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Abstract
Rationale: Neutrophils are key effectors in the host's immune response to sepsis. Excessive stimulation or dysregulated neutrophil functions are believed to be responsible for sepsis pathogenesis. However, the mechanisms regulating functional plasticity of neutrophils during sepsis have not been fully determined.
Objectives: We investigated the role of autophagy in neutrophil functions during sepsis in patients with community-acquired pneumonia.
Methods: Neutrophils were isolated from patients with sepsis and stimulated with phorbol 12-myristate 13-acetate (PMA). The levels of reactive oxygen species generation, neutrophil extracellular trap (NET) formation, and granule release, and the autophagic status were evaluated. The effect of neutrophil autophagy augmentation was further evaluated in a mouse model of sepsis.
Measurements and Main Results: Neutrophils isolated from patients who survived sepsis showed an increase in autophagy induction, and were primed for NET formation in response to subsequent PMA stimulation. In contrast, neutrophils isolated from patients who did not survive sepsis showed dysregulated autophagy and a decreased response to PMA stimulation. The induction of autophagy primed healthy neutrophils for NET formation and vice versa. In a mouse model of sepsis, the augmentation of autophagy improved survival via a NET-dependent mechanism.
Conclusions: These results indicate that neutrophil autophagy primes neutrophils for increased NET formation, which is important for proper neutrophil effector functions during sepsis. Our study provides important insights into the role of autophagy in neutrophils during sepsis.
Excessive stimulation or dysregulation of neutrophil functions is believed to be responsible for the pathogenesis of sepsis. However, the mechanisms regulating neutrophil plasticity during sepsis have not been fully determined.
Our results indicated that the autophagy pathway is responsible for proper neutrophil functions during sepsis and provide further rationale for investigating neutrophil autophagy as a novel therapeutic target in sepsis.
Sepsis is defined as a dysregulated host inflammatory response to life-threatening infections (1). The host's immune response to sepsis is considered to be characterized by an initial hyperinflammatory phase, followed by a protracted immunosuppressive phase (2, 3). Neutrophils are phagocytes that play important roles in the host’s inflammatory response to invading pathogens. Neutrophils can engulf and eliminate microorganisms via oxidative and nonoxidative mechanisms (4). These phagocytes are equipped with proteolytic granules that are highly toxic to invading pathogens (4) and that can release these granules. The granules are intertwined with web-like structures composed of DNA that form neutrophil extracellular traps (NETs) (5). Therefore, neutrophils are considered to be major players in the host's immune response to sepsis (2, 6).
The excessive stimulation of neutrophils is believed to be responsible for the development of multiple organ failures during sepsis (7). Neutrophils from patients with sepsis show an increase in reactive oxygen species (ROS) generation, which is highly toxic to host tissues (8). NETs also have potentially detrimental effects for host tissues during sepsis (9). Although NET formation induces the physical sequestration of pathogens from the host tissues and ensures the elimination of the invading pathogen, it can also deliver dangerous enzymes that can damage host tissues (10). Conversely, several studies have indicated that neutrophils are compromised during sepsis. Marked defects in neutrophil chemotaxis (11–13), ROS generation (14, 15), and bactericidal activity during sepsis have been observed (16, 17). Moreover, the transfer of donor granulocytes to recipient patients with sepsis decreases the sepsis severity and induces a marked improvement in sepsis biomarkers (18). These discrepancies regarding the role of neutrophils during sepsis reflect the functional plasticity of neutrophils during sepsis pathogenesis. However, the precise mechanisms regulating neutrophil plasticity during sepsis have not been fully elucidated.
Autophagy is a cellular degradation system responsible for clearing damaged or dysfunctional intracellular components (19, 20). Because the autophagy pathway is critical for cellular protection against external stress, the augmentation of autophagy can be protective against sepsis-induced dysfunction of organs, including the kidney (21), lung (22, 23), liver (23), and heart (24). Autophagy is also important for the effector functions of neutrophils. NET formation requires autophagy (25, 26); the depletion of NETs resulted in hypersusceptibility to sepsis in a mouse model of polymicrobial sepsis (27). In addition, recent studies indicated that increased autophagy induced the polarization of neutrophils toward the pro-tumoral phenotype (28) and identified a relationship between neutrophil autophagy and NET formation during sepsis (29).
In the present study, we hypothesized that autophagy was responsible for the activated phenotype of neutrophils in patients with community-acquired, pneumonia-induced sepsis. We observed a remarkable enhancement of autophagy in neutrophils isolated from patients who survived sepsis. The up-regulation of neutrophil autophagy was correlated with NET formation and granule release in response to subsequent stimulation. Moreover, neutrophils isolated from nonsurviving patients with sepsis showed impaired autophagy and decreased NET formation. In a mouse model of sepsis, the augmentation of autophagy protected the mice from lethal sepsis via a NET-dependent mechanism. These results suggest that the up-regulation of autophagy in neutrophils during sepsis is a novel mechanism that modulates neutrophil functional plasticity during sepsis. Some of these results have been previously reported in the form of a poster presentation (30).
This prospective study included patients admitted to the intensive care unit (ICU) of Kangdong Sacred Heart Hospital, Kyungpook National University Hospital, Kyung Hee University Medical Center, and Kyung Hee University Hospital at Gangdong between December 2013 and October 2016. Adult patients (age ≥ 18 yr) with sepsis in community-acquired pneumonia were enrolled. All study participants provided informed consent in accordance with the Declaration of Helsinki. Sepsis in community-acquired pneumonia was defined as previously described (31). Venous blood was taken from patients with sepsis within 24 hours after the ICU admission, and neutrophils were purified using histopaque (Sigma-Aldrich, St. Louis, MO) centrifugation followed by Dextran (Pharmacosmos, Holbaek, Denmark) sedimentation as previously described (32). Cells were resuspended in RPMI 1640 (Gibco, Carlsbad, CA) supplemented with 5% fetal bovine serum (Gibco).
Neutrophils were permeabilized and stained with primary monoclonal antibodies (mAbs) both LC3B-specific antirabbit mAb (Abcam, Cambridge, UK) and myeloperoxidase-specific antimouse mAb (Abcam) with secondary mAbs. Cells were counterstained with 4′,6-diamidino-2-phenylindole (Thermo Fisher Scientific Inc., Waltham, MA) and visualized by confocal microscopy (model LSM 710; Carl-Zeiss, Oberkochen, Germany).
Intracellular ROS were determined using a fluorescent probe, 2′7′-dichlorodihydrofluorescein diacetate (Thermo Fisher Scientific Inc.) as previously described (33). Extracellular NET formation was measured using Sytox Green (Thermo Fisher Scientific Inc.) as previously described (33). The fluorescence was measured using Spectramax M2/e fluorescence microplate reader (Molecular Devices, Sunnyvale, CA).
Neutrophils were fixed in 4% glutaraldehyde (Ted Pella Inc., Redding, CA) with 1% paraformaldehyde (EMS, Hatfield, PA) solution. Cells were further dehydrated, treated with propylene oxide (EMS), and embedded in Eponate 812 resin (Ted Pella Inc.). The sections were made using Ultra microtome and examined in a LEO 912AB EF-TEM (Carl Zeiss) at the Korean Basic Science Institute (Chuncheon, Republic of Korea).
BALB/c (male, 4–6 weeks old) mice were purchased from SAMTAKO (Osan, Republic of Korea). Procedures of animal experiments were approved by the Institutional Animal Care and Use Committee of Hallym University and Keimyung University. Experimental sepsis was induced by the cecal-ligation and puncture (CLP) procedure as previously described (34).
Data are presented as the means ± SD for continuous variables and the number (%) for the categorical variables, and were compared using the t test or χ2 test, respectively. A logistic regression model was used to adjust for potential confounding factors in the association between prognostic factors and ICU mortality. All tests were two-sided; P < 0.05 was considered statistically significant. Data were analyzed using the PASW statistics software version 22 (SPSS Inc., Chicago, IL). For the analysis of in vitro studies, statistical data were analyzed by GraphPad Prism 7.0 (GraphPad Software Ltd, San Diego, CA). Comparisons between two groups were performed with either a two-tailed Student's t test (parametric) or Mann-Whitney (non-parametric test). Comparisons between multiple groups were analyzed using one-way analysis of variance. The Mantel-Cox log-rank test was used for survival data. P values < 0.05 were considered statistically significant.
Forty-four patients with sepsis were included in the study. The sepsis etiology was restricted to community-acquired pneumonia. The demographic and clinical characteristics of the patients are summarized in Table 1. Thirty-one patients had comorbidities, and 17 patients had more than two comorbidities. The comorbidities did not significantly affect ICU mortality. The causative pathogens were identified in 27 patients (61.4%). The basic hematological and biochemical parameters of the patients with sepsis at the time of admission are described in Table E1 in the online supplement. Thirty-four patients (77.3%) used mechanical ventilators; among these patients, 17 (38.6%) received a tracheostomy. Three patients (6.8%) received extracorporeal membranous oxygenation therapy, and seven patients (15.9%) received renal replacement therapy (Table E2).
| Characteristics | No. of Patients (%) or Mean ± SD |
|---|---|
| Patients with sepsis (n = 44) | |
| Age, yr | 68.8 ± 15.9 |
| Sex, male | 31 (70.5) |
| SOFA score at admission | 9.5 ± 3.3 |
| APACHE II score at admission | 24.6 ± 8.1 |
| Identification of causative pathogens | |
| Identified | 27 (61.4) |
| Streptococcus pneumoniae | 6 (13.6) |
| Klebsiella pneumoniae | 7 (15.9) |
| Staphylococcus aureus | 3 (6.8) |
| Pseudomonas aeruginosa | 3 (6.8) |
| Escherichia coli | 2 (4.5) |
| Aspergillus | 1 (2.3) |
| Actinetobacter baumannii | 3 (6.8) |
| Pneumocystis jiroveci | 1 (2.3) |
| Adenovirus | 1 (2.3) |
| Unidentified | 17 (38.6) |
| Sepsis | 11 (25.0) |
| Septic shock | 33 (75.0) |
| Comorbidities* | |
| Diabetes | 11 (25.0) |
| Hypertension | 10 (22.7) |
| Chronic alcoholics | 4 (9.1) |
| Old pulmonary tuberculosis | 4 (9.1) |
| Chronic obstructive pulmonary disease | 8 (18.2) |
| Dementia | 5 (11.4) |
| Intracranial hemorrhage | 4 (9.1) |
| Stroke | 3 (6.8) |
| Parkinsonism | 3 (6.8) |
| Ischemic heart disease | 3 (6.8) |
| Solid cancer† | 2 (4.5) |
| Sleep apnea | 1 (2.3) |
Patient survival was monitored for 28 days after admission to the ICU to assess neutrophil functions during the early phases of sepsis. The mortality rate in the ICU was 22.7% (Table E4). Seven patients died due to the progression of the predisposing pneumonia, and three other patients died from acute myocardial infarction, refractory ventricular fibrillation, and acute stroke, respectively. The characteristics of the patients who survived and who did not survive sepsis in the ICU are compared in Table 2. In the univariate analysis, continuous renal replacement therapy and the lactic acid concentration were significantly associated with ICU mortality (Table 2). However, we did not find any factors associated with ICU mortality in the multivariate analysis (Table 3).
| Variables | Survivors (n = 34) | Nonsurvivors (n = 10) | Univariate Analyses OR (95% CI) | P Value |
|---|---|---|---|---|
| Age, yr | 69.5 ± 15.1 | 69.1 ± 19.4 | 0.986 (0.942–1.033) | 0.15 |
| Sex, male | 24 (70.6) | 7 (70) | 1.604 (0.286–9.012) | 0.28 |
| SOFA score | 9.1 ± 3.0 | 11.1 ± 4.1 | 1.192 (0.953–1.491) | 0.11 |
| APACHE score | 25.3 ± 7.6 | 26.7 ± 10.3 | 1.023 (0.934–1.121) | 0.15 |
| Septic shock | 9 (26.5) | 7 (70) | 0.825 (0.144–4.725) | 0.15 |
| CRRT | 3 (8.5) | 4 (40) | 8.533 (1.455–50.045) | 0.02 |
| WBC, 106/μl | 14.2 ± 7.3 | 10.0 ± 9.1 | 0.935 (0.203–4.875) | 0.40 |
| Hb, g/dl | 12.6 ± 1.9 | 12.0 ± 1.8 | 0.836 (0.552–1.265) | 0.39 |
| HCT, % | 37.8 ± 6.3 | 36.2 ± 4.5 | 0.953 (0.836–1.088) | 0.07 |
| Platelet, 105/μl | 2.3 ± 1.2 | 1.6 ± 1.0 | 1.414 (0.986–1.132) | 0.73 |
| Na, mEq/l | 135 ± 6.5 | 135 ± 5.0 | 0.997 (0.885–1.124) | 0.55 |
| K, mEq/l | 4.1 ± 0.8 | 4.3 ± 0.76 | 1.384 (0.533–3.594) | 0.92 |
| BUN, mg/dl | 23.9 ± 12.5 | 37.5 ± 21.5 | 1.053 (1.004–1.103) | 0.15 |
| Cr, mg/dl | 1.2 ± 0.8 | 1.62 ± 1.28 | 1.537 (0.754–3.129) | 0.063 |
| AST, IU/L | 5.95 ± 6.9 | 51.1 ± 1.2 | 1.006 (0.999–1.103) | 0.2 |
| ALT, IU/L | 42.1 ± 3.9 | 52 ± 6.7 | 1.009 (0.996–1.021) | 0.2 |
| Bilirubin, mg/dl | 1.12 ± 0.92 | 1.4 ± 1.2 | 1.328 (0.673–2.618) | 0.16 |
| BNP, pg/ml | 406.1 ± 50.7 | 436 ± 30.5 | 1.000 (0.999–1.002) | 0.57 |
| Lactic acid, mmol/l | 4.03 ± 2.89 | 8.1 ± 6.4 | 1.239 (1.035–1.483) | <0.001 |
| CRP, mg/dl | 99.9 ± 10.4 | 142 ± 36 | 1.003 (0.997–1.010) | 0.38 |
| Procalcitonin, ng/ml | 11.2 ± 3.7 | 21.3 ± 0.6 | 1.008 (0.989–1.028) | 0.18 |
| Arterial blood pH | 7.3 ± 0.14 | 7.18 ± 0.18 | 0.012 (0.000–1.255) | 0.47 |
| PaO2/FiO2 ratio | 136.7 ± 94.9 | 85.9 ± 34.6 | 0.987 (0.971–1.004) | 0.063 |
| Variables | Odds Ratio (CI) | P Value |
|---|---|---|
| CRRT | 8.19 (0.951–70.557) | 0.056 |
| Lactic acid, mmol/L | 1.225 (0.986–1.531) | 0.074 |
First, we evaluated the functions of neutrophils isolated from patients with sepsis. Neutrophils were isolated from surviving patients with sepsis (surviving sepsis neutrophils [SSNs]), nonsurviving patients with sepsis (nonsurviving sepsis neutrophils [NSNs]), age-matched controls (ACs), and healthy donors (healthy neutrophils [HNs]). The isolated neutrophils were exposed to various phorbol 12-myristate 13-acetate (PMA) concentrations for 2 hours, and then NET formation and ROS generation were examined. Compared with neutrophils from ACs and HNs, SSNs showed increased levels of PMA-induced NET formation (Figure 1A). The NSNs showed attenuated levels of NET formation compared with the SSNs (Figure 1A). Notably, compared with the HNs, ACs showed the attenuated NET generation, which was in accordance with the findings of a previous study (35). The SSNs also showed increased ROS generation in response to PMA stimulation compared with the ACs, whereas the NSNs showed attenuated ROS generation compared with the SSNs (Figure 1B).
Figure 1. Neutrophils isolated from surviving patients with sepsis were primed for neutrophil extracellular trap (NET) formation in response to phorbol 12-myristate 13-acetate (PMA) stimulation. (A–C) Neutrophils were isolated from healthy donors (healthy neutrophils [HN]; n = 20), age-matched controls (AC; n = 11), surviving patients with sepsis (surviving sepsis neutrophils [SSN]; n = 20), and nonsurviving patients with sepsis (nonsurviving neutrophils [NSN]; n = 11). The neutrophils were stimulated with various PMA concentrations for 2 hours. (A) NET formation in response to PMA stimulation was determined by Sytox Green staining. (B) Reactive oxygen species generation in response to PMA stimulation was determined by 2’,7’-dichlorofluorescin diacetate (DCF-DA) staining. (C) PMA-induced granule release was determined by surface expressions of granule markers. (D) Percentages of NET-releasing neutrophils (HN, n = 10; AC, n = 10; SSN, n = 10; and NSN, n = 7). (E) Confocal images of basal NET formation. Representative images of more than seven experiments are shown. Blue, 4’,6-diamidino-2-phenylindole (DAPI); green, myeloperoxidase (MPO). Scale bar, 10 μm. All results are expressed as means ± SEMs. *P < 0.05; **P < 0.01; ***P < 0.001. MFI = mean fluorescence intensity.
[More] [Minimize]We further examined whether neutrophils isolated from patients with sepsis were primed for increased granule release in response to PMA stimulation. To assess degranulation, the surface expressions of azurophilic granules (CD63), specific granules (CD66b), and secretory vesicles (CD35) were examined as previously described (32, 36). The basal expression levels of CD63 and CD35 were significantly increased in the SSNs compared with those in the HNs, ACs, and NSNs (Figure 1C). After PMA stimulation, the SSNs had higher levels of CD63 and CD35 surface expression than those of the HNs and ACs (Figure 1C). The NSNs showed attenuated surface expressions of CD63 and CD35 in response to PMA stimulation (Figure 1C). The individual values corresponding to each patient are shown in Figure E1 in the online supplement. The increased basal NET formation in the SSNs was further confirmed using confocal microscopy (Figures 1D and E2). The SSNs, but not the NSNs, showed a significant increase in basal NET formation compared with the HNs and ACs.
Collectively, these findings suggested the presence of different neutrophil functional phenotypes during sepsis and prompted us to further investigate the mechanisms underlying this phenomenon.
Previous studies have shown that autophagy plays a critical role in NET formation (25, 26, 37). Moreover, a recent study demonstrated autophagy of sepsis neutrophils using confocal microscopy (29). Therefore, we first evaluated autophagy in sepsis neutrophils using immunoblotting. The SSNs exhibited higher levels of the autophagy-specific protein light chain 3 (LC3) than the HNs and increased the conversion of LC3-I to LC3-II (Figure 2A). To evaluate the autophagic status in the SSNs, we examined the p62 protein levels in SSNs. The p62 protein level was significantly decreased in the SSNs compared with that in the HNs (Figure 2A). The mammalian target of rapamycin (mTOR) is a serine/threonine kinase that negatively regulates autophagy (28, 37). To determine whether the increase in autophagy in the SSNs was associated with the deactivation of mTOR signaling, we evaluated the phosphorylation of 4EBP-1 and p70S6K, which are the direct downstream substrates of mTOR signaling. However, p-4EBP-1 and p-S6K were not altered in the SSNs (Figure 2A). These results suggested that the increased autophagic status of the SSNs was due to an mTOR-independent pathway.
Figure 2. Autophagic status of sepsis neutrophils. (A) Immunoblotting analysis of the autophagy (light chain 3 [LC3] and p62) and mammalian target of rapamycin pathways (4E-BP1 and S6K) in surviving sepsis neutrophils (SSNs) and healthy neutrophils (HNs). Representative immunoblots. (B and C) Immunoblotting analysis of the autophagy pathways in nonsurviving neutrophils (NSNs), HNs, SSNs, and age-matched controls (ACs). (D) Quantitative densitometric analysis of immunoblots. Each dot represents an individual human. Means ± SDs are shown. (E) Autophagic flux assay. Neutrophils were exposed to 3-methyladenine (3MA) (10 μM), chloroquine (CQ) (10 μM), leupeptin (Leup) (20 μM), or bafilomycin (Baf) (10 μM) for 1 hour. Representative images of more than four experiments are shown (upper panel). Scale bar, 10 μm. The ratios of positive cells to an average of 100 cells are shown (lower panel). The positive cells were defined as cells with more than five LC3 dots. The results are expressed as the means ± SDs. HN, n = 5; AC, n = 5; SSN, n = 4; NSN, n = 4. *P < 0.05; **P < 0.01; ***P < 0.001. PMA = phorbol 12-myristate 13-acetate; Veh = vehicle.
[More] [Minimize]Next, we evaluated the autophagic status of the NSNs. Compared with the SSNs and the positive controls, the NSNs did not exhibit significant changes in either the total amount of LC3 or the conversion of LC3-I to LC3-II (Figure 2B). Moreover, compared with the SSNs, the NSNs exhibited impaired p62 degradation (Figure 2B and 2C). To evaluate the dysregulation of autophagy in the NSNs, we examined the autophagy-associated molecules in the sepsis neutrophils. Both the SSNs and NSNs showed a significant increase in Atg5, and Beclin-1, but there were no significant changes in the levels of Atg5-Atg12 and free-Atg12 levels (Figure 2C). This profile suggested impairment in the autophagosome formation or an increased autophagic flux in the NSNs. Therefore, we evaluated the autophagic flux in sepsis neutrophils using confocal microscopy.
Neutrophils were exposed to either 3-methyladenine (3-MA, 10 μM), chloroquine (CQ, 10 μM), leupeptin (Leup, 20 μM), or bafilomycin (Baf, 10 μM) for 1 hour. Consistent with a previous study (29), the SSNs showed increased LC3 fluorescence with a punctate staining pattern compared with the HNs (Figure 2E). An autophagy sequestration inhibitor, 3-MA, reduced the percentages of LC3 dot-positive cells in SSNs, whereas Leup (an inhibitor of the lysosomal protein degradation) and Baf (an inhibitor of autophagosome-lysosome fusion) increased the percentages of LC3 dot-positive cells (Figure 2E). These results suggested an increase in the induction and flux of autophagy in the SSNs. In contrast, the NSNs had a lower percentage of LC3 dot-positive cells than those in the SSNs (Figure 2E). The autophagosome degradation inhibitors (CQ and Leup) did not significantly enhance the percentages of LC3 dot-positive cells in NSNs (Figure 2E), whereas Baf slightly increased the percentages of LC3 dot-positive cells in NSNs. These results suggested that autophagosome formation was impaired in the NSNs.
We further confirmed autophagy in sepsis neutrophils using transmission electron microscopy. The SSNs had an increased occurrence of autophagic vacuoles (Figure 3, arrows), whereas no significant changes were observed in the NSNs.
Figure 3. Neutrophils isolated from surviving patients with sepsis showed increased autophagic vacuoles. Purified neutrophils were left untreated and examined by transmission electron microscopy. The healthy neutrophils (HNs) and age-matched controls (ACs) were normal in appearance with proper nuclear segmentation. The surviving sepsis neutrophils (SSNs) showed increased autophagic vacuoles with cellular disorganization. The inset shows enlarged images of autophagic vacuoles. The nonsurviving neutrophils (NSNs) did not show any significant changes. Representative images of more than four independent experiments. Arrow, autophagic vacuole; double arrow, isolating membrane extended from the Golgi apparatus (G); black scale bar, 2 μm; scale bar in inset, 500 nm.
[More] [Minimize]Next, we determined whether the induction of autophagy-primed neutrophils for PMA-induced NET formation. Neutrophils isolated from healthy donors were primed with either rapamycin (an mTOR-dependent autophagy inducer, 100 nM) or trehalose (an mTOR-independent autophagy inducer, 10 mM) and stimulated with PMA. Both the rapamycin- and trehalose-treated neutrophils exhibited increased NET formation in response to PMA stimulation (Figures 4A and 4B). Treatment with autophagy inhibitors (3-MA, CQ, Leup, and Baf) abrogated the priming effect of the autophagy inducers on NET formation (Figures 4A and 4B). In addition, diphenylene iodonium (an inhibitor of reduced nicotinamide adenine dinucleotide phosphate oxidase) treatment also completely abrogated the effect of the autophagy inducers on NET formation (Figures 4A and 4B). We also examined the effects of the autophagy inhibitors on LPS-induced NET formation. Consistent with a previous study (5), LPS treatment enhanced NET formation (Figure 4C). The LPS-induced NET formation was inhibited by all of the autophagy inhibitors, except diphenylene iodonium (Figure 4C). These results indicated that autophagic turnover is critical for proper NET formation in neutrophils.
Figure 4. Autophagy induction primes neutrophils for phorbol 12-myristate 13-acetate (PMA)-induced neutrophil extracellular trap (NET) formation. (A and B) Effects of autophagy inducers on PMA-induced NET formation in healthy neutrophils. NET formation in response to PMA stimulation was determined by Sytox Green staining. To inhibit autophagy pathways, the neutrophils were stimulated with PMA (1 μg/ml, 1 h) in the presence of either diphenylene iodonium (DPI) (1 μM), 3-methyladenine (3-MA) (10 μM), chloroquine (CQ) (10 μM), leupeptin (Leu) (20 μM), or bafilomycin (Baf) (10 μM). (A) The effect of rapamycin (Rp) on PMA-induced NET formation. The neutrophils were primed with Rp (100 nM, 1 h) and further stimulated with PMA in the presence or absence of autophagic inhibitors (n = 10 per group). (B) The effect of trehalose (Treh) on PMA-induced NET formation. The neutrophils were primed with Treh (10 mM, 3 h) and further stimulated with PMA in the presence or absence of autophagic inhibitors (n = 10 per group). (C) The effect of LPS on NET formation. The neutrophils were exposed to LPS (100 ng/ml, 2 h) in the presence or absence of autophagic inhibitors. (D) NET-induced autophagy in neutrophils. The neutrophils were exposed to either vehicle (Veh) or isolated NETs for 2 hours. The expression of autophagy-associated molecules (light chain 3 [LC3] and p62) was examined. All results are expressed as means ± SEMs. *P < 0.05; **P < 0.01; ***P < 0.001.
[More] [Minimize]To investigate the relationship between the autophagy machinery and NET formation, we evaluated whether the isolated NETs could induce autophagy in HNs. NETs were isolated from PMA-stimulated neutrophils as previously described (38), with slight modifications. The neutrophils were exposed to the isolated NETs for 2 hours, and then LC3 and p62 expression was examined in the NET-exposed neutrophils. The LC3 expression level and the conversion of LC3-I to LC3-II were significantly increased in the NET-exposed neutrophils (Figure 4D). In addition, the p62 expression level was decreased in the NET-exposed neutrophils (Figure 4D). These results suggested that autophagy primes neutrophils for NET formation and vice versa.
Next, we examined the effect of autophagy on mouse survival in an experimental sepsis model. BALB/c mice were treated with either rapamycin (100 mg/kg, intraperitoneally), trehalose (2 g/kg, intraperitoneally), or vehicle (saline, intraperitoneally) 1 hour before CLP surgery and on days 1 and 3 after CLP surgery. The rapamycin- and trehalose-treated mice showed significant increases in survival compared with the survival rate in the mice treated with the vehicle (Figure 5A). Next, we evaluated autophagy in the neutrophils isolated from mice with sepsis. The mice were treated with either rapamycin or vehicle 1 hour before CLP surgery and killed 18 hours after surgery. Neutrophils were isolated from the peritoneum and peripheral blood. Both peritoneal and blood neutrophils from the rapamycin- and trehalose-treated groups showed increased LC3 fluorescence with a punctate staining pattern compared with the neutrophils from the control CLP groups (Figure 5B). In addition, the blood neutrophils from the rapamycin-treated groups had lower mean lobe counts (Figure 5C). Similar to the neutrophils isolated from patients with sepsis, the blood neutrophils from the rapamycin-treated groups showed increased NET formation (Figure 5D) and decreased ROS generation (Figure 5E) in response to PMA stimulation compared with the neutrophils from the control CLP groups. We confirmed the effect of rapamycin on bone marrow–derived neutrophils from naive mice. Rapamycin-treated, bone marrow–derived neutrophils showed decreased mean lobe counts (Figure 5F) and enhanced NET formation in response to PMA and formylmethionylleucylphenylalanine stimulation (Figure 5G). No significant changes in ROS generation were observed (Figure 5H).
Figure 5. Autophagy augmentation improves sepsis survival in a murine sepsis model via increased neutrophil extracellular trap (NET) formation. (A) Survival rates after rapamycin (Rp), trehalose (Treh), or vehicle (Veh) administration in mice with cecal-ligation and puncture (CLP)-induced sepsis. BALB/c mice were administered an intraperitoneal injection of either Rp (100 mg/kg, intraperitoneally, n = 17), Treh (2 g/kg, intraperitoneally, n = 10), or Veh (saline, n = 24) 1 hour before CLP surgery and on Days 1 and 3 after CLP surgery. Arrows indicate intraperitoneal injections. *P < 0.05. (B–E) BALB/c mice were administered an intraperitoneal injection of Rp, Treh, or Veh 1 hour before CLP surgery. After 24 hours, the mice were killed, and blood and peritoneal neutrophils were isolated. *P < 0.05. (B) The presence of light chain 3 (LC3+ ) puncta in neutrophils from Rp- and Treh-treated CLP-induced mice with sepsis was analyzed using confocal microscopy. Representative images of more than three independent experiments are presented. (C) Mean lobe counts in the blood neutrophils (left panel) (n = 5 per group). Representative Wright-Giemsa images of blood neutrophils (right panel). (D and E) Blood neutrophils isolated from Rp-treated CLP-induced mice with sepsis showed increased NET formation in response to phorbol 12-myristate 13-acetate (PMA) stimulation. The isolated neutrophils were stimulated with PMA (1 μg/ml) for 2 hours, and then (D) NET formation and (E) reactive oxygen species production were quantified. (F–H) The effect of Rp on bone marrow neutrophils isolated from naive mice. Neutrophils were isolated from the bone marrow of naive mice (n = 20), exposed to Rp (100 nM) for 1 hour, and stimulated with various concentrations of PMA or formylmethionylleucylphenylalanine (fMLP). The (F) mean lobe counts, (G) NET generation, and (H) reactive oxygen species production were quantified (n = 5 per group). (I) The effect of NET depletion on the beneficial effect of Rp on the survival of mice with CLP-induced sepsis. The mice were treated intraperitoneally with either vehicle (Control, n = 15), recombinant human DNase (rhDNase) alone (DNase, n = 6), Rp alone (n = 17), or Rp + rhDNase (n = 14). *P < 0.05. Results are expressed as means ± SEMs. Scale bars = 10 μm. *P < 0.05; **P < 0.01; ***P < 0.001. DCF-DA = 2’,7’-dichlorofluorescin diacetate; LyG = lymphocyte antigen 6 complex locus G6D.
[More] [Minimize]Based on these results, we examined whether autophagy-induced NET formation protected the mice against sepsis-induced lethality. We examined the effect of recombinant human DNase (rhDNase) on the survival of the rapamycin-treated mice with sepsis. The mice were simultaneously treated with rhDNase (50 mg/kg, intraperitoneally) and rapamycin (50 mg/kg, intraperitoneally) or vehicle 1 hour before surgery and on days 1 and 3 after surgery. Consistent with a previous finding (27), NET depletion alone did not cause a significant difference in overall survival compared with the control CLP-induced mice with sepsis (Figure 5I). However, NET depletion completely abolished the beneficial effect of rapamycin on the survival of the mice with sepsis (Figure 5I).
Despite recent advances in understanding the role of autophagy in the pathophysiology of sepsis (21, 22, 24), little is known about neutrophil autophagy during sepsis. Recently, Kambas and colleagues described neutrophil autophagy during sepsis and evaluated the role of tissue factors (29). In the present study, we found that autophagy in neutrophils during pneumonia-induced sepsis played an important role in maintaining the innate effector functions of neutrophils. Neutrophils isolated from surviving patients with sepsis showed increased autophagy induction (Figures 2A–2D) through an mTOR-independent pathway (Figure 2A) with an increased autophagic flux (Figure 2E). These changes ensured that neutrophils would increase NET formation in response to subsequent stimulation (Figure 1A). Interestingly, neutrophils from nonsurviving patients with sepsis exhibited dysregulated autophagy (Figures 2B–2E) and impaired NET formation in response to subsequent stimulation (Figure 1A). The importance of autophagy for NET formation was confirmed using various autophagy inducers (Figures 4A–C), which suggested a critical role for autophagic turnover in NET formation. Finally, autophagy augmentation improved survival in a mouse model of sepsis (Figure 5A) in a NET-dependent manner (Figure 5I). Together, these results suggested a critical role for autophagy in the modulation of neutrophil functions during sepsis.
Although we found increased autophagic induction and flux in neutrophils isolated from surviving patients with sepsis, the mechanism by which autophagy was induced in these cells is unclear. Autophagy is a specialized effector mechanism that is activated in response to innate immune receptor activation. Various innate immune receptors ligands (i.e., the ligands for the toll-like receptors and nucleotide-binding oligomerization domain–like receptors), damage-associated molecular patterns, and pathogen-associated molecular patterns induce autophagy in innate immune cells (39, 40). Because neutrophils are activated by activation of these innate immune receptors during sepsis pathogenesis (41, 42), these signals might also be responsible for increased autophagy induction in neutrophils during sepsis.
Interestingly, we found impaired autophagosome formation in neutrophils from nonsurviving patients with sepsis. These cells exhibited increased expression of autophagic components, such as Atg5 and beclin-1 (Figure 2C), which suggested an increase in autophagy induction in neutrophils during sepsis. However, they did not exhibit significant changes in the conversion of LC3-II and p62 degradation (Figures 2B–2D). Moreover, the levels of free-Atg12 and the Atg5-Atg12 complex were not significantly changed (Figures 2C and 2D). The autophagic flux assay also found impaired autophagic flux in the NSNs (Figure 2E). Moreover, we did not observe a significant increase in autophagosomes in neutrophils isolated from the nonsurviving patients with sepsis (Figure 3). These results suggested that neutrophils isolated from nonsurviving patients with sepsis might have defects in sequestration via the elongating phagophores, despite an increased drive for autophagy induction.
Interestingly, a recent study suggested that the insufficiency of integral components of autophagosomes was responsible for frustrated autophagy in animal models of autophagy (43, 44). LC3, which is an integral protein involved in autophagosome formation (20), is required for the formation and elongation of phagophores (45, 46). However, LC3 is also involved in the trafficking of granules in innate immune cells. LC3-II colocalizes with CD63 and is released extracellularly upon stimulation in mast cells (47). Interestingly, we found low basal CD63 expression in the NSNs compared with that in the SSNs (Figure 1C). The NSNs also exhibited attenuated surface expression of CD63 upon PMA stimulation compared with the SSNs (Figure 1C). Moreover, the NSNs had an exhausted functional profile (Figures 1A and 1B). Therefore, the exhaustion of neutrophils during sepsis probably induces an insufficiency in the integral components for autophagy, which results in impaired autophagosome formation.
Neutrophils are a double-edged sword during sepsis pathogenesis. Excessive stimulation of neutrophils is believed to be detrimental to the hosts during sepsis (7, 8). Conversely, a number of studies have found dysregulated or paralyzed neutrophil functions during sepsis (11, 16, 48, 49). We believe that our results might explain the discrepancies in the effector functions of neutrophils during sepsis. In the present study, the NET-releasing capacity correlated with the autophagic state in neutrophils isolated from patients with sepsis (Figures 1 and 2). The induction of autophagy in neutrophils enhanced NET release in response to PMA stimulation (Figures 4A–4C). Moreover, the augmentation of autophagy increased survival in a murine model of sepsis (Figure 5A) via a NET-dependent pathway (Figure 5I). Therefore, the autophagic state of neutrophils might explain the discrepancies in the effector functions of neutrophils during sepsis. Supporting this hypothesis, a recent study found that the autophagic status mediated the neutrophil phenotype in the tumor microenvironment (28).
We also found that the augmentation of neutrophil autophagy in a mouse sepsis model enhanced survival via increased NET formation (Figure 5). Recent studies indicated that the augmentation of autophagy improved survival in a murine model of polymicrobial sepsis. Autophagy induction protected against the development of acute kidney injury (21), cardiomyocyte injury (24), acute lung injury (22), and hepatic injury (50) in a murine sepsis model. Notably, autophagy-modifying drugs improved the survival in a murine model of experimental sepsis (22, 23, 50), and LC3B-deficient mice were susceptible to LPS-induced mortality (51). Because autophagy was responsible for cellular protection by clearing damaged or dysfunctional intracellular components (19), these authors proposed that the beneficial effects of autophagy in sepsis were due to enhanced cellular protection against sepsis-induced tissue injury.
To the best of our knowledge, this study was the first to evaluate neutrophil autophagy in murine experimental sepsis. Unlike human neutrophils, murine neutrophils did not increase autophagy during sepsis (Figure 5B). Treatment with the autophagy-modifying drugs rapamycin and trehalose improved survival in mice with CLP-induced sepsis (Figure 5A) and enhanced autophagy in both blood and peritoneal neutrophils from these mice (Figure 5B). These neutrophils showed increased NET formation in response to subsequent stimulations (Figure 5D). Interestingly, rhDNase treatment completely reversed the beneficial effect of rapamycin on the survival of mice with sepsis (Figure 5I). A previous study investigated the effect of NET depletion in mice with sepsis (27) and showed that the depletion of NETs by rhDNase treatment did not improve the overall survival of mice with sepsis despite a marked improvement in median survival 24 hours after CLP surgery. Consistent with this finding, we observed that rhDNase treatment did not have any effect on the overall survival of the CLP-induced mice with sepsis (Figure 5I). However, rhDNase treatment completely reversed the rapamycin-induced survival enhancement in the mice with sepsis (Figure 5I). These results suggest that neutrophil autophagy is not normally induced in mice with sepsis. Thus, the augmentation of neutrophil autophagy may be critical for improved survival in mice with sepsis.
We found an autophagy-inducing role of NETs. Exposure to isolated NETs induced the conversion of LC3-I to LC3-II, and enhanced the degradation of p62 in HNs (Figure 4D). Emerging evidence suggests a role of NETs in crosstalk between neutrophils and other immune cells. NETs produced in atherosclerotic lesions prime macrophages for a proinflammatory phenotype (52). NETs also directly activated plasmacytoid dendritic cells (53) and prime T lymphocytes (54). Because massive NET formation is expected in the inflammatory environment during sepsis, excessive stimulation of neutrophils may be mediated by NETs in an autocrine and/or paracrine manner. Moreover, NETs released from neutrophils may activate surrounding immune cells, such as macrophages. Further study is needed to examine whether NETs induce autophagy in macrophages.
Although we obtained evidence that nonsurvivors of sepsis had dysfunctional neutrophils with dysregulated autophagy, we failed to find clinically relevant parameters that connected dysregulated neutrophil autophagy to survival in patients with sepsis. In the univariate analysis, we found significant differences in the lactic acid concentration and the continuous renal replacement therapy implementation frequency between the survivor and nonsurvivor groups (Table 2). Creatinine, blood urea nitrogen, and the PaO2/FiO2 exhibited a trend toward significant differences between the survivors and nonsurvivors (Table 2). However, no significant differences were found in our multivariate analysis. We believe that the relatively smaller sample size of our study and the assessment of initial laboratory data without serial follow-up were responsible for these results. Further study is needed to evaluate clinically relevant biomarkers for the evaluation of autophagic defects in sepsis neutrophils.
In conclusion, our study provided important insights into the function of neutrophil autophagy during sepsis. The induction of neutrophil autophagy enhanced NET production, which protected mice against sepsis-induced lethality. These results suggested that the augmentation of neutrophil autophagy is an attractive therapeutic target for the treatment of sepsis.
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*These authors contributed equally to this work.
Supported by grant NRF-2012R1A6A3A04040639 from the National Research Foundation of Korea (NRF).
Author Contributions: S.Y.P. and S.S. designed the research, performed data collection and analysis, and contributed to manuscript preparation. Y.-J.Y., J.-K.K., S.-Y.K., and W.-G.A. performed experiments. S.K. performed in vivo experiments; H.J.K., S.-H.P., M.G.L., K.-S.J., Y.B.P., E.-K.M., Y. Ko, S.-Y.L., Y. Koh, and M.J.P. performed patient enrollment and data collection; D.-K.S. contributed reagents and provided key advice in research design. C.-W.H. conceived and designed the research, analyzed the data, wrote the manuscript, provided financial support, and approved the final paper.
This article has an online supplement, which is accessible from this issue’s table of contents at www.atsjournals.org
Originally Published in Press as DOI: 10.1164/rccm.201603-0596OC on March 30, 2017
Author disclosures are available with the text of this article at www.atsjournals.org.
