Acute phase proteins in racehorses with inflammatory airway disease.

BACKGROUND: Systemic inflammation is observed in horses with heaves and could also be present in horses with a lesser degree of pulmonary inflammation. HYPOTHESIS/OBJECTIVES: It was hypothesized that racehorses with inflammatory airway disease (IAD) have increased concentration of circulating acute phase proteins. The objective of this study was to compare serum acute phase proteins of racehorses with and without lower airway inflammation. ANIMALS: Serum from 21 client-owned Standardbred racehorses with exercise intolerance and lower airway inflammation and serum from 10 client-owned Standardbred racehorses with exercise intolerance without lower airway inflammation. METHODS: In a case-control study, serum samples from previously characterized horses presented for exercise intolerance with or without lower airway inflammation based on bronchoalveolar lavage fluid cytology were analyzed for serum amyloid A protein (SAA), C-reactive protein (CRP), and haptoglobin using commercial ELISAs. RESULTS: There was no significant differences between groups for SAA (non-IAD versus IAD, median (range): 3.47 (0.06-34.94) versus 6.33 (0.06-80) µg/mL, P = .49), CRP (10.87 (2.05-29.03) versus 4.63 (0.02-31.81) µg/mL, P = .23) or haptoglobin (900.36 (607.99-2018.84) versus 749.54 (530.81-1076.95) µg/mL, P = .09). CONCLUSIONS AND CLINICAL IMPORTANCE: In this population of poorly performing racehorses in training, serum SAA, CRP, and haptoglobin were not helpful in distinguishing between horses with IAD from horses with exercise intolerance from other causes.

Inflammatory stimuli differentially modulate the transcription of paracrine signaling molecules of equine bone marrow multipotent mesenchymal stromal cells.

OBJECTIVE: Osteoarthritis (OA) is a degenerative disease of joint tissues that causes articular cartilage erosion, osteophytosis and loss of function due to pain. Inflammation and inflammatory cytokines in synovial fluid (SF) contribute to OA progression. Intra-articular (IA) injections of multipotent mesenchymal stromal cells (MSCs) are employed to treat OA in both humans and animals. MSCs secrete paracrine pro-inflammatory and anabolic signaling molecules that promote tissue repair. The objective of this study was to investigate the effects of OASF on the gene expression of paracrine signaling molecules by MSCs. METHODS: The effects of Lipopolysaccharide (LPS) and interleukin (IL)-1ß as well as both normal (N) and osteoarthritis (OA) SF stimulations on the expression of paracrine pro-inflammatory (tumor necrosis factor (TNF)-α, IL-1ß, IL-8), modulatory (IL-6) and anabolic (vascular endothelial growth factor (VEGF), transforming growth factor (TGF)-ß1 and insulin-like growth factor (IGF)-1) signaling molecules by equine bone marrow multipotent mesenchymal stromal cells (eBM-MSCs) was investigated employing reverse transcriptase-polymerase chain reaction (RT-PCR). RESULTS: In contrast with NSF, OASF significantly up-regulated the expression of VEGF in eBM-MSCs. Both NSF and OASF significantly down-regulated the expression of IL-1ß. LPS and IL-1ß significantly increased the expression of pro-inflammatory cytokines (TNF-α, IL-8 and IL-6; and IL-1ß and IL-8 respectively). DISCUSSION: We conclude that the transcription of paracrine signaling molecules in eBM-MSCs is modulated by SF. Furthermore, OA alters the properties of SF and the response of eBM-MSCs. Finally, the effects of LPS or IL-1ß stimulation are distinct to that observed following stimulations with OASF.

PI3K and MAPKs regulate neutrophil migration toward the airways in heaves.

BACKGROUND: Neutrophils accumulate in the airways of horses with heaves. They likely play an important role in the disease pathogenesis. Understanding the pathways regulating their migration may help identifying new therapeutic targets. HYPOTHESIS: MAPK and PI3K pathways are involved in neutrophil migration toward the airway lumen in heaves. ANIMALS: Twelve heaves-affected horses and 4 healthy horses. METHODS: Migratory activity of bronchoalveolar lavage fluids (BALF) from horses with heaves and healthy horses was compared by means of a Boyden chamber. Involvement of MAPK and PI3K pathways in neutrophil migration was investigated by pretreating neutrophils with inhibitors of p38 MAPK, JNK, MEK1/2, and PI3K. The capacity of a p38 MAPK inhibitor at decreasing neutrophil chemotaxis toward the airways was also evaluated in vivo. RESULTS: BALF from symptomatic heaves-affected horses induced a greater degree of chemokinesis (P = .0004) than BALF from healthy horses. Although all pathways tested were involved in neutrophil migration, inhibition of PI3K was most potent in vitro. An inhibitor of p38 MAPK administered before challenge in horses with heaves did not alter BALF chemokinetic properties. BALF neutrophil percentage and BALF migratory activity were positively correlated after 14 and 35 days of antigen challenge in healthy (P = .05; R(2) = 0.82) and heaves-affected horses (P = .03; R(2) = 0.76), respectively. CONCLUSIONS AND CLINICAL IMPORTANCE: MAPK and PI3K pathways regulate neutrophil migration induced by BALF of horses with heaves. Inhibition of multiple pathways might be required to completely abolish BALF-induced neutrophil migratory activity and possibly inflammation in heaves.

Markers of systemic inflammation in horses with heaves.

BACKGROUND: Systemic inflammation in horses with heaves is poorly characterized. OBJECTIVES: To assess acute phase proteins (APP) and inflammatory cytokine profiles in serum of healthy horses and horses with heaves. ANIMALS: Six healthy horses and 6 heaves-affected horses belonging to the University of Montreal. METHODS: Prospective, observational study. Healthy and heaves-affected control horses were exposed to a 30-day natural challenge with hay and straw to induce clinical exacerbation of heaves. Serum samples were obtained by venipuncture before (T0) as well as after 7 (T7) and 30 days (T30) of stabling. Serum APP (haptoglobin, serum amyloid A protein [SAA] and C-reactive protein [CRP]) and cytokines (IL-2, IL-4, IFN-α, IL-10, IFN-γ, and CCL-2) were measured using singleplex or multiplex ELISA. RESULTS: Serum haptoglobin concentrations were significantly higher in heaves-affected horses at all time points with no overlap with those of healthy controls. They were also significantly increased by antigen challenge in both controls (T7) and horses with heaves (T7 and T30). Serum SAA was detected more frequently in heaves-affected horses compared with healthy controls at T7. There was no difference in serum concentrations of CRP, IL-10, IFN-γ, and CCL-2 between groups, whereas IL-2, IL-4, and IFN-α remained undetectable in all samples. CONCLUSIONS AND CLINICAL IMPORTANCE: In heaves, haptoglobin is a marker of both acute and chronic systemic inflammation, whereas high concentrations of SAA indicate acute inflammation.

Effect of long-term fluticasone treatment on immune function in horses with heaves.

BACKGROUND: Corticosteroids currently are the most effective pharmacological treatment available to control heaves in horses. Systemically administered corticosteroids have been shown to alter immune response in horses, humans, and other species. Aerosolized administration theoretically minimizes systemic adverse effects, but the effect of inhaled corticosteroids on immune function has not been evaluated in horses. OBJECTIVES: To evaluate the effects of prolonged administration of inhaled fluticasone on the immune system of heaves-affected horses. ANIMALS: Heaves-affected horses were treated with inhaled fluticasone (n = 5) for 11 months or received environmental modifications only (n = 5). METHODS: Prospective analysis. Clinical parameters and CBC, lymphocyte subpopulations and function, and circulating neutrophil gene expression were sequentially measured. Primary and anamnestic immune responses also were evaluated by measuring antigen-specific antibodies in response to vaccination with bovine viral antigen and tetanus toxoid, respectively. RESULTS: No clinical adverse effects were observed and no differences in immune function were detected between treated and untreated horses. CONCLUSIONS AND CLINICAL IMPORTANCE: The treatment of heaves-affected horses with inhaled fluticasone at therapeutic dosages for 11 months has no significant detectable effect on innate and adaptive (both humoral and cell-mediated) immune parameters studied. These results suggest that prolonged administration of fluticasone would not compromise the systemic immune response to pathogens nor vaccination in adult horses.

Bronchoalveolar lavage fluid cytology and cytokine messenger ribonucleic Acid expression of racehorses with exercise intolerance and lower airway inflammation.

BACKGROUND: There is limited information relating bronchoalveolar lavage (BAL) cytology and cytokine messenger ribonucleic acid (mRNA) expression in racehorses with inflammatory airway disease (IAD). HYPOTHESIS AND OBJECTIVE: We hypothesize that cytokine expression in BAL cells would correlate with cytology. Thus, we evaluated the mRNA expression of selected cytokines in BAL cells in racehorses with exercise intolerance and lower airway inflammation. ANIMALS: Thirty-one client-owned Standardbred racehorses with exercise intolerance. METHODS: Prospective, observational study. Cells were obtained by BAL, and mRNA expression of interleukin (IL)-1ß, IL-4, IL-8, tumor necrosis factor (TNF)-α, and interferon (IFN)-γ was determined by reverse transcription quantitative polymerase chain reaction (RT-qPCR). RESULTS: Nine horses had normal BAL cell differential cytology (Controls), while 22 horses had evidence of IAD based on BAL fluid cytology. Relative expressions of TNF-α/glyceraldehyde 3-phosphate dehydrogenase (GAPDH; 0.0092 ± 0.010 versus 0.0045 ± 0.005, P= .034), IL-4/GAPDH (0.001 ± 0.002 versus 0.0003 ± 0.0003, P= .029), and IFN-γ/GAPDH (0.0027 ± 0.003 versus 0.0009 ± 0.001, P= .028) were greater in horses with IAD compared with controls. Furthermore, IL-4/GAPDH (0.001 ± 0.002 versus 0.0002 ± 0.0003, P < .0001) and IFN-γ/GAPDH (0.003 ± 0.003 versus 0.001 ± 0.001, P= .002) mRNA expression was increased in horses with increased metachromatic cell counts compared with horses with normal metachromatic cell counts. Only the mRNA expression of IL-1ß/GAPDH (1.1 ± 0.7 versus 0.3 ± 0.3, P= .045) was increased with airway neutrophilia. CONCLUSIONS AND CLINICAL IMPORTANCE: Differences in gene expression were associated with the presence of IAD and with specific cell types present in airway secretions of Standardbred racehorses with poor performance. These findings suggest that different pathophysiological pathways are implicated in IAD.

Cholinergic stimulation attenuates the IL-4 induced expression of E-selectin and vascular endothelial growth factor by equine pulmonary artery endothelial cells.

The endothelium plays a critical role in regulating leukocyte recruitment and migration during inflammation. Recent studies provide evidence that acetylcholine (ACh) and other cholinergic mediators block endothelial cells activation and leukocyte recruitment during inflammation. We thus postulated that the non-neuronal cholinergic system might modulate the recruitment of neutrophils during allergic pulmonary inflammation. In the present study, we examined the effects of cholinergic stimulation on the expression of neutrophil chemokines and adhesion molecules by endothelial cells stimulated by recombinant equine (re) IL-4. Using primary equine pulmonary artery endothelial cells culture and real-time RT-PCR method, we observed that ACh, nicotine, and muscarine inhibit the expression of E-selectin and vascular endothelial growth factor by endothelial cells stimulated by reIL-4. The expression of CXCL-8, a potent neutrophil chemotactic cytokine, remained unaffected however. These findings suggest that the cholinergic anti-inflammatory pathway may modulate pulmonary allergic inflammation and remodeling by the inhibition of selected adhesion molecules and growth factors.

Genomic and non-genomic effects of dexamethasone on equine peripheral blood neutrophils.

BACKGROUND: Glucocorticoids have potent anti-inflammatory properties and are frequently used for the treatment of domestic animal species, including horses. They induce a down-regulation of multiple inflammatory pathways through both genomic and non-genomic effects. Currently, little is known on the effects of glucocorticoids on equine peripheral blood neutrophils. HYPOTHESIS: Dexamethasone (DEX), a potent synthetic glucocorticoid, inhibits the functions of equine peripheral blood neutrophils through both genomic and non-genomic effects. ANIMALS: Six healthy adult mixed breed female horses. METHODS: To assess the genomic effects of DEX, peripheral blood neutrophils were isolated using a gradient technique and incubated 6 h with 100 ng/ml LPS and 10(-6) M DEX alone, or combined with the glucocorticoid receptor (GR) inhibitor RU486 (10(-5) M). Messenger RNA for IL-8, TNF-alpha and TLR-4 were measured using real-time RT-PCR. The non-genomic effects of DEX were studied in neutrophils incubated with 5 microM dichlorodihydrofluorescein (DCF) and 10(-6) M DEX 5, 10 and 15 min prior to being stimulated with 5 ng/ml phorbol myristate acetate. Neutrophils were similarly co-incubated with DEX (10(-6) M, 15 min) and RU486 (10(-5) M) to evaluate the contribution of the GR to these effects. The oxidation of DCF was studied using flow-cytometry. RESULTS: Neutrophils stimulation with LPS resulted in a significant increase in IL-8, TNF-alpha and TLR-4 mRNA expressions (p<0.0001); incubation with DEX significantly down-regulated this process (p<0.0001). DEX significantly reduced oxidation of DCF after 10 and 15 min of incubation (p<0.0001). Those effects were mediated through the GRs. CONCLUSION: DEX exerts anti-inflammatory effects on equine peripheral blood neutrophils through both genomic and non-genomic pathways.