Edited by: Willem Van Eden, Utrecht University, Netherlands
Reviewed by: Angela Ianaro, University of Naples Federico II, Italy; Maritza Romero, Augusta University, United States
†These authors have contributed equally to this work.
Specialty section: This article was submitted to Nutritional Immunology, a section of the journal Frontiers in Immunology
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Dietary proteins can influence the maturation of the immune system, particularly the gut-associated lymphoid tissue, when consumed from weaning to adulthood. Moreover, replacement of dietary proteins by amino acids at weaning has been shown to impair the generation of regulatory T cells in the gut as well as immune activities such as protective response to infection, induction of oral and nasal tolerance as well as allergic responses. Polymeric and elemental diets are used in the clinical practice, but the specific role of intact proteins and free amino acids during the intestinal inflammation are not known. It is plausible that these two dietary nitrogen sources would yield distinct immunological outcomes since proteins are recognized by the immune system as antigens and amino acids do not bind to antigen-recognition receptors but instead to intracellular receptors such as mammalian target of rapamycin (mTOR). In this study, our aim was to evaluate the effects of consumption of an amino acid-containing diet (AA diet) versus a control protein-containing diet in adult mice at steady state and during colitis development. We showed that consumption of a AA diet by adult mature mice lead to various immunological changes including decrease in the production of serum IgG as well as increase in the levels of IL-6, IL-17A, TGF-β, and IL-10 in the small and large intestines. It also led to changes in the intestinal morphology, to increase in intestinal permeability, in the number of total and activated CD4+ T cells in the small intestine as well as in the frequency of proliferating cells in the colon. Moreover, consumption of AA diet during and prior to development of dextran sodium sulfate-induced colitis exacerbated gut inflammation. Administration of rapamycin during AA diet consumption prevented colitis exacerbation suggesting that mTOR activation was involved in the effects triggered by the AA diet. Therefore, our study suggests that different outcomes can result from the use of diets containing either intact proteins or free amino acids such as elemental, semielemental, and polymeric diets during intestinal inflammation. These results may contribute to the design of nutritional therapeutic intervention for inflammatory bowel diseases.
Nutrients are important modulators of the immune system and can act in the differentiation of specific subsets of immune cells and in the maintenance of immunological homeostasis (
The specific effect of dietary proteins on the development of immune system has been addressed using either protein-free diets (
To understand the immune effects of either intact proteins or free amino acids as nitrogen sources is of great importance in clinical practice, especially in cases of intestinal inflammation. It has been reported that nutritional intervention plays a beneficial role in the treatment of inflammatory bowel diseases (IBDs). Polymeric diets, which contain intact proteins, usually yield better results than elemental formulas with only free amino acid (
Several types of diet interventions have been demonstrated to ameliorate inflammation in IBD patients. Suskind et al. showed that a specific carbohydrate diet (SCD) therapy during the course of Crohn’s is associated with improvement in clinical and laboratory parameters as well as with concomitant changes in the fecal microbiome (
Thus, the aim of this study is to clarify the effects of consumption of diets containing free amino acids during adulthood in gut homeostasis at steady state and under inflammatory conditions. Our results may contribute to understand the direct and side effects of elemental, semielemental, and polymeric diets used for the treatment of intestinal conditions such IBDs.
Male C57BL/6 mice used in the experiments were obtained from the Animal Breeding Facility (CEBIO) of Universidade Federal de Minas Gerais (UFMG). Mice were maintained under environmentally controlled conditions (using Alesco® racks with individually ventilated cages) with a 12-h light–dark cycle at the experimental animal facility of Laboratório de Imunobiologia, Instituto de Ciências Biológicas, UFMG, Belo Horizonte, Brazil. Different experimental groups were housed in separate cages. At the end of the experiments, mice were sacrificed by cervical dislocation. All procedures were approved by the Ethics Committee for Animal Use in Research of UFMG (Protocol no. 114/2010, CEUA-UFMG, Brazil).
Experimental diets were prepared using the AIN 93M standard formulation (
Composition of the control diet (CAS diet) and the experimental diet containing free amino acids (AA diet).
Components (g/100 g) | Control diet (casein) | Experimental diet (AA) |
---|---|---|
Casein | 17.9 | – |
Free amino acid | – | 17.9 |
Corn starch | 39.75 | 39.75 |
Dextrinized corn starch | 13.2 | 13.2 |
Sucrose | 10.0 | 10.0 |
Soybean oil | 7.0 | 7.0 |
Cellulose | 5.0 | 5.0 |
Mineral mix | 3.5 | 3.5 |
Vitamin mix | 1.0 | 1.0 |
Supplementation of |
0.3 | 0.3 |
Choline bitartrate | 0.25 | 0.25 |
Tert-butylhydroquinone | 0.0014 | 0.0014 |
Amino acid composition of AA diet (modified AIN 93G).
Amino acid composition (g/100 g) | |||
---|---|---|---|
Alanine | 0.46 | Lysine | 1.3 |
Arginine | 0.64 | Methionine | 0.46 |
Aspartic acid | 1.22 | Phenylalanine | 0.88 |
Cysteine | 0.37 | Proline | 2.05 |
Glutamic acid | 3.63 | Hydroxiproline | <0.1 |
Glycine | 0.32 | Serine | 0.97 |
Histidine | 0.46 | Threonine | 0.67 |
Isoleucine | 0.85 | Tryptophan | 0.21 |
Leucine | 1.54 | Tyrosine | 0.93 |
Valine | 1.0 |
C57BL/6 mice at 7–8 weeks of age were fed either experimental diet or control diet for 5 weeks (Figure
Effects of a diet containing free amino acids (AA diet) on immunoglobulin production.
In the first protocol, C57BL/6 mice at 7–8 weeks were introduced to either experimental diet or control diet during colitis development. Colitis was induced by three cycles of 1% (w/v) dextran sodium sulfate (DSS; 40 kDa) administration in drinking water for 7 days, alternating with 7-day periods of recovery. Diet consumption and the first DSS administration started at the same time. Control group received only water. Liquid consumption was monitored and all mice groups consumed similar volumes of DSS solution daily. After the last DSS cycle, animals were euthanized.
The second protocol was designed to evaluate the lasting effects of these diets when consumed previously to colitis induction. Mice received either amino acid or casein diet for 7 days. Experimental diets were then replaced by commercial chow and colitis was induced by 1% DSS consumption for 7 days.
To evaluate the role of mammalian target of rapamycin (mTOR) activation by AA diet in colitis, mice received daily 200 μl rapamycin (at 10 mg/ml per mice) intraperitoneally for 7 days during diet consumption and prior to colitis induction.
Total serum proteins, albumin, transferrin (Labtest®, Lagoa Santa, MG, Brazil), glycemia (Katal®, Belo Horizonte, MG, Brazil), serum triglycerides, and cholesterol (Doles®, Goiânia, GO, Brazil) were determined using enzymatic kits.
For measurement of secretory IgA (sIgA), intestinal contents of the small and large intestines were collected by carefully washing the intestinal lumen of each portion of the intestine with 10 and 5 ml of cold PBS, respectively. Collected material was transferred to a test tube, vigorously vortexed, and centrifuged for 30 min at 850
Levels of isotype-specific Ig in intestinal lavage samples and serum were determined by ELISA. Briefly, 96-well plates (Nunc®, Sigma-Aldrich, St. Louis, MO, USA) were coated with 0.1 mg/ml goat antimouse Ig in coating buffer, pH 9.8. Wells were blocked with 200 µl PBS contain 0.25% casein for 1 h at room temperature. After washing the plates six times, serial dilutions of samples were added to wells and incubated for 1 h at 37°C. Plates were washed six times again and 100 µl biotinylated goat antimouse heavy chain-specific polyclonal antibodies were added, and then incubated for 1 h at 37°C. After six washes, a detection solution containing a 1/10,000 dilution of horseradish peroxidase-conjugated streptavidin was added and incubated for 45 min. After washing, color reaction was developed at room temperature using 100 μl/well orthophenylenediamine (1 mg/ml), 0.04% H2O2 substrate in sodium citrate buffer. Reaction was interrupted by the addition of 20 μl/well 2 N H2SO4. Absorbance was measured at 492 nm by an ELISA reader (BIO-RAD, Philadelphia, PA, USA) and Ig concentrations were determined using a standard curve.
Colon and small intestine samples were weighed and homogenized in PBS containing 0.05% Tween-20, 0.1 mM phenylmethylsulphonyl fluoride, 0.1 mM benzethonium chloride, 10 mM ethylenediaminetetraacetic acid (EDTA), and 20 KIU Aprotinin A using a tissue homogenizer (100 mg tissue/ml buffer). Suspensions were centrifuged at 12.000
Following euthanasia, small intestine and colon tissues were harvested; mesenteric and adipose tissues were removed. Visible Peyer’s patches were removed. Tissues were then cut open longitudinally and drawn through a pair of curved forceps while applying gentle pressure to remove intestinal contents. Tissues were cut into 2–4 cm fragments, then washed twice to remove feces in calcium- and magnesium-free HBSS containing 2% FCS (at 4°C). Tissues were placed in 50-ml tubes and washed three times in HBSS containing 2% FCS at 4°C. Tissues were transferred to 25-cm3 tissue culture flasks and incubated at 37°C in HBSS containing 10% FCS, 0.2 mmol/l EDTA, 1 mmol/l DTT, 100 U/ml penicillin, and 100 µg/ml streptomycin. After 20 min, flasks were shaken vigorously for 30 s, and the supernatant containing the intraepithelial lymphocytes was separated from the tissue fragments using a stainless steel sieve. To isolate
Histological sections of small intestine and colon were obtained from casein-fed and AA-fed mice. Tissues were fixed by 10% PBS-buffered formalin, embedded in paraffin, and 3-mm thick sections were obtained, stained with hematoxylin and eosin and examined under a light microscope. To measure the thickness of crypts, mucous and muscular layer, villus height and goblet cell numbers, the selected image was focused by an optical microscope and captured by a video camera JVC TK-1270/RGB previously coupled to the microscope body. The captured image was digitalized, transferred to a microcomputer and analyzed using the software
Histological examination was performed using a blind score based on a semiquantitative system described previously (
Clinical evaluation was assessed using a previously defined scoring system, which includes loss of body weight, diarrhea, and the presence of blood in the stools. Each score was determined as follows: change in weight (0: <1%; 1: 1–5%; 2: 5–10%; 3: 11–15%; 4: >15%), diarrhea (0: negative; 2: moderate; 4: severe), and stool blood (0: absence; 2: hidden blood; 3: visible blood) as previously described (
Total and phosphorylated mTOR were evaluated by western blotting analysis. Samples of colon were weighed and homogenized in buffer containing 20 mmol/L Tris-HCl, pH 7.4, 120 mmol/L NaCl, 1 mmol/L EDTA, 5 mmol/L ethylene glycol bis (2-aminoethylether) tetra-acetic acid, 50 mmol/L β-glycerophosphate, 50 mmol/L NaF, 0.3% 3-(3-cholamidepropyl) dimethylammonio-1-propanesulphonate, 1 mmol/L dithiothreitol, 4 mg/mL leupeptin, and 4 mg/mL aprotinin using a tissue homogenizer. The remaining homogenate was centrifuged at 12,000
Intestinal permeability was determined by measuring radioactivity diffusion in the blood after oral administration of diethylenetriamine penta-acetic acid (DTPA) labeled with 99 m-technetium (99mTc) as previously described (
Analysis of proliferating cells in the intestinal mucosal was performed according to a previously reported methodology (
Results were expressed as the mean ± standard error of the mean (SEM). All data were analyzed using Kolmogorov–Smirnov normality test to test its Gaussian distribution. Parametric tests (Student’s
Malnutrition affects several aspects of the activity of the immune system (
Consumption of AA diet did not change the levels of serum immunoglobulins, IgM or IgA (data not shown), but levels of serum IgG were decreased in AA-fed mice after 7 days of diet administration (Figure
For most of the evaluated cytokines, the effects of AA diet were observed mainly after the first week of consumption. At this time point, AA-fed group showed lower concentrations of IL-10, IL-4, IFN-γ, IL-17A, IL-6, and TGF-β in the spleen, when compared to the casein group (Figure
Cytokines levels in the small intestine and colon after dietary consumption. C57BL/6 mice at 7–8 weeks of age were fed either experimental diet [diet containing free amino acids (AA diet)] or control diet [casein-containing diet (CAS diet)] for 5 weeks. Levels of IL-6, IL-17A and IL-10 were measured in the small intestine
Dietary replacement of casein by free amino acids led to alterations in the number of cells in the
Number of cells in the
Since consumption of diet containing free amino acids for 7 days led to changes in cytokine production in the gut, we next investigated whether AA diet could also influence the intestinal permeability and morphology. Intestinal permeability was significantly increased in AA-fed group after 7 days of diet consumption, when compared to casein group (Figure
Histological analysis of the small intestine after dietary consumption. C57BL/6 mice at 7–8 weeks of age were fed either experimental diet [diet containing free amino acids (AA diet)] or control diet [casein-containing diet (CAS diet)] for 5 weeks. Intestinal permeability was determined by% dose/g of 99 m-technetium (99mTc)-diethylenetriamine penta-acetic acid (DTPA) in blood
Our results showed that consumption of a diet containing free amino acids was associated with changes in the intestinal mucosa suggesting a local disruption of homeostasis. Our next step was to test whether AA diet would affect intestinal inflammation using a model of colitis induced by the administration of three cycles of DSS (Figure
Effects of dietary consumption during dextran sodium sulfate (DSS)-induced colitis development. C57BL/6 mice at 7–8 weeks of age were introduced at experimental diet [diet containing free amino acids (AA) diet] or control [casein-containing diet (CAS)] diet. All mice were kept on these diets throughout colitis induction. Colitis was induced by three-cycle administration of 1% (w/v) DSS in drinking water for 7 days, alternated with 7-day recovery periods
AA diet promoted an increase in the severity of colitis. AA-fed mice presented greater weight loss (Figure
We examined next whether AA diet administration prior to colitis induction would also affect disease severity (Figure
Histological evaluation of colitis development after dietary consumption.
Amino acid signaling through the highly conserved serine-threonine kinase mTOR has well-known consequences on proliferation of immune cells and inflammation (
To confirm that mTOR activation by amino acid containing diet (AA diet) was involved in exacerbation of colitis, we treated AA-diet-fed mice daily with the mTOR inhibitor rapamycin during diet consumption and previously to colitis induction by DSS (Figure
Effect of rapamycin administration during experimental diet consumption in colitis induction.
The immunological role of dietary proteins has been already addressed by studies using either a standard rodent diet in which intact proteins were replaced by equivalent amounts of amino acids (
In the present study, we showed that consumption of an AA diet resulted in immunological alterations in adult mice at steady state conditions. These alterations were not due to changes in their nutritional status. Consumption of AA diet was nutritionally equivalent to CAS diet concerning parameters such as weight maintenance and levels of total serum proteins, albumin, transferrin, glycemia, and triglycerides. Previous studies in humans and animals also showed nutritional equivalence between diets containing intact proteins and free amino acids (
Consumption of AA diet resulted in several immunological changes including lower levels of serum IgG after 7 days of diet administration. Reduction in serum IgG and IgA levels have also observed when AA diets were consumed after weaning (
The dietary nitrogen source did not affect secretory IgA (SIgA) levels in the small intestine, but the consumption of diet containing free amino acids induced higher levels of this immunoglobulin in the colon after 5 weeks. It is likely that this is a direct result of the stimulatory effect of amino acids in immune cells as evidenced by the increase in cellular infiltration, reduction of goblet cells and presence of edema in the submucosal layer seen in histological examination of the colonic mucosa after 5 weeks of AA diet consumption. Moreover, AA-fed mice had an augmented frequency of proliferating cells in the small intestine 1 week after diet consumption. Higher sIgA levels were also observed in AA-fed mice during intestinal inflammation. Other studies have also shown that gut inflammation in mice (
Consumption of AA diet resulted in an increase in the number of lamina propria cells of the small intestine, but not of the colon. Since amino acids are known to signal through intracellular receptors such as mTOR that control cell proliferation, we examined whether AA-fed mice had increased number of proliferating cells in the small intestinal mucosa using an anti-PCNA monoclonal antibody. AA-fed mice had an augmented number of PCNA+ cells in the small intestine than control CAS-fed mice suggesting that ingestion of free amino acids had an effect in cell proliferation in the gut mucosa. T cell subsets in the small intestine were specifically affected by AA diet. Numbers of total and activated (CD44+) CD4+ T cells as well as Th17 (CD4+ RORγt+) cells were augmented in the small intestine, but not in the colon, of AA-fed mice. It is known that naïve CD4+ T lymphocytes can differentiate into Th17, Th1, Th2, or Treg cells depending on the cytokines present during their activation. Differentiation into Th17 cells requires the expression of the transcription factor RORγt which is upregulated in the presence of TGF-β and IL-6 (
It is remarkable that AA diet consumption by adult mice led to histopathological changes mostly in their colonic mucosa suggesting that prolonged consumption of diets containing free amino acids can be especially harmful for the final portions of the intestine. Although the small intestine was also affected in these mice, our hypothesis is that intrinsic features of the colonic mucosa makes it more susceptible to inflammatory changes brought about by amino acid ingestion. Since the concentration of autochthonous bacteria in the colon exceeds by large the one in the small intestine, this portion is physiologically primed and more prone to undergo pathological alterations once stimulated by a proinflammatory stimuli.
To further explore the effects of amino acid ingestion in the colon, we examined the impact of AA diet during DSS-induced colitis. Mice were fed AA diet concomitantly to the induction of colitis. AA diet consumption significantly boosted colitis development when three cycles of DSS were administered. AA-fed mice had higher clinical scores and weight loss throughout disease development including at the remission time points that followed each cycle of DSS. The histological index determined after the third cycle was also higher in AA-fed mice when compared to CAS-fed mice. Histological evaluation showed a more intense inflammatory infiltrate in the mucosa and submucosa, loss of mucosal architecture and thickening of the muscle layer. Furthermore, AA diet consumption prior to colitis induction also led to a more severe inflammation of the colonic mucosa. This suggests that the effects induced by dietary free amino acids at steady state exacerbated the development of an experimental IBD and that they would last even after discontinuation of diet consumption.
Elemental diets, which have in its composition only free amino acids, are used in the nutritional treatment of patients with IBDs in case of polymeric diet intolerance. Some studies have shown that elemental diets yielded more beneficial results in active Crohn’s disease (CD) (
There are few studies evaluating the effects of elemental diets in experimental models of colitis. In the colitis model induced by transferring cells from IL-10-deficient C57BL/6 mice to CB-17 SCID mice, elemental diet consumption prevented weight loss and suppressed intestinal inflammation, whereas mice fed standard diet showed a severe form of colitis (
One major factor to be considered when using diets containing a high concentration of free amino acids would be the impact of its increased osmolarity on intestinal absorption and permeability. Increased gut permeability is known to play a key role in triggering bacterial translocation and inflammation during IBDs (
Another mechanism by which AA fed diet could contribute to gut inflammation would be a direct effect of amino acids in the immune cells located in the gut mucosa. The increase in the number of total cells, CD4+ T lymphocytes, CD4+ CD44+ activated T cells and Th17 cells observed in the
To address the putative role mTOR activation in the inflammatory effects triggered by the AA diet, we used rapamycin during AA diet consumption prior to colitis induction. Several studies have demonstrated that blockade of mTOR activation by inhibitors such as rapamycin and everolimus reduces experimentally induced colitis. Rapamycin forms a gain-of-function complex with the intracellular FK506-binding protein protein, and this complex directly interacts with and inhibits mTOR (
In conclusion, our study demonstrates that the consumption of a diet containing free amino acids caused several immune alterations in the intestinal mucosa including increase in SIgA and cytokine secretion in the colon, augmented intestinal permeability and accumulation of proliferating cells and activated CD4+ T lymphocytes in the small intestine. Although these alterations did not lead to spontaneous development of colitis, free amino acids in the diet caused exacerbation of intestinal inflammation when consumed either during or prior colitis development. Blockage of mTOR activation by treatment with rapamycin during AA diet consumption prevented colitis exacerbation suggesting this cellular sensor complex was involved in the effect mediate by the AA diet. These results provide further insight into the immunological effects of elemental versus polymeric diet formulations used as a complementary therapeutic tool for IBDs.
AS and SFA performed the experiments, analyzed the data, and wrote the article; MGM, LL, and MFG performed the experiments and helped with the analysis of data; DR helped with the experiments on flow cytometry; PVB performed the gut permeability analysis; EV and EF performed the cell proliferation analysis; TC and FR performed the blots for mTOR analysis; DC performed the histological analysis and helped discussing the data; AG-S helped designing the experiments and analyzing the data; ACF designed the experiments, supervised the study, analyzed the data, and wrote the manuscript.
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
We would like to thank Ilda Marçal de Souza for the excellent care of the animals. This work was supported by grants from FAPEMIG (APQ 00704-14, RED-00140-16) and by the Pró-reitoria de Pesquisa of Universidade Federal de Minas Gerais (PRPq-UFMG). Some of the authors were recipients of scholarships (AS, MGM, and LL) and research fellowships (DC and ACF) from Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq, Brazil). SFA is a recipient of a PhD scholarship from Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES, Brazil).
The Supplementary Material for this article can be found online at
Body weight, food consumption and biochemical tests. C57BL/6 mice at 720138 weeks of age were fed either experimental diet (AA diet) or control diet (CAS diet) for 5 weeks. Body weight
Cytokine levels in spleen. C57BL/6 mice at 7–8 weeks of age were fed either experimental diet (AA diet) or control diet (CAS diet) for 5 weeks. Levels of IL-10, IL-4, IFN-γ, IL-17A, TGF-β, IL-6, and IL-12 were measured in the spleen by ELISA after 7 days of dietary consumption,