Potential for amino acids supplementation during inflammatory bowel diseases

Abstract

Abstract:

The pathophysiology of inflammatory bowel diseases (IBDs) is multifactorial and involves interactions of gut luminal content with mucosal barrier and especially immune cells. Malnutrition is a frequent issue during IBD flares, especially in Crohn's disease (CD) patients, and nutritional support is frequently used to treat malnutrition but also in an attempt to modulate intestinal inflammation. The use of oral or enteral nutrition intervention in IBDs may be effective, alone or in combination with drugs, to achieve and maintain remission. However, standard diets are less effective than new-generation biotherapies and could be improved by supplementation with specific immunomodulatory amino acids. Experimental studies evaluating glutamine, the preferential substrate for enterocytes, are promising. Some clinical studies with oral glutamine in CD are until now disappointing, but new formulations and targeting could enhance glutamine efficacy at the site of mucosal lesions. The role of arginine, involved in nitric oxide and polyamines synthesis, still remains debated. However, the effects of these amino acids in IBD have been poorly documented in humans. Other candidates like glycine, cysteine, histidine, or taurine should also be evaluated in the future. (Inflamm Bowel Dis 2010)

Inflammatory bowel diseases (IBDs), including ulcerative colitis (UC) and Crohn's disease (CD), are characterized by chronic intestinal inflammation, which is possibly related to an imbalance of immune response to luminal factors. It is believed that the deregulation of pro- and antiinflammatory cytokines production plays a key role in the development and perpetuation of intestinal inflammation in IBD.1 An increased production of proinflammatory cytokines (tumor necrosis factor [TNF]α, IL-1β, IL-6, IL-8) has been demonstrated in patients with IBD. In contrast, the production of antiinflammatory cytokines (IL-4, IL-10) remains only moderate in these patients.1–3

Although the development of highly active drugs such as anti-TNFα antibodies has changed the short-term prognosis of severe IBD, there is still a need to develop alternative approaches or adjuvant therapies able to influence the long-term outcome of the disease with a low-risk profile. This could be achieved by oral or enteral immunonutritional or pharmaconutritional approaches. Historically, nutrition has been viewed as adjunctive care and not as an active therapeutic strategy. However, the pharmaconutrition concept refers to the delivery of high loads of specific nutritional compounds that exert modulatory effects on different targets exceeding their role of metabolic substrate.4, 5 In accordance with this concept, the administration of key nutrients should be dissociated from the provision of nutritional substrates.

We will focus this review on the immunomodulatory effects of specific amino acids such as glutamine and arginine. Both of these amino acids are considered conditionally essential amino acids during metabolic stress, especially when the gut is involved in systemic inflammation.4, 5 We will also briefly indicate some recent data with 4 other amino acids: glycine, histidine, cysteine, and taurine that may be considered good candidates for future evaluation.

GLUTAMINE

In patients with quiescent CD, whole-body glutamine metabolism seems to be unaltered.6 Nevertheless, glutamine mucosal content is decreased in noninflamed and more markedly depleted in inflamed ileum and colon from CD patients undergoing surgery compared with a control population.7 As glutamine is the preferred substrate for both enterocytes and other rapidly dividing cells, such as immune cells, these data suggest a potential beneficial role for glutamine supplementation. Glutamine has been described to have beneficial effects on the clinical outcome of critically ill patients through several mechanisms including antiinflammatory effects, induction of heat shock proteins, support of antioxidant defenses, and modulation of intestinal proteolysis.8 In IBD patients, protein composition such as glutamine enrichment does not influence the effectiveness of enteral nutrition,9 but studies are still limited.

In animals, glutamine protects intestinal mucosa in different models of IBD, i.e., trinitrobenzene sulfonic acid (TNBS),10–12 dextran sulfate sodium (DSS),13 indomethacin,14, 15 or acetic acid16 -induced intestinal inflammation. In TNBS-induced colitis, prophylactic treatment (2 weeks) with glutamine decreased bacterial translocation, intestinal lesions, and cytokine (TNFα, IL-8) production.10 A short-term (3-day) prophylactic treatment also limited TNBS-induced intestinal damage.11, 12 Curative treatment with oral glutamine also demonstrated a beneficial effect in an indomethacin model,14 but failed in a TNBS model.11, 17 These experimental data suggest a beneficial role of glutamine during colitis that could be explained by different mechanisms, summarized in Figure 1.

Figure 1. Putative mechanisms of action of glutamine in intestinal inflammation.

Glutamine could improve intestinal integrity and thus barrier function. Indeed, glutamine stimulates intestinal epithelial cell (IEC) proliferation18 and reduces IEC apoptosis.19 In addition, glutamine improved gut protein balance.20 For instance, glutamine increased the expression of heat shock proteins (hsp) that have protective cellular properties.21 In healthy volunteers we reported that enteral glutamine enhanced the intestinal expression of hsp32, also known as hemoxygenase-1 (HO-1), with potent antioxidant properties.22 In IEC23 or in TNBS-induced colitis,24 the pharmacological induction of HO-1 is respectively associated with an inhibition of inducible nitric oxide (NO) synthase activity23 and a reduction of intestinal damage.24 In TNBS-induced colitis the beneficial effects of glutamine pretreatment are associated with HO-1 induction.12 However, it was recently proposed that HO-1 could have a protective role when it is induced before the onset of inflammation but not in established acute or chronic inflammation,25 and those authors concluded that the induction of HO-1 may not be a promising approach for the acute treatment of active IBD. Another example is the increased expression by glutamine of tight junction proteins such as Zonula occludens-1,26 which is decreased in IBD models.27

An innate immunity defect is involved in IBD. Both dietary components and bacterial products from key luminal contents in the intestine may interact with inflamed epithelium. Supplementation of LPS-treated rats with glutamine is associated with a downregulation of TLR4 expression28 underlying a putative role of glutamine on innate immune response.

Glutamine is also able to modulate inflammatory and oxidative response per se. In vitro data indicate that glutamine influences cytokine production by macrophages,29 lymphocytes,30 and IEC (Table 1). Glutamine deprivation exacerbates proinflammatory cytokine production in IEC,38, 45 whereas glutamine supplementation limits the inflammatory response, both in vitro and in vivo.35, 46 The action of glutamine on cytokine production is mediated by different signaling pathways (Fig. 2), including nuclear factor kappa B (NF-κB),16, 46, 47, 50, 51 signal transducer and activator of transcription (STAT),37, 47 Jun N-terminal kinase (JNK),48 or peroxisome proliferator-activated receptor-γ (PPARγ).49 During IBD, oxidative stress is increased in intestinal mucosa and antioxidative defenses, especially glutathione, are reduced. In CD patients, we previously reported that mucosal glutathione content is reduced as compared with controls, and this depletion was also seen in noninflamed mucosa, and more severely in malnourished patients.52 Glutamine improved intestinal antioxidative defenses like glutathione in different models of injury,53, 54 but it is not yet established in IBD models.11 Thus, it appears that glutamine is able to downregulate proinflammatory and oxidant responses that contribute to intestinal damage.

Figure 2. Intracellular signaling pathways regulated by glutamine in intestinal inflammation. Glutamine inhibits STAT-4,37 STAT-1, 5,47 or JNK48 transcription factors that results in an inhibition of NF-κB signaling pathway. Glutamine may also act through peroxisome proliferator activated receptor γ (PPARγ)49 or inhibit Toll-like-receptor-4 (TLR4).28 Glutamine is also able to inhibit IκBα ubiquitination that limits degradation of the inhibitor of NF-κB, IκBα, avoiding the translocation of NF-κB.38, 46, 47 These regulatory pathways lead to a decreased generation of proinflammatory cytokine and a decreased adhesion molecules expression, resulting in an inhibition of intestinal inflammation. [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]

Table 1. Amino Acids That Have Shown Some Antiinflammatory Effects in Intestinal Epithelial Cells

Nutrient	Model	Main Results
Arginine	Co-culture model of Caco-2 cells with leukocytes and nonpathogenic bacteria	No effect on cytokine and NO production (31).
Arginine	HCT-8 treated with a cocktail of pro-inflammatory cytokines	Downregulation of the CXC-chemokine IL-8 through NO pathway (32).
Arginine	Newborn piglet jejunum IPEC-J2 cells Rat IEC-6	Upregulation of intestinal cell migration through a NO and a focal adhesion kinase (FAK) dependent mechanism (33).
Glutamine	IECs treated with a cocktail of pro-inflammatory cytokines	No effect on iNOS or NO production (34). Downregulation of CXC chemokines (35).
Glutamine	Caco-2 treated with TNFα	Inhibition of translocation of E. coli across Caco-2 monolayers (36).
Glutamine	Caco-2 treated with LPS	A decreased production and mRNA expression of IL-8 through STAT4 (37) and IκB/NFκB (38).
Glutamine	IEC-18, IEC-6	Induction of heat shock proteins (hsp25 (21), hsp70 (39-41)).
Glutamine/ Glycine	In vitro model of wound repair in HT-29	Glutamine significantly increased cell migration compared with controls whereas glycine incubation had no effect (42).
Histidine	Hydrogen peroxide and TNF-α induced Caco-2 and HT-29 cells	Inhibition of IL-8 production and mRNA expression (43)
Taurine	Co-culture model of Caco-2 with THP-1 cells	Inhibitory effect of TNFα-induced IL-8 mRNA expression and secretion from the Caco-2 cells (44).
		Downregulation of THP-1-induced LDH release (44)

Glutamine effects on intestinal inflammation in humans have been less documented. We previously reported that glutamine reduced proinflammatory cytokine (IL-8 and IL-6) production and enhanced antiinflammatory cytokine IL-10 production by human cultured-duodenal biopsies both in basal55, 56 and in vitro inflammatory conditions.57 The same results have been obtained with colonic biopsies from CD patients.51 Clinical studies in patients with CD are limited (Table 2) and provide only controversial symptomatic data,58–60 with no direct evaluation of the immune effects of glutamine supplementation on cytokine production by inflamed gut mucosa. In addition, some limitations to these studies have to be underlined. Moreover, the design of these studies are heterogeneous, since the study by Akobeng et al59 with oral glutamine was performed in children, while Ockenga et al60 used the parenteral route for glutamine administration. Finally, Den Hond et al58 used as a placebo another amino acid, glycine, that seems to have its own immunomodulatory properties.61 Hence, the lack of efficacy reported in these studies should be considered with caution. A pilot study compared butyrate and glutamine suppositories in patients with pouchitis after ileal pouch-anal anastomosis.62 Six of the 10 patients who received glutamine had no recurrence of symptoms, while only 3 of the 9 patients who received butyrate responded similarly. This suggests that delivering a high dose of glutamine at the site of inflammation could be beneficial, as recently shown in an experimental study.47 Indeed, intrarectal administration of glutamine is associated with less intestinal damage and decreased STAT1, STAT5, and NF-κB expression in TNBS-induced colitis.47

Table 2. Studies Evaluating Glutamine Supplementation in Crohn's Patients

Reference	Supplemented / Control Patients	Dose of Gln	Duration, Route of Supplementation	Results
CD, Crohn's disease; CDAI, Crohn Disease Activity Index; CRP, C-reactive protein; Gln, glutamine; PCDAI, Pediatric Crohn's Disease Activity Index; UC, ulcerative colitis.
Den Hond et al (58)	7 / 7 CD with increased permeability	21 g d^-1 (7 g x 3 times daily)	4 weeks, oral route	No difference for intestinal permeability, CDAI, CRP.
Abokeng et al (59)	9 / 9 children witth active CD	Polymeric diet with 42% of amino acid composition as glutamine (approximately 8.5 g d^-1)	4 weeks, 7 with oral and 2 with enteral route in each group.	No difference for weight, remission rate, plasma orosomucoid. PCDAI better in control group (P = 0.002)
Ockenga et al (60)	12 / 12 IBD patients (9 / 3 Crohn / UC in Gln group ; 10 / 2 Crohn / UC in control group)	0.2 g kg^-1 d^-1	24 (17–36) days in gln group and 19 (14–21) days in control group. Total parenteral nutrition.	No difference for intestinal permeability, CDAI (Crohn), CRP, lymphocyte number, length of stay.

Some theoretical limitations to the use of glutamine in IBD should be discussed. IBD often affects the distal small bowel and colon, whereas glutamine is the preferred substrate for enterocytes but not for colonocytes, which prefer butyrate, at least in the physiological situation. In addition, glutamine is rapidly absorbed in the proximal small intestine63 and may not reach the inflamed part of the intestine at a sufficient concentration to achieve its antiinflammatory effects. Innovative approaches of encapsulation and drug delivery to allow a high rate of glutamine supply to the distal ileum and colon should be developed.

In conclusion, the present data, including our most recent results in mucosa of CD patients, indicate that glutamine may have beneficial effects during IBD by achieving adequate dosing and site-targeting. Although currently available data do not support the use of glutamine supplementation,9 the use of glutamine as a therapy for CD has been poorly evaluated. New clinical studies using adapted galenic approaches are warranted to evaluate the effect of glutamine supplementation on clinical outcomes, on mucosal inflammatory response, and on intestinal lesions and to understand the involved mechanisms.

ARGININE

L-arginine is an amino acid that can modulate the immune response by several biochemical pathways.64 The first hypothesis of the immune effects of arginine is related to the NO synthesis, as arginine is the substrate of NO synthase. Deleterious or favorable effects of NO still remain debated65 and could depend on the dose of NO and its location. Excess NO and thus peroxynitrites in mucosa is associated with intestinal damage but at the same time NO production is reduced in microvessels in IBD due to the increase of arginase activity.66 In addition, pharmacological inhibition of NO production in experimental colitis does not affect the severity of experimental colitis.67, 68 In IEC (Table 1), arginine-induced NO production or NO donors decreased proinflammatory cytokines production32 and in stimulated duodenal biopsies, arginine-induced NO production does not affect cytokine production.69 In cultured colonic biopsies from active CD patients, we recently observed that arginine had no effect on NO and cytokine production.51 Similarly, arginine did not exacerbate inflammatory response induced by E. coli in Caco-2 cells.31 We speculate that supplemental arginine during IBD may not enhance NO production but may be metabolized into the arginase pathway.

Thus, arginine can serve as a substrate for polyamine synthesis, which is known to be strongly involved in protein synthesis enhancement70 and could facilitate mucosal healing during gut inflammation.71 In indomethacin-induced intestinal inflammation, dietary arginine decreased the number and the length of ulcers but did not affect villus height or crypt depth.72 In contrast, high doses of arginine were associated with moderately higher inflammation score in TNBS-induced colitis.73 In addition, treatment with arginine or isoleucine upregulated production of antimicrobial peptides called defensins in IEC, suggesting that arginine may improve innate immunity.74 Thus, it appears that arginine supplementation has been poorly documented in IBD models due to its role in NO synthesis that raises concern as to its safety during acute inflammation. In addition, we recently showed that arginine alone has no effect on proinflammatory cytokine release by cultures colonic biopsies from active CD patients, but arginine synergizes the effects of glutamine.51 Thus, arginine may play a key role in healing processes during IBD and should be evaluated at moderate dose in combination with other pharmaconutrients.

OTHER AMINO ACIDS

Glycine, cysteine, histidine, and taurine appear to be possibly interesting amino acids to modulate intestinal inflammation. Glycine has been recently proposed as an immunomodulator amino acid in other models.75, 76 In chemically induced (TNBS and DSS) colitis in rats, prophylactic dietary glycine limited the production of the proinflammatory cytokines and chemokines and reduced intestinal lesions.61 Histidine reduced IL-8 secretion by IEC.43 In addition, dietary histidine has been recently shown to reduce colitis lesions in IL10^−/− mice.77 Cysteine, 1 of the 3 constituent amino acids of glutathione, has been evaluated in experimental colitis as a prodrug and showed preventive effects78 that may be related to its role as glutathione precursor. Concerning taurine, in vitro data showed a protective role on enterocyte-like Caco-2 cell monolayer and a reduction of IL-8 release.44 Prophylactic treatment with taurine in DSS-treated mice limited intestinal damage.44 Interestingly, curative treatment with taurine also reduced the inflammatory response in TNBS-induced colitis, probably by increasing the defenses against oxidative insult.79 In addition, in vivo and in vitro studies evaluated the effects of a prodrug 5-aminosalicyltaurine which had a protective role in TNBS-induced colitis80 and limited inflammatory response in cultured cells.81 This prodrug appears to be more effective than the reference drug 5-ASA.80

CONCLUSION AND PERSPECTIVES

Experimental data evaluating pharmaconutrition with amino acids such as glutamine and arginine in colitis models are promising but further studies are required to evaluate the clinical effects of these amino acids. While modern therapies have improved efficacy, they are expensive and have some side effects. In contrast, amino acid supplementation would have a lower cost, but current clinical data do not support the use of amino acid supplementation in IBD. Future directions of research should be 1) to evaluate the combination of amino acids and to understand how they work; 2) to develop drug delivery and targeting issues to achieve adequate concentrations at the site of inflammation; 3) to study dose response and toxicity; and 4) to address the concept of combined treatment associating nutrient and drug.