Allergic asthma continues to increase despite

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1 originl reserch Bromelin Limits Airwy Inflmmtion in n Ovlbumin-induced Murine Model of Estblished Asthm Eric R. Secor Jr, ND, MPH, MS, LAc; Sonli J. Shh, BS; Lind A. Guernsey, BS; Crig M. Schrmm, MD; Roger S. Thrll, PhD ABSTRACT Context Allergic sthm continues to increse despite new phrmcologicl dvnces for both cute tretment nd chronic-disese mngement. Asthm is multifctoril disese process with genetic, llergic, infectious, environmentl, nd dietry origins. Reserchers re investigting the benefits of lifestyle chnges nd lterntive sthm tretments, including the bility of bromelin to inhibit inflmmtion. Bromelin is commonly used, proteolyticlly ctive pinepple extrct. Objective The present study intended to determine the bility of bromelin to reduce the inflmmtion of preexisting sthm vi n ovlbumin (OVA)-induced murine model of llergic irwy disese (AAD). Design The reserch tem designed study exmining the effects of bromelin in control group of mice tht received phosphte buffered sline (PBS) only nd in n intervention group tht received bromelin in PBS. Setting The study took plce in the Deprtment of Immunology t the University of Connecticut s School of Medicine, Frmington. Intervention The reserch tem sensitized femle C57BL/6J mice with intrperitonel OVA/lum nd then chllenged them with OVA erosoliztion for 10 consecutive dys. On dy 4, the tem begn dministering dily doses of PBS to the control group (n = 10) nd bromelin (6mg/kg) in PBS to the bromelin (intervention) group (n = 10). Outcome Mesures The primry mesures included broncholveolr lvge (BAL) cellulr differentil, cellulr phenotype vi flow cytometry, nd lung histology. Additionl outcomes included testing for serum cytokines nd immunoglobulin. Results Bromelin tretment of AAD mice (bromelin group) resulted in significnt nti-inflmmtory ctivity s indicted by reduced BAL totl leukocytes (P <.05), eosinophils (P <.05), nd cellulr infiltrtes vi lung pthology (P <.005), s compred to the control group. In ddition, bromelin significntly reduced BAL CD4 + nd CD8 + T cells without ffecting cell numbers in the spleen or hilr lymph node. The study found decresed interleukins IL-4, IL-12, IL-17, s well s IFN-α in the serum of bromelin-treted nimls. Conclusions The results suggest tht bromelin hs therpeutic effect in estblished AAD, which my trnslte into n effective djunctive therpy in ptients with similr conditions, such s llergic sthm, who hve chosen to initite tretment fter the onset of symptoms. (Altern Ther Helth Med. 2012;18(5):9-17.) Eric R. Secor Jr, ND, MPH, MS, LAc, is n ssistnt professor; Sonli J. Shh, BS, is doctorl cndidte; Lind A. Guernsey, BS, is reserch ssocite; nd Roger S. Thrll, PhD, is professor, Deprtment of Immunology, University of Connecticut School of Medicine, Frmington. Crig M. Schrmm, MD, is n ssocite professor, Deprtment of Peditrics, University of Connecticut School of Medicine. Corresponding uthor: Eric R. Secor Jr, ND, MPH, MS, LAc E-mil: esecor@uchc.edu Author Disclosure Sttement: This reserch ws supported by NIH grnts: NCCAM K-08-AT001569, AI R01 HL-43573, nd AI R21 AI Authors hve no conflicts of interest regrding this reserch. Allergic sthm continues to increse despite new phrmcologicl dvnces for both cute tretment nd chronic-disese mngement. The keystones of sthm therpy include (1) voidnce of triggers, (2) relief of symptoms with inhled β-drenergic gonists, nd (3) control of irwy inflmmtion with inhled nd orl corticosteroids, inhled chromones, nd orl leukotriene ntgonists. Asthm is multifctoril disese process with genetic, llergic, infectious, environmentl, nd dietry origins. 1 Reserchers re investigting the benefits of lifestyle chnges nd lterntive sthm tretments. 2 They seek evidencedbsed tretments tht might become sfe nd efficcious therpies djunctive to the current tretment formulry. Bromelin Reduces Estblished Asthm ALTERNATIVE THERAPIES, sept/oct 2012, VOL. 18, NO. 5 9

2 According to the 2007 Ntionl Helth Interview Survey, roughly 40% of dults in the US popultion use some form of complementry or lterntive medicine (CAM). 3 Severl studies hve exmined the use of CAM by ptients with sthm, with use estimtes rnging from 4% to 79% in dults nd from 33% to 89% in children. 4-7 Among the most commonly used CAMs re brething techniques, herbl products, homeopthy, nd cupuncture. 4,8 Nevertheless, reserchers hve not found strong evidence for the effectiveness of these modlities. 9 Lck of evidence hs been ttributed to clinicl trils with smll smple sizes, poor methodology, nd pucity of mechnistic dt generted in wellchrcterized preclinicl models. 10,11 Reserchers hve estblished severl murine models of sthm or llergic irwy disese with the im of understnding the immunologicl mechnisms tht underlie this complex disese These models shre severl fetures tht include (1) sensitiztion phse with n djuvnt (lum) nd model ntigen ovlbumin (OVA), (2) n ntigen chllenge phse vi trchel instilltion or nose-only erosoliztion, nd (3) the genertion of lung inflmmtion chrcterized by infiltrtion of white blood cells (eosinophils), lung pthology, nd inflmmtory Th2-skewed cytokines (interleukins IL-4, IL-5, IL-3) ,16,17 Modeling llows for investigtion of the effectiveness of numerous, novel, ntisthm therpeutics tht hve rich history of trditionl use, such s propolis 18 ; Cmelli sinensis 19 ; Chinese-ptent botnicl formuls 20 ; Boswelli serrt (frnkincense), Glycyrrhiz glbr (licorice), nd Curcum long (turmeric) 21 ; Mimos pudic 22 ; nd Anns comosus (common pinepple) A criticl distinction exists, however, between the models nd the ctul use of the therpies (ie, the schedule t which prctitioners deliver the therpy). This schedule includes the periods before or during sensitiztion, fter sensitiztion, before the cute ntigen chllenge, or fter the development of inflmmtory disese. Severl investigtors hve been interested in the ntiinflmmtory mechnisms of specific extrcts of A comosus, such s the commonly used, proteolyticlly ctive pinepplederived bromelin. Reserchers re evluting the extrcts in vriety of both in vivo nd in vitro models. Bromelin is combintion of sulfur-contining cysteine endopeptidses tht hve brod specificity for the clevge of proteins. Some reserchers hve proposed one mechnism of ction to ccount for bromelin s therpeutic ctivity: the clevge of lymphocyte-cell surfce receptors, such s CD4, CD8, CD44, nd CD62L. Receptor clevge cn result in ltered cell communiction, cell trfficking, nd cell signling pthwys, leding to the modultion of pro- nd nti-inflmmtory cytokines such s IL-2, IL-4, IL-6, nd TNF-α This modultion mkes it unique choice for modulting llergic sthm. Reserchers hve shown tht bromelin hs potentil beneficil effects in treting infections, cncer, crdiovsculr disese, musculoskeletl injuries, nd rthritis. 28 The reserch tem for the current study hs shown previously tht bromelin, injected intrperitonelly nd dministered orlly, cn reduce irwy inflmmtion in mouse model of sthm, 29,30 t lest in prt through enzymtic clevge of CD25 from ctivted T cells. 31 The current tem notes, however, tht its previous studies ddressed the bility of bromelin to ttenute the development of sthm when given to sensitized mice prior to nd during exposure to n erosol ntigen. While this model my hve merit for preventing the development of sthm in previously sensitized llergic individuls, it did not ssess whether bromelin could modulte existing sthm when prctitioners pply tretment fter onset. The outcomes for tril studying the second circumstnce hold more relevnce in clinicl setting, nd s the gol of its present study, the reserch tem hs chosen to exmine tht second circumstnce. METHODS Animls The reserch tem purchsed femle C57BL/6J mice, 3 to 6 months of ge with weights from 17 g to 20 g (Jckson Lbortory, Br Hrbor, Mine), nd housed them conventionlly in plstic cges with corncob bedding. The tem mintined the mouse room t 22 C to 24 C with dily light/drk cycle (light from 0600 to 1800 hours). The study supplied food nd wter d libitum. The Animl Cre Committee t the University of Connecticut Helth Center pproved ll protocols for mouse use. Interventions Ovlbumin Sensitiztion nd Aerosol Exposure Protocol. The reserch tem immunized mice with three weekly intrperitonel injections of suspension contining 25 µg of OVA (grde V; Sigm Chemicl, St Louis, Missouri) nd 2 mg of luminum hydroxide (lum) in 0.5 ml of 0.9% sodium chloride (ph 5.5, 308 mosmol/l; Bxter Helthcre, Deerfield, Illinois). One week fter the lst injection, the reserch tem exposed the mice to erosolized 1% OVA in norml sline, 1 hour per dy for 10 dys. 29,30 A BANG nebulizer (CH Technologies, Westwood, New Jersey) generted the erosols into 7.6-L inhltion-exposure chmber with ttched restrint tubes. The mss-medin erodynmic dimeter nd geometric stndrd devitions were 1.4 µm nd 1.6 µm, respectively. The estimted dily inhled OVA dose pproximted 30 µg to 40 µg per mouse. Control nd Bromelin Tretment. The reserch tem mde stock solution of stem bromelin, Lot no (Vitl Nutrients, Middletown, Connecticut), using 60 mg of bromelin dissolved in 250 ml of phosphte buffered sline (PBS). For the bromelin group (n = 10), the reserch tem dministered 6 mg/kg of bromelin in 0.5 ml of PBS. The control group (n = 10) received 0.5 ml of PBS only. The tem delivered the tretments intrperitonelly twice dily for 7 consecutive dys, beginning on dy 4 of the OVA erosol chllenge (Figure 1). The reserch tem optimized the bromelin dose bsed on previous in vivo dose-response studies tht its lbortory hd performed. 29,30 As previously report- 10 ALTERNATIVE THERAPIES, SEPT/OCT 2012, VOL. 18, NO.5 Bromelin Reduces Estblished Asthm

3 Figure 1. Bromelin Tretment Protocol The reserch tem sensitized mice (n = 20) with three ovlbumin-lum intrperitonel injections 1 week prt (dy 21, dy 14, dy 7). From dys 1 to 10, nimls underwent erosol chllenge with 1% OVA in sline for 1 hour dily. From dys 4 to 10, nimls received tretment with either bromelin (6 mg/kg in 0.5 ml phosphte buffered sline [PBS]) or PBS only (0.5 ml) twice dily. The reserch tem scrificed the nimls 24 h fter the lst tretment nd hrvested broncholveolr lvge, lung tissue, hilr nodes, nd spleen from ech mouse to ssess the outcomes of llergic irwy disese. Sensitiztion OVA / lum 3 IP injections Aerosol chllenge 1% OVA/sline 1 h/d AAD Outcomes BAL WBCs, eos, lymphs Lung histology Cytokines nd Immunoglobulins Tretments (dys 4-10) Phosphte buffered sline (PBS) 0.5 ml Bromelin 6 mg/kg in 0.5 ml PBS Administered IP 2x/d Abbrevitions: AAD, llergic irwy disese; BAL, broncholveolr lvge; eo, eosinophil; IP, intrperitonel injections; lymph, lymphocyte; OVA, ovlbumin; PBS, phosphte buffered sline; S, scrificed; WBC, white blood cell (leukocyte). ed, the reserch tem hd tested bromelin independently for uthenticity, potency ( GDU/g), microbil contmintion, residul solvents, hevy metls, fltoxins, nd endotoxin (Vitl Nutrients, Middletown, Connecticut; ChromDex, Clerwter, Florid; nd Phrmline, Florid, New York). 31 Twenty-four hours fter the finl erosol exposure nd the finl bromelin tretment, the reserch tem scrificed the mice for tissue nlysis. Outcome Mesures Broncholveolr Lvge nd Tissue Processing At scrifice, the reserch tem hrvested broncholveolr lvge (BAL) fluid, hilr lymph nodes (HLN), nd spleens nd processed them for the isoltion nd enumertion of leukocytes. For collection of BAL, the tem lvged the lungs in situ, with five 1.0-mL liquots of sterile sline. The tem determined the totl protein concentrtions in BAL fluid using the Pierce Bicinchoninic Acid Protein Assy Kit (Thermo Scientific Pierce, Rockford, Illinois) with OVA s the stndrd. The tem hrvested the lymph nodes nd spleens nd mechniclly disrupted them into single-cell suspensions, using lysis of splenic erythrocytes Tris mmonium chloride (TAC) lysis buffer (9 prts 0.83% w/v NH 4 Cl; 1 prt 2.57% w/v Tris, ph 7.0; TAC solution). For ll tissue smples, the tem obtined counts of the totl nucleted cells using hemocytometer with nigrosin dye exclusion s mesure of vibility. Flow Cytometry Post centrifugtion (1500 rpm x 10 min) the reserch tem resuspended cell pellets vi mnul disruption in PBS contining 0.2% bovine serum lbumin (BSA) nd 0.1% NN 3 t concentrtion of 1 x 10 5 to 1 x 10 6 white blood cells/ml. The tem then incubted 100 µl of the cells with 100 µl mab (diluted per mnufcturers recommendtions for 30 min t 4 o C). After stining the cells, the tem wshed them twice with PBS-0.2% BSA-0.1% NN 3 solution nd mesured their reltive fluorescence intensities on 4-decde log scle. For the mesurement, the reserch tem used flow-cytometric nlysis with LSR II (Becton-Dickinson, Sn Diego, Cliforni) nd BD FACSDiv Softwre v 4.1 (Becton Dickinson, Sn Jose, Cliforni). The tem nlyzed the results with FlowJo (Tree Str, Ashlnd, Oregon) nd used the following fluorescence-lbeled monoclonl ntibodies: CD4-PcOrnge (RM4-5); CD8-APC-780 (53-6.7); CD19-PE (1D3); CD25-AF-700 (PC61.5); F4/80- PECy7 (BM8); CDllc-APC (N418); nd MHCII-FITC (2G-9) (Phrmingen, Sn Jose, Cliforni, or ebioscience, Sn Diego, Cliforni). Bromelin Reduces Estblished Asthm ALTERNATIVE THERAPIES, sept/oct 2012, VOL. 18, NO. 5 11

4 Lung Histology For nimls tht did not undergo BAL (n = 4 per group), the reserch tem fixed the removed lungs with 10% buffered formlin nd processed them in stndrd mnner. The tem stined tissue sections with hemtoxylin nd eosin nd evluted ll specimens with microscope-mounted Nikon Eclipse 400cmer (Tokyo, Jpn). The tem creted digitl imges using Spot RT Slider Softwre (Sterling Heights, Michign) nd evluted them in Microsoft Photo Editor (Redmond, Wshington). Five seprte individuls grded the degree of cellulr infiltrtion (0-5) in blinded mnner. Serum Immunoglobulin nd Cytokine Mesurements The reserch tem thwed ll serum smples nd vortexed them prior to immunoglobulin nd cytokine mesurement. The tem determined immunoglobulin concentrtions using Milliplex Mouse Immunoglobulin Isotype Kit Pnels (Ct #MGAM-300; Millipore, Billeric, Msschusetts) following the mnufcturer s instructions. Assy sensitivities (minimum detectble concentrtions) were IgM 0.3 ng/ml; IgG1 0.3 ng/ml; IgG3 0.4 ng/ml; IgG2 0.4 ng/ml; IgG2b 0.4 ng/ml; nd IgA 0.7 ng/ml. For ssessment of cytokines nd chemokines, the tem processed smples with Milliplex Mouse Cytokine/Chemokine kit (Ct #MPXMCYTO70KPMX22; Millipore, Billeric, Msschusetts) following mnufcturer s instructions. Assy sensitivities were IL-1α 5.1 pg/ml; IL-1β 2.0 pg/ml; IL pg/ml; IL pg/ml; IL pg/ml; IL pg/ ml; IL-12(p40) 4.9 pg/ml; IL pg/ml; IL pg/ ml; IL pg/ml; nd TNFα 1.0 pg/ml. Sttisticl Anlysis The reserch tem mde sttisticl comprisons between groups with nlysis of vrince nd unpired t tests using JMP Softwre (SAS Institute, Cry, North Crolin). Serum cytokine levels were not normlly distributed, nd the tem log-trnsformed them for sttisticl nlysis. The tem expressed ll dt s mens ± stndrd error of the men nd considered differences to be significnt t P <.05. RESULTS Bromelin Tretment Ws Nontoxic As the reserch tem hs reported previously, 29 intrperitonel bromelin ws nontoxic, s ssessed by body weight nd BAL protein concentrtion. The tem noted no Figure 2. Effect of Bromelin Tretment on Totl Leukocytes For mice with llergic irwy disese, bromelin significntly reduced totl broncholveolr (BAL) lvge leukocytes nd eosinophils in the bromelin group (blck brs) s compred to the control group (gry brs) without chnge in BAL mcrophges, neutrophils, or lymphocytes. The reserch tem sw no significnt reduction in leukocytes in the tissue of the spleen or hilr lymph nodes. The dt represent men ± stndrd error of the men vlues. Totl number (x 10 5 ) Totl leukocytes (x10 6 ) Abbrevitions: BAL, broncholveolr lvge; eo, eosinophil; HLN, hilr lymph node; lymph, lymphocyte; mc, mcrophge; PMN, neutrophil; WBC, white blood cell (leukocyte). Indictes P <.05 for the bromelin vs the control group; n = 10 nimls per group. 12 ALTERNATIVE THERAPIES, SEPT/OCT 2012, VOL. 18, NO.5 Bromelin Reduces Estblished Asthm

5 difference in body weight in bromelin-treted mice s compred to PBS-treted control nimls (19.1 ± 0.26 g vs 18.8 ± 0.35 g; P =.54). Also, no significnt chnge in BAL totl protein concentrtions occurred between bromelintreted mice nd PBS controls (143 ± 10 μg/ml vs 174 ± 17 μg/ml; P =.18; n = 10 nimls per group). Bromelin Decresed Pulmonry Eosinophili in Estblished Asthm Exposure of OVA-sensitized mice to dily OVA erosols resulted in AAD, chrcterized by robust increses in BAL leukocytes nd eosinophils (Figure 2). Tretment with bromelin significntly inhibited OVA-induced increses in BAL leukocytes (7.22 ± 1.20 vs ± 3.84 x 10 5 cells in control mice; P =.035; n = 10 ech) nd eosinophils (1.65 ± 0.40 vs 5.80 ± 1.87 x 10 5 cells; P =.044), without chnge in other cell types. In contrst to leukocyte numbers in BAL fluid, bromelin did not chnge those numbers in the spleens nd HLNs of the sensitized nd chllenged mice (Figure 2). Bromelin Selectively Decresed CD4 + T nd CD8 + T Cells in Broncholveolr Lvge From Mice With Asthm Although the bromelin tretment did not significntly reduce the totl number of BAL lymphocytes (Figure 2; control [2.3x10 6 ± 0.6]; bromelin [1.2x10 6 ± 0.4, P =.13]), bromelin did reduce the distribution of BAL CD4 + T cells nd CD8 + T cells in the treted nimls of the intervention group (Tble 1). BAL CD19 + B cells were trended towrd significnce between the two groups P =.05. Nevertheless, bromelin did not ffect the reltive percentges of (1) CD4 + (control [10.3 ± 1.8%], bromelin [7.6 ± 1.4%], P =.26); (2) CD8 + (control [10.8 ± 2.7%], bromelin [7.8 ± 1.4%], P =.35); nd (3) CD19 + lymphocytes (control [9.3 ± 2.3%], bromelin [11.3 ± 2.7%], P =.57) (Tble 1). Similrly, bromelin exerted no effects on the number or percentges of splenic or HLN lymphocytes (Tble 1). In contrst, both the bsolute numbers nd percentges of BAL CD4 + T cells tht expressed the surfce ctivtion mrker CD25 + were lower in the bromelin group compred to control nimls (P =.012; Figure 3). The percentges of CD4 + T cells expressing CD25 were unchnged in the spleen (P =.27) nd HLNs (P =.33; Figure Figure 3. Effect of Bromelin Tretment on CD4 + CD25 + T Lymphocytes In broncholveolr lvge fluid, bromelin tretment significntly decresed the percentge of CD4 + CD25 + T cells (blck brs) s compred to phosphte buffered sline treted control mice (gry brs). In contrst, bromelin hd no effect on CD4 + CD25 + T cell percentges in the spleens or hilr lymph nodes. The dt represent men ± stndrd error of the men vlues; n = 10 nimls per group. % CD4 + T cells Abbrevition: HLN, hilr lymph node. Indictes P <.05 for the bromelin vs the control group. Bromelin Reduces Estblished Asthm ALTERNATIVE THERAPIES, sept/oct 2012, VOL. 18, NO. 5 13

6 Tble 1. Lymphocyte Distributions in Tissues From Bromelintreted nd Control Mice BAL (totl cells x 10 6 ) Control Bromelin P-vlue 3). CD4 + T cell 2.41 ± ± CD8 + T cell 2.29 ± ± CD19 + B cell 2.81 ± ± Spleen (totl cells x10 6 ) CD4 + T cell ± ± CD8 + T cell ± ± CD19 + B cell 3964 ± ± HLN (totl cells x10 6 ) CD4 + T cell 48.8 ± ± CD8 + T cell 42.5 ± ± CD19 + B cell 42.5 ± ± BAL (%) CD4 + T cell 10.3 ± 1.8% 7.6 ± 1.4%.26 CD8 + T cell 10.8 ± 2.7% 7.8 ± 1.4%.35 CD19 + B cell 9.3 ± 2.3% 11.3 ± 2.7%.57 Abbrevitions: BAL, broncholveolr lvge; HLN, hilr lymph node. The distribution of lymphocyte subsets (CD4+ nd CD8+ T cells nd CD19+ B cells) ws mesured in the broncholveolr lvge (BAL), spleen, nd hilr lymph nodes (HLNs) nd % of cells in the BAL fter 10 dys of ovlbumin erosol exposures in mice receiving phosphte buffered sline (control group) nd s compred to bromelin tretment on dys Significnt reductions were observed in totl cell numbers in the BAL between tretments. No differences were noted in totl cells in the spleen nd HLN or in % BAL in ny cells subsets. Dt represent men ± stndrd error of the men; n = 10 nimls in ech group. Bromelin Attenuted Lung Histopthologic Chnges During Allergic Airwy Disese The reserch tem performed histologicl evlutions nd obtined pthology scores on unmnipulted, uninflted, formlin-fixed lungs from seprte groups (n = 4 ech) of PBS-treted control nimls nd bromelin-treted mice with AAD. The tem stined the smples with hemtoxylin nd eosin (Figure 4). As demonstrted in the top pnels, the AAD mice in the control group (Figure 4A) showed substntil peribronchil nd perivsculr inflmmtion comprising lymphocytes, plsm cells, nd eosinophils. Bromelin-treted AAD mice hd less histologicl injury (Figure 4B). Sttisticl comprison of pthologicl scoring by five blinded reviewers demonstrted significntly less pthology in the bromelin-treted mice thn in the control nimls (men score 1.40 ± 0.27 vs 2.35 ± 0.31; P =.0077; Figure 4C). Bromelin Did Not Affect Serum Immunoglobulin Levels but Decresed Selective Serum Cytokines Bromelin tretment did not elicit generlized immune suppression, s evidenced by lck of direct effect on serum immunoglobulin levels (Tble 2) or generlized effect on serum cytokines. As seen in Figure 5, however, bromelin-treted nimls did demonstrte selective decreses in key cytokines, including IL-4 (0.33 vs 1.38 pg/ml; P =.0002), IL-12 (8.67 vs 36.8 pg/ml; P =.003), IL-17 (0.54 vs 3.88 pg/ ml; P =.0004), IFN-g (1.56 vs 86.5 pg/ml; P =.016). In contrst, the chemokine interferon, gmm-induced protein (IP-10) incresed in bromelin-treted nimls (61.6 vs 39.2 pg/ml; P =.02). DISCUSSION The current study is continued chrcteriztion of the use of bromelin, cysteine protese extrcted from pinepple, in sthm. The reserch tem hs previously shown tht intrperitonel nd orl dministrtion of bromelin cn prevent the development of sthm in sensitized mice when given prior to erosolized ntigen exposure. 29,30 Since most people tking bromelin for sthm, however, would hve n estblished disese lredy nd my be experiencing symptoms, more relevnt question is whether bromelin cn ttenute existing sthm. To the reserch tem s knowledge, few reports exist tht study the effects of botnicls in models of estblished or chronic sthm. 20 The present study demonstrted beneficil effect of bromelin when dministered 3 dys fter consecutive OVA erosol chllenge, time when eosinophilic irwy inflmmtion nd irwy hyperresponsiveness were well estblished. 17,29,31 At tht point, bromelin ws cpble of ttenuting the irwy leukocytosis nd eosinophili nd reducing the histopthologicl chnges in the lung tht occur in sthm. This nti-inflmmtory effect ppered to be tissue specific, s no chnges occurred in the spleen or HLN leukocyte popultions or in serum immunoglobulin levels, even though the reserch tem gve the bromelin systemiclly. Brod decreses in CD4 + nd CD8 + T cells nd CD19 + B cells in the BAL fluid ccompnied the decrese in irwy eosinophilic inflmmtion. Such chnges were lso selective to the irwys, nd the reserch tem did not see them in HLNs or spleens. While cell numbers decresed significntly, no chnges occurred in the reltive percentges of mjor lymphocyte subpopultions in BAL for either the control or the bromelin-treted mice. This finding differed from wht the reserch tem hd observed when it gve bromelin before the initition of the OVA ero- 14 ALTERNATIVE THERAPIES, SEPT/OCT 2012, VOL. 18, NO.5 Bromelin Reduces Estblished Asthm

7 Figure 4. Bromelin Tretment Reduces Lung Pthology The reserch tem stined lung sections with hemtoxylin nd eosin, nd five individuls scored the smples blindly on scle from 0 to 5, bsed on the level of pthology. Pnels A nd B illustrte representtive pthology from control, phosphte buffered sline treted mouse (A) nd bromelin-treted mouse (B). The inflmmtion (rrows) surrounding irwys (w) nd blood vessels (bv) ppered significntly reduced in the bromelin (B) niml s compred to the control niml (A). Figures re t 10X mgnifiction. Pnel C demonstrtes men ± stndrd error of the men pthology scores for ech group (n = 4 ech). Men pthology score Abbrevitions: BR, bromelin; PBS, phosphte buffered sline. Indictes P <.05 between groups. Tble 2. Serum Immunoglobulin Levels in Control nd Bromelin-treted Mice IgG1 IgG2 IgG2b IgG3 IgA IgM PBS 1836 ± ± ± ± ± ± 161 BR 2167 ± ± ± ± ± ± 214 P-vlue Abbrevitions: BR, bromelin; Ig, immunoglobulin; PBS, phosphte buffered sline. Serum immunoglobulin levels were mesured fter 10 dys of ovlbumin erosol exposures in mice receiving phosphte buffered sline (control) nd receiving bromelin (intervention) on dys The tem sw no significnt differences in ny immunoglobulin clss. Dt represent men ± stndrd error of the men; n = 4 nimls in ech group. Bromelin Reduces Estblished Asthm ALTERNATIVE THERAPIES, sept/oct 2012, VOL. 18, NO. 5 15

8 Figure 5. Bromelin Reduces Selected Cytokine Levels Bromelin tretment selectively decresed specific cytokine levels in serum of mice with llergic irwy disese. The dt represent men ± stndrd error of the men vlues; n = 4-10 smples per group. Concentrtion (pg/ml) b b Abbrevitions: G-CSF, grnulocyte colony-stimulting fctor; IL, interleukin; IFN, interferon; KC, kertinocyte chemottrctnt; MCP, monocyte chemotctic protein; MIP, mcrophge inflmmtory protein; RANTES, regulted upon ctivtion, norml T-cell expressed nd secreted; TNF, tumor necrosis fctor. Indictes P <.05. b Indictes P <.005 for the bromelin vs the control group. sol chllenges. In tht model, bromelin tretment decresed totl lymphocytes in ddition to BAL lymphocyte subsets. 30 A potentil explntion for the discrepncy could be the difference in the time of bromelin dministrtion. In the tem s model from its other studies, significnt lymphocyte recruitment to the irwys occurred by dy 3 of OVA exposures. 29,30 Crson et l found from dy 3 to dy 10 tht the predominnt Th2-skewed lymphocyte found in the BAL during AAD ws the CD4 + CD25 +, ctivted T effector cells. 14 In the reserch tem s other studies, tretment with bromelin prior to erosol chllenge my hve ltered the pttern of initil CD4+ T-cell recruitment wheres, in the current study, lter dministrtion of bromelin (from dy 3 to 10), occurred fter sthm ws well estblished (eg, CD4 T cells re prominent). While totl CD4 + T cells were unchnged in the current study, bromelin tretment significntly reduced the percentge of CD4 + T cells expressing the ctivtion mrker CD25. Agin, this finding ws specific to BAL, nd the reserch tem did not see it in the spleen or HLN. The generl inhibition of irwy inflmmtion exerted by bromelin my explin the regionl reduction in CD4 + CD25 + T cells nd hence the existence of fewer cells tht hve n ctivted (CD25 + ) phenotype. It my lso reflect selected removl of CD25 (the high ffinity IL2-Rα) from T cells by the bromelin tretment, which the reserch tem previously hs reported occurs with tretment. 31 Reduction of CD25 my limit IL-2 from binding, which corresponds to reduced cell expnsion nd differentition. Ameliortion of disese lso could occur by selective expnsion of regultory T cells. The reserch tem, however, hs shown previously tht bromelin tretment lso reduced CD25 from regultory T cells (which constitutively express CD25) while hving no effect on expression of Foxp3. Bromelin did not ffect serum immunoglobulin levels, further suggesting lck of generlized immunomodultory effect. The bromelin tretment, however, selectively decresed some serum cytokines specificlly, interleukins 4, 12, nd 17 s well s IFN-γ. The tretment did not ffect other Th2 cytokines, such s IL-5 nd IL-13. The role of IL-4 in sthm is well estblished. The differentition of nïve T lymphocytes into Th2 cells in the presence of n llergen requires this cytokine, nd it is the principl stimulus for B-cell isotype switching to IgE. 32 The reserch tem would expect inhibition of this key Th2 cytokine to ttenute the AAD response. IL-17 expression in the irwys is lso upregulted fter sensitiztion nd chllenge with ntigen, nd it is ssocited prticulrly with neutrophil recruitment nd more severe sthm. 33 Of interest, Th17 cells re resistnt to steroids in vitro, nd recent study indicted tht irwys hyperrectivity induced by trnsferred Th17 cells in mouse model is steroid-resistnt. 34 Zho et l hve found tht some humn prticipnts with steroid-resistnt sthm hve elevted levels of Th17 cells compred to helthy controls. 35 Tht bromelin decresed IL-17 in AAD mice rises the intriguing specultion tht it could be useful in some 16 ALTERNATIVE THERAPIES, SEPT/OCT 2012, VOL. 18, NO.5 Bromelin Reduces Estblished Asthm

9 humns with refrctory, Th17-driven sthm. Additionlly, bromelin significntly incresed serum levels of interferon γ-inducible protein (IP-10). Reserchers hve found tht IP-10 expression occurs in bronchil epithelil cells nd believe tht the effect is ssocited with humn immune defense ginst pthogens. 36 It is uncler, however, if incresing levels of IP-10 in nonvirl, llergic, irwy-disese model would provide dded support. Likewise, reserchers would not expect bromelin s inhibition of IFN-γ nd IL-12 to be therpeutic trget in sthm nd tht inhibition my demonstrte some broder immunomodultory effects of the gent. Conclusion In summry, dministrtion of bromelin fter the onset of AAD (sthm) inhibited progressive irwy eosinophili by ~70%. This effect ws similr to the ~55% reduction in irwy eosinophils seen when the current reserch tem gve bromelin to sensitized mice before initition of OVA erosol chllenges in prior study. 29,30 Thus, bromelin my be s effective in treting existing sthm s it is in preventing the development of llergic irwy inflmmtion. The ttenuted irwy eosinophili ws ssocited with fewer irwy CD4 + nd CD8 + T cells, decresed numbers of ctivted CD4 + CD25 + T cells, lower levels of Th2 cytokines, nd reduced lung histopthology. This modultory role of bromelin ppered to be specific to the tissue or site of inflmmtion, s tretment did not ffect lymphocyte numbers in the spleen or HLN. These observtions suggest tht bromelin my be effective in humn sthmtics with existing disese, perhps prticulrly in those with more steroidresistnt sthm. Acknowledgements The reserch tem thnks the following people for their technicl ssistnce: Enrico P. Liv, ND, RPh, of Vitl Nutrients for providing the Bromelin extrct nd offering his qulity control expertise nd Justin Hummel for ssisting with niml hndling. REFERENCES 1. Miller AL. The etiologies, pthophysiology, nd lterntive/complementry tretment of sthm. Altern Med Rev. 2001;6(1): Kopnin H, Hfkens J. Necessry lterntives: ptients views of sthm tretment. Ptient Prefer Adherence Jun 24;4: Brnes PM, Bloom B, Nhin RL. Complementry nd lterntive medicine use mong dults nd children: United Sttes, Ntl Helth Stt Report Dec 8;12: Slder CA, Reddel HK, Jenkins CR, Armour CL, Bosnic-Anticevich SZ. Complementry nd lterntive medicine use in sthm: who is using wht? Respirology. 2006;11(4): Mrks G, Cohen M, Kotsirilos V, et l. Asthm nd Complementry Therpies: A Guide for Helth Professionls. Ntionl Asthm Council of Austrli. Accessed June 19, Mrino LA, Shen J. Chrcteristics of complementry nd lterntive medicine use mong dults with current sthm. J Asthm. 2006;47(5): Argüder E, Bvbek S, Sen E, et l. 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Inhibitory effects of lbuterol nd fenoterol on RANTES nd IP-10 expression in bronchil epithelil cells. Peditr Allergy Immunol. 2011;22(4): Bromelin Reduces Estblished Asthm ALTERNATIVE THERAPIES, sept/oct 2012, VOL. 18, NO. 5 17