Fenretinide prevents inflammation and airway hyperresponsiveness in a mouse model of allergic asthma.

AJRCMB Articles in Press. Published on 02-June-2014 as 10.1165/rcmb.2014-0121OC

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Fenretinide prevents inflammation and airway hyperresponsiveness in a mouse model of allergic asthma Cynthia Kanagaratham1, Alžběta Kalivodová2, Lukáš Najdekr2, David Friedecký2, Tomáš Adam2, Marian Hajduch2, Juan Bautista De Sanctis3, Danuta Radzioch1,4 1.

Department of Human Genetics, McGill University, Montreal, Quebec, Canada

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Institute of Molecular and Translational Medicine, Faculty of Medicine and Dentistry, Palacky University and University Hospital in Olomouc, Olomouc, Czech Republic

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Institute of Immunology, Faculty of Medicine, Universidad Central de Venezuela, Sabana Grande, Caracas, Venezuela

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Faculty of Medicine, Division of Experimental Medicine, McGill University, Montreal, Quebec, Canada

Running Title: Fenretinide protects against asthma phenotypes

This article has an online data supplement, which is accessible from this issue's table of content online at www.atsjournals.org

To whom correspondence should be addressed: Dr. Danuta Radzioch 1650 Cedar Ave. L11-218, Montreal, Qc, Canada, H3G 1A4 Tel: (514) 934-1934 ext. 44517, Fax: (514) 934-8260, E-mail: [email protected]

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Copyright © 2014 by the American Thoracic Society


Funding This work was supported by an award from the Sandler Foundation for Asthma Research and a grant from the Canadian Institutes of Health Research (CIHR) awarded to D.R. (MOP-106544). J.B.D.S. is a recipient of a grant from the Foundation of Science and Technology (FONACIT) (Project G2005000389). A.K, L.N., D.F., T.A., M.H. are recipients of the grant from the Czech Ministry of School and Education (CZ.1.05/2.1.00/01.0030, LM2011024) and Technological Agency of the Czech Republic (TE01020028). C.K. is a recipient of doctoral awards from CIHR, Fonds de recherché Santé Québec (FRQ-S), and the AllerGen Network of Centres of Excellence.

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ABSTRACT Arachidonic and docosahexaenoic acids (AA and DHA) play important roles in inflammation and disease progression, where AA is viewed as pro-inflammatory and while DHA is antiinflammatory. We observe in our model of allergic asthma that the ratio of AA to DHA is significantly skewed in a pro-inflammatory direction. Fenretinide, a vitamin A derivative, has been shown to correct fatty acid imbalances in other diseases. Therefore, we wanted to explore if fenretinide can have a protective effect in allergic asthma. To accomplish this, we measured the levels of AA and DHA in the lungs of non-allergic, ovalbumin induced allergic, and fenretinide treated allergic mice. We also investigated the effect of allergic asthma and fenretinide treatment on markers of oxidative stress, levels of metabolites, IgE production, airway hyperresponsiveness and histological changes. Our data demonstrates that treatment of allergen sensitized mice with fenretinide prior to allergen challenge is able to prevent ovalbumin induced changes in the ratio of AA to DHA. The levels of several metabolites, such as serotonin, and markers of cellular stress which are increased after ovalbumin challenge are also controlled by fenretinide treatment. We observed the protective effect of fenretinide against ovalbumin induced airway hyperresponsiveness and inflammation in the lungs, illustrated by a complete block in the infiltration of inflammatory cells to the airways and dramatically diminished goblet cell proliferation, even though IgE remained high. Overall, our results demonstrate that fenretinide is an effective agent targeting inflammation, oxidation, and lung pathology observed in allergic asthma. Keywords: airway hyperresponsiveness, inflammation, arachidonic acid, docosahexaenoic acid, metabolomics, lipid mediators, serotonin, fenretinide, allergic asthma

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INTRODUCTION Allergic asthma is a chronic and heterogeneous disease of the respiratory and immune systems affecting nearly 300 million people worldwide (1). Common treatments include inhaled corticosteroids and beta-agonists; however, they are not helpful to all patients. Therefore, new treatments for asthma are of considerable demand. The hallmark phenotype of asthma is airway hyperresponsiveness (AHR). Airways naturally respond to irritating compounds by constricting; however, airways of hyperresponsive individuals are more sensitive and this reflex occurs at lower doses of the constricting agent than in healthy individuals (2;3). AHR increases resistance to the air flowing into the airways causing difficulty breathing. High level of plasma IgE is also be observed in allergic asthma. Exposure to IgE specific allergen causes crosslinking of IgE-FCεRI molecules found on the surface of granulocytes leading to degranulation and release of inflammatory mediators. Other phenotypes include airway inflammation and mucous metaplasia, caused by the recruitment of inflammatory cells and cytokine secretion in the airways. Recruitment of inflammatory cells to the airways can result in tissue destruction and airway remodeling. Identification of disease biomarkers is necessary to accurately design new therapies and to better understand the sequence of events in the diseased host. The goal of our study was to profile the changes in levels of omega (n)-3 and n-6 polyunsaturated fatty acids (PUFAs), docosahexaenoic acid (DHA) and arachidonic acid (AA) respectively, in our mouse model of ovalbumin induced allergic asthma. Imbalance in the amounts of PUFAs plays an important role in the emergence and progression of unresolved inflammatory diseases, such as asthma, rheumatoid arthritis, cardiovascular disease and cancer (4). n-3 and n-6 PUFAs play complementary roles in inflammation and compete for the same desaturation and oxidation enzymes in order to produce 4


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their downstream inflammatory mediators; therefore, a steady state balance should be maintained for good health. n-3 PUFAs, which include DHA and its precursor eicosapentaenoic acid (EPA), have been shown to have anti-inflammatory properties such as suppressing leukocyte chemotaxis and the generation of reactive oxygen species (5;6). On the contrary, metabolism of n-6 PUFAs is known to produce more pro-inflammatory metabolites. Oxidation of AA by 5-lipoxygenase (5-LOX) produces 4-series leukotrienes that can enhance the synthesis of immunoglobulins by B lymphocytes (7), secretion of mucus (8), recruitment of neutrophils into the airways (9). Fenretinide [N-(4-hydroxyphenyl)retinamide, 4-HPR, Figure E1] is a semi-synthetic analogue of vitamin A that has been shown to restore inflammation associated changes in AA and DHA levels in mouse models of cystic fibrosis and spinal cord injury (10-12). Along with modulating the levels of AA and DHA, in both cases fenretinide also conferred protection against a wide range of disease associated phenotypes (10-12). Therefore, we hypothesized that fenretinide might also prevent the lipid imbalance from occurring in allergic asthma, leading to better control of the inflammatory reaction in the lungs of sensitized animals exposed to allergen.

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MATERIALS AND METHODS Mice Seven-week-old male A/J mice (20-25 g) were purchased from Jackson Laboratories (Maine, USA). Experiments were approved by the Animal Care Committee of the McGill University Health Center (Montreal, QC, Canada) and were in compliance with the guidelines set by the Canadian Council of Animal Care (CCAC, Ottawa, ON, Canada). Sensitization, treatment, challenge protocols, and experimental groups Mice were sensitized weekly for three consecutive weeks by intraperitoneal injections with ovalbumin allergen (OVA). Following sensitization the mice were split into three groups: nonallergic (PBS), and allergic (OVA), fenretinide treated and allergic (FEN-OVA). Treatment began one week after the third sensitization. Allergic-treated mice received fenretinide daily at 60mg/kg. Mice in non-allergic and allergic groups were treated with drug vehicle. All mice were vehicle or drug treated for four weeks. During the final week of treatment, the allergic (OVA) and allergic-treated mice (FEN-OVA) were challenged with 1% ovalbumin solution for 30 minutes, while non-allergic mice (PBS) were exposed to PBS. Further details are provided in the online data supplement. Measurements of lipids and markers of oxidation PUFAs, AA and DHA, were measured as previously described (10). Approximately 20mg of mashed lung tissue was collected three hours after final ovalbumin challenge and preserved in 1mL of 1nM butylated hydroxyanisole solution to prevent oxidation of fatty acids. Nitrotyrosine and malonyldialdehyde (MDA) were measured as previously described (12).

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Metabolomics analysis Plasma metabolites were measured by HPLC-MS. More information is provided in the online data supplement. Lung protein analysis CXCL1 and CXCL10 were measured in mouse lung homogenates by ELISA. Cyclooxygenase-2 (COX-2) and 5-LOX expression was measured by western blot analysis. More information is provided in the online data supplement. Measurement of airway resistance Airway resistance was measured using a Buxco plethysmograph system and Harvard Apparatus ventilators. The mice were anesthetized, tracheotomized and connected to a ventilator. A nebulizer was used to administer ascending doses of methacholine (PBS, 20, 40, and 80 and 160 mg/mL). The maximum resistance value for each mouse at each dose of methacholine was determined using a Buxco plethysmograph system and Harvard Apparatus ventilators (Harvard Apparatus, Holliston, Massachusetts, USA). More information is provided in the online data supplement. IgE measurements Blood was collected into EDTA coated tubes from mice by intracardiac puncture 48 hours after the final allergen challenge. To isolate plasma, blood samples were centrifuged at 2000rpm for seven minutes at 4oC. Total IgE in the plasma was measured by ELISA using the BD OptEIA kit (BD Biosciences, Mississauga, ON, Canada) following manufacturer’s instructions.

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In vitro experiment 1.5x105 MLE-12 cells were treated with 1.25µM of fenretinide for 21 hours, and then stimulated with 50ng/mL of LPS (in combination to 1.25µM of fenretinide) for three hours. These time points were chosen based on previous studies (13). RNA was extracted and gene expression was measured by real-time qPCR. More information and primer sequences are provided in the online data supplement. Lung histopathology Lungs were prepared by formalin fixation and paraffin embedding. Hematoxylin and eosin stain or Periodic acid Schiff (PAS) stain was done to quantify inflammation or goblet cell hyperplasia of the airways, respectively. More information is provided in the online data supplement. Statistical analysis Metabolomics data was analyzed using R software. All other data was analyzed with GraphPad Prism 5 (version 5.02, GraphPad Software Inc., San Diego, CA). More information is provided in the online data supplement.

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RESULTS Profiling changes in levels of AA, DHA, and of metabolites in ovalbumin induced allergic asthmatic mice. The ratio of AA to DHA (AA/DHA) is used as a marker of inflammation. An increase in AA/DHA compared to baseline value is usually associated with a pro-inflammatory reaction, while a decrease in AA/DHA is often correlated with better control of inflammatory reaction. As shown in Figure 1A, our data demonstrated that ovalbumin challenged mice (OVA) had significantly greater ratio of AA to DHA than PBS challenged mice. There was a slight increase in AA after ovalbumin challenges (Figure 1B); however, the imbalance in the AA to DHA ratio was primarily mediated by a significant decrease in DHA (Figure 1C). Since COX-2 was shown to convert AA into pro-inflammatory mediators such as prostaglandins and thromboxanes, which can induce bronchoconstriction, we tested if the aberrant AA/DHA ratio was also associated with the modulation of COX-2 (14). As shown in Figures 1D and 1E, ovalbumin challenge caused an increase in the expression of COX-2 in the lung by 2.5 folds relative to non-allergic mice in our mouse model of asthma (OVA vs PBS groups). Similarly, the expression of 5-LOX, another AA converting protein, is also increased post-ovalbumin challenge (Figures 1F and 1G). We wanted to know if changes in metabolite levels could be observed in allergic mice which might allow us to better understand the mechanism of allergic asthma development. To build a metabolomics signature of allergic asthma we measured the levels of 89 metabolites by HPLCMS in the plasma of our mouse model of allergic asthma. We compared the levels of these metabolites between non-allergic PBS challenged and allergic ovalbumin challenged mice (PBS vs OVA) by principle component analysis (PCA), discriminant function analysis (DFA), and hierarchical clustering. Ellipses in PCA represent 75% confidence intervals for principle 9



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component distribution of the groups (Figure E2A). Quality control samples are grouped in the center of the plot with narrow dispersion which shows good performance of metabolite measurements. Although there is relatively large dispersion within the groups, non-allergic and allergic groups tend to be separated most substantially. Hierarchical clustering shows that many metabolites differ between non-allergic and allergic groups (Figure E3). We applied DFA supervised statistical analysis to visually show that allergic and non-allergic mice can be separated (Figure E2B). As shown in Figure 2A, our data demonstrated that there are significant differences in quantity of 19 metabolites between allergic animals and control sensitized animals. The most significant change was the increased in serotonin following ovalbumin challenge. Fenretinide corrects allergen challenge induced changes in AA, DHA, metabolites and markers of oxidative stress. Since fenretinide was shown to normalize disease associated imbalance in AA and DHA levels in cystic fibrosis and spinal cord injury mouse models, we tested the effect of fenretinide treatment on the levels of AA, DHA, metabolites, as well as markers of oxidative stress in our ovalbumin induced model of allergic asthma. No significant difference was observed in the ratio of AA to DHA between fenretinide treated and vehicle treated non-allergic mice (Table E2). As shown in Figure 1A, treatment with fenretinide prior to antigen challenge decreased the ratio of AA to DHA (FEN-OVA vs OVA). This corrective effect of fenretinide resulted from fenretinideinduced normalization of both AA and DHA levels. As shown in Figures 1B and 1C, following the treatment with fenretinide AA was decreased while DHA was increased, respectively. Treatment with fenretinide prior to ovalbumin challenge also prevented the increase in COX-2 but not 5-LOX (Figures 1D-G).

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Subsequently, we also checked if the 19 metabolites we had identified to be altered in allergic animals might also get corrected by the treatment with fenretinide. PCA, DFA and hierarchical clustering of metabolomics data show that fenretinide treated allergic mice can be distinguished from those that were non-treated and allergic (FEN-OVA vs OVA) (Figures E1 and E2). As shown in Figure 2B, the levels of 14 metabolites were significantly changed in fenretinide treated allergic mice compared to non-treated allergic mice. Of these 14 metabolites, five of them (arginine, C3DC+C4OH, carnitine, serotonin, and tyrosine) were overlapping with the 19 metabolites altered by allergen challenge (Figure 2C). Fenretinide treatment normalized the level of these five metabolites to levels observed in non-allergic PBS challenged mice. MDA is a marker of lipid oxidation and nitrotyrosine is a marker of protein oxidation, and both are markers of cellular stress and damage. We observed that ovalbumin challenge increased MDA and nitrotyrosine in the lungs of allergic mice (Figures 3A and 3B). DHA has anti-oxidant properties and therefore we explored whether fenretinide can reduce the oxidative stress in the lungs associated with ovalbumin challenge (15). The levels of MDA and nitrotyrosine in fenretinide treated non-allergic mice were comparable to vehicle treated non-allergic mice (Table E2). The treatment with fenretinide prevented the increase in MDA and nitrotyrosine in the lungs of allergen sensitized and challenged mice. Fenretinide protects from allergen induced AHR but not IgE production. The protective effect of fenretinide in normalizing levels of several important allergic response mediators prompted us to also evaluate its potential efficiency in inhibiting other phenotypes associated with allergic asthma, such as AHR and IgE production.

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Lung responsiveness to methacholine was measured by the gold standard invasive method for measuring airway resistance (Figure 4A). A statistically significant increase in lung resistance was observed in the allergic mice (OVA) compared to the PBS challenged mice at 80 and 160 mg/mL of methacholine. The drug alone does not cause any significant change in airway responsiveness (Figure E4). Fenretinide treated allergic mice (FEN-OVA) had significantly lower lung resistance than vehicle treated and challenged mice (OVA) at both 80 and 160mg/mL methacholine. We explored if a similar protective effect as seen for AHR can also be seen for IgE production following treatment with fenretinide. Unsensitized A/J mice have a baseline plasma IgE concentration of approximately 500 ng/mL (data not shown). All mice in the three study groups were intraperitoneally sensitized with ovalbumin and their IgE concentrations were greater than those of naïve mice (Figure 4B). Between vehicle treated groups (PBS vs OVA), ovalbumin challenges caused IgE titer to increase compared to PBS challenged mice. Unlike airway responsiveness, fenretinide did not prevent an increase in the production of IgE in response to allergen challenge. The protection against AHR could result from fenretinide affecting the balance between pro-inflammatory and anti-inflammatory chemokines and cytokines; therefore, we next assessed levels of several important allergic asthma mediators. Fenretinide prevents changes in inflammatory mediators. Decrease in DHA following antigen challenge, can increase the availability of AA to lipid converting enzymes resulting in a pro-inflammatory signaling cascade. AA can induce the production of IL-8 through the actions of COX-2 and NF-κΒ (16). In vitro studies have shown that fenretinide is capable of preventing the production of IL-8 from LPS stimulated human lung epithelial cells (17). Therefore, we measured the murine counterpart of IL-8, CXCL1, in the 12



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lungs of our allergic, allergic-treated, and control mice. CXCL1 was upregulated post-ovalbumin challenge compared to PBS challenged mice (OVA vs PBS) (Figure 5A). Pre-treatment with fenretinide prevented the increase in expression of CXCL1 in the lungs (FEN-OVA vs OVA) (Figure 5A). Similarly, we also observed a decrease in the macrophage chemoattractant, CXCL10, in the ovalbumin challenged mice that were treated with fenretinide compared to those who were vehicle treated and ovalbumin challenged (FEN-OVA vs OVA) (Figure 5B). We also explored the effect of fenretinide on the response of mouse lung epithelia cells when exposed to LPS to assess what other cytokines important in allergic asthma are affected by the treatment with this drug. We found that LPS stimulation augments the expression of a majority of small signaling molecules in mouse lung epithelial cells. Compared to unstimulated cells, mRNA expression of Ccl2, Ccl5, Ccl7, Ccl11, Cxcl1, Cxcl2, Cxcl9, Cxcl10, Il-6, iNOS, Pdgf and Tnf-α were all increased following a three hour exposure to LPS (MEDIUM vs LPS) (Figure 5C and Figure E5A-L). Pretreatment of cells with fenretinide prior to LPS stimulation (FEN-LPS) dampened the transcription of all measured cytokines and chemokines; however significance was not reached for Ccl2 and Pdgf (Figure 5C and Figure E5A-L). Fenretinide prevents recruitment of inflammatory cells to the airways after ovalbumin challenge. Compared to PBS challenged animals (Figure 6A), untreated and ovalbumin challenged mice (Figure 6B) display a marked influx of inflammatory cells to the area surrounding the airway and blood vessels. A dramatic reduction in inflammatory cell recruitment was observed in mice treated with fenretinide prior to ovalbumin challenge (Figure 6C). Inflammation was quantified by counting the number of inflammatory cells in the area surrounding the airways normalized by the square of the perimeter of the airway basement membrane (Figure 6D). Again untreated 13



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allergen challenged animals had a greater number of inflammatory cells surrounding the airways when compared to PBS challenged mice. Treatment with fenretinide prevented the increase of inflammatory cells to the airways caused by allergen challenge. Fenretinide prevents goblet cell hyperplasia in the airways of allergic mice. The PAS method was used to identify mucous producing goblet cells. In PBS challenged mice, very few to almost no PAS positive cells were identifiable (Figure 7A). Challenge with ovalbumin caused a visible increase in PAS positive cells (Figure 7B). Treatment with fenretinide prior to challenge with ovalbumin resulted in only a few PAS positive cells to be identified in the airways (Figure 7C). Conclusions drawn from visual inspection of airways were validated by quantifying goblet cell hyperplasia by counting PAS positive epithelial cells in the airways and dividing by the perimeter of the airway basement membrane. As initially observed ovalbumin challenge caused a marked increase in hyperplasia that was attenuated by pretreatment with fenretinide (Figure 7D).

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DISCUSSION In human studies, asthmatics have been shown to have varying levels of PUFAs compared to healthy controls (18;19). However, it is not certain whether this is a consequence of the disease or if this feature predisposes an individual to the disease. High dietary intake of n-6 PUFAs in westerners has been proposed as one of the reasons for the increased prevalence of complex diseases (20). In our mouse model of allergic asthma, we show that allergen challenge causes changes in lipid and metabolic mediators. Yokoyama et al also observed a significant decrease in DHA after antigen challenge (21). Perhaps a significant increase in AA would have been observed in our study if we had done more challenges or used a stronger antigen than ovalbumin. Using a guinea pig model of allergic asthma, Morin et al also observed a distortion in AA/DHA in the pro-inflammatory direction after antigen challenge, however in their case the observed difference in the ratio was caused by an increase in AA (22). Our changes in AA and DHA levels were concordant with an increase in inflammatory mediators, such as COX-2. COX-2 converts AA into prostaglandins which could cause bronchoconstriction (14). DHA can inhibit upregulation of COX-2 by decreasing the degradation of IκBα and activity of NF-κΒ (23-25). MacLean et al show that ovalbumin activated splenocytes from DHA fed mice produce less IL-4 and IL-13 than mice fed with a normal diet, and DHA inhibits the production of IL-13 from Th2 cells (26). AA is also converted into proinflammatory leukotrienes through 5-LOX, however in our study fenretinide did not have any effect on the allergen induced increase in 5-LOX expression in the lungs. Fenretinide could therefore be a potential candidate drug for asthmatic patients who are not helped with 5-LOX inhibitors or can be used in combination with 5-LOX inhibitors.

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We have previously explored the changes induced at the RNA and protein level in our ovalbumin induced mouse model of allergic asthma; therefore, we were interested to see the effects of ovalbumin challenge on metabolites. We observed changes in an array of metabolites and the largest change was observed in serotonin. Provocation with allergen challenge has been shown to upregulate this neurotransmitter in bronchoalveolar lavage fluid of allergic mice and asthmatic patients (27). Serotonin is used as a bronchoconstrictor, chemoattractant for eosinophils, and can induce smooth muscle cell proliferation (28-30). DHA can reduce the expression of serotonin receptor (30). Blocking serotonin receptors in mice is protective against airway inflammation and remodeling in mice (31;32). Following allergen challenge we also observed increases in markers of cellular stress, MDA and nitrotyrosine. MDA is a marker of lipid peroxidation and pulmonary oxidative stress. MDA can be measured in exhaled breath and levels of MDA are inversely correlated with forced expiratory volume and directly correlated with IL-8 and TGF-β1 in asthmatic children (33;34). Nitrotyrosine is a marker of protein oxidation and is also elevated in exhaled breath of asthmatics (35). Concordant with the increase in nitrotyrosine after ovalbumin challenge is the observed decrease in tyrosine in our metabolites screen. However, the results from studies where PUFA levels in allergic asthma are controlled by pharmacological or dietary supplementation with PUFAs are not all consistent. In ovalbumin induced mouse models, groups have shown administration of DHA, EPA, or their derivatives prior to ovalbumin challenge reduce eosinophil and lymphocyte count and levels of proinflammatory mediators in bronchoalveolar lavage fluid and hyperresponsiveness associated with allergen challenge (21;22;36;37). However, other researchers show that ovalbumin sensitized mice treated with diets enriched with DHA or EPA, or supplemented with fish oils, 16



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have worsened airway responsiveness phenotypes in addition to higher levels of IL-4, lL-5, IL-6, and IL-12 in the lung environment (38;39). Fenretinide is a synthetic retinoid that has been widely explored as an anticancer drug in numerous clinical trials due to its pro-apoptotic properties (40). Recent studies have shown that fenretinide can modulate the levels of PUFAs, AA and DHA (10-13). The above mentioned phenotypes, AA/DHA, nitrotyrosine, MDA, and serotonin, have all been shown to be modulated by fenretinide (12). Furthermore, the anti-inflammatory properties of fenretinide are also presented through in vitro and in vivo models of bacterial infection (41;42). We used this knowledge to launch our investigation on a possible role of this drug to have a protective effect in allergic asthma. To date, no other research group has explored the benefits of fenretinide in allergic asthma. Our findings show that a pre-treatment with fenretinide is capable of preventing the ovalbumin induced increase in ratio of AA to DHA, by increasing the levels of DHA and decreasing the levels of AA. This correlates with a significant reduction in COX-2 expression. In addition, allergen induced increases in markers of cellular oxidative state, MDA and nitrotyrosine, were also prevented. We present 14 metabolites whose levels are modulated by fenretinide of which five incurred changes due to allergen challenge: arginine, carnitine, C3DC+C4OH, tyrosine and serotonin. Decreases in arginine and carnitine in allergic asthmatics have been reported in previous studies (43;44). Both metabolites are connected to allergic asthma via nitric oxide production (43;45). Tyrosine is necessary for the synthesis of thyroid hormone, dopamine and melanin, all of which play key roles in inflammatory processes and in immune response (46). To balance the observed

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decrease in tyrosine, the level of the tyrosine nitration product, nitrotyrosine, is increased following ovalbumin challenge. All five metabolites are important, however, we chose to focus on serotonin in our discussion. Interestingly the effect of fenretinide on serotonin levels in our asthma model is contrary to the effects seen in spinal cord injury. Here we show that fenretinide prevents the allergen induced increase in serotonin, while in spinal cord injury fenretinide increases the number of serotogenic fibers in the spinal cord (12). The role of serotonin in both diseases is different and fenretinide has a different effect depending on the disease and signaling pathways. Fenretinide also hinders cytokine production induced by external stimuli. Epithelial cell lines derived from cystic fibrosis patients and healthy controls release IL-8 when treated with TNF-α, and pre-treatment with fenretinide prevented the release of the neutrophil chemoattractant, IL-8, from cells of cystic fibrosis patients only (17). In FXR1-WT and FXR1-KO cell lines, pretreatment with fenretinide interferes with LPS induced production of IL-6, CCL2 and CCL5 along with changes in the AA and DHA levels (13). To add to these results, fenretinide effectively inhibited LPS induced expression of various Th1 and Th2 cytokines, including Ccl5, Ccl7, Ccl11, Cxcl1, Cxcl2, Cxcl9, Cxcl10, Il-6, Tnf-a, and iNOS in mouse lung epithelial cells. In vivo, we show that allergen induced production of the Th17 neutrophil chemoattractant CXCL1 in mouse lungs was inhibited by treatment with fenretinide. Those treated with fenretinide also had decreased concentration of circulating CXCL10 in the lung. Elevated levels of CXCL10, a chemoattractant for lymphocytes, monocytes, and natural killer cells, have been detected in asthmatic children (47). As mentioned earlier, the inhibition of NF-κΒ activation by DHA could provide a possible explanation for the observed results. DHA also attenuates NF-κΒ activation

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by decreasing the response of toll like receptors to signals such as LPS, preventing expression of inflammatory mediators downstream of NF-κΒ (48). Our data demonstrate very potent protective property of fenretinide against allergic asthma. We have systematically evaluated several classical phenotypes associated with allergic asthma, including AHR, IgE production and histological features. We demonstrated that fenretinide prevents allergen induced AHR, airway eosinophil and goblet cell hyperplasia, but not the production of IgE. It is likely that the production of IgE may have not been affected because in our model the treatment with fenretinide commences only after allergen sensitization, but this is a more relevant physiologically model since most of the patients suffering from allergic asthma are already pre-exposed to allergen and sensitized at the time of treatment. We believe that starting the treatment after sensitization was established is representative of what occurs to allergic patients. Interestingly, even in the presence of high levels of IgE, the influx of inflammatory cells and hyperplasia of the airways associated with allergen exposure was prevented. Our most impressive findings are the ovalbumin induced histological changes that are prevented by fenretinide pre-treatment. The recruitment of inflammatory cells to the airway is nearly completely halted which is in accord with fenretinide’s ability to inhibit cytokine production in vitro and in vivo as seen in our study and other studies (41;42). Further investigation can be done to verify if airway structural changes associated with a chronic model of allergic asthma can also be prevented by fenretinide. In conclusion, fenretinide represent an interesting candidate drug for treating allergic asthma that deserves attention especially in the cases of allergies which are very difficult to treat with

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corticosteroids. The exact molecular mechanism explaining how fenretinide modulates PUFA levels which in turn have profound effects on allergic asthma phenotypes certainly deserves more studies. Nevertheless, administration of fenretinide prior to allergen challenge protected mice from AHR and airway inflammation, in addition to directing levels of AA and DHA, oxidation markers, and metabolites like serotonin, towards healthy levels.

The anti-

inflammatory properties of fenretinide are also showcased through its ability hinder the increase in expression of many cytokines induced by antigen challenge. Overall, given the broad range of effects on various endpoints, fenretinide may be a potential novel compound for controlling the inflammation associated phenotypes observed in allergic asthma.

ACKNOWLEDGEMENTS We would like to thank Dr. Marie-Christine Guiot at the Montreal Neurological Institute for allowing us to use her histology laboratory for processing and staining our histological samples. We would also like to thank Dr. Robert Smith from the National Institute of Health for generously providing the fenretinide powder.

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FIGURE LEGENDS

Figure 1: Fenretinide (FEN) prevents changes in ratio of arachidonic acid (AA) to docosahexaenoic acid (DHA), and cyclooxygenase-2 (COX-2) expression in lungs of ovalbumin challenged mice. (A) Animals challenged with ovalbumin (OVA) have a significant change in ratio of AA to DHA. (B) While no significant change is observed in the level of AA, DHA is significantly decreased (C) in OVA challenged mice compared to PBS challenged mice. Ovalbumin challenge causes an increase in the expression of COX-2 (Figures D and E) and 5lipoxygenase (5-LOX) (Figures F and G). Treatment with FEN prior to antigen challenge maintained the levels of AA/DHA, DHA, and COX-2 similar to non-allergic mice (PBS group), while 5-LOX expression remained elevated In addition, fenretinide also significantly decreased the levels of AA (B). Data are presented as median ± interquartile range and statistical significance was calculated by the means of a one-way ANOVA. Figures A, B, C: n = 9, 12 and 12 for PBS, OVA, and FEN-OVA, respectively; Figures D and F: n >= 3 for PBS, OVA and FEN-OVA; **p < 0.01, and *** p < 0.001. Figure 2: Metabolic changes in plasma associated with allergic asthma. (A) The levels of 19 metabolites were changed following challenge with ovalbumin (OVA) compared to non-allergic group (PBS). (B) The levels of 14 metabolites were significantly different between vehicletreated allergic mice (OVA) and fenretinide treated allergic mice (FEN-OVA). (C) Five metabolites overlap between PBS vs OVA and OVA vs FEN-OVA comparisons representing metabolites that are altered by OVA challenge and corrected by FEN treatment. Data are presented as heatmap of log transformed values and statistical significance was calculated by the means of t-test.; n = 12, 11 and 12 for PBS, OVA, and FEN-OVA, respectively; *p < 0.05, **p

P2Y12 antagonist attenuates eosinophilic inflammation and airway hyperresponsiveness in a mouse model of asthma.

Interleukin 33 exacerbates antigen driven airway hyperresponsiveness, inflammation and remodeling in a mouse model of asthma.

A novel synthetic mycolic Acid inhibits bronchial hyperresponsiveness and allergic inflammation in a mouse model of asthma.

Suppression of heme oxygenase-1 activity reduces airway hyperresponsiveness and inflammation in a mouse model of asthma.

Asthma and airway hyperresponsiveness.

Concomitant exposure to ovalbumin and endotoxin augments airway inflammation but not airway hyperresponsiveness in a murine model of asthma.

Novel genes in Human Asthma Based on a Mouse Model of Allergic Airway Inflammation and Human Investigations.

Adipose-derived stem cells ameliorate allergic airway inflammation by inducing regulatory T cells in a mouse model of asthma.

Effect of a retinoid X receptor partial agonist on airway inflammation and hyperresponsiveness in a murine model of asthma.

Biosignature for airway inflammation in a house dust mite-challenged murine model of allergic asthma.

Glutathione modulation during sensitization as well as challenge phase regulates airway reactivity and inflammation in mouse model of allergic asthma.

Oroxylin A Inhibits Allergic Airway Inflammation in Ovalbumin (OVA)-Induced Asthma Murine Model.

Cyclic nitroxide radicals attenuate inflammation and Hyper-responsiveness in a mouse model of allergic asthma.

Suhuang antitussive capsule at lower doses attenuates airway hyperresponsiveness, inflammation, and remodeling in a murine model of chronic asthma.

Vaccination against IL-33 Inhibits Airway Hyperresponsiveness and Inflammation in a House Dust Mite Model of Asthma.

Molecular Mechanisms of Airway Hyperresponsiveness in a Murine Model of Steroid-Resistant Airway Inflammation.

Pericytes contribute to airway remodeling in a mouse model of chronic allergic asthma.

Compartmental and temporal dynamics of chronic inflammation and airway remodelling in a chronic asthma mouse model.

Chimeric Antigen Receptor-Redirected Regulatory T Cells Suppress Experimental Allergic Airway Inflammation, a Model of Asthma.

Sinomenine attenuates airway inflammation and remodeling in a mouse model of asthma.

Beta-agonists, airway hyperresponsiveness, and asthma.

Thuja orientalis reduces airway inflammation in ovalbumin-induced allergic asthma.

Syk Regulates Neutrophilic Airway Hyper-Responsiveness in a Chronic Mouse Model of Allergic Airways Inflammation.

Stigmasterol Modulates Allergic Airway Inflammation in Guinea Pig Model of Ovalbumin-Induced Asthma.