The novel compound Sul-121 inhibits airway inflammation and hyperresponsiveness in experimental models of chronic obstructive pulmonary disease

COPD is characterized by persistent airflow limitation, neutrophilia and oxidative stress from endogenous and exogenous insults. Current COPD therapy involving anticholinergics, β2-adrenoceptor agonists and/or corticosteroids, do not specifically target oxidative stress, nor do they reduce chronic pulmonary inflammation and disease progression in all patients. Here, we explore the effects of Sul-121, a novel compound with anti-oxidative capacity, on hyperresponsiveness (AHR) and inflammation in experimental models of COPD. Using a guinea pig model of lipopolysaccharide (LPS)-induced neutrophilia, we demonstrated that Sul-121 inhalation dose-dependently prevented LPS-induced airway neutrophilia (up to ~60%) and AHR (up to ~90%). Non-cartilaginous airways neutrophilia was inversely correlated with blood H2S, and LPS-induced attenuation of blood H2S (~60%) was prevented by Sul-121. Concomitantly, Sul-121 prevented LPS-induced production of the oxidative stress marker, malondialdehyde by ~80%. In immortalized human airway smooth muscle (ASM) cells, Sul-121 dose-dependently prevented cigarette smoke extract-induced IL-8 release parallel with inhibition of nuclear translocation of the NF-κB subunit, p65 (each ~90%). Sul-121 also diminished cellular reactive oxygen species production in ASM cells, and inhibited nuclear translocation of the anti-oxidative response regulator, Nrf2. Our data show that Sul-121 effectively inhibits airway inflammation and AHR in experimental COPD models, prospectively through inhibition of oxidative stress.


Effects of Sul-121 on LPS-induced AHR.
In line with earlier reports 24,25 , we observed increased airway responsiveness to histamine 2 and 3 hours after LPS instillation, with PC100 values (the provocation concentration of histamine causing a 100% increase in pleural pressure (P pl )) at these time points significantly lower than that for LPS-naive animals that received only saline instillation (Fig. 1B). Although inhaled Sul-121 did not affect airway responsiveness in LPS-naive control animals (Fig. 1B), it did prevent LPS-induced AHR in a dose-dependent manner up to 90% (Fig. 1C). At 30 mM (nebulizer concentration), Sul-121 almost fully prevented LPSinduced AHR (0.89 ± 0.09, p < 0.01; Fig. 1C). In line with our observation that Sul-121 did not affect airway responsiveness in LPS-naive, saline-challenged animals, it also did not affect methacholine-induced contraction of bovine tracheal smooth muscle strips ( Supplementary Fig. 1A). This finding strongly suggests that Sul-121 does not have a direct effect on airway smooth muscle tone. [24][25][26] , LPS challenge significantly increased the number of lung neutrophils in both cartilaginous (from 3.0 ± 0.8 to 19.8 ± 4.2 cells/mm basement membrane) and non-cartilaginous airways (from 4.7 ± 0.5 to 8.008 ± 1.099 cells/mm basement membrane), which was largely prevented by 30 mM Sul-121 in both airway categories (cartilaginous airways: 7.340 ± 2.402 cells/mm basement membrane; non-cartilaginous airways: 3.834 ± 0.824 cells/mm basement membrane; p < 0.01 both; Fig. 2A-C). Similar to the neutrophil changes in the airways, LPS induced a significant increase of neutrophils in bronchoalveolar lavage (BAL) fluids (BALFs) (from 0.07 ± 0.02 to 2.28 ± 0.66 × 10 7 cell number retrieved from the BAL, Fig. 2D). Inhalation of Sul-121 dose-dependently prevented LPS-induced neutrophilia by up to 60% using 30 mM Sul-121 (to 0.91 ± 0.30 × 10 7 cell number retrieved from the BAL; p < 0.001; Fig. 2D). Sul-121 was without effect on basal neutrophil numbers in the lungs of saline challenged, LPS-naive animals (Fig. 2D).

Blood H 2 S level and Lung CBS expression. H 2 S may have a protective role in inflammatory airway
diseases 16 . LPS challenge tended to decrease levels of serum H 2 S (from 342 ± 99 to 134 ± 56 × 10 −7 M), an effect prevented in guinea pigs pretreated with 30 mM Sul-121 (360 ± 157 × 10 −7 M; Fig. 3A). As shown in Fig. 3B, in both LPS-naive, saline challenged and LPS challenged animals we found an inverse correlation between serum H 2 S levels and the number of neutrophils in non-cartilaginous airways (p = 0.041, r = 0.45). Neither immunohistochemistry nor western blotting revealed any difference in the abundance of the H 2 S producing enzyme, CBS 15 , in the lungs of animals in any of our study groups (Fig. 3C,D; Supplementary Fig. 2). Overall, our data suggest that Sul-121 protects concomitant loss of serum H 2 S and against the accumulation of lung neutrophils that are otherwise induced by LPS challenge.
Lung Nrf2 expression and MDA Levels. Neither LPS nor Sul-121, alone or in combination altered the expression of the anti-oxidant transcription factor Nrf2 expression in lung homogenates ( Fig. 4A; Supplementary  Fig. 3) or lung sections (Fig. 4B). Polyunsaturated lipids can be degraded under oxidative stress leading to the formation of MDA 27 . We therefore analyzed oxidative stress in lung tissue by measuring total MDA levels. LPS induced a 2-fold increase in MDA abundance (from 0.06 ± 0.01 to 0.12 ± 0.01 μ mol/g protein; p < 0.01), confirming that oxidative stress had developed, and 30 mM Sul-121 fully prevented the LPS-induced MDA in lungs (0.07 ± 0.01 μ mol/g protein; p < 0.05; Fig. 4C). Moreover, we found a positive correlation between lung MDA level and airway neutrophil number (p = 0.0324, r = 0.457; Fig. 4D). Taken together, these findings indicate that Sul-121 may -at least partially, exert its protective effects in the lung by normalization of LPS-induced oxidative stress.
Scientific RepoRts | 6:26928 | DOI: 10.1038/srep26928 IL-8 Release and p65 Nuclear Translocation in hTERT Cells. The mechanisms by which Sul-121 may decrease airway neutrophilia were further studied in vitro using human ASM cells, which we have previously shown, and confirm here, to be capable of expressing and releasing abundant IL-8 in response to challenge with CSE by about 5-fold above basal (34 ± 13 pg/ml) 9,10 (Fig. 5A). Treatment of cells with Sul-121 dose-dependently reduced 15% CSE-induced IL-8 release up to 90%, with 300 μ M Sul-121 almost fully abrogating IL-8 release (p < 0.001); an effect on par with that elicited by 1 μ M fenoterol, a first line COPD therapeutic 28 (Fig. 5A). Cell viability was not compromised by any concentrations of Sul-121 (Fig. 5B). CSE exposure significantly increased nuclear translocation of the NF-κ B subunit, p65, an effect that was fully prevented by Sul-121 (from 255 ± 46 to 126 ± 12 fluorescence % of vehicle; p < 0.01; Fig. 5C,D). Guinea pigs were intranasally instilled with LPS (5 mg/ml in saline, 300 μ l; t = 0 h) to induce AHR, or with saline (control) as outlined in the Material and Methods. At 30 min before LPS or saline instillation, animals were treated by inhalation of aerosolized vehicle or Sul-121 (3 or 30 mM nebulizer concentration). Inset, structure of Sul-121 (6-hydroxy-2,5,7,8-tetramethylchroman-2-yl (piperazin-1-yl) methanone. At 25 h after LPS challenge, animals were terminated (A). Airway responsiveness to histamine in the different treatment groups was assessed by determining the provocation concentration of histamine causing a 100% increase in P pl (PC100). Data are expressed as ratio between the PC100-value at the different time points over the PC100-value at baseline (t = − 24 h), with a value of 1 representing normoresponsiveness (B,C). N = 4-6 animals per group. (C) # p < 0.05, ## p < 0.01, compared with baseline (t = − 24 h); one way ANOVA repeated measurement with bonferroni's multiple comparison tests. (B,C) * p < 0.05, * * p < 0.01, * * * p < 0.001 compared with the LPS control group at the same time point; two way ANOVA with bonferroni post-tests.

Discussion
COPD is characterized by persistent and progressive airflow limitation associated with chronic pulmonary inflammation 1 . Although often underestimated in clinical assessment, AHR is an important characteristic of COPD 29 that associates with accelerated lung function decline 30,31 . There is a strong association between AHR and accumulation of lung neutrophils 29 , which are associated with production of ROS and a number of potent pro-inflammatory cytokines 3,32 . In our present study, using a guinea pig model of LPS-induced neutrophilia that mimics that in COPD and promotes the development of AHR, we show that Sul-121 dose-dependently prevents AHR induced by intranasal instillation of LPS in vivo. The acute LPS-challenge model we employ induces marked accumulation of lung neutrophils, a feature that correlates strongly with clinical outcomes 2,33,34 . Here we report from in vitro and in vivo studies that though Sul-121 has no direct bronchodilatory effects, it does strongly inhibit the development of AHR, primarily by inhibiting LPS-induced lung inflammation. Indeed, inhaled Sul-121 prevents LPS-induced lung neutrophilia, confirmed in BALF and in both cartilaginous and non-cartilaginous airways, as well as the induction of markers of oxidative stress in the lungs, demonstrating that Sul-121 possesses significant anti-oxidant and anti-inflammatory properties.
As a potent neutrophil chemoattractant and activator 2 , levels of IL-8 in the lungs are strongly correlated with neutrophil number in COPD patients 35,36 . We report that Sul-121 dose-dependently reduces CSE-induced IL-8 release from human ASM cells in vitro, potentially explaining the capacity for inhaled Sul-121 to block LPS-induced lung neutrophilia in vivo. NF-κ B is involved in the transcription of a variety of pro-inflammatory genes, including IL-8, and is activated by CSE 9,10 . The activation of NF-κ B is associated with the translocation of its p65 subunit to the nucleus, subsequently triggering transcription of inflammatory cytokines and chemokines 37 . We show that Sul-121 pre-treatment effectively prevents CSE-induced p65 nuclear translocation in human ASM cells, an effect that parallels suppression of CSE-induced IL-8 release. Taken together, our current findings indicate that Sul-121 may prevent airway neutrophilic inflammation by decreasing IL-8 release upon inhibition of NF-κ B activation and subsequent nuclear translocation.
Oxidative stress plays a central role in inflammatory responses in COPD 7 . NF-κ B can be activated by oxidative stress, leading to downstream inflammatory responses 38 . Therefore, the anti-inflammatory effect of Sul-121 could be explained by an anti-oxidant effect. Nrf2 is a nuclear factor that controls cellular anti-oxidative responses 39 . Under favorable physiological conditions, the Nrf2 activity is suppressed by Keap1, a cytosolic protein binding partner that prevents Nrf2 nuclear translocation. Under conditions of oxidative stress, Keap1 dissociates from, and permits nuclear translocation of Nrf2 40 . Subsequently, Nrf2-induced transcription of anti-oxidant genes initiates adaptive responses that can counteract oxidative stress 39 . We now report that CSE, as well as LPS, exposure of human ASM cells induces Nrf2 nucleus translocation, and that Sul-121 significantly decreases Nrf2 nucleus translocation induced by both CSE and LPS. Although these findings at first seem contradictory, they actually do support an anti-oxidant role for Sul-121 as they diminish the requirement for endogenous pathways to increase the transcription of anti-oxidative stress genes. In support of an anti-oxidant property for Sul-121, we report that it significantly reduces both exogenous ROS levels in pro-oxidant treatments (e.g. CSE and H 2 O 2 ), as well as endogenous ROS produced by ASM cells in response to PMA. Taken together, our findings support a hypothesis that Sul-121 exerts anti-inflammatory effects through normalization of oxidative stress. Accordingly, LPS-induced elevation of MDA, a product of peroxidative breakdown of polyunsaturated fatty acids 27 , is effectively reduced in guinea pigs pretreated with Sul-121. Importantly, lung MDA levels correlate with lung neutrophil infiltration induced by LPS in vivo. The anti-oxidant properties of Sul-121 may explain, at least in part, its capacity in the present study to suppress LPS-induced AHR, as oxidative stress has been directly implicated to underpin pathogenesis leading to decreased lung function 41,42 . In addition, peroxidative breakdown of polyunsaturated fatty acids contributes to impairment of the epithelial integrity 43,44 , which can contribute to increased transmigration of neutrophils to the airway lumen 45 . Since CSE has been shown to impair airway epithelial integrity in vitro 46,47 , it is tempting to speculate that the prevention of LPS-induced airway neutrophilia by Sul-121 may be associated with maintenance of epithelial integrity due to its anti-oxidative properties. Our work supports future studies to fully investigate this possibility.
Notably, H 2 S reportedly protects against LPS-induced lung injury 16 . Several studies have focused on the effects of exogenous H 2 S donors on inflammation 20,48,49 . Nonetheless, little is known about the relationship between airway inflammation and endogenous H 2 S production. Serum H 2 S levels may be decreased in COPD patients during acute exacerbation 50 , and patients requiring antibiotics due to lower respiratory tract infections exhibit significantly reduced serum H 2 S levels compared subjects not requiring antibiotics 51 . In line with these findings, we report that LPS challenge induces a trend towards reduced serum H 2 S, and serum H 2 S levels negatively correlate with neutrophil number in non-cartilaginous (p = 0.0406). The oxidation of blood H 2 S to its pro-inflammatory sulfite form occurs during oxidative stress generated upon exposure of neutrophils to LPS 17 . Therefore, we speculate that LPS-induced oxidative stress leads to loss of serum H 2 S in the guinea pig model we have employed. Interestingly, resistance to corticosteroid therapies in COPD patients has been attributed to the imbalance of acetylation-deacetylation states of histones due to the impact of oxidative stress on histone deacetylase 13,52 . Thus, it would be interesting to study whether Sul-121 prevents or reverses corticosteroid resistance in experimental models of COPD.
In conclusion, we show that Sul-121 reduces neutrophilic inflammation and AHR in an LPS-induced experimental model of COPD in vivo, probably due to the reduction of oxidative stress as well as inhibition of NF-κ B and Nrf2 activation. These findings support future work to determine the potential for Sul-121 as a candidate for treatment of COPD.

Methods
Animals. Outbred male, specified pathogen-free Dunkin Hartley guinea pigs (Harlan, Heathfield, UK) weighing 350-450 g were used. Guinea pigs were randomly divided into indicated experimental groups with 4-6 guinea pigs per group. All in vivo protocols described in this study were approved by the University of Groningen (Groningen, The Netherlands) Committee for Animal Experimentation. All the methods were carried out in accordance with the approved guidelines. Animal Model of COPD. Guinea pigs were intranasally instilled with LPS (Sigma, L-2880) to induce neutrophilic airway inflammation and AHR [24][25][26] . Guinea pigs were held in upright position while 300 μ l LPS (5 mg/ml in sterile saline) was slowly instilled intranasally and kept in the upright position for an additional 2 min to allow sufficient spreading of the fluid throughout the airways. Control animals were instilled with 300 μ l sterile saline. Experimental In Vivo Protocols. As shown in Fig. 1A, 30 min before LPS or saline instillation animals were treated by inhalation of aerosolized vehicle (2% dimethyl sulfoxide and 0.2% Tween80 in saline) or Sul-121 solutions (3 or 30 mM, nebulizer concentrations) for 3 min in a 9-liter Perspex cage 53 . A DeVilbiss nebulizer (type 646) driven by an airflow of 8 l/min provided the aerosol with an output of 0.33 ml/min. Airway responsiveness to histamine was measured 24 hours before (basal) and 1, 2, 3, 6 and 24 hours after LPS/saline instillation by lung function measurements as described below. At 25 hours after LPS challenge, BAL was performed to assess inflammatory cell infiltration in the airways.
In a separate protocol, guinea pigs inhaled Sul-121 (30 mM, 3 min) or vehicle 30 min before LPS or saline challenge, followed by blood and lung tissue collection at 25 hours later. Blood was collected by heart puncture and stored in EDTA coated tubes. Neutrophil numbers were determined in blood. Serum was obtained by centrifuging (2000 rcf, 10 min) to quantify levels of Sul-121 and H 2 S. Lung tissue was snap frozen and used for histology (neutrophils) and immunohistochemistry (CBS, Nrf2) using transverse frozen cross-sections (5 μ m) of the upper right lung lobe, and for Western blotting (CBS, Nrf2) and malondialdehyde (MDA) measurements using homogenates of the other parts of the lungs.
Measurement of Airway Responsiveness to Histamine. Lung function was assessed by online measurement of pleural pressure (P pl ) under conscious and unrestrained conditions 53 . In short, a small fluid-filled latex balloon catheter was surgically implanted inside the thoracic cavity and connected to a pressure transducer (TXX-R; Viggo-Spectramed, Bilthoven, The Netherlands) via an external saline-filled cannula. P pl was continuously measured using an online computer system. Changes in P pl are linearly related to changes in airway resistance and serves as a sensitive index for stimulus-induced bronchoconstriction 53 .
Histamine provocations were performed by inhalation of stepwise increasing concentrations of histamine (Sigma, H-7250) in saline (0, 25, 50, 75, 100, 125 and 150 μ g/ml). Solution were nebulized for maximally 3 minutes with intervals of 7 minutes, until the P pl was increased by more than 100% above baseline for at least 3 consecutive minutes. The provocation concentration of histamine causing a 100% increase in P pl (PC100) was derived by linear interpolation of the concentration-P pl curve and used as an index for airway responsiveness. Animals were habituated to the experimental conditions as previously described 53 . Neutrophil Counting in BAL Fluid and Lung Tissue. BAL was performed as previously described 54 .
After anesthesia with pentobarbital (Euthasol 20% i.p.), the trachea was exposed and cannulated, and the lungs were gently lavaged using 5 ml of sterile saline (37 °C), followed by three subsequent aliquots of 8 ml of saline. The recovered BALFs were kept on ice and centrifuged at 200 rcf for 10 min at 4 °C. Pellets were re-suspended into a final volume of 1 ml phosphate-buffered saline (PBS), and a CASY cell counter (Model TT; Innovatis, Reutlingen, Germany) was used to count total cell numbers. For neutrophil determination, cytospin preparations were stained with May-Grünwald and Giemsa stain 54 . Cell differentiation was performed by counting at least 400 cells in duplicate.
Tissue nonspecific alkaline phosphatase (TNAP) staining was used to identify neutrophils on frozen lung sections 26,55 . Sections were rinsed in a TRIS-base buffer (pH 7.6) for 2 min, and then incubated for 5 min in a TRIS-base buffer (pH 9.0) containing 1 mg/ml naphthol AS-BI phosphate (Sigma, N2125) and 1 mg/ml Fast Red TR Salt hemi (zinc chloride) salt (Sigma, F8764). Sections were rinsed in a TRIS-base buffer (pH 7.6) for 2 min, counterstained with haematoxylin (Sigma, GHS3) for 1 min, and mounted in Kaiser's glycerol gelatin. Airway neutrophils were counted in the adventitia and sub-mucosa, and expressed as the number of positively stained cells per mm basement membrane length.
Scientific RepoRts | 6:26928 | DOI: 10.1038/srep26928 MDA Measurement. MDA concentrations in guinea pig lung homogenates was measured by the thiobarbituric acid reactive substances assay 56 . Lung lysates were mixed with 10% trichloroacetic acid (Sigma, T9159; 1:1, v/v) and centrifuged at 2,200 x g for 15 min for protein removal. Samples were mixed with 6.7 g/l thiobarbituric acid (Sigma, T5500; 1:1, v/v) and heated at 95 °C. MDA levels were determined by measuring absorption at 550 nm. The standard curve was made by measuring a series of gradually diluted 1,1,3,3,-tetramethoxypropane (Sigma, 108383) solutions. MDA levels in lung homogenates were expressed as μ mol MDA per g protein. ASM Cell Culture. Three human airway smooth cell lines, immortalized by human telomerase reverse transcriptase (hTERT) 57 were used for all the experiments. The primary cultured human airway smooth cells used to generate each hTERT immortalized cell line were prepared as described previously 57 . Informed consent was obtained from all subjects. All procedures were in accordance with the relevant guidelines and approved by the Human Research Ethics Board of the University of Manitoba. The cells were maintained in Dulbecco's modified Eagle's medium (DMEM, Life technologies, 11965-092) containing heat-inactivated fetal bovine serum (10% vol/vol), streptomycin (50 U/ml), penicillin (50 mg/ml) in a humidified atmosphere at 37 °C in air/CO 2 (95%:5% vol/vol).

Cigarette Smoke Extract Preparation.
Cigarette smoke extract (CSE) was freshly prepared by pumping the smoke from two combusted 3R4F research cigarettes (Reference Cigarette Program, University of Kentucky) through 25 ml of serum-free DMEM 10,28 . This was designated as 100% CSE.

IL-8 Measurement.
hTERT-ASM cells were plated on 24-well plates. After grown to confluence, cells were treated with the indicated concentrations of the Sul-121 in the absence and presence of 15% CSE for 24 hours in serum-free DMEM. Culture medium was collected to measure IL-8 concentrations using an IL-8 enzyme-linked immunosorbent assay (ELISA) kit (Pelikine, M1918) according to the manufacturer's instructions. Fenoterol (Boehringer Ingelheim, 217-742-8, 1 μ M) treatment was used as a positive control. Cells were trypsinized for trypan blue cell counting to determine cell viability 10 . Data represent from 7-24 experiments.
Nrf2 and p65 Immunofluorescence. ASM cells were plated on cover slips in 12-well plate. After grown to confluence, cells were treated with 300 μ M Sul-121, with or without 15% CSE or 10 μ g/ml LPS (Sigma, L-2880), for 2 hours. Cells were then fixed with a solution containing 4% paraformaldehyde and 4% sucrose for 15 min at room temperature, followed by treatment with 0.3% Triton X-100 for 5 min at room temperature. Cells were blocked for 1 hour at room temperature with PBS containing 5% bovine serum albumin and 2% donkey serum. Cells were then incubated overnight at 4 °C with primary antibodies against Nrf2 (Abcam, ab31163, 1:100) and p65 (Cell Signaling, #3033S, 1:20). The next day, cells were washed with PBS and incubated with secondary antibodies (1:500) for 1 hour at room temperature. After wash with PBS, nuclei were stained with Hoechst (Invitrogen, H3570, 1:10000) for 5-10 sec, immediately followed by two quick and four 10 min washing steps with ultra-pure water. After staining, coverslips were mounted using ProLong ® Gold Antifade Mountant reagent (Life Technologies, P36930) and imaged using an Olympus AX70 microscope equipped with digital image capture system (ColorView Soft System with Olympus U CMAD2 lens, Olympus Corporation, Tokyo, Japan). The background corrected fluorescence measurements were performed with Image J 1.48v 58  To examine the production of ROS, ASM cells were plated on 96-well plates. After grown to confluence, cells were treated with 300 μ M Sul-121 in the absence and presence of 0.1 μ M PMA (Sigma, P-8139) as control 59 for 2 hours. 10 mM NAC (Sigma, A9165) served as positive control. After removal of the supernatant, cells were incubated with 0.1 μ M carboxy-H2DCFDA for 1 hour and ROS production was measured as described above. Data represent 4 experiments.

Statistics.
Data represent means ± SEM, from n experiments. Statistical significance of differences was evaluated by one-way or two way ANOVA with Bonferroni post-hoc tests, or by two tailed Student's t-test using Prism 5 software. Pearson's correlation tests were also performed by using Prism5. Differences were considered to be statistically significant when p < 0.05.
All the figures and pictures were created by authors of this paper.
Scientific RepoRts | 6:26928 | DOI: 10.1038/srep26928 All in vivo protocols described in this study were approved by the University of Groningen (Groningen, The Netherlands) Committee for Animal Experimentation. All the methods were carried out in accordance with the approved guidelines.
For experiments involving human samples, informed consent was obtained from all subjects. All procedures were in accordance with the relevant guidelines and approved by the Human Research Ethics Board of the University of Manitoba.