Bromelain treatment reduces CD25 expression on activated CD4+ T cells in vitro

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Abstract

Bromelain (Br), an extract from pineapple stem with cysteine protease activity, exerts anti-inflammatory effects in a number of inflammatory models. We have previously shown that Br treatment decreased activated CD4+ T cells and has a therapeutic role in an ovalbumin-induced murine model of allergic airway disease. The current study was designed to determine the effect of Br on CD4+ T cell activation, specifically the expression of CD25 in vitro. CD25 is up regulated upon T cell activation, found as a soluble fraction (sCD25) and is a therapeutic target in inflammation, autoimmunity and allergy. Br treatment of anti-CD3 stimulated CD4+ T cells reduced CD25 expression in a dose and time dependent manner. This reduction of CD25 was dependent on the proteolytic action of Br as the addition of E64 (a cysteine protease inhibitor) abrogated this response. The concentration of sCD25 was increased in supernatants of Br treated activated CD4+ T cells as compared to control cells, suggesting that Br proteolytically cleaved cell-surface CD25. This novel mechanism of action identifies how Br may exert its therapeutic benefits in inflammatory conditions.

Introduction

The hallmark of many immune mediated disorders is the dysregulated activation of T cells [1], [2]. Primary events in the activation of T cells are the presentation of an antigenic stimulus via an antigen presenting cell (APC) to the T cell [3] and augmentation by co-stimulatory signals (CD80, CD86) on the APC [4]. Following this unique interaction the T cell up-regulates activation markers such as CD25, the high affinity alpha chain of the IL-2 receptor [5]. Once IL-2 binds to CD25 it triggers a signaling cascade, resulting in T cell proliferation and additional IL-2 production. CD25 is constitutively expressed on CD4+ CD25+ Foxp3+ T regulatory cells, up-regulated on CD4+ T cells in response to antigen and a fraction of CD25 can be released in soluble form (sCD25) [6]. Increased levels of sCD25 have been shown to correlate with disease severity in individuals with allergic asthma [7]. Serum levels of sCD25 rise in accordance with the IL-2 receptor and are currently being investigated as biomarkers in allergy and asthma. Therapeutic interventions that target T cell mediated diseases are often designed to either boost (cancer, infectious diseases) or lower (autoimmune, allergy) the threshold of T cell activation via alteration of cell surface receptors.

A number of natural products such as essential fatty acids [8], Ginseng extracts [9], Curcumin [10] and Bromelain (EC 3.4.22.32) an extract from the common pineapple [11], have been identified as therapeutic targets for inflammation due to their ability to modulate T cell activation and expansion. Bromelain (Br) is a combination of proteins which contain cysteine protease activity [12]. Br has been shown to enhance the expression of CD11c and CD86 [13], [14] or suppress other surface markers such as CD44, B7-1, and CD80 [15], [16] on distinct cell populations in a variety of immunological systems. Br can modulate T cell production of inflammatory cytokines IL-4, IFN-γ [15], [16] and IL-13 [17] and down-regulate inflammatory responses in animal models of inflammatory bowel disease [18], experimental allergic encephalomyelitis [16] and allergic airway disease [19].

It has been previously demonstrated that Br inhibits downstream TCR signaling in CD4+ T helper cells [20] but the effect of Br on the expression of CD25 has been largely unexplored. The current study was designed to better understand the effect of Br on activated CD4+ T cells. In this manuscript we report that Br inhibits CD25 expression on activated CD4+ T cells, possibly via proteolytic cleavage.

Section snippets

Mice

Studies were performed on female C57BL/6J mice (Jackson Laboratory, Bar Harbor, ME), 8–10 weeks of age and weighing 17 to 20 g. Mice were housed conventionally in the animal facility at the University of Connecticut Health Center in accordance with institutional and Office of Laboratory Animal Welfare guidelines. All experimental procedures were approved by the institution's Center for Laboratory Animal Care.

Reagents

To directly reflect the natural products utilized in the marketplace, we chose a

Bromelain treatment reduces CD25 expression on activated CD4+ T cells

Previous work in our laboratory suggests that Br may reduce murine allergic asthma by preferentially modulating CD4+CD25+ T cells [17]. This experiment was designed to evaluate the effect of Br on CD25+ expression on activated CD4+ T cells, independent of TCR stimulus and therefore Br treatment was initiated on CD4+CD25+ T cells after the TCR stimulus was removed. As anticipated, CD25 expression was greatly increased on anti-CD3 stimulated naïve CD4++ T cells with 78% of the cells positive for

Discussion

We and others have previously demonstrated that Br exerts immuno-modulatory effects in CD4+ T cell mediated inflammation [16], [18], [21]. Our previous observations utilizing a murine model of ovalbumin-induced allergic asthma demonstrated that in vivo Br administration significantly reduced activated CD4+ T cells in particular CD4+CD25+ cells. There was no effect on the Foxp3 expressing T regulatory cells which constitutively express CD25. This observation led us to the current experiments

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    Grant support: This research was supported by NIH/AI R01 HL-43573, NIH/NCCAM FG32-AT001569, NIH/NCCAM K08 and Competitive Technologies Inc.

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