SOD2 ameliorates pulmonary hypertension in a murine model of sleep apnea via suppressing expression of NLRP3 in CD11b⁺ cells

This study was ethic approved by the Institutional Review Board of Zhongshan Hospital and written informed consent was obtained from all patients. Blood were obtained from patients diagnosed with OSA in sleep center of Shanghai Zhongshan Hospital affiliated to Fudan University and healthy donators.

Mice were maintained and bred in Fudan University animal facility according to the National Institutes of Health Guidelines for the Humane Treatment of Laboratory Animals. All animal procedures have been approved by Fudan University institutional animal care and use committee in accordance with the Helsinki Declaration of 1975. Because Sod₂^−/− mice are neonatal lethal, heterozygous SOD2^−/+ mice were adopted in this study. C57bl/6 and SOD₂^−/+ mice were obtained as described from Nanjing model animal center [20, 21]. Mice were maintained on a 12:12- h night-day cycle, with standard mice chow and water available ad libitum. All experiments were conducted 6-week-old male mice in pathogen-free facilities.

The animals were weighed and randomly divided into two groups. One group was exposed to CIH. The other group was exposed to air and used as a control. An established rodent model of CIH was utilized [22]. Briefly, CIH mice were placed into a specially designed chamber, which contained a gas control delivery system to regulate the flow of oxygen and nitrogen into the chamber. During each 1-min period of intermittent hypoxia, the oxygen concentration in the chamber was adjusted between 5 and 21%. Nitrogen was introduced at a rate sufficient to achieve a fraction of inspired oxygen (FiO2) of 5% within 30 s and to maintain this level of FiO2 for 10 s; then, oxygen was introduced at a rate to achieve a FiO2 of 21% within 20 s. C57bl/6 mice were placed into this chamber for 9 h daily, 7 days per week, for 6 consecutive weeks. At all other times, the mice were kept in chambers with an oxygen concentration of 21%. The oxygen concentration in these chambers was continuously observed by an oxygen analyzer, and the levels were under feedback control by a computerized system connected to a gas valve outlet. Deviation from the determined settings was corrected by the addition of pure nitrogen or oxygen through solenoid valves. The control group was handled in the same manner as the CIH group, except oxygen concentration in the control chambers was maintained at a constant 21% throughout the experiment. The experimental protocol was approved by the local Animal Care and Use Committee of Fudan University.

Echocardiographic measurement is a valuable parameter to reflex the degree of PH. Briefly, mice were slightly anesthetized using isoflurane and analyzed within 2 h. Hair of the mice were moved for better scanning. Inhaled isoflurane was administered at 3% induction and 1–1.5% maintenance. After anesthetization, each mice was placed in the supine position on a temperature-controlled pad. All these echocardiography was handled by the skilled animal technician in animal heart ultrasonic laboratory. The probe of a Vivid S5 echocardiography system (Vivid S5, GE Healthcare, Chicago, IL) was gently be tilted laterally to obtain a view of the PH crossing over the aorta. After that, the ultrasound was switched to the color Doppler mode, parallel to the direction of blood flow in the vessel to obtain a flow waveform. Right ventricle cross-sectional area (RVCSA) and tricuspid annular plane systolic excursion (TAPSE), parameters correlating well with right ventricular function, were collected for further analysis.

Right ventricular systolic pressure (RVSP), conventionally used as an indicator of mean pulmonary arterial pressure, was measured by closed-chest puncture of the right ventricle (RV). After measurement through echocardiography, a longitudinal skin incision was made on the right side of the neck, and blunt layer-by layer separation of the tissues was performed until the right external jugular vein was exposed. A polyethylene catheter was gradually inserted into the pulmonary artery through an incision in the right external jugular vein, and the RV systolic pressure was recorded using a pressure transducer, which was interfaced to a BL-420S Bio Lab System (Chengdu TME Technology Co., Ltd., Chengdu, China). At the end of the experiment, the mice were anesthetized with an intraperitoneal injection of 3% sodium pentobarbital, and various organs were harvested. RV hypertrophy was evaluated as the ratio of the weight of the RV wall to that of the left ventricle plus septum.

For RNA extraction and quantitative real time-polymerase chain reaction (RT-PCR) analysis, total RNA was extracted from tissues using Trizol (Invitrogen, California, USA). High fidelity cDNA was generated from each RNA samples with Superscript III cDNA amplification System (Invitrogen). Quantitative RT-PCR reaction samples were prepared as a mixture with Quantitect SYBR Green PCR kit (Qiagen, Dusseldorf, Germany) following the manufacturer’s instructions. Reactions were performed using an Applied Biosystems Prism 9700 PCR machine. The PCR conditions were as follows: 95 °C for 30 s followed by 45 cycles of 95 °C for 15 s, 55 °C for 30 s and 72 °C for 30 s. For protein analysis, Western blot was carried out as previous described [23]. SOD₂ antibody was purchased from abcam Co. USA.

Formalin-fixed paraffin-embedded tissue sections were dewaxed, hydrated, heated for 10 min in a conventional pressure cooler, treated with 3% H₂O₂ for 30 min, and then incubated with normal fetal bovine serum for 30 min. Sections were then incubated with antibodies overnight (anti-NLRP3 diluted 1:200) after washing and then were incubated with biotin-labeled secondary antibody for 60 min at 37 °C after washing. The multiplicative quick score method (QS) were applied to assess the intensity and the extent of cell staining of NLRP3. In brief, the proportion of positive cells was estimated and given a percentage score on a scale from 1 to 6 (1 = 1–4%; 2 = 5–19%; 3 = 20–39%; 4 = 40–59%; 5 = 60–79%; and 6 = 80–100%). The average intensity of the positively staining cells was given an intensity score from 0 to 3 (0 = no staining; 1 = weak, 2 = intermediate, and 3 = strong staining). The QS was then calculated by multiplying the percentage score by the intensity score to yield a minimum value of 0 and a maximum value of 18. HE staining was conducted as previously described [24]. Masson staining was conducted as previously described [14].

PE-conjugated lineage specific antibodies (Ly6G), APC-conjugated CD11b antibody, PercPcy5.5-conjugated Ly6C were used in FACS (BD Pharmingen, USA). For FACS analysis, isolated cells were washed with phosphate-buffered saline and then incubated with anti-mouse antibody described above for 40 min at 4 °C to achieve specific binding. Single-cell suspensions were evaluated by multi-color flow cytometry using a LSRII flow cytometer (BD Biosciences). Data was analyzed using FlowJo 10 software (BD bioscience Inc. USA).

Antibody CD11b modification was followed by previous described [25]. MAG3-anti-CD11b was labeled with ^99mTc following the developed method [25]. Briefly, 50 μl lgMAG3-anti-CD11b conjugate was added to a solution of mixed 45 μl of 0.25 M ammonium acetate and 15 μl tartrate buffer, getting rid of physical movement, and then ^99mTc-pertechnetate generator eluate was added. Using acetone as the developing solvent, the labeling rate was measured by radio- TLC (Bioscan, Washington, DC, USA): 99mTc-MAG3-anti-CD11b antibody, Rf = 0; Na99mTcO4, Rf = 0.73. PD-10 columns were used for products purification with 0.25 M ammonium acetate as eluent, and the first radioactive product peak was recorded. Mice were prepared for SPECT/CT scanning. The parameter of FOV (Transaxial) is 80 mm, spatial resolution is less than 1.3 mm, FOV (Axial) is 60 mm, peak value is 226Kcps and sensitivity is 4.3% (380~640Kev), content rate of scattered radiation is 6 .3%.

CD11b⁺ cells were separated from C57BL/6 and SOD₂^−/+ mice using magnetic beads. Bone marrow cells and lung cells from C57BL/6 and SOD₂^−/+ mice was collected and made into single-cell suspensions. Anti-CD11b magnetic microbeads were used to obtain CD11b⁺ cells.

Brdu staining in this study was applied to measure the level of cell proliferation. Cells were covered with FBS for 3 days and then treated with Brdu (20 mM, CAT:558599) for 1 h in 37 C, and then washed. Cells in the plates were then treated with paraffin and incubated in HCl (1 M) for 10 min on ice, followed by HCL (2 M) for 10 min at room temperature and 20 min at 30 C. Neutralize by borate buffer solution 10 min at room temperature. After washed with phosphate buffer solution for three to five times, cells was observed by immunofluorescence microscope for brdu positive staining.

The following antibodies were used in current studies: CD11b antibody (abcam, Cambridge, USA); NLRP3 antibody (abcam, Cambridge, USA); Tissue samples from SOD₂^−/+/WT mice were fixed with 4% paraformaldehyde for 12 h followed by 30% sucrose overnight. Texas Red -conjugated rabbit-specific secondary antibody and FITC-conjugated mouse-specific secondary antibody (Biolegend, USA) were used for immunofluorescence analysis of NLRP3 and CD11b respectively. NLRP3 and CD11b fluorescence expression were observed and captured by Leica TCS SP8 (Germany).

Data from at least three independent experiments or five mice per group are represented as mean ± SD. Normality distribution was measured before using unpaired Student’s t tests. Comparisons between multiple groups were performed using ANOVA with the Bonferroni test. A P value < 0.05 was considered statistically significant. * meant p < 0.05; ** meant p < 0.01; ***meant p < 0.001.

We analyzed the expression of SOD2 of 117 participants in the blood to compare the deficiency of SOD2 in OSA groups and the control group. Blood samples were obtained from total 117 donators. Among them, 39 samples are from healthy donators, and 29 are diagnosed with mild OSA, 31 with moderate OSA, 18 with severe OSA. Male participants are more than the female in the group of OSA compared to the control group, while no difference in the age (Table 1). SOD2 was found to be decreased in the plasma in OSA patients, and the difference amplified expression of SOD2 was decreased in CIH mice (Fig. 1b), and the expression of SOD2 molecular in the blood was also decreased (Fig. 1c, d). We used heterozygous SOD2-/+ mice in this study, in which SOD2 expression was knocked down.

Six-week of CIH successfully induced PH just as our study declared before. In adult mice, RVSP was higher in SOD2-/+ by exposure to CIH for 6 weeks than WT mice (Fig. 2a, Additional file 1: Figure S1). Right ventricle hypertrophy index (ration of right ventricle (RV) to the left ventricle (LV) and septum (S)) in SOD2-/+ mice were significantly increased compared to those in WT mice (Fig. 2b). Echocardiographic analysis showed elevation in RVCSA in SOD2 knock-down mice, suggesting that diastolic dysfunction resulted from PH (Fig. 2c). Transthoracic echocardiography showed that exposure to CIH further increased the RV wall thickness (Fig. 2d, e), while transthoracic echocardiography confirmed this and also revealed further reduction of the peak velocity of blood flow in the pulmonary artery (Fig. 2f, g). The changes were exacerbated in the SOD2-/+ mice compared with the WT mice following exposure to CIH (Fig. 2d-g). TAPSE correlates well with other standardized parameters of right ventricular function. Echocardiographic analysis in this study showed that TAPSE was attenuated in SOD2-/+ mice compared to WT mice (Fig. 2h-i). Collectively, these findings showed that CIH induced PH with a relative RV hypertrophy in adult mice, and deficiency of SOD2 aggravated CIH by affecting RVSP, ratio of RV/(LV+S), RVCSA, thickness of the RV, blood velocity of pulmonary artery and TAPSE, which might lead to PH.

It is well known that remodeling of pulmonary blood vessels remains to be the main character of PH. Morphometric analyses of lung tissues showd by H&E staining of the pulmonary arterioles indicated that CIH induced thickness of vessel wall compared with the mice in the control group, whereas knock-down of SOD2 aggravated the CIH‐induced morphometric changes in the pulmonary arterioles (Fig. 3a). The hyperplasic state of collagen was observed by Masson collagen stain. Collagen volume fraction was greater in the CIH group, furthermore, this change was amplified in SOD2-/+ group (Fig. 3b). α‐SMA is a positive marker of smooth muscle cells and von Willebrand factor (VWF) is a key molecular in blood vessel formation. Double-immunofluorescence staining of α‐SMA and VWF was shown in Fig. 3c to indicate the classification of vessels muscularized. Percentage of area of α‐SMA+/(α‐SMA++VWF+) indicated the muscularization degree. Zero-20% represented for no muscularization (N), 20%-40% for partly muscularization (P), and 40%-100% for fully muscularization (M). Pulmonary vessel mascularization was showed in Fig. 3d statistically. CIH induced pulmonary vessel muscularization, and deficiency of SOD2 magnified this muscularization in pulmonary arterioles. Heart weight to tibia length ration showed the relative myocardial hypertrophy after deficiency of SOD2 (Fig. 3e). Medial vascular wall thickness was higher in the group of SOD2-/+ mice compared to WT mice under CIH condition showed by elastica van gieson staining (Additional file 1: Fig S2). Taken together, SOD2 could be an important factor of attenuating CIH‐induced pulmonary vessel muscularization, collagen hyperplasia, and smooth muscle progression in the lung.

Given that SOD2 modulated CD11b+ cells proliferation or mobilization, we analyzed the percentage of CD11b+ cells in the lung, blood and bone marrow. Results showed that percentage of CD11b+ cells was elevated in the blood, the lung as well as the bone marrow by flow cytometry analysis (Fig. 4a-j, e, g, i), which demonstrated that CD11b+ cells were largely proliferated in bone marrow and mobilized to the lung and peripheral blood under the condition of SOD2 deficiency. Among CD11b+ cells, monocytic myeloid cell line-Ly6C+Ly6G-cells were increased, while granulocytic myeloid cell line-Ly6C-Ly6G+ cells were decreased (Fig. 4f, h, j).

In vivo, MAG3-anti-CD11b was adopted to analyze the macroscopic distribution of CD11b+ cells in WT and SOD2 deficiency mice under CIH condition. Micro-PET scanning of in vivo 99mTc-labelled-MAG3-anti-CD11b showed that elevated statistically lung mean-SUV value and total reserved SUV in the lung of SOD2-/+ mice (Fig. 5a and Fig. 6c), which indicated more CD11b+ cells mobilized to the lung. Furthermore, more 99mTc accumulation in the heart of SOD2 deficiency mice compared to WT mice under CIH condition (Fig 5d-e). Taken together, CD11b, as an inflammatory marker, indicated deeper inflammatory injury under CIH in SOD2 deficiency mice. The visual change using radioactive marker is expected to be a biomarker in vivo to measure the degree of CIH related PH.

Deficiency of SOD2 aggravated CIH induced PH. Previous study revealed elevated activation of NLRP3 in PH and CIH model. We next indicated that NLRP3 was increased in SOD2 deficient mice under CIH condition. Figure 6a showed increased mRNA expression of NLRP3 in the group of SOD2-/+ after CIH. Figure 6b showed the measurement of histology score of IHC staining of NLRP3 in the group of SOD2-/+ was higher than the group of WT mice. IHC staining of NLRP3 in the lung showed that NLRP3 was mainly expressed in interstitial cells of the lung (Fig. 6c, e). Co-staining of CD11b and NLRP3 indicated that NLPR3 was mainly expressed in CD11b+ cells. Comparing to the group of WT mice, NLPR3+CD11b+cells was increased in the group of SOD2 deficiency mice under CIH condition (Fig. 6d, f). Thus, we put forward that SOD2 in CD11b+ might regulate CIH induced PH through NLRP3 pathway.

How do these elevated CD11b+ cells act on PASMC cells? We separated CD11b+ cells from bone marrow of SOD2 deficiency mice and WT mice under normal air and CIH condition using anti-CD11b magnetic beads. The CD11b+ cells gain rate was beyond 99% (Fig. 7a-c). To further ascertain the role of SOD2-deficient-CD11b+ cells involved in CIH induced PH model, we co-cultured SOD2-deficient-CD11b+ myeloid cells and PASMC cells using transwell plates (CD11b+ cells in the upper side and PASMCs in the lower side). Marked reduction of tunnel positive PASMCs after co-culture with SOD2-deficient-CD11b+ myeloid cells compared to the group of SOD2+/+ -CD11b+ cells (Fig. 7). Under condition of co-culture with SOD2-deficient-CD11b+ cells, Brdu positive cells were increased in PASMC cells compared with control (Fig. 7e). Expression of mRNA of NLRP3 in these CD11b+ cells and PASMCs were analyzed. It was further verified that NLRP3 was elevated in SOD2-deficient-CD11b+ myeloid cells but not in PASMCs (Fig. 7f-g). Secretion of IL‐1β and IL-18 were increased in the supernatant following the endogenous NLRP3 protein activation (Fig. 7h, i). Activation of the NLRP3 inflammasome is well known to be associated with the oxidative stress. The results indicated that SOD2-deficient-CD11b+ myeloid cells could activate NLRP3 inflammasome, aggravate proliferation of PASMC, and promote release of IL‐1β and IL-18, which led to the apoptosis resistance and over-proliferation of PASMC.