Abstract
Overweight/obesity has been suggested as a risk factor for asthma development, and prospective studies have confirmed that high body weight precedes asthma symptoms. However, the nature of the association between overweight/obese status and asthma remains unclear. Animal models of obesity-related asthma are very useful for understanding disease pathophysiology. Although C57/B6J mice are the most widely used animal model for researching obesity-related asthma, gender differences are not always taken into consideration. Therefore, to explore the effect of gender on the development of obesity-related asthma, both female and male C57/B6J mice were used in this study. The mice were fed with a high-fat diet or a low-fat diet as control. Body weight, body length, liver weight, and Lee’s Index were used to evaluate obesity status, and lung histology, lung inflammatory cells infiltration, and inflammatory cytokines in bronchoalveolar lavage fluid (BALF) were examined for asthma evaluation. We found that the mean body weight of male mice on a high-fat diet gradually increased and was significantly higher than control male mice on a low-fat diet (P < 0.01), while no significant differences were found between female mice at the end of 12 weeks of feeding. Furthermore, the obese asthma group female and male mice exhibited significantly high inflammatory cells infiltration than normal weight or obese female and male mice (P < 0.01). However, the obese asthma group presented higher Neu infiltration, Th1 cytokine, and interferon gamma (IFNγ) concentrations in BALF than the asthma group in both the genders (P < 0.01). In conclusion, both female and male mice are suitable for the obesity-related asthma model, although male mice might be more stable. Besides, obesity-related asthma is not Th2 type asthma.
Introduction
Obesity has been steadily increasing in developed and developing countries, 1 and is associated with several related diseases including gastroesophageal reflux disease, sleep apnea, and asthma. The prevalence of overweight or obesity in the 2–19–years’ age group increased from 6% in 1980 to 16% in 2008. In 2010, 43 million children worldwide, with 35 million in developing countries alone, were estimated to be overweight or obese, and this number is expected to double by 2020. 2 Meanwhile, an estimated 300 million people are affected by asthma worldwide, with children constituting almost half of the cases. 3
Several studies have shown a significant association between obesity and asthma in children and adults. 4 Prospective studies support that a high body weight precedes asthma. 5 However, the underlying mechanisms remain unclear. There are very few cross-sectional or prospective studies on the potential pathogenesis underlying obesity-related asthma. Animal models are excellent to study the link between obesity and asthma. Although C57/B6J mice were used in nearly all the animal studies on obesity-related asthma, the gender effect was not considered. 6 Thus, this study explored the effect of gender on airway inflammation in obesity-related asthma mice model.
Materials and methods
Mice
A total of 80, 3- to 4-week-old, specific pathogen-free C57/B6J mice (Shanghai Slac Laboratory Animal Center, Shanghai, China), including 40 males and 40 females, were used in this study. Female and male mice were randomly divided into four groups (normal weight, obesity, asthma, and obese asthma), respectively. All the animal procedures were approved by the Institutional Animal Care and Ethics Committee, and were consistent with the standards established by the Guide for the Care and Use of Laboratory Animals. Briefly, mice were maintained on a 12:12 h light cycle within individual static isolation cages with bedding that were autoclaved prior to use. Mice received autoclaved, acidified water from autoclaved water bottles. The high fat or ordinary diet was purchased from Medicine Ltd (Yangzhou, China). Cotton squares were provided in all cages for enrichment. Mice were housed in same-gender groups of ≤5 per cage. Body weight, liver weight, body length, and Lee’s Index were used to evaluate the obesity status. Body length was detected from apex nasi to anus. Lee’s Index was calculated as the cubic root of body weight multiplied by 1000 and divided by body length.
Obesity-related asthma mice model
The obesity and obese asthma groups were fed with 45% high-fat food, while the normal weight and asthma groups were fed with regular mice food. After 8 weeks of feeding, on days 1 and 13, the asthma and obese asthma groups were sensitized by intraperitoneal (i.p.) injection of 0.01% ovalbumin (OVA)/Al(OH)3 suspension. Then, they were challenged in an enclosed box filled with 1% OVA aerosol for 30 min from days 25 to 32. Normal weight and obesity groups were sensitized and challenged by normal saline on the same schedule.
Bronchoalveolar lavage fluid collection and detection
All mice were anesthetized by chloral hydrate and sacrificed within 24 h after the last challenge. After sacrifice, one side of the bronchus was ligated and bronchoalveolar lavage fluid (BALF) was collected by flushing one side of the lungs with three separate normal saline flushes through the trachea. The rate of recovery was >80%. Different cell counts were determined for each BALF sample using a cytometer. The cytokines were detected by enzyme-linked immunosorbent assay (ELISA).
Isolation and staining of lung tissues
The non-lavaged portions of the lungs were stored in an ultralow temperature freezer or fixed in 10% formalin and cut into 4-μm-thick paraffin-embedded tissue sections, which were stained with hematoxylin and eosin (HE). The degrees of allergic airway inflammation were scored by a person who was blinded to the treatment, and were assessed according to the following histological grading system (scored 0–4): absence of peribronchial inflammatory cells (0), a few scattered peribronchial inflammatory cells involving <25% of the circumference of the bronchus (1), focal peribronchial inflammatory cell infiltrate not completely surrounding the bronchus (2; that is, involving approximately 25%–75% of the circumference of the bronchus), one definite layer of peribronchial inflammatory cells completely surrounding the bronchus (3), and two or more layers of peribronchial inflammatory cells completely surrounding the bronchus (4).
Statistical analysis
All the results were expressed as mean ± SEM and analyzed by one-way analysis of variance (ANOVA), with SPSS 17.0. For samples with unequal variances, Dunnett’s T3 test was used. Statistical significance was determined as P < 0.05.
Results
Physical appearance of mice
The hair of both female and male mice in the obesity and obese asthma groups were glossier than the normal weight and asthma groups. No obvious differences in appearance were found between the four groups among females, while noticeable obesity was seen in the obesity and obese asthma groups among males. During the process of OVA challenge, the asthma and obese asthma groups presented symptoms such as restlessness, polypnea, and abdominal muscle twitch.
Weight gain
The baseline mean body weight of male and female mice from each group showed no significant differences. Compared to the normal weight and asthma groups, the mean body weight of female mice in the obesity and obese asthma groups increased rapidly during the first 4 weeks of feeding (P < 0.01) and increased slowly thereafter, with no significant differences between the two groups after 12 weeks of feeding (P > 0.05). In contrast, the mean body weight of male mice in the obesity and obese asthma groups increased gradually, and was significantly higher than the male mice in the normal weight and asthma groups at the end of 12 weeks of feeding (P < 0.01). The mean body weight of male mice in the obesity and obese asthma groups was 20% higher than that of the normal weight and asthma groups. There were significant differences in the mean body weight of male mice in all the four groups, compared to the female mice (P < 0.01) (Table 1).
Weight status of mice in each group (g, n = 10).
P < 0.05 compared with normal weight group of the same gender.
P < 0.05 compared with obesity group of the same gender.
P < 0.05 compared with asthma group of the same gender.
P < 0.05 compared with the same group of female mice.
Body length, liver weight, and Lee’s Index of mice
The body length of female mice in the obesity group was significantly higher than that of the normal weight and asthma groups (P < 0.05), but no significant differences were found among the obese asthma, normal weight, and asthma groups (P > 0.05). Moreover, there were no significant differences in the liver weight and Lee’s Index of female mice among the four groups (P > 0.05).
Significant differences were observed in the body length, liver weight, and Lee’s Index of male mice between the obesity and obese asthma groups, and the normal weight and asthma groups (P < 0.01). The differences in body length, liver weight and Lee’s Index between the normal weight and asthma groups, and the obesity and obese asthma groups were not significant (P > 0.05). The body lengths of female mice in the four groups were not significantly different (P > 0.05), while the Lee’s Indexes were all higher than female mice (P < 0.01). The liver weights of female mice in the obesity and obese asthma groups were significantly lower than in male mice in the same groups (P < 0.01; Table 2).
Status of body length, liver weight, and Lee’s Index of mice in each group (n = 10).
P < 0.05 compared with normal weight group of the same gender.
P < 0.05 compared with obesity group of the same gender.
P < 0.05 compared with asthma group of the same gender.
P < 0.05 compared with the same group of female mice.
Lung histology and airway inflammatory score
As shown in Figure 1, both female and male mice in the normal weight group presented a complete lung structure, regular bronchial lumen, and intact mucous epithelium, with little or no inflammatory cells infiltration. The inflammatory cells infiltration of both female and male mice in the obesity group were significantly higher than in the normal weight group (P < 0.01), but lower than in the asthma and obese asthma groups (P < 0.01). The median inflammatory scores of female and male mice in the obesity group were 1.5 and 2, respectively. Both female and male mice in the asthma and obese asthma groups showed significantly higher inflammatory cells infiltration compared to the normal weight and obesity groups (P < 0.01), while no significant differences were found in female and male mice between the asthma and obese asthma groups, respectively (Figure 1).
Increased lung inflammatory cells infiltration in the asthma and obese asthma groups of both female and male mice. (a) Representative photomicrographs of HE staining in sections of lungs from mice of each group (×100). Calibration bars = 100 μm. (b) Lung inflammatory cells infiltration score of mice.
Cell counts in BALF
The total cell counts in BALF of both female and male mice in the obesity groups were significantly increased compared to both the normal weight groups (P < 0.01), but were lower than the asthma and obese asthma groups (P < 0.01). When comparing male mice with female mice, no significant differences were found between the same groups other than the obesity groups (Figure 2(a)).
Comparison of inflammatory cell counts and cytokines in BALF of mice in each group (×104/mL, n = 10). (a) Total cell counts in BALF. (b–d) Differential cell counts in BALF. (e–g) Concentrations of IL-5, IL-13, and IFNγ in BALF.
Similar changes in differential cell counts in BALF were found in female and male mice. The lymphocytes (Lym) were significantly increased in the asthma and obese asthma groups (P < 0.01). The asthma group showed the highest eosinophil (Eos) counts, while the obesity group showed the highest neutrophil (Neu) counts (P < 0.01). Except between female and male mice of the obesity groups, there were no significant differences between female and male mice in the other groups. The Neu counts in the obese asthma group were significantly higher than in the asthma group in both female and male mice (Figure 2(b)–(d)).
Concentrations of IL-5, IL-13, and interferon gamma in BALF
Changes in concentrations of Th2 cytokines, interleukin (IL)-5, and IL-13 in BALF were similar to the changes in Eos counts in BALF, while Th1 cytokine and interferon gamma (IFNγ) were similar to Neu counts. The concentrations of IL-5 and IL-13 in the asthma group were the highest, while the concentration of IFNγ in the obesity group was the highest compared to the other groups (P < 0.01). Except for the obesity groups, no significant differences in IL-5 and IL-13 were found between female and male mice in the same groups (P > 0.05). There were no significant differences in IFNγ between female and male mice in all the groups (P > 0.05; Figure 2(e)–(g)).
Discussion
Animal models can provide valuable information on several features of disease pathogenesis and treatment. Although they do not represent all clinical characteristics, proper use of animal models is valuable to understand the mechanisms of the disease and therapeutics. Several epidemiological surveys have demonstrated that asthma is more prevalent in obese children and adults. However, the mechanism of the association between obesity and asthma has not been fully clarified. Animal models of obesity-related asthma would be an ideal choice for exploring the potential mechanism. There is a wide range of asthma and obesity models in different animals.7,8 BALB/c mice, C57BL/6 mice, guinea pigs, or Sprague-Dawley rats are always used to induce asthma models,7,9 while C57BL/6 mice and rats are generally used for diet-induced obesity or morbid obesity, such as ob/ob, db/db mice. 10 Given the relatively clear genetic background and the abundant molecular reagents and antibodies for mice, C57BL/6 mice are the most frequently used animal model to study obesity-related asthma.
Gender differences have been described to play a role in various diseases. Matsubara et al. 11 found that endogenous estrogen might regulate the neurokinin 1-dependent prejunctional activation of airway smooth muscle in allergen-exposed mice, suggesting a role of gender differences in the asthma airway responsiveness. Although controversial, gender effect on the onset of obesity-related asthma has been reported.12,13 However, none of these studies mentioned or analyzed gender-specific differences. 14 Therefore, male and female C57BL/6 mice were used in this study to identify any gender-specific differences in obesity-related asthma.
First, a high-fat diet was fed to induce weight gain. The body weight of female mice increased significantly during the first 4 weeks and then grew slowly afterward. After 12 weeks of feeding, no significant differences were found between high-fat diet and low-fat diet groups of female mice. The body weight of male mice grew gradually and there were significant differences between high-fat diet and low-fat diet groups of male mice at the end of 12 weeks of feeding. Besides, there were significant differences in the mean body weight of all the four groups of male mice compared to the same groups of female mice. Furthermore, the body length, liver weight, and Lee’s Index were significantly higher in high-fat diet male mice compared to the low-fat diet male mice, while no significant differences in the body length, liver weight, and Lee’s Index were found among the female groups. These results indicated that male mice were more suitable as an obesity animal model, which was consistent with previous studies that used male C57BL/6 mice. 15
Next, to study the effect of gender on obesity-related asthma, mice were sensitized and challenged by OVA. The female mice in the obesity + asthma group showed significantly higher inflammatory cells infiltration than those in the normal weight and obesity groups, and were comparable with the asthma group. Moreover, the inflammatory cell numbers and inflammatory cytokines in BALF in female mice of the obesity + asthma group were significantly increased compared to the normal weight and obesity groups. The male mice showed the same trend as the female mice, suggesting that both female and male mice on a high-fat diet stimulated by OVA exhibited airway inflammation. However, the degree of airway inflammation in male mice was significantly higher than in female mice in the obesity group, which confirmed that male mice were more suitable for the obesity animal model.
Furthermore, the obesity + asthma group presented higher Neu infiltration, Th1 cytokine, and IFNγ concentrations in BALF than that of the asthma group in both the genders, indicating that obesity-related asthma was non-Th2 type asthma, which needs to be further investigated.
Footnotes
Declaration of conflicting interests
The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.
Funding
This work was supported by the Zhejiang Medical Technology & Education project (2016KYB198) and the Wenzhou Science & Technology project (Y20160019).
