Myeloid-Specific SIRT6 Deletion Protects Against Particulate Matter (PM_2.5)-Induced Airway Inflammation

Introduction

Airborne particulate matter (PM_2.5) pollution exposure is a major risk factor for global public health and known to cause many adverse health effects.¹ Epidemiological studies have documented that there is a close correlation between PM_2.5 exposure and increased incidence of respiratory diseases, such as asthma and chronic obstructive pulmonary disease.² PM_2.5 with aerodynamic diameter ≤ 2.5μm (PM_2.5) is a leading contributor to pulmonary inflammation because its major target organ is lung and can penetrate deep into the alveolar regions. PM_2.5-induced pulmonary inflammation might be seen as a critical process in mediating systemic adverse effects.³ Therefore, it is urgently needed to explore the molecular mechanisms responsible for PM_2.5-induced airway inflammation.

As a fundamental component of the respiratory immune system, alveolar macrophages (AMs), which reside at the boundary between the human body and outside world, are the most abundant macrophage in the lung. They clear dead cells and foreign antigen or airborne particles through the release of anti-inflammatory cytokines and the high phagocytic activity. Thus, AMs play a critical role in host defense, tissue homeostasis and control of airway inflammation.^4–6 However, the underlying mechanisms of AMs in regulation of PM_2.5-induced airway inflammatory remain unknown.

Acetylation is a widely occurring epigenetic modification of proteins that are involved in diverse biological processes.^7,8 In recent years, the physiological functions of the sirtuin deacetylase family (SIRT1-SIRT7) have been studied. Among the seven sirtuins, SIRT6 plays a leading role in regulating aging, inflammation, cancer and metabolic homeostasis.⁹ Accumulating evidences have revealed that SIRT6 widely participates in respiratory diseases, such as allergic airway inflammation,¹⁰ acute respiratory distress syndrome (ARDS),¹¹ chronic obstruction pulmonary diseases,⁹ and lung fibrosis.¹² Moreover, SIRT6 regulated macrophage polarization by activating the AMPK pathway and subsequent autophagy.^13,14 Although AMs are the first responders to ambient air pollution exposure in the airway, the possible role of SIRT6 in airway inflammation of macrophages following PM_2.5 exposure remains unclear.

In the present study, we sought to determine the underlying mechanisms of SIRT6 in the regulation of PM_2.5-induced airway inflammation in macrophages. Our findings demonstrated that myeloid-specific SIRT6 deletion protects against PM_2.5-induced airway inflammation and might suggest that inhibition of SIRT6 could prevent airway disorders induced by PM_2.5.

Materials and Methods

Animal Studies

The LysMcre mice were kindly donated by Dr G. Feng (University of California at San Diego, CA, USA). Using Sirt6^fl/fl mice, obtained from Jax Lab., we developed Myeloid cell-specific Sirt6 conditional knockout mice (Sirt6^fl/fl-LysMCre) by crossing Sirt6^fl/fl mice with the LysMCre mice. The primer sequences for the LysMCre and Sirt6 genes are shown in Table 1. A specific pathogen-free environment was maintained in the Laboratory Animal Center of Guangdong Medical University for all mice. A mouse model for short-term exposure to PM_2.5 was established according to a previous study.⁸ Standard reference airborne PM_2.5 was purchased from National Institute of Standards and Technology (NIST) Company. Briefly, PM_2.5 was suspended and sonicated in saline at 100 μg PM (in 50 μL saline) per day by intratracheal instillation for 3 days. The same volume of saline was given to control mice. Experiments were conducted under protocols using experimental procedures and anesthesia methods according to the ethical review of laboratory animal welfare People’s Republic of China National Standard GB/T 35892–2018. Experiments were also conducted under protocols using experimental procedures and anesthesia methods approved by the Animal Ethical Committee of Guangdong Medical University (No. GDY2003027).

Table 1 Sequence of Primers Were Used in This Study

Cell Cultures

Isolation and culture of BMDMs were carried out similarly to previous study.⁷ Briefly, from mice aged 6–8 weeks, BM cells were harvested from femurs and tibiae with PBS. To promote differentiation of bone marrow-derived macrophages, BM cells were cultured in DMEM medium supplemented with 10% FBS (vol/vol) and 10 ng/mL recombinant murine M-CSF for 7 days.

The human monocyte-derived macrophages cell line, THP1 cells were purchased from the Cell Bank of the Chinese Academy of Science (Shanghai, China). THP-1 cells were cultured in RPMI-1640 medium supplemented with 10% FBS (vol/vol) and 20 nM PMA over 48 h as primary human macrophages. Water-saturated atmosphere containing 5% CO₂ was routinely maintained at 37°C for the cell cultures above. PM_2.5, which contains polycyclic aromatic hydrocarbons, was purchased from National Institute of Standards and Technology (NIST) Company. We suspended and sonicated PM_2.5 in PBS or saline at a final concentration of 2 μg/μL (mass/vol), harvested BMDMs and THP-1 cells were treated with four concentrations of PM_2.5 (25, 50, 75, 100 μg/mL) for 24 h or with PM_2.5 (100 μg/mL) for four times (3, 6, 12, 24 h).

Collection of Bronchoalveolar Lavage Fluid (BALF)

BALF was performed according to our previous study.⁷ In brief, the lungs were instilled with 0.8 mL PBS after exsanguination. Cytospin slides were prepared by Wright-Giemsa staining, and cell counts in BALF were counted under the microscope in a blinded method.

RT-PCR Analysis

Total RNA of lung tissue or cells was isolated using the Trizol reagent (Invitrogen). Reverse transcription was performed via PrimeScript RT reagent Kit with gDNA Eraser (TAKARA) according to the manufacturer’s instructions. Quantitative PCR was carried out with SYBR Green PCR Master Mix (TAKARA). The primers are presented in Table 1.

Western Blot

Cells were lysed in RIPA buffer. Proteins in lysates of cells were separated by SDS-PAGE. Western Blot (WB) analyses were performed as described with antibody to SIRT6 (Santa Cruz), antibody to GAPDH, Tubulin and β-actin (Beyotime Biotechnology, China).

Enzyme-Linked Immunosorbent Assay (ELISA)

The levels of mouse IL-6, TNF-α, CXCL1, and CXCL2 in lung homogenate were measured using ELISA kit (Elabscience).

siRNA Studies

siRNA for knockdown of Sirt6 was synthesized by GenePharma, and the sequence (5’-3’) was as follows: UCCAUCACGCUGGGUACAUTT. siRNA was transfected to cells using lipofectamine^®3000 (Invitrogen), according to the manufacturer’s protocols with the following minor modifications. After 4–6 h of siRNA transfection, the transfected cells were exposed to PM_2.5.

Statistical Analysis

Results are presented as mean with SEM. We used the GraphPad Prism software (version 8.0, San Diego, CA) for all calculations and graphing. Comparisons between two groups were made using the Student’s t-test or Mann–Whitney U-test. One-way ANOVA was used for comparisons between more than two groups. Statistical significance was defined as a value of P less than 0.05.

Results

SIRT6 Expression is Increased in Macrophages Following PM_2.5 Exposure

To address the possible role of SIRT6 in PM_2.5-induced airway inflammation, we first assessed SIRT6 expression in the lung tissue of mice exposed to PM_2.5. A mouse model for short-term exposure to PM_2.5 was established as described in method. We found that the expression of SIRT6 was significantly increased in lung tissue of mice following PM_2.5 exposure compared with those in control groups (Figure 1A and Figure 1). To identify whether SIRT6 is macrophage specific, we first examined the expression of SIRT6 in THP1 derived macrophages exposed to PM_2.5. We found that treating THP1 cells with PM_2.5 resulted in a dose- or time-dependent increase of SIRT6 (Figure 1C–). Furthermore, we generated bone marrow–derived macrophages (BMDMs) from Sirt6^fl/fl mice. Consistent with the above findings, the expression of SIRT6 was significantly increased in PM_2.5-induced BMDMs in a dose- and time-dependent manner (Figure 1F–). Collectively, these findings suggested that macrophages are involved in SIRT6-mediated airway inflammation induced by PM_2.5.

Figure 1 PM2.5 exposure induced SIRT6 expression in macrophages. The expression of SIRT6 in lung tissues of mice was analyzed by RT-PCR (A) and Western blot (B). THP1 Cells were exposed to PM2.5 at indicated times or concentrations, SIRT6 expression was analyzed by RT-PCR and Western blot (C–E). BMDMs were exposed to PM2.5 at indicated times or concentrations, SIRT6 expression was analyzed by RT-PCR and Western blot (F–H). Data are presented as the mean ± SEM of three independent experiments. *P<0.05, **P<0.01, ***P<0.001, and ****P<0.0001. Abbreviations: PM, Particulate matter; SIRT6, Sirtuin 6; BMDMs, bone marrow–derived macrophages.

Inhibition of SIRT6 Attenuated PM_2.5-Induced Inflammatory Cytokines in vitro

Previous studies showed that PM_2.5 induces inflammatory cytokine secretion. In the present study, we also found that mRNA levels of tumor necrosis-alpha (TNF-α), interleukin 6 (IL6), C-X-C ligand 1 (CXCL1), and CXCL2 were significantly increased in PM_2.5-induced THP1 cells and BMDMs (Figure 2). We further investigated the role of SIRT6 gene silencing in THP1 cells on PM2.5-induced inflammatory cytokines (Figure 3A and Figure 3). SIRT6 gene silencing further increased PM_2.5 induced inflammatory cytokines compared with the control cells that received control siRNA (Figure 3C–).

Figure 2 PM2.5 induced inflammatory cytokines production in macrophages. THP1 cells and BMDMs were treated with PM2.5 for 4 hours at indicated concentrations (25, 50, 75, 100μg/mL). The mRNA expression of Il-6, Tnf-α, Cxcl1, and Cxcl2 were increased in THP1 cells (A–D) and BMDMs (E–H) was analyzed by RT-PCR. Data are presented as the mean ± SEM of three independent experiments. *P<0.05, **P<0.01, ***P<0.001, and ****P<0.0001. Abbreviations: PM, Particulate matter; BMDMs, bone marrow–derived macrophages; Il-6, interleukin 6; Tnf-α, tumor necrosis-alpha; Cxcl, C-X-C ligand.

Figure 3 Knockdown of SIRT6 reduced PM2.5-induced inflammatory cytokines in THP1 cells. THP1 cells were transfected with control siRNA and Sirt6 siRNA for 24 h, and then were treated with PM2.5 at 100 μg/mL for 4 h. SIRT6 expression was measured using Western blot (A) and RT-PCR (B). The mRNA expression of Il6, Tnf-α, Cxcl1, and Cxcl2 in THP1 cells were determined using RT-PCR (C–F). Data are presented as the mean ± SEM of three independent experiments. **P<0.01, ***P<0.001, and ****P<0.0001. Abbreviations: PM, Particulate matter; Il-6, interleukin 6; Tnf-α, tumor necrosis-alpha; Cxcl, C-X-C ligand.

To further confirm this effect, we generated mice with myeloid conditional deletion of Sirt6 (Sirt6^fl/fl-LysMCre). The genotyping results were determined by WB and RT-PCR analysis (Figure 4A and Figure 4). Representative genotyping results as shown in Supplementary Figure 1A. Moreover, double staining of macrophage (F4/80) and SIRT6 was also performed. Compared with the Sirt6^fl/fl mice, the expression of SIRT6 in macrophages was significantly decreased in PM2.5-exposed Sirt6^fl/fl-LysMCre mice (Supplementary Figure 1B and C). BMDMs were isolated from Sirt6^fl/fl-LysMCre and Sirt6^fl/fl mice. Consistent with the above findings, we found that the levels of IL-6, TNF-α, CXCL1, and CXCL2 were significantly decreased in Sirt6-deficient BMDMs compared with the control group (Figure 4C–). Collectively, these findings imply that the loss of SIRT6 in macrophages attenuated PM_2.5-induced inflammatory cytokine.

Figure 4 Sirt1-deficient BMDMs attenuated PM2.5-induced inflammatory cytokines. Genotyping was assessed in BMDMs from Sirt6fl/fl and Sirt6fl/fl-LysMCre mice using Western blot (A) and RT-PCR analysis (B). BMDMs were treated with PM2.5 at 100 μg/mL for 4 h to measure the levels of Il6, Tnf-α, Cxcl1, and Cxcl2 using RT-PCR (C–F). Data are presented as the mean ± SEM of three independent experiments. ***P<0.001 and ****P<0.0001. Abbreviations: PM, Particulate matter; Il-6, interleukin 6; Tnf-α, tumor necrosis-alpha; Cxcl, C-X-C ligand.

Sirt6^fl/Fl-LysMCre Mice Attenuated PM_2.5-Induced Airway Inflammation

Next, we further elucidate the role of SIRT6 in PM_2.5-induced airway inflammation in vivo. As described in method, a mouse model for short-term exposure to PM_2.5 was established using Sirt6^fl/fl littermates and Sirt6^fl/fl-LysMCre mice. As described in Figure 5A, compared with PM_2.5-induced Sirt6^fl/fl mice, SIRT6 expression in lung tissue was significantly decreased in Sirt6^fl/fl-LysMCre mice after PM2.5 exposed. The number of total cells, macrophage, neutrophil, lymphocytes and eosinophil were significantly decreased in BALF of PM2.5-induced Sirt6^fl/fl-LysMCre mice compared with those in Sirt6^fl/fl-LysMCre mice after PM2.5 exposed (Figure 5B–). Moreover, airway inflammation was significantly decreased in Sirt6^fl/fl-LysMCre mice exposed to PM_2.5 (Figure 5H and Figure 5). Compared with the Sirt6^fl/fl mice, both mRNA and protein expression of IL-6, TNF-α, CXCL1, and CXCL2 in the lung tissue were significantly decreased in PM2.5-exposed Sirt6^fl/fl-LysMCre mice (Figure 5J and Figure 5).

Figure 5 Conditional knockout of SIRT6 in macrophages attenuated PM2.5-induced inflammatory responses in mice. The expression of SIRT6 in the lung tissue was assessed by Western blot (A). The number of total cells (B), macrophage (C), neutrophil (D), lymphocytes (E) and eosinophil (F) in the BALF were measured by Flow cytometry. Representative images of macrophages and neutrophils in the BALF were analyzed using Giemsa’s staining (G, Scale bar: 100 μm). Representative images of lung sections with hematoxylin and eosin (H&E) staining (H, Scale bar: 100 μm). Semi quantification of inflammation expression in the lungs were performed using Image Pro 6.1 software (I). Il6, Tnf-α, Cxcl1 and Cxcl2 mRNA expression in lung homogenate were measured by QPCR (J–M). Protein concentrations of cytokines above in lung homogenate were measured by ELISA (N–Q). Data are presented as Mean ± SEM of three independent experiments (n=6 for each group). *P<0.05 and **P<0.01. Abbreviations: BALF, Bronchoalveolar lavage fluid; Il-6, interleukin 6; Tnf-α, tumor necrosis-alpha; Cxcl, C-X-C ligand.

Discussion

In the present study, we showed that SIRT6 expression was increased in PM_2.5-induced BMDMs, but Sirt6-deficient BMDMs decreased cytokine secretion when treated with PM_2.5. Moreover, mice with myeloid cells specific knockdown of SIRT6, display decreased airway inflammation. Collectively, our data suggested that inhibition of SIRT6 in macrophages may represent therapeutic strategy for PM_2.5-induced airway inflammation.

Sirtuin family, type III histone deacetylase (HDAC), consists of seven members. They participate in aging, inflammation, cancer and metabolic homeostasis by regulating cell cycle, cell differentiation, cell metabolism, cell senescence and death. Previous study revealed that SIRT6 might act as a double-edged sword in respiratory inflammatory diseases. SIRT6 has been found to play a protective role in allergen-induced inflammation by inhibiting IL-4-associating TH2 immune response, attenuating inflammatory cell recruitment, decreasing mucin production.^10,15 To identify whether SIRT6 is macrophage specific, we first examined the expression of SIRT6 in THP1 derived macrophages exposed to PM_2.5. Furthermore, we generated bone marrow–derived macrophages (BMDMs) from Sirt6^fl/fl mice. The expression of SIRT6 was significantly increased in PM_2.5-induced BMDMs and THP1 cells in a dose- and time-dependent manner. Double staining of macrophage (F4/80) and SIRT6 was also performed and confirmed that SIRT6 gene knockout is macrophage-specific. We found that inflammatory cytokines were decreased in BMDMs with myeloid-specific deletion of SIRT6 after stimulation of PM_2.5. In vivo, Sirt6^fl/fl-LysMCre mice substantially decreased airway inflammation in response to PM_2.5 exposure. According to previous studies, we found that the profiles of sirtuin deacetylase family in the myeloid cells are different in different diseases. Some sirtuin such as SIRT1, SIRT4, SIRT5, and SIRT7protect again airway inflammation, hepatocellular carcinoma development, pro-inflammatory response, or mycobacterial clearance in macrophages.^7,16–18 However, some sirtuin such as SIRT2 and SIRT3 promote chronic staphylococcal infection or inflammasome activation in macrophages.^19,20 Therefore, the exact functions and mechanisms of sirtuin members in different diseases need to be further elucidated.

Limitations

Our study does have some limitations. First, the use of simulated PMs in our study was purchased from National Institute of Standards and Technology (NIST) Company, which do not represent airborne PMs that contain more chemicals. Although we found that PMs could induce airway inflammation in vivo and in vitro experiments, it is true that the use of simulated PMs in our study does not represent airborne PMs that contain more chemicals. Second, macrophages are highly phagocytic that may affect the experimental results in our study. Therefore, the role of SIRT6 on PM_2.5-induced airway inflammation in macrophages should be interpreted cautiously due to limitations in our study.

Conclusion

Collectively, our study indicated that SIRT6 promoted PM_2.5-induced airway inflammation in macrophages and suggested that inhibition of SIRT6 in macrophages might be a strategy for treating the airway inflammation induced by PM_2.5.