Bullone, M.

De Lagarde, M.

Lavoie, J. -P.

Vargas, A.

English

Institutional Research Information System University of Turin

Serum Surfactant Protein D and Haptoglobin as PotentialBiomarkers for Inflammatory Airway Disease in HorsesM. Bullone*, M. de Lagarde*, A. Vargas, and J.-P. LavoieBackground: The identification of serum biomarkers of lung inflammation would facilitate the diagnosis of inflammatoryairway disease (IAD) in horses.Hypothesis: Horses with IAD have higher serum concentrations of markers of inflammation compared to controls.Animals: Twelve horses with IAD and 10 control horses.Methods: This was a prospective case–control study. Blood and BALF were collected from horses with IAD and controls.Serum concentration of surfactant protein D (SP-D), haptoglobin, serum amyloid A (SAA) and of the soluble form of trig-gering receptor expressed on myeloid cells 1 (sTREM-1) was measured using commercial ELISA tests.Results: Horses with IAD had higher serum concentration (log-transformed values) of SP-D (mean  SD: 1.773  0.51),haptoglobin (6.657  0.202) and SAA (0.128  0.396) compared to controls (0.942  0.226, 6.38  0.22, 0.398  0.319,respectively; P < .01 for all). Furthermore, the concentrations of SP-D and haptoglobin combined allowed differentiating the2 groups (IAD: 8.43  0.564, controls: 7.322  0.249, P < .0001) with a sensitivity and specificity of 100% when a cut-off of7.70 (log value) was employed.Conclusions and Clinical Importance: Surfactant protein D and haptoglobin serum concentrations could be a diagnostic aidin IAD. Further studies are necessary to establish the specificity of our findings before they can be applied in everyday practice.Key words: Acute phase proteins; Biomarker; Lung; sTREM-1.Inflammatory Airway Disease (IAD) includes clinicalentities characterized by cough, decreased perfor-mance and delayed recovery after exercise, with lowerairway inflammation detected at bronchoalveolar lavagefluid (BALF) cytology.1 As the bronchoalveolar lavageis a procedure not routinely performed in the field, IADremains underdiagnosed. The identification of serumbiomarkers for lung inflammation would facilitatedisease recognition.Biomarkers are “substances, structures or processesthat can be measured in the body or its products whichinfluence or predict the incidence of an outcome or dis-ease”.2 Biomarkers of lung inflammation have beenstudied, but few are pathognomonic for a given condi-tion or specific for single organs. Both specific (surfac-tant protein D, SP-D) and nonspecific (acute phaseproteins) inflammatory biomarkers are elevated inblood of horses suffering from lower airway inflamma-tory diseases (heaves and IAD). In heaves, circulatinghaptoglobin concentrations are increased compared tocontrols, with no overlapping measures betweengroups.3 There is a significant increase in serum SP-Dconcentrations in horses with IAD.4We hypothesized that the protein expression of 4biomarkers of pulmonary (SP-D) and nonspecificinflammation haptoglobin, serum amyloid A, SAA andthe soluble form of triggering receptor expressed onmyeloid cells 1, sTREM-1, is higher in the serum ofhorses with IAD. We aimed at investigating the concen-trations of these biomarkers in the serum of horses withIAD and controls, and at studying whether their distri-bution pattern is associated with the inflammatoryphenotype detected at BALF cytology.Material and MethodsAnimalsHorses with IAD were selected among the cases referred to theequine hospital of the Universite de Montreal for respiratory prob-lems between September 2011 and 2013. The study was performedin accordance with the guidelines of the Canadian Council on Ani-mal Care and the protocol approved by the Ethics Committee ofthe Universite de Montreal (#Rech-1647). Horses with IAD wereincluded in the study if they presented clinical signs of cough,nasal discharge or increased breathing effort during/after exercise,together with increased neutrophil (>5%), mast cell (>2%) orFrom the Department of Clinical Sciences, Faculty of VeterinaryMedicine, Universite de Montreal, St-Hyacinthe, QC Canada(Bullone, de Lagarde, Vargas, Lavoie)This work was performed at the Faculty of Veterinary Medicine ofthe Universite de Montreal.*These authors contributed equally to this work.Corresponding author: J.-P. Lavoie, Department of ClinicalSciences, Faculty of Veterinary Medicine, Universite de Montreal,3200 rue Sicotte, St-Hyacinthe, J2S2M2 QC, Canada; e-mail: jean-pierre.lavoie@umontreal.ca.Submitted April 1, 2015; Revised June 8, 2015; AcceptedJuly 22, 2015.Copyright © 2015 The Authors. Journal of Veterinary InternalMedicine published by Wiley Periodicals, Inc. on behalf of AmericanCollege of Veterinary Internal Medicine.This is an open access article under the terms of the CreativeCommons Attribution-NonCommercial License, which permits use,distribution and reproduction in any medium, provided the originalwork is properly cited and is not used for commercial purposes.DOI: 10.1111/jvim.13602Abbreviations:BAL bronchoalveolar lavageBALF bronchoalveolar lavage fluidELISA enzyme-linked immunosorbent assayIAD inflammatory airway diseaseSAA serum amyloid ASP-D surfactant protein DTREM-1 triggering receptor expressed on myeloid cells 1sTREM-1 soluble form of triggering receptor expressed onmyeloid cells 1J Vet Intern Med 2015eosinophil (>1%) percentage at the BALF cytology.1 Controlhorses were either selected among horses referred to the hospitalfor nonrespiratory elective procedures and did not have signs ofdiseases or inflammation detected at BALF cytology (n = 5), orthey were owned by the Universite de Montreal (n = 5). Exclusioncriteria for both groups of horses were the presence of systemicinflammatory or infectious processes based on clinical examinationor results. Racehorses in training/racing were excluded, as werehorses with a history of increased respiratory effort at rest, or anydrug administration within the 15 days before admission. All theclient-owned horses were transported to the hospital on the day ofexamination. The horses owned by the Universite de Montrealwere not trailered before sampling. All horses underwent completeclinical and endoscopic examination of the respiratory system. Allowners signed an informed consent form to store samples forresearch purposes.Bronchoalveolar LavageBronchoalveolar lavage procedure was performed as previouslydescribed.5 Cytopreparations from unfiltered BALFa with ProtocolHema 3b for differential counting of 400 cells.Blood CollectionBlood was collected from the jugular veins by means of 18Gneedles into sterile tubes. Ninety millilitres of blood were collectedfrom each horse. Within 2 hours from collection, plasma andserum were harvested from the sample and 1.5 mL aliquots werestored at -80°C until used. Serum samples were used for ELISA.Protein Expression StudiesSurfactant protein D, SAA, haptoglobin and sTREM-1 werequantified in serum samples using commercially available ELISAkits. The cross-reactivity of the antibody used in the SurfactantProtein D human Elisa kitc with the equine SP-D was validated bythe Western blot technique. The Phase Serum Amyloid A Mul-tispecies Elisa,d the Equine Haptoglobulin Elisa,d and horseTREM-1 Elisae were previously validated by the manufacturer andused accordingly to the instructions. Plates were washed using anautomatic plate washerf and absorbance was obtained using amicroplate reader.g The assay standard curves ranged from 1.56 to100 ng/mL for SP-D (serum dilution 1:10), 9.375 to 600 ng/mLfor haptoglobin (serum dilution 1:32000), 1.25–20 ng/mL for SAA(serum dilution 1:2000) and 62.5 pg/mL-2000 pg/for sTREM.When SAA concentrations exceeded the assay quantificationrange, there were attributed the value 3.30 lg/mL (higher limit ofthe linear part of the curve).StatisticsStatistical analyses were performed with Prism 6.h All data wereanalysed after Log10 transformation to reduce intergroup variabil-ity. Normal distribution of data within each group was assessedwith Kolmogorov-Smirnov tests. Student t-tests or one-wayANOVA with Tukey’s post-tests were used to compare the vari-ables studied in different groups. Pearson tests were used for detect-ing correlations between serum biomarker concentrations andother variables assessed in serum or BALF in horses with IAD.ResultsTwelve horses with IAD and 10 controls wereincluded. The IAD group consisted of 4 Warmbloods, 5Quarter-Horses and associated breeds, and 3 Drafthorses. Their average age was 6 years old (range 2–14).There were 4 females, 6 geldings and 2 males. Horseswere performing light to moderate work. Disease dura-tion was less than 2 years for most of them except one(4-year duration). They were fed hay while beingstabled (8/12) or kept in paddock/pastures (4/12). Thecontrol group consisted of 2 Draft horses, 1 Haflinger,2 Quarter-horses and 5 Standardbreds that were part ofthe University’s herd. Their average age was 6 years old(range 2–9); there were 5 females, 4 geldings and 1male. All control horses were performing light work,and were stabled and fed hay.All IAD horses had increased BALF neutrophil per-centage (range 6–53%), 7 horses also had BALF masto-cytosis (range 3–9%), 1 horse had BALF eosinophilia(3%) and 1 horse had both BALF mastocytosis (3%)and eosinophilia (3%). Control horses had normalBALF neutrophil (range 1–3.5%) and mast cell percent-age (<2%). Blood neutrophil (P = .3), leucocyte(P = .4), and fibrinogen (P = .6) concentrations weresimilar between the 2 groups of horses.The concentrations of SP-D (P = .0001), haptoglobin(P = .006) and SAA (P = .003), but not sTREM-1(P = .87), were significantly increased in the sera ofhorses with IAD (Fig 1). Data overlap between the 2groups was minimal for serum SP-D concentrations.Raw data ranged from 3.88 to 288 ng/mL for SP-D(min-max: 3.88–21.98 in controls and 14.8–288 in IAD),from 1.18 9 106 to 10.6 9 106 ng/mL for haptoglobin(1.18 9 106–5.52 9 106 in controls and 2.59 9 106–10.6 9 106 in IAD), from 0.69 to 36.14 ng/mL forsTREM-1 (0.69–17.34 in controls and 1.56–36.14 inIAD) and from 0.21 to 3.30 lg/mL for SAA (0.21–1.55in controls and 0.28–3.30 in IAD). Serum samples from4 horses with IAD had SAA concentrations exceedingthe assay quantification range. When combined, thesum of the log-transformed values of SP-D and hap-toglobin (P < .0001), of SP-D and sTREM-1(P = .003), of SAA and SP-D (P < .0001), of SAA andhaptoglobin (P = .0001) and of SAA and sTREM-1(P = .025), were significantly higher in the sera ofhorses with IAD compared to controls. The sum of theconcentrations of haptoglobin and sTREM-1 was notdifferent between both groups (P = .24) (Fig 2C). Nooverlap was observed between the 2 groups when thesum of SP-D and haptoglobin concentrations was stud-ied (Fig 2A). A cut-off point of 7.70 (Log-transformedvalue) allowed complete distinction of horses with IADfrom controls. A cut-off value of 0.80 for the sum ofSAA and SP-D (Fig 2D) permitted differentiation ofhorses with IAD from controls (sensitivity = 100%,specificity = 90%). In horses with IAD, SAA concentra-tion was significantly correlated with neutrophil per-centage in BALF (r = 0.56, P = .05). The two horseswith BALF eosinophil percentage >1% had the highestserum concentrations of SP-D. Haptoglobin values werenegatively correlated with serum neutrophil concentra-tions (r = 0.69, P = .01), neutrophil percentage inBALF (r = 0.75, P = .005) and with serum leucocyteconcentrations (r = 0.61, P = .04). Haptoglobin2 Bullone et alconcentrations were significantly affected by the pres-ence of haemosiderophages (P = .0009) in BALF.Horses with haemosiderophages detected at BALFcytology (there were no haemosiderophages found inthe BALF of the control group (Fig 3) had higher val-ues of haptoglobin concentration in the serum com-pared to the IAD-affected horses with nohaaemosiderophages and to the controls (P < .05 andP < .001 respectively, Fig 3).DiscussionInflammatory Airway Disease is highly prevalent inthe equine population. However, diagnosis remains achallenge, as clinical signs are not pathognomonic. Theimplementation of biomarkers specific for lung inflam-mation could facilitate its diagnosis. Our results demon-strate that the serum concentrations of SP-D,haptoglobin and SAA are increased in horses with IADcompared to controls, moreover when combined, theserum concentrations of SP-D and haptoglobin allowdifferentiating horses with IAD from healthy horseswith no overlap between groups.Surfactant protein D is a collectin mainly synthesizedin the lungs by alveolar type II cells. It plays an impor-tant role in protecting the lungs against various inflam-matory processes, allergies and infections. In our study,and in agreement with a previous report,4 horses withFig 1. Serum concentration of SP-D (A), haptoglobin (B), sTREM-1 (C) and SAA (D) in horses with IAD and controls. Values were logtransformed for analysis.Fig 2. Serum concentration of combined SP-D and haptoglobin (A), SP-D and sTREM-1 (B), haptoglobin and sTREM-1 (C), SAA andSP-D (D), SAA and haptoglobin (E), and sTREM-1, and SAA (F) in horses with IAD and controls. Values were log transformed for anal-ysis.Serum Biomarkers for IAD 3IAD had higher serum concentrations of SP-D. DespiteSP-D being also expressed in the synovial fluid, diseasesof these organ systems tend to lower circulating SP-Dlevels in man,6 whereas increases in serum SP-D con-centrations have been associated to lung pathologies.4Whether serum SP-D concentration also decreases inhorses with joint diseases has not been studied to date,and would require investigation.Serum amyloid A is a major acute phase proteinreported to range from <0.5 to 20 lg/mL. Serum SAAlevels are low in healthy individuals, and increase from10 to 1000 times within a few hours from tissue injury,to reach peak values within 24–28 h.7 Serum amyloid Ahas immunoregulatory functions, linked to the develop-ment of chronic diseases. In this study, and in agree-ment with findings in heaves,3 serum SAAconcentrations had a 3.5-fold increase in horses withIAD when compared to controls. As SAA increases areproportional to the extent of tissue damage,7 it indicatesthat horses with IAD sustain lung injuries of similarmagnitude to those found in heaves. Serum amyloid Aincreases after transportation.8 In our study, horseswith IAD and 5 of the control horses underwent trans-portation. The SAA values of control horses, whethertransported or not, were not significantly different(Mann–Whitney t-test, P = .8), suggesting that trailer-ing unlikely affected our results.Circulating haptoglobin concentrations increase 1–10times in the 12–24 h after an injury, and peaks 72 hlater.7 Haptoglobin is mainly produced to scavenge hae-moglobin. In our study, haptoglobin was increased onaverage 2-fold in horses with IAD, whereas its serumconcentration has been reported to increase 5–10-foldafter inflammation and infection in horses.7 Serum hap-toglobin was increased in 3/4 horses with IAD present-ing haemosiderophages in their BALF, whereas horseswith IAD with no haemosiderophages have a concentra-tion of serum haptoglobin similar to the controls. Thesefindings suggest that the specificity of serum haptoglobinas biomarkers for IAD should be ascertained in the pres-ence of exercise-induced pulmonary haemorrhage. It hasbeen hypothesized that peripheral blood and airway neu-trophil degranulation may constitute a source of hap-toglobin in horses with inflammatory lung diseases suchas heaves.3 The negative correlations we observedbetween haptoglobin serum concentrations and bothblood and BALF neutrophils would not support thishypothesis, and was unexpected. Haemosiderophagesmay have a less potent neutrophil chemo-attractiveactivity than other inflammatory cells, but more studiesare needed to corroborate this assumption.Comparing results from this study to a previousreport,3 horses with IAD have higher haptoglobin valuesthan those of heave-affected horses, which would supportthe hypothesis of disease progression from IAD toheaves. However, a study evaluating both diseases in thesame conditions is needed to confirm that hypothesis.The TREM-1 is a membrane receptor expressed onblood neutrophils and monocytes, and also on alveolarmacrophages, which has a role in lung inflammatoryresponse against infections by amplifying the TLR-asso-ciated response.9 As IAD is clinically comparable tomild to moderate human asthma, not surprisinglysTREM-1 serum concentrations were similar betweenhorses with IAD and controls.ConclusionsMeasuring several inflammation-related blood pro-teins increases their sensitivity as blood biomarkers forthe diagnosis of IAD. The combination of an organ-specific biomarker (SP-D) and of an acute phase protein(haptoglobin) yielded the best diagnostic results. How-ever, the effect of other causes of reduced performancesin horses, whether affecting the lungs or other organson SP-D is still unknown, even though our grouprecently reported no difference in SAA and haptoglobinconcentrations in blood of racehorses with and withoutIAD.10 Also, as healthy horses were studied as controls,the effect of other pulmonary conditions on acute phaseproteins could represent a potential source of bias (de-creasing sensitivity and specificity of the test). Prospec-tive studies investigating these aspects are needed beforethese biomarkers can be employed as a diagnostic toolin the field.AcknowledgmentsThis work was supported by the Natural Sciencesand Engineering Research Council of Canada (RGPIN-2014-06-198), Le Fonds du Centenaire and by an unre-stricted grant from the Pfizer Clinical Research Fund.Conflict of Interest Declaration: Authors disclose noconflict of interest.Off-label Antimicrobial Declaration: Authors declareno off-label use of antimicrobials.Footnotesa Cytospin, Rottorfix Hettish, Beverly, MA.b Fisher Scientific, Ottawa, ON, Canada.c Biovendor, Ashville, NC.Fig 3. Effect of the presence of BALF haemosiderophages onserum haptoglobin concentrations. Haptoglobin levels>6 9 106 ng/mL (log-transformed values ≥6.8) were associatedwith the presence of haemosiderophages in the BALF.4 Bullone et ald Cederlane, Burlington, ON, Canada.e MyBioScience, SanDiego, CA.f ELX50 Auto Strip washer; Bioteck-Instruments Inc., Winooski,VT.g Power Wave X340; Bioteck-Instruments Inc.h GraphPad Software Inc., La Jolla, CA.References1. Couetil LL, Hoffman AM, Hodgson J, et al. Inflammatoryairway disease of horses. J Vet Intern Med 2007;21:356–361.2. Strimbu K, Tavel JA. What are biomarkers? Curr Opin HIVAIDS 2010;5:463–466.3. Lavoie-Lamoureux A, Leclere M, Lemos K, et al. Markersof systemic inflammation in horses with heaves. J Vet Intern Med2012;26:1419–1426.4. Richard EA, Pitel PH, Christmann U, et al. Serum concen-tration of surfactant protein D in horses with lower airway inflam-mation. Equine Vet J 2012;44:277–281.5. Jean D, Vrins A, Beauchamp G, et al. Evaluation of varia-tions in bronchoalveolar lavage fluid in horses with recurrentairway obstruction. Am J Vet Res 2011;72:838–842.6. Christensen AF, Sorensen GL, Horslev-Petersen K, et al.Circulating surfactant protein -D is low and correlates negativelywith systemic inflammation in early, untreated rheumatoid arthri-tis. Arthritis Res Ther 2010;12:R39.7. Jacobsen S, Andersen PH. The acute phase protein serumamyloid A (SAA) as a marker of inflammation in horses. EquineVet Educ 2007;9:38–46.8. Casella S, Fazio F, Giannetto C, et al. Influence of trans-portation on serum concentrations of acute phase proteins inhorse. Res Vet Sci 2012;93:914–917.9. Habibzay M, Saldana JI, Goulding J, et al. Altered regula-tion of Toll-like receptor responses impairs antibacterial immunityin the allergic lung. Mucosal Immunol 2012;5:524–534.10. Leclere M, Lavoie-Lamoureux A, Lavoie JP. Acute phaseproteins in racehorses with inflammatory airway disease. J VetIntern Med 2015;29:940–945.Serum Biomarkers for IAD 5

Serum Surfactant Protein D and Haptoglobin as Potential Biomarkers for Inflammatory Airway Disease in Horses

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Serum Surfactant Protein D and Haptoglobin as Potential Biomarkers for Inflammatory Airway Disease in Horses

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