This study found that short- and long-term dietary supplementation with fresh frozen pineapple juice was both safe and effective for the treatment of chronic colitis in Il10−/− mice. Mice that received fresh, enzymatically active juice had decreased colitis-associated mortality, decreased colon inflammation, and decreased inflammation-associated neoplasia compared with mice that received boiled, proteolytically inactive juice. Taste preferences of the mice prevented an analogous dietary supplementation with bromelain purified from stem. Oral administration of bromelain purified from stem once daily was found to have no long-term effect on inflammation and inflammation-associated neoplasia in this model. Variables that may explain the observed differential effects of fresh pineapple juice vs. bromelain purified from stem include the manner of administration, the total dose achieved, and/or differences in enzyme composition or activity against critical cell surface molecules.
Our studies showed that fresh pineapple juice has anti-inflammatory activity while boiled juice does not. This demonstrates definitively that the anti-inflammatory components of juice are heat-sensitive, since no components are added or subtracted from these samples. By using a variety of methods of chemical inactivation, we and others previously showed that the anti-inflammatory activity of bromelain required proteolytically active bromelain enzymes (3
). In separate studies, we showed the proteolytic activities of both pineapple juice and purified bromelain were also abrogated by heating (14
). For these reasons, we feel that our results strongly implicate proteolytic effects of the bromelain enzymes present in the fresh juice as the mechanism for the anti-inflammatory activity that we observed. It is important to note, however, that our study design cannot rule out the possibility that the beneficial anti-inflammatory effects are due to a heat-sensitive non-proteolytic component of pineapple juice.
We have previously shown that the amount of any given cell surface molecule that is removed by bromelain enzymes is a function of both the enzyme concentration and the length of exposure (4
). The time required for re-expression of cell surface molecules following proteolysis varies according to the cell surface molecule and the cell on which it is expressed, ranging from <30 minutes in the case of CD62L on murine peripheral blood leukocytes (8
) to >48 hours for CD44 or CD45RA on human lymphocytes (3
). The proteolytic spectrum of fruit bromelain, the major enzyme present in pineapple fruit, toward cell surface molecules is different than that of stem bromelain, the major enzyme present in bromelain purified from stem (14
). However, we do not currently know which molecule(s) must be removed to achieve a long-term decrease in inflammation. Indeed, the full spectrum of cell surface molecules that are sensitive to proteolysis by either of these bromelain enzymes remains to be determined. This is further complicated by our lack of knowledge of whether the critical proteolytic effects are on leukocytes, epithelial cells, colonic bacteria, or other cell types that are present within the colonic microenvironment. Therefore, the question of whether fruit bromelain or stem bromelain proteolytic activity or some other heat-sensitive component of juice is most critical for anti-inflammatory efficacy remains unresolved. The enthusiastic consumption of pineapple juice by the mice allowed achievement of a higher daily dose of fruit bromelain (equivalent to the fruit bromelain content in 36 mg of bromelain derived from stem) than could be achieved by a single daily administration of bromelain derived from stem. It is possible that these differences in dose account for the lack of long-term effects associated with treatment with bromelain derived from stem as compared with fresh juice. The more frequent exposure to proteolytic enzymes allowed by dietary ad libitum
consumption in drinking water may have further enhanced the efficacy of juice compared with bromelain administered once daily. Mice that received fresh juice developed anti-bromelain IgG responses that were substantial and were only slightly lower than those developed by mice treated with bromelain purified from stem. Our studies showed that these antibodies did not affect the proteolytic activity of bromelain. Thus, differential immune responses against bromelain in mice treated with fresh juice vs. bromelain purified from stem probably contribute minimally to the differences in efficacy observed between these 2 groups.
The lack of long-term efficacy of bromelain purified from stem was somewhat surprising, given our prior findings that long term oral bromelain treatment decreased the incidence of spontaneous colitis in Il10−/−
mice and that short term (16 day) treatment with oral bromelain decreased the severity of piroxicam-triggered colitis in this model (5
). Resistance to the proteolytic effects of bromelain via mutation is not expected. The data do suggest that mechanisms by which long-term bromelain treatment may prevent
onset of spontaneous colitis differ from its effects on established colitis. The differences between the short-term (16 day) efficacy of oral bromelain on severity of piroxicam-triggered colitis and its lack of efficacy in the model when given long-term (6 months) is more difficult to reconcile. It is possible, however, that continuous or episodic absence of bromelain-sensitive molecules on leukocytes may affect gene expression patterns in a manner that favors ongoing inflammation. It is also possible that the pathways that maintain chronic inflammation long-term differ from those that predominate early in the course of colitis. Daily bromelain treatment also lacked long-term efficacy in the helicobacter-triggered colitis model, where helicobacter organisms remain present and provide a continuous stimulus for inflammation. Further studies of interim time points will be needed to begin to address these mechanisms. However, our results do highlight the importance of carrying out long-term studies of efficacy prior to adopting long-term use of anti-inflammatory treatments previously proven to be efficacious only in short-term studies.
It is important to note that although consumption of fresh juice increased survival of these mice with colitis (), decreased their median severity of colon inflammation (), and also decreased the incidence of inflammation-associated neoplasia ), a few mice treated with fresh juice continued to exhibit severe inflammation. The neoplasias observed in the fresh juice group were limited to those mice with histologic scores higher than the median. This suggests that fresh juice primarily inhibits the development of inflammation-associated colon cancers by decreasing inflammation; however our studies cannot rule out an additional effect of fresh juice on tumor growth.
We previously showed that in vitro
bromelain treatment of human colonic tissue resulted in decreased secretion of pro-inflammatory cytokines (7
). The results presented here additionally show that exposure to juice or bromelain purified from stem can increase the degradation of some chemokines that may be secreted into the lumen. This provides an additional mechanism by which consumption of pineapple juice may decrease inflammatory activity in vivo
In addition to determining the efficacy of fresh pineapple juice treatment on chronic colitis, we also demonstrated that, similar to bromelain purified from stem, bromelains present in fresh pineapple juice can also remove cell surface molecules known to affect leukocyte migration and function. CD44, CD45R, CD62L, and CD8 were found to be at least partially sensitive to removal by exposure to fresh juice (). Note that the bromelain concentration in the diluted juice used for these assays (340 µg/ml) is less than the 1 mg/ml used in prior reports using bromelain purified from stem (4
). Differences in juice bromelain concentration, amino acid-based differences in the sensitivity of murine cell surface molecules to bromelain proteolysis, and/or differences in the epitopes of the specific antibody clones used for this study can explain quantitative differences in bromelain sensitivity of between the current and previously published studies (4
The bromelain-sensitive and partially-sensitive molecules identified in this study are important in leukocyte adhesion, migration, and activation. CD8α is a co-activation molecule for MHC class I-restricted cellular immune responses and also regulates the activation threshold for intestinal intraepithelial lymphocytes (22
). CD45R is a tyrosine phosphatase that regulates T cell activation threshold (23
). CD44 and CD62L were shown to be homing molecules for leukocytes (25
). CD44 has also been shown to affect lymphocyte adhesion (3
) and activation (3
). Thus full or partial removal of these molecules by bromelains in juice could affect leukocyte trafficking and activation.
In summary, this study shows that long-term dietary supplementation with fresh frozen pineapple juice containing proteolytically active bromelain enzymes does not negatively affect the health or body weight of mice with colitis. Consumption of fresh juice decreases inflammation severity and the incidence and multiplicity of inflammation-associated colonic neoplasia in the commonly used Il10−/− murine model of inflammatory bowel disease. In contrast, long-term once-daily treatment with bromelain purified from pineapple stem was not effective in decreasing inflammation or neoplasia in this model. Additional studies will be required to determine how the differential effects of fresh pineapple juice vs. bromelain purified from stem are related to the manner of administration, the total dose achieved, or to the differences in enzyme activity between their formulations, since the factors that limited bromelain dosing in these murine studies can be mitigated in humans through use of enteric coated pills taken multiple times daily. Studies to better understand the mechanisms by which bromelain affects colon inflammation and inflammation-associated neoplasia, including identification of the full range of bromelain-sensitive molecules and the cell signaling pathways affected, will aid translation of these findings to effective IBD therapies. In the interim however, the safety and efficacy of fresh pineapple juice in this commonly used murine model of inflammatory bowel disease provides a strong rationale for trials of dietary supplementation with fresh pineapple in humans with inflammatory bowel disease.