Josiane Lefebvre-Lavoie

IL-4 activates equine neutrophils and induces a mixed inflammatory cytokine expression profile with enhanced neutrophil chemotactic mediator release ex vivo

Neutrophils are potent contributors to the lung pathophysiological changes occurring in allergic airway inflammation, which typically involve T helper type 2 (Th2) cytokine overexpression. We have previously reported that equine pulmonary endothelial cells are activated by the Th2 cytokine IL-4 and express chemotactic factors for neutrophils after stimulation. We have further explored the possible mechanisms linking Th2-driven inflammation and neutrophilia by studying the effects of recombinant equine IL-4, a prototypical Th2 cytokine, on peripheral blood neutrophils (PBN) isolated from normal animals and from horses with asthmatic airway inflammation (equine heaves). We found that IL-4 induced morphological changes in PBN, dose- and time-dependent expression of IL-8 mRNA, as well as the release of chemotactic factors for neutrophils in culture supernatants. Also, IL-4 induced a mixed inflammatory response in PBN from control and asthmatic-animals with increased expression of proinflammatory IL-8 and TNF-α but a marked inhibition of IL-1β. IL-4 type I receptor (IL-4Rα) and CD23 (FcεRII) expression were also upregulated by IL-4. Importantly, disease as well as chronic antigenic exposure modified gene expression by PBN. Finally, we found that activation of equine neutrophils with IL-4 involved STAT6 phosphorylation and p38 MAPK and phosphatidylinositol 3-kinase (PI3K); the pharmacological inhibitors, SB-203580 and LY-294002, respectively, significantly reversed IL-4-induced gene modulation in PBN. Overall, results from this study add to the link between Th2-driven inflammation and neutrophilia in the equine model and further extend the characterization of IL-4 effects on neutrophils.

Efficacy of oral prednisolone and dexamethasone in horses with recurrent airway obstruction in the presence of continuous antigen exposure.

REASONS FOR PERFORMING STUDY Orally administered prednisolone and dexamethasone are used commonly in the treatment of recurrent airway obstruction (RAO) in horses. However, the efficacy of prednisolone in improving pulmonary function during continuous antigen exposure has not been evaluated critically and there is little evidence supporting the efficacy of low-dose oral dexamethasone in the same conditions. HYPOTHESIS Oral prednisolone and dexamethasone improve pulmonary function in RAO under conditions of continuous antigen exposure, and dexamethasone is more effective than prednisolone at commonly used dosages. METHODS Using a randomised crossover design, prednisolone (2 mg/kg bwt) and dexamethasone (0.05 mg/kg bwt) were administered per os, s.i.d. for 7 days, to 7 horses during clinical exacerbation of the disease. Maximal difference in transpulmonary pressure (DeltaP(L)), lung resistance (R(L)) and elastance (E(L)) were measured before and after 3 and 7 days of treatment. RESULTS Prednisolone and dexamethasone improved pulmonary function significantly. However, the improvement was of greater magnitude after 3 and 7 days of treatment with dexamethasone compared to prednisolone. Also, after 7 days of treatment with dexamethasone, DeltaP(L) and R(L) were not different from values obtained when horses were on pasture, while all 3 pulmonary function parameters remained different from pasture values after prednisolone treatment. CONCLUSIONS Both corticosteroids improve pulmonary function, in spite of continuous antigen exposure. However, oral dexamethasone at 0.05 mg/kg bwt is more effective than prednisolone at 2 mg/kg bwt in the treatment of RAO. POTENTIAL RELEVANCE Prednisolone was shown, for the first time, to our knowledge, to improve the pulmonary function of horses with RAO in the presence of continuous antigen exposure. This study also demonstrates the efficacy of low-dose oral dexamethasone in reversing airway obstruction in these conditions.

Profiling of differentially expressed genes using suppression subtractive hybridization in an equine model of chronic asthma.

Background Gene expression analyses are used to investigate signaling pathways involved in diseases. In asthma, they have been primarily derived from the analysis of bronchial biopsies harvested from mild to moderate asthmatic subjects and controls. Due to ethical considerations, there is currently limited information on the transcriptome profile of the peripheral lung tissues in asthma. Objective To identify genes contributing to chronic inflammation and remodeling in the peripheral lung tissue of horses with heaves, a naturally occurring asthma-like condition. Methods Eleven adult horses (6 heaves-affected and 5 controls) were studied while horses with heaves were in clinical remission (Pasture), and during disease exacerbation induced by a 30-day natural antigen challenge during stabling (Challenge). Large peripheral lung biopsies were obtained by thoracoscopy at both time points. Using suppression subtractive hybridization (SSH), lung cDNAs of controls (Pasture and Challenge) and asymptomatic heaves-affected horses (Pasture) were subtracted from cDNAs of horses with heaves in clinical exacerbation (Challenge). The differential expression of selected genes of interest was confirmed using quantitative PCR assay. Results Horses with heaves, but not controls, developed airway obstruction when challenged. Nine hundred and fifty cDNA clones isolated from the subtracted library were screened by dot blot array and 224 of those showing the most marked expression differences were sequenced. The gene expression pattern was confirmed by quantitative PCR in 15 of 22 selected genes. Novel genes and genes with an already defined function in asthma were identified in the subtracted cDNA library. Genes of particular interest associated with asthmatic airway inflammation and remodeling included those related to PPP3CB/NFAT, RhoA, and LTB4/GPR44 signaling pathways. Conclusions Pathways representing new possible targets for anti-inflammatory and anti-remodeling therapies for asthma were identified. The findings of genes previously associated with asthma validate this equine model for gene expression studies.

Equine CTNNB1 and PECAM1 nucleotide structure and expression analyses in an experimental model of normal and pathological wound repair.

BackgroundWound healing in horses is fraught with complications. Specifically, wounds on horse limbs often develop exuberant granulation tissue which behaves clinically like a benign tumor and resembles the human keloid in that the evolving scar is trapped in the proliferative phase of repair, leading to fibrosis. Clues gained from the study of over-scarring in horses should eventually lead to new insights into how to prevent unwanted scar formation in humans. cDNA fragments corresponding to CTNNB1 (coding for β-catenin) and PECAM1, genes potentially contributing to the proliferative phase of repair, were previously identified in a mRNA expression study as being up-regulated in 7 day wound biopsies from horses. The aim of the present study was to clone full-length equine CTNNB1 and PECAM1 cDNAs and to study the spatio-temporal expression of mRNAs and corresponding proteins during repair of body and limb wounds in a horse model.ResultsThe temporal pattern of the two genes was similar; except for CTNNB1 in limb wounds, wounding caused up-regulation of mRNA which did not return to baseline by the end of the study. Relative over-expression of both CTNNB1 and PECAM1 mRNA was noted in body wounds compared to limb wounds. Immunostaining for both β-catenin and PECAM1 was principally observed in endothelial cells and fibroblasts and was especially pronounced in wounds having developed exuberant granulation tissue.ConclusionThis study is the first to characterize equine cDNA for CTNNB1 and PECAM1 and to document that these genes are expressed during wound repair in horses. It appears that β-catenin may be regulated in a post-transcriptional manner while PECAM1 might help thoracic wounds mount an efficient inflammatory response in contrast to what is observed in limb wounds. Furthermore, data from this study suggest that β-catenin and PECAM1 might interact to modulate endothelial cell and fibroblast proliferation during wound repair in the horse.

Equine ANXA2 and MMP1 expression analyses in an experimental model of normal and pathological wound repair.

BACKGROUND Wounds on horse limbs can develop exuberant granulation tissue which resembles the human keloid. Clues gained from the study of over-scarring in horses might help control fibro-proliferative disorders. OBJECTIVES The aim of the present study was to clone full-length equine ANXA2 cDNA then to study spatio-temporal expression of ANXA2 and MMP1 mRNA and protein, potential contributors to remodeling, during repair of body (normal) and limb (fibro-proliferative) wounds in an established horse wound model. METHODS Cloning of ANXA2 was achieved by screening size-selected cDNA libraries. Expression was studied in intact skin and in biopsies of 1, 2, 3, 4 and 6-week-old wounds of the body and limb. Temporal gene expression was determined by semi-quantitative RT-PCR while protein expression was mapped immunohistochemically. RESULTS ANXA2 mRNA was up-regulated only in body wounds, corroborating the superior and prompt tissue turnover at this location. Immunohistochemistry partially substantiated the mRNA data in that increased staining for ANXA2 protein was detected in neo-epidermis which formed more rapidly and completely in body wounds. MMP1 mRNA levels in body wounds significantly surpassed those of limb wounds in week one biopsies. The protein was abundant in migrating epithelium of limb wounds at weeks two and four; conversely, body wounds in which epithelialization was near complete showed diminished staining of MMP1. CONCLUSION We conclude that ANXA2 and MMP1 might participate in remodeling during wound healing in the horse, and that differences in expression may contribute to the excessive proliferative response seen in the limb.

CONTRIBUTION OF SRF, ELK-1 AND MYOCARDIN TO AIRWAY SMOOTH MUSCLE REMODELING IN HEAVES, AN ASTHMA-LIKE DISEASE OF HORSES

Myocyte hyperplasia and hypertrophy contribute to the increased mass of airway smooth muscle (ASM) in asthma. Serum-response factor (SRF) is a transcription factor that regulates myocyte differentiation in vitro in vascular and intestinal smooth muscles. When SRF is associated with phosphorylated (p)Elk-1, it promotes ASM proliferation while binding to myocardin (MYOCD) leading to the expression of contractile elements in these tissues. The objective of this study was therefore to characterize the expression of SRF, pElk-1, and MYOCD in ASM cells from central and peripheral airways in heaves, a spontaneously occurring asthma-like disease of horses, and in controls. Six horses with heaves and five aged-matched controls kept in the same environment were studied. Nuclear protein expression of SRF, pElk-1, and MYOCD was evaluated in peripheral airways and endobronchial biopsies obtained during disease remission and after 1 and 30 days of naturally occurring antigenic exposure using immunohistochemistry and immunofluorescence techniques. Nuclear expression of SRF (P = 0.03, remission vs. 30 days) and MYOCD (P = 0.05, controls vs. heaves at 30 days) increased in the peripheral airways of horses with heaves during disease exacerbation, while MYOCD (P = 0.04, remission vs. 30 days) decreased in the central airways of control horses. No changes were observed in the expression of pElk-1 protein in either tissue. In conclusion, SRF and its cofactor MYOCD likely contribute to the hypertrophy of peripheral ASM observed in equine asthmatic airways, while the remodeling of the central airways is more static or involves different transcription factors.

Inhaled corticosteroids modulate the (+)insert smooth muscle myosin heavy chain in the equine asthmatic airways

Rationale Overexpression of the (+)insert smooth muscle myosin heavy chain (SMMHC) isoform could contribute to airway bronchospasm by increasing the velocity of contraction. Whether the (+)insert isoform is present in the small airways and its expression is reversible in asthma are unknown. Objectives To determine the anatomical location and the expression kinetics of the (+)insert SMMHC isoform in airways of horses with heaves and to evaluate its modulation in response to disease status. Methods We evaluated the (+)insert SMMHC isoform in the airways of horses with heaves during disease exacerbation and remission, and in controls. The expression kinetics of the SMMHC (+)insert was then assessed at multiple time points in two studies: first, in horses with heaves treated for a 1-year period with antigen avoidance alone, inhaled corticosteroids alone or both; second, in horses with heaves before and after a 30-day natural antigen exposure. Gene expression analysis was assessed by quantitative PCR and protein expression was confirmed by targeted mass spectrometry. Measurements and main results The (+)insert SMMHC isoform was significantly increased in central and peripheral airways, but not in the trachea of heaves-affected horses in clinical exacerbation when compared horses with heaves in remission and controls. Both corticosteroid administration and antigen avoidance led to a significant reduction of the (+)insert expression in the airways. The (+)insert SMMHC isoform was not significantly increased in airways after 1 month of antigenic re-exposure. Conclusions The (+)insert SMMHC expression is increased throughout the bronchial tree in horses with heaves and reversible by corticosteroids administration and antigen avoidance.

OB-cadherin cloning and expression in a model of wound repair in horses.

REASON FOR PERFORMING STUDY Horses suffer from a debilitating impediment in repairing wounds located on the lower limb that leads to the development of a fibroproliferative disorder (exuberant granulation tissue). This condition is a source of wastage since it often forces retirement from competition. Treatments that resolve or prevent this condition are still lacking, maybe due to deficient knowledge of the underlying molecular mechanisms. Fibroblast-to-myofibroblast conversion is an essential step allowing contraction during wound repair and is accompanied by an increase in OB-cadherin expression. OBJECTIVES To clone equine cadherin-11 (CDH11) cDNA and to study its spatiotemporal expression profile during the repair of body and limb wounds, thereby contributing to a better understanding of the repair process. METHODS Cloning was by a PCR technique. Expression was studied in intact skin and in 1, 2, 3, 4 and 6-week-old wounds of the body and limb. Temporal CDH11 gene expression was determined by RT-PCR while OB-cadherin protein expression was mapped immunohistochemically. RESULTS Equine CDH11 is a highly conserved gene and protein. mRNA was not expressed in equine skin whereas the wound repair process was characterised by a significantly higher expression in the thorax than in limb samples. mRNA expression pattern was paralleled by protein data as confirmed by immunohistochemistry. CONCLUSIONS The data suggest that deficient OB-cadherin expression in the first phases of wound repair contributes to the excessive proliferative response seen in horse limb wounds. POTENTIAL RELEVANCE Future studies should verify the quantitative, temporal expression of this protein in order to provide the basis for targeted therapies that might prevent the development of EGT in horse wound repair.

Evaluation of contractile phenotype in airway smooth muscle cells isolated from endobronchial biopsy and tissue specimens from horses

OBJECTIVE To develop a method to maintain the initial phenotype of airway smooth muscle (ASM) cells isolated from equine endobronchial biopsy specimens in long-term cell culture. SAMPLE Endobronchial tissue specimens (8 to 10/horse) collected from the lungs of previously healthy horses at necropsy (n = 12) and endobronchial biopsy specimens collected from standing, sedated, heaves-affected horses in clinical remission of the disease (5) and control horses (4). PROCEDURES A sampling protocol was developed to recover and maintain a contractile phenotype in ASM cells from endobronchial specimens from freshly harvested equine lungs and from healthy and heaves-affected horses. Immunologic techniques were used to evaluate the contractile phenotype of ASM cells in culture. RESULTS Characteristic ASM cells were successfully cultured from endobronchial tissue or biopsy specimens from both healthy and heaves-affected horses, and their contractile phenotype was maintained for up to 7 passages. Moreover, the capacity of cells at the seventh passage to contract in a collagen gel in response to methacholine was maintained. CONCLUSIONS AND CLINICAL RELEVANCE ASM cells isolated from equine endobronchial tissue and biopsy specimens were able to maintain a contractile phenotype in long-term cell cultures, suggesting they could be used for tissue engineering and in vitro studies of equine ASM cells.

Efficacy of inhaled budesonide for the treatment of severe equine asthma

Summary Background Corticosteroids are the most potent drugs for the control of severe equine asthma, but adverse effects limit their chronic systemic administration. Inhaled medications allow for drug delivery directly into the airways, reducing the harmful effects of these drugs. Objectives To evaluate the efficacy of inhaled budesonide specifically formulated for the equine use and administered by a novel inhalation device in horses with severe asthma. Study design Experimental studies in horses with naturally occurring asthma with cross‐over, randomised, blinded experimental designs. Methods In Study 1, budesonide (1800 μg twice daily) administered using a novel Respimat® based inhaler was compared to i.v. dexamethasone (0.04 mg/kg). In Study 2, 3 doses of budesonide (450, 900, and 1800 μg) were compared to oral dexamethasone (0.066 mg/kg). Lung function, bronchoalveolar fluid cytology (Study 1), CBC, serum chemistry, and serum cortisol and adrenocorticotropic hormone (ACTH) values were evaluated. Results In Study 1, there was a marked and significant improvement in the lung function of all horses treated with budesonide and dexamethasone. Neutrophil percentages in bronchoalveolar fluid decreased in all horses treated with dexamethasone and in four of six horses treated with budesonide. Serum cortisol and blood ACTH concentrations decreased with both treatments. In Study 2, there was a significant improvement in the lung function with all dosages of budesonide, and the effects of higher dosages were comparable to those of dexamethasone. Dexamethasone and budesonide at the two higher dosages induced a significant decrease of cortisol concentrations. Main limitations The Respimat® based inhaler is not currently commercially available. Conclusions Administration of budesonide with the Respimat® based inhaler provided dose‐dependent relief of airway obstruction in horses with severe asthma, but also a suppression of serum cortisol.