INTRODUCTION
Acute lung injury (ALI) causes significant morbidity and mortality (1) (2, 3) (4) (5) (6)
Reports have suggested that various proresolving mediators, such as lipoxins, resolvins, and protectins, can regulate the inflammatory response (7) (8) (9) (8) in vivo and in vitro (9) (10) (11)
As we have known, neutrophils seem to play a major role in the development of ALI (12) (13) (14)
Apoptosis is believed to be a major mechanism for clearing neutrophils from sites of inflammation (15) (16) (17) (18) (19)
MATERIALS AND METHODS
Neutrophil isolation and culture
The human experimental protocol was approved by the Clinical Research Committee of Union Hospital and was in compliance with the Helsinki Declaration. All participants have signed an informed consent before blood collection. Blood from healthy volunteers was fractionated using a discontinuous density gradient of Percoll (MP Biochemicals, Calif) (20) 6 cells/mL, purity >95%, viability >98%) were resuspended in RPMI 1640 medium supplemented with 10% fetal bovine serum and containing 1% penicillin and streptomycin, preincubated with various concentrations of 7R,14S-dihydroxy- 4Z,8E,10E,12Z, 16Z,19Z–docosahexaenoic acid MaR1 (1–100 nM; Cayman Chemicals, Ann Arbor, Mich) and the pan-caspase inhibitor z-VAD-fmk (20 μM; InvivoGen, San Diego, Calif) for 30 min and then challenged with LPS (100–500 ng/mL; Sigma-Aldrich Co, St. Louis, Mo) at 37°C in 5% CO2 atmosphere.
Assessment of neutrophil apoptosis in vitro
Apoptosis was assessed by flow cytometry using annexin V–fluorescein isothiocyanate (FITC) apoptosis detection kit (eBioscience, San Diego, Calif). Activated caspase-3 in neutrophils was measured by flow cytometry using caspase-3 detection kit (FITC-DEVD-FMK; Calbiochem, Darmstadt, Germany).
Western blotting analysis
Protein was extracted from neutrophils using the protein extraction reagent kit (KeyGEN BioTECH, Nanjing, China) according to the manufacturer's protocol. Extracted proteins were separated by electrophoresis on 10% polyacrylamide sodium dodecyl sulfate gels and then transferred onto polyvinylidene fluoride membrane. The membranes were blocked with 5% nonfat milk for 60 min and then probed with antibodies to p-p38, Bcl-2, p-ERK1/2 (1:500; all from Santa Cruz Biotechnology, Santa Cruz, Calif), Mcl-1, p-AKT (1:1,000; both from Epitomics, Lausanne, Switzerland), and β-actin (1:1,000; Antgene Biotechnology, Wuhan, China). After overnight incubation with the primary antibodies, the membranes were incubated with goat-antirabbit or goat-antimouse antibody (1:3,000; Antgene Biotechnology) for 1 h. Protein was detected using chemiluminescence reagents. Images were scanned using UVP imaging system and analyzed by ImageJ software (Version 1.45s; National Institutes of Health, Bethesda, Md).
Murine ALI model
Ten-week-old male BALB/c mice (Hua Fu Kang Co, Beijing, China) weighing 20 to 25 g were housed at four per cage and maintained in a specific pathogen-free room with controlled temperature (22°C–24°C) and humidity (60%–65%) under a 12-h light/dark cycle. The mice were given standard laboratory chow and water ad libitum . All animal experiments were approved by the Animal Care and Use Committee of Tongji Medical College of Huazhong University of Science and Technology. Mice were anesthetized with 2% sodium pentobarbital (80 mg/kg, intraperitoneally; Sigma-Aldrich Co). Heart rate, body temperature, and toe pinch were consistently monitored to determine the depth of anesthesia. The animal experiments were divided into two parts. To investigate the effects of MaR1 on the resolution of the LPS-induced ALI, 80 mice were randomly assigned to the LPS-treated group (LPS) or LPS plus MaR1–treated group (MaR1). Acute lung injury was induced by intratracheal instillation of LPS at a dose of 3 mg/kg (11) normal saline (NS, 0.1 mL/mouse) were injected via the tail vein. The concentration of MaR1 used was based on the results of our preliminary experiments (11)
To study the mechanism of MaR1 on the resolution of the LPS-induced ALI, 128 mice were randomly assigned to the sham group (sham), LPS group (LPS), LPS plus MaR1 group (MaR1), and LPS plus MaR1 and pan-caspase inhibitor z-VAD-fmk group (MaR1 + z-VAD-fmk). After endotracheal intubation, mice were instilled with LPS (3 mg/kg) or NS (1.5 mL/kg). After 24 h, NS (0.1 mL/mouse) or MaR1 (1 ng/mouse) (11) (21)
Lung injury score assessment
At indicated times, mice were sacrificed by sodium pentobarbital overdose. Then, the right main bronchus was tied. The left lung was inflated at 15 cm H2 O with 4% paraformaldehyde and removed for paraffin embedding. Sections were stained with hematoxylin and eosin. Lung injury scores were determined by an investigator blinded to the treatment groups using recently published criteria (22)
Bronchoalveolar lavage analysis
Bronchoalveolar lavage fluid (BALF) was collected by flushing the lungs. Total cell counts in the BALF were measured by a hematocytometer. One part of each BALF sample was placed on a microscopic slide and centrifuged for 5 min at 1,000 rpm using the cytospin (Thermo Fisher Scientific, Waltham, Mass), and then BALF cells were stained with Giemsa (23, 24)
Measurements of BALF cytokines and pulmonary myeloperoxidase activity
The levels of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), IL-10, monocyte chemoattractant protein 5 (MCP-5), and macrophage inflammatory protein-1γ (MIP-1γ) in BALF were measured by enzyme-linked immunosorbent assays (RayBiotech, Norcross, Ga) according to the manufacturer's instructions. Lung tissues were homogenized in isotonic sodium chloride for analysis of myeloperoxidase (MPO) activities. Pulmonary MPO activity was assessed using the MPO kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China).
Assessment of neutrophil apoptosis in BALF
To assess neutrophil apoptosis in BALF, cells were labeled with APC-conjugated antimouse ly-6G antibody and FITC-conjugated anti–annexin-V antibody (eBioscience) for 30 min. The population of annexin-V+ ly-6G+ cells was determined by flow cytometry. Activated caspase-3 in BALF neutrophils was evaluated using APC-conjugated antimouse ly-6G antibody and caspase-3 detection kit. DNA cleavage was assayed by detection of cytoplasmic histone-associated DNA fragments (Roche, Laval, Quebec, Canada).
Statistical analysis
Data are expressed as mean ± SEM and analyzed using SPSS software version 16.0 (SPSS Inc, Chicago, Ill). Results grouped by time and treatment were analyzed by two-way analysis of variance. Other data were analyzed by one-way analysis of variance and followed by the least significant difference post hoc test. A value of P < 0.05 was considered statistically significant.
RESULTS
MaR1 prevented LPS-mediated inhibition of apoptosis in isolated human neutrophils
Experiments were performed with different concentrations of MaR1 to determine whether MaR1 directly affects the apoptosis of isolated neutrophils. The present data suggested that, at lower concentrations (1–10 nM), MaR1 had no effect on the apoptosis of isolated neutrophils. However, at a high concentration (100 nM), MaR1 prolonged neutrophil survival. To exclude the impact of MaR1 on neutrophil apoptosis, we used MaR1 concentration of less than 10 nM in subsequent experiments (Fig. 1, A and B). As shown in Figure 1, C and D, LPS suppressed neutrophil apoptosis in a concentration-dependent manner. The concentration of LPS, apparently necessary to inhibit neutrophil apoptosis, was 500 ng/mL. Furthermore, we investigated the role of MaR1 on the regulation of LPS-induced neutrophil survival. Although MaR1 prevented the inhibition of neutrophil apoptosis by LPS at concentrations up to 1 nM, apparent maximum inhibition was achieved at a concentration of 10 nM (Fig. 1, E and F). To assess the role of caspase-3 in neutrophil apoptosis, we used the pan-caspase inhibitor z-VAD-fmk. Preincubation of neutrophils with z-VAD-fmk attenuated the proapoptotic effect of MaR1 (Fig. 2, A and B). In addition, z-VAD-fmk inhibited the activation of caspase-3 in neutrophils (Fig. 2C).
Fig. 1: Maresin 1 prevented LPS-mediated inhibition of apoptosis in isolated human neutrophils.Neutrophils were isolated from whole blood and labeled with FITC–annexin-V and propidium iodide to determine neutrophil apoptosis by flow cytometry. A, The effect of MaR1 on neutrophil apoptosis. Human neutrophils were incubated with MaR1 (1, 10, or 100 nM) for 24 h. B, The percentage of apoptotic neutrophils in samples pretreated with MaR1 (1, 10, or 100 nM) for 24 h. C, The inhibitory effect of LPS on neutrophil apoptosis. Human neutrophils were incubated with LPS (100, 200, or 500 ng/mL) for 24 h. D, The percentage of apoptotic neutrophils in samples incubated with LPS (100, 200, or 500 ng/mL) for 24 h. E, MaR1 reversed LPS-mediated inhibition of neutrophil apoptosis. Human neutrophils were cultured with MaR1 (1 or 10 nM) for 30 min and then incubated with LPS (500 ng/mL) for 24 h. F, The percentage of apoptotic neutrophils in samples pretreated with MaR1 and incubated with LPS (500 ng/mL) for 24 h. Data are means ± SEM (n = 3 per group). **P < 0.01 vs. control group. ## P < 0.01 vs. LPS group. & P < 0.05 vs. LPS + MaR1 (1 nM) group.
Fig. 2: The pan-caspase inhibitor z-VAD-fmk overrode the neutrophil proapoptotic effects of MaR1.Human neutrophils were cultured with MaR1 (10 nM) and z-VAD-fmk (20 μM) for 30 min and then incubated with LPS (500 ng/mL) for 24 h. A, Z-VAD-fmk attenuated the proapoptotic effects of MaR1. Samples were labeled with FITC–annexin-V and propidium iodide to determine neutrophil apoptosis by flow cytometry. B, Percentage of apoptotic neutrophils. C, Z-VAD-fmk inhibited the activation of caspase-3. Samples were labeled with FITC–caspase-3 to determine the expression of caspase-3 by flow cytometry. MFI indicates mean fluorescence intensity. Data are means ± SEM (n = 3 per group). *P < 0.05, **P < 0.01 vs. control group. # P < 0.05, ## P < 0.01 vs. LPS group. ^ P < 0.05, ^^P < 0.01 vs. MaR1 group.
MaR1 inhibited the activation of signaling pathways that regulate neutrophil apoptosis
To investigate the effect of MaR1 on the intracellular signaling pathways that mediate neutrophil apoptosis, we studied the activation of mitogen-activated protein kinase (MAPK) and phosphoinositide 3-kinase (PI3K). The present data suggested that LPS could trigger the phosphorylation of AKT, ERK, and p38 to generate the survival signals for neutrophils. In contrast, MaR1 inhibited the LPS-induced phosphorylation of AKT, ERK, and p38, triggering proapoptotic signals for neutrophils (Fig. 3, A–D). Next, we also examined the roles of the antiapoptotic regulators Bcl-2 and Mcl-1 in neutrophil apoptosis. As shown in Figure 3, E to G, the expressions of Bcl-2 and Mcl-1 increased rapidly in neutrophils cultured with LPS, but this effect was abolished by pretreatment with MaR1.
Fig. 3: Maresin 1 inhibited the activation of signaling pathways that regulate neutrophil apoptosis.Human neutrophils were cultured with MaR1 (10 nM) for 30 min and then incubated with LPS (500 ng/mL). A, Western blot analysis showing inhibition of AKT, ERK, and p38 phosphorylation by MaR1 within 30 min. Beta-actin was used as a loading control. Representative results from three independent experiments were shown. Density analysis revealed that LPS enhanced the phosphorylation of AKT (B), ERK (C), and p38 (D), which was blocked by MaR1 treatment. E, Western blot analysis showing the effect of MaR1 in downregulating Bcl-2 and Mcl-1 expression within 2 h. Beta-actin was used as a loading control. Representative results from three independent experiments were shown. Density analysis revealed that LPS enhanced the expression of Bcl-2 (F) and Mcl-1 (G), which was inhibited by MaR1 treatment. Data are means ± SEM (n = 3 per group). *P < 0.05, **P < 0.01 vs. control group.# P < 0.05, ## P < 0.01 vs. LPS group.
MaR1 accelerated the resolution of LPS-induced ALI
Broncheoalveolar lavage fluid cytology showed significant changes in the numbers of several cell types (Fig. 4, A–C). Before LPS instillation, most of the cells in BALF were alveolar macrophages. The number of neutrophils in BALF increased significantly after LPS administration, peaking at 24 h, and then declining dramatically until they were effectively absent by day 7 (Fig. 4B). Pathohistological changes included disrupted alveoli, hemorrhage, thickness of alveolar septum, and infiltration of inflammatory cells after LPS administration (Fig. 4D). At day 7, cytological analysis indicated that alveolar structures and the degree of inflammatory infiltration had normalized somewhat, consistent with the observed changes in the number of BALF neutrophils. Lung injury scores in the LPS group were significantly increased on days 1 and 2 (Fig. 4E). In addition, we also evaluated the degree of pulmonary edema by measuring the amount of BALF protein and the lung wet/dry weight ratio. These data indicated similar degrees of lung injury in LPS-treated mice (Fig. 4, F and G). Overall, neutrophil infiltration, alveolar disturbance, and pulmonary edema peaked at day 1 and gradually recovered to normal by day 7. Hence, LPS instillation resulted in neutrophil-mediated ALI, which resolved within 7 days without any treatment.
Fig. 4: Maresin 1 accelerated the resolution of LPS-induced ALI.At 24 h after intratracheal instillation of LPS (3 mg/kg), mice were administered NS or MaR1 (1 ng/mouse) intravenously and sacrificed on days 0, 1, 2, 4, and 7. Lung tissues were evaluated for pathohistological changes and lavaged for analysis of BALF. A, Total BALF cells, (B) BALF neutrophils, (C) BALF monocytes/macrophages, (D) lung tissue sections from control mice (day 0), mice with inflammation induced by LPS (day 1), and mice with inflammation induced by LPS and treated with MaR1 or saline (days 2, 4, and 7). Hematoxylin and eosin stain, (magnification, 200 ×). E, Lung injury scores, (F) BALF protein, (G) Lung wet/dry weight ratio. Data are means ± SEM (n = 8 per group). *P < 0.05; **P < 0.01 vs. LPS group.
To confirm the proresolving effects of MaR1, MaR1 was given at the peak of inflammation in the LPS-induced ALI model. Our data showed that the number of alveolar neutrophils in MaR1-treated mice peaked on day 1 and then declined to normal by day 4 (Fig. 4B). Furthermore, we found that the number of macrophages increased gradually in MaR1-treated mice until day 2, after which the number declined to normal by day 7. The number of alveolar macrophages was higher in MaR1-treated mice than that in LPS-treated mice on days 2 and 4 (Fig. 4C). Histological changes in MaR1-treated mice differed from those in LPS-treated mice and included disturbed alveoli and neutrophil infiltration, which gradually alleviated and recovered to normal at day 4. Lung injury scores correlated with the pathohistological changes (Fig. 4E). Pulmonary edema was attenuated by MaR1 (Fig. 4, F and G). These data suggested striking evidence of accelerated resolution in MaR1-treated mice.
MaR1 prevented LPS-induced neutrophil survival in the ALI model
To measure neutrophil apoptosis in BALF, cells were labeled with FITC–annexin-V and APC–ly-6G antibodies, a specific cell surface marker for mouse neutrophils. Maresin 1 treatment significantly increased neutrophil apoptosis in comparison with the LPS and MaR1 + z-VAD-fmk groups (Fig. 5, A and B). Apoptosis was also quantified using a cytoplasmic histone-associated DNA fragment assay, which also revealed a marked increase in neutrophil apoptosis in MaR1-treated mice (Fig. 5C). Activation of caspase-3, which is crucial for the initiation, propagation, and execution of apoptosis, was assessed to elucidate the mechanism of neutrophil apoptosis. Activation of caspase-3 by MaR1 correlated with neutrophil apoptosis (Fig. 5, D and E).
Fig. 5: Maresin 1 promoted neutrophil apoptosis in LPS-induced ALI.At 24 h after LPS intratracheal instillation, MaR1 (1 ng/mouse) or NS was administered intravenously. At the same time, some mice were injected intraperitoneally with the pan-caspase inhibitor z-VAD-fmk (10 μg/kg in 0.2 mL of saline) followed by two additional doses of z-VAD-fmk 4 and 8 h later. Broncheoalveolar lavage fluid was collected by flushing the lungs, and mice were sacrificed at 24 h after MaR1 treatment. A, Flow cytometric analysis of neutrophil apoptosis. Cells in BALF were labeled with APC–ly-6G and FITC–annexin-V antibodies. Apoptotic neutrophils (ly-6G+ Annexin-V+ ) were indicated in the upper right quadrant. Numbers in each quadrant represented the percentage of cells within each quadrant. B, Percentage of apoptotic neutrophils. C, Apoptosis of BALF cells was assayed by detection of cytoplasmic histone-associated DNA fragments. D, Expression of activated caspase-3 in neutrophils. Cells in BALF were labeled with APC–ly-6G and FITC–caspase-3 antibodies. E, Representative bar graph illustrating caspase-3 activity in neutrophils. MFI indicates mean fluorescence intensity. F, MaR1 increased the number of apoptotic neutrophils available for the macrophages to phagocytize (arrows). Asterisks denoted the neutrophils in BALF. G, Percentage of macrophages containing apoptotic bodies in BALF. Data are means ± SEM (n = 8 per group). *P < 0.05, **P < 0.01 vs. sham group. ## P < 0.01 vs. LPS group. && P < 0.01 vs. MaR1 group.
Next, we evaluated whether MaR1 promotes macrophage phagocytosis. Maresin 1 treatment increased the number of macrophages containing apoptotic bodies, as determined by light microscopy (Fig. 5, F and G). In contrast, treatment with the pan-caspase inhibitor z-VAD-fmk suppressed neutrophil apoptosis, caspase-3 activation, and macrophage phagocytosis of apoptotic bodies. This series of experiments demonstrated that MaR1 promoted the resolution of inflammation in caspase-dependent neutrophil apoptosis.
The protective effects of MaR1 were partially abolished by the pan-caspase inhibitor z-VAD-fmk
To investigate whether neutrophil apoptosis plays a pivotal role in enhancing the resolution of LPS-induced ALI, we applied the pan-caspase inhibitor z-VAD-fmk in the LPS-induced ALI models. Intraperitoneal administration of z-VAD-fmk attenuated the protective effects of MaR1 in reducing neutrophil accumulation (Fig. 6B), mitigating histopathological changes (Fig. 6, D and E) and alleviating pulmonary edema (Fig. 6, F and G) in LPS-induced lung inflammation. As shown in Figure 6C, the elevation in the number of BALF monocytes/macrophages in MaR1-treated mice correlated with the number of macrophages containing apoptotic cells and with neutrophil apoptosis. Myeloperoxidase activity in lung tissues is considered an index of neutrophil infiltration. Consistent with the number of neutrophils in BALF, pulmonary MPO activity in MaR1-treated mice was lower than that in LPS-treated mice, but the MaR1-associated suppression of MPO activity was partially abolished by treatment with z-VAD-fmk (Fig. 6H).
Fig. 6: The protective effects of MaR1 were partially abolished by the pan-caspase inhibitor z-VAD-fmk.At 24 h after LPS intratracheal instillation, MaR1 (1 ng/mouse) or NS was administered intravenously. At the same time, some mice were injected intraperitoneally with the pan-caspase inhibitor z-VAD-fmk (10 μg/kg in 0.2 mL of saline) followed by two additional doses of z-VAD-fmk 4 and 8 h later. Broncheoalveolar lavage fluid was collected by flushing the lungs, and mice were sacrificed at 24 h after MaR1 treatment. Total BALF cells (A), BALF neutrophils (B), BALF monocytes/macrophages (C), and lung tissue sections (D) from the sham group, LPS group (induced by LPS), MaR1 group (induced by LPS and treated with MaR1), and MaR1 + z-VAD-fmk group (induced by LPS and treated with MaR1 and z-VAD-fmk). Hematoxylin and eosin stain (magnification, 200 ×). E, Lung injury scores. F, BALF protein. G, Lung wet/dry weight ratio. H, Lung MPO activity. Data are means ± SEM (n = 8 per group). **P < 0.01 vs. sham group. ## P < 0.01 vs. LPS group. && P < 0.01 vs. MaR1 group.
In addition, we detected the production of both proinflammatory and anti-inflammatory cytokines in BALF, which suggests that the balance between opposing cytokine mediators plays an important role in determining the course of LPS-induced ALI. The levels of most of the proinflammatory cytokines examined (e.g., including TNF-α, IL-1β, MCP-5, and MIP-1γ) were significantly lower in MaR1-treated mice compared with those in LPS-treated mice (Fig. 7, A, B, D, and E). The level of the anti-inflammatory cytokine IL-10 slightly increased in the MaR1-treated mice relative to the level in LPS-treated mice (Fig. 7C). In contrast, z-VAD-fmk enhanced the production of the proinflammatory cytokines and inhibited the production of the anti-inflammatory cytokine IL-10 (Fig. 7, A–E).
Fig. 7: Maresin 1 reduced the production of proinflammatory cytokines and upregulated the production of anti-inflammatory cytokine IL-10.At 24 h after LPS intratracheal instillation, MaR1 (1 ng/mouse) or NS was administered intravenously. At the same time, some mice were injected intraperitoneally with the pan-caspase inhibitor z-VAD-fmk (10 μg/kg in 0.2 mL of saline) followed by two additional doses of z-VAD-fmk 4 and 8 h later. Broncheoalveolar lavage fluid was collected by flushing the lungs, and mice were sacrificed at 24 h after MaR1 treatment. A, BALF TNF-α. B, BALF IL-1β. C, BALF IL-10. D, BALF MCP-5. E, BALF MIP-1γ. Data are means ± SEM (n = 8 per group). **P < 0.01 vs. sham group. ## P < 0.01 vs. LPS group. & P < 0.05, && P < 0.01 vs. MaR1 group.
DISCUSSION
Using a combined in vitro and in vivo approach, we demonstrated that MaR1 overcomes the LPS-induced suppression of neutrophil apoptosis by inhibiting the activation of survival signal pathway in vitro and accelerated the resolution of LPS-induced ALI through promoting the caspase-dependent neutrophil apoptosis in vivo .
The control of neutrophil apoptosis is central to homoeostasis and resolution of inflammation (25, 26) in vitro using isolated human neutrophils. Our results showed that LPS, a toll-like receptor-4 agonist, delayed neutrophil apoptosis. Toll-like receptors play a central role in innate immunity by mediating pathogen-associated molecular pattern recognition, as reflected by the increased susceptibility to infections of toll-like receptor transduction deficiency (27) (16) in vitro study indicated that treatment with MaR1 prevented LPS-mediated suppression of neutrophil apoptosis.
Neutrophil apoptosis is controlled by a complex network of signaling pathways, including the PI3K, MAPK pathways, and Bcl-2 family (28–31) (32, 33) c and other proapoptotic proteins (25) (34)
In present study, we used an intratracheal LPS instillation murine model that exhibited some physiological and biological similarities with human ALI (22) (35) (36)
Apoptosis of inflammatory neutrophils is a critical control point for termination of the inflammatory responses (16) (17) (37)
The pan-caspase inhibitor z-VAD-fmk partially blocked the protective effects of MaR1 in our experiments, supporting the hypothesis that MaR1 promotes the resolution of LPS-induced ALI by enhancing neutrophil apoptosis. Because of the partial blockade by z-VAD-fmk, we speculated that MaR1 would have other biological effects on resolution in addition to promotion of neutrophil apoptosis, which did not depend on apoptotic signaling pathways. Considering all findings, we concluded that MaR1 promoted the resolution of LPS-induced ALI primarily through promotion of caspase-dependent neutrophil apoptosis.
In conclusion, our in vitro data demonstrated that MaR1 protected against LPS-mediated suppression of neutrophil apoptosis by inhibiting phosphorylation of AKT, ERK, and p38, downregulating the expression of Bcl-2 and Mcl-1, and activating caspase-3. Our in vivo study also revealed that MaR1 promoted the resolution of LPS-induced ALI by enhancing caspase-3–dependent neutrophil apoptosis. Taken together, our results indicate that MaR1 is a potentially useful new therapeutic agent for treating ALI.
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