Background
Crohn’s disease and ulcerative colitis are chronic inflammatory disorders of the gut that cause major life-long disability. Afflicting mostly young people at an age when they are most active both in their private and professional life, inflammatory bowel disease (IBD) represents an important public health problem affecting the patients education, working abilities, social life and quality of life. The cause of IBD is multiple and so far not completely understood. However, genetic factors, environmental factors and the gut bacteria play a role in disease development.
Conventional therapy of active IBD pre-dominantly target anti-inflammatory immune responses, largely due to cytokine release within the intestine. Thus, therapeutic treatment mainly includes anti-inflammatory drugs, immunosuppressants, biologic agents, and antibiotics [
1]. However, these agents may cause severe adverse effects and are therefore not suitable for long-term treatment of IBD. Moreover, conventional drugs block manifestation or consequences of inflammation in acute disease. Thus, there is a necessity for therapeutic strategies that target improvement of impaired barrier function in remission. Suitable candidates are dietary supplements and food components such as colostrum. Although natural therapies are commonly associated with lower toxicity and fewer side effects than conventional drugs, the scientific proof of their effectiveness and safety is demanded [
2].
Colostrum, the secretion produced by the mammary glands during the first three days post-partum, contains many functional nutrients. These include immunoglobulins, growth factors, and antimicrobial peptides. The potential of colostrum to affect gastrointestinal infections and to reduce the incidence of immune-mediated diseases is well established [
3,
4]. In accordance with this, we recently demonstrated the protective effect of orally applied colostrum in a murine colitis model [
5]. Beneficial effects were characterized by improvement of clinical colorectal inflammation symptoms and by the induction of immunoregulatory mechanisms, predominantly of the innate immune system arm. Thus, identification of factors responsible for preventing experimental colitis might provide the basis for developing a long-term treatment regimen.
In IBD patients, local production of polymeric IgA (pIgA) is altered and this affects both immunological homeostasis and humoral immune responses [
6,
7]. IgA does not activate the classical complement pathway and inhibits the production of pro-inflammatory cytokines in response to lipopolysaccharide (LPS), thereby maintaining mucosal integrity [
8]. SIgA preparations from colostrum are active against various microbial antigens and inhibit adhesion and invasion by enteropathogenic
Escherichia coli in vitro[
9,
10]. Colostral sIgA influences the development of the gastrointestinal immune system in milk-fed infants; however, little is known about the effectiveness of oral sIgA in maintaining gut barrier functions in adults.
Another colostral peptide with a broad range of immunomodulatory properties is lactoferrin (Lf), a member of the iron-binding glycoprotein family. Lf is predominately found in mucosal secretions and neutrophilic granules [
11]. Lf concentration and iron saturation differ among species; bovine (bLf) and human (hLf) Lfs show strong sequence homology [
12]. BLf has been reported to stimulate mucosal and systemic immune responses when given orally [
13,
14]. Moreover, there is experimental evidence that Lf reduces the severity of colitis in rodents [
15,
16].
In the present study, we explored the potential of orally applied colostral bLf and sIgA for modulating immune responses and recovery from dextran sodium sulfate (DSS)-induced murine colitis. Whole bovine colostrum (BC) improved the clinical severity of colitis, whereas bLf had no effect. SIgA influenced DSS-mediated immune cell redistribution and specifically altered colonic cell infiltration. Therefore, it might be an interesting candidate for promoting regeneration from acute colitis.
Discussion
As shown in a previous study, oral BC prophylaxis accelerates recovery from DSS-induced colitis [
5]. However, most treatment regimens for IBD patients are not available before the onset of disease. Therefore, the present study was undertaken to evaluate the potential of whole BC and selected colostral compounds such as sIgA and bLf in a therapeutic setting. Both compounds display immunomodulatory potential [
6,
11], are resistant against proteolytic degradation, and accumulate in blood and intestinal tissue of mice [
18,
19].
The major finding of the study was that whole BC and in part colostral sIgA improved clinical recovery from colitis when applied therapeutically. This conclusion was confirmed by the enhanced weight gain of sIgA-fed mice and the lower disease activity (occult blood, diarrhea) in the BC-fed treatment group. Although BC and sIgA prevented neither the initial weight loss after induction of colitis nor the histological outcome of colitis, sIgA- and BC-fed mice recovered more quickly than control animals. In striking contrast, bLf failed to improve the clinical and histological course of colitis after both therapeutic and prophylactic applications (data not shown). This was surprising since bLf showed anti-inflammatory potential in DSS-induced colitis and stimulated mucosal and systemic immune responses when given orally [
13,
14]. In addition, reduced LPS-induced epithelial damage and barrier destruction have been reported [
20], as well as decreased rectal bleeding and an improved histological score [
15]. These findings, seemingly contradictory to our study, could be attributed to the fact that in these studies, human Lf with 7% iron saturation level was applied perorally twice a day. In contrast, we used Lf derived from bovine colostrum, and the level of iron saturation of bovine Lf was in range of 5 to 30%. Therefore, the discrepancy might be due to the therapeutic regimen rather than to the iron saturation level. Thus, increasing the number of Lf injections per day might have beneficial effects on the course of colitis. However, this was beyond the scope of the present study.
Another remarkable finding of our study was the effect of immunoglobulins on those immunological effector cells that most probably promote disease recovery. Mature DCs (CD11c+CD83+) and immunoregulatory γδ TCR+ T cells were almost normalized in lymph nodes and spleens in the sIgA and IgG groups. Similar data were obtained in the prophylactic setting. SIgA almost normalized the DSS-mediated cell redistribution (CD4+ T helper cells, γδ TCR+ T cells, CD11c+CD83+ mature dendritic cells) in lymph nodes and spleen.
Although
γδ T cells are important in intestinal inflammation, the functional role of this cell population remains unknown. Under physiological conditions, they represent a minor subset within lymph nodes and spleens; however, they are induced in mice and men by inflammatory responses [
21,
22]. Peripheral human
γδ T cells exert regulatory functions and suppress T helper cell proliferation [
23]. Thus, the specific effects of sIgA on immune cell distribution can be regarded as favorable immune modulation of DSS-induced inflammation. In addition, colostral sIgA but not IgG influenced colonic cell infiltration. Just as whole BC is effective in preventing DSS-induced colitis, so sIgA treatment was characterized by massive increases in colonic CD11c
+ and CD83
+ DCs as well as
γδ TCR
+ T cells. Thus, the increase of local regulatory cells after BC prophylaxis was most likely to have been due to colostral sIgA rather than IgG. Activated
γδ T cells have been shown to promote epithelial cell growth via the production of keratinocyte growth factor [
24] and to regulate intestinal homeostasis [
25], while depletion of
γδ T cells worsened inflammation in mouse models of colitis [
26,
27]. Therefore, in accordance with these studies, the enhanced recovery from colitis was most probably mediated by immune cell modulation through colostrum and in particular colostral sIgA.
In our previous study, we hypothesized that MDSCs contributed to the clinical benefit of prophylactically applied colostrum [
5]. Here again, sIgA prophylaxis specifically increased the number of splenic CD11b
+Gr-1
+ MDSCs. Although the function of CD11b
+Gr-1
+ MDSC in intestinal inflammation remains unclear, MDSCs accumulated in the bone marrow and spleen of DSS-exposed mice, and intravenous transplantation of DSS-induced splenic CD11b
+Gr-1
+ cells in C57Bl/6 mice resulted in advanced mucosal healing [
17]. We therefore conclude that the strong induction of CD11b
+Gr-1
+ cells by sIgA contributed to the enhanced recovery from weight loss at later time points.
Methods
Mice
Female outbred NMRI mice were purchased from Harlan Winkelmann GmbH (Melderslo, Netherlands) at the age of four to six months. They received standard food and water ad libitum. Mice aged eight to twelve months (average body weight 30 g) were randomly assigned to different experimental groups. All animal experiments were performed in compliance with the German animal protection law (TierSchG BGBI S. 1105; 25.05.1998) and were approved by the local commitee on the ethics of animal experiments of the University of Rostock (Landesamt für Landwirtschaft, Lebensmittelsicherheit und Fischerei Mecklenburg-Vorpommern) under permit number 2007/06/14;LALLFM-V/TSD/7221.3-1.1-059/08.
Colostrum, lactoferrin and secretory immunoglobulin a
For the therapeutic setting, BC powder (SANIMALIS, Heinsberg, Germany) was skimmed, pasteurized, and freeze-dried to ensure minimum denaturation of immunoglobulins and nutrients. The total protein content of the colostrum preparation was 60–70 g/100 g.
The BC (SANIMALIS) solution used for prophylactic treatment was skimmed, casein-free and sterile-filtered. The concentrations of immunoglobulin and other major ingredients were as previously described [
5]. The total protein content was 7.25 g/100 ml (carbohydrate 0.2 g/100 ml, fat < 0.01 g/100 ml).
BLf was purified from bovine, and sIgA from human colostrum. IgG was isolated from human serum. All therapeutic agents were purchased from Sigma-Aldrich (Taufkirchen, Germany). Bovine BSA (Sigma-Aldrich) served as control.
Induction of colitis
Acute colitis was induced by DSS, as previously described [
28]. Briefly, DSS (36–50 kDa; MP Biomedicals, Eschwege, Germany) was dissolved in sterilized tap water and presented to the mice at a final concentration of 5% for seven days. Fresh DSS solution was provided every second day.
Therapeutic treatment protocol
All therapeutic interventions were initiated from day 3 after starting DSS exposure until the end of the experiment (day 15). Either 20 mg/kg bw BC powder (BC, n = 6), 1 mg/kg bw sIgA (sIgA, n = 6), IgG (IgG, n = 6), 150 mg/kg bw bLf (bLf, n = 5) or protein control (BSA, n = 5) were dissolved in physiological sodium chloride solution (0.9% NaCl) and given to each mouse in a final volume of 100 μl daily by oral gavage. DSS-treated negative controls received 100 μl NaCl (DSS, n = 8). Physiological values were obtained from animals with no intervention (untreated, n = 6-8).
Prophylactic treatment protocol
One hundred μl of pure BC (n = 6) was given to the mice orally by gavage daily for 14 days. All prophylactic treatments were initiated two weeks prior to colitis induction. SIgA and IgG were dissolved in NaCl. Each mouse received 2 mg/kg bw sIgA (n = 8) or IgG (n = 8) in final volume of 100 μl daily for 14 days by oral gavage.
Clinical evaluation of colitis
After starting DSS exposure at day 1, the mice were weighed daily until the end of the experiment at day 15. Weight loss during the induction phase of colitis was observed from day 1 to day 7 (day 1 = 100%), whereas the recovery phase was defined as weight gain after stopping DSS until the end of the experiment from day 8 to day 14 (day 8 = 100%). Stool consistency was checked for diarrhea and feces were screened for blood during the acute inflammation phase of colitis from day 1 to day 7 (therapeutic protocol) or from day 1 to day 14 (prophylactic protocol) using a HemoCARE occult blood detection kit (Care Diagnostica, Voerde, Germany). DAI was determined according to Cooper et al. (1993) with minor modifications, by scoring changes in fecal blood, and stool consistency, as shown in Additional file
3[
29].
Colon histology
Mice were killed on day 15 after starting DSS. At autopsy, whole colons were removed and their lengths were measured. The colons were unrolled and divided longitudinally into two parts. One part was fixed and embedded in paraffin. Sections were stained with hematoxylin and eosin. The degree of colonic inflammation was analyzed by a pathologist in a blinded fashion, according to the scoring system originally described by Cooper et al. (1993) as shown in Additional file
4[
29].
Flow cytometry
Flow cytometry was performed on leukocytes from peripheral blood, spleens, and mesenteric lymph nodes as described previously [
5]. Briefly, leukocytes were labeled using the following fluorescein isothiocyanate (FITC)- and phycoerythrin (PE)-conjugated anti-mouse monoclonal antibodies (mAbs) at the appropriate concentrations: CD3ε FITC, CD19 FITC,
γδ TCR PE (ImmunoTools, Fiesoythe, Germany), CD11b FITC, CD11c FITC, CD4 PE, CD8 PE; Gr-1 PE (Miltenyi Biotech, Bergisch-Gladbach, Germany), CD83 PE (eBioscience, Frankfurt, Germany). Negative controls consisted of lymphocytes stained with the appropriate isotype controls. Myeloid-derived suppressor cells (MDSC) were defined as CD11b
+Gr-1
+. Subsets of these cell populations were detected by gating on the CD11b
+Gr-1
+high, CD11b
+Gr-1
+intermediate, or CD11b
+Gr-1
+low fraction, definable by varying expression intensity. Samples were analyzed on a FACSCalibur Cytometer (BD Pharmingen) using CellQuest software and gating on total leukocytes (BD Biosciences, Mountain View, CA USA). Relative numbers are given.
Statistics
Values are reported as the mean ± standard deviation (SD). To ensure reproducibility, experiments were repeated twice with three to four mice per group (except therapeutic experiment group DSS + bLf and DSS + BSA). Data from separate experiments were combined for statistical analysis and presentation in figures and tables. Owing to small sample sizes, group and time effects of clinical parameters (weight loss, DAI) were analyzed by a nonparametric one-way ANOVA model for repeated measurements using SAS 9.2 software [
30]. To analyze the impact of DSS-colitis on histological parameters (cell infiltration and distribution, colon length) we first compared between untreated and NaCl groups. To analyze the influence of the treatment during the course of colitis, comparison was made between NaCl and the susceptive treatment group (BC, sIgA, and bLf). For the impact of BC and sIgA, we used BSA as appropriate protein control. To analyze whether observed immunoglobulin-induced effects are specific for sIgA, we used IgG as control. Analyses were performed with the parameter-free Mann Whitney
U-test using SPSS software [
31]. P < 0.05 was considered the criterion for statistical significance.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
PB and CK designed the research; PB and EZ conducted the research and analyzed the data. PB and SK performed the statistical analyses. PB, CM and CK wrote the paper and have primary responsibility for its final content. All authors read and approved the final manuscript.