Modulation of Aire regulates the expression of tissue-restricted antigens

Intrathymic expression of tissue-restricted antigens (TRAs) has been viewed as the key element in the induction of central tolerance and recently, a central role for the autoimmune regulator (Aire) has been suggested in this process. The aim of this study was to establish whether down or up-regulation of Aire leads to alterations in TRA expression and whether this is limited to thymic epithelial cells. This study also characterized whether TRAs follow Aire expression during normal development, and whether thymic microenvironment plays a role in the expression of Aire and TRAs. We did several in vivo and in vitro experiments to manipulate Aire expression and measured expression of four TRAs (Trefoil factor-3, Insulin-2, Major urinary protein-1 and Salivary protein-1) by real-time RT-PCR. Aire had an allele dose-dependent effect on TRA expression in the thymuses of mice from two strains, C57BL/6J and Balb/c, but had no effect on TRA expression in the lymph nodes. In the thymus, Aire and TRAs were both localized in the medulla and were co-expressed during normal development and involution. In the primary stromal cells as well as thymic epithelial cell line, the adenoviral over-expression of Aire resulted in an increase in TRA expression. By manipulating in vitro organ-cultures we showed that thymic microenvironment plays a dominant role in Aire expression whereas TRAs follow the same pattern. The data underline a direct role for Aire in TRA expression and suggest that modulation of Aire has a potential to control central tolerance and autoimmunity.
1
Introduction
Thymus has an essential role in establishing immune tolerance. Previous studies have demonstrated that tissue-restricted antigens (TRAs) are expressed in thymus and that this expression is needed for the deletion of self-reactive T cells (Kyewski and Klein, 2006). A central feature in this process is the promiscuous expression of TRAs by epithelial cells in thymic medullary region, where the TRAs are presented and encountered by the thymocytes, leading to the induction of tolerance either by clonal deletion or functional inactivation (Derbinski et al., 2001). In this context, medullary thymic epithelial cells (mTEC) highly express MHC molecules with costimulatory signals and act as professional antigen presenting cells (APCs) in thymus. A detailed study of the gene expression pattern in mTEC revealed that many of the TRAs and, in particular, almost all putative autoantigen targets of experimental animal models and human diseases are expressed by mTEC (Derbinski et al., 2001; Gotter and Kyewski, 2004). Altogether, the pool of promiscuously expressed genes in thymus appears to be highly diverse including tissue and sex-specific genes and genes specifically involved in development (Kyewski and Klein, 2006).
An important molecule in regulation of TRA expression in mTEC is autoimmune regulator (Aire) (Nagamine et al., 1997). The Aire protein has several features such as SAND and PHD finger domains that are characteristic to proteins involved in transcriptional control and has been reported to bind directly to DNA (Kumar et al., 2001) and to a common transcriptional regulator and histone acetyltransferase, CREB binding protein (CBP) (Pitkanen et al., 2000). In the thymus and cell lines, the Aire protein is subcellularly located to the nuclear bodies (Bjorses et al., 1999; Heino et al., 1999), which have been associated with several functions, including modulation of chromatin structure, transcriptional control, DNA repair and antiviral response (Everett and Chelbi-Alix, 2007). Initial studies have shown the Aire protein to be predominantly expressed in mTECs and suggest it has a role in regulation of immune tolerance (Blechschmidt et al., 1999; Heino et al., 1999). In humans, mutations in AIRE cause autoimmune-polyendocrinopathy-candidiasis ectodermal dystrophy (APECED), a syndrome characterized by the presence of autoantibodies to multiple self antigens and lymphocytic infiltration of endocrine glands, leading to autoimmune endocrine disorders (Perheentupa, 2006; Peterson and Peltonen, 2005). In agreement with the human disease, the Aire deficient mice have autoantibodies and tissue infiltration, although the full development of autoimmune disease appears to depend on the genetic background of the mouse (Anderson et al., 2002; Kuroda et al., 2005; Ramsey et al., 2002). The Aire deficiency affects negative selection since there is a complete failure to delete the organ-specific thymocytes in this mouse model (Liston et al., 2003). More importantly, the microarray analysis of mTEC population shows a decreased or abolished expression of multiple tissue specific genes in the Aire deficient mouse suggesting thus that Aire plays a role in modulating TRAs in the mTEC (Anderson et al., 2002; Derbinski et al., 2005; Jiang et al., 2005).
This study aims to further clarify whether Aire can directly regulate the TRA expression by analyzing the expression of four antigens in several experimental settings where Aire's expression has been modulated. The study aims to establish whether there is a dose-dependent correlation between the number of Aire allele copies and TRA expression level in thymic epithelial cells, and whether TRAs are co-expressed with Aire during thymic development and involution. We also studied whether the over-expression of Aire as a sole factor is sufficient to induce TRA expression and whether thymic microenvironment plays a role in the expression of Aire and TRAs.
2
Material and methods
2.1
Mice and cell cultures
Aire deficient mice (C57BL/6J and Balb/c background) were generated at The Walter and Eliza Hall Institute (Melbourne, Australia). The inserted targeting construct containing LacZ gene replaced mouse Aire exon 8. For genotyping, the genomic DNA was extracted using JetQuick Tissue DNA Spin Kit (Genomed), and wild-type (WT) and knockout (KO) alleles were amplified using primers: 1042 5′-cagaagaacgaggat-3′, 1045 5′-cagactgccttggga-3′ or 1043 5′-ctgtcttctgtgaaggcttctagg-3′. As shown in Fig. 1A, primers pair 1042/1043 and 1043/1045 detect WT and KO alleles, respectively. Thymuses from 4- to 6-week-old WT, Aire HET (heterozygote) and Aire KO mice were used. Embryonic (E13.5, E15.5 and E17.5), newborn, neonatal D11 and adult (6 weeks, 6 months and 12 months) mouse tissues were used in developmental dynamics analysis. Mice were maintained at the mouse facility of the Institute of Molecular and Cell Biology, Tartu University. TEC 1C6 cell line (Mizuochi et al., 1992) was kindly provided by G. Holländer (University of Basel, Switzerland). Human embryonic kidney (HEK293) cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal calf serum (FCS), 100 U/ml penicillin, 100 μg/ml streptomycin and 0.25 μg/ml amphotericin B (Gibco BRL).
2.2
EGFP and Aire adenovirus construction and infection
The pAdTrack-CMV (Stratagene) vector expressing enhanced green fluorescence protein (EGFP) gene was used as pAd-GFP plasmid. The mouse Aire gene was amplified from pcAire vector (Heino et al., 2000) using the primers: mAire-5-SalI 5′-tttgtcgac agatggcaggtggggatggaatg-3′ and mAire-3-NotI_stop 5′-tttgcggccgctcaggaagagaagggtggtgtc-3′ and cloned into SalI and NotI sites of pAdTrack-CMV resulting in AdAire-GFP. HEK293 cells (Invitrogen), which constitutively express AdEasy deleted E1 genes in-trans, were used for expression analysis of adenoviral vectors and for virus growth. To make recombinant adenoviruses, pAd-GFP and pAdAire-GFP plasmids were recombined with pAdEasy-1. The amplification and harvest process was repeated to generate higher titer viral stocks. Subsequently, the lyzates from several amplification steps were purified by CsCl gradient centrifugation (He et al., 1998). Virus bands was collected and mixed with 2× preservation buffer (10 mM Tris pH 8.0, 100 mM NaCl, 0.1% BSA, 50% glycerol). We quantified the Ad-GFP and AdAire-GFP virus particles by absorbance measurement at 260 nm to be equivalent to 1012 particles with virus titers >2 × 108 pfu/ml. The adenoviruses were next verified for their expression by infection of HEK293 cells for 48 h and analyzed by immunoblotting with rabbit polyclonal anti-mouseAire and mouse monoclonal anti-G3PDH (Ambion). The signal was detected by Supersignal® West Pico Chemoluminescent Substrate (Pierce Biotechnology) according to the manufacturer's instructions. For adenoviral infection of thymic primary culture and TEC 1C6, the cells were infected with Ad-GFP and AdAire-GFP at approximately 70% confluence. Cells were incubated in 500 μl serum-free OptiMEM (100 U/ml penicillin, 100 μg/ml streptomycin and 0.25 μg/ml amphotericin B) and infected with equal amounts of virus for 1 h. The infection rate was further assessed by quantitative real-time PCR analyzing intake of viral genomic DNA.
2.3
Thymic stromal cell isolation
Small cuts were made into the capsules of thymi dissected from eight to twelve 4-week-old mice and thymocytes were released by repetitive pipeting. The remaining thymic fragments were incubated in 0.5 mg/ml dispase/collagenase (Roche) and 1.5 μg/ml DNase I (AppliChem) in PBS at 37 °C for 20 min, with gentle agitation using a Pasteur pipette every 5 min. Released cells were collected to separate fractions and fresh enzyme solution was added four times. The cells were resuspended in 5 mM EDTA in 10 ml of RPMI-1460. Isolated cells were used either for stromal cell isolation or for growing thymic primary culture. For thymic primary culture, the isolated cells were cultured for 4 days in DMEM supplemented with 10% FCS. For stromal cell isolation cells were counted in each fraction. To obtain 2 × 108 cells required for isolation, the final fraction was used first and then collected backwards through the collected fractions. The fractions were pooled and passed through a 100 μm mesh to remove clumps.
2.4
Cell sorting
For CD45 depletion CD45 MicroBeads (Miltenyi Biotec) were used according to manufacturers instructions. For cortical epithelial cell (cTEC) isolation, the CD45− cells were stained with H213-HB Ab (anti-CDR1) followed by goat anti-rat IgG Microbeads (Miltenyi Biotech) and AutoMACS separation (isolation mode: Possel-S). The positive fraction (CDR1+) contained magnetically bound cTEC-s. For mTEC isolation CDR1− cells were stained with G8.8 (anti-EpCAM, generated from a G8.8 hybridoma cell line) followed by goat anti-rat IgG Microbeads (Miltenyi Biotec) and separation as previously described. The purity of mTEC and cTEC was >80% as assessed by staining with anti-CD45 FITC (30F11, Miltenyi Biotech) and anti-I-Ab PE (AF6-120.1, BD Biosciences) using FACSCalibur flow cytometer (BD Biosciences).
2.5
Immunofluorescence and microscopy
Cryostat sections (5μm) of fresh-frozen 10-day-old mouse thymus were thaw-mounted onto SuperFrost Plus microscope slides (Menzel–Gläzer) and fixed in cold acetone (−20 °C) for 5 min. Sections were permeabilized in PBS/0.5% Triton X-100/1% normal goat serum (DAKO) for 15 min. Slides were blocked with 1% normal goat sera for 20 min at room temperature and incubated with rat monoclonal G8.8 (1:100) or rabbit polyclonal anti-mAIRE (1:2000) and then incubated with Alexa Fluor 594-conjugated goat anti-rat IgG (H + L) or Alexa Fluor 488-conjugated goat anti-rabbit (Fab)2 (both from Molecular Probes, Eugene), followed by washing six times in PBS. The slides were incubated with 15 μg/ml DAPI (Roche) and mounted with fluorescent mounting medium (DAKO). The images were acquired by fluorescence microscopy (Eclipse TE2000-4; Nikon, Melville, NY).
2.6
Thymic reaggregate organ culture
Reaggregated thymic organ-cultures were done as described previously (Jenkinson et al., 1992). Briefly, thymic stromal cells from E17.5 C57BL/6 mice were prepared by disaggregating fetal thymic lobes, which had been previously cultured for 7 days in 1.45 mM deoxyguanosine (Sigma, St. Louis, MO) using 1× trypsin (Life Technologies, Grand Island, NY). Reaggregates were formed by mixing together stromal cells and thymocytes at a 3:1 cell ratio and cultured for 3 days at 37 °C.
2.7
Real-time PCR
RNA was isolated using TRIzol (Invitrogen, Life Technologies) and reverse-transcribed to cDNA using the SuperScript™ III Reverse Transcriptase (Invitrogen, Life Technologies). Real-time PCR was performed with the ABI Prism 7900HT Fast Real-Time PCR System instrument (Applied Biosystems) using qPCR SYBR Green Core Kit (Eurogentec) according to the manufacturer's instructions except that 2 mM MgCl2 concentration was used. The amplification program included an initial denaturation step at 95 °C for 10 min, followed by denaturation at 95 °C for 15 s, and annealing and extension at 60 °C for 1 min, for 45 cycles. SYBR Green fluorescence was measured after each extension step, and the specificity of amplification was evaluated by melting curve analysis. Primers used to amplify specific gene products from murine cDNA were K2-8 sense, 5′-aggagctcattccgtagctg-3′; K2-8 antisense, 5′-tctgggatgcagaacatgag-3′; Aire 11/12, 5′-ccccgccggggaccaatctc-3′; Aire 12/13, 5′-agtcgtcccctaccttggcaagc-3′; Tff3 sense, 5′-tacgttggcctgtctccaag-3′; Tff3 antisense, 5′-cagggcacatttgggatact-3′; Ins2 sense, 5′-gacccacaagtggcacaac-3′; Ins2 antisense, 5′-tctacaatgccacgcttctg-3’, Mup1 sense, 5′-tctgtgacgtatgatggattcaa-3’; Mup1 antisense, 5′-tctggttctcggccatagag-3′; Spt1 sense, 5′-aacttctggaactgctgattctg-3′; Spt1 antisense, 5′-gaggcctcattagcagtgttg-3′. The relative gene expression levels were calculated using the comparative Ct (ΔΔCt) method (according to Applied Biosystems), where the relative expression is calculated as , and where Ct represents the threshold cycle. Every sample was run in three parallel reactions.
3
Results
3.1
Decrease in Aire expression down-regulates the TRA expression in a dose-dependent manner
We initially set out to confirm by real-time PCR the previously published array results (Derbinski et al., 2005) demonstrating decreased TRA mRNA levels in the Aire deficient mouse. In order to study whether the self antigen expression is dependent on Aire we chose four TRAs; Tff3, Ins2, Mup1 and Spt1, which were downregulated in the Aire deficient mouse according to publicly available microarray data (Derbinski et al., 2005). Throughout the study, we normalized our data to the expression level of keratin 8 (K8) mRNA, which in thymus is specifically expressed in epithelial cell fraction and is not influenced by Aire gene expression (Anderson et al., 2002; Derbinski et al., 2005). The real-time PCR analysis showed almost complete absence of TRA mRNA signal in C57BL/6 Aire KO thymus samples, which was uniformly seen with all four antigens studied (Fig. 1B). Furthermore, Aire allele dose-dependency was observed, as heterozygous mouse thymus consistently showed lower expression levels compared to the WT thymus levels. The expression level of all four TRAs in heterozygous mice thymus was approximately 10–20% of the expression level in WT mice. In order to determine whether Aire's effect on TRA expression depends on the genetic background, we also measured expression levels of the four TRAs in Aire KO and Aire HET mice backcrossed to Balb/c WT mice (Fig. 1C). Again, we observed a clear allele dose-dependency for all TRAs studied and almost no expression of the TRAs in the Aire KO mouse.
We next determined whether Aire has a similar effect on TRA expression in the lymph nodes and quantified the expression of TRAs in the lymph nodes from C57BL/6 mice. Although we could clearly detect Aire mRNA in the lymph nodes at level that was even higher than the one of the whole thymus (Fig. 2A), most of the analyzed TRAs were undetectable or close to the detection limit. The higher expression of Aire in lymph nodes was relative to the epithelial cell marker K8, limiting the detection of Aire mRNA signal to the epithelial cell fraction. However, the mRNA signal for the Ins2 was clearly present in lymph node samples but, interestingly, did not depend on the presence of Aire (Fig. 2B).
In order to establish whether Aire co-localizes in the thymus with TRAs, we purified the thymic mTEC based on the cell-surface marker EpCAM (Fig. 3A) and analyzed the expression of the TRA genes. As seen in Fig. 3B, the expression of the Tff3, Ins2, Mup1 and Spt1 antigens was limited to the mTEC population, i.e. the cell population of Aire expression. The cTEC population showed a very low expression for all four TRAs both in the WT as well as Aire KO mouse. Collectively these data show that Aire dose-dependently regulates TRA expression in thymus but not in the lymph nodes, and confirms by real-time PCR the previously published microarray data, suggesting that both Aire and TRAs are predominantly expressed in thymus medullary epithelium.
3.2
TRAs follow the expression of Aire during normal development and involution
Thus far, the expression of Aire and TRAs has been studied in fetal or neonatal mice using WT versus Aire KO mice. If the expression of self antigens is directly dependent on Aire, this should be evident throughout the development of thymic tissue. However, the thymic cell content and volume changes significantly during development. To limit our analysis to the epithelial cell subsets only, we normalized our data again to the K8 gene. Thymuses from different embryonic, neonatal, young or adult developmental stages were analyzed for the Aire and TRA expression (Fig. 4). Very low Aire expression was detectable already at day E13.5 but showed a significant increase at E15.5. Thus, the start of Aire expression coincides with the influx of the first wave of the hematopoietic cells to the thymus. We observed the highest expression level at postnatal D11 and a gradual decrease thereafter until the very last time-point studied. Aire expression was, however, clearly present even in 12-month-old mice. The expression of TRAs closely followed the pattern of Aire reaching their peak at D11 followed by an obvious decrease, suggesting a role for Aire in their regulation.
3.3
Over-expression of Aire results in an increase in TRA expression
Although the lack of Aire has been shown to have a negative effect on TRA expression, the ability of Aire as a single factor to up-regulate TRA expression has not been demonstrated. We used an adenoviral expression system (AdAire-GFP versus Ad-GFP) to determine whether the specific over-expression of Aire has any effect on TRAs. Infection with the AdAire-GFP resulted in an increased production of Aire protein (Fig. 5A). The AdAire-GFP infection also resulted in an increase of expression of all four TRAs studied, which was detected in primary thymic stromal cells (Fig. 5B) as well as in thymic medullary epithelial cell line TEC 1C6 (Fig. 5C). The results demonstrate that Aire can indeed act as a single inducer of TRAs in thymic epithelial cells even in the absence of signals from other cell-types normally present in thymus.
3.4
Thymic microenvironment is needed for the expression of Aire and TRAs
Although the precise mechanism is not fully understood, it has been demonstrated that the complex 3D structure of thymus is essential for mTECs to function properly (Anderson et al., 2006). To study whether the microenvironment plays a role in Aire and TRA expression, we used ex vivo culture of thymocyte-depleted dissaggregated and reaggregated thymic stromal cells. As shown in Fig. 6, the disaggregation of thymus to 2D culture resulted in a dramatic decrease in expression of Aire as well as most of the TRAs. This effect was, at least partly, reversed by reaggregation suggesting a critical role for the thymic microenvironment in Aire as well as TRA expression.
4
Discussion
In this paper, we report Aire dependent expression of four TRAs in mouse thymus. TRA expression was previously reported to be substantially decreased in Aire deficient mTEC subpopulations by microarray analysis (Anderson et al., 2002; Derbinski et al., 2005), which prompted us to follow the expression of four TRAs as marker antigens. All four genes have highly selective tissue specific expression. For example, Tff3 is restricted to mucin producing epithelial cells, with high expression in stomach and intestine (Hoffmann and Jagla, 2002; Karam et al., 2004), Spt1 is expressed in salivary and lacrimal glands (Dickinson et al., 1989) and Mup1 is expressed in liver but also in salivary, lacrimal and mammary glands (Shaw et al., 1983). Of the two insulin genes present in the mouse genome and encoded from separate loci, we selected Ins2 due to its high expression in the thymus as well as pancreatic beta cells, and previously reported Aire dependent expression pattern (Chentoufi and Polychronakos, 2002; Derbinski et al., 2005). Furthermore, it has been reported that Ins2 deficient mouse with low insulin expression in thymus has T cell reactivity to proinsulin (Chentoufi and Polychronakos, 2002).
The expression signal of all four TRAs tested was readily detectable in whole thymus and sorted mTEC samples by real-time PCR analysis. Interestingly, the TRA expression in heterozygous mouse thymus, both on C57Bl/6 and Balb/c background, was repeatedly only 10–20% and not 50% of the expression level seen in WT mouse, which would be expected when one of the Aire genomic alleles remains intact. This expression at lower levels than expected in the heterozygous mouse suggests Aire haploinsufficiency in regulation of target TRA genes. In haploinsufficiency of transcriptional regulators, only one intact gene copy is not sufficient for the functional activity of the regulated target gene product. The phenomenon has been explained by a stochastic expression model where diploid cells have a higher probability than haploid cells in maintaining the abundance of an expressed gene product above a low threshold level (Cook et al., 1998; Kaern et al., 2005). Further support to Aire haploinsufficiency comes from the report by Liston et al (Liston et al., 2004) demonstrating that the loss of one copy of the Aire allele in TCR-insHEL double transgenic mice caused severe functional defects in negative selection of autoreactive T cells and resulted in pancreatic cell insulitis, with diabetes incidence comparable to the Aire KO mouse. It should be noted, however, that the activation of target genes by a transcriptional factor in vivo can be dependent on the specific gene and the physiological context, such as in the case of cardiac transcription factor Nkx2–5 (Jay et al., 2005).
A recent study reported that lymph node stroma can also express Aire as well as a range of TRAs and contributes accordingly to tolerance induction (Lee et al., 2007). Here we report that, although Aire as well as Ins2 expression is clearly present in the lymph nodes of WT mice, the lack of Aire does not lead to a decrease in Ins2 expression in the Aire KO mice. The data suggests that, unlike in the thymus, other Aire-independent factors are likely to control Ins2 expression in the lymph nodes. Regarding the rest of the TRAs studied, we found very low, if any, expression by real-time PCR, which did not allow us to quantify the changes.
In order to determine whether the thymic expression of Aire and TRAs follow the same pattern during development, we monitored the expression throughout mouse development from E13.5 to 12 months. We detected an increase in Aire expression at day E15.5, which is in concordance with earlier reported results (Sousa Cardoso et al., 2006). The expression was at its highest at D11 and decreased thereafter, but was still present even in 12-month-old mouse tissue. The expression of TRAs followed a similar pattern to Aire expression, indicating a correlation between the amount of Aire and TRAs. The data demonstrate that the dynamics of Aire and TRA expression closely follow the dynamics of thymic function in general, being most active during the postnatal period and followed by a gradual decline in activity (Gray et al., 2006).
Aire dependent TRA expression is further illustrated by adenoviral experiments enforcing Aire expression in thymic epithelial cells. We show that over-expression of Aire as a single factor is sufficient to induce the expression of all four TRAs studied, providing evidence that modulation of Aire can directly lead to alterations in TRA levels and may thus also affect the maintenance of central tolerance. The finding that Aire, in addition to primary thymic stromal cells, can also modulate TRA levels in the thymic medullary epithelial cell line, suggests that there is no need for other cell types for the Aire-induced up-regulation of TRAs to occur.
The disruption of normal thymic architecture is known to affect the expression pattern and functionality of thymus, and it has been suggested that interactions between epithelial cells and thymocytes control the development of the thymic microenvironment and T cell development (Van Ewijk et al., 1994). Although the maturation of thymic epithelial stroma during the fetal period apparently occurs independently of thymocyte-derived signals (Jenkinson et al., 2005) and is mainly regulated by thymic mesenchyme (Jenkinson et al., 2003), thymocytes deliver signaling molecules, which are needed to maintain the normal adult thymic microenvironment. For example, lymphotoxin that signals through the lymphotoxin receptor and directs the alternative NfkappaB pathway, is needed for development of the thymic medullary compartment. Consequently, lymphotoxin receptor deficient mouse thymus had subnormal levels of Aire and TRAs (Boehm et al., 2003; Chin et al., 2003). Thymic medullary atrophy and lower expression of Aire and TRAs have been reported in mouse models deficient in several genes involved in the NFkappaB pathway, such as TRAF6, NIK, RelB or p52 suggesting an important role of this pathway in development of thymic medulla (Akiyama et al., 2005; Burkly et al., 1995; Kinoshita et al., 2006; Zhang et al., 2006). A recent study suggests that Aire deficiency may also cause changes in the organization and composition of the medullary epithelial compartment (Gillard et al., 2007). Thus, it is presently unknown whether the reduced levels of TRA expression seen in Aire KO mice are predominantly the result of changes in transcriptional activity or changes in thymic epithelial cell development. In this study, we show a sustained Aire and TRA expression in 2-deoxyguonosine treated FTOC, which rapidly disappeared after the disruption of the three-dimensional thymic meshwork into two-dimensional culture. Aire as well as TRA expression was regained in RTOC, however, the presence of thymocytes did not further augment this effect. These results show that Aire and TRA expression is dependent on the three-dimensional structure of epithelial microenvironment. This expression, however, seems to be independent of the presence of thymocytes being in line with previous data demonstrating the Aire expression signal in RAG deficient and CD3etg26 transgenic mice, in which T-cell development is blocked (Jenkinson et al., 2005; Zuklys et al., 2000).
In conclusion, we show that Aire has a dose-dependent effect on TRA expression in thymus but not in the lymph nodes. Both, Aire as well as TRAs localize in the thymic medulla and are co-expressed during normal development and involution. We also show that Aire can directly induce TRA expression in medullary epithelial cells although the thymic microenvironment plays a crucial role for the maximal expression to occur. Our data suggest a clear correlation between the expression of Aire and TRAs and indicate that approaches to stimulate Aire expression in thymic epithelium could be considered to modulate tolerance induction to peripheral antigens.

tissue-restricted antigens
autoimmune regulator
autoimmunity
thymus


Diet supplementation for 5 weeks with polyphenol-rich cereals improves several functions and the redox state of mouse leucocytes

Background Cereals naturally contain a great variety of polyphenols, which exert a wide range of physiological effects both in vitro and in vivo. Many of their protective effects, including an improvement of the function and redox state of immune cells in unhealthy or aged subjects come from their properties as powerful antioxidant compounds. However, whether cereal-based dietary supplementation positively affects the immune function and cellular redox state of healthy subjects remains unclear.
Introduction
Nowadays, consumption of natural products is a matter of major importance as regards health. Indeed, consumption of fruit and vegetables was already associated with a reduced risk of chronic diseases and age-related functional decline several years ago [1]. Nevertheless, less attention has been focused on the importance of phytochemicals from cereals in maintaining health. It is well known that the nutritional status is a central factor contributing to immune system competence [2] and that an adequate function of the immune system is related to health promotion and disease prevention [3]. Nutrients that have traditionally been shown to be required for correct immune system function include different types of vitamins, zinc, copper, and selenium. However, there is an increasing amount of data showing the implication of the so-called “non-nutrients”, i.e., phytochemicals, in the immune system responses [4]. Further, the phytochemicals found in natural products have been involved in protecting against a wide range of oxidative stress-related chronic diseases [5]. The overproduction of oxidants and the overwhelming of the antioxidant defence lead to oxidative stress, that can cause oxidative damage to large biomolecules, such as proteins, DNA, and lipids, resulting in an increased risk of disease [5, 6]. For this reason, to forestall an excessive production of reactive oxygen species (ROS), sufficient amounts of antioxidants need to be consumed. However, the generation of ROS is a key part of normal immune system function [7]. These molecules play a key role in the defence against infectious agents and seem to be involved in regulating many biological processes, serving as second messengers [8]. Thus, it is important to maintain a balance between ROS generation and antioxidant defences within immune cells to ensure a correct functioning of the immune system and help to prevent the onset of chronic diseases.
Cereals naturally contain a wide variety of polyphenols such as the hydroxycinnamic acids, ferulic, vanillic, and p-coumaric acids [9], which show a strong antioxidant power and may help to protect from oxidative stress and, therefore, can decrease the risk of contracting many diseases. Flavonoids are present in small quantities, even though their numerous biological effects and their implications for inflammation and chronic diseases have been widely described [10, 11]. The mechanisms of action of polyphenols go beyond the modulation of oxidative stress-related pathways [6]. Cereal phytochemicals exert their health benefits through multifactorial physiological mechanisms, including enhancement of the immune system and hormones, facilitation of substance transit through the digestive tract, metabolism, modulation and antioxidant, antibacterial and antiviral activities [12].
It is known that unhealthy or aged subjects are more susceptible to infection, showing an impairment of function and redox state of immune cells and that such impairment can be counteracted by antioxidant administration [13, 14]. However, whether dietary antioxidant supplementation affects the functional and redox state of immune cells of healthy subjects remains unclear. Not all subjects are likely to benefit in the same way from supplementation, i.e., there are certain groups within the apparently healthy, general population that benefit from adequate intakes of certain compounds [15].
Since there is an increasing interest in consuming products naturally rich in phytochemicals able to provide health benefits and prevent a wide variety of diseases, in this context we have investigated in the present work the effects of diet supplementation for 5 weeks with different types of cereal fractions naturally rich in polyphenols on certain parameters of immune function and redox state of peritoneal leukocytes from adult healthy mice.
Material and methods
Animals
One hundred-fifty female ICR (CD-1) mice (Mus musculus) (Harlan Ibérica, Barcelona, Spain), which were 24 weeks old on arrival to our laboratory, were used. Mice were checked periodically by animal care personnel and were specific pathogen free, as tested by Harlan according to FELASA recommendations, and did not show any sign of malignancy or other pathological processes. The animals were randomly housed in polyurethane boxes, at a constant temperature (22 ± 2°C) in sterile conditions inside an aseptic air negative-pressure environmental cabinet (Flufrance, Cachan, France), on a 12/12 h reversed light/dark cycle. All animals were fed water and standard Sander Mus (A04 diet from Panlab L.S., Barcelona, Spain) pellets ad libitum. The diet was in accordance with the recommendations of the American Institute of Nutrition for laboratory animals. Mice were treated according to the guidelines of the European Community Council Directives 86/6091 EEC. Although we have previously observed that the oestrous cycle phase of mice has no effect on this experimental assay, all females used in the present work were at the beginning of dioestrous.
Experimental groups
The animals were marked for their individual follow-up. Five experimental groups, one control (n = 30) and four supplemented (30 mice per group), were used. The supplementation started at 28 weeks of age. Then, the supplemented groups were fed for a period of 5 weeks, and at the age of 33 ± 2 weeks samples were obtained to evaluate certain parameters of function and redox state of peritoneal leukocytes. The experiments were carried out in January at a constant laboratory temperature of 25 ± 2°C. Food intake was measured weekly at the same time (9:00–11:00 h). Each animal was fed weekly 50 g of diet (control or supplemented) and, after 1 week, the uneaten remains were weighed. Body weights were determined once every week. No significant differences on food consumption and body weight among diet groups during the study were observed.
Cereal products and preparation of extracts
All cereal products (wheat germ, buckwheat flour, fine rice bran and wheat middlings, named B, C, D and E; respectively) used in this study were provided by Danone Vitapole (France). The samples were stored and kept at −20°C prior to analysis. Moisture content of the cereal samples was evaluated by drying at 130°C for 90 min. The analyses were done in duplicate. The different extracts were analyzed by Danone Vitapole (France). Briefly, the cereal products were sequentially extracted with water at 50°C followed by 80% methanol and ethyl acetate. Extraction with each solvent was carried out twice for 30 min under N2, in the dark and constant agitation (200 rpm). The ethyl acetate extracts were evaporated under vacuum and re-dissolved in 80% methanol. The final insoluble pellet was dried. The soluble fractions and the insoluble pellet were stored at 4°C (in the dark and under N2) prior to analysis of polyphenols and determination of antioxidant activity.
Analysis of total polyphenols and antioxidant activity
The amount of total phenolic compounds in all extracts was determined by Danone Vitapole (France), according to the Folin–Ciocalteu method [16]. The total phenolic concentration is calculated from a standard curve using a range of concentrations of gallic acid. The results are expressed as milligrams of gallic acid equivalents per gram of material. The antioxidant in vitro activity of the extracts obtained from selected cereal products was evaluated using an Oxygen Radical Absorbance Capacity (ORAC) procedure (Brunswick Laboratories, Wareham, MA, USA). Values are expressed as μmol of Trolox equivalents (TE) per g of dry matter. This assay was conducted in triplicate. There was also a positive correlation between ORAC and total polyphenol content (r2 = 0.843).
Preparation of diets
The control group only received a standard maintenance diet (AO4 diet from Panlab L.S. Barcelona, Spain; see Table 1) and the remaining groups received 80% of control diet plus 20% of cereal fractions naturally rich in polyphenols (B, C, D and E; see Table 1). Control and supplemented diets were prepared by mixing the standard maintenance diet or one of the cereal fractions with water, forming a biscuit-shape preparation. Both types of preparations were daily turned upside down and maintained in a dark room for 3 days to obtain a dry preparation. Then, the humidity of the different diets was measured and each of them was stored at 4°C for less than 3 weeks, avoiding exposure to light in order to prevent their oxidation.
Table 1Nutritional composition of standard (control) and experimental (B, C, D and E) dietsDiet ingredientsa (mg/100g)ControlBCDEHFLFbHFLFbLFbLFbHFLFbPolyphenols  Gallic acid————0.49————  Catechin———10.80.192.043.34——  p-HB acid—0.270.99—0.78——1.04—  Vanillic acid—0.5413.191.82—1.93—0.860.46  p-coumaric acid———0.210.161.2818.910.060.68  Sinapic acid————0.454.73———  Ferulic acid—25.9755.600.420.0911.7569.1923.8363.06  Quercetin——0.17a—2.42a————  Rutin——3.94a—11.85a————  Oryzanol—————4.03a——Vitamins  Vitamin A0.45————  Vitamin D30.0038————  Vitamin E213.650.21.335.55  Thiamin0.11.870.411.811.48  Riboflavin0.4————  Niacin2————  Pantothenic acid1.5————  Pyridoxin0.40.870.20.180.68  Biotin0.01————  Folacin0.12914569103  Cobalaminec2————  Choline50————Fatty acids  Palmitic acid460————  Palmitoleic acid39————  Stearic acid130————  Oleic acid650————  Linoleic acid1,390————  Linolenic acid130————Minerals  Calcium88025107862  Phosphorum590————  Chloride310————  Sodium250————  Potasium600————  Magnesium140259174910203Proteinsd15.433.611.521.316.85Carbohidratesd101.521.167.47.428Fibred3.97.73.140.429.3Each control (non-supplemented) animal received a 100% of A04 maintenance diet (Panlab, Barcelona, Spain). Each supplemented animal received an 80% of A04 maintenance diet plus 20% of cereal fractions (B, C, D or E) naturally rich in polyphenols. B = wheat germ; C = buckwheat flour; D = fine rice bran; E = wheat middlings. ORAC (μTrolox Equivalents/g): B = 106; C = 81; D = 121; E = 49. Total polyphenols content (eq mg gallic acid/g): B = 11.15; C = 8.48; D = 15.24; E = 9.35. HF (mg/100g) = hydrosoluble fraction; LF (mg/100mg) = lyposoluble fraction; p-HB = p-hydroxybenzoic. amg/100 g. bmg/100 mg. cμg/100 g. dg/100 g
Collection of peritoneal leukocytes
Peritoneal suspensions were obtained between 8:00 and 10.00 h, without sacrificing the animals, following the method previously described [17]. Peritoneal leukocytes (macrophages and lymphocytes) were collected, identified, quantified by their morphology using optical microscopy (40×) and their concentration was adjusted depending on the function assayed [14, 18]. The cellular viability, determined in each experiment using the trypan-blue exclusion test, was in all cases higher than 95%.
Leukocyte functions
The chemotaxis assays were performed according to a technique previously described [14]. The results were expressed as chemotaxis index (C.I.) by counting the number of macrophages and lymphocytes at the lower face of the filters of the Boyden’ chambers [14]. The intracellular superoxide anion (O2·−) levels were evaluated as described elsewhere [17]. The results were expressed as nmol of O2·− per 106 cells. The lymphoproliferation assay and the determination of interleukin-2 (IL-2) levels were carried out following the methods previously described [14]. The proliferation results were expressed as percentage of stimulation by the mitogens Concanavalin A (Con A) or lipopolysaccharide (LPS) with respect to spontaneous proliferation that was considered to be 100. In the case of IL-2 the results were expressed as pg/ml.
Leukocyte redox state
Total glutathione and its oxidized form (GSSG) were spectrophotometrically evaluated according to [17]. The results are expressed as nmol/106cells. To calculate the GSSG/GSH ratio, the GSH (reduced form) was obtained by substracting the GSSG values from the total glutathione values. The secretion of mouse tumor necrosis factor alpha (TNFα) was measured in culture supernatants stimulated with LPS, using an ELISA kit (HyCult Biotechnology, Uden, The Netherlands). The results were expressed as pg/ml, being the minimum detectable value 8 pg/ml. Catalase activity (CAT) was carried out according to [19], slightly modified. Samples of peritoneal suspension (adjusted to 106 cells/ml) were resuspended in 50 mM phosphate buffer, sonicated and centrifuged at 3200 g (4°C) for 20 min. The reaction was initiated by the addition of H2O2 and spectrophotometrically measured at 240 nm (25°C) for 50 s. The results were expressed as U/106 cells. Malondialdehyde (MDA) levels were evaluated according to a technique previously described [20], slightly modified, and quantified by high performance liquid chromatography (HPLC). Aliquots of peritoneal suspension (adjusted to 106 cells/ml) resuspended in 50 mM KH2PO4 (Sigma) pH 6.8 were sonicated and added to a mixture reaction that contained 0.44 M ortophosphoric acid (H3PO4, Fluka, Steinheim, Germany), 2-thiobarbituric acid (0.6%), (TBA, Sigma) and 3 mM butylated hydroxytoluene (BHT, Sigma). It was heated at 95°C for 30 min and after cooling on ice, the samples were extracted adding n-butanol (Panreac), centrifugated at 13,000 rpm (4°C) for 5 min and 50 μl of supernatants (organic phase) were injected in the HPLC column (Novapack C18, 15 cm × 3.9 mm, Waters). As mobile phase 50 mM KH2PO4 pH 6.8/methanol (90/10 v/v) was used. The flow rate of the mobile phase was adjusted to 0.4 ml/min. MDA was monitored at λ = 532 nm in a WATERS 486 ultraviolet detector. Standard curves were prepared fresh daily using MDA bis (dimethyl acetal, Sigma). The results were expressed as nmol/106cells.
Statistical analysis
Statistical analysis was performed using the software SPSS 11.5 (Chicago, IL, USA). The data are expressed as the mean ± standard deviation (S.D.) of the eight values corresponding to the number of experiments. Each value is the mean of the data from assays performed in duplicate. The normality of the samples was confirmed by the Kolmogorov–Smirnov test and the homogeneity of variances by the Levene test. The data were analysed by the one-way analysis of variance (ANOVA) followed by the Tukey test. The Kruskall Wallis test for non-parametric data was used. The minimum level considered statistically significant was P < 0.05.
Results
Immune system response
Figure 1 shows the chemotaxis capacity, lymphoproliferative response to the T-cell mitogen Concanavalin A (ConA) or the B-cell mitogen LPS and release of IL-2 of peritoneal lymphocytes from adult female mice supplemented for 5 weeks with cereal fractions naturally rich in polyphenols. Mice supplemented with cereals showed a strongly significant increase of lymphocyte chemotaxis compared with that of control mice (P < 0.001 with B, D and E and P = 0.031 with C) (Fig. 1a). As regards lymphoproliferative response to Con A (Fig. 1b), mice supplemented with any of the cereals B (P = 0.027), C (P = 0.002), D (P < 0.001) or E (P < 0.001) showed a significantly higher capacity compared with that of control mice. The supplementation with the cereals C (P < 0.001), D (P = 0.024) or E (P < 0.001), but not with B, was able to increase lymphoproliferation in response to LPS with respect to that of non-supplemented mice (Fig. 1c). The secretion of IL-2 by peritoneal leukocytes from mice supplemented with cereal B (P < 0.001), C (P = 0.004) or E (P = 0.006) was higher than that of control mice (Fig. 1d). The chemotaxis capacity of macrophages increased significantly in cereal E-supplemented mice in comparison with that of control mice (434 ± 39 versus 290 ± 42, P < 0.001). The intracellular superoxide anion (O2·−) levels in non-stimulated peritoneal leukocytes significantly increased after supplementation for 5 weeks with cereal B and C with respect to non-supplemented mice (Controls: 24 ± 5; B: 42 ± 7, P < 0.001; C: 41 ± 7, P < 0.001). The release of intracellular O2·− from peritoneal leukocytes stimulated with latex beads was significantly higher in mice supplemented with cereals B, C and E (Controls: 52 ± 5; B: 63 ± 4, P = 0.045; C: 67 ± 8, P = 0.002 E: 64 ± 7, P = 0.021).
Fig. 1(a) Chemotaxis capacity of peritoneal lymphocytes, (b) lymphoproliferation in ConA (1 μg/ml)-stimulated 48 h-culture supernatants, (c) lymphoproliferation in LPS (1 μg/ml)-stimulated 48 h-culture supernatants and (d) release of interleukin-2 (IL-2) in supernatants of 48 h-cultures of peritoneal leukocytes stimulated with Con A (1 μg/ml) from adult female ICR (CD-1) mice supplemented for 5 weeks with cereal fractions (B, C, D or E) naturally rich in polyphenols. Bars show the mean ± SD of eight values, corresponding to the same number of experiments, being each value the mean of duplicate assays. *P < 0.05 **P < 0.01 and ***P < 0.001 versus the control group. Lymphoproliferation in response to both mitogens, Con A and LPS, was expressed as % of stimulation with respect to spontaneous proliferation, the latter considered as 100. Spontaneous lymphoproliferation (in counts per min): Control = 1245 ± 253; B = 1348 ± 239; C = 1071 ± 171; D = 1186 ± 166; E = 1052 ± 169
Cellular redox state
The content of oxidized glutathione (GSSG) and the GSSG/GSH ratio, the levels of TNFα, CAT activity and MDA levels in peritoneal leukocytes are shown in Fig. 2. All cereal fractions studied decreased significantly the GSSG levels (P < 0.001) compared to those of control mice (Fig. 2a). The GSSG/GSH ratio (Fig. 2b) was significantly decreased after supplementation with any of the cereals B (P < 0.001), D (P = 0.002) or E (P < 0.001) compared with that of non-supplemented mice. Supplementation for 5 weeks with any of the cereals did not cause changes in the GSH levels of peritoneal leukocytes with respect to the values of controls (2.29 ± 0.23). The secretion of TNFα decreased in leukocytes from mice supplemented with cereal C (P = 0.005), D (P = 0.007) and E (P = 0.004) in comparison with that of non-supplemented mice (Fig. 2c). The CAT activity of peritoneal leukocytes from mice supplemented with cereal C (P < 0.001), D (P = 0.003) and E (P = 0.040) was higher than the activity of this antioxidant enzyme in control mice (Fig. 2d). Finally, the lipid oxidative damage in peritoneal leukocytes, evaluated as MDA levels (Fig. 2e), was lower (P < 0.001) in mice supplemented for 5 weeks with cereals than in the control mice.
Fig. 2(a) Oxidized glutathione (GSSG) content, (b) GSSG/GSH ratio, (c) tumor necrosis factor (TNFα) levels in LPS-stimulated (1 μg/ml) culture supernatants, (d) catalase (CAT) activity and (e) malondialdehyde (MDA) levels in peritoneal leukocytes from adult female ICR (CD-1) mice supplemented for 5 weeks with cereal fractions (B, C, D or E) naturally rich in polyphenols. Bars show the mean ± SD of eight values, corresponding to the same number of experiments, being each value the mean of duplicate assays. *P < 0.05 **P < 0.01 and ***P < 0.001 versus the control group
Discussion
It is widely accepted that antioxidant compounds may help to protect against oxidative stress, with resulting health promotion and disease prevention [21]. Moreover, an adequate intake of these compounds can enhance certain aspects of immune function, which is related to health [4]. Thus, it is becoming clear that diet supplementation with appropriate amounts of antioxidants enhances the immune function and improves the redox state of unhealthy subjects [15]. However, whether antioxidant supplementation has these effects in healthy subjects, still remains unknown. In the present study the cereal fractions used were able to improve several aspects of immune function. Thus, important events of innate immune response, i.e., migration capacity (chemotaxis) and microbicidal activity (intracellular superoxide anion levels), appear to be effectively affected by the supplementation with cereals naturally rich in polyphenols. Akbay et al. [22] showed that the three flavonoid glycosides from Urtica dioica L., quercetin-3-O-rutinoside, kaempherol-3-O-rutinoside and isorhamnetin-3-O-glucoside increased, in vitro, the chemotaxis and the intracellular killing activity of neutrophils, using methods similar to those of the present work. In our study, the fraction E is only able to enhance macrophage chemotaxis, whereas all the cereal fractions were able to show a strong stimulatory effect on this function in lymphocytes. This fact suggests that the polyphenols present in cereal fractions could exert a cell-dependent effect on migration capacity. Since a higher NF-κB expression is linked to an increased production of migratory inhibitory factor (MIF), thus being responsible for a decreased migration capacity [13], and several polyphenols are able to inhibit NF-κB activation [23], it seems plausible that some of the polyphenols present in the cereals used in our study could enhance the chemotaxis capacity through their action on NF-κB activation. All cereal fractions used in the present work, with the exception of D, have been shown to increase the microbicidal activity of peritoneal leukocytes. Since p-hydroxybenzoic acid is not present in fraction D, this compound could be responsible for that effect. However, to date, there is no evidence of a microbicidal activity increase following p-hydroxybenzoic acid administration.
Lymphoproliferation is a crucial event of the acquired immune response. Several studies have demonstrated a strong stimulatory effect on this capacity and on the cytokine well-known as enhancer of proliferative response, namely the IL-2, after antioxidant supplementation. Thus, Bub et al. [4] showed an increase in both functions in peripheral blood mononuclear cells (PBMC) from healthy subjects after diet supplementation with fruit juices rich in polyphenolic compounds. In ethanol-fed mice, Cheshier et al. [24] demonstrated an increased IL-2 production by mitogen-stimulated splenocytes after in vitro administration of Pycnogenol®, a commercial combination of phenolic compounds from pine bark. To date, no studies on the effect of the isolated polyphenolic compounds of the cereals used in the present research on these functions have been carried out.
Glutathione (GSH) plays important roles in antioxidant defence, nutrient metabolism and regulation of many cellular events, and its deficiency contributes to the oxidative stress-related pathogenesis of several chronic diseases [25]. Both the oxidized glutathione (GSSG) content and the GSSG/GSH ratio, the latter being considered a sensitive marker of cellular oxidative stress [26], were significantly decreased after supplementation with all the cereal fractions, although fraction C only showed a tendency to diminish the GSSG/GSH ratio. However, the GSH content of peritoneal leukocytes was not changed after cereal-based supplementation, suggesting that the lower GSSG/GSH ratio observed is primarily due to diminutions of the GSSG content. Rosenblat et al. [27] demonstrated that isoflavonoid glabridin (25 μg/kg/day) supplementation was able to reduce the GSSG content in murine peritoneal macrophages. However, the same authors also observed increased GSH levels in these immune cells.
Tumor necrosis factor is one of the most common proinflammatory cytokines produced predominantly by macrophages that has been reported to increase in many oxidative stress-related chronic diseases [28]. Our study showed that each of the cereal fractions, except B, was able to decrease the secretion of TNFα by peritoneal leukocytes. In agreement with Wang and Mazza [29], catechin at a concentration of 31 μM inhibited the TNFα production of LPS/IFNγ-activated RAW 264.7 macrophages. However, our results suggest that p-coumaric acid could also have an important anti-inflammatory role because this common phenolic acid is present in each cereal fraction, except the B. Moreover, in rats with endotoxemia, two different doses (75, 150 mg/kg, i.p.) of a water extract of propolis, a natural product rich in p-coumaric acid, were able to inhibit TNFα production in plasma [30]. Nevertheless, other authors did not observe any effect on TNFα production in whole blood cultures in the presence of phenolic compounds like p-coumaric acid at the concentrations of 10−7–10−4 M [31]. The most probable mechanism triggered by phenolic compounds for blocking LPS-induced production of TNFα by macrophages could be the inhibition of NF-kB activation [23].
Catalase plays a fundamental role in protecting cells against oxidative damage [32]. Decreased CAT activity may compromise the overall enzymatic defence system. In the present study, each of the cereal fractions increased CAT activity in peritoneal leukocytes, although fraction B only showed a tendency to increase this enzymatic activity. In other study, the renal CAT activity was significantly increased in rats fed black rice, one of the most important cereals rich in phenolic acids [33]. Khan et al. [34] showed that following oral feeding (0.2% w/v) for 30 days of polyphenols from green tea, a popular beverage rich in catechin, CAT activity significantly increased in the small bowel, liver and lungs of mice. In fact, fractions C and D (the fractions rich in catechin) showed the greatest effect. The present data suggest that p-coumaric acid, which is present in almost all the cereal fractions (C, D and E) might also have a relatively important role in modulating the CAT activity. Indeed, an oral administration of p-coumaric acid (100 mg/kg) for five days ameliorated the CAT activity in the heart from rats treated with doxorubicin, an anticancer antibiotic [35]. This potential protective action of p-coumaric acid is likely to be due to its free radical scavenging power.
ROS generation is able to promote oxidative damage to fatty acids present in biological membranes via an autocatalytic process known as lipid peroxidation. Malondialdehyde is one of the end-products of this process [36], which is related to the onset of chronic diseases [37]. In the present work, all the cereal fractions strongly decreased the MDA levels in peritoneal leukocytes. It has been observed that highly reactive and destructive hydroxyl radicals are effectively scavenged by oral administration of p-coumaric acid (317 mg/day) for 30 days in rats [38], which might decrease the potential damage to biological membranes by lowering MDA production. Several authors have shown that rutin, quercetin or catechin, which are some of the flavonoids present in the cereals used in the present work, are able to decrease lipid oxidative damage [39, 40]. The same happens with ferulic acid, the most common phenolic acid in cereal cell walls, which inhibit peroxidation [41]. Red wine polyphenols, together with ascorbic acid, inhibit lipid peroxidation in human muscle tissue [42]. The same authors demonstrated that, in the presence of catechin, ascorbic acid at pH 3.0 works in a sinergystic way preventing lipid oxidative damage and β-carotene cooxidation. This fact might explain the central role that consuming natural products rich in polyphenolic antioxidants, like cereals, has in promoting health and preventing the onset of chronic diseases.
The present data demonstrate the importance of polyphenol-rich cereal consumption because not only has shown to be very effective for modulating several parameters of immune function, but also is able to improve to a large extent the redox state of immune cells from mice. More importantly, we have demonstrated that cereal-based antioxidant supplementation can exert beneficial effects in healthy mice and thus we suggest that normal healthy populations may benefit from this supplementation. Indeed, a recent study has provided evidence for potential protective effects of moderate consumption of polyphenol-rich red wine in healthy subjects [43]. It has been reported that subjects susceptible to infections or suffering from chronic inflammatory processes showing an impairment of function and redox state of immune cells likely to benefit from antioxidant administration [13, 14]. However, it is difficult to know whether there is changes or not in the healthy mice from the present study may be a reliable predictor for the nutrient-immune requirements in disease-associated, unhealthy mice. To confirm this possibility we should know the optimal combination and quantities of nutrients, the potential interactions that might occur between these nutrients and the most appropiate time at which that immunonutrition should be provided. In words of Berger [15], “the efficiency of supplementation is a question of timing. Antioxidant nutrients cannot cure an installed disease, but they may prevent its promotion”.
It is important to point out that the cereal fractions used by us contain other compounds, both nutrients and non-nutrients, such as vitamins, minerals or fibre and, therefore, we cannot rule out that they can result in health benefits. However, the present cereal fractions show a good correlation coefficient between total antioxidant capacity and content of phenolic compounds, thus suggesting that polyphenols are largely responsible for the antioxidant activity of the different cereal fractions, which ultimately may help to protect from oxidative stress and, consequently, can decrease the risk of many potential diseases. Nevertheless, it is important to take into account some issues. First, the doses used play a crucial role, since it is known that excessive amounts of antioxidant compounds can impair the immune function [44] or even becomes potentially prooxidants [45]. Yet, we have not used “megadoses”, but nutritional doses of polyphenols. Second, the present recommendation is not a single polyphenol-based supplementation to modulate the antioxidant status or the immune system, but it is the combination of different substances as the best way to provide protective action [46, 47]. Therefore, the cereals used by us contain a complex natural mixture of polyphenols of different size, polarity and solubility. Third, we can speculate that one of the reasons for the protective effects of cereals could be that the metabolism of polyphenols carried out by the gut microflora [48] is able to provide immune protection beyond the gut. Fourth, it should not be ruled out the possibility that synergistic and/or additive effects occur. Several studies have shown that polyphenols can work synergistically with other antioxidant compounds [42, 49]. Thus, it is of great physiological importance that many polyphenols of different solubility will interact in the body cells and compartments, with antioxidants recharging adjacent antioxidants in an integrated manner [50]. Finally, we consider the present nutritional intervention in animals as preventive, but not therapeutic, since it is able to maintain the normal functional and redox status of the body cells from a general population of mice. Therefore, we do not deliver a cereal-based antioxidant supplement to unhealthy subjects, i.e., in conditions caused by excessive free radicals generation. In conclusion, we strongly believe that polyphenols naturally present in cereals may have important implications for health preservation, by acting, al least partly, as modulators of immune function and redox state. Although we emphasize the importance of further studies on this subject, we suggest the use of this type of supplementation for the general population since “prevention is a much more effective approach that treatment of disease”.

polyphenols
health promotion
peritoneal leukocytes
leukocyte functions
leukocyte redox state