Introduction

The epithelium of the small intestine has the paradoxical task of facilitating digestive and absorbent processes on the one hand while establishing an immunologically efficient barrier against commensal and pathogenic microorganisms on the other. Intestinal epithelial cells provide the first physical barrier to microbial invasion through mechanisms of both the innate and adaptive immune response. Among the mechanisms of the innate immune response are the secretion of mucous, the production of antimicrobial molecules, and the production of certain cytokines, and among those of the adaptive immune response is the production of immunoglobulin A (IgA) [24, 35].

Paneth cells, a key type of intestinal epithelial cell in the innate immune response, produce peptides and proteins with strong local antimicrobial activity, the most abundant being defensins, lysozyme, and phospholipase A2. The secretion of these molecules into the intestinal lumen is continuous and can increase in the presence of bacteria and their antigens or as a result of the interaction of cholinergic agonists with their receptors [1, 22, 52].

Plasma IgA+ cells, important in the adaptive immune response, are found throughout the lamina propria underlying mucosal surfaces of the intestinal epithelium. These cells produce IgA with the incorporated joining (J) chain to produce polymeric IgA (pIgA), which is transported across the epithelial barrier by pIgR. Once this complex reaches the apical membrane of epithelial cells, proteases cleave the extracellular region of pIgR, inducing the release of S-IgA into the lumen. By binding to antigens, S-IgA protects intestinal mucosal surfaces against colonization and invasion by pathogens [18, 30, 41, 46].

Communication between the intestinal epithelium and immune cells is mediated by cytokines. Cytokines produced by immune cells maintain the normal homeostasis of the epithelium and regulate the expression of pIgR and antimicrobial peptides. On the other hand, cytokines produced by epithelial cells modified the expression of antibodies by plasmatic cells. Thus, the balance of cytokines determines a healthy or pathogenic inflammatory response, as well as an effective or ineffective barrier to microbial invasion [8, 42].

It is well known that at a certain level (without malnutrition) caloric restriction (CR) improves immune responses and prevents several diseases, such as cancer. Among the several types of CR used in animal models, the every-other-day diet, which alternates ad libitum feeding and fasting, was used in the present study. Regardless of the model used, several studies have demonstrated that CR diminishes pro-inflammatory cytokines, free radical production, blood glucose levels, and insulin resistance. Regarding cancer, CR decreases cellular proliferation and neuronal damage. Moreover, this dietary regime is known to slow or prevent illnesses associated with aging [14, 40, 49, 50].

Studies examining the effect of CR on the immune function have focused on the production of T cells, antibodies, and cytokines [11, 12, 15, 16, 33] in the spleen, lymphatic nodes, peripheral blood, thymus, and salivary glands. Recently, we reported that CR decreased duodenal IgA levels in mice. However, there are still no reports on the effects of CR in the ileum or on the activity of antimicrobial peptides in either compartment.

The aim of the present study was to assess the effects of CR on IgA levels, gene expression encoding for cytokines, α chain of IgA and pIgR in the ileum, and on lysozyme and phospholipase A2 production in both the duodenum and ileum.

Materials and methods

Animals, experimental groups, and CR protocol

Three-week-old male Balb/c mice were obtained from Harlan Sprague Dawley Inc. and were randomized in two groups, ad libitum (AL) and CR, each one of n = 6. All of them were individually housed in plastic cages, on a 12 h (12:12) light:dark cycle (lights on at 8 a.m.) and with the NIH-31 diet ad libitum for 5 weeks to let them adapt to our animal breeding unit. Animal handling was in accordance with Mexican federal regulations for animal experimentation and care (NOM-062-ZOO-1999, Ministry of Agriculture, Mexico City, Mexico) and approved by the Institutional Animal Care and Use Committee.

The CR protocol, which began during the ninth week of life, involved alternating ad libitum feeding and fasting (every other day) for 4 months. Food was always removed and replaced at 1 p.m., and food intake was measured daily. Body weight was determined every week on the same day and time.

Collection of biological samples

Food was removed at 7 a.m. on week 27, and at 1 p.m., mice from all groups were anaesthetized with diethyl-ether and exsanguinated by cardiac puncture. Plasma was collected and stored at −80°C for stress hormone quantification by ELISA. A section of distal small intestine was removed from each animal, and 5 ml of PBS containing 0.02% sodium azide was passed through it in order to collect the intestinal fluid. The washout material was centrifuged at 10,000×g for 20 min at 4°C. After the supernatant was harvested, it was stored at −70°C to await analysis. Total IgA was measured by enzyme-linked immunosorbent assay (ELISA). Small segments from duodenum and ileum were homogenized with 1 mL of RIPA lysis buffer plus a protease inhibitor cocktail (Roche Diagnostics GmbH, USA, 11836145001) and then centrifuged at 10,000 rpm for 10 min at 4°C. The supernatant was stored in 50 μL aliquots at −80°C until protein analysis. The Lowry method was used for quantification of proteins in one of the 50-μL aliquots [28].

Enzyme-linked immunosorbent assay for IgA

We determined IgA antibody concentration in fluids from the ileum using enzyme-linked immunosorbent assay (ELISA), according to a previously reported method [13].

Detection of alpha chain, J-chain, pIgR, lysozyme, and A2-phospolipase proteins by chemiluminescent Western blotting

Soluble proteins in aliquots were denatured by immersing Eppendorf tubes in boiling water for 10 min in the presence of sodium dodecylsulfate and β-mercaptoethanol. Fifty micrograms of total protein, in a denaturing 10% to 15% polyacrylamide gel, were separated by electrophoresis at 88 V for 2 h, using the Laemli method [25].

The proteins were transferred to a PVDF membrane at 15 V during 45 min for lysozyme, A2-phospolipase and J-chain, and 18 V and 20 V for IgA and pIgR, respectively, during 1 h in the Trans-Blot SD Semi-Dry Transfer Cell (Bio-Rad). Membranes were blocked with 5% (w/v) nonfat dry milk in Tris buffer (pH 7.4), containing 0.1% (v/v) Tween 20, for 2 h at RT with constant agitation. Then, membranes were incubated overnight at 4°C, under constant agitation, with one of the following antibodies: (a) goat polyclonal antibody raised against secretory phospholipase A2 of rat origin (Santa Cruz: sc-14472) at a dilution 1:800, (b) goat polyclonal antibody raised against lysozyme C of human origin (Santa Cruz: sc-27958) at a dilution 1:800, (c) goat polyclonal antibody raised against J-chain at a dilution 1:400 (Santa Cruz: sc-34654), (d) goat anti-mouse IgA HRP (Invitrogen: 62-6720) at a dilution 1:10,000, or (e) rabbit polyclonal serum produced against a peptide of secretory component (H-GNSVSITYYPPTSVNRHTRKYWCOH) at a dilution of 1:200.

Membranes were washed and incubated with rabbit anti-goat IgG HRP or goat anti-rabbit-IgG HRP (Invitrogen, Carlsbad, Cal, USA, G21234) at a dilution of 1:10,000 for 2 h at RT under constant agitation. After washing, membranes were incubated for 1 min with Western Blotting Luminol Reagent (Santa Cruz), and bands were detected using photographic film (Amersham Hyperfilm ECL) with an exposure of 30 s for IgA and J-chain, 2 min for pIgR, 4 min for phospholipase, and 3 min for lysozyme. Membranes were stripped and reblotted with an anti-β-actin antibody at a dilution 1:400. Densitometric measurements of protein bands were analyzed and quantified with the Quantity One 1-D Analysis Software.

Real time polymerase chain reaction assays

A small segment of duodenum and ileum were cut and stored at −70°C to await analysis. RNA extraction, DNA synthesis, and real time polymerase chain reaction assays were all performed according to a previously reported method [38]. Specific oligonucleotide primers were originally generated by using the online assay design software (ProbeFinder: http://www.universal-probelibrary.com) and the primer sequence for each gene that is shown in Table 1. mRNA levels were calculated by using the comparative parameter threshold cycle (Ct) method and normalized to ribosomal RNA.

Table 1 Primer sets used for real-time PCR (designs based on ensemble transcript ID of the Mouse Probe Library)
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Corticosterone and epinephrine assays

Serum corticosterone and norepinephrine levels from individual mice were determined using commercially available corticosterone EIA kits (Assay designs, Ann Arbor, MI, USA, cat. # 901–097), and epinephrine (Alpco Immunoassays, Salem NH, USA, cat.# 17-Epihu-E01). The corticosterone and epinephrine concentration in the serum samples was calculated from a standard curve and expressed in nanograms per milliliter.

Statistical analysis

Data are presented as the mean ± SD. The comparison of two groups was analyzed by using the Student’s unpaired two-tailed t test. All analyses were performed using the statistical program Sigma Stat for Windows Version 2.03 software (SPSS Inc.).

Results

CR affects expression of molecules of the innate intestinal immunity

Whereas CR caused a significant threefold increase in lysozyme mRNA levels in duodenum (*P < 0.001), there was no effect in ileum (P > 0.05) (Fig. 1a). Phospholipase A2 mRNA levels were significantly greater in the CR than control group, in both duodenum (twofold) and ileum (threefold) (P < 0.05) (Fig. 1b).

Fig. 1
figure 1

Effect of caloric restriction on the mRNA expression of lysozyme (a) and phospholipase A2 (b). Mice were sacrificed and samples of intestinal gut tissue were obtained. The mRNA expression of lysozyme and phospholipase was measured by real time RT-PCR for caloric restricted (CR) and ad libitum fed (AL) mice, as mentioned in “Materials and methods”. mRNA levels were calculated by using the comparative parameter threshold cycle (Ct) method and normalized to ribosomal RNA. CR induced a significant threefold increase in lysozyme mRNA levels in duodenum (*P < 0.001), but there was no effect in ileum (P < 0.05) (a). Phospholipase A2 mRNA levels were significantly greater in the CR than control group, in both duodenum and ileum (P < 0.05) (b)

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Determination of a possible CR effect on the protein expression for lysozyme and phospholipase A2 was done with chemiluminescent Western blotting, finding that CR: (1) significantly increased the synthesis of lysozyme in intestinal duodenum (P = 0.012), and (2) had no effect on the synthesis of lysozyme in ileum (P > 0.05) (Fig. 2a) or on the synthesis of phospholipase A2 in either the intestinal duodenum or ileum (P < 0.05) (Fig. 2b). These results show that lysozyme gene expression is correlated with protein expression in duodenum. In contrast, there was no relationship between lysozyme gene and protein expression in ileum, nor did the expression of the phospholipase A2 gene correlate with that of proteins in the whole intestine (P < 0.05).

Fig. 2
figure 2

Effects of CR on protein synthesis of lysozyme (a) and phospholipase A2 (b). Intestinal mucosa of mice was obtained and processed with RIPA buffer and protease inhibitors. 50 μg per well samples were analyzed through Western blot by adding antibodies against lysozyme or phospholipase A2. Synthesis of lysozyme in duodenum of CR mice was significantly increased (P = 0.012), although there was no observed effect in ileum (P > 0.05) (a). CR did not induce any changes in the synthesis of phospholipase A2 in either the proximal or distal intestine (P < 0.05) (b)

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CR modifies levels of ileum IgA and the expression of alpha-chain, J-chain, and pIgR

Because the expression of genes for alpha chain, J-chain, and pIgR in the intestinal mucosa is important for IgA secretion to the intestinal lumen, we determined this gene expression in the ileum by RT-PCR. Real-time RT-PCR demonstrated that mRNA levels of alpha chain and J-chain were significantly greater in CR than AL mice in the intestinal ileum (P < 0.001, Fig. 3). The levels of pIgR mRNA also underwent a greater increase in ileum of CR than AL mice (P = 0.002, Fig. 3). These results demonstrate that CR modified the gene expression for IgA and pIgR in ileum.

Fig. 3
figure 3

Real-time RT-PCR analysis of α-chain, J-chain, and polymeric Ig receptor (pIgR) mRNA in intestinal samples of CR mice. Samples of the ileum were collected from CR mice. The expression of mRNA for α-chain, J-chain, and for the polymeric Ig receptor (pIgR) was measured by real-time RT-PCR. Data represent the mean ± SD (n = 6). In ileum, CR significantly increased alpha chain and J-chain mRNA levels (P < 0.001) and pIgR mRNA levels (P = 0.002)

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Surprisingly, Western blot analysis showed low levels of alpha chain and J-chain, in contrast with high mRNA levels (P < 0.001, Fig. 4). Therefore, the decrease in the intestinal mucosal IgA is not related to the normal behavior of the alpha chain and J-chain gene expression. Similarly, low levels of pIgR were found in ileum, in contrast with high mRNA levels (Fig. 4).

Fig. 4
figure 4

Western blot analysis of α-chain, J-chain, and pIgR. Equal amounts (50 μg) of total protein from each sample were blotted with specific antibodies, and then the membrane was stripped and reblotted with an anti-β-actin antibody. Densitometry of Western blot showed that in ileum of CR mice, α-chain, J-chain, and pIgR were significantly decreased (two to threefold)

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CR affects levels of intestinal S-IgA

We recently reported [26] that the intestinal IgA concentration was slightly but significantly lower in duodenum of CR than AL mice. In the present work, it was observed that CR reduced IgA levels in ileum (P < 0.001). Therefore, CR affects the secretion of IgA in the entire small intestine (Fig. 5).

Fig. 5
figure 5

Effect of CR on intestinal S-IgA. After a 6-h fasting period, the mice were sacrificed and the intestinal fluid from ileum was obtained. The IgA concentration was determined by ELISA and is expressed as microgram per milliliter. Data were obtained from six mice/group and are presented as the mean ± SD. In ileum, the concentration of intestinal IgA was lower in the CR than AL group (P < 0.001, Bonferroni t test)

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CR alters the transcription of genes for several cytokines

Since the expression of alpha-chain and pIgR mRNA is upregulated by cytokines that mediate innate and adaptive immunity [17, 18, 27, 32, 46], we quantified the expression of genes for TNF-α, TGF-β, IFN-γ, IL2, IL-4, IL-6, IL-10, and IL-12 by using real time PCR in ileum.

In terms of innate immunity, it was found that CR: (1) augmented TNF-α gene expression threefold (P < 0.001, Fig. 6a), (2) significantly increased the gene expression for IL-12 (threefold; P < 0.025, Fig. 6a) and IL-10 (fivefold; P = 0.007, Fig. 6a), and (3) had no effect on IL-6 gene expression (P = 0.02, Fig. 6a).

Fig. 6
figure 6

Real-time RT-PCR analyses of cytokines in intestinal samples of CR mice. Samples of ileum from mice were processed as mentioned in “Materials and methods”. CR increased the mRNA levels for TNF-α (threefold, P < 0.001), IL-12 (threefold; P < 0.025) and IL-10 (fivefold; P = 0.007). mRNA levels for IL-6 remained unaffected by CR (P = 0.02) (a). CR increased mRNA levels of IFN-γ, TGF-β, and IL-2 in ileum, while such levels of IL-4 remain unchanged (b)

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In terms of adaptive immunity, it was found that CR: (1) significantly increased the gene expression for IL-2 (2.5-fold) (P < 0.001), (2) induced a fourfold increase in the gene expression for IFN-γ and threefold increase in TF-β gene expression P < 0.001) (Fig. 6b), and (3) did not affect IL-4 gene expression in ileum (Fig. 6b).

Corticosterone and epinephrine levels were not modified by CR

To determine if our CR protocol induced stress in mice, we measured the serum concentration of corticosterone and epinephrine by ELISA. Neither stress marker was changed in CR compared to control mice (Table 2).

Table 2 Effect of caloric restriction on serum corticosterone and epinephrine levels
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CR does not modify the body weight of mice

By the end of this study, mice subjected to CR by intermittent fasting were consuming essentially the same amount of food as did those fed AL. On the days they had access to food, the CR mice ate roughly twice as much as did mice fed AL (Fig. 7).

Fig. 7
figure 7

Comparison of body weight between CR and AL mice. Male Balb/c mice of CR group compensate for periods of fasting by increasing their food intake. Thus, they gain weight at rates similar to mice fed AL. Body weight was determined weekly. The fasting day started at the second week and DR continued until 20th week, at which time body weights of the AL and CR groups were not significantly different (P > 0.05)

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Discussion

Although the effects of nutritional manipulation on the immune function have been extensively studied in animals, there are few reports about such effects on innate and adaptive mucosal immunity. In the current contribution, the effects of CR on both the innate and adaptive immune response in the small intestine were measured.

The greater expression of the genes for lysozyme and phospholipase A2 that were found in the duodenum of CR compared to ad libitum mice could indicate a higher production of the corresponding proteins. The fact that the phospholipase A2 protein was not found at higher concentrations in the intestinal mucosa might be due to its diffusion to the intestinal lumen [20]. On the other hand, the lysozyme protein is more cationic than phospholipase A2, meaning that it could have a greater tendency to bind to mucous, which would impede its diffusion [37]. However, we cannot discard the possibility of post-transcriptional regulation. In any case, the increased production of lysozyme and perhaps of phospholipase A2 resulting from CR in the present study would tend to increase resistance to microbial invasion.

Moreover, the current results show that CR increased the expression of the genes for alpha-chain, J-chain of IgA, and its receptor (pIgR) but decreased the expression of the corresponding proteins and S-IgA levels in ileum. Recently, we reported [26] that CR reduces IgA levels in duodenum, apparently as a consequence of reduced numbers of IgA+ cells in the LP, and a reduced synthesis of pIgA. In this case, transport was not a factor, since CR increased pIgR synthesis in this intestinal section. The fact that CR decreased the expression of pIgR in ileum means that transport could be a related factor for the decreased S-IgA levels found.

According to a recent study [47] using long-term CR (4 months), lower levels of free radicals were found in uninfected CR mice than uninfected control animals (with an ad libitum diet). However, after Salmonella typhimurium infection, higher levels of free radicals were observed in mice from the CR than the ad libitum-fed group. It is possible that upon studying IgA expression after infection, the same effect will be found. If so, the higher gene expression would indicate a potential for production of IgA that is not activated in the absence of infection. It would be interesting in a future study to test this parameter of the immune response in the intestine after infection and with a CR regimen.

There have been studies about the effects of CR on cytokine production in serum, spleen, adipose tissue, and salivary glands, usually reporting a decrease in pro-inflammatory cytokine production. However, to the best of our knowledge, there have been no reports on the expression of genes encoding for cytokines in the intestinal mucosa. We are reporting the raw data, which do not lend themselves to conclusions about mechanisms. Owing to the lack of any other comparable data in the literature about the effects of CR on gene expression in the intestinal mucosa, we mention data about the effects of CR on gene expression for cytokines in plasma while recognizing that the production of cytokines in plasma and the mucosa are not really comparable. In plasma, there is a greater production of IL-10 and TGF-β by regulatory T lymphocytes as well as of Th2 needed for oral tolerance induction [34, 51, 54].

The gene expression in ileum increased for almost all cytokines, both pro-inflammatory (TNF-α, IL-12, IFN-γ, and IL-2) and anti-inflammatory (IL-10 and TGF-β). The exceptions were the unchanged expression of IL-6 and IL-4. Recently, we reported more varied results of the effect of CR in duodenum [26], finding: (1) an increase in the expression of the genes for TNFα, IL-6, and TGF-β, and (2) no change in the expression of the genes for IL-10, IL-4, and IFN-γ. This difference could be related to the kind of antigens present in either compartment—that is, food antigens in duodenum vs. microbial antigens in ileum.

Besides their pro- or anti-inflammatory role, TGF-β, IL-6, and IL-10 induce IgA B-cell differentiation [3, 4], while IFN-γ, TNF-α, TGF-β, and IL-4 upregulate pIgR expression [24, 32]. In the current contribution, CR did not modify the expression of the gene for IL-4 in ileum. In duodenum and plasma, a lack of modification has also been reported for IL-4 [48]. However, whereas our results show that CR did not modify IL-6 in ileum, previous studies in mice and rats have shown that CR has diverse effects on the production of IL-6 in plasma [19] in activated macrophages [7], adipose tissue [53], and duodenum [26]. This suggests that the effect of CR on the production of cytokines may be tissue specific.

We found that CR increased mRNA levels for IL-12, IFN-γ, and TNF-α (pro-inflammatory cytokines) in ileum. There are previous reports that CR decreases the gene expression for these cytokines in plasma [9, 44]. In another study on the effect of CR in duodenum, levels of IFN-γ and IL-12 remain unchanged [26]. Our results also show that CR increased mRNA levels for anti-inflammatory cytokines (IL-10 and TGF-β). This same tendency has been reported in plasma [9, 48] and duodenum [26].

In the present study, the production of IL-2 was higher in ileum of CR than AL mice, contrary to the reported effect in duodenum [26]. Several studies have shown that CR enhances IL-2 gene expression in spleen cells of mice and rats stimulated by concanavalin A [2, 11, 23, 29, 36]. It has been demonstrated that IL-2 is essential for expansion of Treg cells that attenuate chronic colitis in a mouse model [21, 31].

Whereas TNF-α, INFγ, IL-6, and IL-12 contribute to inflammation, TGF-β and IL-10 are regulatory cytokines with a pivotal role in limiting the inflammatory immune response [27]. Thus, the balance between TNF-α on the one hand, and the combined effect of TGF-β and IL-10 on the other hand, must greatly influence the resulting pro- or anti-inflammatory effect. The current assumption is that dietary restriction without malnutrition exerts a potent anti-inflammatory effect [5, 6, 10], which if true would imply in part a greater effect of TGF-β and IL-10 than TNF-α.

Since CR is considered a biological stressor [45], and the effect of stress hormones (catecholamines and glucocorticoids) on IgA alpha-chain mRNA expression and IgA secretion was recently reported [13, 39, 43], in the present study, corticosterone and epinephrine were measured by ELISA in plasma of mice. However, no significant differences between CR and AL groups were found in this respect (Table 2). A possible explanation is that mice adapted rapidly to the intermittent fasting regimen, did not lose weight, and maintained a steady state (Fig. 7). Hence, it is likely that such a regimen was no longer a stressor.

In conclusion, CR modified the expression of molecules that regulate both innate and adaptive intestinal immunity. We observed increased gene expression of lysozyme and phospholiphase A2 in intestinal mucosa of CR mice. It is possible that the production of both corresponding proteins was increased, and that the phospholipase A2 protein diffused to the intestinal lumen. The CR-induced increase in the production of lysozyme, and perhaps that of phospholipase A2 as well, would tend to increase resistance to microbial invasion. It would be interesting to quantify protein levels in intestinal fluid.

Whereas CR increased the expression of genes for α-chain, J-chain, and the pIgR, the corresponding proteins were diminished, and the S-IgA levels in intestinal fluid were also reduced. It is possible that plasmatic cells reduced the production of pIgA, and epithelial cells reduced its transport because of a reduction in pIgR.

CR modified the expression of the genes for IL-2, IL-10, IL-12, TNF-α, and TGF-β, all of which regulate the synthesis of IgA and pIgR, the inflammatory response, and the immune response in the intestine. Whereas TNF-α, INFγ, and IL-12 contribute to inflammation, TGF-β and IL-10 are regulatory cytokines with a pivotal role in limiting the inflammatory immune response [27]. The balance between TNF-α (pro-inflammatory) on the one hand, and TGF-β and IL-10 (anti-inflammatory) on the other hand, must greatly influence the resulting effect. Further research is needed to determine the influence of the complex balance of cytokines in the changes produced by CR on IgA levels and on the overall immune response. Also, future studies are necessary to determine whether the daily CR model has the same effects on the intestinal immune system as the alternate day CR model. As yet, there are no reports that compare the results of both interventions in the same animal model.