mice correlated with age To answer whether progressive loss of FoxN1 + TECs with age accelerates thymic involution, we observed thymic size changes in uCreER T -fx/fx mice and found that the size was gradually reduced at an accelerated rate, showing significant differences from 3 months of age when compared to fx/fx-only (without Cre-recombinase) and uCreER T -fx/+ littermates ( Fig. 3B and data not shown). The 3-month-old uCreER T -fx/fx mice showed a thymic size equivalent to that of ∼ 14-month-old WT mice ( Fig. 3A and data not shown). By 5–6 months old, the uCreER T -fx/fx mouse thymus was as small as the thymus of a 22-month-old WT mouse ( Fig. 3A,B ). The absolute number of total CD45 − TSCs, total thymocytes, particularly DP and CD4 + SP thymocytes ( Fig. 3C,D ), was found to negatively correlate with ages and showed an accelerated decline in the uCreER T -fx/fx mice. The age-related decline of DP and CD4 + SP absolute cell numbers were steeper in uCreER T -fx/fx mice providing proof that loss of FoxN1 + TECs accelerated thymic aging. However, the proportions of thymocytes expressing CD4 and CD8 in all groups of mice were less affected (data not shown). This is consistent with the changes in the naturally aged thymus, where there is a reduction in absolute thymocyte number, but no alterations in the proportions of major thymocyte subpopulations ( Thoman, 1995 ; Aspinall, 1997 ). Because in the uCreER T - fx/+ mice, one copy of floxed- FoxN1 gene undergoes a spontaneous gradual deletion over time ( Fig. 1B ), this also produces thymic involution with age. This is consistent with prior findings of mildly reduced thymic size in FoxN1 +/nu heterozygous nude mice with a congenital loss of one FoxN1 copy, despite normal thymic structure ( Scheiff et al. , 1978 ; Kojima et al. , 1984 ), and reflects a haploinsufficient feature of the FoxN1 gene. But the involution in uCreER T - fx/+ heterozygous mice did not occur as early or to as severe a degree as was seen in uCreER T - fx/fx homozygous mice (samples in Fig. 3B middle column, and Fig. 3C,D ), which suggests that the thymic phenotype is sensitive to FoxN1 gene dosage ( Chen et al. , 2009 ). Fx/fx-only mice that lacked Cre-recombinase showed normal thymic size, similar to WT mice ( Fig. 3A,B ), indicating that the phenotype was attributed to FoxN1 gene recombination and not to the loxP -floxed (fx) allele. Accelerated changes in thymic microarchitecture in uCreER T -fx/fx mice resembled that of the naturally aged thymus In our previous report ( Gui et al. , 2007 ), we analyzed the architecture of the naturally aged thymus with antibodies to Keratin-5 (K5) and K8 and found that the aged thymus (> 18 months old) showed a disorganized cortex and medulla, with poorly defined boundaries between cortical and medullary regions (corticomedullary junction, CMJ) and a mosaic pattern of intermingled medullary TECs (mTECs, presenting K5 + ) and cortical TECs (cTECs, presenting K8 + ). The uCreER T - fx/fx mice also showed these phenotypes with age, but it significantly occurred as early as at age of 3–6 months of age in these mice ( Fig. 4 ), including the changes in gross and microarchitectures observed by H&E staining ( Fig. 4A ), and sparse K5 + mTECs and mingled K5 and K8 staining with an indistinct CMJ ( Fig. 4B ). The results enhanced the fact that the uCreER T -fx/fx mice undergo accelerated thymic aging because of the loss of FoxN1 + TECs with age. Reduced MHC-II hi UEA-1 hi mTECs in uCreER T -fx/fx mice resembled those found in the naturally aged thymus The thymus in C57BL/6 WT mice normally undergoes initial involution from ∼ 3 months of age, but the observable shrinkage is pronounced from ∼ 12 months of age and is characterized by reduced thymic size and decreased cellularity of thymocytes and TECs. Detailed information on specific thymic phenotypes during aging is limited, because in the aged thymus, except for decreased cellularity, all T-cell subsets are present and the proportions of T-cell subsets do not change ( Thoman, 1995 ; Aspinall, 1997 ; Chidgey et al. , 2007 ). Furthermore, although the percentage of double negative 1 thymocytes has been reported to be relatively increased by some investigators ( Thoman, 1995 ; Aspinall, 1997 ), it is not a specific thymic aging phenotype. Thymic aging has also been associated with development of a poorly defined CMJ ( Takeoka et al. , 1996 ), but evaluation of this feature is somewhat various. As we found that the percentage of MHC-II hi UEA-1 hi mature mTECs was reduced in the conditional FoxN1 knockout mice in our parallel study ( Cheng et al. , 2010 ), and this subpopulation was reported to have a high FoxN1 expression in WT mice ( Chen et al. , 2009 ), as well as aged mice are known to have gradually reduced FoxN1 expression ( Ortman et al. , 2002 ), we hypothesized that reduction in MHC-II hi UEA-1 hi subpopulation may be related to natural thymic aging and should show in the naturally aged thymus. Indeed, flow cytometry analysis showed that the percentage of MHC-II hi UEA-1 hi mature mTECs did decline with age in WT mice ( Fig. 5A,B ). Coinciding with the flow cytometry results, our immunohistology results showed a similar reduction in UEA-1 + TECs during the thymic aging process ( Fig. 5C ). These changes paralleled the steady reduction in thymic size with age in WT mice ( Fig. 3A ). Mice of the fx/fx-only, without Cre-recombinase, had thymic size and MHC-II hi UEA-1 hi mTEC numbers that were similar to those of WT mice ( Fig. 5C,D ), indicating again that the loxP -floxed allele will not induce phenotype changes, and the fx/fx-only mice can be used as controls in place of WT mice. To determine whether the decline of MHC-II hi UEA-1 hi mTECs in naturally aged mice is more closely related to the gradual reduction in FoxN1 expression, we observed the changes in MHC-II hi UEA-1 hi mTECs in uCreER T -fx/fx mice, which have a spontaneous FoxN1 deletion. We found that the loss of MHC-II hi UEA-1 hi subset in uCreER T -fx/fx mice was accelerated, which was significantly reduced at 3–6 months of age ( Fig. 6A,C ). The slope of decline was steeper than those observed in fx/fx-only and uCreER T -fx/+ mice, ( Fig. 6B ). We noticed that onset was various in individual mice and complete penetrance of this phenotype needs a span of several months, but this is a valuable phenotype related to thymic aging and FoxN1 + TEC reduction. Exogenous FoxN1 could partially, but significantly, rescue natural aging-related thymic involution, increase thymocyte number and elevate peripheral CD4 + T-cell function If gradual decline of FoxN1 expression is a potential cause of age-related thymic involution, then, enhancement of FoxN1 expression in the middle-aged and/or aged thymus should retard further thymic involution and/or rejuvenate thymic function. For a rapid test of this hypothesis, we performed intrathymic injection of either the FoxN1 -cDNA–bearing vector or the empty vector into middle-aged (9–12 months old) and aged (18 months old) age-matched paired WT mice. The results showed the following: (i) Nonviral polyethylenimine (PEI)-mediated FoxN1 -cDNA plasmid delivery ( Boussif et al. , 1995 ) can transform ∼ 20–30% of thymic cells based on GFP expression (data not shown). Although GFP + cells included both thymocytes and TECs, FoxN1 only provides functionality in TECs, but not in thymocytes. (ii) Injection of FoxN1 -cDNA plasmid into thymic anlage and/or peri-thymus of nude mice, which have a germline mutation of FoxN1 with athymia phenotype, could also partially rescue the thymic phenotype. Number of thymocytes was significantly increased, and mature CD4 + SP and CD8 + SP T cells could be found in the FoxN1 -cDNA-infused nude mice ( Fig. 7A ). The results suggest that a FoxN1 -cDNA plasmid, when injected using a PEI in vivo delivery approach, can work well as an intrathymic injection system. (iii) Comparing the age-matched paired FoxN1 -cDNA plasmid- and empty vector-infused groups, the thymic size and number of thymocytes in the FoxN1 -cDNA plasmid-infused group were always larger or more than that in the empty vector-infused age-matched mice for both ages tested (two examples shown in Fig. 7B ). (iv) Total thymocyte number either in middle-aged or in aged mice was significantly increased after infusion of FoxN1 -cDNA plasmid ( Fig. 7C,D ). (v) The percentage of intracellular IL-2 + peripheral CD4 + T-cell population cannot be elevated in response to costimulation of CD3 and CD28 antibodies in the aged mice ( Zhu et al. , 2007 ) ( Fig. 8 A, the second row), whereas it was significantly elevated in aged mice following two infusions of the FoxN1 -cDNA plasmid, given every 3 weeks, for a total incubation of 6 weeks, compared with the group infused with empty vector. The partial rescue of natural aging-related thymic involution and improved thymic function, thereby partially rejuvenating peripheral CD4 + T-cell function in the aged mice resulting from supplying aged WT mice with exogenous FoxN1 confirms that shortage of FoxN1 in the thymus is a key issue related to T-cell immune system aging. Discussion Under the physiological condition, thymic aging is regulated by changes in gene expression. These changes should be accounted for as epigenetic changes including chromatin remodeling and DNA modifications, rather than genetic changes, because there is no DNA sequence change. However, by studying which genes are expressed and how changes in expression affect rates of thymic aging by using a genetic model should provide insights into single gene function in aging. A conventional gene knockout model cannot be used in this type of aging study because suddenly shutting down a gene does not mimic a natural aging scenario, whereas the conditional spontaneous gene deletion model can be used for this study because, like natural aging, this deletion model generates a process with progressive/gradual declining gene expression. We, herein, established such a genetic model by using a loxP -uCreER T approach to study changes in expression of the FoxN1 gene with thymic aging, and found that loss of epithelial cell-autonomous gene FoxN1 is a key to induction and acceleration of age-related thymic involution. This was confirmed not only by spontaneously deleting FoxN1 with age to accelerate thymic aging phenotypes via the loss-of-function approach, but also by supplying exogenous FoxN1 cDNA to partially, but significantly, rescue naturally aging-related thymic involution and improve naturally aged thymic function, through a rapid test of the gain-of-function approach. Although FoxN1 mRNA has been reported to decrease with age ( Ortman et al. , 2002 ), it has long been speculated whether decline of FoxN1 expression is a cause of age-related thymic involution or an effect arising from thymic epithelial cell deterioration during the thymic aging process. The results in this study demonstrate that gradual loss of FoxN1 through uCreER T spontaneous activation caused accelerated thymic aging, including atrophic thymic size, disrupted thymic microarchitecture – especially an indistinct CMJ, accelerated reduction in number of TECs – prominent decline in MHC-II hi UEA-1 hi mTEC subset in percentage and numbers, and sharply reduced number of thymocytes – particular in DP and CD4 + SP subsets. All these phenotypes resemble those of the naturally aged thymus. On the other hand, input of exogenous FoxN1 cDNA locally rescued the naturally aged thymic phenotype, including reversed thymic involution, increased number of total thymocytes, and rescued peripheral CD4 + T-cell function in response to costimulation by CD3 and CD28 antibodies. All these results provided evidence that loss of FoxN1 is a potential cause of age-related thymic involution in mice. Age-related thymic involution may be through several potential mechanisms, in which there is defect in interactions between TECs and LPCs. Thymic atrophy/involution can be triggered by defects in hematopoietic cell function, such as knockout of T-cell receptor rearrangement gene and depletion of T-cell progenitors ( Gruver et al. , 2007 ), or defects in nonhematopoietic cell function, such as alteration of cytokines produced by TECs of the stroma ( Lynch et al. , 2009 ) and knockout of FgfR2-IIIb gene ( Revest et al. , 2001 ). There are two opposing views of the mechanisms that mediate the initial changes in age-related thymic involution. One holds that LPCs, including hematopoietic stem cells (HSCs), and their downstream multipotent progenitors (MPPs) and early thymic progenitors (ETPs), in aged animals develop cumulative intrinsic defects with age to trigger thymic involution ( Min et al. , 2004 ). The other maintains that aging causes dysfunction of basic thymic microenvironmental cells, composed primarily of TECs, causing secondary changes in T-cell precursors – thymocytes, thereby triggering thymic involution ( Min et al. , 2007 ; Zhu et al. , 2007 ). Based on our studies in this report, we favor the hypothesis that the dominant defect in age-related thymic involution is primarily derived from TEC dysfunction in the thymus, which in turn cause age-related thymic lymphopoietic insufficiency, but we do not rule out that LPCs will eventually develop an irreversible intrinsic defect during aging. Indeed, we found that aged (> 22 months old) murine bone marrow cannot efficiently compete with its young counterpart to develop equivalent mature T cells under the same microenvironment in an in vivo competitive model (data unpublished), which consists with other group’s experiment ( Zediak et al. , 2007 ), although it has the capacity to do so in a young microenvironment with a noncompetitive style ( Zhu et al. , 2007 ). However, the intrinsic defect in bone marrow occurs chronologically later than the thymic involution. We believe that aging causes TEC dysfunction at first, and then additionally causes the LPC intrinsic defect. Both TEC and LPC defects amplify the age-related thymic involution. Generally, there is not one specific phenotype which can be used to define thymic aging except for thymic involution. In our studies, we found that decreases in one of the mTEC subsets – MHC-II hi UEA-1 hi population and FoxN1 + TECs can be used to define thymic aging, because both TEC subsets were found to decrease in inverse proportional to an increase in the age of the thymus. The MHC-II hi UEA-1 hi TECs are a mature mTEC subset. The loss of this subset may be because these cells being more sensitive to loss of FoxN1 and/or they fail to be continuously replaced by TEC progenitors in situ . This is related to FoxN1 function in the postnatal thymus, which we reported in our parallel study ( Cheng et al. , 2010 ). Recently, there has been an increased interest in rejuvenation strategies for the immune system ( Chidgey & Boyd, 2006 ; Lynch et al. , 2009 ). Restoration of dysfunctional thymic epithelial cells is an effective strategy for rejuvenation of the T-cell immune system. Administration of keratinocyte growth factor can promote mitosis in differentiated TECs; administration of a nonredundant cytokine Interleukin-7, mainly produced by TSCs in the thymus, can augment thymocyte proliferation, whereas administration of FoxN1 cDNA may have potential benefits for promoting TEC progenitor differentiation and maintaining mature mTEC survival. Incorporation of multiple strategies may succeed in creating rejuvenating conditions that more closely resemble conditions found in the natural physiological state, resulting in a more effective strategy. In summary, age-related thymic involution may occur and be regulated through several potential mechanisms including defects in hematopoietic and nonhematopoietic cells. However, alteration of epithelial cell-autonomous gene expression, such as expression of FoxN1 , in TECs may be considered as a key factor of age-related thymic involution for several reasons: (i) decrease in FoxN1 expression with age causes, while providing exogenous FoxN1 prevents progressive TEC impairment and thymic involution (ii), TEC defect occurs earlier than hematopoietic stem cell intrinsic deterioration (iii), and infusion of young LPCs into the aged thymus cannot reverse aged TEC defects, while replacement of aged TEC meshwork with young one can promote aged LPCs to produce normal levels of T cells ( Zhu et al. , 2007 ). However, it is unlikely that changes in only one or two genes can fully explain the complex process of age-related thymic involution, in which there may be multiple defects in the interactions between TECs and LPCs. Therefore, we will continue to decipher these mechanisms from understanding how aging impairs epithelial cell-autonomous gene expression and determining what are the downstream genes targeted by FoxN1. Experimental procedures Mice and genotyping The FoxN1 fx (fx) mice were generated as described in our parallel study ( Cheng et al. , 2010 ), in which exons 5 and 6 (DNA binding domain) of the FoxN1 locus are flanked by two loxP sites. We crossbred fx/fx homozygous mice with pCAGG-CreER™ mice (Jackson Lab, Bar Harbor, ME, USA; #004682) ( Hayashi & McMahon, 2002 ), carrying a tamoxifen(TM)-inducible ubiquitous Cre (which we refer to here as uCreER T ), then backcrossed them with fx/fx mice to obtain fx/fx and fx/+ carrying uCreER T mice. All young mice were genotyped by PCR at 3–4 weeks of age, using DNA obtained from the tails. Multiple-primer PCR (PCR was performed with three primers of A, B, and C, in one reaction) was used for genotyping and to check fx locus deletion ( Fig. 1 ). Primer locations are shown in Figure 1A (arrows): 5′-primer-A: 5′-cca acc tcc tgg gga cat ga-3′ and 3′-primer-B: 5′-tag gag gag ggg agc gcc ta-3′, which produce 566-bp and 648-bp amplicons for the WT and floxed- FoxN1 (fx) loci, respectively, as well as 5′-primer-C: 5′-gtg ggc ttt tca cca tcc ta-3′, which produces a ∼ 444-bp amplicon in the mutant locus with deleted exons 5 and 6 of FoxN1 (denoted as ‘खE5&6′), with primer-B. Aged (≥ 18 months old) WT mice were purchased from National Institutes for Aging (Bethesda, MD, USA). All animal experiments were performed according to the protocols approved by the Institutional Animal Care and Use Committee of the University of Texas Health Science Center at Tyler, in accordance with guidelines of the National Institution of Health, USA. FoxN1 + TECs and microarchitecture determined by histological and immunofluorescent analysis For immunofluorescent staining, cryosections (6 ॖm) were fixed in cold acetone and blocked with donkey serum in TBS containing 0.5% BSA or with an endogenous biotin-blocking kit (Invitrogen, Carlsbad, CA, USA) if a biotinylated antibody was used. Primary antibodies were rabbit anti-mouse FoxN1 ( Itoi et al. , 2007 ) or rabbit anti-mouse K5 (Covance, Berkeley, CA, USA), and rat anti-mouse K8 (Troma-1 supernatant) or biotinylated-anti-UEA-1 (Vector Labs, Burlingame, CA, USA). Secondary reagents were Cy3-donkey anti-rabbit IgG, FITC-donkey anti-rat IgG, Cy3-streptavidin (all from Jackson Immunoresearch), or AlexaFluor-488-anti-rabbit IgG (Invitrogen), respectively. For hematoxylin and eosin (H&E) staining, thymi were fixed, cut into 5-ॖm-thick paraffin sections, and stained with H&E. Number and proportions of subpopulations of thymocytes and TECs analyzed by flow cytometry Single-cell suspension of thymocytes, prepared with cell strainers ( Zhu et al. , 2007 ), was stained with combinations of either fluorochrome-conjugated anti-mouse-CD3, -CD4, and -CD8 ( Zhu et al. , 2007 ). Enzymatically (Collagenase-V/DNase-I) dissociated thymic cells ( Gui et al. , 2007 ) were stained with combinations of fluorochrome-conjugated anti-mouse-CD45, -MHC-II (M5/114), -Ly51 (6C3), and either FITC-anti-mouse-EpCam (G8.8) (BioLegend, San Diego, CA, USA) or FITC-UEA-1 (Vector Labs). Data were acquired with a dual-laser FACS Calibur system and analyzed using CellQuest (BD Biosciences, San Jose, CA, USA) and FlowJo (Tree Star Inc., Ashland, OR, USA) software. Intrathymic transformation of FoxN1 cDNA FoxN1 -cDNA, preceded by a Kozak sequence to enhance expression and followed by the internal ribosome entry site (IRES) and the green fluorescent protein (GFP) reporter gene, is driven by the CMV promoter in pADTrack vector [kindly provided by Dr. Brissette ( Prowse et al. , 1999 ; Weiner et al. , 2007 )]. The control vector is an empty pADTrack plasmid. FoxN1 -cDNA plasmid was delivered in vivo by a nonviral PEI-mediated method ( Boussif et al. , 1995 ; Neu et al. , 2005 ). A mixture of 40-ॖg plasmid and 6.4 ॖL of PEI (Genesee Scientific Corp., San Diego, CA, USA) at ionic balance N/P ratio = 8, in ∼ 25-ॖl volume was intrathymically injected into each middle-age or aged mouse at three sites of the thymic lobes under anesthesia and suprasternal notch surgery ( Zhu et al. , 2007 ). The middle-aged mice were injected once, and 1 month after injection the thymi were analyzed by flow cytometry, whereas the aged mice were injected twice at 3-week intervals, and 3 weeks after the follow-up injection the thymi were analyzed by flow cytometry. Analysis of intracellular IL-2 in peripheral CD4 + T cells in response to costimulation of CD3 and CD28 antibodies Spleen cells were freshly isolated from either FoxN1 -cDNA plasmid- or empty vector-infused 18-month-old mice, which were injected intrathymically every 3 weeks over a 6-week period for a total of two injections. The erythrocytes were depleted with ACK (Ammonium-Chloride-Potassum) lysis buffer (pH7.2, 0.15 m m NH 4 Cl/1.0 m m KHCO 3 /0.1 m m Na 2 EDTA). The spleen cells (2 × 10 6 /well) were cultured with anti-mouse CD3ॉ and CD28 antibodies (2 ॖg/mL each) supplemented with GolgiStop (4 ॖg/mL) for 5 h in a 48-well plate. The harvested cells were stained for surface CD4, then fixed with 1% PFA (Paraformaldehyde)/PBS overnight, permeabilized with 0.1% Triton-X100 in 0.1% sodium citrate, pH 7.2, then stained with IL-2 antibody intracellularly. The results were analyzed by a FACS Calibur. All Antibodies and GolgiStop were purchased from BD Pharmingen (San Diego, CA, USA). Statistics Comparisons were made by the paired Student’s t -test. P < 0.05 was considered statistically significant. Correlations were analyzed by linear regression using Prism-4 software (GraphPad).

Journal

Aging Cell – Wiley

Published: Jun 1, 2010

Keywords: loxP /CreER T system; thymic aging; thymic epithelium; spontaneous FoxN1 gene recombination

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