Increases in peripheral SIRT1: A new biological characteristic of asthma

Study design and clinical subjects

Ninety-seven patients with asthma, who were referred to the Affiliated Hospital of Guangdong Medical College (Zhanjiang, China) from January to December 2013, were included in our study. The diagnosis of asthma was based on the Global Initiative for Asthma guidelines.14 The exclusion criteria included the following: oral corticosteroid use or the occurrence of a respiratory tract infection within 4 weeks of enrolment in the study, as well as any chronic cardiopulmonary disease other than asthma (including chronic obstructive pulmonary disease (COPD)), or pregnancy. A total of 161 healthy volunteers were recruited; 118 age-matched and sex-matched subjects among them were included in the study's control group (Table 1).

This study was approved by the ethics committee of our institution (Affiliated Hospital of Guangdong Medical College, Zhanjiang, China), and written informed consent was provided by each of the participants.

Pulmonary function tests

Spirometry was measured using standard spirometric techniques (POS Instrumentation, Inc., Louisville, USA) at least 6 h after each patient's most recent treatment with albuterol, according to the American Thoracic Society guidelines.14

Measurement of serum SIRT1 and total IgE levels

Human: Venous blood samples were obtained, and serum samples were subsequently prepared. The serum SIRT1 concentrations were measured using a SIRT1 enzyme-linked immunosorbent assay (ELISA) kit (USCN Life Science Inc. Wuhan, China) specific for SIRT1 of human or mouse according to the manufacturer's instructions. The minimum detection limit of the SIRT1 assay was 0.055 ng/mL. The total serum IgE levels and the percentages of peripheral blood eosinophils were each determined using a routine blood test in each patient and control subject.

Mice: 0.5 mL blood was collected from the abdomen of each mouse, and serum samples were prepared. Serum SIRT1 was measured using an ELISA kit as described above.

Mice sensitization and the airway challenge

All animal experimental processed in this study were approved by the Institutional Animal Care and Use Committee of the Guangdong Medical School. Twelve 8-week-old female BALB/c mice (Vital River, Peking, China) were divided into an experimental group and a control group (n = 6 each).11 Mice were sensitized and challenged as reported.15 All mice were sacrificed at day 28, and the appropriate samples were subsequently obtained.

Bronchoalveolar lavage fluid collection, cell counting and cytokines measurement

Forty-eight hours following the last challenge, the mice were sacrificed with 50 mg/kg of pentobarbital (Sigma P3761). Tracheotomies were performed, and 0.5 mL of pre-chilled phosphate buffer saline was instilled into the lungs; bronchoalveolar lavage fluid (BALF) was subsequently collected. The BALF was centrifuged, and the pellets were re-suspended in Hanks. To evaluate the differential cell count in BALF, we prepared BALF cells by cytospin. To determine the cell differentials, the smears were stained with a Diff-Quik solution (Propbs Scientific Ltd. China). Cells were counted under a microscope. Cells of four random sights were counted in each sample. The mean of the values from the four random sights was used for each cell count. The BALF supernatants were also collected; the levels of the cytokines interleukin 4 (IL-4), IL-5 and IL-13 in supernatants were measured via ELISA.

Lung tissue histopathology

Following BALF collection, the right lungs were removed and fixed in 4% formalin, processed and embedded in paraffin, and cut into three microns thick sections. The treated sections were stained with SIRT1 antibodies (Rabbit anti-mouse; 2028s, Cell Signaling Technology; 1:100 diluted) by streptavidin-peroxidase technique, and the nuclei were stained by hematoxylin.

Ribonucleic acid isolation and real-time reverse-transcriptase polymerase chain reaction analysis

The mouse left lungs were removed and washed with saline. The tissues were lysed using TRIzol reagent (Invitrogen); the total ribonucleic acid was purified according to the manufacturer's instructions. The primer sequences for SIRT1 were as follows: forward, 5′-TTCCAGCCGTCTCTGTGTCA-3′ and reverse, 5′-ATTCCTGCAACCTGCTCCAA-3′.11 The primer sequences for GAPDH were as follows: forward, 5′-TGAAGCAGGCATCTGAGGG-3′ and reverse, 5′-CGAAGGTGGAAGAGTGGGAG-3′.16

Statistical analysis

The Kolmogorov–Smirnov test was performed to determine the normality of distribution. Comparisons between two visits were analysed with the paired t-test. The interrelationships between different parameters were determined using the Pearson correlation. Data were presented either as the means ± standard error of the means or as n (%), unless otherwise stated. The analyses and graphs were completed using GraphPad Prism 6.0 software (http://www.graphpad.com/scientific-software/prism/). Statistical significance was set at a P-value < 0.05.

Patient characteristics

The clinical features and laboratory findings of the patients enrolled in this study are summarized in Table 1. The mean age of the patients with asthma was 43.78 years. There was no significant difference in gender composition between the patients with asthma and the controls or healthy participants. In regard to factors associated with asthma, there were significant differences between asthmatic patients and control subjects; for example, the asthmatic patients exhibited increased total serum IgE levels and percentages of peripheral blood eosinophils (500.3 ± 77.27 IU/mL and 4.586 ± 0.35%, respectively, P < 0.0001). The forced expiratory volume in 1 s (FEV₁) and FEV₁/forced vital capacity values of patients with asthma patients were also lower than that of control subjects (both P < 0.0001). There were no significant differences in smoking habits between the patients with asthma and the control subjects.

Serum SIRT1 in both the healthy individuals and the asthmatic patients

Among the 22 healthy teenagers we examined, the serum levels of SIRT1 were significantly higher than those of the adults. Among the 20∼60-year-old adults, there were no significant differences in the serum SIRT1 levels between different age groups (Fig. 1). Additionally, the serum SIRT1 levels were significantly higher in the patients with asthma patients than in the control subjects (2.819 ± 0.158 ng/mL vs 1.026 ± 0.022 ng/mL, respectively, P < 0.0001) (Fig. 2a). In the patients with asthma, the serum SIRT1 levels were not affected by smoking (Fig. 2b).

The correlation between serum SIRT1 and other clinical parameters

The total serum IgE levels and the predicted pulmonary function parameter of FEV₁/FVC%, two characteristics closely associated with asthma,17-19 were measured. In the patients with asthma, the Spearman's rank correlation analysis demonstrated that the serum SIRT1 levels correlated positively with the total IgE levels (r = 0.267, P = 0.008) (Fig. 3a; Table 2). The SIRT1 levels were also correlated negatively with pulmonary function (r = −0.29, P = 0.004) (Fig. 3b; Table 2). However, we did not observe a significant correlation between serum SIRT1 levels and other clinical parameters such as the levels of peripheral blood eosinophilia, gender or smoking habits (Table 2).

Cytokines expression and SIRT1 distribution in the OVA-sensitized and challenged mice

In the BALF of the OVA-sensitized and challenged mice, the numbers of eosinophils and lymphocytes increased; the expression levels of IL-4, IL-5 and IL-13 also increased significantly compared with those of the control group (Fig. 4a,b). We found that SIRT1 levels also increased in the serum of the OVA-sensitized and challenged mice (6.112 ± 0.751 ng/mL vs 2.459 ± 0.221 ng/mL, respectively, P < 0.01) (Fig. 4c), which was consistent with our findings in the human samples. However, both the immunohistochemistry and quantitative polymerase chain reaction (PCR) results demonstrated a decrease in the expression of SIRT1 in the lung tissues of the OVA-sensitized and challenged mice compared with those of the control group (Fig. 5).

SIRT1 is associated with aging and inflammation

Aging is an important contributing factor to the pathogenesis of many chronic inflammatory diseases.20, 21 In this study, serum SIRT1 levels remained elevated among healthy adolescents but decreased significantly among adults. We have not collected serum from individuals older than 60; however, in a recent study, Rahul Kumar et al. tested serum SIRT1 levels in young adults among post-graduate students and subjects between 65 and 75 years of age; they found that the serum SIRT1 levels of the elderly subjects were much lower than those of the younger subjects.22 SIRT1 is an anti-aging factor and has been proven to delay aging.23 SIRT1 expression decreases with increasing age; therefore, the protective effect it exerts on the tissue also decreases. Asthma is common among all age groups and has emerged as a significant problem for elderly people. Morphologic and physiologic abnormalities have been observed in aging human lungs, including decreased mucociliary function, air space dilatation, elastic recoil loss and elastin fibre loss, diminished diffusing capacity and evidence of low-grade inflammation of the respiratory tract.24 These changes may superimpose their effects upon those induced by asthma-related chronic inflammation of the airways, making aging a harmful factor in the evolution of airway inflammation.

SIRT1 is an effective deacetylase that contributes to the maintenance of normal lung function. SIRT1 was recently found to inhibit pro-inflammatory mediator release via Nuclear Factor Kappa-light-chain-enhancer of activated B cells in the setting of sustained inflammation in aging lungs.25 Transgenic mice with modest SIRT1 over expression that were receiving a high-fat diet demonstrated reduced levels of pro-inflammatory cytokines such as IL-6 and tumour necrosis factor-alpha.12 Conversely, myeloid cell-specific SIRT1 knockout mice demonstrated increased cytokine section when challenged with lipopolysaccharide.13 The SIRT1 activator, resveratrol, which exerts potent anti-inflammatory effects in cell culture (A549 lung epithelial cells),26 was also found to suppress asthmatic reactions;9 another SIRT1 activator, SRT1720, was found to exert similar effects in an OVA-induced asthmatic mouse model.11 These results suggest that SIRT1 may exert both protective effects and potent anti-inflammatory effects in mouse models of allergic airway diseases.

Serum SIRT1 correlated with IgE levels and pulmonary function and may serve as a diagnostic marker and therapeutic target for asthma

In our study, serum SIRT1 levels correlated positively with serum IgE levels and negatively with pulmonary function. The interaction between IgE and antigens causes a series of immunological reactions in asthma. Cross-linked IgE binds to mast cells via the high-affinity IgE receptor and triggers the subsequent transcription of cytokines, the synthesis of prostaglandins and leukotrienes and the release of preformed vasoactive mediators. These mediators rapidly induce mucosal oedema, mucous production and smooth muscle constriction, and result in the production of asthmatic inflammatory infiltrates and in bronchial hyperresponsiveness.27 Serum IgE levels increase in patients with severe asthma compared with those in patients with non-severely asthma, independent of the levels of allergic sensitization, which is considered an important asthma biomarker.28, 29 Additionally, a recent study suggested that the intake of resveratrol was associated with higher FVC levels;30 the authors of this study explained the mechanism by hypothesizing that resveratrol mediated SIRT1 activation. SIRT1 is helpful in sustaining normal lung function. When SIRT1 expression decreased, the ability of the lungs to resist damage reduced. High-serum SIRT1 may have an adverse effect on pulmonary function in the setting of asthma. Serum SIRT1 correlated with both IgE levels and pulmonary function, suggesting that serum SIRT1 possibly correlate with asthma severity.

SIRT1 decreasing in lungs aggravates the inflammation of asthma

In the present study, we demonstrated that serum SIRT1 levels increased in patients with asthma patients. In the OVA-sensitized and challenged mice, SIRT1 increased in the serum but decreased in the lung tissues coupled with eosinophils, lymphocytes, macrophage and neutrophils infiltration. We speculate that the high-serum SIRT1 levels were due to airway inflammation and the subsequent release of SIRT1 from inflammation cells. Ichikawa et al. reported that SIRT1 decreased in the lung homogenates of asthmatic mice,11 which is consistent with our findings. SIRT1 is a multifunctional molecule and is involved in a variety of pathways. The loss of SIRT1 may lead to multiple molecular pathway disorders and weaken the anti-inflammatory functions of various types of cells. A recent study demonstrated that lung SIRT1 levels are decreased in other lung inflammatory diseases such as COPD and emphysema.31, 32 As an anti-aging and anti-inflammatory factor, lung SIRT1 decreases may be responsible for the uncontrolled airway inflammation of asthma.

In summary, our findings indicated that serum SIRT1 varies with age. The change may be the result of changes in protein expression or the leakage of SIRT1 from the lung tissues. The anti-aging and anti-inflammatory roles of SIRT1 have been proven in other studies; decreased SIRT1 expression may result in adverse events in asthmatic lungs. Perspective studies will be necessary to determine whether the serum levels of SIRT1 decline in patients with asthma following an appropriate treatment. Although the exact function of SIRT1 in the setting of asthma remains unclear, our study indicates that high levels of serum SIRT1 are a biological characteristic of asthma; modulating SIRT1 with activators may be an attractive therapeutic strategy in the treatment of asthma.