Fenretinide Prevents Inflammation and Airway Hyperresponsiveness in a Mouse Model of Allergic Asthma

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Fenretinide prevents inflammation and airway hyperresponsiveness in a mouse model of

allergic asthma

Cynthia Kanagaratham1, Alžběta Kalivodová

2, Lukáš Najdekr

2, David Friedecký

2, Tomáš

Adam2, Marian Hajduch

2, Juan Bautista De Sanctis

3, Danuta Radzioch

1,4

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

2. Institute of Molecular and Translational Medicine, Faculty of Medicine and Dentistry,

Palacky University and University Hospital in Olomouc, Olomouc, Czech Republic

3. Institute of Immunology, Faculty of Medicine, Universidad Central de Venezuela,

Sabana Grande, Caracas, Venezuela

4. 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]

Page 1 of 57 AJRCMB Articles in Press. Published on 02-June-2014 as 10.1165/rcmb.2014-0121OC

Copyright © 2014 by the American Thoracic Society

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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 anti-

inflammatory. 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



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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: non-

allergic (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 HPLC-

MS 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



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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 fenretinide-

induced 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



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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



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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 pre-

treatment 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 pro-

inflammatory 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 pro-

inflammatory 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,



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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, pre-

treatment 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



20

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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29

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 5-

lipoxygenase (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 vehicle-

treated 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 <



30

0.01, and ***p < 0.001 between PBS and OVA groups, and †p < 0.05, ††p < 0.01, and †††p <

0.001 between OVA and FEN-OVA groups.

Figure 3: Fenretinide (FEN) prevents increase in markers of oxidative stress in lungs. Cellular

oxidation markers malonyldialdehyde (A) and nitrotyrosine (B) are increased in the lungs of

mice following ovalbumin (OVA) challenge. By treating mice with FEN prior to OVA challenge

(FEN-OVA), the increase in cellular oxidation markers was prevented. Data are presented as

median ± interquartile range and statistical significance was calculated by the means of a one-

way ANOVA. n = 9, 12 and 12 for PBS, OVA, and FEN-OVA, respectively; ***p < 0.001.

Figure 4: Fenretinide (FEN) prevents airway hyperresponsiveness (AHR) but not IgE

production in allergic asthma. (A) Ovalbumin challenge (OVA group) causes a dose response

increase in AHR compared to non-allergic mice (PBS group). Treatment with FEN prior to OVA

challenge (FEN-OVA group) prevented AHR to methacholine in allergic mice. Data are

presented as mean ± SEM from two independent experiments and statistical significance was

calculated by one-way ANOVA. n = 8, 10 and 10 for PBS, OVA, and FEN-OVA, respectively;

*p < 0.05 between PBS and OVA groups, †p < 0.05 and ††p < 0.01 between OVA and FEN-

OVA groups. (B) IgE level was measure in the plasma by ELISA. Concentration of IgE in the

plasma is increased after OVA challenge and is unaffected by treatment with FEN. Data are

presented as median ± interquartile range and statistical significance was calculated by the means

of a one-way ANOVA. n = 11, 9 and 11, for PBS, OVA and FEN-OVA, respectively; *p < 0.05.



31

Figure 5: Fenretinide (FEN) prevents increase in markers of inflammation. CXCL1 (A) and

CXCL10 (B) levels were measured in lung homogenates by ELISA. Comparison of untreated

ovalbumin challenged allergic mice (OVA) and FEN treated allergic mice (FEN-OVA), shows

that CXCL1 and CXCL10 levels in the lungs of treated mice are lower than in untreated mice.

Data are presented as median ± interquartile range and statistical significance was calculated by

the means of a one-way ANOVA. n = 6 per group for CXC1 and n = 5 per group for CXCL10;

*p < 0.05 and ***p < 0.001. (C) Heatmap of fold change in mRNA expression of small

molecules in MLE-12 cells after stimulation with lipopolysaccharide (LPS) with or without

fenretinide (FEN) pretreatment. Expressions in stimulated (LPS) and FEN treated and stimulated

(FEN-LPS) cells are normalized to untreated and unstimulated cells (MEDIUM). Values

represent fold increase from untreated group. For all genes, LPS stimulated group was

significantly different from MEDIUM group. Data are from two experiments done in triplicates

and statistical significance was calculated by the means of a one-way ANOVA. *p < 0.05, **p <

0.01, and ***p < 0.001 and represent difference between LPS and FEN-LPS groups. For box

plots, see Figure E5.

Figure 6: Fenretinide (FEN) prevents recruitment of inflammatory cells to the airways. Panels A

and B represent lung sections from drug vehicle treated animals challenged with either PBS or

ovalbumin (OVA), respectively. Panel C represents lung section from FEN treated animal

challenged with OVA (FEN-OVA). In panel D, inflammation surrounding at least four airways

for each mouse from each group was quantified by counting the number of inflammatory cells

and dividing by the square of the perimeter of the basement membrane (Pbm2). Data are

presented as median ± interquartile range and statistical significance was calculated by a one-

way ANOVA. n = 4, 7 and 6 for PBS, OVA, and FEN-OVA, respectively; *p < 0.05, **p < 0.01.



32

Figure 7: Fenretinide (FEN) prevents goblet cell hyperplasia. Panels A and B represent lung

sections from drug vehicle treated animals challenged with either PBS or ovalbumin (OVA),

respectively. Panel C represents lung section from FEN treated animal challenged with OVA

(FEN-OVA). In panel D, the number of periodic acid Schiff positive cells in at least four airways

from each animal in each group was counted and normalized by the perimeter of the airway

basement membrane (Pbm). Data are presented as median ± interquartile range and statistical

significance was calculated by the means of a one-way ANOVA. n = 6 for all groups; *p < 0.05,

**p < 0.01.



A

PBS OVA FEN-OVA0

5

10

15

AA

/DH

A

Ratio

*** ***

PBS OVA FEN-OVA0

1

2

3

4

Fold

change f

rom

PB

S

** **

PBS OVA FEN-OVA

COX-2

β-actin

D

E

PBS OVA FEN-OVA

5-LOX

β-actin

F

G

0

1

2

3

4

5

PBS OVA FEN-OVA0

1

2

3

4

5

Fold

change f

rom

PB

S

** **

Figure 1

B

PBS OVA FEN-OVA20

25

30

35

40

45

Ara

chid

onic

acid

(nm

ol/m

g o

f phospholip

id) **

C

PBS OVA FEN-OVA2.5

3.0

3.5

4.0

4.5

5.0

5.5

Docosahexa

enoic

acid

(nm

ol/m

g o

f phospholip

id) *** ***

**



PBS

OVA

*** Serotonin

* C3DC+C4OH

* MethylbutyrylGly.+isovalerylGly

** C10

* Proline

* Guanidinoacetate

* Histidine

** Citrate+isocitrate

** Asparagine

* Glutamine

* Betaine

** Tthreonine+homoserine

** Lysine

* Citrulline

** Serine

* Homocysteine

* Carnitine

* Tyrosine

** Arginine

OVA

FEN-O

VA

† Serotonin

† Kynurenine

†† C3DC+C4OH

† Taurine

†† C6:1

†† Carnitine

† C20:2

† Tyrosine

† C18:2

†† C20:1

††† C18

† C20

† C22

† Arginine

PBS

OVA

FEN-O

VA

*** † Serotonin

* † C3DC+C4OH

* †† Carnitine

*** † Tyrosine

** † Arginine

PBS vs OVA OVA vs FEN-OVA

19 145

PBS OVA

*** Serotonin

* C3DC+C4OH

* MethylbutyrylGly.+isovalerylGly

** C10

* Proline

* Guanidinoacetate

* Histidine

** Citrate+isocitrate

** Asparagine

* Glutamine

* Betaine

** Tthreonine+homoserine

** Lysine

* Citrulline

** Serine

* Homocysteine

* Carnitine

* Tyrosine

** Arginine

-1.5 0.5

A B

C

Figure 2



A

PBS OVA FEN-OVA0

5

10

15

20

Malo

nyld

eald

ehyde

(nm

ole

s/m

g o

f pro

tein

)

*** ***

B

PBS OVA FEN-OVA0

5

10

15

Nitro

tyro

sin

e

(ng/m

g p

rote

in)

*** ***

Figure 3



A B

Bas

eline

Saline 20 40 80 16

0

0

2

4

6

8PBS

OVA

FEN-OVA * **

Methacholine dose (mg/ml)

Resis

tance

(cm

H2O

*s/

ml)

PBS OVA FEN-OVA0

2000

4000

6000

IgE

(ng/m

L)

* *

Figure 4



PBS OVA FEN-OVA0

50

100

150

200

CX

CL1

(pg/m

g o

f pro

tein

)

* ***

PBS OVA FEN-OVA0

200

400

600

800

1000

CX

CL10

(pg/m

g o

f pro

tein

)

*

C A

B

MEDIU

M

LPS

FEN-L

PS

1 71 28 *** Cxcl9

1 43 26 * Cxcl2

1 40 26 * Cxc1

1 25 9 *** iNOS

1 16 9 * Tnf-α

1 14 12 Ccl2

1 11 6 *** Ccl7

1 9 7 *** Cxcl10

1 3 1 * Ccl11

1 3 2 *** Il-6

1 2 1 ** Ccl5

1 2 1 Pdgf

Increase in expression

Figure 5



B A PBS OVA FEN-OVA D C

PBS OVA FEN-OVA0

50

100

150

Cells

/Pb

m2

(cells

/mm

2)

* **

Figure 6



B A C

PBS OVA FEN-OVA0

10

20

30

40

50

PA

S+ C

ells

/Pb

m

(cells

/mm

)

** *

D PBS OVA FEN-OVA

Figure 7



Fenretinide prevents inflammation and airway hyperresponsiveness in a mouse model of

allergic asthma

Cynthia Kanagaratham, Alžběta Kalivodová, Lukáš Najdekr, David Friedecký, Tomáš Adam,

Marian Hajduch, Juan Bautista De Sanctis, Danuta Radzioch

Online data supplement



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, and challenge protocol, and experimental groups

All mice were sensitized weekly for three consecutive weeks by intraperitoneal injection with

100μg of ovalbumin (OVA) adsorbed to 1.5mg of aluminum hydroxide (Imject Alum, Pierce,

Rockford, IL) in 0.2mL of sterile PBS. Following sensitization the mice were split into three

groups: fenretinide treated and allergic (FEN-OVA), non-allergic (PBS) and allergic (OVA).

Treatment began one week after the third sensitization. Allergic-treated mice were orally treated

with fenretinide at a daily dose of 60mg/kg. Fenretinide powder (NIH, Bethesda, MD, USA)

was resuspended in 95% ethanol to a 20 mg/mL stock concentration in a sterile manner and

protected from light. The appropriate volume of fenretinide necessary for each mouse based in

its weight was incorporated into 12.5 mL of Peptamen liquid diet (Nestle, Brampton, ON,

Canada). During sensitization, mice were also fed with Peptamen to make them accustomed to

the diet. Mice in the non-allergic and allergic groups received the drug vehicle, ethanol, in their

Peptamen rather than the 60mg/kg volume of fenretinide. The maximum volume of ethanol

diluted in Peptamen never exceeded 90ul. All mice were vehicle or drug treated for four weeks.

All mice included in the study consumed their daily volume of 12.5mL of Peptamen

supplemented with drug or vehicle. During the fourth week of treatment, the allergic (OVA

group) and allergic-treated mice (FEN-OVA group) were exposed to 1% OVA solution for 30



minutes, while non-allergic mice (PBS group) were exposed to PBS. A fourth group of

fenretinide treated and PBS challenged mice (FEN-PBS) was also done to test the effects of

fenretinide alone on lipids, markers of oxidative stress and airway responsiveness. .

Metabolomic analysis

Measurements, data processing and statistical analysis were done at the Laboratory of

Metabolomics at the Institute of Molecular and Translational Medicine, Medical Faculty,

Palacký University in Olomouc. Plasma samples collected from mice three hours after final

antigen challenge were treated as follows: 2 µL of each sample was mixed with 8 µL of LC-MS

quality water and 40 µL of LC-MS methanol (tempered to -80°C). The final solutions were

vortexed and stored overnight at -80°C (1). After centrifugation (14 000 g, 15 min, 4°C) the 35

µL of supernatant was transferred into the glass vials. Part of supernatant (10 ul) was pooled and

used for quality control (QC) purpose (2).

All samples were analyzed by HPLC (UHPLC Dionex Ultimate 3000 RS, Thermo Fisher

Scientific, MA, USA) using modified method previously published (3). Triple quadrupole mass

spectrometer 5500 QTrap (AB Sciex, CA, USA) was used as a detector. Normal phase LC

system with conditions of HILIC separation was used with column Luna 3 µm NH2 100 Å, 150

x 2 mm (Phenomenex, Torrance, USA). The solvent was aqueous buffer: 20 mM ammonium

acetate (pH 9.45), solvent B: acetonitrile. The gradient with flow rate of 0.3 mL/min for HPLC

was: t=0.0, 95% B; t=15.0, 30% B; t=17.0, 5% B; t=23.0, 5% B; t=23.1, 95% B; t=28.0 min

95% B. MS method settings were as follows: acquiring samples were done in both ionization

modes (positive, negative) with ionspray voltage of +/- 4500 V, capillary temperature 400°C,

Curtain Gas – 30 arb, Ion Source Gas (GS1) – 40 arb and Ion Source Gas (GS2) – 40 arb.



Metabolomic data were processed by software MultiQuant 2.1.1. (AB Sciex, CA, USA). All

peaks were manually corrected.

Preparation of lung homogenates for measuring cytokine concentration and western

blotting

Three hours after the final ovalbumin challenge animals were sacrificed by CO2 exposure, lungs

were harvested and snap frozen in liquid nitrogen. We previously showed that the differences in

cytokine levels between the allergic and non-allergic group is best observed three hours after the

final antigen challenge (4). Lungs were homogenized with a polytron in PBS containing

protease inhibitor (Roche). Homogenates were spun at 15000 rpm for 15 minutes to discard any

debris. Protein concentration of lung homogenates was determined using Bio-Rad protein assay

(Bio-Rad) according to the manufacturer’s instructions.

Lung protein analysis

Mouse lung homogenates were used to measure CXCL1 and CXCL10 by ELISA (R and D

Systems) following the manufacturer’s instructions. For western blotting, 30ug of total protein

was resolved on a 4-12% Bis-Tris Gel (Life Technologies) and electrophoretically transferred to

a PVDF membrane (Millipore, Bedford, MA). Membranes were blocked overnight at 4oC with

5% solution of dehydrated milk powder prepared in Tris buffered saline-0.1% Tween 20.

Membranes were probed with primary antibodies, either 1:500 diluted anti-cyclooxygenase-2 or

1:250 diluted anti-5-lipoxygenase antibodies, overnight at 4oC (Cayman Chemical, Ann Arbor,

MI, USA). As a control, membranes were incubated one hour at room temperature with anti β-

actin diluted 1:5000 (Sigma, St-Louis, Missouri, USA). Appropriate secondary antibodies

conjugated with horse radish peroxidase were used. Membranes were developed with western



lightening plus – enhanced chemiluminescence substrate (PerkinElmer, Inc. Waltham, MA,

USA).

Measurement of airway resistance

Forty-eight hours after the third allergen challenge, the resistance of the respiratory system was

measured. The mice were first anesthetized with a cocktail of ketamine-xylazine-acepromazine

(100/10/3 mg/kg), 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). Once data collection was complete, anesthetized animals were

euthanized by cardiac puncture.

Cell experiments

MLE-12 cells (ATCC #: CRL-2110) were cultured in DMEM (Wisent) supplemented with 2%

inactivated fetal bovine serum (Multicell), 1% Penicillin/Streptomycin (Multicell), and 2 mM L-

glutamine (Invitrogen). Cells were plated in 24 well plates (Corning) at 1.5x105

cells/mL in a

total volume of 0.5 mL and left to adhere overnight. The following day, cells were treated with

fenretinide at a concentration of 1.25 μM. 21 h after treatment, media was replaced, again

containing 1.25 μM fenretinide in addition to 50 ng/mL LPS from Escherichia coli (Sigma).

Stimulation with LPS was stopped after 3 h and RNA was extracted from the cells using the

RNeasy Mini kit (Qiagen) following the manufacturer’s instructions. These time points were

chosen based on previous studies (5). RNA concentration was measured using a Nanodrop 2000.

200 ng of RNA was reverse transcribed into cDNA using the QuantiTect Reverse Transcription



kit according to the manufacturer’s instructions. Expression of cytokine mRNA was measured

using a StepOne Plus Real-Time qPCR instrument (Life Technologies) and Fast SYBR Green

Master Mix (Life Technologies). Efficiency and specificity of primers (IDT DNA, see Table E1

in the online data supplement) was tested and expression of all cytokines was normalized to

Gapdh and calculated using the cycle threshold (CT) values and the 2-ΔΔCT

method.

Lung histopathology

Forty eight hours after final ovalbumin challenge, lungs were removed from the mice, inflated

with 10% buffered formalin (Fisher Scientific, Nepean, ON, Canada) and immersed therein for a

minimum of 36 hours. The lungs were then trimmed, dehydrated, embedded in paraffin and cut

into 3 μm thick sections using a Reichert-Jung microtome. The sections were deparaffinized,

hydrated and stained with the hematoxylin and eosin (H&E) and periodic acid Schiff (PAS)

stains by standard procedures.

Measurement of inflammation

H&E stained slides were used to quantify the inflammation in the lungs. Inflammatory cells

found within the peribronchial space surrounding the airways were counted. Data was

normalized by dividing the cell counts by the square of the perimeter of the basement membrane

(Pbm2). Per mouse, at least four airways cross sections coming from the same depth were counted

and four to six mice per group were quantified.

Measurement of goblet cell hyperplasia

PAS stained slides were used to quantify goblet cell hyperplasia in the lungs. PAS positive cells

within the airway were counted. Data was normalized by dividing the counts by the perimeter of



the basement membrane (Pbm). Per mouse, at least four airways cross sections coming from the

same depth were counted and four to six mice per group were quantified.

Statistical analysis

The metabolomics dataset was interpolated and statistically evaluated in R software (6). First

step in the data processing was reduction of systematic error by data interpolation by means of

quality control samples. LOESS method was applied. Coefficient of variation was calculated for

all compounds in quality control samples. Compounds with values higher than 30 % were

rejected from further processing (2). Zero values in dataset were imputed by two thirds of the

minimum of appropriate feature within a sample group (2). Metabolomic data were considered

as compositional data and centered log ratio (clr) transformation and centering was applied on

the dataset (7). For statistical evaluation several approaches were chosen - unsupervised

principal component analysis (PCA) method, cluster analysis and supervised discriminant

function analysis method (DFA). Box plots were generated for all measured metabolites (after

clr transformation).

All other data was analyzed with GraphPad Prism 5 (GraphPad Software Inc., San Diego, CA).

One-way ANOVA followed by a Bonferroni post-test procedure was used for comparing all

three experimental groups. Comparisons between two groups for metabolites were done using

unpaired t-test with Welch's correction.



References

(1) Yuan M, Breitkopf SB, Yang X, Asara JM. A positive/negative ion-switching, targeted

mass spectrometry-based metabolomics platform for bodily fluids, cells, and fresh and

fixed tissue. Nat Protoc 2012 April 12;7(5):872-81.

(2) Dunn WB, Broadhurst D, Begley P, Zelena E, Francis-McIntyre S, Anderson N, Brown M,

Knowles JD, Halsall A, Haselden JN, Nicholls AW, Wilson ID, Kell DB, Goodacre R.

Procedures for large-scale metabolic profiling of serum and plasma using gas

chromatography and liquid chromatography coupled to mass spectrometry. Nat Protoc

2011 June 30;6(7):1060-83.

(3) Bajad SU, Lu W, Kimball EH, Yuan J, Peterson C, Rabinowitz JD. Separation and

quantitation of water soluble cellular metabolites by hydrophilic interaction

chromatography-tandem mass spectrometry. J Chromatogr A 2006 August 25;1125(1):76-

88.

(4) Camateros P, Tamaoka M, Hassan M, Marino R, Moisan J, Marion D, Guiot MC, Martin

JG, Radzioch D. Chronic asthma-induced airway remodeling is prevented by toll-like

receptor-7/8 ligand S28463. Am J Respir Crit Care Med 2007 June 15;175(12):1241-9.

(5) Lachance C, Wojewodka G, Skinner TA, Guilbault C, De Sanctis JB, Radzioch D.

Fenretinide Corrects the Imbalance between Omega-6 to Omega-3 Polyunsaturated Fatty

Acids and Inhibits Macrophage Inflammatory Mediators via the ERK Pathway. PLoS One

2013 September 12;8(9):e74875.



(6) R: A language and environment for statistical computing. R Foundation for Statistical

Computing. [computer program]. Vienna, Austria: 2013.

(7) Pawlowsky-Glahn V, Buccianti A. Compositional Data Analysis: Theory and

Applications. 1 ed. Sussex, United Kingdom: Wiley; 2011.



TABLES

Table E1: Forward and reverse primer sequences

Gene Forward Reverse

Ccl2 5’‐TCA CCT GCT GCT ACT CAT TCA CCA‐3’ 5’‐TAC AGC TTC TTT GGG ACA CCT GCT‐3’

Ccl5 5’‐TCG TGC CCA CGT CAA GGA GTA TTT‐3’ 5’‐TCT TCT CTG GGT TGG CAC ACA CTT‐3’

Ccl7 5’‐TCT GTG CTG AAG CCC ATC AGA AGT‐3’ 5’‐TTG CTT CTT GGC TCC TAG GTT GGT‐3’

Ccl11 5’‐TAT TCC TGC TGC TCA CGG TCA CTT‐3’ 5’‐GGC TTT CAG GGT GCA TCT GTT GTT‐3’

Cxcl1 5’‐TGC TAG TAG AAG GGT GTT GTG CGA‐3’ 5’‐TCC CAC ACA TGT CCT CAC CCT AAT‐3’

Cxcl2 5’‐CCT CAA CGG AAG AAC CAA AGA G‐3’ 5’‐AGG CAC ATC AGG TAC GAT CCA‐3’

Cxcl9 5’‐ATC TTC CTG GAG CAG TGT GGA GTT‐3’ 5’‐AGT CCG GAT CTA GGC AGG TTT GAT‐3’

Cxcl10 5’‐TGC CCT TGG TCT TCT GAA AGG TGA‐3’ 5’‐TCG CAC CTC CAC ATA GCT TAC AGT‐3’

Gapdh 5'‐ATG TGT CCG TCG TGG ATC TGA‐3' 5'‐TTG AAG TCG CAG GAG ACA ACC T‐3'

Il‐6 5’‐TCG GAG GCT TAA TTA CAC ATG TTC‐3’ 5’‐AAT CAG AAT TGC CAT TGC ACA A‐3’

iNOS 5’‐GGC AGC CTG TGA GAC CTT TG‐3’ 5’‐CAT TGG AAG TGA AGC GTT TCG‐3’

Pdgf 5’‐TGT GTC TTC TTC CTC ATG TGC CCT‐3’ 5’‐TCC CAT TAC AAC CTT GCT CAC CCT‐3’

Tnf‐α 5’‐AGA CCC TCA CAC TCA GAT CAT CTT C‐3’ 5’‐CCT CCA CTT GGT GGT TTG CT‐3’

Table E2: Mean values of lipids and markers of oxidative stress in non-allergic (PBS)

allergic (OVA), treated-non-allergic (FEN-PBS), and treated-allergic (FEN-OVA) mice.

PBS (n = 9) OVA (n = 12) FEN-PBS (n = 12) FEN-OVA (n = 12)

AA (nmol/mg of phospholipid)

32.56 ± 1.020 33.67 ± 0.8643 30.16 ± 0.6990 ** 29.53 ± 0.6700 **

DHA (nmol/mg of phospholipid)

4.267 ± 0.1130 *** 3.292 ± 0.09959 3.742 ± 0.04680 *** 3.783 ± 0.03860 ***

AA/DHA 7.680 ± 0.3223 *** 10.36 ± 0.4537 8.073 ± 0.2024 *** 7.826 ± 0.2346 ***

Nitrotyrosine (ng/mg of protein)

5.600 ± 0.2635 *** 9.575 ± 0.5849 6.425 ± 0.2652 *** 6.192 ± 0.1905 ***

Malondialdehyde (nmol TBARS/mg of protein)

9.622 ± 0.7448 *** 13.48 ± 0.6468 11.31 ± 0.6478 * 9.250 ± 0.2407 ***

Abbreviations:

OVA: ovalbumin, FEN: fenretinide, AA: arachidonic acid, DHA: docosahexaenoic acid



Data are presented as mean ± SEM. Statistical significance was calculated by the means of a

one-way ANOVA (*p < 0.05, **p < 0.01 and ***p<0.001 between group in respective column

and OVA group.



FIGURE LEGENDS

Figure E1: Structure of fenretinide molecule.

Figure E2: Principal component analysis (PCA) and discriminant function analysis (DFA) of

metabolomics data. Unsupervised PCA (A) and supervised DFA (B) of plasma samples used to

assay 89 metabolites by HPLC-MS. Ellipses in PCA represent 75% confidence intervals for

principle component distribution of the groups. Quality control samples are grouped in the

center of the PCA plot. DFA plot shows that allergic and non-allergic mice can be separated.

Figure E3: Heatmap of comparing levels of 89 metabolites in non-allergic (PBS), ovalbumin

challenged allergic (OVA), and fenretinide treated allergic (FEN-OVA) mice.

Figure E4: Fenretinide (FEN) prevents airway hyperresponsiveness (AHR) in allergic asthma.

Ovalbumin challenge (OVA group) causes a dose response increase in AHR compared to non-

allergic mice (PBS group). Treatment with FEN prior to OVA challenge (FEN-OVA group)

prevents AHR to methacholine in allergic mice. Fenretinide alone (FEN-PBS) does not cause

any significant changes in airway responsiveness compared to non-allergic mice. Data are

presented as mean ± SEM from two independent experiments and statistical significance was

calculated by one-way ANOVA. n = 8, 10, 8 and 10 for PBS, OVA, FEN-PBS and FEN-OVA,

respectively; *p < 0.05. At 80 and 160 mg/ml of methacholine, PBS, FEN-PBS and FEN-OVA

groups are significantly different from OVA group.

Figure E5: mRNA expression of small molecules in MLE-12 cells after stimulation with

lipopolysaccharide (LPS) with or without fenretinide (FEN) pre-treatment. Expressions in

stimulated (LPS) and FEN treated and stimulated (FEN-LPS) cells are normalized to untreated



and unstimulated cells (MEDIUM). Data are presented as median ± interquartile range from two

experiments done in triplicates (n=6) and statistical significance was calculated by the means of

a one-way ANOVA. *p < 0.05, **p < 0.01, and ***p<0.001.



FIGURES

Figure E1



Figure E2



PB

S

OVA

FEN

.OVA

C14

C16:2

C12

C10

C16OH

C3:OH

Creatinine

Serine

C12:1

C6:1

C18:1OH

Isobutyrylglycine+ButyrylGly

Taurine

C14:1

C5DC

Glycerol 3−phosphate

2−aminoadipate

3−phosphoglycerate

C10:1

C14:2

Proline

C8:1

C4DC+C5OH

2−hydroxyisovalerate

Cytosine

3−methylhistidine+N−methylhistidine

Tryptophan

Asparagine

Alanine+Sarcosine+Beta−Alanine

Lactate

Serotonin

Glycine

Ribitol+Arabitol

Citrate+Isocitrate

Porphobilinogen

Citrulline

5−oxoproline

Phenylserine

FGAr

Lysine

2−methylbutyrylGly+IsovalerylGly

C8

Indoxyl sulfate

Homocysteine

C6

Succinate+Methylmalonate

Pseudouridine

Tetrahydrobiopterin

Fucose

Valine

Choline

Palmitic acid

Glutamine

C18:1

N−methylhistidine+3−methylhistidine

C16

C18:3

Guanidinoacetate

4−guanidinobutanoate

Kynurenine

C20:1

C2

Galactitol

Oxalate

Malate

C16:1

C3

C5

C18:2

C20:2

C18

Phenylalanine

C20

C12:DC

Hexoses

Tyrosine

N.N−dimethylglycine+2−aminoisobutyric acid

Threonine+Homoserine

Betaine

C3DC+C4OH

C4

Mannitol

Histidine

Arginine

Glutamate

C22

Cystine

Carnitine

Methionine

−1 0.5Row Z−Score

Color Key

Figure E3



Figure E4



Figure E5



Documents

Fenretinide Prevents Inflammation and Airway Hyperresponsiveness in a Mouse Model of Allergic Asthma