Modulatory role of regulatory T cells in a murine model of severe equine asthma

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1 Henríquez et l. BMC Veterinry Reserch (2017) 13:117 DOI /s RESEARCH ARTICLE Open Access Modultory role of regultory T cells in murine model of severe equine sthm Cludio Henríquez 1, Griel Morán 1*, Cristin Crrsco 3, José Srmiento 4, Miguel Brrí 2, Hugo Folch 2 nd Benjmin Uerti 5 Astrct Bckground: It is ccepted tht T regultory cells (Treg) control different types of immune responses. In connection with this role, we hve recently descried n importnt increse in CD4+, CD25 high, Foxp3+ lymphocytes in the irwy system of horses coursing with n excertion of severe equine sthm (EA). To explore the potentil role of this popultion in the resolution of EA inflmmtion, we used murine experimentl model in which irwy neutrophilic inflmmtion, which is similr to tht oserved in EA, is induced in mice y continul exposure to Aspergillus fumigtus contminted hy. This model hs the dvntge tht in mice we my induce reduction of the Treg popultion using low doses of cyclophosphmide (Cy). Results: The results indicted tht the percentge of Treg cells incresed with llergen exposure, s in horses; nd nimls prtilly depleted of Treg cells y tretment with Cy showed incresed irwy inflmmtion, demonstrted y n incresed percentge of neutrophils nd specific immunogloulins in roncholveolr lvge fluid (BALF). Furthermore, histopthologic study of nimls tht were pretreted with Cy efore ntigenic chllenge showed higher cellulr infiltrtion in the lung nd deeper remodeling chnges in the ronchi, including epithelil nd golet cell hyperplsi s well s irwy smooth muscle hypertrophy. Conclusion: In this murine model of EA, the reduced numer nd function of Treg induced y low doses of Cy, which directly correltes with incresed irwy inflmmtion nd lung infiltrtion, indictes tht Treg my ply mjor role in the regultion nd resolution of EA. Keywords: Equine sthm, Recurrent irwy ostruction, Regultory T cells, Airwy inflmmtion Bckground Equine sthm (EA) [1 3] formerly clled inflmmtory irwy disese (mild sthm) nd recurrent irwy ostruction (severe sthm) is chrcterized y periods of cute ronchoconstriction, ronchil hyper-responsiveness, pulmonry neutrophili, decresed lung complince nd n increse in totl lung resistnce. Acute episodes of excertion re commonly triggered y inhltion of pollutnts such s Feni rectivirgul, Thermoctinomyces vulgris nd Aspergillus fumigtus [4, 5]. Resolution of irwys inflmmtion is likely medited vi multiple mechnisms, with Tregs potentilly plying mjor contriutory role. * Correspondence: gmorn@uch.cl 1 Phrmcology nd Morphophysiology Deprtment, Fculty of Veterinry Sciences, Universidd Austrl de Chile, Vldivi, Chile Full list of uthor informtion is ville t the end of the rticle It is generlly ccepted tht regultory T cells ply n importnt role in tumor growth [6], control of utoimmunity [7] nd llergic-medited processes [8]. Since the puliction of the seminl pper y Shimon Skguchi [9] pointing out the role of CD4+, CD25+ s the T lymphocytes responsile for suppression of the immune response, nd ecuse of the importnt contriution of Fontenot [10], who descried the role of trnscription fctor Foxp3 s second mrker for this lymphoid popultion, CD4+, CD25+, Foxp3+ cells hve een the most studied Treg supopultion, nd their relevnce hs een evluted in mice nd humns in the context of different immune-medited diseses [11 18]. However, in domestic nimls, informtion out the role of these cells in immune regultion is scrce. In this context, we hve recently demonstrted tht in horses with severe EA excertion fter chllenge with moldy hy contminted with Aspergillus fumigtus, the numer of Treg cells is gretly The Author(s) Open Access This rticle is distriuted under the terms of the Cretive Commons Attriution 4.0 Interntionl License ( which permits unrestricted use, distriution, nd reproduction in ny medium, provided you give pproprite credit to the originl uthor(s) nd the source, provide link to the Cretive Commons license, nd indicte if chnges were mde. The Cretive Commons Pulic Domin Dediction wiver ( pplies to the dt mde ville in this rticle, unless otherwise stted.

2 Henríquez et l. BMC Veterinry Reserch (2017) 13:117 Pge 2 of 9 incresed in lymphocytes recovered from BALF [19]. This increse occurs in prllel with irwy inflmmtion, ut, currently, we my only speculte out the importnce of Treg in the control nd resolution of the cute severe EA process. An pproch to nswer this question my e ttempted through the use of recently descried murine EA model tht we hve set up in our lortory [20]. The essence of this model is the use of hy contminted with Aspergillus fumigtus s edding for experimentl mice. After eing housed in this condition, Rockefeller (RK) mice show n increse in the percentge of neutrophils in BALF, higher levels of specific ntiodies ginst the fungus in the respirtory trct, nd histology tht demonstrtes intense polymorphonucler infiltrtion of the irwy: these re importnt fetures tht lso pper in EA [1]. Moreover, in our experimentl model, the disese is induced in nimls over 5 months of ge ecuse younger mice re less sensitive or refrctory to the induction of irwy inflmmtion; the sme ge dependence occurs in the equine species, where the disese is usully oserved in horses older thn 8 yers. In the mice of this experimentl model, s lso occurs in horses with excertion of severe EA, irwy inflmmtion is resolved when nimls return to non-contminted environment. It is worthy of note tht the murine strin used here does not hve incresed individul susceptiility to irwy inflmmtion, s does occur in EA-susceptile horses [21]. Despite this limittion of the experimentl model, the description of immunologicl processes could e pplicle to EA-susceptile nd non-susceptile individuls. This murine model of severe EA presented us with the opportunity to modulte the Treg popultion using low doses of cyclophosphmide (Cy), s descried erlier y Bron [22]. In this context, Askense s work [23] showed tht Cy given t doses tht do not suppress the immune response my increse delyed-type hypersensitivity rections in mice; our lortory lso reported -in tht low doses of Cy rogte the phenomenon of ntigenic competition [24]; t tht time, this effect ws ttriuted to the ctivtion of T suppressor cell. It since ecme evident tht low doses of Cy potentite the immune response in severl niml models of cncer [25 27]. Methods Animls RK femle mice, ged 2 months (young) or 8 months (old), were used in ll experiments. All nimls were otined nd mintined t the Animl Fcility of the Universidd Austrl de Chile. During exposure to A. fumigtus contminted hy, the nimls were plced in n isoltion room with pproprite ventiltion nd filtering systems. This study ws pproved y the Bioethics Committee for the Use of Animls in Biomedicl Reserch of the Universidd Austrl de Chile. The effect of exposure to the fungus ws evluted in 4 groups of nimls: non-exposed young nimls (n = 8), exposed young nimls (n = 8), non-exposed old nimls (n = 8), exposed old nimls (n = 8). The mesured outcome for fungus exposure ws reltive Treg content (CD4+, CD25+, Foxp3+) in spleen. The effect of cyclophosphmide on irwy inflmmtion ws evluted on three groups of 2 month old nimls: not exposed to fungus nd not treted with cyclophosphmide (n = 6), exposed to fungus ut not treted with cyclophosphmide (n = 6), nd treted with cyclophosphmide nd exposed to fungus (n = 6). Aspergillus fumigtus culture, protein extrction nd hy contmintion Culture of Aspergillus fumigtus ws crried out in two steps. The fungus ws first cultured in peptone gr for 7 dys t 37 C nd then in mlt roth for 7 dditionl dys. The resulting fungi were used for solule protein extrction nd to contminte the mouse edding. For protein extrction, fungi were lended nd susequently sonicted (Ultrsonic Homogenizer, series 4710 Cole Prmer, USA). The resulting suspension ws centrifuged t g for 10 min, nd the superntnt contining solule proteins ws stored t 20 C for lter use. To expose mice to Aspergillus fumigtus, hy ws sterilized nd sprinkled with wter contining fungus spores. The contminted hy ws then introduced into plstic gs nd incuted for 48 h or more t 37 C until mold contmintion ecme visile. At this point, the contminted hy ws used s mice edding. During the experiment, nimls were mintined under these conditions ccording to the design of ech study protocol. Cyclophosphmide dministrtion nd induction of ronchil inflmmtion Groups of mice were intrperitonelly treted with Cy t dose of 20 mg/kg of ody weight, for three consecutive dys immeditely efore strting the ntigenic chllenge. As controls, groups of mice were injected with sline nd exposed or not exposed to Aspergillus fumigtus contminted hy. Experimentl groups were mintined in this condition for different lengths of time, nd were then euthnized with n overdose of pentoritl t dys 5, 11 nd 17. In ech cse, lood smples, BALF, spleen nd lung tissue were otined for nlysis. Otention, processing nd nlysis of smples Smples of lood, BALF, spleen nd lung were otined 5, 11 nd 17 dys fter the chllenge conditions egn. Blood (200 μl) ws otined ech time from the treted nd control mice nd llowed to clot; the collected ser were stored t 20 C until use. BALF ws otined from previously euthntized mice; for this purpose, the

3 Henríquez et l. BMC Veterinry Reserch (2017) 13:117 Pge 3 of 9 trche ws exposed nd cnnulted with 24G Teflon I.V. ctheter. BALF ws collected with sline solution (3 0.5 ml), nd cells were centrifuged once t 500 g for 7 min. Pelleted BALF cells were resuspended in PBS nd the totl numer of leucocytes ws counted using Neuuer chmer. A totl of cells in cytocentrifuged preprtion of BALF stined with My- Grünwld s Giems were differentited under light microscopy, ccording to clssicl cell morphology. Lung nd spleen were otined fter scrifice with no previous intervention. Lungs were fixed in 4% formlin nd processed for histology, while spleen ws tesed in cold-culture medium, nd the cell suspension ws filtered through guze nd djusted to the desired concentrtion for flow cytometry. Flow cytometry nlysis To determine the impct of exposure to Aspergillus fumigtus contminted hy, ge nd Cy tretment on the Treg cell popultion, suspension of splenocytes djusted to concentrtion of cells per ml ws wshed with PBS (ph 7.2) nd then resuspended in flow cytometry stining uffer (ebioscience). For the detection of different mrkers, cells were incuted with the following ebioscience monoclonl ntiodies: APC- efluortm 780-leled nti mouse CD45, PE- Cy5-leled nti mouse CD3 (145-2C11), FITC-leled nti mouse CD4 (RM4 5), APC-leled nti mouse CD25 (PC61.5) nd PE-leled nti mouse Foxp3 (FJK16s), ccording to the mnufcturer s recommendtions. At lest events were ssyed using Beckmn Coulter, CyAn ADP Anlyzer, nd dt were nlyzed using Summit v4.3 softwre. To determine different cell popultions, lymphocytes were gted using SSC nd the CD45 mrker; then, the CD4 popultion ws visulized nd the CD25 positive-cell content ws nlyzed. Lter, Foxp3-positive cells were determined within the totl CD4 supopultion, nd the co-expression of CD25 nd Foxp3 ws determined (Fig. 1). c CD25 CD25 CD4 CD25 CD4 CD45 Foxp3 SSC CD45 d e f CD4 Foxp3 Isotype control Fig. 1 Representtive figures of gting strtegy for Treg determintion in mice spleen. For this purpose gte ws positioned round lymphocytes (), nd the CD4+ popultion ws gted () in order to determinte the percentge of CD4+ cells tht express mrkers CD25 (c) nd Foxp3 (d) within the CD4 suset. Finlly, co-expression of CD25 nd Foxp3 were mesured within CD4 cells (e); isotype control for Foxp3 ws used s control (f)

4 Henríquez et l. BMC Veterinry Reserch (2017) 13:117 Pge 4 of 9 Anlysis of ntiody production The levels of Aspergillus-specific immunogloulins in BALF nd serum were determined y ELISA. Briefly, 96-well polystyrene micropltes (Srstedt, USA) were coted with 10 μg/ml of Aspergillus fumigtus totl protein tht ws dissolved in cronte uffer (ph 9) nd incuted overnight. The pltes were locked with solution of 5% non-ft milk followed y the ddition of 100 μl of the pproprite dilution of BALF (1:1) or serum (1:100) into ech well. The pltes were incuted t 37 C for 2 h, wshed three times nd incuted with peroxidse-conjugted nti-mouse IgE, IgG1 or IgG2 ntiodies (Snt Cruz, Inc., USA) for n dditionl 2 h t 37 C. After incution, the pltes were thoroughly wshed, nd the rection ws reveled with o-phenylenedimine (OPD; Sigm, USA). The colorimetric rection ws evluted t 450 nm using microplte reder (ELx800, Bio-Tek Instruments, USA). Histologicl nlysis For histologicl sections study, control or experimentl lung tissues were fixed in 4% formldehyde, emedded in Prplst, cut into 5-μm sections nd stined with hemtoxylin & eosin. Sttisticl nlysis SigmPlot 11.0 ws used for sttisticl nlyses. Descriptive sttistics y group were run on the dt, nd the Kolmogorov Smirnov test showed tht the dt were normlly distriuted. ANOVA ws performed to compre the differences in CD4+, CD25+, Foxp3+ supopultion percentges, nd this ws followed y Tukey test. Overll, p vlues <0.05 were regrded s significnt. Results Splenic Treg cell percentges increse in mice s function of ge nd exposition to Aspergillus fumigtus contminted hy As first pproch, we determined the reltive percentge of CD4+, CD25+, Foxp3+ cells in the spleens of two nd 8 month old mice y flow cytometry. The results indicted tht younger mice hd significntly less Treg (5.19% ± 0.45) thn older mice (6.77% ± 0.89) prior to Aspergillus exposure, nd tht the percentge of Treg cells significntly increses only in young mice upon 5 dys of exposure to Aspergillus fumigtus (from 5.56% ± 0.53 to 6,91% ± 0,94 fter mold chllenge) (Fig. 2). Low doses of cy induce reduction of splenic Treg in mice exposed to Aspergillus fumigtus contminted hy Knowing tht exposure to Aspergillus fumigtus contminted hy induces n increse of the CD4+, CD25+, Foxp3+ popultion in mice, s next step, we tested the % of CD4+CD25+Foxp * * 2 2 Months 8 Months Fig. 2 Percentge of CD4, CD25, Foxp3 positive cells in spleen of 2 nd 8montholdmiceexposed( ) or non-exposed ( )toaspergillus fumigtus. Dt re presented s Medin ± SD. p <0.05(n =8) effect of low doses of Cy in prtilly reducing the splenic Treg popultion, following Bron et l. s protocol [22]. In short, efore ntigenic chllenge, the mice were injected dily three times with 20 mg/kg, nd the results, which re shown in Fig. 3, indicte tht Cy reduced the percentge of CD4+ cells tht crried the regultory mrkers Foxp3 nd CD25. In this experiment, the Treg popultion ws reduced from 7.59% ± 1.17 to 5.90% ± dys fter the lst dose of the cytosttic drug, indicting tht Cy does not induce chnges in the percentge of totl CD4+ cells compred to the CD3+ popultion, in totl ccordnce with the results reported previously y Bron et l. [22]. The reduction of Treg popultion -induced y low doses of cy- showed positive correltion with n incresed irwy inflmmtory response in mice exposed to Aspergillus fumigtus contminted hy Next, to evlute if the reduction of Treg induced y Cy influenced the inflmmtory llergic irwy response in mice exposed to Aspergillus fumigtus contminted hy, nd knowing tht the chllenge in this murine experimentl model could replicte mny clinicl findings of horses with cute excertion of severe sthm, we evluted neutrophil percentges in BALF nd specific ntiodies levels ginst Aspergillus fumigtus in BALF. As oserved in Fig. 4, BALF neutrophil percentge ws gretly incresed in mice pretreted with Cy efore exposure to Aspergillus fumigtus contminted hy, compred to nimls tht were only exposed to the mold. This increse of neutrophils in the Cy-treted group ws evident nd sttisticlly significnt t dys 5 nd 11 post ntigenic chllenge. Since the irwy inflmmtory response to Aspergillus fumigtus in horses nd in the nimls of our murine model is medited t lest in prt y the onset of hypersensitivity type I nd III, it ws interesting to determine specific ntiodies ginst the

5 Henríquez et l. BMC Veterinry Reserch (2017) 13:117 Pge 5 of 9 % of CD4+CD25+Foxp3+ 10 * % of CD4+CD25+Foxp NCl Cy NCl Cy NCl Cy Dy 5 Dy 11 Dy 17 Fig. 3 Effect of tretment with low doses of cyclophosphmide on the reltive percentge of CD4+, CD25+, Foxp3+ positive cells in the spleens of mice exposed to Aspergillus fumigtus, compred to nimls injected with the sme volume of isotonic sline solution nd exposed to the fungus, 5, 11 nd 17 dys fter tretment. p <0.05(n =6) % of CD4+CD25+Foxp llergen in BALF otined from nimls chllenged with the mold, either treted or not treted with the cytosttic drug. Figure 5 shows Aspergillus fumigtus-specific IgE, IgG1 nd IgG2 levels in mice tht were not exposed nd in mice exposed to contminted hy nd either treted or not treted with Cy. IgE levels were significntly higher in the Cy-treted nd Aspergillus-exposed group only on dy 5 (Fig. 5). IgG1 levels were significntly different etween ll three experimentl groups on dy 5 (i.e. control group, Aspergillus-exposed group, ndcy-tretedndaspergillus-exposed group); on dy 11, IgG1 levels were only significntly higher in the Cytreted nd Aspergillus-exposed group; y dy 17, there were no significnt differences etween groups (Fig. 5). IgG2 levels were significntly higher only in the Cytreted nd Aspergillus-exposed group on dy 5; on dy 11 nd 17, IgG2 levels were significntly higher in oth Aspergillus-exposed groups thn in the control group (Fig. 5c). Lstly, lung histopthology of nimls chllenged with Aspergillus fumigtus (Fig. 6) showed tht the group of nimls pretreted with Cy presented significnt perironchiolr nd perivsculr mononucler nd neutrophilic infiltrtion, ccumultion of intrluminl ronchiolr mucus nd serofirinous exudtes. Moreover, there ws extensive folding of the irwy epitheli, which is sign of irwy smooth muscle contrction nd smooth muscle hypertrophy. This inflmmtory response ws oserved predominntly in the perironchiolr interstitil tissues nd often extended into the lmin propri nd luminl surfce epithelium of the ffected irwys. We lso oserved infiltrtion of inflmmtory cells into the lveolr spce. % Neutrphils * * 20 0 Dy 5 Dy 11 Dy 17 Fig. 4 Effect of tretment with low doses of cyclophosphmide on the reltive percentge of neutrophils otined from BALF in nonexposed controls ( ), compred with nimls treted with isotonic sline solution nd exposed to Aspergillus fumigtus ( ), nd nimls tht were pretreted with cyclophosphmide nd exposed to Aspergillus fumigtus ( ). Dt re presented s Medin ± SD. p < 0.05 (n =8) Discussion In this pper, we descrie tht the trnsient decrese of CD4+, CD25+, Foxp3+ splenic T regultory cells, induced y Cy, correltes with incresed irwy inflmmtion in mice exposed to hy contminted with Aspergillus fumigtus. The results presented here demonstrte tht, in our experimentl model, older nimls (8 months old) showed higher percentge of CD4+, CD25+, Foxp3+ cells in the spleen when compred to younger mice (2 months old). Similr findings were reported y Zho [28], who lso compred the cpcity of CD4+, CD25+ cells from older nd younger mice to inhiit delyed hypersensitivity, mixed lymphocyte rection or cytokine production. From their results, they concluded tht CD4 +, CD25+ cells present in the older mice, were less effective in suppressing the ove-mentioned ssys. These fcts my explin why ging produces progressive decline in immune regultion, nd in the cse of horses nd in the mice of our experimentl model [20],

6 Henríquez et l. BMC Veterinry Reserch (2017) 13:117 Pge 6 of c OD 450 nm OD 450 nm Dy 5 Dy 11 Dy Dy 5 Dy 11 Dy 17 c 0.4 OD 450 nm Dy 5 Dy 11 Dy 17 Fig. 5 IgE (), IgG1 () nd IgG2 (c) levelsincontrolnimls( ), nimls chllenged with Aspergillus fumigtus nd injected with sline ( )nd nimls pretreted with cyclophosphmide nd chllenged with Aspergillus fumigtus ( ). Dt re presented s Medin ± SD. p <0.05(n =8) why ge fcilittes the occurrence of irwy immunemedited inflmmtion. On the other hnd, our results indicte tht upon stimultion only young mice show significnt increse of CD4+, CD25+, Foxp3+ cells percentge in the spleen, llowing us to propose tht ging mkes these cells less prone to proliferte or e recruited in response to ntigenic chllenge. In ddition, we demonstrte tht Cy t low doses induces sttisticlly significnt decrese in CD4+, CD25+, Foxp3+ cells in the spleens of mice chllenged with Aspergillus fumigtus, s descried in norml mice y Bron et l. [22]. This tretment does not ffect the reltive percentges of other T supopultions, nd this effect hs een explined y the presence of lower ATP levels in Treg cells thn in conventionl T cells. This low ATP content, in turn, ttenutes the synthesis of glutthione, which results in lower cpcity for Cy detoxifiction [28, 29]. Moreover, Lutsik et l. [30] reported tht Cy decresed Treg cell numers nd led to decresed functionlity y decresing GITR nd Foxp3 expression, fct tht dded to the percentge diminution of Treg nd could ccount for the exggerted irwy immune response oserved in nimls tht received Cy. The fct tht low dose of Cy tretment prior to chllenge with Aspergillus fumigtus correlted with ugmented inflmmtion nd n increse in specific immunogloulins ginst the fungus in BALF, my indicte the regultory role of Treg in this process. In this respect, IgE nd IgG1 isotypes re especilly importnt ecuse oth hve the ility to ind to the highffinity IgE Fc receptors on mst cells nd sophils; therefore, their enhnced production correltes with incresed sensitiztion of the irwys nd more ggrvted hypersensitivity type I rection. On the other hnd, the IgG2isotype,whichistheonlyIgGsuclssthtfixes complement in mice, ppers to e responsile for type III hypersensitivity ecuse, upon forming immunocomplexes with the ntigen, complement is ctivted nd pro-inflmmtory fctors such s C3 nd C5 re relesed. Therefore, we my conclude tht the increse in immunogloulins tht re responsile for type I nd III hypersensitivity, could directly influence the pthogenesis of irwy immune-medited inflmmtion. As consequence of ntigen-ntiody complex formtion in the irwys, complement ctivtion nd the production of other chemottrctnts y resident cells in our experimentl model mice nd in severe equine sthm, neutrophils migrte to the irwys, producing n increse of these cells in BALF, which serves s n importnt dignostic finding for this disese. Our results show tht there is n importnt increse in BALF neutrophils of mice exposed to Aspergillus fumigtus, nd this phenomenon is even greter in mice pretreted with low doses of Cy, which indictes tht the irwy immune-medited inflmmtion process induced y the fungus is ggrvted y Cy. This conclusion is corroorted y the histopthologicl study tht showed tht the highest degree of lung infiltrtion ws found in nimls exposed to the ntigen nd pretreted with Cy

7 Henríquez et l. BMC Veterinry Reserch (2017) 13:117 Pge 7 of 9 c d e f Fig. 6 Representtive histologicl fetures of norml mice lung () nd norml ronchus () compred with microgrphs of lungs otined from mice 17 dys fter the initil chllenge with Aspergillus fumigtus (c & d) nd of lungs from nimls pretreted with cyclophosphmide nd exposed to Aspergillus fumigtus (e & f) efore chllenge. Finlly, considering tht the nturl thymus-derived, or peripherlly induced dptive Treg cell popultions hve een ssocited with the outcome of processes such s utoimmunity, trnsplnttion rejection, cncer nd mucosl tolernce [31]; it is not surprising tht locl irwy-induced inflmmtion correltes with n increse of splenic Treg cells, nd regultory role of this popultion s ws descried here. One of this study s limittions is the lck of informtion out specific Treg chnges t the site of inflmmtion (the irwys); Treg supopultions should idelly e evluted in the lungs nd in BALF. However, these results do revel the systemic effect of Cy on Treg popultions, nd strongly suggest prticiption of this cell suset in inflmmtory processes. Another limittion of this study is tht Cy hs een shown to induce lung inflmmtion t high doses in rts [32]. While in our experience this is not noticele through the prmeters evluted in this study (histology or BALF reltive cellulr content, dt not shown), this does not exclude the possiility tht potentil dmge induced y Cy my hve influenced the inflmmtory response ginst Aspergillus fumigtus. Our study group hs previously reported similr Treg cell supopultion in horses with excerted severe EA [19]. Treg supopultions my well ply regultory role in inflmmtory processes in different tissues, nd our findings here certinly show tht inhiition of Treg (through pre-tretment with Cy) coincides with severe irwy inflmmtion fter inhltory chllenge. However, trnsltion of these results to the equine species is limited y horses individul susceptiility to EA. Multiple fctors my ffect horses susceptiility to

8 Henríquez et l. BMC Veterinry Reserch (2017) 13:117 Pge 8 of 9 inhltory llergens, including heritle or genetic trits. Fmilil trits hve een proven in sid species, nd the sme hs een shown in humns [21, 33]. The murine strin used in this study doesn t hve n incresed individul susceptiility to irwy inflmmtion, nd s such does not relte specificlly to EA-susceptile horses; however, the immunologicl processes descried here re vlid cross species, nd could e pplicle to equine susceptile nd non-susceptile individuls, leit the necessry limittions inherent to ny reserch model. Conclusion In this murine model of sthm, the reduced numer nd function of Treg induced y low doses of Cy nd which directly correltes with incresed irwy inflmmtion nd lung infiltrtion, indictes tht Treg my ply role in the regultion nd resolution of severe equine sthm. Arevitions BALF: Broncholveolr Lvge Fluid; Cy: Cyclophosphmide; EA: Equine sthm; OPD: O-phenylenedimine; RK: Rockefeller; Treg: T regultory cells Acknowledgements Not pplicle. Funding This work ws prtilly supported y FONDECYT grnt Doctorl Scholrship MESECUP AUS-601, nd Grdute School Fculty of Veterinry Sciences, Universidd Austrl de Chile. Avilility of dt nd mteril All dt generted nd nlyzed for this study re included in this pulished rticle. Supplementry informtion files in extenso re ville upon request to the corresponding uthor. Authors contriutions CH conceived the study, prticipted in the design of the study, smple otining nd processing. GM prticipted in the design of the study nd coopertes with the niml model. CC crried out the flow cytometry nlysis. JS prticipted in the design of the study nd helped to drft the mnuscript. MB prticipted in the design of the study nd helped to drft the mnuscript. HF prticipted in design nd coordintion of the study nd helped to drft the mnuscript. BU helped to drft the mnuscript nd dt interprettion. All uthors red nd pproved the finl mnuscript. Authors informtion Not pplicle. Competing interests The uthors declre tht they hve no competing interests. Consent for puliction Not pplicle. Ethics pprovl nd consent to prticipte This study ws pproved y the Bioethics Committee for the Use of Animls in Biomedicl Reserch of Universidd Austrl de Chile; resolution numer Pulisher s Note Springer Nture remins neutrl with regrd to jurisdictionl clims in pulished mps nd institutionl ffilitions. Author detils 1 Phrmcology nd Morphophysiology Deprtment, Fculty of Veterinry Sciences, Universidd Austrl de Chile, Vldivi, Chile. 2 Immunology Deprtment, Fculty of Medicine, Universidd Austrl de Chile, Vldivi, Chile. 3 Pthology Deprtment, Regionl Hospitl of Vldivi, Vldivi, Chile. 4 Physiology Deprtment, Fculty of Medicine, Universidd Austrl de Chile, Vldivi, Chile. 5 Veterinry Clinicl Sciences Deprtment, Fculty of Veterinry Sciences, Universidd Austrl de Chile, Vldivi, Chile. Received: 29 Septemer 2015 Accepted: 24 April 2017 References 1. Bullone M, Lvoie J-P. Asthm, of horses nd men how cn equine heves help us etter understnd humn sthm immunopthology nd its functionl consequences? Mol Immunol. 2015;66: Couetil LL, Crdwell JM, Gerer V, Lvoie JP, Leguillette R, Richrd EA. Inflmmtory irwy disese of horses revised consensus sttement. J Vet Intern Med. 2016;30: Pirie RS, Couetil LL, Roinson NE, Lvoie JP. Equine sthm: n pproprite, trnsltionl nd comprehendile terminology? 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9 Henríquez et l. BMC Veterinry Reserch (2017) 13:117 Pge 9 of Askense PW, Hyden BJ, Gershon RK. Augmenttion of delyed-type hypersensitivity y doses of cyclophosphmide which do not ffect ntiody responses. J Exp Med. 1975;141: Folch H, Lopetegui F, Stegmeier E. Arogtion of ntigenic competition phenomenon y low dose of cyclophosphmide. Zentrll Veterinrmed B. 1980;27: Audi S, Nicols A, Cthelin D, Lrmonier N, Ferrnd C, Foucher P, Fnton A, Bergoin E, Myndie M, Arnould L, Btemn A, Lorcerie B, Solry E, Chuffert B, Bonnotte B. Increse of CD4+ CD25+ regultory T cells in the peripherl lood of ptients with metsttic crcinom: phse I clinicl tril using cyclophosphmide nd immunotherpy to eliminte CD4+ CD25+ T lymphocytes. Clin Exp Immunol. 2007;150: Rech AJ, Vonderheide RH. Clinicl use of nti-cd25 ntiody dclizum to enhnce immune responses to tumor ntigen vccintion y trgeting regultory T cells. Ann N Y Acd Sci. 2009;1174: Schowsky RH, Mdireddi S, Shrm R, Yolcu ES, Shirwn H. Trgeting CD4 +CD25+FoxP3+ regultory T-cells for the ugmenttion of cncer immunotherpy. Curr Opin Investig Drugs. 2007;8: Zho L, Sun L, Wng H, M H, Liu G, Zho Y. Chnges of CD4+CD25+Foxp3 + regultory T cells in ged Bl/c mice. J Leukoc Biol. 2007;81: SchenkU,FrscoliM,ProiettiM,GeffersR,TrggiiE,BuerJ,RicordiC, Westendorf AM, Grssi F. ATP inhiits the genertion nd function of regultory T cells through the ctivtion of purinergic P2X receptors. Science Signling 4. Sci Signl. 2011;4(162):r Lutsik ME, Semnni RT, De Psclis R, Kshmiri SV, Schlom J, Szevri H. Inhiition of CD4(+)25+ T regultory cell function implicted in enhnced immune response y low-dose cyclophosphmide. Blood. 2005;105: Curotto de Lfille MA, Lfille JJ. CD4(+) regultory T cells in utoimmunity nd llergy. Curr Opin Immunol. 2002;14: Cooper JA Jr, Merrill WW, Reynolds HY. Cyclophosphmide modultion of roncholveolr cellulr popultions nd mcrophge oxidtive metolism. Possile mechnisms of pulmonry phrmcotoxicity. Am Rev Respir Dis. 1986;134(1): Pirie RS. Recurrent irwy ostruction: review. Equine Vet J. 2014;46(3): Sumit your next mnuscript to BioMed Centrl nd we will help you t every step: We ccept pre-sumission inquiries Our selector tool helps you to find the most relevnt journl We provide round the clock customer support Convenient online sumission Thorough peer review Inclusion in PuMed nd ll mjor indexing services Mximum visiility for your reserch Sumit your mnuscript t