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Nutritional Immunology

Combined Glutamine and Arginine Decrease Proinflammatory Cytokine Production by Biopsies from Crohn's Patients in Association with Changes in Nuclear Factor-B and p38 Mitogen-Activated Protein Kinase Pathways^1–3,

⁴ Appareil Digestif Environnement Nutrition EA4311, Institute for Biomedical Research, IFRMP23, Rouen University and Rouen University Hospital, Rouen, France and ⁵ Department of Gastroenterology and Nutrition, Rouen University Hospital, Rouen, France

^* To whom correspondence should be addressed. E-mail: pierre.dechelotte{at}chu-rouen.fr.

	ABSTRACT

TOP ABSTRACT Introduction Subjects and Methods Results Discussion LITERATURE CITED

 
Glutamine (Gln) and arginine (Arg) are conditionally essential amino acids with immunomodulatory properties. The aim of the study was to assess the effects of Gln and Arg alone or in combination on cytokine release by cultured colonic biopsies from patients with active Crohn's disease (CD). Ten consecutive patients [mean (range) age 26 (18–39) y] with active colonic CD (mean CD activity index: 383.7 ± 129.8) were prospectively included in the study. Eight colonic biopsies were obtained via a colonoscopy and incubated during 18 h with low (physiological) or high (pharmacological) doses of Arg (0.1 or 2 mmol/L designated as Arg^low or Arg^high, respectively) and Gln (0.6 or 10 mmol/L designated as Gln^low or Gln^high, respectively). The concentrations of cytokines [interleukin (IL)-4, IL-10, IL-8, IL-6, tumor necrosis factor- (TNF), IL-1β, interferon-) were assessed by ELISA, and nitric oxide (NO) production was evaluated by Griess assay. Nuclear factor (NF)-B p65 subunit, inhibitor of NFB-, and p38 mitogen-activated protein kinase (MAPK) were assessed by immunoblotting. Arg^high/Gln^high decreased the production of TNF, IL-1β, IL-8, and IL-6 (each P < 0.01). Arg^low/Gln^high decreased IL-6 and IL-8 production (both P < 0.01), whereas Arg^high/Gln^low did not affect cytokine and NO production. Arg^low/Gln^high and Arg^high/Gln^high decreased NF-B p65 subunit expression, whereas p38 MAPK was decreased only by Arg^high/Gln^high. Combined pharmacological doses of Arg and Gln decreased TNF and the main proinflammatory cytokines release in active colonic CD biopsies via NF-B and p38 MAPK pathways. These results could be the basis of prospective studies evaluating the effects of enteral supply of combined Arg and Gln during active CD.

	Introduction

TOP ABSTRACT Introduction Subjects and Methods Results Discussion LITERATURE CITED

Parallel to the development of specific pharmacological drugs such as anti-TNF antibodies, enteral nutrition and immunonutrition taken orally to decrease gut inflammation has been proposed for patients with active CD (6). Pharmaconutrients such as glutamine and arginine are both considered conditionally essential amino acids during metabolic stress, especially when the gut is involved in systemic inflammation (7,8).

Glutamine has a protective role in intestinal injury models, such as experimental enterocolitis (9,10). Glutamine likely protects the gut via mucosal healing and a decrease in bacterial translocation (11). Moreover, we and others have reported that glutamine downregulated the intestinal inflammatory response in experimental models (9,12,13) by modulating the nuclear factor-B (NFB) pathway (14–17). However, data in patients with CD are limited and controversial (18–20) and have not shown the immune effects of glutamine supplementation on cytokine production by inflamed gut mucosa during active CD.

Moreover, L-arginine can modulate the immune response via different biochemical pathways (21). The first hypothesis of the immune effects of arginine is the immunomodulation via nitric oxide (NO). In intestinal epithelial cells, arginine decreased proinflammatory cytokine production in parallel with some increase in NO production (22) or did not exacerbate inflammatory response (23). Second, it has also been shown that microvessels in CD exhibited microvascular endothelial dysfunction, characterized by the loss of NO-dependent dilation that may contribute to the poor wound healing and maintenance of chronic inflammation (24). This endothelial dysfunction may be partially corrected by arginine supply. Third, arginine can also serve as a substrate for polyamine synthesis, which is strongly involved in protein synthesis enhancement (25) and could facilitate mucosal healing (26,27).

However, to our knowledge, no data are available about the effects of both arginine and glutamine on gut inflammation in patients with active CD. Thus, this study was undertaken on the hypothesis that glutamine and/or arginine supplementation could decrease mucosal gut inflammation in patients with severe active CD.

	Subjects and Methods

TOP ABSTRACT Introduction Subjects and Methods Results Discussion LITERATURE CITED

 
Subjects

Ten consecutive patients with active colonic CD (Table 1) were prospectively included in this study when admitted to the Department of Hepato-Gastroenterology, Rouen University Hospital. All patients had a previous endoscopic and histological examination to establish the diagnosis of CD. Activity of CD at hospitalization was assessed according to the CD activity index (CDAI). We assessed the C-reactive protein (CRP) concentration in plasma samples obtained at admission and recorded the current treatment for CD (Table 1). No patient had associated disease or complained of extraintestinal manifestations of CD or extraintestinal infection. Stool cultures for Yersinia enterocolitica, Salmonella sp, Shigella, Campylobacter jejuni, and Clostridium difficile were negative in all cases, as well as the serological test for cytomegalovirus. During a planned colonoscopy performed to assess the endoscopic extent and severity of the colonic lesions and to obtain biopsies for histology, bacteriology, and virology, 8 additional colonic biopsies were taken for the study in inflammatory areas surrounding mucosal ulcerations. Endoscopic evaluation of the activity of CD was assessed using the CD endoscopic index of severity (CDEIS) (28), which could range from 0 to 44, with higher scores indicative of greater severity of disease. Written consent of patients to participate in the study was obtained in all cases. This study was approved by the Ethical Committee of Caen University Hospital and complies with the International Declaration of Helsinki.

    Plasma CRP. In plasma samples obtained at admission of patients, we assessed CRP by immunonephelemetry (BN II, Dade Behring Nephelemeter Analyzer).

    Biopsy culture. Colonic biopsies (6.5 ± 0.2 mg, mean ± SEM) were immediately transferred in preweighed tubes containing cooled Arg and Gln-free culture medium (DMEM, Eurobio) supplemented with glucose (4.5 g/L), penicillin (0.1 U/L), streptomycin (10^–4 U/L), 10% of fetal calf serum, 0.1 mmol/L Arg, and 0.6 mmol/L Gln. Biopsies were then processed within 1 h for organ culture as previously described (12,29–31). Briefly, biopsies were placed in plastic 24-well culture plates and bathed in 1 mL of Gln-free and Arg-free DMEM supplemented with glucose (4.5 g/L), penicillin (0.1 U/L), streptomycin (10^–4 U/L), 10% of fetal calf serum, and Arg and Gln as follows: 1) Arg 0.1 mmol/L/Gln 0.6 mmol/L (Arg^low/Gln^low); 2) Arg 2 mmol/L/Gln 0.6 mmol/L (Arg^high/Gln^low); 3) Arg 0.1 mmol/L/Gln 10 mmol/L (Arg^low/Gln^high); 4) Arg 2 mmol/L/Gln 10 mmol/L (Arg^high/Gln^high). Each condition was conducted in duplicate. Culture plates were placed in a chamber equilibrated with 5% CO₂ and incubated at 37°C during 18 h. Culture media and biopsies were then transferred into vials, frozen in liquid nitrogen, and stored at –80°C until cytokine analysis.

    Cytokine immunoassays. Culture media were assessed for concentrations of IL-1β, IL-4, IL-6, IL-8, IL-10, interferon (IFN)-, and TNF using a specific sandwich ELISA (R&D Systems), as previously reported (12). These assays have a sensitivity of 1, 10, 0.7, 3.5, 3.9, 8, and 1.6 ng/L for IL-1β, IL-4, IL-6, IL-8, IL-10, IFN, and TNF, respectively. Assays in samples and standards were conducted simultaneously according to the manufacturer's recommendations. The optical density was read at 450 nm for all cytokines in a 960 Photometer (Metertech). Immunoassays were performed on duplicate samples with a CV <10%. Results of cytokine concentration in culture media were expressed as pg/mg wet tissue.

    Nitrite assays. Culture media were assessed to determine nitrite production as an index of NO production. NO production was estimated based on the sum of nitrites and nitrates (total NO) determined by using the total NO kit according to the manufacturer's instructions (R&D systems). Briefly, in the first stage, nitrate was initially converted to nitrite by the enzyme nitrate reductase. The reaction was followed by a colorimetric detection of nitrites via the dye product of the Griess reaction. The resultant color change was quantified by spectrophotometry at 540 nm. Nitrite levels were expressed as µmol/mg tissue. The detection limit was <0.25 µmol/L.

    Immunoblotting. Cultured biopsies were homogenized in ice-cold lysis buffer containing 50 mmol/L HEPES (pH 7.5), 150 mmol/L NaCl, 10 mmol/L EDTA, 10 mmol/L β-glycerophosphate, 100 mmol/L NaF, 2 mmol/L sodium orthovanadate, 1 mmol/L phenylmethylsulfonyl fluoride, 20 µmol/L leupeptin, 0.1 UI/L aprotinin, and 1% Triton X-100. Vials were placed on ice for 15 min and then centrifuged for 15 min at 4°C and 10,000 x g. The supernatant containing proteins was collected and stored at –80°C until analysis. Proteins (30 µg) were separated on 4–12% Tris-glycine resolving gels (Invitrogen) and transferred to a nitrocellulose membrane (GE Healthcare), which was blocked for 1 h at room temperature with 5% (wt:v) nonfat dry milk in Tris-buffered saline (10 mmol/L Tris, pH 8; 150 mmol/L NaCl) plus 0.05% (wt:v) Tween 20 followed by an overnight incubation at 4°C with rabbit polyclonal anti-inhibitor of NFB (IB) (1:1000, SantaCruz Biotechnology, Tebu-bio), rabbit polyclonal anti-p65 (1:1000, SantaCruz), rabbit polyclonal anti-p38 (1:1000, SantaCruz), or mouse monoclonal anti-β-actin (1:1000, Sigma Aldrich) antibodies. After 3 washes in a blocking solution of 5% (wt:v) nonfat dry milk in Tris-buffered saline/0.05% Tween 20, 1 h incubation with peroxydase-conjugated goat anti-rabbit or anti-mouse IgG (1:5000, SantaCruz) was performed. After 3 additional washes, immunocomplexes were revealed by using the Enhanced Chemiluminescence detection system (GE Healthcare). Protein bands were quantified by densitometry using IRIS 5.10 software.

    Statistical analysis. For cytokine concentrations, values are individual values and medians. For IB, NF-B p65 subunit, and p38 mitogen-activated protein kinase (MAPK), values are means ± SEM. Results were compared using 1-way ANOVA for repeated measures if comparable variances were observed (assessed using a Bartlett test). In other cases, a nonparametric test was used (Friedman test). A P-value < 0.05 was considered significant. Pairwise multiple comparisons were conducted using the paired t test or Wilcoxon's test as appropriate. As 6 post hoc tests were performed, Holm's corrections were applied with = 0.05.

	Results

TOP ABSTRACT Introduction Subjects and Methods Results Discussion LITERATURE CITED

 
    Patients. Median CDAI and CRP were 412 and 147 mg/L, respectively, which classified our patients as having severe active CD (Table 1). Endoscopic assessment of the activity of CD evaluated using the CDEIS revealed a median score of 13.6.

    Cytokine and NO production. The NO production evaluated by the assessment of total nitrites was not modified by the administration of pharmacological doses of Arg and Gln (data not shown).

The TNF concentration was lower in the Arg^high/Gln^high condition than the other 3 conditions (P < 0.01; Fig. 1A). Neither the Arg^high/Gln^low nor the Arg^low/Gln^high condition affected TNF production compared with the Arg^low/Gln^low condition considered as control.

View larger version (10K): [in this window] [in a new window]   FIGURE 1  Effects of combinations of 2 levels of Arg and Gln on TNF (A), IL-1β (B), IL-6 (C), IL-8 (D), and IL-4 (E) production by cultured colonic biopsies from active CD patients. Values are individual data and medians, n = 10. Medians without a common letter differ, P < 0.05. P-values for specific comparisons are in Supplemental Table 1.

The IL-6 concentration was reduced by the Arg^low/Gln^high and Arg^high/Gln^high conditions compared with the Arg^low/Gln^low condition (P < 0.01; Fig. 1C). In contrast, Arg^high/Gln^low did not affect IL-6 release compared with Arg^low/Gln^low.

The IL-8 concentration the decreased in Arg^high/Gln^high condition compared with the other 3 conditions (P < 0.01; Fig. 1D). In addition, the Arg^low/Gln^high condition decreased IL-8 release compared with Arg^low/Gln^high and Arg^high/Gln^low conditions (P < 0.01). In contrast, the Arg^high/Gln^low condition did not affect IL-8 release compared with Arg^low/Gln^low.

The release of IL-4 was not modified by Arg^high/Gln^low, Arg^low/Gln^high, and Arg^high/Gln^high conditions compared with Arg^low/Gln^low (Fig. 1E) but was reduced by Arg^high/Gln^high compared with Arg^low/Gln^high.

IL-10 and IFN concentrations were not modified in the tested conditions (data not shown).

    Immunoblotting for IB and NFB p65 subunit. IB expression was not significantly modified by culture conditions (Fig. 2A,B). In contrast, treatment affected NFB p65 subunit expression (P < 0.001; 1-way ANOVA). In fact, p65 expression was reduced by 32 and 20% in Arg^low/Gln^high and Arg^high/Gln^high conditions, respectively, compared with Arg^low/Gln^low (Fig. 2C). The difference between Arg^low/Gln^high and Arg^high/Gln^high conditions was not significant (P = 0.044).

View larger version (22K): [in this window] [in a new window]   FIGURE 2  Effects of combinations of 2 levels of Arg and Gln on IB (A,B), NFB p65 subunit (A,C), and p38 MAPK (A,D) expression in cultured biopsies from Crohn's patients. Values are means ± SEM, n = 10. Means without a common letter differ, P < 0.05. Exact P-values for specific comparisons are in Supplemental Table 1.

	Discussion

TOP ABSTRACT Introduction Subjects and Methods Results Discussion LITERATURE CITED

 
The main result of our study was the decrease in the release of several proinflammatory cytokines, including TNF, by colonic biopsies of patients with active CD treated with pharmacological concentrations of glutamine and arginine. Our results suggest that this antiinflammatory effect could be mediated by a reduced expression of p65 NF-B subunit and p38 MAPK. To a lesser degree, an antiinflammatory action was also observed with glutamine alone, whereas arginine alone did not modify the production of any of the proinflammatory cytokines tested in our study.

Glutamine has been reported to support the trophicity of intestinal mucosa in animal and human models (25). We have previously shown that glutamine used at a pharmacological level (10 mmol/L) decreased the production of 2 proinflammatory cytokines, namely IL-6 and IL-8, and increased the production of the antiinflammatory cytokine IL-10 in an experimental model of human gut inflammation (12). These encouraging results prompted us to perform the present study in biopsies from patients with active CD. We also found a significant decrease in IL-6 and IL-8 with a pharmacological level of glutamine (10 mmol/L). Interestingly, we also found that a pharmacological level of glutamine was associated with a significant decrease in the NFB p65 subunit expression. We and others have previously reported a downregulation of the NFB p65 subunit expression by glutamine in experimental models of gut inflammation (14–17). The downregulation of IL-6, IL-8, and NFB p65 subunit by a glutamine-enriched enteral nutrition could be of major interest during active CD. In fact, NFB is one of the most important transcription factors for the induction of genes mediating inflammatory response in CD (32). It has been reported that an aberrant activation of NFB is involved in the pathogenesis of inflammatory bowel disease (33–36). Moreover, in murine models of inflammatory bowel disease, inhibition of NFB attenuated the intestinal inflammatory response (37). In the present study, glutamine alone had no statistical effect on TNF, IL-1β, and IFN production and did not affect the p38 MAPK expression. This could explain in part the disappointing results of previous studies with glutamine-enriched enteral nutrition in active CD (19). Indeed, it has been widely described that the main proinflammatory cytokine involved in the onset and the perpetuation of gut inflammation in CD is TNF (38,39). Moreover, p38 MAPK regulates the expression of proinflammatory cytokines in CD (40). In a murine model of CD, the inhibition of p38 MAPK attenuated inflammatory response by limiting p38 activity but also NFB activity (41). Considering these experimental results, a downregulation of the expression of p38 MAPK could be associated with decreased expression of the main proinflammatory cytokines involved in active CD. Our study suggests that a pharmacological level of glutamine may exert some antiinflammatory effect in active CD via decreased IL-6 and IL-8 production and NFB p65 subunit expression, but does not influence TNF production and p38 MAPK expression.

In contrast, high glutamine combined with high Arg concentration significantly decreased the production of TNF and reduced IL-6 and IL-8 production. Similarly, this combination decreased the expression of NFB p65 subunit and p38 MAPK. To our knowledge, this is the first report of the antiinflammatory synergistic effects of Gln and Arg in active CD. The simultaneous decrease of NFB p65 subunit and p38 MAPK expression observed in our study could explain the sharp decrease of proinflammatory cytokines in biopsies treated with both high Gln and Arg compared with those treated with high Gln alone. Our results also suggest that Gln and Arg may have cooperative effects to regulate both p38 MAPK expression and TNF and IL-1β production. In accordance with our results, inhibition of p38 MAPK by different drugs reduced the TNF release by cultured biopsies from CD patients (40) and the mRNA levels of proinflammatory cytokines as well as histological alterations during trinitro-benzene sulphonic acid-induced colitis (42). The present study is the first, to our knowledge, to report on the cooperative effects of Gln and Arg in gut inflammation. This result extends 2 previous experimental animal studies suggesting a potentially beneficial effect of Gln associated with Arg by an enhanced phagocytic activity against bacteria (43,44). In the latter study, the combination of Gln and Arg was superior to Gln alone or Arg alone (43).

Lastly, Arg alone was unable to induce an antiinflammatory effect in biopsies from active CD patients. This result confirms our previous finding in intestinal biopsies from healthy subjects with an induced in vitro inflammation (31). Data on Arg effects on gut inflammation are controversial. In an experimental enterocolitis, a high concentration of Arg increased the inflammatory response (45). However, we showed that Arg did not enhance gut inflammation during active CD, as it had been suspected in previous reports (22,23). In the same way, Parlesak et al. (23) recently reported that the addition of Arg in an experimental sepsis-like condition did not affect the integrity of enterocyte layers. In our study, the high Arg concentration did not increase the production of NO by active CD patient biopsies. These results are consistent with those of Boughton-Smith et al. (46) and Guihot et al. (47), who found a correlation between the inflamed gut and the production of inducible NO synthase activity in ulcerative colitis but not in CD. Our study exclusively included patients with active CD. Thus, the addition of arginine in the culture media may have led toward the arginase and polyamines synthesis pathways rather than the inducible NO synthase induction and NO production. Targeting the Arg-polyamines pathway with Arg in vivo could be of clinical interest for mucosal repair (48).

In conclusion, our results suggest that Gln combined to Arg decrease the production of TNF and proinflammatory cytokines probably through a regulation of NFB and p38 MAPK expression in colonic biopsies of active CD patients. In contrast, Gln alone only decreases IL-8 and IL-6 via NFB and has no effect on TNF production and p38 MAPK expression. To achieve adequate concentrations of these amino acids at the site of inflammation, future studies should include drug delivery and targeting issues.

This study suggests that specific nutrients exerting an antiinflammatory effect may be beneficial in patients with active CD in addition to conventional therapies. Considering our findings, a high Gln- and Arg-containing oral or enteral diet should be evaluated in patients with active CD.

	ACKNOWLEDGMENTS

 
We thank Richard Medeiros, Rouen University Hospital Medical Editor, for his valuable help in editing the manuscript.

	FOOTNOTES

 
¹ Supported by Appareil Digestif Environnement Nutrition EA4311, Rouen University.

² Author disclosures: S. Lecleire, A. Hassan, R. Marion-Letellier, M. Antonietti, G. Savoye, C. Bôle-Feysot, E. Lerebours, P. Ducrotté, P. Déchelotte, and M. Coëffier, no conflicts of interest.

³ Supplemental Table 1 is available with the online posting of this paper at jn.nutrition.org.

⁶ Both authors equally contributed to this work.

⁷ Abbreviations used: Arg^low, arginine at 0.1 mmol/L; Arg^high, arginine at 2 mmol/L; CD, Crohn's disease; CDAI, Crohn's disease activity index; CDEIS, Crohn's disease endoscopic index of severity; CRP, C-reactive protein; Gln^low, glutamine at 0.6 mmol/L; Gln^high, glutamine at 10 mmol/L; IFN, interferon; IB, inhibitor of nuclear factor-B; IL, interleukin; MAPK, mitogen-activated protein kinase, NFB, nuclear factor B; NO, nitric oxide; TNF, tumor necrosis factor.

Manuscript received 3 September 2008. Initial review completed 24 September 2008. Revision accepted 29 September 2008.

	LITERATURE CITED

TOP ABSTRACT Introduction Subjects and Methods Results Discussion LITERATURE CITED

 

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