Food bioactive peptides

There is a series of specific food peptides referred to as bioactive peptides that, besides their nutritional value, modulate biological processes. Bioactive peptides can be present in food as such or can result from in vivo or in vitro protein digestion⁽Reference Hayes, Stanton and Fitzgerald¹⁾. To obtain the latter, proteolytic enzymes from animal or bacterial origin can be used. Many of the bioactive peptide enriched functional foods currently marketed are obtained by bacterial fermentation. The low cost of these products and the positive image associated with fermented drinks and foods make them very attractive⁽Reference Hayes, Stanton and Fitzgerald^1, Reference Hayes, Ross and Fitzgerald²⁾. Any source of food proteins can produce bioactive peptides. Thus, they have been isolated from sardine, corn, soya, egg, gelatin, etc.⁽Reference Korhonen and Pihlanto³⁾. Nevertheless, the main source of food bioactive peptides is milk⁽Reference Hayes, Stanton and Fitzgerald¹⁾. Several studies have demonstrated that food bioactive peptides can reach the intestine, resisting digestion, and go through the intestinal barrier, and hence exert effects both at the intestinal and systemic levels⁽Reference Hayes, Ross and Fitzgerald²⁾. Accordingly, intestinal⁽Reference Hayes, Stanton and Fitzgerald^1, Reference Politis and Chronopoulou^4, Reference Baldi, Ioannis and Chiara⁵⁾, cardiovascular⁽Reference Erdmann, Cheung and Schroder^6, Reference Saito⁷⁾ and immunological⁽Reference Politis and Chronopoulou⁴⁾ effects have been described⁽Reference Hayes, Stanton and Fitzgerald^1, Reference Korhonen and Pihlanto^3, Reference Martinez Augustin and Martinez de Victoria Munoz⁸⁾. In addition, a group of peptides can also act as biocarriers for Ca and other minerals, increasing their bioavailability⁽Reference Martinez Augustin and Martinez de Victoria Munoz^8, Reference Shimizu⁹⁾. The activities of milk peptides and products containing them have been recently reviewed elsewhere⁽Reference Hayes, Stanton and Fitzgerald^1, Reference Hayes, Ross and Fitzgerald²⁾.

Inflammatory bowel disease

Inflammatory bowel disease (IBD) is a chronic and relapsing condition that severely affects the quality of life of patients. The term IBD refers to a group of diseases such as microscopic colitis, collagenous colitis, pseudomembranous colitis, ulcerative colitis (UC) and Crohn's disease (CD). UC and CD are the most relevant conditions, both in incidence and prevalence⁽Reference Bernstein, Fried and Krabshuis¹⁰⁾. They share histological and clinical similarities, but there are clear differences between them; whereas in UC only the colonic mucosa is damaged, in CD any part of the gastrointestinal tract from the mouth to the anus can be affected (although mostly the ileum and colon), and damage involves not only the intestinal mucosa but the whole intestinal wall⁽Reference Bernstein, Fried and Krabshuis¹⁰⁾. On the other hand, differences in the pathophysiology of these diseases have been observed that indicate a preponderance of T helper (Th) 1 response in CD patients, with an increased production of IL-12, interferon (IFN)-γ and IL-18, whereas a Th2 response seems to be operating in UC patients⁽Reference Baumgart and Carding¹¹⁾.

The aetiology of IBD is unknown but an interplay of genetic⁽Reference Achkar and Fiocchi¹²⁾, environmental and lifestyle factors (dietary habits, smoking and stress) has been associated with this condition⁽Reference Bernstein, Fried and Krabshuis^10, Reference Gassull, Mane and Pedrosa^13–Reference Levenstein, Prantera and Varvo¹⁵⁾. Genetic variants have been linked to intestinal microflora and intestinal permeability alterations in IBD patients, and currently the most accepted theory proposes that intestinal inflammation results from an inappropriate immune response by the host (driven by genetic and environmental factors) to the intestinal microflora.

IBD can be currently treated with drugs, among which immunosuppressors, like azathioprine and corticoids, and aminosalicylates, together with anti-TNF antibodies, are the treatment of choice in acute episodes and/or for delaying relapses of the disease⁽Reference Bernstein, Fried and Krabshuis¹⁰⁾. Unfortunately, most of these treatments can lead to severe side effects⁽Reference Schiffrin, El Yousfi and Faure¹⁶⁾. Therefore, the search for new treatments that could alleviate or treat the disease is in progress.

Pathophysiology of inflammatory bowel disease

Every type of intestinal cell is involved in the pathophysiology of IBD. Epithelial cells constitute the first line of defence. Studies in which the NF-κB response in the intestinal epithelium of mice was specifically ablated showed that colonic inflammation and pancolitis developed at 3 weeks of age⁽Reference Nenci, Becker and Wullaert¹⁷⁾. The authors of this study indicated that the inhibition of the NF-κB response favours the disruption of the intestinal barrier integrity, resulting in bacterial translocation and intestinal inflammation⁽Reference Nenci, Becker and Wullaert¹⁷⁾. In accordance with this observation, low levels of defensins in intestinal epithelial cells have been observed in IBD patients⁽Reference Ramasundara, Leach and Lemberg¹⁸⁾.

Macrophages and neutrophils are the first cells attracted to the focus of inflammation. There macrophages produce mainly TNF, IL-1β, IL-6 and IL-12. These cytokines attract neutrophils, T and B lymphocytes and regulate the inflammatory response. At least four different types of CD4⁺ Th lymphocytes (Th1, Th2, Th17 and Treg) are involved in the immune response in IBD. These are the result of naive Th cell (Th0) differentiation that ultimately depends on the cytokines produced in the inflammatory site (Fig. 1). Differentiated Th cells in turn produce cytokines that regulate the immune response activating or directing B lymphocytes, macrophages, neutrophils and cytotoxic cells.

Fig. 1. Molecular requirements for T helper (Th) cell differentiation. Adapted from Dennick and Tangye⁽Reference Deenick and Tangye⁷⁶⁾. IFN, interferon; ROR, retinoic acid-related orphan receptor; Stat, signal transducers and activator of transcription; T-bet, T-cell specific T-box transcription factor; Treg, regulatory T cells.

Treg are CD4⁺Foxp3⁺regulatory T cells that play an essential role in intestinal homoeostasis. Treg are characterized by the expression of Foxp3, which is considered to confer their regulatory activity⁽Reference Gupta, Shang and Sun¹⁹⁾. The expression of Foxp3 in peripheral Th0 is dependent on transforming growth factor (TGF)-β. Treg appear in the intestine after the oral administration of antigens and produce IL-10 and TGF-β1, which, in general terms, down-regulate inflammation. Several pro-inflammatory cytokines inhibit Treg induction, including IL-6, IL-21, IL-23 and IL-27. Among them, IL-23 is the key for the inhibition of Treg during inflammation⁽Reference Boden and Snapper²⁰⁾. IBD patients appear to have lower numbers of Treg both in blood and colon; nevertheless it is important to bear in mind that Treg isolated from these patients are functionally active in vitro ⁽Reference Boden and Snapper²⁰⁾.

Th17 cells are a newly described subpopulation of Th cells involved in the pathogenesis of IBD. These pro-inflammatory lymphocytes are characterized by the expression of the master transcription factor retinoic acid-related orphan receptor-γt as well as IL-17A, IL-17F, IL-21, IL-22 and IL-26⁽Reference Korn, Bettelli and Oukka²¹⁾. Th17 cells differentiate under the influence of IL-1β, IL-6, IL-21, IL-23 and TGF-β. The latter has been shown to be essential for differentiation⁽Reference Korn, Bettelli and Oukka²¹⁾. Th17 cells appear to be involved in the pathophysiology of IBD, particularly in CD, in which Th1 cells are also implicated⁽Reference Brand^22–Reference Leppkes, Becker and Ivanov²⁴⁾.

Th1 cells express STAT-4 and produce IL-2, IL-12, IL-18 and IFN-γ. While IFN-γ, IL-12 and IL-18 drive Th1 differentiation, IL-10, TGF-β and IL-4 inhibit it. IL-2 acting in an autocrine fashion induces Th1 proliferation. The Th1 response implicates mainly the activation of macrophages, increasing the production of IFN-γ by lymphocytes and in turn the production of IL-12 by macrophages and dendritic cells. Th2 cells express GATA-3 and produce IL-4, IL-5, IL-6 and IL-13. Th2 cells proliferate in response to IL-4, while IFN-γ inhibits them. The activation of the Th2 response implicates B cells, increasing antibody production. As indicated above, the Th1 response is predominant in CD patients, while in UC colitis the Th2 response seems to dominate⁽Reference Bernstein, Fried and Krabshuis¹⁰⁾.

Animal models of inflammatory bowel disease

In general, animals do not suffer from chronic intestinal inflammation spontaneously. Therefore, several animal models that mimic different aspects of the disease have been developed, including gene knockout, transgenic, chemical and adoptive transfer models⁽Reference Jurjus, Khoury and Reimund²⁵⁾. Chemical models use different substances that disrupt the intestinal barrier or induce an immune reaction to elicit colonic inflammation⁽Reference Izcue, Coombes and Powrie²⁶⁾. Among these the models of murine colitis induced by the administration of trinitrobenzene sulfonic acid (TNBS) or dextran sodium sulfate (DSS) are the most widely used⁽Reference Wirtz, Neufert and Weigmann^27, Reference Sanchez de Medina, Martinez-Augustin and Gonzalez²⁸⁾. The first is a simple and convenient model based on one single rectal administration of TNBS dissolved in ethanol. TNBS is a hapten that elicits an immune response when bound with high-molecular-weight tissue proteins, while ethanol contributes by the disruption of the intestinal barrier. The result is a severe and prolonged degenerative inflammation of large parts of the colon that shares several clinical and molecular characteristics with CD. In particular, the inflammation produced by the administration of TNBS-ethanol involves all the layers of the intestinal mucosa and produces long-lasting damage, with cell infiltration and ulcers⁽Reference Martinez-Augustin, Merlos and Zarzuelo²⁹⁾. Furthermore, the administration of TNBS-ethanol to mice, as observed in human CD, produces a Th1-driven inflammation characterized at the initial stage by the infiltration of macrophages and neutrophils, releasing high levels of pro-inflammatory cytokines such as TNF, IL-1β or IL-6, followed by infiltration of T cells, mainly of the CD4⁺ phenotype, that produce IL-2 and IFN-γ⁽Reference Wirtz, Neufert and Weigmann^27, Reference Martinez-Augustin, Merlos and Zarzuelo²⁹⁾. It is interesting to note that, when administered directly into the ileum of rats, TNBS also induces ileitis⁽Reference Requena, Daddaoua and Martinez-Plata³⁰⁾. The administration to mice or rats of DSS in the drinking water gives rise to an inflammation characterized by bloody diarrhoea, ulcerations and granulocyte infiltration. DSS is thought to be directly toxic to epithelial cells, disrupting the intestinal mucosal barrier. Both the TNBS and DSS models can be reproduced successfully in the absence of adaptive immunity, although this does not mean that the latter does not contribute to pathology in normal conditions⁽Reference Wirtz, Neufert and Weigmann²⁷⁾. The involvement of Th17 cells in these models is unclear or controversial at this time. Besides their similarities to multiple aspects of human IBD, the DSS and the TNBS murine models have several outstanding characteristics: the onset and duration of inflammation are immediate and controllable and there are no artificial genetic deletions or manipulations that are not found in human IBD⁽Reference Alex, Zachos and Nguyen³¹⁾.

The most used knockout model of IBD is the Il10^−/− mice. These animals develop a spontaneous caecal inflammation and colitis at 2–4 months of age that features many characteristics observed in human IBD. The inflammation in this model is Th1 driven and is similar, therefore, to CD inflammation in human subjects. Transgenic models of IBD are well represented by the HLA-B27 model of rat colitis. In this model human HLA-B27/microglobulin 2 transgenic rats develop chronic colitis that, among other characteristics, is accompanied by some extraintestinal complications resembling those seen in IBD patients, like spondyloarthropathy involving peripheral and axial joints, dermatological complications and male genital inflammation⁽Reference Sartor³²⁾. Finally, in the transference model CD4⁺ CD45RB^high T cells (or equivalent) from healthy wild-type mice are transferred to mice lacking functional B and T cells⁽Reference Ostanin, Bao and Koboziev³³⁾. After 5–8 weeks, pancolitis and intestinal inflammation are observed in recipient mice with features that are similar to those of human CD. This model presents the advantages that both early symptoms of inflammation and the perpetuation of the disease can be studied⁽Reference Ostanin, Bao and Koboziev³³⁾.

Bioactive peptides and inflammatory bowel disease

So far, the evidence indicates that at least two milk bioactive peptides could be useful in the treatment of IBD: TGF-β and bovine glycomacropeptide (BGMP), also known as bovine casein macropeptide.

TGF-β is a growth factor present in high concentrations in the milk of several species, including human milk, and it is also produced in small amounts in the intestine of newborn infants. There are at least three isoforms of TGF-β (TGF-β1, TGF-β2 and TGF-β3). These isoforms have high homology among themselves (70% TGF-β1 v. TGF-β2 and 74% TGF-β3 v. TGF-β2), and the amino acid sequence is also highly conserved in different species (<94%)⁽Reference Massague³⁴⁾. It has been described that TGF-β1 and TGF-β2 are generally equivalent in their functionality both in vivo and in vitro ⁽Reference Ozawa, Miyata and Nishimura³⁵⁾. Human milk contains mainly TGF-β1 and TGF-β2, the last being the major isoform (95%)⁽Reference Massague^34, Reference Cheifetz, Hernandez and Laiho³⁶⁾. Recent studies indicate that TGF-β is expressed as a pre-pro-factor that gives rise to the secretion of the pro-factor after intracellular hydrolysis, included with other proteins in a latent complex. The latent form in milk is finally activated by gastric acid⁽Reference Nakamura, Miyata and Ando³⁷⁾.

TGF-β has pleiotropic functions, including the regulation of the immune function as well as functions related to cellular growth and differentiation. TGF-β1 transferred from the mother via placenta or milk is so biologically relevant that mice in which this gene is disrupted survive until weaning and then succumb to a wasting syndrome accompanied by a multifocal, mixed inflammatory cell response and tissue necrosis, leading to organ failure and death⁽Reference Letterio, Geiser and Kulkarni³⁸⁾. It has been widely demonstrated in rodents that the oral administration of TGF-β induces oral tolerance and inhibits allergic reactions⁽Reference Ozawa, Miyata and Nishimura³⁵⁾. When TGF-β is orally administered to TGF-β-null mice it prevents their death and can be localized in internal organs such as the lung, indicating that it can be active at extraintestinal levels⁽Reference Letterio, Geiser and Kulkarni^38, Reference Letterio³⁹⁾. The fast absorption in newborns together with the detection of high TGF-β levels in the plasma of healthy human volunteers reinforces this hypothesis⁽Reference Ozawa, Miyata and Nishimura^35, Reference Donnet-Hughes, Duc and Serrant⁴⁰⁾.

Alterations in TGF-β signalling can play an important role in IBD. In fact, in a study carried out in mice in which a dominant-negative mutant form of the TGF-β type II receptor was overexpressed specifically in the intestine, the animals spontaneously developed diarrhoea and under specific pathogen-free conditions they were more susceptible to the induction of colonic inflammation by DSS⁽Reference Hahm, Im and Parks⁴¹⁾. In these mice, treatment with DSS produced an increased expression of MHC class II, an exacerbated generation of autoantibodies against intestinal goblet cells, and an augmented activity of matrix metalloproteinase in intestinal epithelial cells, compared with wild-type littermates⁽Reference Hahm, Im and Parks⁴¹⁾. On the other hand, there is strong evidence that indicates the importance of TGF-β in the development of IBD. The induction of TGF-β brought about by the oral administration of haptenated colonic proteins (to induce oral toleration) protects mice from the induction of colitis by the administration of TNBS. This preventive effect is also obtained after the systemic administration of TGF-β. Conversely, the administration of anti-TGF-β antibodies reverses the protective effect of oral haptenated colonic proteins⁽Reference Neurath, Fuss and Kelsall⁴²⁾. These studies indicate that TGF-β is a major regulator of intestinal mucosal homoeostasis, acting in fact by regulating suppressive cells in an autocrine fashion, preventing the expression of IFN-γ and therefore inhibiting the activation of T (mainly Th1) and B cells, resulting in protection of the epithelial monolayer from the permeability-enhancing effects of TNF⁽Reference Donnet-Hughes, Duc and Serrant⁴⁰⁾.

Several authors have shown that the oral administration of TGF-β has anti-inflammatory effects at the intestinal level in animal models of colitis. Recently, a study has been published in which animals were fed cow's milk (500 μl) containing TGF-β (3 μg/l) daily for 2 weeks, before the induction of colitis by the administration of DSS. The authors observed that the animals that received TGF-β lost less weight and had a decreased degree of intestinal inflammation⁽Reference Ozawa, Miyata and Nishimura³⁵⁾. Furthermore, in 2005 Schiffrin et al. carried out a study to determine the effect of a casein-based formula containing TGF-β (1 ng/mg protein) to treat inflammation in human leucocyte antigen (HLA)-B27 transgenic rats⁽Reference Schiffrin, El Yousfi and Faure¹⁶⁾. These authors described that the administration of TGF-β reduced leucocytes and the acute phase reactants fibrinogen and orosomucoid. In addition, colonic weight and wall thickness as well as the mRNA for IFN-γ were reduced. Finally, there was an increase in mucin-2 production in the caecum of the animals that received the TGF-β containing formula and a normalization of the muscle proteolytic activity. A formula with the same protein composition has been proven to be anti-inflammatory in paediatric patients with CD, associated with a reduction in the expression of IFN-γ⁽Reference Beattie, Schiffrin and Donnet-Hughes⁴³⁾. In this study, seven children with CD received a TGF-β2-enriched formula for 8 weeks. In spite of the small number of patients, the results were quite clear since all the children showed a significant improvement of the disease activity, with C-reactive protein returning to normal, an increase in serum albumin and a substantial weight gain. Furthermore, ileal biopsies of these children showed reduced mucosal inflammation in six of seven children, with complete healing in two⁽Reference Beattie, Schiffrin and Donnet-Hughes⁴³⁾. However, the lack of a control group limits the usefulness of this study. This was followed by another study with 29 children with CD in which a 79% remission rate was achieved, with complete healing in ten cases, after 8 weeks administration of the TGF-β-enriched formula⁽Reference Fell, Paintin and Arnaud-Battandier⁴⁴⁾. In this study, Fell et al. described a fall in ileal IFN-γ, indicating a decrease in Th1 response, and a strong fall in ileal and colonic IL-1β and in colonic IL-8 mRNA⁽Reference Fell, Paintin and Arnaud-Battandier⁴⁴⁾.

These and several other studies have indicated that formulas enriched in TGF-β could induce clinical remission associated with mucosal healing in IBD patients⁽Reference Beattie, Schiffrin and Donnet-Hughes^43–Reference Afzal, Van Der Zaag-Loonen and Arnaud-Battandier⁴⁶⁾. These formulas are casein-based, lactose-free formulas that are enriched in TGF-β and are currently in the market. Although several studies have shown that the formulas are less effective than corticosteroids, they have proven to be more active in paediatric patients than in adults⁽Reference Fell⁴⁵⁾. As a result, TGF-β-enriched formulas are used in paediatric patients in which the administration of corticosteroids could have deleterious effects on linear growth⁽Reference Fell⁴⁵⁾.

The other bioactive peptide potentially useful in IBD is BGMP, a 64-amino-acid peptide derived from the digestion of milk k-casein. This peptide is produced physiologically in vivo, as a result of k-casein hydrolysis, and in vitro. Actually, BGMP is one of the components that elute with the whey that results after chymosin digestion during the cheese-making process. This peptide can be easily purified and is found in the market with a high degree of purity. BGMP is heavily glycosylated and many of its actions depend on glycosylation⁽Reference Aimutis⁴⁷⁾.

A wide variety of actions and applications have been described for BGMP. Because it does not contain phenylalanine in its sequence, BGMP has been proposed to be useful in the making of products for individuals with phenylketonuria⁽Reference Laclair, Ney and MacLeod^48–Reference Ney, Hull and van Calcar⁵⁰⁾. On the other hand, this peptide has anticarcinogenic properties and has been proposed to be used in tooth pastes⁽Reference Aimutis⁴⁷⁾. Its mechanism of action is related to the inhibition of bacterial growth, the prevention of tooth demineralization and the promotion of enamel remineralization⁽Reference Aimutis⁴⁷⁾. In addition, BGMP may combat infection since it has been reported to bind to cholera and Escherichia coli enterotoxins, to inhibit bacterial and viral adhesion, to promote bifidobacteria growth and to modulate the immune system response⁽Reference Hayes, Ross and Fitzgerald^2, Reference Erdmann, Cheung and Schroder^6, Reference Saito⁷⁾. Because of its facilitation of mineral absorption⁽Reference Bruck, Kelleher and Gibson⁵¹⁾ and its possible antimicrobial and prebiotic effects, BGMP is added to infant formulas⁽Reference Sandstrom, Lonnerdal and Graverholt⁵²⁾. It is important to indicate that, although as indicated earlier there are some studies showing that this peptide promotes the growth of bifidobacteria in vitro ⁽Reference Idota and Nakajima^53, Reference Yakabe and Idota⁵⁴⁾, so far there is not definitive evidence of the prebiotic effect of BGMP, since the studies carried out with rhesus monkeys and human infants were hampered by a high initial level of bifidobacteria⁽Reference Bruck, Kelleher and Gibson^51, Reference Bruck, Redgrave and Tuohy⁵⁵⁾.

BGMP has been shown to affect both innate and adaptive immunity. Thus BGMP increases the proliferation of concanavalin A-stimulated rat splenocytes⁽Reference Requena, Gonzalez and Lopez-Posadas⁵⁶⁾ and induces the expression and production of inducible nitric oxide synthase, cyclooxygenase, IL-10 and Foxp3 at the concentration of 1 g/l. However, there is also evidence that BGMP inhibits mouse splenocyte proliferation induced by lipopolysaccharide and phytohaemagglutinin⁽Reference Otani, Kawasaki and Kawakami⁵⁷⁾, while an inhibition in the production of IFN-γ has been observed in concanavalin A-stimulated splenocytes (P Requena, A Daddaoua, A Zarzuelo, MD Suárez, F Sanchez de Medina and O Martinez-Augustin, unpublished results).

In macrophages BGMP seems to have also a stimulatory effect. For instance, BGMP increased the proliferation and phagocytic activity in a human macrophage cell line⁽Reference Li and Mine⁵⁸⁾. Our research group has shown, both in THP-1 cells (a monocyte/macrophage cell line) and in human peripheral blood macrophages, that BGMP stimulates the production of several cytokines (TNF, IL-1β and IL-8)⁽Reference Requena, Daddaoua and Guadix⁵⁹⁾. In these experiments the mechanism of action of this peptide was further studied, showing that the whole peptide is needed, since it was active when protease inhibitors were added to the culture medium⁽Reference Requena, Daddaoua and Guadix⁵⁹⁾, and no effect was observed when it was hydrolysed (P Requena, A Daddaoua, A Zarzuelo, MD Suárez, F Sanchez de Medina and O Martinez-Augustin, unpublished results). The effect of BGMP was prevented by the addition of mitogen-activated protein kinase or NF-κB inhibitors, and BGMP induced the phosphorylation of NFκB inhibitor-α. Therefore, an involvement of these signal transduction pathways in the stimulatory effect of BGMP on cytokine production by macrophages was hypothesized⁽Reference Requena, Daddaoua and Guadix⁵⁹⁾. In addition, studies by Monnai and Otani⁽Reference Monnai and Otani⁶⁰⁾ indicated that BGMP increases the expression of an IL-1 receptor antagonist-like component in mouse spleen cells, involving probably monocytes.

In general, these results suggest that BGMP modulates the immune/inflammatory response by the activation of macrophages, favouring the differentiation of Treg cells and hampering the activation of Th1 cells.

As stated above, an exacerbated immune response and an imbalance in the intestinal flora have been blamed, among other factors, for the aetiology of IBD. The fact that BGMP exerts immunomodulatory, antimicrobial and possibly prebiotic activities makes it a very good candidate to modulate intestinal inflammation. In fact, our research group has described that the oral administration of BGMP results in substantial anti-inflammatory effects in experimental colitis and ileitis⁽Reference Requena, Daddaoua and Martinez-Plata^30, Reference Daddaoua, Puerta and Zarzuelo⁶¹⁾.

In a first study we used the model of colitis induced by the administration of TNBS to rats. The animals were administered BGMP daily starting either 2 d before (pre-treatment) or 3 h after (post-treatment) colitis induction⁽Reference Daddaoua, Puerta and Zarzuelo⁶¹⁾. The effect of BGMP (500 mg/kg per d) was compared with that of sulfasalazine, an established drug used in the treatment of IBD. We found that pre-treatment with glycomacropeptide had a dose-dependent anti-inflammatory effect, characterized by lower body-weight loss, decreased anorexia (57%), colonic damage (65%) and weight; length ratio (32%), as well as a reduction in colonic alkaline phosphatase activity (42%) and IL-1β, trefoil factor 3 and inducible nitric oxide synthase mRNA levels (P<0·05)⁽Reference Daddaoua, Puerta and Zarzuelo⁶¹⁾. The magnitude of the anti-inflammatory effect was generally comparable with that of sulfasalazine⁽Reference Daddaoua, Puerta and Zarzuelo⁶¹⁾.

To further ascertain the mechanism involved in the anti-inflammatory effect of BGMP and to find new beneficial applications for this peptide, we studied its effect in a model of ileitis induced by the injection of TNBS in the ileum of rats⁽Reference Requena, Daddaoua and Martinez-Plata³⁰⁾. Since CD affects any part of the intestine, ileitis frequently affects CD patients. Furthermore, ileitis can be also the result of ileal infections⁽Reference Sands^62, Reference Navarro-Llavat, Domenech and Masnou⁶³⁾ and it is a frequent complication of the ileal pouch-anal anastomosis interventions practised to treat UC⁽Reference Alexander⁶⁴⁾. While lymphocyte Th1 and Th2 responses are predominant in colonic CD and UC patients, respectively, it seems that at least in Crohn's ileitis both types of T cells are involved in the inflammatory response. Our results indicated that BGMP pre-treatment (500 mg/kg per d) results in a marked reduction of inflammatory injury, as assessed by lower extension of necrosis and diminished damage score, myeloperoxidase, alkaline phosphatase, inducible nitric oxide synthase, IL-1β and IL-17. Again, the effects of BGMP were similar to those observed in the same experiment for 5-aminosalicylic acid (200 mg/kg per d). 5-Aminosalicylic acid is the active part of sulfasalazine, which is released from the prodrug by colonic bacteria⁽Reference Nielsen and Munck⁶⁵⁾.

From the above-mentioned studies with BGMP, we can conclude that BGMP has an anti-inflammatory effect in animal models of ileitis and colitis induced by TNBS and that this effect is comparable with that of drugs frequently used to treat IBD. We have shown that BGMP decreases the expression of IL-1β, IL-1ra, TNF and IL-17 in ileitis/colitis and therefore we can conclude that the mechanism of action of BGMP probably involves Th17 cells, macrophages and T cells but not Th1 cells⁽Reference Requena, Daddaoua and Martinez-Plata^30, Reference Daddaoua, Puerta and Zarzuelo⁶¹⁾.

In a third study, we obtained mRNA from the colon of control and TNBS colitic rats administered vehicle or BGMP (500 mg/kg per d) as described earlier and carried out microarrays, allowing the simultaneous measurement of over 30 000 genes and real-time PCR for 96 selected genes (P Requena, A Daddaoua, A Zarzuelo, MD Suárez, F Sanchez de Medina and O Martinez-Augustin, unpublished results). The use of powerful genomic and postgenomic techniques gave us further insight into the mechanism of action of this peptide. The processing of our results from microarrays and real-time PCR with Ingenuity Pathway Analysis software indicated that the Il1b (IL-1β) and Il6 (IL-6) genes, both of which are down-regulated by BGMP, are key points in its mechanism of action. We observed also a decrease in IL-17, in neutrophil and macrophage infiltration and in tissue remodelling genes, as well as an increase in the Il10 gene and in a set of genes related to lymphocyte infiltration.

IL-6 is a key factor in the development of inflammation and specifically in the uncontrolled inflammatory process in IBD⁽Reference Mudter and Neurath^66–Reference Carey, Jurickova and Ballard⁶⁸⁾. In fact, increased blood and mucosal levels of this cytokine have been described in UC and CD patients, correlated with the severity of the disease⁽Reference Mudter and Neurath⁶⁶⁾. Accordingly, antibodies against IL-6 or its soluble receptor have been shown to be useful in the treatment of IBD in human subjects⁽Reference Mitsuyama, Sata and Rose-John⁶⁷⁾. There is growing evidence indicating that this cytokine, produced mainly by macrophages and CD4⁺ T cells, is one of the main ones in the chronic phase of colitis⁽Reference Mudter and Neurath⁶⁶⁾. IL-6 has three main roles in inflammation: (i) the recruitment of neutrophils to the inflammatory site and their activation to produce matrix metalloproteinases, thus contributing to severe tissue damage; (ii) the regulation of CD4⁺ leucocyte apoptosis: IL-6, together with IL-12 and TNF, is an important antiapoptotic cytokine involved in the pathogenesis of CD, whose inflammatory response, like that of the TNBS model of colitis, is driven by Th1 (and possibly Th17) cells; and (iii) the induction of Th17 cell differentiation⁽Reference Mudter and Neurath^66–Reference Carey, Jurickova and Ballard⁶⁸⁾.

From our results we may hypothesize that down-regulation of Il6 gene expression could: (1) specifically decrease Th17 cell populations, (2) reduce neutrophil infiltration at the site of inflammation, (3) lower the expression of tissue remodelation genes, probably as a consequence of the decreased neutrophil infiltration, and (4) probably allow the expression of Il10, which could also contribute to the resolution of colitis. In this study, control rats were fed with BGMP and Il6 gene expression was also down-regulated in these rats, reinforcing the idea that IL-6 down-regulation could be a key point in the anti-inflammatory effect of BGMP.

It is known that in acute inflammation, leucocyte recruitment to the site of inflammation is characterized by an initial infiltration of neutrophils, which are later replaced by a more sustained influx of mononuclear cells, initially macrophages and then lymphocytes⁽Reference Levenstein, Prantera and Varvo¹⁵⁾. Therefore, the presence of increased amounts of lymphocytes together with a decrease in neutrophils and macrophages may be interpreted as indicative of an advanced stage of recovery from inflammation. Our results show just that, i.e. a decrease in the expression of genes related to macrophage and granulocyte activation, movement, recruitment and/or accumulation, with BGMP treatment, together with an up-regulation in both B and T cell marker genes, indicating that the inflammatory state of BGMP-treated animals was closer to resolution (P Requena, A Daddaoua, A Zarzuelo, MD Suárez, F Sanchez de Medina and O Martinez-Augustin, unpublished results). These data are further sustained by the finding that Il1b (mainly produced by macrophages)⁽Reference Requena, Daddaoua and Guadix⁵⁹⁾ and Il6 (a neutrophil recruitment cytokine)⁽Reference Ramasundara, Leach and Lemberg¹⁸⁾ are key genes in the effect of BGMP, as indicated by the analysis of microarray data using Ingenuity Pathway Analysis software.

Because of the nature of our studies we cannot ascertain whether BGMP prevents the inflammatory damage induced by TNBS administration or accelerates the recovery from injury. Nevertheless, when BGMP was administered to TNBS rats before (pre-treatment) or after (post-treatment) the TNBS challenge, we found that the pre-treatment was more effective, stressing the importance of preventive actions. As alluded to earlier, studies carried out with splenocytes have shown that the addition of BGMP to culture medium increases the production of macrophage-produced cytokines (IL-1β, TNF and IL-8) in a fashion that seems to be dependent on the activation of mitogen-activated protein kinase and NF-κB⁽Reference Requena, Daddaoua and Guadix⁵⁹⁾. These results are apparently at odds with the anti-inflammatory effect of BGMP. However, the very complexity of IBD pathology makes it possible that monocyte activation may be involved in the anti-inflammatory activity of BGMP. Thus several studies suggest that defective response of the mucosa to harmful stimuli may actually worsen the outcome, at least in some cases⁽Reference Nenci, Becker and Wullaert^17, Reference Qualls, Kaplan and van Rooijen⁶⁹⁾. We have already described the study of Nenci et al.⁽Reference Nenci, Becker and Wullaert¹⁷⁾ in which reduced epithelial activation of the NF-κB pathway results in spontaneous severe colonic inflammation. Similarly, the absence of monocytes and dendritic cells aggravates rather than ameliorates experimental colitis⁽Reference Qualls, Kaplan and van Rooijen⁶⁹⁾. Because it is well established that intestinal inflammation is strongly dependent on the presence of non-pathogenic bacteria, it may be concluded that the microbiota constitutes the challenge that must be met by the intestinal mucosa in just adequate terms: not too soft, not too hard⁽Reference Seksik, Sokol and Lepage⁷⁰⁾. Thus, defects in immune function may impair a prompt resolution of intestinal injury, triggering a more robust reaction to a normally trivial challenge. If so, monocyte stimulation would result in a more efficient and prompt response to luminal antigens as they gain access to the subepithelial milieu. In this regard, it is tempting to speculate that BGMP may work at least in part via monocyte/macrophage-stimulating actions.

Other biopeptides with anti-inflammatory potential

As indicated above, a number of milk-derived peptides with antimicrobial and immunomodulatory effects have been described. These properties could be very useful in the amelioration of intestinal inflammation. The properties of these peptides have been discussed in several reviews⁽Reference Hayes, Stanton and Fitzgerald^1, Reference Martinez Augustin and Martinez de Victoria Munoz⁸⁾. Recent studies have shown in the HLA-B27 model of intestinal inflammation that Ca-supplemented diets ameliorate several important aspects of colitis, reducing IL-1β expression and histological scores and increasing the expression of extracellular matrix remodelling genes⁽Reference Schepens, Schonewille and Vink⁷¹⁾. Since many casein phosphopeptides have been shown to enhance Ca²⁺ absorption in vivo ⁽Reference Martinez Augustin and Martinez de Victoria Munoz^8, Reference Shimizu⁹⁾, these could be good anti-inflammatory candidates. On the other hand, several peptides and fermented milk products have been reported to have antioxidant effects. Antioxidants including glutathione and flavonoids⁽Reference Mane, Loren and Pedrosa^72–Reference Sanchez de Medina, Vera and Galvez⁷⁵⁾ have proven to be effective in the treatment of IBD in several animal models, indicating that perhaps antioxidant peptides derived from milk could also be beneficial.