Abstract
Background
Equine asthma (EA) is a chronic lower airway inflammation that leads to structural and functional changes. Hyaluronic acid (HA) has crucial functions in the extracellular matrix homeostasis and inflammatory mediator activity. HA concentration in the lungs increases in several human airway diseases. However, its associations with naturally occurring EA and airway remodelling have not been previously studied. Our aim was to investigate the association of equine neutrophilic airway inflammation (NAI) severity, airway remodelling, and HA concentration in horses with naturally occurring EA. We hypothesised that HA concentration and airway remodelling would increase with the severity of NAI. HA concentrations of bronchoalveolar lavage fluid supernatant (SUP) and plasma of 27 neutrophilic EA horses, and 28 control horses were measured. Additionally, remodelling and HA staining intensity were assessed from endobronchial biopsies from 10 moderate NAI horses, 5 severe NAI horses, and 15 control horses.
Results
The HA concentration in SUP was higher in EA horses compared to controls (p = 0.007). Plasma HA concentrations were not different between the groups. In the endobronchial biopsies, moderate NAI horses showed epithelial hyperplasia and inflammatory cell infiltrate, while severe NAI horses also showed fibrosis and desquamation of the epithelium. The degree of remodelling was higher in severe NAI compared to moderate NAI (p = 0.048) and controls (p = 0.016). Intense HA staining was observed in bronchial cell membranes, basement membranes, and connective tissue without significant differences between the groups.
Conclusion
The release of HA to the airway lumen increases in naturally occurring neutrophilic EA without clear changes in its tissue distribution, and significant airway remodelling only develops in severe NAI.
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Background
Equine asthma (EA) is characterised by hyperresponsive airways and chronic lower airway inflammation of variable degree. Severe EA is associated with structural changes in the airways and apparent clinical signs, such as frequent coughing and respiratory effort at rest, while in mild/moderate EA the inflammation is milder without marked structural changes, and it is less distinct both in diagnostics and clinical signs. Typical clinical signs in mild/moderate EA are decreased performance and chronic, intermittent or occasional cough usually observed during exercise [1].
EA and human asthma share some etiological, pathophysiological, and immunological similarities, such as hyperresponsive airways, sensitivity to certain antigens and endotoxins, and pulmonary remodelling. Thus, horses can provide a naturally occurring translational model for human asthma [2]. While about 50% of severe human asthma is characterised by eosinophilia, this is unusual for severe EA, which typically shows neutrophilic response, and mild/moderate EA displays mildly increased neutrophils, eosinophils, or metachromatic cells [1]. Analysing histopathological findings in bronchial biopsies, horses with naturally occurring EA showed bronchial epithelial and submucosal inflammation, thickening of the basal membrane, airway smooth muscle hypertrophy, and fibrosis, expanding the understanding of the pathophysiology of EA [3,4,5,6].
Hyaluronic acid (HA) is a critical component of the healthy, effectively working extracellular matrix (ECM) with dual functions in inflammatory processes. HA is a non-sulfated glycosaminoglycan, produced in the inner leaflet of plasma membrane by the activity of HA synthases [7]. HA synthases and hyaluronidases are responsible for the diversity of HA molecular size. Different HA synthases produce HA molecules of various sizes, and hyaluronidases degrade HA to smaller fragments [8]. The biological activity of HA depends on its size: low-molecular-weight (LMW) HA (100–500 kDa) stimulates cell migration and inflammatory protein production, while high-molecular-weight (HMW) HA (1000–6000 kDa) has anti-inflammatory and tissue protecting functions [7, 9]. HA serves as a critical component in the ECM of most tissues, including airways and lungs [7, 10]. Elevated levels of HA in bronchoalveolar lavage fluid (BALF) have been detected in asthma in animal and human studies, and the presence of LMW HA has been suggested to indicate inflammation [11,11, 13, 14, 26,27,28,29]. In these previous studies, the increased HA was suggested to derive from its increased production in lung interstitium fibroblasts, epithelial cells, and alveolar cells, and to be associated with tissue fibrosis. This could indicate an attempt of the organism to protect the tissues and to provide hydration during chronic inflammation. However, in this study, increased HA content was not associated with fibrosis. HA is synthesised in the inner surface of cell membrane by HA synthases, and after synthesis it is transported to the extracellular space [30]. Therefore, as shown in the present study, HA is abundant in cell membranes, extracellular structures, and connective tissue.
In this study, the tissue distribution of HA, the intensity of HA staining, and plasma HA concentration were similar between the groups, unlike in a study on human patients with induced asthma exacerbation [31]. Lauer et al. [31] found that controls had less HA in lung tissues due to lack of smooth muscle hypertrophy and basement membrane thickening. Additionally, in their study serum HA concentration peaked in asthmatic patients five hours after an allergen challenge, which was not performed in our study. Moreover, human allergic asthma is often eosinophilic, while we studied neutrophilic airway inflammation [32].
HA molecules can have both pro- and anti-inflammatory actions depending on their size, and during inflammation the production of LMW HA increases, which leads to the activation of inflammatory pathways and intensive cytokine production [7, 33]. It has been suggested that local production of LMW HA contributes to airway bronchoconstriction, inflammation, and fibrosis, and is associated with chronic asthma and remodelling [41].
Conclusion
In this study, we show for the first time that HA concentration increases in BALF in naturally occurring neutrophilic EA. However, the HA tissue distribution and plasma concentration remained similar in control horses and horses with EA. Airway remodelling was the highest in horses with severe NAI, but it was not associated with airway HA content. Identifying the association between EA and increased HA concentration in lungs can offer new insights to EA therapy modalities and further research. In the future, the roles of HMW and LMW HA in EA need further investigations with the focus on therapy.
Data availability
The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.
Abbreviations
- ANCOVA:
-
analysis of covariance
- BALF:
-
bronchoalveolar lavage fluid
- bHABC:
-
biotinylated hyaluronic acid binding complex
- EA:
-
equine asthma
- EBB:
-
endobronchial biopsy
- ECM:
-
extracellular matrix
- HA:
-
hyaluronic acid
- HABC:
-
hyaluronic acid binding complex
- HE:
-
haematoxylin and eosin
- HMW:
-
high-molecular-weight
- LMW:
-
low-molecular-weight
- Masson:
-
Masson Trichrome
- NAI:
-
neutrophilic airway inflammation
- PaO2 :
-
arterial oxygen content
- rs :
-
Spearman’s rank correlation
- SD:
-
standard deviation
- SUP:
-
supernatant
- TW:
-
tracheal wash
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Acknowledgements
We kindly thank Merja Ranta, Ninna Koho, Eija Rahunen, and Taija Hukkanen for their expertise in laboratory work. Laura Hänninen is greatly acknowledged for her expertise in statistical analyses.
Funding
Open Access funding provided by University of Helsinki (including Helsinki University Central Hospital). Financial support for the study was provided by the Finnish Veterinary Foundation and the Finnish Foundation of Veterinary Research.
Open Access funding provided by University of Helsinki (including Helsinki University Central Hospital).
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AM and NH designed and coordinated the study. NH, AM, and HR collected the samples and conducted the sample processing. H-MJ and NA performed the biopsy histological scoring and prepared the images of histological sections. PN and A-MM carried out confocal microscopy, SO performed the HA analyses, and VK assessed the biopsy HA staining. NH, AM, PN, and A-MM conducted the statistical analyses and prepared the images and tables. NH drafted the manuscript. All authors revised the draft critically and read and approved the final submitted manuscript.
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The animal study was reviewed and approved by the Project Authorization Board in the Regional State Administrative Agency (ESAVI/3285/2020), and all experiments were performed in accordance with EU regulations and ARRIVE guidelines. Written informed consent was obtained from the owners for the participation of their animals in this study.
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12917_2024_4136_MOESM1_ESM.png
Supplementary Material 1: Endobronchial biopsy from a control horse stained with (A) or without (B) the biotinylated hyaluronic acid binding complex
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Höglund, N., Rossi, H., Javela, HM. et al. The amount of hyaluronic acid and airway remodelling increase with the severity of inflammation in neutrophilic equine asthma. BMC Vet Res 20, 273 (2024). https://doi.org/10.1186/s12917-024-04136-2
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DOI: https://doi.org/10.1186/s12917-024-04136-2