The current study is a continued characterization of the use of bromelain, a cysteine protease extracted from pineapple, in asthma. We have previously shown that the i.p. administration of bromelain exerts anti-inflammatory effects in an OVA-induced murine model of AAD (23
). The present study was designed to determine the efficacy of oral bromelain treatment in a murine model of asthma. The data presented in this study demonstrate that oral bromelain therapy can attenuate inflammation in a well-characterized murine model of asthma (23–25
) and support the existing literature that oral enzymes are bioavailable and efficacious for the treatment of inflammatory conditions (14
The quality control and analytical verification of botanical products such as bromelain remain a concern as the use of these products continues to increase (6,31–33
). Many botanicals are now undergoing intensive testing to ensure safety and quality for consumers and reproducibility for basic science and clinical research (34–36
). The bromelain utilized for these experiments was obtained from a professional natural product manufacturing company that produces products for clinical use. The bromelain extract was independently evaluated for purity, potency and contamination to ensure quality and reproducibility (). There were no solvent residues, microbial contamination, or greater than acceptable heavy metals or pesticide residues. There was no observed toxicity of oral bromelain treatment in either naive or AAD animals (as assessed by body weight, BAL protein concentrations and lung pathology).
The presence of increased BAL eosinophils is a hallmark of asthma. Compared with saline-treated AAD mice, bromelain-treated AAD mice had significantly reduced lung eosinophilia as observed by BAL differential and flow cytometry (). This observed effect of bromelain treatment on eosinophils may be due to a reduction in lymphocyte populations or a decrease in the Th2 cytokines that are responsible for the recruitment of eosinophils into the lung during asthma. Therefore, we examined the effect of oral bromelain treatment on Th2 cytokines (IL-5, IL-13) and lymphocyte subsets (CD4+, CD8+ and CD19+). Bromelain treatment reduced the percentages of CD8+ T cells and CD19+ B cells in the BAL () and BAL IL-13 concentrations (). CD19+ B cells can act as primary antigen presenting cells in Th2 conditions such as asthma. A bromelain-mediated reduction in this subset may also diminish T cell driven inflammation thereby inhibiting the progression of allergic disease. Though, primarily a CD4+ T cell mediated condition, CD8+ T cells which secrete Th2 cytokines, have also been implicated in asthma (37
). A reduction in these CD8+ T cells via bromelain may have additional protective effects by modulating lymphocyte-mediated cytotoxic asthma mechanisms such as granzymes or perforins. The effects of bromelain on the lymphocyte subpopulations were observed locally as represented by the BAL and not systemically (in peripheral blood or spleen, data not shown) suggesting that bromelain activity is focused at sites of inflammation.
Interestingly, the percentages of CD4+ T cells were not significantly reduced. This may be due to bromelain altering the ratio of CD4+ T cell subsets, mainly the effector cells (CD4+CD25−Foxp3−) which cause airway inflammation, to regulatory cells (CD4+CD25+ Foxp3+) which suppress it. Bromelain may enhance regulatory cell numbers and their function leading to the resolution of disease. The characterization of the effects of bromelain on these regulatory T cells is currently being investigated in our laboratory.
In addition, we examined the effects of bromelain treatment in asthma on both pulmonary function (PFT) and pathology. As a measure of pulmonary function, both saline and bromelain treatment groups were challenged to increasing doses (0–300
mg/ml) of methacholine, a common lung smooth muscle irritant, for assessment of airway reactivity and sensitivity (). During the baseline challenge, there was no difference between the groups. After OVA aerosols, the saline-treated group became more sensitive to methacholine whereas the bromelain group did not vary from baseline. This protective effect on lung function correlates well with the observed reductions in eosinophils, lymphocytes and cytokines that have been implicated in the pathogenesis of airway reactivity (38–41
). In regards to lung pathology (), bromelain-treated mice appeared to qualitatively have reduced pulmonary injury as compared with saline-treated controls; however, independent blinded scoring did not reveal significant differences between the groups.
Future studies will evaluate the effects of bromelain in a 7–10
day AAD model with more advanced disease and bromelain administration will be varied after the onset of asthma (3 days of aerosol) to better simulate a clinical presentation and given during or before OVA sensitization to evaluate its effect on the priming response. Experiments are currently being designed to evaluate the effect of bromelain treatment on key immunoregulatory CD4+ T cell subsets such as Foxp3+ T regulatory cells which are known to modulate allergic responses (42
) and to measure the retained enzymatic activity of bromelain in the serum of naïve and AAD mice after oral administration. In conclusion, the results obtained from this study suggest that the oral administration of bromelain attenuates inflammation in an OVA-induced murine model of asthma. These results may translate well in clinical trials.