Download Anti-inflammatory potential of a malleable matrix composed of

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Anti-inflammatory potential of a malleable matrix composed of
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fermented whey proteins and lactic acid bacteria in an atopic dermatitis
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model.
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Josée Beaulieu1,2, Claude Dupont1 and Pierre Lemieux2*.
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1. Institut national de la recherche scientifique, INRS-Institut Armand-
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Frappier, 531 boul. des Prairies, Laval, Québec, Canada, H7V 1B7.
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2. Technologie Biolactis, 500 boul. Cartier suite 218, Laval, Québec,
Canada, H7V 5B7.
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* Corresponding author: Pierre Lemieux, Ph.D
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e-mail adress:
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JB: [email protected]
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CD: [email protected]
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PL: [email protected]
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ABSTRACT
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Background :
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Over the last 10 years, whey proteins have received considerable attention in the area of
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functional foods and nutraceuticals. In this paper, a novel fermented whey protein-based
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product described as a gel-like malleable protein matrix (MPM) has been tested for its
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anti-inflammatory activity. Preliminary in vitro results have already indicated that MPM
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could exert such an anti-inflammatory activity.
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Methods :
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The systemic anti-inflamatory activity of the MPM was explored using the oxazolone-
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induced atopic contact dermatitis mouse model (ACD). Many parameters including ear
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thickness, side effects as well as neutrophil extravasation were monitored.
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Results:
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The MPM exhibited anti-inflammatory effect, in the ACD model, comparable to
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hydrocortisone (control).
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inflammation while no side effects, as compared to hydrocortisone, were observed. The
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MPM seemed to reduce neutrophil extravasation in tissue as evidenced by blood
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polymorphonuclear cells as well as ear myeloperoxidase content.
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Conclusion:
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The anti-inflammatory activity demonstrated in the ACD model suggests that the
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mechanism of action of the MPM is different than that of hydrocortisone and could
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become a relevant product for people suffering from dermatological manifestations
Mice fed with MPM showed strong reduction in the ear
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associated with immune dysfunctions such as allergies, eczema, dermatitis, and
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autoimmune diseases like psoriasis.
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KEYWORDS:
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DERMATITIS (ACD)
WHEY
PROTEINS,
PROBIOTICS,
INFLAMMATION,
ATOPIC
CONTACT
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Background
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Modern life-styles which leads to obesity, stress and inactivity, is the major cause
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of immunologic diseases, particularly those associated with chronic inflammation are on
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the upswing during the last decade [1-3]. Much evidence exist that functional foods have
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protective effects on immune deficiency [4-6] including whey proteins which modulate
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some immune functions [5]. Other studies revealed that the whey proteins possess a
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myriad of activities including antioxidant activity attributed to increasing glutathione
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content [7, 8], anti-allergic activity [9] anti-inflammatory activity [9-11] and also possess
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immunomodulatory activities [12-19]. Whey proteins such as β-lactoglobulin (b-LG),
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bovine serum albumin (BSA) and α-lactalbumin (a-LA) have been shown to stimulate
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splenocyte proliferation, increase interleukin-1 production by macrophages and increase
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GSH production [18, 19].
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immunomodulatory activities [5]. Peptides derived from whey have shown stimulation
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of lymphocytes, increased phagocytosis and increased secretion of immunoglobulin A
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(IgA) by Peyer’s patches [13, 17, 20].
Whey peptides have recently been shown to possess
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Lactoferrin (LF), a minor whey protein, has been extensively studied. LF assists
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the phagocytosis process in neutrophils, increases production of interleukin-8 (IL-8) [13]
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and stimulates immune cells production [15, 19, 21]. Moreover, LF has also
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demonstrated anti-inflammatory effects in animal models by inhibition of pro-Th1
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cytokines and increasing production of regulatory cytokine IL-10 [9, 11].
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specifically, LF exerts its anti-inflammatory effect during mouse atopic contact dermatitis
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(ACD) by reducing ear skin thickness and infiltration of inflammatory cells following a
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direct topical contact [11].
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In addition, some probiotic Lactic acid bacteria (LAB) have shown
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immunomodulatory and anti-inflammatory activities.
The genus Lactobacillus
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commonly used in many fermented dairy products [23] is the most studied of these
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probiotics [22]. The effects of LAB are very strain-dependent but many lactobacilli act
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on Peyer’s patches to stimulate IgA production, stimulate phagocytosis and possess anti-
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inflammatory and anti-allergic activity by reducing the production of cytokines and
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immunoglobulin E (IgE) [24-27].
Cytokine production is strain-dependent: some
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lactobacilli are able to increase Th1 while others increase Th2 [28]. These results
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suggest that lactobacilli could act both as immunostimulating and anti-inflammatory
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agents. It also indicates that the effect of probiotics acting in synergy with food
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ingredients can be more intense than the probiotics alone [29].
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Considering the positive effects on the immune system of both whey proteins and
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probiotic lactobacilli, a novel fermented whey protein-based ingredient, called Malleable
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Protein Matrix (MPM) [30], was tested for its immunomodulatory activities [31]. From
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this study, it appeared that the MPM exerted an anti-inflammatory activity in vivo due to
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its ability to reduce ear thickness during the ACD model by the diminution of neutrophils
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extravasation in ear without side effects. Moreover, vitamins present in the MPM (niacin
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and riboflavin) as well as calcium also possess immunomodulatory effects [32-34].
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The objective of this present study is to evaluate the systemic anti-inflammatory
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potential of MPM and to determine how its complex composition may lead to synergistic
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effects. For this purpose, the oxazolone-induced atopic contact dermatitis mouse model
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(ACD) was used. This ACD mouse model requires two distinct phases [35]. First, the
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sensitization phase is initiated by topical application of oxazolone, which permits the
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activation of T cells through Langerhans antigen acting as an antigen presenting cells.
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The elicitation phase is next achieved by a subsequent topical application of oxazolone,
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which initiate the inflammation process by recruiting activated T effector cells which in
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turn attract inflammatory cells [36-38]. The inflammatory cells recruited in this ACD
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model are principally macrophages, which attract neutrophils in the early inflammatory
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phase and monocytes as well as dendritic cells in the early and late inflammatory phases.
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CD4+ T cell are not effector cells in an ACD model but are regulatory cells that can
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control the intensity of inflammatory reaction [39, 40]. This ACD model has recently be
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used to evaluate the anti-inflammatory activity of LF [11] and a milk fermented by
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Lactobacillus casei [27].
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Methods
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REAGENTS
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The Malleable Protein Matrix (MPM) was obtained from Technologie Biolactis inc.
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(LaBaie, Qc, Canada). Briefly, the MPM is obtained by a protein specific recuperation
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procedure following the fermentation of sweet whey by a proprietary Lactobacillus
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kefiranofaciens strain (R2C2) isolated from kefir grains and adapted to grow in whey
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[30].
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MPM contains 80% water, 8% protein, 6% minerals (2% calcium), 5% carbohydrate
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(2.7% lactose) and less than 1% of fat. Lyophilized MPM requires reconstitution in
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water: 20 g of lyophilized MPM was blended with 80 mL of water for 2 minutes at
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maximum speed The final reconstituted product is stable at 4°C for at least 1 month.
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Water-soluble hydrocortisone (HC) was obtained form Sigma-Aldrich Canada (Oakville,
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On, Canada) and was diluted in deionized, double-distilled water to a final concentration
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of 10 mg/mL. For the mouse ACD model, the 4-ethoxy-methylene-2-phenyloxazol-5-
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one (oxazolone) (Sigma-Aldrich, Canada) was required at 5% in acetone to cause
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The composition of MPM is shown in Table 1. On a humid basis (wt/wt), the
inflammation.
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ANIMALS
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CD-1 female mice were obtained from Charles River Laboratories (St-Constant, Qc,
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Canada) and were used at 20 days of age for studies in a mouse ACD model. The
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animals were housed in filter top isolator cages in a room kept at 20-23°C with humidity
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maintained between 35-45% with a 12 hour light-dark cycle and free access to a standard
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laboratory pelleted Rodent Lab Diet 5001 (Ren's Feed & Supplies Limited, Oakville, On).
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The experimental protocols used were approved by the Animal Care Committee of the
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INRS- Institut Armand-Frappier (Comité institutionnel des soins aux animaux et de leur
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utilisation (CISAU)) and were performed in accordance with the recommendations of the
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Canadian Council on Animal Care as specified in the Guide to the Care and Use of
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Experimental Animals (CISAU # 0306-01 and # 0410-01).
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MOUSE ATOPIC CONTACT DERMATITIS (ACD)
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The murine model of ACD was based on those firstly described by Garrigue et al. [41]
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and modified as follows: abdomen hair of CD-1 mice were removed and the sensitization
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phase was done with the application of 100 µL of oxazolone 5 % in acetone on the
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hairless abdomen (Sigma-Aldrich, Oakville,On). After 4 days, the elicitation phase (first
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challenge) was initiated with application of 50 µL of oxazolone 5% in acetone on the
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right ear (25 µL each side of the ear). The second challenge was done 7 days after the
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first challenge with the same procedure. The ear thickness of the mice was measured
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every day with a digital caliper (VWR, Mont-Royal, Canada).
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PROPHYLACTIC PROTOCOL - MOUSE ACD
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The prophylactic anti-inflammatory potential of MPM was evaluated by the
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administration of MPM 7 days prior to sensitization. Groups of 10 CD-1 mice received
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each day by gavages (per os (p.o)), 100 µL of reconstituted lyophilized MPM, 100 µL of
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water or 100 µL of water-soluble hydrocortisone (10 mg/mL). The mouse ACD was
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performed as described previously and ear thickness was measured every day. The
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mice’s weight was measured twice a week. The spleen’s weight was measured at the end
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of the protocol and was randomized in accordance to each mouse’s weight.
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THERAPEUTIC PROTOCOL - MOUSE ACD
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The therapeutic anti-inflammatory potential of MPM was evaluated by the administration
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of either MPM, soluble hydrocortisone or water as in the prophylactic protocol only after
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the first challenge.
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EVALUATION OF PERIPHERAL WHITE BLOOD CELL COUNTS
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At the end of the prophylactic protocol of mouse ACD, the blood of each mouse was
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taken and white blood cell counts were evaluated by flow cytometry. Briefly, the red
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blood cells were lysed with Optilyse C (Beckman-Coulter, Fullerton) in accordance with
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manufacturer’s instructions. The cell counts were obtained by passage of 20 µL of
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preparation in a Flow Cytometry Epics XL cytometer (Beckman Coulter, Fullerton). The
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lymphocytes, monocytes and polymorphonuclears (PMN) were separated in accordance
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with cell size and cell granulometry.
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EVALUATION OF EARS-MYELOPEROXIDASE (MPO) CONTENT
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The method for the evaluation of MPO content was adapted from those developed by
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Bradley et al. [42] and Xia and Zweier [43]. The mice were sacrificed at the end of
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prophylactic protocol by CO2 and the ears were immediately remove and frozen quickly
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in liquid nitrogen. The ears were chop up and put in 50 mM phosphate potassium buffer,
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pH 6.0 supplemented with 0.5% hexadecyltrimethylammonium bromide (HTAB). The
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ears were treated with three cycles of sonication (10 sec.) in water-ice bath followed by
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three freeze-thaw cycles in methanol-dry ice bath.
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sonication (10 sec.) in water-ice bath finished the MPO extraction. The homogenates
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were centrifuged at 10 000 g for 15 min at 4 oC and the supernatants were conserved at –
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80 oC until analyses. For the quantification of MPO content, a MPO standard was used
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(Sigma-Aldrich, Oakville, On). Hundred µL of homogenates (or standard) were mixed
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with 2.9 mL of 50 mM phosphate potassium buffer containing 0.117 mg/mL of o-
Finally, another three cycles of
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dianisidine (Sigma-Aldrich, Oakville, On), and 0.0005% hydrogen peroxide. The kinetics
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was followed during 5 min at 460 nm with spectrophotometer Varian Cary 300 (Varian,
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St-Laurent, QC).
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STATISTICAL ANALYSIS
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The inflammatory mouse ACD experiments were performed with a minimal amount of
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10 mice/group and two independent experiments. The statistical analysis of data were
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done using the software JMP IN version 5.1 (SAS Institute, Montréal, Canada).
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RESULTS
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MPM is a whey-fermented product that should possess, by its composition, an
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anti-inflammatory potential. To demonstrate this potential the atopic contact dermatitis
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(ACD) model was used. Figure 1 shows an important reduction of ear thickness in mice
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consuming MPM as compared to hydrocortisone. In the prophylactic protocol (Figure
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1A), the maximal reduction of ear thickness was approximately 26% in the MPM group
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and 35% in the hydrocortisone group as compared to the control group. This reduction
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was observed immediately after the first challenge and increases after the second
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challenge. In the therapeutic protocol (Figure 1B), the reduction of ear thickness is
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higher only after the second challenge for the MPM group. This maximal reduction was
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37% and 40% for MPM and hydrocortisone respectively compared to the control. No
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statistical difference between these both group indicates that the anti-inflammatory effect
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of MPM is comparable to hydrocortisone.
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The consumption of hydrocortisone is associated with a negative effect on mice
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growth which is clearly demonstrated by the cessation of growth in the mice who
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received hydrocortisone (Figure 2). The mice consuming MPM demonstrated no side
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effects on growth. Mice consuming hydrocortisone show a 50% reduction in spleen
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weight as compared to water or MPM cosumption (Figure 3) showing spleen atrophy
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caused by an immunosuppression of immune cells. No difference was observed between
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the water and MPM group. Moreover, cell counts (Figure 4) showed a reduction of
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approximately 50% in circulating lymphocytes with hydrocortisone consumption
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compared to water. On the contrary, the consumption of MPM showed a tendency for
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increased lymphocyte numbers. These results demonstrate an absence of side effects
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caused by the MPM consumption.
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The polymorphonuclear (PMN) cell counts was higher in MPM and
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hydrocortisone fed groups than in the water group (Figure 4). The PMN counts are 1.86
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and 2.35 fold higher in MPM and hydrocortisone respectively indicating a possible
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diminution of PMN extravasation. Figure 5 shows that the neutrophil content in ear is
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62.4% and 82.6% reduced by the MPM and hydrocortisone consumption respectively as
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measured by myeloperoxidase (MPO) ear analysis. These MPO results confirm that the
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PMN extravasation, and particularly, neutrophil extravasation is reduced by the MPM
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consumption.
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Discussion
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MPM contains a variety of ingredients including whey proteins and peptides,
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lactic acid bacteria and their related exopolysaccharides, group B vitamins and calcium
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(Table 1).
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interesting anti-inflammatory potential [5, 14, 15, 34, 44, 45].
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components, the MPM should possess an anti-inflammatory potential, which may be
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greater than its individual components, which is confirmed by the murine ACD model.
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This model of inflammation has proven to be a sensitive and useful tool to determine
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efficacy and potency of several anti-inflammatory and immunosuppressive drugs used in
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dermatological disorders such as psoriasis. Glucocorticoids, such as hydrocortisone, are
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commonly used to relieve skin and joint inflammation [35]. This model comprises two
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important phases in order to examine inflammation: 1) Sensitization phase by application
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of oxazolone on the abdomen, allowing the recruitment of antigen presenting cells, which
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capture and presents the antigen (oxazolone) to naive T lymphocytes that afterwards
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become active. 2) Elicitation phase with application of oxazolone on the ear, which
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allows activation of T lymphocytes to move to the ear and recruit inflammatory cells [35,
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46].
All these ingredients possess known effects on the immune system and
In light of these
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MPM and hydrocortisone administered p.o. either in a prophylatic (Figure 1A) or
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therapeutic fashion (Figure 1B) reduced the inflammation with the same level of efficacy
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as shown with a reduction of ear redness and thickness. In the prophylactic protocol
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(Figure 1A), a reduction of ear inflammation is observed as soon as one day after the first
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challenge and this protective effect is conserved throughout the entire course of the
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experiment. For therapeutic protocol, however, the anti-inflammatory effect of MPM
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consumption is apparent only after the second challenge (Figure 1B). The effect of MPM
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in this model (therapeutic protocol) showed that time is required to overcome exisiting
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inflammation. Comparison between these two protocols suggests that the MPM should
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be consumed prior to challenge so that an anti-inflammatory effect can be observed. This
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therapeutic effect is interesting because those who suffer from such disease can consume
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MPM during crisis and will benefit of the effect.
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reduction of inflammation as demonstrated by the comparison with hydrocortisone and
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probably, a reduction of skin itching and pain.
This study shows an acceptable
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These results suggest that the MPM has a strong anti-inflammatory effect as
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demonstrated by its ability to reduce dermatological inflammation to the same extent than
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hydrocortisone. However in contrast to hydrocortisone, the MPM shows no side effects
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including spleen atrophy, reduced lymphocyte circulating cells or deleterious effect on
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body weight gain (Figures 2, 3 and 4). Hydrocortisone exerts its anti-inflammatory
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potential by suppression of immune cells. The reduction of inflammation observed by
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hydrocortisone treatments correspond to a suppression of total immune cells (not only
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those implicated in inflammation), which is seen by the reduction in blood lymphocytes
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(Figure 4) and in spleen weight (Figure 3) for the mice consuming hydrocortisone.
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Consequently, people consuming hydrocortisone are in a general immunosuppressed state
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and are more susceptible to other diseases. No reduction of immune cells or spleen
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weight differences where observed for the mice who consumed MPM in comparison with
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mice who consumed only water. In fact, there is a trend that shows immune stimulation
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by the MPM consumption.
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Atopic dermatitis is a disease that effects young children, but the use of
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hydrocortisone would not be advisable because of its inhibitory properties on growth
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[47]. This study showed this inhibitory effect on young mice (Figure 2) but growth was
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not affected by MPM consumption. Consequently, consumption of MPM by children
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and young adult in replacement of hydrocortisone as an anti-inflammatory product would
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be a good alternative.
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The absence of these detrimental effects by MPM consumption suggests that the
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mechanism of its anti-inflammatory action is different than that of hydrocortisone.
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However, both hydrocortisone and MPM seem to inhibit neutrophil extravasation and
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accumulation in inflamed tissues as shown with a higher polymorphonuclear cells (PMN)
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in circulation as well as a reduced MPO content in ear (Figures 4 and 5). Results in
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figure 4 demonstrate an inverse correlation between inflammation and PMN counts
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where in the hydrocortisone and MPM groups, the blood PMN counts is higher while the
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ear thickness is lower than reference water group. These results are consistent with those
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observed for ear MPO content (Figure 5). The MPO is an enzyme exclusively present in
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neutrophil granules and its enzymatic activity measured in a tissue is in direct correlation
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of the levels of neutrophils in a tissue [42]. The MPO results showed that the neutrophil
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infiltration in ear of mice that received hydrocortisone and MPM is reduced compared to
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the mice receiving water. The blood PMN count parameter and ear MPO content could
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be explained by the fact that in ACD, the neutrophils (the most important group in PMN)
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move from blood to ear because these cells are principally responsible for inflammation
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[36-38].
The hydrocortisone as well as the MPM seems to prevent the neutrophil
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extravasation from blood to ear, reducing the ear inflammation.
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mechanism causing this inhibition of neutrophil extravasation is different between these
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two groups because of the absence of immunosuppression in MPM group as seen by an
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absence of side effects (Figures 2, 3, and 4).
However, the
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These results demonstrate that, as a new product, the Malleable Protein Matrix
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reduces inflammation and immune dysfunctions when consumed orally while
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maintaining an appropriate immune system threshold.
Experiments to demonstrate the
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mechanism of action responsible for the anti-inflammatory effect of the MPM
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consumption and other parameters to determine how specific cells are implicated and
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influenced by MPM consumption in this ACD model are underway.
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Conclusion
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MPM possesses a strong anti-inflammatory effect comparable to hydrocortisone
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when examined in the ACD model. The anti-inflammatory effects of consumption of
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MPM occur without the undesirable side effects normally associated with
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hydrocortisone. Therefore, MPM would be an alternative of choice for children and
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young adult suffering from chronic inflammatory of various diseases such as ACD. The
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consumption of the MPM could act as a preventive or a therapeutic nutraceutical in the
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case of inflammatory diseases like atopic dermatitis or related diseases such as, psoriasis.
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Psoriasis is an autoimmune disease with similar effects on the immune system to that
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observed for ACD.
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Competing Interests
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Technologie Biolactis (TB) is the industry sponsor of the National Science and
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Engineering Research Council of Canada (NSERC) grant obtained by INRS (CD).
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Research conventions signed between TB and INRS made over the year INRS a minor
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shareholder of TB (less than 1%). The findings of the present study are covered by a
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patent application (PCT CA2002/001899). JB is an on-site scholar of Fond de recherche
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en santé du Québec (FRSQ) and part of the scholarship is covered by TB.
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Authors’ contributions
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JB design the animal studies, carried out the animal and other experiments, perform the
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statistical analysis and drafted the manuscript. CD participated in the design of animal
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studies, data interpretation and the statistical analysis. CD revised the manuscript for the
13
intellectual content and language. PL participated in the design of animal studies, data
14
interpretation and revised the manuscript for the intellectual content and language. All
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authors read and approved the final manuscript.
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Acknowledgements
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The authors wish to thank M. Roger Dubuc for the help in adaptation of the MPO
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enzymatic assay and Lilianne Gueerts for the help in animal studies. François Shareck,
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Alain Lamarre and Denis Girard for the intellectual help in animal design as well as
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results interpretation. Jean-François Lapointe revised the manuscript for the intellectual
22
content and language and participated in data interpretation. This study was funded by the
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National Science and Engineering Research Council of Canada (NSERC) Strategic Grant
16
1
STP 246405-1. JB was a Ph.D. scholar of « Fond de recherche en santé du Québec
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(FRSQ) »
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4
5
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Table legend
1
2
Table 1. Composition of MPM
3
4
Figure Legends
5
Figure 1. Ear thickness of mice administered p.o. with the MPM, hydrocortisone or
6
water. Fig 1A. 7 days prior sensitization and challenges with oxazolone (p<0.07 for
7
MPM and hydrocortisone groups compared with water reference group during the entire
8
experiment) Fig 1B. only after sensitization but during oxazolone challenges (p<0.05 for
9
MPM and hydrocortisone groups compared to water reference group during the entire
10
experiment). Legend: ■ Water, ■ MPM, ■ Hydrocortisone (n=10)
11
Figure 2. Mice weight gain during the ACD model.
12
Legend: ■ Water, ■ MPM, ■ Hydrocortisone (* p < 0.05) (n=10)
13
Figure 3. Spleen weight of mice at the sacrifice of the ACD model.
14
Legend: ■ Water, ■ MPM, ■ Hydrocortisone (** p < 0.01) (n=10)
15
Figure 4. Circulating cell counts 17 days after the first oxazolone challenge.
16
Legend: ■ Lymphocyte counts, ■ PMN counts
17
(* p < 0.05; ** p < 0.01) (n=10)
18
Figure 5. Myeloperoxidase (MPO) contents in ears 18 days after the first oxazolone
19
challenge.
20
(* p < 0.05) (n=10)
24
Table 1. Composition of MPM
Composition (g/100 g)
Humidity
Protein
Fat
Ash (minerals)
Carbohydrates
Lactose
Galactose
80.0
8.3
1.2
5.9
4.7
2.7
0.2
Minerals (mg/100g)
Potassium
Sodium
Calcium
Phosphorus
Selenium
Magnesium
142.9
175.2
1507.4
730.3
< 0.1
5.4
Oligo-elements (mg/100g)
Copper
iron
Manganese
Zinc
Riboflavine (B2)
Niacin (B3)
Pyridoxine (B6)
Cobalamine (B12)
Ascorbic acid (C)
Folic acid
0.07
0.24
0.05
0.13
Vitamins (mg or µg/100g)
0.32 mg
1.00 mg
0.04 mg
Not detected
Not detected
5 µg
Bacterial count (CFU/100g)
LAB
6X1011