Increased hypoxia-inducible factor 1α expression in lung cells of horses with recurrent airway obstruction
© Toussaint et al.; licensee BioMed Central Ltd. 2012
Received: 6 February 2012
Accepted: 7 May 2012
Published: 23 May 2012
Recurrent airway obstruction (RAO, also known as equine heaves) is an inflammatory condition caused by exposure of susceptible horses to organic dusts in hay. The immunological processes responsible for the development and the persistence of airway inflammation are still largely unknown. Hypoxia-inducible factor (Hif) is mainly known as a major regulator of energy homeostasis and cellular adaptation to hypoxia. More recently however, Hif also emerged as an essential regulator of innate immune responses. Here, we aimed at investigating the potential involvement of Hif1-α in myeloid cells in horse with recurrent airway obstruction.
In vitro, we observed that Hif is expressed in equine myeloid cells after hay dust stimulation and regulates genes such as tumor necrosis factor alpha (TNF-α), interleukin-8 (IL-8) and vascular endothelial growth factor A (VEGF-A). We further showed in vivo that airway challenge with hay dust upregulated Hif1-α mRNA expression in myeloid cells from the bronchoalveolar lavage fluid (BALF) of healthy and RAO-affected horses, with a more pronounced effect in cells from RAO-affected horses. Finally, Hif1-α mRNA expression in BALF cells from challenged horses correlated positively with lung dysfunction.
Taken together, our results suggest an important role for Hif1-α in myeloid cells during hay dust-induced inflammation in horses with RAO. We therefore propose that future research aiming at functional inactivation of Hif1 in lung myeloid cells could open new therapeutic perspectives for RAO.
KeywordsHypoxia inductible transcription factor-1 Recurrent airway obstruction Inflammation Lung Horse
Recurrent airway obstruction (RAO) or heaves is a well-known respiratory disease in horses that shares any pathophysiological similarities with asthma in humans [1–3]. RAO is a severe, potentially debilitating, chronic inflammatory airway disease typically affecting middle-aged horses. Acute exacerbations are characterized by neutrophilic airway inflammation, coughing, periods of labored breathing at rest and exercise intolerance due to bronchospasm and mucus accumulation in the airways . It is initiated following exposure to organic dusts, molds, and lipopolysaccharides (LPS) in hay . Periods of acute exacerbation are interspersed by periods of remission, when horses are kept away from the causative environment . The immunological processes responsible for the persistent airway inflammation are still largely unknown . RAO is thought to result from an aberrant immune response orchestrated by antigen-specific T lymphocytes via the secretion of pro-inflammatory cytokines. Whether these T lymphocytes have a type 1 or type 2 phenotype and cytokine secretion profile is still a matter of debate [7–11]. Although little studied so far, innate immune mechanisms, which constitute a central interface between external stimuli and the adaptive immune system, may also play an important role in the pathophysiology of RAO.
Hif1 is an essential regulator of adaptation to low oxygen levels . Hif1 is a heterodimer composed of an oxygen-regulated α-subunit and a constitutively expressed β-subunit. In general the abundance of α-subunits is primarily regulated by a family of prolyl hydroxylases (PHD). In normoxia, PHD is activated and directs the degradation of the α-subunit by the ubiquitin-proteasome pathway .
Under hypoxia, PHD activity decreases, which leads to the stabilization and translocation to the nucleus of the α-subunits which heterodimerize with the β-subunit. This dimer recognizes the hypoxia response elements (HREs) to induce target gene expression. Hif1 activity is primarily regulated by the abundance of the Hif1-α subunit . Since Hif1 is highly involved in the adaptation of cellular metabolism to hypoxic condition, it was also proposed that Hif1 is an important promoter of inflammatory responses, which most often require innate immune cells to adapt to oxygen-deprived inflammatory environments [15, 16]. Indeed, it was found that Hif1 regulates many pro-inflammatory genes such as tumor necrosis factor alpha (TNF-α), interleukin-8 (IL-8) and vascular endothelial growth factor A (VEGF-A) . Hif1 was furthermore found to engage in cross-talk with another major pro-inflammatory pathway, the Nuclear Factor (NF)- κB pathway . Although most studies focused on hypoxic conditions, Hif may promote inflammatory functions of myeloid cells also in normoxic conditions [19, 20]. Indeed, Hif1-α may be indirectly activated in myeloid cells by pro-inflammatory stimuli such as LPS and the pro-inflammatory cytokine TNF-α [21, 22]. After LPS or cytokine stimulation, NF-κB promotes Hif1-α gene transcription, promoting its accumulation in spite of post-translational degradation [18, 23]. In myeloid cells, hypoxia through decreased Hif1-α degradation and inflammatory stimuli through increased Hif1-α transcription may synergistically potentiate the activation of Hif1.
We hypothesized that, because myeloid cells are implicated in RAO, and because Hif1 is a major regulator of the pro-inflammatory functions of myeloid cells, Hif1 could play a role in the pathophysiology of RAO. To test this hypothesis, complementary in vitro and in vivo approaches were undertaken.
Firstly we assessed in vitro expression of Hif1 mRNA and its target genes in horse myeloid cells in response to hay dust. Secondly, in vivo, we analyzed whether hay dust-induced lung inflammation in RAO-affected horses correlated with increased Hif1 expression in lung myeloid cells. Finally, we tested whether increased Hif1 expression in BALF myeloid cells correlated with clinical variables in RAO-affected horses.
Hif1- is expressed basally and upregulated by hay dust in horse myeloid cells
Hay dust induced pro-inflammatory genes associated with Hif1-α activity in equine monocytes
Horse clinical status assessment
Individual results of clinical evaluation
Epithelial cells (%)
Increased Hif1-α mRNA expression following hay dust challenge in BALF cells of healthy and RAO-affected horses
Hif1-α-regulated cytokines are overexpressed in the BALF cells of RAO-affected horses during crisis
Correlation between Hif1-α expression in lung myeloid cells and lung dysfunction
Correlation between Hif1-α mRNA expression and clinical parameters
Hif1-α mRNA/Neutrophils (%)
Hif1-α mRNA/PaO2 (mmHg)
Hif1-α mRNA/X5HZ (kPa/L-1/s)
Healthy(n = 4)
r = −0.519
r = 0.231
r = −0.634
RAO-affected(n = 5)
r = 0.863 (*)
r = −0.881 (*)
r = −0.844 (*)
Hif1 is increasingly recognized as a master regulator of inflammatory responses. Recently, it has been demonstrated that Hif1 activity is increased in the lung of mouse models of asthma, as well as in BALF cells from asthmatic patients [24, 25]. In the present work, we report similar findings in horses suffering from RAO, a disease that shares many similarities with human asthma. We demonstrated that the level of Hif1-α mRNA expression was significantly increased in BALF cells of hay dust-challenged horses and that this effect was more pronounced in RAO-affected than in healthy animals.
The increase in Hif1-α mRNA expression in BALF cells of challenged horses may be explained by the presence of LPS in hay dust. Indeed, it has been previously demonstrated that LPS induces Hif1-α mRNA expression through NF-κB activation in human monocytes and macrophages under normoxic conditions . While administration of aerosolized LPS to RAO-affected horses induces airway neutrophilia, it is not sufficient to elicit an RAO crisis . At the present time the contribution of LPS to disease severity remains unclear. Yet, emerging evidence suggests that a synergistic effect may exist between inhaled LPS and organic dust particulates. Hif1 activation could also be enhanced by other components present in the hay dust, such as Alternaria spores, β-Glucans, which also could activate NF- κB [27, 28], even though this hypothesis will require further testing. From this first study, we suggest that LPS or other components present in hay dust may upregulate Hif1-α gene expression via NF-κB activation in both healthy and RAO-affected horses, with a more pronounced effect in RAO-affected horses. It is well known that NF-κB activation and hypoxia synergistically activate Hif1 . Whereas NF-κB increases Hif1-α mRNA expression through fixation on a NF-κB site in the promoter of the Hif1-α gene, hypoxia post-translationally stabilizes the Hif1-α protein . Accordingly, it has previously been shown that NF-κB activity is significantly increased in the lungs of RAO-affected horses during crisis when compared to unchallenged healthy horses . Moreover, during RAO crises, inflammation induces a hypoxic environment in the lung tissues, which is a consequence of decreased perfusion, bronchial edema and increase in metabolic activity of recruited inflammatory cells. Furthermore, as illustrated by our clinical results, inflammation-induced local tissue hypoxia may be reinforced by the fact the horses in crisis usually become hypoxemic. Given that Hif1-α mRNA expression was significantly higher in challenged RAO-affected horses than in healthy subjects, it is possible that during the challenge, healthy horses, unlike RAO-affected horses, do not develop enough lung inflammation to induce hypoxia and thereby to increase Hif1 activity. A synergy between NF-κB activation and hypoxia in inflamed airways might thereby potentiate Hif1 activation in RAO. Further studies should be conducted to directly verify this hypothesis.
The observed increase in Hif1-α mRNA expression in BALF cells of hay dust-exposed horses did not necessarily imply that Hif1 transcriptional activity was increased. To estimate in vivo whether Hif1 was transcriptionally active, we could evaluated the expression of known Hif1 target genes. Hay dust challenge induced a significant increase in IL-8 and TNF-α mRNA expression in healthy and RAO-affected horses, and this increase was significantly more pronounced in RAO-affected horses. However, only challenged RAO-affected horses showed increased VEGF-A expression. The use of a Hif1-α inhibitor indicated that VEGF-A mRNA and IL-8 mRNA were highly regulated by Hif1 in hay dust stimulated-monocytes in vitro, in line with reports that Hif1 is able to regulate VEGF-A and IL-8 in human and murine macrophages . However, the regulation of TNFα- mRNA expression in monocytes after hay dust stimulation was only weakly Hif1 dependent. It is likely that hay dust-induced TNF-α mRNA expression is mainly under the control of other transcription factor such as NF-κB. Following LPS stimulation, Hif1 can induce TNF-α transcription by direct fixation to the TNF-α gene promoter . Yet NF-κB may also induce the transcriptional activation of TNF-α independently of Hif1 .
In an in vitro study, Laan and coll.  reported that IL-8 and TNFα- play a role in RAO. In equine AM stimulated with a hay dust solution, the expression of these two cytokines is significantly increased in RAO horses compared to healthy horses [34, 35]. In rabbits, lung hypoxia was shown to promote IL-8 production from AM . Moreover Franchini et al.  have suggested that AM are implicated in the initial inflammatory reaction, since they demonstrated that release of IL-8 by AM was required for neutrophil attraction. Taken together, these findings may suggest that in RAO-affected horses, the exacerbation of Hif1 activation in AM might be the initial response to airborne challenges, followed by an increase of IL-8 cytokine production and neutrophil invasion.
Currently, we have no information about the role played by VEGF-A in heaves. VEGF-A is a potent stimulator of vascular angiogenesis, permeability, and remodeling that also plays important roles in wound healing and tissue cytoprotection . The regulation of vascular permeability is essential for inflammatory processes in airways, especially in the course of chronic lung diseases . In humans beings and mice, VEGF-A is involved in the pathophysiology of bronchial asthma [39, 40]. Given the similarities between RAO and asthma it may be speculated that VEGF-A could play an important role in RAO pathophysiology.
The expression of these three cytokines positively correlated in vitro and in vivo with that of Hif1-α. Even though our in vitro observations suggest that Hif1 activity in monocytes may contribute to expression of these cytokines in vivo, we do not rule out a possible involvement of Hif1 in other cell types.
Moreover even if monocytes are precursors of AM, they are not terminally differentiated cells. Therefore, some differences may exist in terms of regulation of Hif1 expression and activity between monocytes and AM. For instance, unlike AM, monocytes are unable to phagocyte and cytokine production and expression of surface molecules is sometimes different . However, it is well known in others species that Hif1-α is expressed by AM  and that LPS can induce Hif1-α in macrophage [34, 43]. It is thus highly likely that, like in monocytes, Hif1-α may be expressed by equine AM after hay dust challenge. Given the causative role proposed for AM in RAO, increased Hif1-α activity in AM following hay dust exposure might thus be responsible for crisis induction in RAO affected- horses.
It could also be worthwhile testing Hif1-α activity in equine neutrophils after hay dust stimulation. Indeed in mice, LPS stimulation can induce Hif1 activation in lung neutrophils . After hay dust exposure, BALF neutrophil percentage increases only in RAO-affected horse and positively correlate with Hif1-α mRNA expression. Thus, it is possible that in RAO-affected horses, hay dust-induced neutrophil influx in the lung could also contribute to upregulated Hif1 activity.
Finally, we have also shown that in challenged RAO-affected horses the level of Hif1-α expression in BALF cells correlated with the degree of lung dysfunction. It will be worthwhile testing whether pharmacological inhibition of Hif1 in RAO-affected horses may impact on disease parameters. Supporting the relevance of this approach, it was shown that inhibition of Hif1 in mice using either pharmacological inhibition or conditional gene deletion significantly reduces airway inflammation in models of asthma [24, 25].
In summary, this study demonstrates that hay dust may activate Hif1 in horse myeloid cells. It further shows that lung myeloid cells display increased Hif1 activation upon hay dust challenge in RAO-affected horses compared to healthy horses, and that this activation correlates with disease severity. Our results thus suggest that it would be worthwhile testing the potential therapeutic benefit of pharmacological Hif1 inhibition in RAO.
Five RAO horses with a history of recurrent episodes of respiratory distress when exposed to dusty stable environment (3 females and 2 geldings, median; ranges, 14.5; 9–22 years) and four healthy controls (1 female and 3 geldings, median; ranges, 17.5; 11–28 years) were investigated in the study. All horses affected by RAO showed recurrent clinical signs for several years and were therefore defined as chronically affected. Several months before the experiment began, horses were dewormed using ivermectin and vaccinated against tetanus and equine influenza. All experiments were conducted with approval of the Institutional Animal Care and Use Committee of the University of Liège.
Experimental protocol in vivo
Pulmonary function tests
Pulmonary function was evaluated at rest with an equine impulse oscillometry system (IOS) Master Screen (VIASYS Healthcare GmbH, Höchberg, Germany), validated for the horse [47, 48]. Only the reactance at 5 Hz was used because it is the most sensitive for measuring changes in mechanical ventilation in the lower respiratory system. Before each experiment, the system was calibrated and the tests were performed without any prior sedation.
Arterial blood gas analysis
Arterial blood was anaerobically collected in heparinized syringes by arterial puncture of the carotid artery with a 20 G 0.9 x 40 mm needle (Terumo Europe, Leuven, Belgium). The blood was immediately analyzed for partial pressure of oxygen and carbon dioxide (PaO2 and PaCO2) with the use of an autocalibrated blood gas analyzer (OPTI CCA; Osmetech, Roswell, Georgia, USA). The results were corrected for the patient’s body temperature.
Bronchoalveolar lavage fluid cell isolation
The horses were sedated with IV romifidine, (0.01 mg/kg; Sedivet; Boehringer Ingelheim, Ingelheim, Germany), and butorphanol (0.02 mg/kg; Torbugesic; Fort Dodge, Wyeth, Madison, New Jersey, USA). Bronchoalveolar lavage was performed by instillation of six boluses of 60 mL of sterile saline with EDTA (0.6 mM) at 37°C into the working channel of a fiberoptic endoscope (250 cm x 9 mm, Pentax, Breda, The Netherlands) wedged into a bronchus. The samples recovered from the six syringes were pooled and immediately placed on ice. An aliquot was collected for cytological analysis and differential cell counts.
Bronchoalveolar lavage fluid cells were collected by centrifugation for 7 min at 300 g. The pellet was filtered and washed twice in phosphate buffered saline (PBS). Cells were resuspended in lysis buffer RA1 (NucleoSpin® RNAII; Macherey-Nagel, Düren, Germany) according to the manufacturer’s instructions and stored at −80°C until RNA isolation.
Production of hay dust suspension
Hay dust suspensions were prepared as previously described by Pirie et al.. Briefly, visibly moldy patches of hay were manually agitated and the dust collected. Fine dust (1 g) was suspended in 1 mL isotonic saline (0.9% NaCl) solution and assayed for LPS content. The quantity of LPS present in the hay dust suspension (48234 EU/mL) was measured by ELISA (ENdoLISA®; Hyglos, Bernried am Starnberger See, Germany). The hay dust suspension was diluted in culture medium to a concentration of 1 μg/mL (LPS content: 48.234 EU/mL).
Isolation of peripheral blood monocytes
Because they required large numbers of cells, these experiments were conducted on equine monocytes as a model of equine myeloid cells. Blood was collected from three healthy adult horses into 2 mM EDTA. The blood was diluted twice with PBS and layered on Histopaque-1077 (Sigma Diagnostics, St Louis, MO). After centrifugation at 400 g for 25 min at 4°C, the mononuclear cells were aspirated and washed twice with ice-cold PBS-EDTA. The cell pellet was suspended in RPMI-1640 with 10% inactivated fetal bovine serum (FBS), penicillin (50 U/mL), and streptomycin (50 μg/mL) and after was incubated at 37°C in 5% CO2 for two hours to permit adherence of monocytes. Nonadherent cells were removed by three washes with PBS. The adherent cell population consisted of >85% monocytes. The monocytes were re-suspended in RPMI-1640 (1×106cells/mL) and seeded in a 6-well culture plate. Each well received 1 of the following 4 treatments: PBS, hay dust suspension (1 μg/mL), Noscapine hydrochloride (150 μM; Sigma-Aldrich, Saint Louis, MO, USA) or Noscapine hydrochloride (150 μM) combined with a hay dust suspension (1 μg/mL). In order to model a stimulation as close as possible to the in vivo situation, we chose to stimulate monocytes with hay dust suspensions.
RNA isolation from blood monocytes and BALF cells and real-time RT-PCR
Total RNA from blood monocytes and BALF cells was extracted using NucleoSpin®RNAII (Macherey-Nagel, Düren, Germany) in presence of Dnase.
Total RNA quantity was measured using a Nanodrop® ND-1000 spectrophotometer (Isogen Life Science B.V., Netherlands). One microgram of RNA was used for each reverse transcription using the First Strand cDNA Synthesis kit (Roche, Basel, Switzerland).
Quantitative PCR reactions were performed with IQ Sybr Green Supermix (Bio-Rad Laboratores, Inc., Marne-la-Coquette, France). The reaction master mix was prepared as recommended by the manufacturer.
Primer sequences used for RT-qPCR validation procedure
Equine accession number
Product size (bp)
5′--------------------------------- > 3′
The cDNA were amplified and quantified in the ICycler iQ real-time PCR detection system (Bio-Rad Laboratories N.V., Nazareth Eke, Belgium). At the end of each qPCR, a melt curve was generated to confirm that a single sequence was amplified for each primer pair. Agarose gel electrophoresis and the melting curve were used to assess the specificity of gene amplification. To confirm reproducibility, all samples were analyzed in duplicate on the plate. The entire experiments were performed in triplicate on independent samples. For each qPCR data analysis, reference genes were analyzed in parallel.
Reference genes stability according to geNorm
in vitro (M)
in vivo (M)
All data are presented as least square means (LSmeans) ± associated S.E.M. (standard error of the mean) for quantitative PCR results. The residual distribution was normally distributed.
During the in vitro study, all four genes were analyzed using the same mixed model (SAS, Cary, NC). The models included the “time” factor (with levels T0, T1, T2 and T4), the “treatment” factor (PBS, dust, noscapine, dust + noscapine) and the interaction between these two factors. There was no effect of “time” in the in vitro kinetic. Correlations between successive measurements made on the same individual were taken into account using an auto-regressive residuals structure. The in vitro experiments were repeated at least three times. P < 0.05 was considered significant.
The clinical values, were analyzed using the PROC GLM procedure of the SAS package (SAS Institute, Cary, NC) and a model including “disease status” (healthy vs RAO), “treatment” (no challenged vs challenged), and their interactions as fixed effects. Separate estimates of the least square means of the states, the treatments and the interaction between the disease status and treatments effects have been obtained for each clinical parameter. P <0.05 was considered significant.
During the in vivo experiment, relative expression values, as obtained from qBasePlus software, were analyzed using the PROC GLM procedure of the SAS package (SAS Institute, Cary, NC) and a model including “disease status” (healthy vs RAO), “treatment” (no challenged vs challenged), "gene" (IL-8, Hif1, TNF-α, VEGF-A) and their interactions as fixed effects. Separate estimates of the least square means of the states, the treatments and the interaction between the disease status and treatments effects have been obtained for each gene". P <0.05 was considered significant.
To study the correlation between the genes expression and the physiological parameters (the percentage of neutrophils, X5Hz and PaO2) standard least-square linear regressions were carried out. Coefficients of correlation of pearson (r) were presented as measures of linear association for regression relationships. Significant differences of the slopes from zero were determined using a two-tailed Student's t test. P <0.05 was considered significant.
Bronchoalveolar lavage fluid
Glycéraldhéyde-3 phosphate dehydrogenase
Hypoxia response element
Impulse oscillometry system
- NF- B:
Nuclear factor B
Arterial blood pressure oxygen
Phosphate buffered saline
Recurrent airway obstruction
Ribosomal protein L32
Succinate dehydrogenase complex subunit
Tumor necrosis factor alpha
We thank Dr Audrey Fraipont, Dr Eve Ramery and Jean-Clément Bustin for their help with horse management, and Raja Fares, Cédric François and Ilham Sbai for technical and secretarial assistance. M. Toussaint is a research fellow at the Fonds pour la formation à la Recherche dans l’Industrie et dans l’Agriculture (FRIA; Belgium).
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