Oral Bromelain Attenuates Inflammation in an Ovalbumin-induced Murine Model of Asthma

Abstract

Bromelain, a widely used pineapple extract with cysteine protease activity, has been shown to have immunomodulatory effects in a variety of immune system models. The purpose of the present study was to determine the effects of orally administered bromelain in an ovalbumin (OVA)-induced murine model of acute allergic airway disease (AAD). To establish AAD, female C57BL/6J mice were sensitized with intraperitoneal (i.p.) OVA/alum and then challenged with OVA aerosols for 3 days. Mice were gavaged with either (phosphate buffered saline)PBS or 200 mg/kg bromelain in PBS, twice daily for four consecutive days, beginning 1 day prior to OVA aerosol challenge. Airway reactivity and methacholine sensitivity, bronchoalveolar lavage (BAL) cellular differential, Th2 cytokines IL-5 and IL-13, and lung histology were compared between treatment groups. Oral bromelain-treatment of AAD mice demonstrated therapeutic efficacy as evidenced by decreased methacholine sensitivity (P </= 0.01), reduction in BAL eosinophils (P </= 0.02) and IL-13 concentrations (P </= 0.04) as compared with PBS controls. In addition, oral bromelain significantly reduced BAL CD19+ B cells (P </= 0.0001) and CD8+ T cells (P </= 0.0001) in AAD mice when compared with controls. These results suggest that oral treatment with bromelain had a beneficial therapeutic effect in this murine model of asthma and bromelain may also be effective in human conditions.

Keywords: CD19+ B Cells; CD8+ T Cells; IL-13; airway inflammation; asthma; cysteine protease; eosinophils; immuno-modulation.

Figure 1.

Bromelain treatment protocol. Each animal was sensitized with 3 OVA-Alum i.p. injections 1 week apart (−21 days, −14 days, −7 days). Six days after the third injection (−1 day) each animal received either Bromelain (200 mg/kg) in 0.5 ml of phosphate buffered saline (PBS) or 0.5 ml PBS alone via gavage (orally). Treatment was administred twice daily (6–8 h apart) for 4 consecutive days. Animals were challenged with 1% OVA in saline for 1 h per day (0–3). All animals were sacrificed 12 h after the last treatment (day 3) and the primary AAD outcomes assessed.

Figure 2.

The effect of oral Bromelain treatment on BAL eosinophils. Total BAL eosinophils were significantly elevated in AAD mice as compared to naïve or naïve oral Bromelain-treated mice (P ≤ 0.0001; A). There was a significant reduction in total BAL eosinophils with Bromelain treatment of AAD mice as compared to saline treated AAD mice. Similar trends were noted on evaluation of BAL samples with Flow cytometry (B). A representative FACS plot showing gated eosinophils on forward and side scatter from; naïve-saline treated (upper left), naïve-Bromelain treated (upper right), AAD-saline treated mice (lower left), and AAD-Bromelain treated mice (lower right) are shown. Statistical comparisons were made by un-paired t-test, *P ≤ 0.02; Data represent means ± SEM, n = 8 animals per group.

Figure 3.

The effect of oral Bromelain treatment on BAL Lymphocytes. After 3 OVA aerosol exposures, there was a significant increase in the percentages of CD19 + B cells (A) CD8 + T cells (B) and CD4 + T cells (C) in the BAL of AAD mice, as compared to naïve controls. Oral Bromelain treatment significantly reduced CD19 + B cells (P ≤ 0.00003; A), and CD8 + T cells (P ≤ 0.00002; B). There was no change in the percentages of CD4 + T cells in the BAL between AAD and Bromelain treated AAD mice (P ≤ 0.095; C). Statistical comparisons were made by un-paired t-test, *P ≤ 0.0001; Data represent means + SEM, n = 8 animals per group.

Figure 4.

The effect of oral Bromelain treatment on IL-5 and IL-13 concentrations in BAL. There were no significant changes in BAL IL-5 concentrations with oral Bromelain treatment of AAD mice as compared to saline treated AAD mice. A significant decrease in IL-13 concentration was observed in oral Bromealin treated AAD mice as compared to the saline treated AAD mice. Statistical comparisons were made by un-paired t-test, *P ≤ 0.04; Data represent means ± SEM, n = 8 animals per group.

Figure 5.

The effect of oral Bromelain treatment on airway reactivity and methacholine sensitivity. At baseline (open circles), Bromelain-treated and saline-treated mice had similar reactivity (P = 0.50) and sensitivity to methacholine (p2 values 48 ± 13 mg/ml in Bromelain treated versus 64 ± 15 mg/ml in saline treated; P = 0.47). After 3 OVA aerosol exposures (filled circles), saline treated mice tended to be more reactive to methacholine (P = 0.069; A) and were twice as sensitive to methacholine (P = 0.01; B) whereas no change occurred in Bromelain treated mice in reactivity (P = 0.52; A) and sensitivity (P = 0.45; B). Comparisons were made by paired t-test and repeated measures ANOVA; n = 7–8 animals per group.

Figure 6.

The effect of oral Bromelain treatment on lung pathology. Top panels: No evidence of histological injury was noted in naïve animals (A) or oral Bromelain treated naïve animals (B). Lower panels: As previously described, 3 day AAD mice develop pathological changes characterized by perivascular and peribronchiolar infiltration of lymphocytes, eosinophils and plasma cells (C), which was reduced in oral Bromelain treated AAD mice (D).