Glutamine attenuates the inhibitory effect of methotrexate on TLR signaling during intestinal chemotherapy-induced mucositis in a rat

The family of mammalian Toll-like receptors (TLRs) are homologs of the Drosophila Toll protein, which was originally identified as a receptor that plays a central role in the establishment of the embryonic dorsal-ventral pattern in fly embryos [1]. Growing evidence suggests that TLRs have a critical role in the recognition of bacterial lipoproteins, major components of bacterial cell walls. Their signaling pathway is known to activate target genes such as nuclear factor-κB (NF-κB) and cytokines that are involved in inflammation and immune responses [2‐4]. In 1997, Medzhitov et al. demonstrated that a Toll-like receptor now known as TLR4 could, when artificially ligated using antibodies, induce the activation of certain genes necessary for initiating an adaptive immune response [5]. When activated, TLRs recruit adapter molecules within the cytoplasm of cells in order to propagate a signal. Four adapter molecules are known to be involved in signaling. These proteins are known as myeloid differentiation primary response gene-88 (MyD88), Mal/TIRAP (Mal is also called TIR domain-containing adaptor protein [TIRAP]), TIR domain-containing adaptor-inducing interferon-β (TRIF) and TRIF-related adaptor molecule (TRAM) interacting with TLRs in response to ligand stimulation. When engaged by gram-negative lipopolysaccharide (LPS), this complex transduces a signal detected by MyD88, which is passed onward by a cascade of the receptor-associated kinase (IRAKs), receptor-associated factor 6 (TRAF6), and NF-kappa B inducing kinase (NIK), resulting in activation of NFκB which amplifies the signal, and ultimately leads to the induction or suppression of genes that orchestrate the inflammatory response [2, 4].

Recent studies have shown that there is an overexpression of TLR2 and TLR4 in intestinal epithelial cells which is correlated with the severity of mucosal damage, together with an increase of apoptotic cells and markedly impaired proliferation [5]. The increased expression of TLRs was found in the intestinal tissues during experimental necrotizing enterocolitis (NEC) which is a well known pediatric intestinal inflammatory disease [6]. TLRs when activated by bacterial ligands signal intracellularly to up-regulate the expression of various cytokines in the intestine, including pro-(IL-1β, IL-6, IL-8) and anti-inflammatory cytokines (IL-10) in human and different NEC models [7, 8].

Oral and gastrointestinal mucositises are common complications of chemotherapy, in particular with drugs affecting DNA synthesis (S-phase-specific agents such as fluorouracil, methotrexate, and cytarabine). Mucositis occurs in 40% of patients after standard dose chemotherapy, and in 100% of patients undergoing high dose chemotherapy and stem cell or bone marrow transplantation and contributes not only to the morbidity of treatment but also to its cost [9, 10]. The pathogenesis of chemotherapy induced gastrointestinal mucositis has been described by Sonis et al. [11] and includes five phases: initiation by chemotherapy, up-regulation and generation of messenger signals, signaling by pro-inflammatory cytokines and amplification of mucosal injury, ulceration of the mucosa and finally, healing. The initial stages of inflammation in mucositis include increased pro-inflammatory cytokine levels, which act as a homing marker for inflammatory immune cells in the submucosa.

Glutamine is a nonessential amino acid which is beneficial in the prevention of infectious morbidity and mortality in seriously ill patients, in part due to its ability to maintain the integrity of intestinal mucosal epithelium. Extensive studies in various experimental models have established that glutamine is an essential respiratory substrate for cells in the small intestinal mucosa, accounting for over one-third of the total CO₂ produced in the small intestine [12]. While in the large bowel the short chain fatty acid butyrate is the preferred fuel for the colonic mucosa, many experimental studies have indicated that glutamine is the preferred fuel source for the small intestine [13, 14]. In addition, glutamine exerts a positive effect on the gut-associated lymphoid tissue and enhances gut barrier function. We have demonstrated recently that administration of enteral glutamine prevents intestinal mucosal damage and accelerates intestinal recovery following methotrexate-induced mucositis in a rat [15]. In this study, we showed that treatment with glutamine attenuates alterations in TLR-4, MyD88 and TRAF6 intestinal expression during LPS endotoxemia in a rat [16].

In the current study we hypothesized that TLR signaling may be involved in the development of MTX-induced intestinal mucositis and that treatment with glutamine might reduce the alterations in this pathway.

Mucositis is an important side effect of methotrexate therapy for which there is no definitive prophylaxis or treatment. This is due in part to the lack of understanding of its pathogenesis and its impact on intestinal structure and function. Over the last decades, significant progress has been made in understanding the underlying mechanisms of mucositis development. The pathobiology of mucositis is complex and includes up-regulation of a range of stress response cytokines and subsequent activation of mitogen activated protein kinase (MAPK) signaling, nuclear factor κB (NFκB) signalling, Fos/Jun signalling and Wnt signaling [19, 20]. The current hypothesis for the development of mucositis includes five biological phases, namely: initiation, which encompasses the primary damage occurring following administration of cytotoxic chemotherapy; message generation, involving the up-regulation of transcription factors including NFκ B, MAPK, matrix metalloproteinases and subsequent activation of cytokine and stress response genes; signalling and amplification producing proteins such as tumour necrosis factor, interleukin-1β and interleukin-6 which cause direct tissue damage and provide positive feedback to amplify the process; ulceration, which resuls in painful ulcers, bacterial infiltration and an influx of macrophages and other inflammatory cells and finally healing, which spontaneously occurs upon cessation of chemotherapy [11].

Possible mediators of mucositis which have not been well studied are related to toll-like receptor signaling. TLR signaling has been shown to function in several of the pathways which mediate mucositis development and tissue injury in general. Therefore, TLRs deserve careful consideration as possible mediators of chemotherapy-induced mucositis. Toll-like receptors are an ancient conserved receptor family that regulate antimicrobial host defense in plants, invertebrates, and mammals [21]. Individual TLRs recognize distinct pathogen-associated molecular patterns (PAMPs) that have been evolutionarily conserved in specific classes of microbes. Interaction of PAMPs with TLRs triggers a complex signaling pathway that leads to the activation of the immune system. There are more than 10 identified members of the TLR family, with well-defined specificities to various components of bacteria, viruses, and fungi [22]. The best characterized member of this family is toll-like receptor 4 (TLR-4), the receptor for lipopolysaccharide, which is the best known and first discovered bacterial cell wall component that can elicit cellular responses. TLR-4 is responsible for the recognition of bacterial endotoxin or LPS and for initiation of the gram-negative bacillary septic shock syndrome [23, 24]. Interaction between LPS and TLR-4 leads to formation of an LPS signaling complex consisting of surface molecules, such as CD14 and MD2, as well as intracellular adaptor molecules, including myeloid differentiation primary response gene 88 (MyD88), TNF-α receptor association factor 6 (TRAF6), and activation of transcription factors, such as nuclear factor κB (NFκB), which then induce the activation of the inflammatory genes, such as tumor necrosis factor-α, interleukin (IL)-1, IL-6, and IL-8 [5].

Epithelial cells of the gastrointestinal tract (GIT) undergo continuous renewal. The process of cell turnover is tightly regulated by mucosal as well as submucosal signalling in order to maintain homeostasis and to compensate for disturbances which may occur in the GIT. The distribution and expression of TLRs in the basement membrane plays a predominant role in regulating epithelial cell kinetics. Therefore, it is important that TLRs levels are continuously regulated to achieve a balance in tissue degradation and fibrogenesis. Furthermore, a skewed level of tissue TLRs has been implicated in the development of acute and chronic intestinal diseases.

The TLR signaling following chemotherapy has not received a great deal of attention. Previous studies have shown a role for ROS and the pro-inflammatory cytokines TNF and IL-1β in the message generation phase of mucositis [25] and in the induction of TLR signaling [26], separately, with no studies documenting a direct relationship between TLR signaling and the development of intestinal mucositis.

Glutamine is a non-essential amino acid which plays an important role in many physiologic and biologic processes. Growing evidence suggests that glutamine is an important nutrient for rapidly dividing cells such as those from the immune system and the gut [12]. We have shown that treatment with oral glutamine prevents mucosal injury and improves intestinal recovery following MTX- injury in the rat [14]. In this other experiment, we demonstrated a correlation between gut trophic effects of glutamine and its stimulating effect on TLR signaling during lipopolysaccharide endotoxemia in a rat [15].

We hypothesized in the present study that this TLR4 signaling is involved during MTX-induced mucositis and that glutamine could prevent intestinal mucosal injury or/and improve intestinal recovery by affecting this pathway. Our data has shown that a single dose of MTX can cause a severe mucosal injury in the small bowel. This is evident from the increased intestinal injury score. Histologically, MTX-animals exhibited degeneration and shortening of the villus length, severe villous epithelial atrophy, significant loss of crypt architecture, signs of crypt remodeling, and polymorphonuclear leukocyte infiltration in the lamina propria. In addition, MTX rats showed intestinal mucosal hypoplasia. The observed decreased bowel and mucosal weight, decreased mucosal DNA, decreased villus height and crypt depth support this conclusion. As a folic acid analogue, the action of MTX primarily is to inhibit DNA synthesis by binding to the enzyme dihydrofolate reductase. This leads to an inhibition of proliferation in the crypts of the small intestine. Our data support this concept. Mucosal DNA content as well as enterocyte proliferation index decreased significantly in both jejunum and ileum following MTX administration. Cell death via apoptosis increased significantly in intestinal mucosa after MTX administration. The loss of small intestinal epithelial cells leading to villous atrophy caused by MTX has been well described and has been appreciated since the 1970s [27, 28].

We found that administration of MTX was associated with down-regulation of TLR-4 Myd88 and TRAF-6 mRNA expression in the jejunal and ileal mucosa. The elevation of TLR-4 and Myd88 mRNA expression in the ileum was found to be statistically significant. Changes in protein levels were in agreement with mRNA expression. Both TLR-4 and Myd88 protein levels were significantly down-regulated in MTX-treated rats compared to control animals. The down-regulation expression of TLR-4, Myd88 and TRAF-6 mRNA and protein expression were poorly correlated with severe mucositis, which leads to induction of inflammatory cytokines such tumor necrosis factor- α, IL-1, and IL-6; and, therefore, cannot explain the developed widespread intestinal mucosal injury. Previous imunohistochemical techniques have shown that TLR4 is expressed at low levels by intraepithelial cells in normal human colon tissues and predominantly in the crypt epithelial cells [29, 30]. In the current study, both TLR4 and TRAF6 expressions stained with a stronger intensity along the entire villus-crypt axis and were expressed on intraepithelial lymphocytes and lymphocytes in submucosa. In addition, weak positive staining was observed in the crypt region. MTX-induced intestinal damage was associated with a significant decrease in TLR-4 and TRAF-6 staining in jejunum and ileum compared to control animals. These changes were in agreement with changes in mRNA and protein levels. Since TLR4 signaling is pro-inflammatory as well as administration of MTX causes inflammatory response, it should be emphasized that the down-regulation of components of the TLR signaling cascade by MTX is presumably compensatory. Such compensation mechanisms are common, activated receptors mediate their own downregulation to limit/stop the response to the stimulus.

We demonstrated previously the beneficial effects of glutamine on methotrexate induced mucositis [15]; however, the exact mechanism of this positive effect remains unclear. Consistent with these data, in the current study enteral glutamine produced various beneficial effects. The results of this study have shown that glutamine given during 72 hours to control animals did not significantly change intestinal mucosal parameters. However, pretreatment with glutamine protected the intestinal mucosa from damage caused by MTX. 80% of rats showed a significant decrease in intestinal mucosal injury grade compared to MTX animals, suggesting lesser degree of intestinal damage. In addition, exposure to enteral glutamine accelerated intestinal mucosal repair and enhanced enterocyte turnover. This is evident from the significant increase in bowel and mucosal weight, increased DNA content and increased villus height and crypt depth in this model. Treatment with glutamine led to a rapid increase in the uptake of bromdioxyuridine (BrdU) and the size of the proliferative compartment in the crypts. After exposure to glutamine supplementation, an abnormally “rich” supply of glutamine entering the small intestine might directly stimulate mucosal hyperplasia. The epithelial cells lining the intestinal canal probably use this amino acid for their own nutrition. The increased absorption of glutamine from the lumen may also stimulate the release and circulation of enteric hormones which have trophic effects on small bowel mucosa. An increased cell proliferation rate was accompanied by increase in villus height, suggesting an increased absorptive surface area. Increased cell proliferation following glutamine administration was accompanied by decreased cell apoptosis. The specific mechanism for the apparent anti-apoptotic effects of glutamine has not yet been elucidated fully. Glutamine may attenuate the enterocyte injury that precedes apoptosis, possibly as a membrane stabilizer or as a free-radical scavenger, thereby preventing or delaying the initiation of the apoptotic cascade. Alternatively, glutamine may act intracellularly to alter the expression of genes related to apoptosis or interfere with specific caspase functions.

Treatment with glutamine in the current study attenuated the inhibitory effect of MTX on TLR-4 signaling. MTX-GLN rats demonstrated a significant increase in ileal TLR-4 mRNA, MyD88 mRNA and TRAF 6 mRNA expression compared to MTX-rats, which was correlated with a trend toward increase in TLR-4 and MyD88 protein, and with the increased number of TLR-4 and TRAF-6 positive cells (by immunohistochemistry). These findings suggest that glutamine stimulates TLR signaling pathways, which is in agreement with other experiments. It has been reported that TLR2 and TLR4 expression is up-regulated under inflammatory conditions [31], which could provide a mechanism for certain inflammatory diseases of the intestine. Indeed, there is a report of increased TLR4 expression in the intestinal epithelium of patients with Crohn’s disease and ulcerative colitis [32]. Whether TLR4 expression is a cause or a result of the disease is unclear, but it does underscore the importance of the gastrointestinal epithelium in preventing excessive and uncontrolled inflammation. Current data suggests that TLRs are differentially expressed in intestinal mucosa during chemotherapy-induced mucositis on both leukocytes and mucosal epithelial cells while serving to modulate leukocyte-epithelial interactions. It should be emphasized that aberrant TLR-4 expression may play an important role in the loss of tolerance to the enteric bacteria during chemotherapy-induced mucositis. Glutamate, by increasing expression of components of the TLR signaling pathway, would be expected to amplify the inflammatory response and further increase damage tissue. That it has the opposite effect suggests that glutamate may be acting independently of TLR signaling.

The mechanism of the positive effect of glutamine on gut barrier function is poorly understood. Recent evidence suggests that glutamine prevents MTX-induced gut barrier disruption by regulating occludin and claudin-1 probably through erk and NF-κB pathways [33]. Since TLR4-MyD88/Mal-NF-kB signaling axis plays an important role in gut barrier function, further experiments are required to understand crosstalk between TLR-signaling and NF-kB-signaling during chemotherapy-induced mucositis.

In summary, methotrexate inhibits TLR-4 signaling pathways. Glutamine improves intestinal recovery and attenuates this inhibitory effect. The positive effect of glutamine on intestinal structure and gut barrier function in intestinal mucositis may be considered as a mechanism by which immunonutrition helps in the recovery of oncologic patients receiving chemotherapy.

This work was supported by a research grant from Israeli Ministry of Health No 3/6164 from 01/06/10 and Hedson Fund for Medical Research.