Background
Epidemiological studies have reported an inverse association between asthma and the intake of vitamin A. Dietary vitamin-A intake and serum vitamin-A concentrations are significantly lower in patients with asthma than in healthy control subjects or in people with severe asthma than in those with mild asthma [
1‐
3]. All-trans retinoic acid (ATRA) is a biologically active metabolite of vitamin A with profound effects on T-cell activation [
4,
5], differentiation [
6], and function [
7,
8]. ATRA binds to retinoic acid receptors in the nucleus leading to the activation of transcription of many target genes [
9‐
11]. Emerging evidence demonstrates that ATRA signaling is critical for T cells differentiation and function. ATRA is an early mediator in the development of CD4
+ T cell-mediated immunity, and also plays a pivotal role in optimal effector and effector memory CD8
+ T cell differentiation in which vitamin A supplementation is used to augment effector responses [
12,
13]. Meanwhile, ATRA promotes Foxp3
+ regulatory T-cell (Treg) differentiation and sustains the stability and function of natural Tregs in an inflammatory milieu. In addition, it suppresses Th17 differentiation. ATRA and rapamycin can synergistically increase TGF-β-mediated Foxp3 induction in antigen-specific Th2 memory cells when effector cytokines are neutralized. These Foxp3
+ T cells converted from Th2 memory cells possess Treg activity and can ameliorate Th2 memory-mediated airway hyperreactivity and eosinophilic inflammation [
14‐
20]. Moreover, ATRA suppresses Th2-related chemokine expression in vitro by down-regulating the expression of interleukin (IL)-5 receptor and inhibiting eosinophil and basophil differentiation [
21‐
23]. In addition to modulating T cells, ATRA plays an important role in the maintenance of the normal epithelial mucociliary phenotype [
21]. However, the role of ATRA on airway inflammation after allergenic challenge has not been established. The goal of this study is to determine whether ATRA alters Th2 response to modulate the severity of airway inflammation in an ovalbumin (OVA)-induced allergic airway inflammation animal model.
Discussion
Previous reports showed that ATRA is the biological active metabolite of vitamin A and has an important immunomodulatory effect by inhibiting Th17 polarization and enhancing Foxp3 expression [
14‐
16]. By using a murine Th2-mediated airway inflammation model, we demonstrated that the administration of ATRA attenuated OVA-induced airway inflammation by decreasing Th2 and Th17 related cytokines and inflammatory cells in the airway and ATRA mediated reduction of Th2 cytokines was via inhibiting GATA-3 expression. Our findings provide further supports for the anti-inflammatory role of ATRA in the treatment of lung diseases.
OVA-induced allergic asthma is recognized as a pathological condition that results from chronic airway inflammation characteristically associated with the infiltration of lymphocytes, eosinophils, macrophages, and neutrophils into the bronchial lumen. An aberrant Th2-type response to allergens is characterized by the overproduction of IL-4, IL-5, and IL-13, which are critical for the maintenance of ongoing IgE-mediated eosinophilic inflammation [
24‐
26]. Accumulating evidence has suggested that aberrant IL-17 production is a key determinant of severe forms of asthma. IL-17A induces lung-structural cells to secrete proinflammatory cytokines (TNF, IL-1β, granulocyte colony-stimulating factor, and IL-6) and chemokines (CXCL1, CXCL2, and CXCL8/IL-8), thereby triggering neutrophil infiltration [
27‐
29].
We showed that the administration of ATRA inhibited OVA mediated secretion of IL-4 and IL-5 in the lung, draining lymph nodes, and the spleen. Consistent with the decreased levels of Th2-related cytokines, the recruitment of inflammatory cells, especially eosinophils, neutrophils, lymphocytes, and macrophages, was markedly decreased in the BALF and the lung after ATRA administration. These results indicated that ATRA may alleviate airway inflammation by reducing Th2 cytokines. Meanwhile, T-bet and GATA-3 are responsible for the regulation of the cytokine genes during Th1/Th2 differentiation. GATA-3 has been shown to promote the expression of several Th2 cytokines, including IL-4, IL-5, and IL-13 [
30,
31]. It is well known that overexpression of GATA-3 predisposes for Th2-mediated diseases such as allergic asthma and suppression of GATA-3 expression in the lung reduces IL-4, IL-5, and IL-13 productions concurrently [
30,
31]. Compared with the vehicle group, the GATA-3 mRNA level in the lung in the ATRA treated group was significantly decreased, which may partially explain the reduction of IL-4 and IL-5 production in these mice. In addition, a recent study performed in chronic asthma model showed that prolonged ATRA treatment tends to inhibit Th17 cell infiltration and neutrophilia without obvious alteration of Th2 cell numbers [
32]. The different observations are probably due to different Th-dominate asthma models used between the two studies, in which the immune responses are mediated by different Th subsets.
The concentration of retinoic acid is likely to be critical to its effect on the airway responses. Previous studies have demonstrated that ATRA reverses the airway hyperresponsiveness (AHR) and exogenous administration of retinoic acid is capable of attenuating the asthma phenotype [
32‐
34]. However, it has also been reported that ATRA promotes Th2 development to exacerbate allergic immune and inflammatory responses during systemic sensitization [
35]. The differences in the responses of the airway are probably linked to the different concentrations and time point used in those studies. Indeed, the study performed by Mateu et al. supports the above notion. They have found that retinoic acid directly enhances allergic responses in vivo, but higher doses effectively decrease AHR by inhibiting IL-5 production [
36]. Therefore, the concentration and time point of retinoic acid should be carefully considered in the applications.
Of note, ATRA was not able to affect Th2 differentiation in a Th2 skewing condition in vitro. The discrepancy between the effects of ATRA on in-vitro and in-vivo Th2 responses suggested that ATRA might not intrinsically control Th2 differentiation. Rather, it might do so by inhibiting the Th17 response or by modulating the function of antigen presenting cells. In addition, Bidad K et al. have reported that ATRA can significantly decrease Th17 cells in patients with ankylosing spondylitis. The effect of ATRA in such patients serves as an immunomodulator on deviated immune cells, which is associated with decreased inflammatory cytokine TNF-α secretion [
36]. In our study, we also found that the level of IL-17A was slightly decreased in the draining lymph nodes and lung, while IFN-γ and T-bet mRNAs were slightly increased in the lung after the administration of ATRA. Unlike the in-vitro results previously reported by Elias et al. [
15], our in vivo data showed that ATRA moderately increased IL-10 expression without affecting IL-10 production, Foxp3 expression and Treg numbers in the lung or spleen. The different responses of Treg cells might be attributed to the difference between the in-vivo environment and in-vitro condition [
32]. Additionally, ATRA effects on Treg cells in vivo might be also influenced by the exposure time of ATRA. For example, Zhao et al. found that the proportion of Foxp3
+ CD4 Treg cells in the lymph nodes is temporarily increased after a week of ATRA treatment [
32]. The data suggest that ATRA may affect Treg cells in a context- and time-dependent manner.
Methods
Animals
A total of 90 female BALB/c mice and 3 DO11.10 mice at 6 ~ 8 weeks of age were purchased from the Shanghai SLAC Laboratory Animal Company. All mice were maintained under specific pathogen-free conditions in our animal facility. BALB/c mice were randomly divided into 3 groups: a control group, OVA plus vehicle (vehicle group), and OVA-plus ATRA (ATRA group). Each group included 10 mice, and 3 independent experiments were performed. Animal experiments were performed according to the Ethics Committee of Ruijin Hospital, Shanghai Jiaotong University School of Medicine.
Ag sensitization and challenge protocol and administration of ATRA
The mouse-asthmatic model was established as described previously [
37,
38]. In both vehicle and ATRA groups, mice received i.p. injections of 100 μg OVA (Sigma-Aldrich) in 0.2 ml Al(OH)
3 adjuvant suspension on days 0 and 14. On days 14, 25, 26, and 27, mice were anesthethized with isoflurane and intranasally received 100 μg of OVA in 0.05 ml phosphate buffer saline (PBS) (days 14) and 50 μg of OVA in 0.05 ml PBS (days 25, 26, and 27). The control group was sensitized with the same volume of Al(OH)
3 and challenged with normal saline instead of OVA. ATRA was dissolved in dimethyl sulfoxide (DMSO) and diluted in corn oil. Mice in the ATRA group received i.p. injections of 400 μg ATRA on days –1, 13, 24, and 26, the latter being one day prior to OVA sensitization. The mice in the vehicle group were injected with the same volume of corn oil.
Bronchoalveolar lavage fluid (BALF)
Twenty-four hours after the final challenge (day 28), BALB/c mice were sacrificed by CO
2. BALF was obtained by the slow injection of 0.4 ml ice-cold PBS into the trachea using a 22-inch i.v. with cathetering three times (total 1.2 ml). This procedure recovered 80 to 90% of the infused fluid. The total number of cells in BALF was counted using a hemacytometer. After cytospin, the cells were fixed and stained by hematoxylin-eosin (H&E). A total of 200 cells were randomly selected to calculate eosinophils, neutrophils, lymphocytes and macrophages under the microscope (Olympus AX70, Japan). Different cell counts were calculated by the following equation:
Histology
The lung lobe was fixed in 10% formalin, embedded in paraffin, and sectioned in 4 μm slices. Sections were stained with H&E and examined microscopically.
Detection of soluble cytokine levels in lung
To determine soluble cytokine levels in the lungs, a snap-frozen right lung lobe from each mouse was thawed on ice and homogenized in a buffer containing a protein-protease inhibitor and 0.1% Triton X-100. The samples were centrifuged, and 50 μl of cell-free supernatant was analyzed by ELISA according to the manufacturer’s instructions. Cytokine levels including IL-4, IL-5, interferon (IFN)-γ, IL-10 and IL-17 in the culture supernatants of mediastinal lymph nodes, splenocytes and lung homogenates were quantified. All the antibodies for the assay were purchased from Biolegend.
Cells stimulation and cytokines assay in vitro
Lymphocytes and splenocytes were used to determine the immune-regulatory effects of ATRA. On day 28, the mice were sacrificed and mediastinal lymph nodes and spleens were isolated. The cell clumps were disaggregated into single-cell suspensions using nylon mesh (30 μm) filtration. Red blood cells were removed by a red-blood-cell lysis buffer. The isolated lymphocytes and splenocytes were cultured in a 0.5 ml RPMI 1640 medium (Sigma-Aldrich) supplemented with 2 mmol/L L-glutamine, 10% fetal calf serum (FCS), 100 mg/ml streptomycin, 100 IU/ml penicillin, 10 mm HEPES, and 20 mm sodium hydrogen carbonate (Invitrogen Life Technologies, Carlsbad, California, USA). Lymphocytes and splenocytes were cultured at the density of 2 × 106/ml in 48 well plates under the stimulation with 10 μg/ml OVA323–339 peptide for 72 hours. The cells were harvested and intracellular staining for IFN-γ, IL-4, and IL-17A were performed for flow cytometry. The cell supernatants were collected and analyzed for IFN-γ, IL-4, IL-5, IL-17A, and IL-10 using mouse ELISA kits (Biolegend).
Real-time PCR analysis
For the total RNA isolation, lung tissue was removed from the animal, immediately froze in liquid N2, and stored at –70°C for assaying. The frozen lung tissue was then homogenized in TRIzol reagent (Invitrogen Life Technologies, Carlsbad, California, USA), and the total RNA was isolated according to the manufacturer’s instructions. Reverse transcription was performed using 40 ng of the total RNA with Superscript III RT kit (Qiagen) and oligo(dT) primers (Invitrogen Life Technologies, Carlsbad, California, USA) as recommended by the manufacturer. The Bio-Rad quantitative PCR SYBR Green Master Mix (Bio-Rad Laboratories) was used and performed the following program: 95°C for 5 min and 40 cycles of amplification at 95°C for 15 seconds and 58°C for 60 seconds. Relative levels of target mRNA were compared with β-actin using the 2−ΔΔCt method. Primer sequences were obtained from Integrated DNA Technologies. Sequences were as follows: β-actin Forward: 5′-GGC TGT ATT CCC CTC CAT CG-3′, Reverse: 5′-CCA GTT GGT AAC AAT GCC ATG T-3′; IL-4 Forward: 5′-GGT CTC AAC CCC CAG CTA GT-3′, Reverse: 5′-GCC GAT GAT CTC TCT CAA GTG AT-3′; IL-10 Forward: 5′-GCT CTT ACT GAC TGG CAT GAG-3′, Reverse: 5′-CGC AGC TCT AGG AGC ATG TG-3′; IL-17A Forward: 5′-TTT AAC TCC CTT GGC GCA AAA-3′, Reverse: 5′-CTT TCC CTC CGC ATT GAC AC-3′; IFN-γ Forward: 5′-ATG AAC GCT ACA CAC TGC ATC-3′, Reverse: 5′-CCA TCC TTT TGC CAG TTC CTC-3′; Foxp3 Forward: 5′-CAC AAT ATG CGA CCC CCT TTC-3′, Reverse: 5′-AAC ATG CGA GTA AAC CAA TGG TA-3′.
In-vitro Th2 differentiation assays
Naïve CD4+ T cells were isolated from 3 DO11.10 mice, and a single-cell suspension was prepared by grinding spleen against a 70-μm nylon cell strainer. After lysis of red cells by red-blood-cell lysis buffer, naïve CD4+ T cells (CD4+ CD62Lhigh CD25-) were first purified by a Mouse CD4+ T Cell Isolation Kit (Miltenyi Biotech) and followed by FACS-sorting. Cells were seeded in 48-well plates for further experiments. The seeding density was 2 × 105 naïve T cells/well.
Naïve T cells were maintained in RPMI 1640 (Invitrogen Life Technologies, Carlsbad, CA) supplemented with 2 mmol/L L-glutamine, 10% fetal-calf serum (FCS), 100 mg/ml streptomycin, 100 IU/ml penicillin, 10 mm HEPES, and 20 mm sodium hydrogen carbonate (Invitrogen Life Technologies, Carlsbad, California, USA). The cells were activated with plate-bound anti-CD3 (2 μg/ml), and soluble anti-CD28 (2 μg/ml) antibodies (BD PharMingen). Th-neutral conditions (Th0) contained no exogenous cytokines or anti-cytokines. Th2 conditions contained 10 ng/ml IL-4 (R&D Systems) with 10 μg/ml anti-IFN-γ (BD PharMingen). Where indicated, IL-2 was added at 100 IU/ml. All ATRA (Sigma, St Louis, MO) were dissolved in dimethyl sulfoxide (DMSO) at stock concentrations of 0.01 M and stored at −80°C in light-proof containers. Stocks were thrown away after 4 freeze-thaw cycles. Cultures containing ATRA were protected from light throughout the time of culture; unless stated otherwise, ATRA was used at different concentration from 0.01, 0.05, 0.1, 0.5, 1, 2.5, 5 to 10 μmol/L.
Statistics
Data are presented as mean ± SEM. The differences between mean values were calculated using student’s t test, and a p value of less than 0.05 was considered significant. All experiments were repeated at least 3 times, and n = 10 in each experimental group.
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
JW performed experimental studies, carried out the immunoassays and drafted the manuscript. YZ participated experimental studies and analyzed data. QL and WZ performed the statistical analysis and helped to draft the manuscript. ZX conceived of the study, designed experiments, analyzed data, and helped to draft the manuscript. All authors have read and approved the final manuscript.