Metformin alleviates allergic airway inflammation and increases Treg cells in obese asthma

Abstract Obesity increases the morbidity and severity of asthma, with poor sensitivity to corticosteroid treatment. Metformin has potential effects on improving asthma airway inflammation. Regulatory T cells (Tregs) play a key role in suppressing the immunoreaction to allergens. We built an obese asthmatic mouse model by administering a high‐fat diet (HFD) and ovalbumin (OVA) sensitization, with daily metformin treatment. We measured the body weight and airway inflammatory status by histological analysis, qRT‐PCR, and ELISA. The percentage of Tregs was measured by flow cytometry. Obese asthmatic mice displayed more severe airway inflammation and more significant changes in inflammatory cytokines. Metformin reversed the obese situation and alleviated the airway inflammation and remodelling with increased Tregs and related transcript factors. The anti‐inflammatory function of metformin may be mediated by increasing Tregs.


Abstract
Obesity increases the morbidity and severity of asthma, with poor sensitivity to corticosteroid treatment. Metformin has potential effects on improving asthma airway inflammation. Regulatory T cells (Tregs) play a key role in suppressing the immunoreaction to allergens. We built an obese asthmatic mouse model by administering a high-fat diet (HFD) and ovalbumin (OVA) sensitization, with daily metformin treatment. We measured the body weight and airway inflammatory status by histological analysis, qRT-PCR, and ELISA. The percentage of Tregs was measured by flow cytometry. Obese asthmatic mice displayed more severe airway inflammation and more significant changes in inflammatory cytokines. Metformin reversed the obese situation and alleviated the airway inflammation and remodelling with increased Tregs and related transcript factors. The anti-inflammatory function of metformin may be mediated by increasing Tregs.

K E Y W O R D S
Metformin, Obese asthma, Tregs

| INTRO DUC TI ON
Asthma is one of the most frequent chronic respiratory diseases, which affects almost 25 million people around the world 1 ; Obesity is a significant health problem and the prevalence of obesity was 42.4% in 2017. 2 Obese asthma increases morbidity with worsening symptoms, with high frequencies and severe exacerbations and is insensitive to medical treatment. 3 Therefore, an appropriate drug to treat obese asthma is urgently needed.
Metformin is regarded as a safe and curative medicine for reducing inflammation in several diseases, including asthma. 4 A retrospective cohort study of the Taiwan National Health Insurance Research Database lasting 11 years indicated that metformin reduced the risk of asthma and diabetes mellitus (DM) compared to that in untreated patients. 4 Metformin may be a potential therapeutic agent for treating obese asthma.
Tregs are important immune-regulatory cells in the maintenance of immune tolerance. Tregs inhibit the activation and proliferation of effector T (Teff) cells by regulating and modulating the immune system which plays a key role in maintaining the immune tolerance of allergens. STAT5 regulates the Treg transcription factor Foxp3 and modulates the Tregs development. Tregs also activate mast cells, eosinophils, and basophils to suppress allergic inflammation and airway remodelling by activating IL-6 or OX40/OX40L. 5 Metformin-elevated Tregs have been shown to alleviate inflammation and enhance the immunomodulating potential in autoimmune diseases. The therapeutic efficacy of metformin for treating obese asthma is scarce. Calixto found that metformin reduced airway eosinophilic inflammation in obese asthma. 6 However, the changes in Tregs, a vital immune-regulation call, have not been elucidated.
In the present study, we proved that metformin increased Tregs significantly and alleviated airway inflammation in obese asthma, which is more severe than non-obese asthma.

| MATERIAL S AND ME THODS
See Supplementary File.

| Metformin treatment reduced body weight and alleviated the airway inflammation in obese asthma
Mice fed with a high-fat diet for 30 weeks exhibited a significant increase in their body weight, while the blood glucose showed non-

| D ISCUSS I ON
Obesity increases the severity and risk of asthma. Obese asthmatic patients are generally found to be unresponsive to ICS treatment, which greatly increases treatment difficulties. 3 Metformin may be a potential treatment option because it not only reduces the weight and blood glucose, but also provides an anti-inflammatory function in several diseases, including asthma. 4 In the present study, we found that metformin alleviated the airway inflammation and remodelling with increased Tregs and its related transcript factors.
The anti-airway inflammation role of metformin may be mediated by increased Tregs.
Histological analyses demonstrated that obese asthma showed more severe airway inflammation, while airway remodelling was In summary, we found that (a) obese asthmatic mice showed more severe airway inflammation; (b) metformin alleviated airway inflammation and increased Tregs in obese asthma. Metformin may be a therapeutic treatment for obese asthma and Tregs may be a therapeutic target.

ACK N OWLED G EM ENT
We would like to thank Dr. Tailin Li (Sun Yat-sen University Second University Hospital) for providing us the CD1 mice.

CO N FLI C T O F I NTE R E S T
The authors claim they have no conflicts of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are openly available

O RCI D
Qiujie Wang https://orcid.org/0000-0003-4942-8166 F I G U R E 2 Effect of metformin on Treg cells and Treg related transcript factor Foxp3 and STAT5. (A) Flow cytometry analysis of Tregs (CD4 + Foxp3 + ) from the spleen of mice from different groups. (B) Quantitative calculation of Tregs from the total CD4 + cells in each group. (C) ELISA analysis of Treg related cytokine Foxp3 in BALF and plasma, from different groups of mice. (D) RT-qPCR analysis of Treg related factors Foxp3 and STAT5 in BALF cells and spleen, from different groups of mice. All values are represented in the form of M ± SD (n = 5 ~ 8/group, P < 0.05 was statistically significant. * P < 0.05, ** P < 0.01, and *** P < 0.001 were compared with the indicated groups)