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Mitochondrial Autophagy and NLRP3 Inflammasome in Pulmonary Tissues from Severe Combined Immunodeficient Mice after Cardiac Arrest and Cardiopulmonary Resuscitation.

Byline: Jing-Jun. Lyu, Jawahar. Mehta, Yi. Li, Lu. Ye, Sheng-Nan. Sun, Hong-Shuang. Sun, Jia-Chang. Li, Dong-Mei. Zhang, Jie. Wei

Background: The incidence of cancer, diabetes, and autoimmune diseases has been increasing. Furthermore, there are more and more patients with solid organ transplants. The survival rate of these immunocompromised individuals is extremely low when they are severely hit-on. In this study, we established cardiac arrest cardiopulmonary resuscitation (CPR) model in severe combined immunodeficient (SCID) mice, analyzed the expression and activation of mitochondrial autophagy and NLRP3 inflammasome/caspase-1, and explored mitochondrial repair and inflammatory injury in immunodeficiency individual during systemic ischemia-reperfusion injury. Methods: A potassium chloride-induced cardiac arrest model was established in C57BL/6 and nonobese diabetic/SCID (NOD/SCID) mice. One hundred male C57BL/6 mice and 100 male NOD/SCID mice were randomly divided into five groups (control, 2 h post-CPR, 12 h post-CPR, 24 h post-CPR, and 48 h post-CPR). A temporal dynamic view of alveolar epithelial cells, macrophages, and neutrophils from bronchoalveolar lavage fluid (BALF) was obtained using Giemsa staining. Spatial characterization of phenotypic analysis of macrophages in the lung interstitial tissue was analyzed by flow cytometry. The morphological changes of mitochondria 48 h after CPR were studied by transmission electron microscopy and quantified according to the Flameng grading system. Western blotting analysis was used to detect the expression and activation of the markers of mitochondrial autophagy, NLRP3 inflammasome, and caspase-1. Results: (1) In NOD/SCID mice, macrophages were disintegrated in BALF, and many alveolar epithelial cells were shed at 48 h after resuscitation. Compared with C57BL/6 mice, the ratio of macrophages/total cells peaked at 12 h and was significantly higher in NOD/SCID mice (31.17 [+ or -] 4.13 vs. 49.69 [+ or -] 2.43, t = 14.46, P = 0.001). After 24 h, the results showed a downward trend. Furthermore, a large number of macrophages were disintegrated in the BALF. (2) Mitochondrial autophagy was present in both C57BL/6 and NOD/SCID mice after CPR, but it began late in the NOD/SCID mice. Compared with C57BL/6 mice, phos-ULK1 (Ser327) expression was significantly lower at 2 h and 12 h after CPR (2 h after CPR: 1.88 [+ or -] 0.36 vs. 1.12 [+ or -] 0.11, t = −1.36, P < 0.01 and 12 h after CPR: 1.52 [+ or -] 0.16 vs. 1.05 [+ or -] 0.12, t = −0.33, P < 0.01), whereas phos-ULK1 (Ser757) expression was significantly higher at 2 h and 12 h after CPR in NOD/SCID mice (2 h after CPR: 1.28 [+ or -] 0.12 vs. 1.69 [+ or -] 0.14, t = 1.7, P < 0.01 and 12 h after CPR: 1.33 [+ or -] 0.10 vs. 1.94 [+ or -] 0.13, t = 2.75, P < 0.01). (3) Furthermore, NLRP3 inflammasome/caspase-1 activation in the pulmonary tissues occurred early and for only a short time in C57BL/6 mice, but this phenomenon was sustained in NOD/SCID mice. The expression of the NLRP3 inflammasome increased modestly in the C57 mice, but the increase was higher in the NOD/SCID mice than in the C57BL/6 mice, especially at 12, 24, 48 h after CPR (48 h after CPR: 1.46 [+ or -] 0.13 vs. 2.97 [+ or -] 0.19, t = 5.34, P = 0.001). The expression of caspase-1-20 generally followed the same pattern as the NLRP3 inflammasome. Conclusions: There is a regulatory relationship between the NLRP3 inflammasome and mitochondrial autophagy after CPR in the healthy mice. This regulatory relationship was disturbed in the NOD/SCID mice because the signals for mitochondrial autophagy occurred late, and NLRP3 inflammasome- and caspase-1-dependent cell injury was sustained.


Cardiac arrest is the leading cause of death in the world. Its incidence is far higher than that of sepsis. Postcardiac arrest syndrome (PCAS) is the pathological process of a systemic ischemia-reperfusion injury (I/R injury) induced by cardiopulmonary resuscitation (CPR) after cardiac arrest. PCAS, which manifests as systemic inflammatory response syndrome, ends in multiple organ dysfunction syndrome, especially in immunosuppressed participants.[1] This phenomenon occurs relatively infrequently in healthy participants who receive CPR. However, the mechanisms underlying multiple organ dysfunction syndrome in immunosuppressed participants have yet to be fully characterized.

Mitochondria are extremely sensitive to I/R injury. Sudden elevation of reactive oxygen species (ROS) induces the mitochondrial permeability transition pore (mPTP) opening and mitochondrial polarization, which determines the fate of the cell.[2],[3],[4],[5],[6],[7],[8] Minor injuries lead to decreases in the mitochondrial membrane potential and mPTP opening, which subsequently causes mitochondrial autophagy. Mitochondrial autophagy not only selectively removes impaired mitochondria but also prevents the production of ROS by damaged mitochondria. Mitochondria autophagy ameliorates the inflammatory response.[9],[10],[11],[12],[13],[14],[15],[16],[17],[18],[19] However, the mechanism of mitochondrial autophagy in the progressive development of Cardiac Arrest-Systemic Inflammatory Response Syndrome (CA-SIRS) to Cardiac Arrest-Multiple Organ Dysfunction Syndrome (CA-MODS) after cardiac arrest remains unclear.

Clinical studies have shown that the survival rate after CPR in immunocompromised patients is significantly lower than that in individuals with normal immune function. We investigated mitochondrial autophagy and the NLRP3 inflammasome in the pulmonary system of NOD/SCID mice for 48 h after CPR.


Animal protocol

One hundred healthy adult male C57BL/6 mice of specific pathogen-free grade (weighing 22 [+ or -] 2 g, age 56-63 days) and 100 nonobese diabetic/severe combined immuno-deficient (NOD/SCID) adult male mice of surface plasmon resonance grade (weighing 24 [+ or -] 2 g, age 56-63 days) were provided by Beijing HuaFukang Bioscience Co., Inc. (Beijing, China), Production License Number: SCXK (Beijing) 2009-0004.

NOD/SCID mice were established in The Jackson Laboratory (Bar Harbor, ME, USA). SCID mice possess an autosomal recessive mutation developed in C.B-17 mice, which are a congenic and inbred strain of BALB mice. SCID mice share a similar genetic background with BALB/c mice. NOD/SCID mice are developed by reciprocal crossing with NOD/Lt mice at the Jackson Laboratory to decrease the natural killer (NK) cell activity. Thus, NOD/SCID mice, also known as immunodeficient mice, lack T lymphocytes and B lymphocytes and exhibit low NK cell activity.

The animals were maintained in the Experimental Animal Center of Renmin Hospital of Wuhan University (license number: S0271402213A). All experimental procedures were performed according to protocols approved by the Institutional Animal Care and Use Committee.

Experimental groups

C57BL/6 (100 mice) and NOD/SCID (100 mice) groups were each randomly divided into the control group (8 mice) and model groups (92 mice). After the return of spontaneous circulation (ROSC), the mice in the model group were further divided into four groups including groups for 2 h after resuscitation, 12 h after resuscitation, 24 h after resuscitation, and 48 h after resuscitation.

Cardiac arrest and cardiopulmonary resuscitation mouse model

Mice were weighed and anesthetized by an intraperitoneal injection of 3% pentobarbital sodium. The trachea was intubated with a 22-gauge catheter, and the mice were subjected to mechanical ventilation (respiratory rate: 100-130/min, tidal volume: 0.2 ml, and inspiratory-expiratory ratio: 1:1). During the surgical procedure, electrocardiogram (ECG) was recorded.

A mouse model of cardiac arrest induced by high potassium chloride was established. After the tracheal tube position was secured and the heart rate (HR) was >300 beats/min, a potassium chloride solution (50 [micro]l and 0.5 mol) at 1.6-2.0 [micro]l/g body weight was injected through the jugular vein to induce immediate cardiac arrest. Then, after the ventilator stopped for 150 s, chest compression was given manually for 3 min. If ROSC failed to appear at 3 min after CPR, resuscitation was discontinued. After CPR and ROSC, HR was continuously monitored, and assisted respiration was performed using a ventilator. After ROSC, the tracheal catheter and the ventilator were removed.

Cardiac arrest was characterized by (1) ventricular fibrillation, electromechanical dissociation, or ventricular standstill as shown by ECG, (2) disappearance of beats in the apical region of the heart, and (3) cyanosis of lips or limbs.

ROSC was determined by (1) normal ECG, (2) HR >200 beats/min, recovered respiration, and physiological reflex, (3) obvious cardiac impulse in the apical region of the heart, and (4) alleviated cyanosis of lips or limbs. Animals undergoing ROSC exhibited abdominal respiration even 5 min after removing the ventilator.

Bronchoalveolar lavage fluid analysis and measurement of cellular reactive oxygen species

The bronchoalveolar lavage fluid (BALF) was collected through an intratracheal cannula with three sequential 1 ml aliquots of 0.9% sterile saline (unclear), and the samples were centrifuged at 1500 rpm/min for 10 min at 4[degrees]C. The cell pellet was resuspended in 0.9% sterile saline for differential cell counts including identifying alveolar epithelial cells, macrophages, and neutrophils through Giemsa staining.

Phenotypic analysis of pulmonary interstitial macrophages by flow cytometry

The right lower lobe of lavaged lungs was minced with fine scissors and enzymatically digested with 0.1% Type I collagenase (Sigma-Aldrich, USA) for 1 h at 37[degrees]C with gentle agitation every 20 min. The single cell suspension was refiltered through a 40 [micro]m nylon mesh after digestion to remove connective tissue and then was washed with D-Hanks. Cell viability was determined with Trypan blue staining.

Phenotypic analysis of pulmonary interstitial macrophages was performed using flow cytometry. The gating strategy for analyzing interstitial macrophages was the same as in previous reports with some modifications.[20],[21],[22] In brief, the isolated cells were incubated with PE/Cy5-F4/80, PE-CD206, PE/Cy7-CD11c antibodies, and CD11b antibodies (BioLegend, San Diego, CA, USA) at the concentrations recommended in the manufacturer's protocol [Supplementary Figure 1 [SUPPORTING:1]]. After 15 min of incubation, flow cytometric analysis was performed to detect migrating macrophages (CD11b + CD11C −), macrophages in situ (CD11b − CD11C +), and macrophage debris (CD11b − CD11C −). Phenotypic analysis of the pulmonary interstitial macrophages was performed with a Cytomics FC500 Flow Cytometer (Beckman Coulter, USA) and analyzed with FlowJox 10.0.7r2 software (Treestar, Ashland, OR, USA).{Figure 1}

Cellular ROS was measured with a DCFDA Cellular ROS Detection Assay Kit (Abcam, Cambridge, MA, USA). Fluorescence of DCFDA was measured on the FL-1 channel with an excitation wavelength of 485 nm and an emission wavelength of 535 nm (FACS Vantage SE, Becton Dickinson, USA).[23] The data were recorded with the use of Flowing Software 2.0 as the 'M2 percentage' fluorescence variation, which indicates the percentage of cells with enhanced ROS production.[23]

Mitochondrial morphology and semiquantitative analysis in the lung tissues

Transmission electron microscopy (TEM) specimens were prepared as follows. Each cubic millimeter of fresh pulmonary tissue was fixed with 4% glutaraldehyde followed by osmium tetroxide. After being washed with phosphate-buffered saline, the tissues were dehydrated in an alcohol series. The embedding agent and 100% acetone were added at a 1:1 ratio for 1 h. The tissue pieces were placed into capsules for embedding and placed in an oven. Ultrathin sections were obtained, and lead citrate staining was performed. The resulting sections were subjected to examination for ultrastructure using a transmission electron microscope (H-7700, Hitachi, Japan).

The mitochondrial structure was semi-quantified using TEM and the Flameng grading system. In each animal, 100 mitochondria were randomly selected in five fields (20 mitochondria in each field). The mitochondrial structure was scored from 1 to 4 (the higher the level, the more severe the injury of mitochondrial structure), and the average score for each animal was determined from 100 scores.

The Flameng grading system is as follows: Score 0, intact mitochondria with mitochondrial granules; Score 1, the structure of the mitochondria remains intact, but the mitochondrial granules are absent; Score 2, swollen mitochondria with cleared matrix but intact cristae and membrane; Score 3, severe mitochondrial swelling, broken cristae with matrix clearing but membrane intact; and Score 4, the mitochondrial cristae are broken and the intact mitochondrial membranes disappear.

Western blotting analysis

Mitochondrial autophagy markers and regulatory molecules, i.e., ULK1, beclin-1, LC3-II, and their phosphorylated forms, were measured in the pulmonary tissues by Western blotting. NLRP3 inflammasome, pro-Casp-1, caspase-1p20, and cell apoptosis molecules from the mitochondria were released including Smac/Diablo, cytochrome c (cyto c), and BAX as well as glyceraldehyde-3-phosphate dehydrogenase (GAPDH) or beta-actin, which were also measured by Western blotting.

Primary antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA) or Abcam. The densities of protein bands were quantified using the Odyssey two-color infrared laser imaging system (Millipore, Billerica, MA, USA), and the band intensities were normalized to GAPDH or beta-actin.

Statistical analysis

The data were analyzed using SPSS 20.0 software (SPSS, Chicago, IL, USA). Measurement data verified for normality and homogeneity of variance are presented as the mean [+ or -] standard deviation and analyzed by t -test. Otherwise, the data are described as the median (25/75 percentiles) and analyzed by the Mann-Whitney U -test. The enumeration data were analyzed using the Chi-square test. P < 0.05 was considered to indicate statistical significance.


Return of spontaneous circulation and survival rates during 48 h after cardiopulmonary resuscitation

The ROSC rates and survival rates during the 48 h after CPR in C57BL/6 and NOD/SCID mice are shown in [Table 1]. Compared to C57BL/6 mice, NOD/SCID mice had a higher body weight, even at the same age, and they were resistant to tracheal intubation and intravenous administration. These animals had a higher ROSC rate that was not significantly different. Importantly, the survival rate of NOD/SCID mice was significantly lower than C57BL/6 mice at 48 h post-CPR (55.88% vs. 75.80%; ? 2 = 5.688; P < 0.01).{Table 1}

Giemsa staining of bronchoalveolar lavage fluid after cardiopulmonary resuscitation

The ratio of macrophages/total cells peaked at 2 h postresuscitation in C57BL/6 mice, and the ratio returned to baseline levels at 48 h after CPR. The ratio of neutrophils to total cell numbers continued to significantly increase. Compared to C57BL/6 mice, the ratio of macrophages/total cells peaked at 12 h and was significantly higher in NOD/SCID mice (31.17 [+ or -] 4.13 vs. 49.69 [+ or -] 2.43, t = 14.46, P = 0.001). After 24 h, the results showed a downward trend. Furthermore, a large number of macrophages were disintegrated in the BALF.

The ratio of neutrophils/total cells dramatically increased and remained elevated in C57BL/6 mice for 48 h [Figure 1]. The ratio of neutrophils to total cell numbers also significantly increased in NOD/SCID mice, but the increase was much smaller than in the C57BL/6 mice (9.12 [+ or -] 0.75 vs. 22.73 [+ or -] 1.34, t = 18.92, P = 0.001).

The ratio of alveolar epithelial cells/total cells also increased at all time points after ROSC in C57BL/6 mice [Figure 1]. The ratio of alveolar epithelial cells to total cell numbers also increased in NOD/SCID mice, but the increase was much smaller than in the C57BL/6 mice, especially at 24 and 28 h after ROSC (24 h after CPR: 42.53 [+ or -] 5.91 vs. 60.79 [+ or -] 5.30, t = 10.88, P = 0.001 and 48 h after CPR: 45.73 [+ or -] 4.41 vs. 71.55 [+ or -] 6.47, t = 15.91, P = 0.001).

Phenotypic analysis of macrophages in lung interstitial tissue with flow cytometry during 48 h after cardiopulmonary resuscitation

In the C57BL/6 mice, the number of migrating macrophages (CD11b + CD11c −) showed a gradual and sustained increase (25.75 [+ or -] 1.02 vs. 59.08 [+ or -] 4.24, t = 29.07, P = 0.001) for 48 h after CPR. Compared to C57BL/6 mice, the number of migrating macrophages derived from the bone marrow in the NOD/SCID mice peaked at 12 h after CPR and then gradually decreased at 48 h [Figure 2].{Figure 2}

Macrophage debris (CD11b − CD11c −) and macrophages in situ in the lung interstitium (CD11b − CD11c +) increased gradually in the NOD/SCID mice (macrophages in situ 4.00 [+ or -] 0.31 vs. 29.25 [+ or -] 2.72, t = 29.01, P = 0.001) after CPR, but the increase was short lived (up to 12 h) and then began to decline in the C57BL/6 mice [Figure 2].

Mitochondria morphological changes in pulmonary functions for 48 h after cardiopulmonary resuscitation

Representative TEM images of mitochondria in C57BL/6 and NOD/SCID mice are shown in [Figure 3]. The mitochondria were intact, the cristae were smooth and clear, and the stroma was homogeneous in the control groups of C57BL/6 mice and NOD/SCID mice. In C57BL/6 mice, mitochondrial swelling, cristae rupture, and vacuole formation were observed at 2 h after resuscitation. The mitochondria were swollen, the density of the matrix was reduced, and the ridge was rough at 12 h after resuscitation. At 24 h post-CPR, autophagosomes and autolysosomes were present. At 48 h after CPR, autolysosomes were observed, and mitochondrial damage was better than it was before in the C57BL/6 mice.{Figure 3}

No differences were observed in the morphology, size, and structure of mitochondria from C57BL/6 and NOD/SCID mice before CPR. In the NOD/SCID mice, mitochondria were condensed at 2 h post-CPR. At 12 h post-CPR, there was mitochondrial swelling, broken cristae, decreased matrix density, and mitochondrial vacuolation. The number of mitochondria with injury (less than Score 2) was significantly higher in NOD/SCID mice than that in C57BL/6 mice. At 24 h after CPR, a large number of mitochondria were destroyed and autophagosomes were rare. At 48 h after CPR, autophagosomes and extensive autolysosomes were present, and the number of damaged mitochondria was higher in the NOD/SCID mice than in the C57BL/6 mice [Figure 4].{Figure 4}

Semiquantitative evaluation (Flameng index) of lung tissues revealed that the mitochondrial injury was significantly higher in NOD/SCID mice than in C57BL/6 mice during the 48 h after CPR (at 12, 24, and 48 h after CPR) [Table 2].{Table 2}

Protein expression and activation of ULK1 during the 48 h after cardiopulmonary resuscitation

ULK1, which is a key protein for mitochondrial autophagy initiation, is regulated by the adenosine 5'-monophosphate-activated protein kinase (AMPK) and mammalian target of rapamycin (mTOR2) signaling pathway. It is phosphorylated by AMPK at Ser 327 and by mTOR at Ser 757. In addition, AMPK promotes autophagy in mitochondria, while mTOR2 inhibits autophagy.[24],[25],[26],[27],[28],[29],[30],[31]

Compared to the control group, the expression of ULK1 in C57BL/6 mice significantly increased at 2 h and reached its highest level 24 h after CPR. It is noteworthy that autophagy signals were triggered later in NOD/SCID mice than in the C57BL/6 mice.

Specifically, compared to C57BL/6 mice, phos-ULK1 (Ser 327) expression was significantly lower at 2 h and 12 h after CPR (2 h after CPR: 1.88 [+ or -] 0.36 vs. 1.12 [+ or -] 0.11, t = −1.36, P < 0.01 and 12 h after CPR: 1.52 [+ or -] 0.16 vs. 1.05 [+ or -] 0.12, t = −0.33, P < 0.01), whereas phos-ULK1 (Ser 757) expression was significantly higher at 2 h and 12 h after CPR in NOD/SCID mice (2 h after CPR: 1.28 [+ or -] 0.12 vs. 1.69 [+ or -] 0.14, t = 1.7, P < 0.01 and 12 h after CPR: 1.33 [+ or -] 0.10 vs. 1.94 [+ or -] 0.13, t = 2.75, P < 0.01).

These data indicate that the pro-autophagy signals were triggered later in NOD/SCID mice as compared to C57BL/6 mice, and the anti-autophagy signals were triggered earlier in NOD/SCID mice as compared to C57BL/6 mice [Figure 4]a.

Protein expression and activation of beclin-1 during 48 h after resuscitation

Beclin-1 is an important regulator of mitochondrial autophagy that is located downstream of the ULK1 promoter.[31],[32] AMPK phosphorylates beclin-1 to promote mitochondrial autophagy [Figure 5]a, [Figure 5]c.{Figure 5}

Compared to the control group, phos-Beclin-1 (Ser 93) increased gradually in both the C57BL/6 and NOD/SCID groups during the 48 h after resuscitation. No significant difference was observed in phos-Beclin-1 (Ser 93) between the C57BL/6 and NOD/SCID groups ( P > 0.05).

Detection of the mitochondrial autophagic flux for 48 h after cardiopulmonary resuscitation

We detected autophagic flux by Western blotting analysis including the ratio of LC3II/LC3I and LC3II/p62. The elevation of LC3II/LC3I represents the initiation of autophagy. LC3 II rises and p62 decreases simultaneously, indicating that the autophagy can be clear up; if LC3II and p62 rise, indicating that autophagy starts normally, but the downstream does not pass and phagosomes and lysosomes cannot fuse.

A Western blot revealed no difference in the expression of LC3B-II in C57BL/6 and NOD/SCID mice at baseline. Compared to the control groups, LC3B-II protein expression in lung tissues was significantly elevated at 12 h after CPR in C57BL/6 mice, and in NOD/SCID mice, and it further increased at 24 h and 48 h after CPR. At 12 and 24 h post-CPR, LC3B-II protein expression in lung tissues from NOD/SCID mice was lower than in C57BL/6 mice (12 h after CPR: 1.20 [+ or -] 0.21 vs. 2.42 [+ or -] 0.12, t = 4.24, P = 0.001 and 24 h after CPR: 1.04 [+ or -] 0.19 vs. 3.45 [+ or -] 0.11, t = 8.80, P = 0.001). Moreover, meanwhile, p62 protein expression was significantly elevated at 2 and 12 h after CPR in C57BL/6 mice, and in NOD/SCID mice, and it decreased dramatically at 24 h and 48 h after CPR [Figure 5]b, [Figure 5]d. Therefore, the mitochondrial autophagy pathway including the initial of autophagy and the formation of phagolysosomes were activated in both groups after CPR. However, mitochondrial autophagy was not fully activated in NOD/SCID mice.

Expression of Bax, Smac/Diablo, and cytochrome c during 48 h after cardiopulmonary resuscitation

Smac/Diablo, which is a 21-kDa mammalian protein released from mitochondria, competitively binds to inhibitor of apoptosis proteins (IAPs) and suppresses the inhibitory role of IAPs on the caspases. Thus, Smac/Diablo could promote apoptosis.

Cyto c is found in the inner membrane of the mitochondrion and is an essential component of the electron transport chain. During mitochondrial stress, cyto c is released from the mitochondrion and interacts with procaspase-9/Apaf 1. This complex activates caspase-9, which leads to caspase-3 activation and apoptosis.

Bax, which is an important pro-apoptotic factor, interacts with Bcl-2 to regulate apoptosis. A Western blot revealed that BAX protein expression peaked at 2 h after CPR and gradually decreased at 24 and 48 h after CPR in C57BL/6 mice. Bax protein levels gradually increased for 48 h after CPR in NOD/SCID mice [Figure 6]. In NOD/SCID mice, cyto c expression increased at 12, 24, and 48 h after CPR, peaking at 24 h post-CPR. The difference in cyto c expression in the two groups of mice was significant (1.10 [+ or -] 0.10 vs. 1.61 [+ or -] 0.19, t = 1.89, P < 0.01). Smac/Diablo expression increased in the lung tissues from NOD/SCID mice at 24 and 48 h after CPR but not in the C57BL mice ( P < 0.01).{Figure 6}

Activation of the NLRP3 inflammasome and caspase-1 after cardiopulmonary resuscitation

The expression of the NLRP3 inflammasome increased modestly in the C57 mice, but the increase was higher in the NOD/SCID mice than in the C57BL/6 mice, especially at 12, 24, and 48 h after CPR (48 h after CPR: 1.46 [+ or -] 0.13 vs.2.97 [+ or -] 0.19, t = 5.34, P = 0.001). The expression of caspase-1-20 generally followed the same pattern as the NLRP3 inflammasome [Figure 7].{Figure 7}

After CPR, the NLRP3 inflammasome and caspase-1 were continuously activated in NOD/SCID mice starting at 12 h post-CPR, leading to caspase-1-dependent cell pyroptosis, which is an event that can take place between apoptosis and necrosis. Consistent with these data, the amount of macrophage debris (CD11b − CD11c −) and the number of disintegrated macrophages were greater in the BALF at 24 and 48 h after CPR in the NOD/SCID mice than in C57BL/6 mice.


In rodent, porcine, and canine cardiac arrest models, respiratory failure occurs soon following CPR and the ROSC. The pulmonary function then recovers in 48-72 h after CPR in healthy animals. In immunocompromised animals, CPR induces a long period of severe respiratory failure with irreversible injury, which significantly decreases survival.[1],[33],[34] Immunocompromised patients also suffer from a high incidence of systemic inflammatory responses and severe multiorgan dysfunction as well as high mortality after CPR.

In our study, although a higher ROSC rate was observed in NOD/SCID mice, their survival rate was significantly lower than that of C57BL/6 mice over the 48 h post-CPR ( P < 0.05). These findings are similar to previous findings in animals and humans.[1],[33],[34]

We show that the lung tissue had a self-healing inflammatory response during the 48 h after CPR in C57BL/6 mice, which manifested as increase in the number of migrating macrophages and neutrophils. This outcome was similar to the response in participants with normal immune function that showed a Th1 inflammatory pattern of granulocyte mobilization from the bone marrow to the peripheral circulation, migration of neutrophils, and recruitment of monocytes to the lung tissue. All these exert anti-inflammatory effects. However, SCID mice showed a Th2 inflammatory pattern in which the macrophages proliferated in situ in the early stages of postresuscitation, followed by the disintegration of these macrophages. This disintegration was accompanied by the shedding of a large number of alveolar epithelial cells, which produced extensive injury to organs. Neutrophils are key effectors of the innate immune response. Reduced neutrophil migration to infection sites leads to lethal sepsis.[1],[33],[34] We have demonstrated a failure of neutrophil migration in SCID mice with poor outcomes after CPR.

In addition, our study demonstrated that the mitochondrial autophagy signaling pathway was initiated in mice with normal immune and immune-deficient mice after CPR. Compared to C57BL/6 mice, the mitochondrial autophagy signal was activated much later in NOD/SCID mice; however, the specific reasons are not clear.[35],[36],[37],[38],[39],[40] We further explored the reasons for delayed initiation of mitochondrial autophagy in the early stages of I/R injury in immunocompromised mice. Competitive phosphorylation of ULK1 is critical for the regulation of mitochondrial autophagy. AMPK directly phosphorylates ULK1 at Ser 327 and Ser 777 to induce autophagy in mitochondria with minor injuries. mTOR is a negative regulator of mitochondrial autophagy. Previous evidence suggests that stimulation with insulin-like growth factor leads to mTOR activation mediated by P13K/AKT signals to inhibit autophagy. Specifically, enhanced activity of mTOR phosphorylates ULK1 at Ser 757 to block the interaction between ULK1 and AMPK in energy-sufficient conditions.[24],[25],[26],[27],[28],[29],[30],[31] Our results showed that, compared to C57BL/6 mice, phos-ULK1 (Ser 327), which promotes autophagy, had significantly lower expression and phos-ULK1 (Ser 757), which inhibits autophagy, had significantly higher expression in NOD/SCID mice in the early stage of postresuscitation. Obviously, beclin-1, which is another important regulator of mitochondrial autophagy, was not as effective as we expected.

Our study demonstrated that, at the early stages of postresuscitation, C57BL/6 mice initiated mitochondrial autophagy and inhibited the overexpression of the NLRP3 inflammasome. Therefore, it reduced the excessive activation of caspase-1 in the late stages of postresuscitation. Thus, the immune-normal individuals showed a transient self-repair inflammation response in PCAS.

The NOD/SCID mice did not properly initiate mitochondrial autophagy in the early stages of postresuscitation, which resulted in mitochondrial ROS → NLRP3 inflammasome → caspase-1 → mitochondrial ROS, and this signal loop continued to amplify the stress signal, continued to the expand inflammatory response, and led to caspase-1-dependent cell pyroptosis.[41],[42],[43],[44],[45],[46],[47],[48],[49],[50],[51],[52],[53],[54] Specifically, the NLRP3 inflammasome continued to express caspase-1 and continued to activate the late stage of postresuscitation in NOD/SCID mice [Figure 8]. This result may partially explain why the survival rate of immunodeficient mice was significantly lower than that of mice with intact immune function in the 48 h following CPR.{Figure 8}

In conclusion, our research confirmed that (1) there is a negative regulatory relationship between the NLRP3 inflammasome and mitochondrial autophagy. For individuals with normal immune function, this relationship is the core part of PCAS that exhibits reversible self-repairable inflammatory responses, and (2) we did not thoroughly explain why NOD/SCID mice did not initiate mitochondrial autophagy properly in the early stages of postresuscitation. That was a limitation of this study.

Part of the reason for that limitation is that we are using NOD/SCID mice without using single gene knockout mice in a CPR model. The original intention is that we encountered immunodeficient patients, such as individuals with long-term use of glucocorticoids, organ transplant patients using anti-rejection drugs, or cancer patients receiving chemotherapy or radiotherapy, and immunodeficiency itself does not occur due to a single genetic defect leading to cellular immunodeficiency or humoral immunodeficiency.

Supplementary information is linked to the online version of the paper on the Chinese Medical Journal website.

Financial support and sponsorship

This work was supported by grants from the National Natural Science Foundation of China (No. 81372020), the Foundation of Young Talent Physician Training Project in Wuhan City (No. 2014ZX0001) and Guiding Foundation of Renmin Hospital of Wuhan University (No. RMYD 2018Z15).

Conflicts of interest

There are no conflicts of interest.


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Title Annotation:Original Article
Author:Lyu, Jing-Jun; Mehta, Jawahar; Li, Yi; Ye, Lu; Sun, Sheng-Nan; Sun, Hong-Shuang; Li, Jia-Chang; Zhan
Publication:Chinese Medical Journal
Article Type:Report
Date:Oct 1, 2018
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