Azithromycin ameliorates sulfur dioxide-induced airway epithelial damage and inflammatory responses

Environmental pollutants are often associated with exacerbation of disease, including those linked to cardiac, skin and respiratory conditions [1, 2]. Air pollution is comprised of several components, one of the major ones being sulfur dioxide (SO₂), a gas commonly produced from combustion of sulfur-containing fuels such as coal as well as from volcanic eruptions [3]. Fine particles formed from fugitive dust, SO₂ and oxygen result in particulate matter (PM), a primary pollutant and irritant which, as air pollution increases, leads to an increase in respiratory and cardiovascular diseases [1].

The lungs, lined with an epithelial layer, are complex structures responsible for filtering and humidifying air, facilitating gas exchange, and acting as the first line of defense against the external environment [4]. The airway epithelium from the nose, trachea and bronchi through to the alveoli, together with the underlying basement membrane form an effective barrier to prevent pollutants and infectious agents such as bacteria and viruses from entering the body [5]. Exposure to air pollutants, both gaseous and PM, can weaken the epithelial barrier, predispose the respiratory system to infections, and facilitate both acute and chronic respiratory disorders [6]. Indeed, fine PM have been shown to lead to a weakened epithelium [7, 8] and are now accepted as a leading contributor to chronic respiratory and cardiovascular diseases [9, 10].

Patients suffering from asthma, COPD and CF are at greatest risk of exacerbations caused by air pollution such as SO₂ and PMs, along with viruses and bacteria. These patients often have compromised lung barrier functions [6]. Treatment of patients with acute exacerbations of respiratory diseases often involves administration of antibiotics such as macrolides including AZM [11]. Indeed, a range of studies have revealed beneficial effects of AZM beyond its anti-microbial effects that has led to this macrolide being used for chronic maintenance treatment to prevent exacerbations of respiratory diseases, a treatment approach that whilst effective, increases the risk of bacterial resistance [11]. AZM has been shown to have multiple disease-modifying effects of relevance to treating and preventing exacerbations of respiratory diseases, including immunomodulation (reviewed by Parnham et al. [12]).

In addition to the anti-microbial and immunomodulatory effects of AZM, previous studies by our group and others have shown that this drug enhances the respiratory epithelial barrier when cultured in air-liquid interface (ALI) culture [13‐15]. Most recently, we demonstrated that AZM induces a partial epidermal differentiation program in bronchial epithelial cells in ALI culture [16]. Histological analysis demonstrated formation of lamellar bodies (LBs). LBs are found in lungs, where they contribute to production and release of pulmonary surfactants, and in skin, where they contribute to the water barrier, presumably facilitating the increased barrier function observed in vitro. However, to date there have been no observations demonstrating similar barrier enhancing effects of AZM in vivo. In this study, we have established an in vivo model of epithelial barrier dysfunction in the airway epithelium using SO₂ exposure to investigate whether AZM has barrier protective functions. We demonstrate that pretreating mice with AZM prior to SO₂ exposure ameliorates AE damage including barrier leakiness, reduces the expression of GST detoxification enzymes and dampens the interferon–alpha associated inflammatory responses.

In this study, we have administered SO₂ gas to mice in a controlled manner to expose all test animals evenly and reproducibly. We have demonstrated that SO₂ can be used as a challenging agent in mice to induce subtle airway epithelial damage and leakiness measured by increased epithelial shedding and blood-derived human serum albumin (HSA) extravasation into BALF. Pre-treating mice with AZM 2 weeks prior to SO₂ exposure reduced epithelial shedding, concentrations of HSA in BALF and mitigated pro-inflammatory gene responses, demonstrating the barrier enhancing role of AZM beyond the antibiotic activity of this drug, as has been previously shown in vitro [13, 14, 16].

Air pollution from natural disasters (i.e. volcanic emissions), and those caused by human influences (i.e. forest fires) are a major concern to governments throughout the world [24]. Sulfuric oxides are continuously produced in vast quantities through the burning of fossil fuels for electricity and transportation [25, 26], as well as in sporadic disasters such as volcanic eruptions. Sulfur dioxide (SO₂) is a major component of air pollution and is transformed into sulfuric acid in water, and when inhaled can cause damage to the respiratory system, leading to inflammation and barrier failure [27, 28]. This makes the study of SO₂ and how it affects pulmonary function highly important and clinically relevant. With the increasing evidence for the lung protective capabilities of AZM [29‐32], we decided to pre-treat mice with AZM before applying short term SO₂ exposure and analyze the effect of AZM on prevention or attenuation of initial damage responses and the subsequent immunomodulatory effects on the epithelial barrier.

Intravenous administration of HSA into experimental animals has been widely used as a marker for vascular permeability and to study other biological problems such as drug delivery [33]. Albumin, for instance, has been used to target clarithromycin to the lung through albumin-derived microsphere carriers [33]. There has, however, been a lack of surrogate markers to measure airway epithelial barrier leakiness and in this study, we used HSA as a marker for barrier failure. By injection of HSA into the tail vein 1 h prior to BALF harvesting, we were able to measure concentrations of HSA that had leaked into BALF using ELISA. Exposing mice to SO₂ resulted in increased concentrations of HSA in BALF which were reduced when mice were pre-treated with AZM.

We also analyzed whether native markers of oxidative stress were changed during SO₂ exposure and performed mass spectrometric analysis of BALF which showed increased expression of several GSTs. GSTs are an integral part of the glutathione redox system, mitigating oxidative damage to tissues, including damage induced by sulfuric compounds [34]. The observation that these proteins were upregulated on SO₂ exposure indicated that while the gross histology of the mouse lungs was unaltered, there was an active biochemical response to oxidative damage in the lungs of SO₂ exposed mice. In addition, we were able to reduce the GST responses by pre-treating mice with AZM for 2 weeks. A reduction in the GST responses indicates that the SO₂-induced oxidative damage was lowered in the AZM treated mice. This is in keeping with reports that AZM inhibits hypoxic lung injury in neonatal rats and ozone-induced lung inflammation in healthy human volunteers [35, 36].

SO₂ is a well-known inducer of inflammation in the respiratory system [37]. Thus, SO₂ exposure has been shown to increase expression of pro-inflammatory cytokines such as interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) in mouse lungs [38]. Furthermore, nitric oxide synthase and intercellular cell adhesion molecule-1 (ICAM-1) levels have been shown to be increased in the lungs of rats following SO₂ inhalation [39]. These data are in line with our findings that increased expression of genes positively correlated with inflammation-related pathways and were mitigated when mice were pretreated with AZM. Expression analysis of RNA from lung tissue indicates that SO₂ exposure resulted in increased expression of inflammatory related gene sets and/or pathways. The pathway with the highest normalized enrichment score was the interferon alpha response. By focusing on the genes that were positively correlated with the interferon alpha response pathway on SO₂ exposure, we noticed that these genes were not as highly expressed when mice had been pre-treated with AZM before SO₂ exposure. Interestingly, expression of several additional immune-related genes followed the same trend, that is, increased expression as a result of SO₂ challenge, but close to control expression in AZM pre-treated mice, regardless of SO₂ exposure, as observed previously in epithelial cells stimulated with purulent exudate [40]. This indicates immunomodulatory effects of AZM, where the pre-treated mice show attenuated inflammatory responses to the SO₂ challenge. Our results indicate that AZM should be considered as a candidate for treatment of lung injury similar to that which occurs in conditions such as acute respiratory distress syndrome (ARDS). In fact, AZM used as adjunctive therapy in patients with ARDS has been shown to be beneficial [41]. Collectively, these data demonstrate that a single exposure of SO₂ can induce inflammation in mouse lung tissue and that this inflammatory response is dampened if mice are pretreated with AZM. As excessive inflammatory responses are hallmarks of progressive lung diseases such as ARDS, it is important to find drugs that dampen the response [42]. Further investigation into the molecular function of AZM in the airway epithelium is of great importance for several lung diseases including both life-threating conditions such as ARDS and COPD.

Using ALI cultured human bronchial epithelial cells, we recently demonstrated that AZM treatment induces epidermal differentiation, as shown by the expression of skin associated markers evaluated by gene expression analysis [16]. This study was based on the investigation of bronchial epithelial cell lines cultured in air-liquid interface conditions where AZM was added directly to the culture media, whereas the in vivo conditions we evaluated in the present study are much more complicated and represent changes following AZM administrated p.o. Nonetheless, the findings presented here show that after p.o. administration, AZM appears to protect the integrity of the respiratory epithelial barrier. In ALI cultures, AZM induces formation of multivesicular bodies and LBs [16], structures which are only found in vivo in keratinocytes generating the water barrier in skin [43]; and in the lungs, associated with club and alveolar type II cells [44]. Interestingly, an increase in the formation of LBs was confirmed in mice pre-treated with AZM, where alveolar type II cells were seen to form accumulations of LBs and in the tracheal epithelial layer, where an accumulation of vacuoles were observed. Whether formation of LBs contributes to the barrier enhancement of AE is currently not known. Pre-treated mice that had been exposed to SO₂ showed less cellular shedding than untreated mice exposed to SO₂, leading us to the conclusion that AZM confers barrier enhancement in the lungs.

Of interest, AZM has been previously shown to attenuate tobacco smoke-induced oxidative stress-related cell viability reduction in cultured human lung cells [45], thus supporting a protective role in an in vitro application.

An important limitation of our model is that in the current study, we only mimicked intermittent exposure of SO₂, as opposed to prolonged exposure. As such, we represent the protective effects of AZM over a short SO₂ exposure time period, resembling a volcanic exposure or a forest fire, but not chronic exposure like that associated with city pollution. Our data indicate however that AZM could have protective effects for individuals with weakened lung function with a foreseeable risk of exposure to SO₂, such as during seasonal changes in city pollution or during travel. The results presented here would benefit from supporting studies using prolonged SO₂ exposure.

Collectively, we have demonstrated in this study that pre-treating mice with AZM before exposure to SO₂ gas reduces the damage to the respiratory epithelium, including reduced epithelial leakiness and oxidative stress, and a dampening of the inflammatory response. This study and previous in vitro studies demonstrate the need for non-antibacterial drugs that maintain the disease modifying effects of AZM with less risk of causing bacterial resistance. The airway epithelium is the first line of defense against various pathogens and external hazardous particles such as SO₂ and therefore strengthening the epithelium in patients with barrier dysfunction is of great importance, particularly for protection of immunodeficient or otherwise compromised patients from hazardous components in the air, such as pollutants and infectious agents like bacteria and viruses. Our data corroborate the notion that AZM provides this sort of protection, and that prophylactic administration of AZM to patients suffering from respiratory diseases, or preferably modified derivatives of AZM lacking antibacterial activity may be a feasible treatment option to reduce exacerbations.